CN1183810A - 具有降低吸附增强水解作用的枯草杆菌蛋白酶dy变异体 - Google Patents
具有降低吸附增强水解作用的枯草杆菌蛋白酶dy变异体 Download PDFInfo
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Classifications
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L12/00—Methods or apparatus for disinfecting or sterilising contact lenses; Accessories therefor
- A61L12/08—Methods or apparatus for disinfecting or sterilising contact lenses; Accessories therefor using chemical substances
- A61L12/12—Non-macromolecular oxygen-containing compounds, e.g. hydrogen peroxide or ozone
- A61L12/124—Hydrogen peroxide; Peroxy compounds
- A61L12/126—Hydrogen peroxide; Peroxy compounds neutralised with catalase or peroxidase
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- A—HUMAN NECESSITIES
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- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D17/00—Detergent materials or soaps characterised by their shape or physical properties
- C11D17/0047—Detergents in the form of bars or tablets
- C11D17/0065—Solid detergents containing builders
- C11D17/0069—Laundry bars
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D17/00—Detergent materials or soaps characterised by their shape or physical properties
- C11D17/0047—Detergents in the form of bars or tablets
- C11D17/0065—Solid detergents containing builders
- C11D17/0073—Tablets
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D17/00—Detergent materials or soaps characterised by their shape or physical properties
- C11D17/0047—Detergents in the form of bars or tablets
- C11D17/0065—Solid detergents containing builders
- C11D17/0073—Tablets
- C11D17/0078—Multilayered tablets
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/0005—Other compounding ingredients characterised by their effect
- C11D3/0052—Gas evolving or heat producing compositions
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/0005—Other compounding ingredients characterised by their effect
- C11D3/0078—Compositions for cleaning contact lenses, spectacles or lenses
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/38—Products with no well-defined composition, e.g. natural products
- C11D3/386—Preparations containing enzymes, e.g. protease or amylase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
- C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
Landscapes
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- General Chemical & Material Sciences (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Birds (AREA)
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Abstract
本发明涉及枯草杆菌蛋白酶DY变异体,其具有对野生型枯草杆菌蛋白酶DY氨基酸序列进行过改良的氨基酸序列,该野生型氨基酸序列包括第一环区、第二环区、第三环区、第四环区、第五环区和第六环区;其中改良的氨基酸序列是在一个或多个环区的特定识别位置上包括与野生型枯草杆菌蛋白酶DY不同的氨基酸(即取代),由此,枯草杆菌蛋白酶DY变异体与野生型枯草杆菌蛋白酶DY相比对不溶性底物具有降低的吸附作用和增强的水解作用。本发明还涉及编码这种枯草杆菌蛋白酶DY变异体的DNA序列。本发明也涉及包含该枯草杆菌蛋白酶DY变异体的用于清洗各种表面的组合物。
Description
技术领域
本发明涉及用于各种清洗组合物的新的酶变异体，以及编码这些酶变异体的DNA序列。
发明背景
酶构成了最大一类的天然存在蛋白质。每种类型的酶通常催化(促进反应而本身不被消耗)不同种类的化学反应。已知一类称为蛋白酶的酶能水解(将化合物分解为两个或多个简单化合物，并在裂解的化学键的每一端摄取水分子的H和OH部分)其它蛋白质。已通过将天然存在的和蛋白质工程化的蛋白酶作为添加剂搀入到洗衣洗涤剂中对这种水解蛋白质的能力加以利用。衣物上所许多污渍是蛋白质性的污渍，特异性广的蛋白酶能基本上除去这些污渍。
遗憾的是，通常这些蛋白在它们天然细菌环境中的效力水平不能转移到相对非天然的洗涤环境中。特别是当不在这些酶的天然环境中使用时，蛋白酶的性质如热稳定性、pH稳定性、氧化稳定性和底物特异性不是需要的最佳状态。
蛋白酶的氨基酸序列决定了蛋白酶的性质。蛋白酶氨基酸序列的改变可以不同程度地改变酶的性质、甚至可使酶失活，这取决于氨基酸序列中改变的位置、性质和/或程度。已采取了一些措施改变野生型蛋白酶的氨基酸序列以试图改善它们的性质，目标在于增强蛋白酶在洗涤环境中的效力。这些措施包括改变氨基酸序列以在非常多样的环境中增强热稳定性和改善氧化稳定性。
尽管现有技术中描述了各种方法，仍继续需要新的有效的蛋白酶变异体，用于清洗各种表面。
发明目的
本发明的一个目的是提供枯草杆菌蛋白酶DY酶变异体，它们与野生型酶相比具有改善的水解作用。
本发明的另一个目的是提供包含这些枯草杆菌蛋白酶变异体的清洗组合物。
概述
本发明涉及枯草杆菌蛋白酶DY变异体，它们是对野生型枯草杆菌蛋白酶DY氨基酸序列中某些氨基酸序列进行改良，野生型氨基酸序列包含第一环区、第二环区、第三环区、第四环区和第五环区；其中改良的氨基酸序列是在一个或多个环区内特定识别位置上包括与野生型枯草杆菌蛋白酶DY不同的氨基酸(即取代)，由此，枯草杆菌蛋白酶DY变异体与野生型枯草杆菌蛋白酶DY相比对不溶性底物具有降低的吸附作用和增强的水解作用。本发明还涉及编码这种枯草杆菌蛋白酶DY变异体的DNA序列。本发明也涉及包含该枯草杆菌蛋白酶DY变异体的用于清洗各种表面的组合物。
详述
I.枯草杆菌蛋白酶DY变异体
本发明涉及枯草杆菌蛋白酶，特别是枯草杆菌蛋白酶DY，其通过突变编码该酶的各种核苷酸序列改变酶的氨基酸序列而已得到改良。本发明改良的枯草杆菌蛋白酶(下文即“枯草杆菌蛋白酶DY变异体”)与野生型枯草杆菌蛋白酶相比对不溶性底物具有降低的吸附作用和增强的水解作用。本发明还涉及编码这些枯草杆菌蛋白酶DY变异体的DNA序列。
本发明的枯草杆菌蛋白酶属于蛋白酶类。蛋白酶是裂解肽键的催化剂。蛋白酶中的一类是丝氨酸蛋白酶。丝氨酸蛋白酶的特征是在活性位点有一个必需的丝氨酸残基。
酶对可溶性底物的水解速率随酶浓度而增加，有许多文件资料证明了这个观察结果。因此看来，对于如在许多清洗情况中遇到的表面结合的底物而言，水解速率会随表面浓度的增加而增加似乎是合理的。已经证明事实是这样的。(Brode，P.F.III和D.S.Rauch，LANGMUIR，“枯草杆菌蛋白酶BPN′：对固定化底物的活性(Subtilisin BPN′：Activity onan immobilized Substrate)”，第8卷，1325-1329页(1992))。事实上，对于不溶性底物来说，当酶的表面浓度改变时发现水解速率与表面浓度呈线性依赖关系。(Rubingh，D.N.和M.D.Bauer，“蛋白酶在蛋白-溶液界面的水解催化作用(Catalysis of Hydrolysis by Proteases atthe Protein-Solution Inerface)”，聚合物溶液，混合物与界面(InPolymer Solutions，Blends and Interfaces)，I.Noda和D.N.Rubingh，Elsevier编，464页(1992))。令人惊讶的是，在试图应用这一原理寻找具有更好清洗效果的变异蛋白酶时，我们没有发现吸附越好性能越好的酶。事实上，我们意外地测定到了相反的情况：酶对底物吸附作用降低导致底物的水解作用增加(即清洗效果更好)。
当不拟受理论的限制时，认为当一种变异体与另一种比较时，改善的性能是源于这样一个事实，即酶吸附作用越小结合紧密性也越小，由此在不溶性蛋白底物要被除去的表面上的流动性更大。在相当的酶溶液浓度下，这种增强的流动性足以胜过任何通过对表面施加更高的酶浓度而赋予的优越性。
此处所述的突变被指定为改变(即降低)酶对表面结合污物的吸附作用。在枯草杆菌蛋白酶DY中，某些氨基酸形成了酶分子的外部环。为便于讨论，这些环被称为第一、第二、第三、第四和第五环区。具体而言，位置58-65形成第一环区；位置94-106形成第二环区；位置125-132形成第三环区；位置153-166形成第四环区；位置186-190形成第五环区；位置199-219形成第六环区(位置编号与野生型枯草杆菌蛋白酶DY的氨基酸序列(SEQ ID NO：1)中的位置相同)。
据信，这些环区在酶分子对表面结合肽的吸附中起重要作用，在一个或多个这些环区中的特定突变对这种吸附作用会有重要影响。当不拟受理论限制时，认为环区对枯草杆菌蛋白酶DY分子的吸附作用是重要的，至少有两个原因：第一，构成环区的氨基酸可以与酶分子作用的任何表面紧密接触。第二，环区与枯草杆菌蛋白酶DY分子的活性位点和结合空穴间的邻近使得它们在酶对表面结合底物(肽/蛋白质污物)的催化产生的吸附中起作用。
这里所用的“变异体”指具有与野生型不同的氨基酸序列的酶。
这里所用的“突变型枯草杆菌蛋白酶DY DNA”指编码枯草杆菌蛋白酶DY变异体的DNA序列。
这里所用的“野生型枯草杆菌蛋白酶DY”指SEQ ID NO：1表示的酶。枯草杆菌蛋白酶DY的氨基酸序列由Nedkov，P.，Oberthur，W.和Braunitzer，G.，在生物化学第366卷第421-430页(BIOL.CHEM.，Vol.366，pp.421-430(1985))作了进一步描述，在此引作参考。
这里所用的“枯草杆菌蛋白酶DY野生型氨基酸序列”包括SEQ IDNO：1以及除下列任一位置之外具有改变的氨基酸序列的SEQ IDNO：1，所指位置包括58、59、60、61、62、64、65、94、95、96、97、98、99、100、101、102、103、104、105、106、125、126、127、128、129、130、131、132、153、154、155、156、157、158、159、160、161、162、163、164、165、166、186、187、188、189、190、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218或219。
这里所用的“亲水性更强的氨基酸”指比目标氨基酸具有更强亲水性的任一其他氨基酸，参考下表所示的亲水性。下面亲水性表(表1)中以亲水性增强的次序由上向下列出氨基酸(参见Hopp，T.P.，和Woods，K.R.，“由氨基酸序列推测蛋白抗原决定簇(Prediction of ProteinAntigenic Determinants from Amino Acid Sequences)”，美国国家科学院汇编(Proceedings of The National Academy of Science USA)，第78卷，3824-3828页，1981，在此引为参考)。表1氨基酸 亲水性值
Trp -3.4
Phe -2.5
Tyr -2.3
Leu，Ile -1.8
Val -1.5
Met -1.3
Cys -1.0
Ala，His -0.5
Thr -0.4
Pro，Gly -0.0
Gln，Asn 0.2
Ser 0.3
Arg+，Lys+，Glu-，Asp- 3.0
表1还指明了哪一个氨基酸带电荷(该特征基于pH为约8-9)。带正电荷的氨基酸为Arg和Lys，带负电荷的氨基酸为Glu和Asp，剩下的氨基酸均为中性。在本发明优选实施方案中，取代的氨基酸是中性的或是带负电荷的，更优选带负电荷的(即Glu或Asp)。
因此，例如“用中性或带负电荷的亲水性相同或更强的氨基酸取代Gln”这句话的意思是Gln会被Asn(亲水性与Gln相同)或Ser、Glu或Asp(亲水性比Gln强)取代；其中每种氨基酸均为中性或带负电荷，并且与Gln相比具有更高的亲水性值。与此相似，“用中性或带负电荷的亲水性更强的氨基酸取代Pro”这句话的意思是Pro会被Gln、Asn、Ser、Glu或Asp取代。
在本发明一个优选实施方案中，枯草杆菌蛋白酶DY变异体具有对枯草杆菌蛋白酶DY野生型氨基酸序列进行改良的序列，其中，在野生型氨基酸序列的第一环区、第二环区、第三环区、第四环区、第五环区或第六环区中的一个或多个环区中的一个或多个位置处包括一个取代；由此枯草杆菌蛋白酶DY变异体与野生型枯草杆菌蛋白酶DY相比对不溶性底物具有降低的吸附作用和增强的水解作用。
在本发明优选实施方案中，参照表1对一个或多个环区中的一个或多个位置取代的氨基酸是中性的或带负电荷的，与野生型氨基酸序列的目标位置上的氨基酸相比亲水性相同或更强，优选亲水性更强。
A.在第一环区的取代
当取代发生在第一环区时，在第58、59、60、61、62、64或65位的一个或多个位置上发生取代。
当取代发生在58位时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
当取代发生在59位时，取代氨基酸是Glu。
当取代发生在60位时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在61位时，取代氨基酸是Asp、Gln、Glu或Ser。
当取代发生在62位时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在64位时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在65位时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
B.在第二环区的取代
当取代发生在第二环区时，在第94、95、96、97、98、99、100、101、102、103、104、105或106位的一个或多个位置上发生取代。
当取代发生在94位时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Met、Pro、Ser或Thr。
当取代发生在95位时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Met、Pro、Ser、Thr或Val。
当取代发生在96位时，取代氨基酸是Asp、Gln、Glu或Ser。
当取代发生在97位时，取代氨基酸是Asp或Glu。
当取代发生在98位时，取代氨基酸是Asp或Glu。
当取代发生在99位时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在100位时，取代氨基酸是Asp或Glu。
当取代发生在101位时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在102位时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
当取代发生在103位时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Pro、Ser、Thr或Val。
当取代发生在104位时，取代氨基酸是Asp或Glu。
当取代发生在105位时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、His、Pro、Ser或Thr。
当取代发生在106位时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Leu、Met、Pro、Ser、Thr或Val。
C.在第三环区的取代
当取代发生在第三环区时，在第125、126、127、128、129、130、131或132位的一个或多个位置上发生取代。
当取代发生在125位时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Met、Pro、Ser、Thr或Val。
当取代发生在126位时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在127位时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在128位时，取代氨基酸是Asn、Asp、Gln、Glu、Gly或Ser。
当取代发生在129位时，取代氨基酸是Asp或Glu。
当取代发生在130位时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在131位时，取代氨基酸是Asp或Glu。
当取代发生在132位时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
D.在第四环区的取代
当取代发生在第四环区时，在第153、154、155、156、157、158、159、160、161、162、163、164、165或166位的一个或多个位置上发生取代。
当取代发生在153位时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在154位时，取代氨基酸是Asp、Gln、Glu或Ser。
当取代发生在155位时，取代氨基酸是Asp或Glu。
当取代发生在156位时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在157位时，取代氨基酸是Asp或Glu。
当取代发生在158位时，取代氨基酸是Asp或Glu。
当取代发生在159位时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在160位时，取代氨基酸是Asp或Glu。
当取代发生在161位时，取代氨基酸是Asn、Asp、Glu、或Ser。
当取代发生在162位时，取代氨基酸是Asp、Gln、Glu或Ser。
当取代发生在163位时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
当取代发生在164位时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Leu、Met、Pro、Ser、Thr或Val。
当取代发生在165位时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在166位时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Pro、Ser、Thr或Val。
E.在第五环区的取代
当取代发生在第五环区时，在第186、187、188、189或190位的一个或多个位置上发生取代。
当取代发生在186位时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、His、Pro、Ser或Thr。
当取代发生在187位时，取代氨基酸是Asp或Glu。
当取代发生在188位时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Leu、Ile、Met、Pro、Ser、Thr、Tyr或Val。
当取代发生在189位时，取代氨基酸是Asp或Glu。
当取代发生在190位时，取代氨基酸是Asp或Glu。
F.在第六环区的取代
当取代发生在第六环区时，在第199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218或219位的一个或多个位置上发生取代。
当取代发生在199位时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、His、Pro、Ser或Thr。
当取代发生在200位时，取代氨基酸是Asn、Asp、Gln、Glu、Gly或Ser。
当取代发生在201位时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在202位时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Met、Pro、Ser或Thr。
当取代发生在203位时，取代氨基酸是Asp或Glu。
当取代发生在204位时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Met、Pro、Ser或Thr。
当取代发生在205位时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Pro、Ser、Thr或Val。
当取代发生在206位时，取代氨基酸是Asp或Glu。
当取代发生在207位时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
当取代发生在208位时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Phe、Pro、Ser、Thr或Val。
当取代发生在209位时，取代氨基酸是Asn、Asp、Gln、Glu、Gly或Ser。
当取代发生在210位时，取代氨基酸是Asp或Glu。
当取代发生在211位时，取代氨基酸是Asp、Gln、Glu或Ser。
当取代发生在212位时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
当取代发生在213位时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Met、Pro、Ser、Thr或Val。
当取代发生在214位时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
当取代发生在215位时，取代氨基酸是Asp或Glu。
当取代发生在216位时，取代氨基酸是Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Met、Pro、Ser、Thr或Val。
当取代发生在217位时，取代氨基酸是Asp、Gln、Glu、或Ser。
当取代发生在218位时，取代氨基酸是Asn、Asp、Gln、Glu、Pro或Ser。
当取代发生在219位时，取代氨基酸是Asn、Asp、Gln、Glu、Gly、Pro或Ser。
G.酶变异体的制备
实施例1
突变型枯草杆菌蛋白酶DY DNA
构建含野生型枯草杆菌蛋白酶DY基因的噬菌粒(“DYP”)。质粒PUC119(Vieira，J.和Messing，J.，“单链质粒DNA的生产(Prodution of Single-Stranded Plasmid DNA)”，153METHODS INENZYMOLOGY3-11(1989))的2.8 Kbp PvuII限制酶片段被克隆入质粒pUB110(芽孢杆菌基因存储中心(Bacliius Genetic Stock Center)，Columbus，OH1E9)的PvuII位点。此pUC119-pUB110杂交质粒称为pJMA601。在pJMA601的BamHI限制位点克隆入聚合酶链反应扩增的来自染色体DNA的枯草杆菌蛋白酶DY基因得到噬粒DYP。噬粒DYP被转化入大肠杆菌(Escherichia coli)Ung-株CJ236中，并且使用VCSM13辅助噬菌体制备含尿嘧啶的单链DNA模板(Kunkel，T.A.，J.D.Roberts和R.A.Zakour，“无需表型选择的快速有效的位点特异诱变(Rapid ang efficient site-specific mutagenesiswithout phenotypic selection)”，METHODS IN ENZYMOLOGY，Vol.54，pp367-382(1987)；如Yuckenberg，P，D.，F.Witney，J.Geisselsoder和J.McClary所改进的，“使用含尿嘧啶的DNA及噬菌粒载体体外定位诱变(Site-directed in vitro mutagenesis using uracil-containingDNA and phagemid vectors)”，DIRECTED MUTAGENESIS-APRATICAL APPROCH，M.J.McPherson编，pp27-48，(1991)；这两篇文献在此均引作参考)。Zoller和Smith的单链引物定位诱变修饰方法(Zoller，M.j.，和M.Smith，“使用M13-衍生的载体进行寡核苷酸定向诱变：在DNA任何片段中产生点突变的有效和通用方法(Oligonucleotide-directed mutagenesis using M13-derivedvectors：an efficient and general procedure for the production of pointmutations in any fragment of DNA)”，NUCLEIC ACIDSRESEARCH，VOL.10，pp6487
-6500，(1982)，在此引为参考)被用于生产所有的突变体(基本上与Yuckenberg等人在1991年上述文献中所述相同)。使用AplliedBiosystem Inc.380B DNA合成仪制备寡核苷酸。诱变反应产物被转化到大肠杆菌株MM294(American Type Culture CollectionE.coli.33625)中。所有的突变体由DNA测序来确证，分离的DNA被转化入枯草芽孢杆菌(Bacillus subtilis)表达型菌株BG2036中(Yang，M.Y.，E.Ferrari和D.J.Henner，(1984)，“枯草芽孢杆菌中性蛋白酶基因的克隆及使用克隆基因产生体外衍生的缺失突变(Cloning ofthe Neutral Protease Gene of Bacillus subtilis and the Use of ClonedGene to Create an In Vitro-derived Deletion Mutation)”，JOUNALOF BACTERIOLOGY，Vol.160，pp15-21)。对于一些环区突变体使用在相应环中具有移码终止子突变的经修饰的DYP生产尿嘧啶模板。设计寡核苷酸用于恢复合适的阅读框及编码以下位置的随机取代氨基酸：58、59、60、61、62、64、65、94、95、96、97、98、99、100、101、102、103、104、105、106、125、126、127、128、129、130、131、132、153、154、155、156、157、158、159、160、161、162、163、164、165、166、186、187、188、189、190、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218或219(这些密码子的三个碱基均使用等摩尔和/或不同量的四种核苷酸的混合物)。校正移码终止并产生功能酶的突变通过消化酪蛋白的能力进行鉴别。通过DNA测序确定随机取代。
实施例2
发酵
含目的枯草杆菌蛋白酶突变体的枯草芽孢杆菌细胞(BG2036)在一升LB-葡萄糖肉汤中生长至中对数期，并接种于Biostat ED发酵罐(B.Braun Biotech，Inc.，Allentown.Pennsylvania)，总体积为10升。发酵培养基含酵母提取物(Yeast Extract)、淀粉、消泡剂、缓冲剂和痕量矿物质(参见FERMENTATION：APRACTICAL APPROACH，Ed.B.McNeil和L.M.Harvey，1990)。在进行发酵的过程中，肉汤保持恒定的PH值为7。加入氯霉素用于诱变质粒的抗生素选择。细胞在37℃培养一昼夜至A600约60，收集细胞。
实施例3
纯化
将发酵肉汤通过以下步骤获得纯化的酶。离心使肉汤不含枯草杆菌细胞，并使用100K截留膜除去细小的颗粒使肉汤澄清。然后在一10K的截留膜上浓缩，流动透析以降低离子强度，并用0.025M的MES缓冲液(2-(N-吗啉代)乙烷磺酸)调pH至5.5。将酶加样于阳离子交换色谱柱或亲和吸附色谱柱上并用NaCl或丙二醇梯度从柱中洗脱(参见Scopes，R.K.，蛋白质纯化原理及实践(PROTEIN PURIFICATIONPRINCIPLES AND PRACTICE)，Springer-Verlag，New York(1984)，此处引为参考)使酶进一步纯化。
使用pNA分析(DelMar，E.G.，C.Largman，J.W.Brodrick和M.C.Geokas，ANAL.BIOCHEM.，Vol.99，pp.316-320，(1979)，此处引为参考)测定梯度洗脱中收集级分的活性酶浓度。此分析测定当酶水解可溶性合成底物琥珀酰-丙氨酰-丙氨酰-脯氨酰-苯丙氨酰-对硝基苯胺(sAAPF-pNA)时对硝基苯胺的释放速率。在分光光度计上410nm下测量水解反应中黄色物质的产生速率，此速率与活性酶的浓度成比例。另外，在280nm下测量的吸光度被用于确定总蛋白浓度。由活性酶/总蛋白的比率得出酶的纯度，并用来鉴别要合并作为存储溶液的级分。
为避免存储中酶的自身水解，向从色谱柱获得的收集级分中加入等重量的丙二醇。在纯化步骤完成时，用SDS-PAGE(十二烷基硫酸钠聚丙烯酰胺凝胶电泳)检测存储酶溶液的纯度，并且使用胰蛋白酶抑制剂II-T型：购自Sigma Chemical Company(St.Louis，Missouri)的火鸡卵白通过活性位点滴定法确定绝对酶浓度。测得的转化系数将表明在酶分子不同位置上进行的哪些变化会导致酶变异体与野生型相比对可溶性底物pNA具有增强的活性。
准备使用时，将酶存储溶液通过一个Sephadex-G25(Pharmacia，Piscataway，New Jersey)分子排阻柱洗脱以除去丙二醇并交换缓冲液。将酶存储溶液中的MES缓冲液换为含0.01MCaCl2并用HCl调节pH为8.6的0.1MTris缓冲剂(三(羟甲基-氨基甲烷))。所有实验均在pH8.6恒温于25℃的Tris缓冲液中进行。
H.酶变异体的特征
实施例4
典型表面制备
购自CPG Inc.(Fairfield，New Jersey)的氨丙基可控多孔玻璃(CPG)被用作共价连接sAAPF-pNA底物的载体，此底物购自Bachem，Inc.(Torrence，California)。反应在二甲基亚砜中进行，(1-乙基-3-〔3-(二甲基氨基)丙基〕碳二亚胺盐酸盐)(EDC)用作偶联剂。完成(用pNA分析监测)时，除去过量的溶剂，并用二甲基亚砜(DMSO)和双蒸水洗涤CPG：sAAPF-pNA。然后，在约70℃下的干燥箱中用N2吹干。反应方案和固定化底物的制备根据Brode，P.F.III和D.S.Rauch“Subtilisin BPN’：Activity on anImmobilized Substrate，”LANGMUIR，Vol.8，p.1325-1329，(1992)(此处引为参考文献)中描述的方法进行。
CPG表面将有62,000±7,000pNA分子/μm2。其表面积与CPG公司为我们所购的CPG提供的值50.0m2/g相比没有改变。这表明将sAAPF-pNA加入到CPG的方法不会损坏多孔结构(平均直径为486)。
实施例5
表面水解作用分析
使用CPG：sAAPF-pNA，可在一项实验中测量酶变异体的吸附作用和CPG-结合肽的水解。向含Tris缓冲液和已抽气的CPG：sAAPF-pNA的烧瓶中加入少量的酶变异体存储溶液。在作曲形运动的摇床上摇动烧瓶90分钟，在此过程中摇床在不同的时间间隔内停机(例如，在吸附水解作用早期如起初的20分钟内每2分钟为一间隔，至实验结束时每10分钟为一间隔)。使CPG：sAAPF-pNA固定并由溶液取样。实验方法及吸附作用和水解的计算方法均按照上述Brode等人于1992年描述的方法进行。
监测所有酶抵抗自身水解的稳定性，在实验过程中应没有明显的自身水解造成的损失。因此，可通过测量溶液的消耗量确定酶的吸附作用。初始酶变异体浓度与每一个时间点测量的浓度间的差给出酶变异体吸附的量。通过在410nm下读取等分样品的吸光度测量pNA从表面的水解量。将取样量和存在于烧瓶中的量相加计算水解的pNA总量。通过减去酶不存在时由pH 8.6的Tris缓冲液水解的pNA的量校正该值。根据酶的效力，这个本底水解作用占总水解作用的7-29％。
实施例6
可溶性底物动力学分析
通过在DU-70分光光度计上于410nm下测量吸收升高与时间的函数来监测可溶性底物sAAPF-pNA的水解速率。酶浓度保持不变，且对于每一次动力学测定底物浓度在90-700μM sAAPF-pNA变化，同时制备的酶浓度范围为6-10nmol。在900秒的期间内每秒测一个吸光度数据，且将该数据转移至LOTUSTM座标纸(LotusDevelopment corporation，Cambridge，Massachusetts)。动力学参数分析通过标准Lineweaver Burk分析进行，其中过程的初始部分(通常第一分钟)数据与线性回归曲线吻合给出V0。以标准倒数形式将V0和S0作图，给出KM和kcat。
I.枯草杆菌蛋白酶DY变异体的例子
下表2-36例举了本发明的对表面结合底物具有降低吸附作用和增强水解作用的枯草杆菌蛋白酶DY变异体。在描述特定的突变体时，野生型中存在的原始氨基酸写在第一位，位置编号写在第二位，取代氨基酸写在第三位。
表2环1-单一突变的变异体Thr58AsnThr58AspThr58GlnThr58GluThr58GlyThr58ProThr58SerAsp59GluGly60AsnGly60AspGly60GlnGly60GluGly60ProGly60SerAsn61AspAsn61GlnAsn61GluAsn61SerGly62AsnGly62AspGly62GlnGly62GluGly62ProGly62SerGly64AsnGly64AspGly64GlnGly64GluGly64ProGly64SerThr65AsnThr65AspThr65GlnThr65GluThr65GlyThr65ProThr65Ser表3环1-双点突变的变异体Thr58Gln + Asn61GluThr58Asn + Asn61AspAsp59Glu + Gly64ProThr58Glu + Gly62ProAsp59Glu + Gly60SerGly60Asn + Gly64AsnAsn61Asp + Thr65GlnThr58Asp + Asn61SerThr58Asp + Thr65ProGly64Glu + Thr65GlyGly64Asp + Thr65AsnThr58Glu + Gly62GlnGly60Asn + Asn61GluThr58Asn + Gly64SerThr58Pro + Asp59GluGly64Glu + Thr65GlnThr58Pro + Gly62SerThr58Gly + Gly64AsnAsn61Gln + Gly64GlnThr58Gly + Asn61AspGly60Pro + Asn61AspGly62Asn + Gly64ProGly62Asp + Thr65AsnThr58Asn + Gly62AsnThr58Pro + Gly62AsnGly64Glu + Thr65SerGly60Pro + Gly62GluGly60Glu + Thr65GlnGly60Pro + Gly62SerThr58Asp + Asn61GlnGly60Asn + Asn61AspThr58Ser + Gly62GluGly60Glu + Gly64SerGly60Pro + Asn61GluThr58Gln + Asn61SerGly60Asp + Thr65ProAsn61Asp + Thr65SerAsn61Ser + Gly64Asp表4环1-三点突变的变异体Thr58Asn + Gly60Gln + Gly64ProThr58Gly + Asn61Ser + Thr65SerAsp59Glu + Gly60Pro + Thr65SerAsn61Glu + Gly62Pro + Gly64AsnThr58Asp + Gly60Asn + Thr65GlnThr58Ser + Gly62Ser + Thr65GlyThr58Pro + Asp59Glu + Gly62ProGly60Glu + Asn61Gln + Thr65GlnGly60Glu + Gly64Asn + Thr65ProGly60Asn + Gly62Asp + Thr65SerThr58Asn + Asn61Glu + Thr65GlnThr58Asn + Asp59Glu + Gly62ProGly60Gln + Gly62Gln + Gly64GluAsn61Gln + Gly62Pro + Thr65SerAsn61Gln + Gly62Asn + Thr65GlnAsn61Ser + Gly62Gln + Thr65GlyGly60Ser + Gly64Gln + Thr65GlyThr58Pro + Gly60Pro + Gly64GlnThr58Asn + Gly60Asn + Thr65ProAsp59Glu + Gly62Gln + Thr65AsnThr58Ser + Gly60Gln + Gly62AspThr58Asn + Gly64Ser + Thr65AsnGly60Pro + Gly62Asp + Thr65GlnAsn61Glu + Gly64Ser + Thr65GlyThr58Pro + Asn61Asp + Gly64GlnAsp59Glu + Asn61Ser + Gly62SerThr58Asn + Gly60Asp + Thr65AsnAsp59Glu + Asn61Gln + Gly62AsnThr58Asn + Gly60Asp + Asn61AspThr58Asn + Gly60Glu + Asn61Glu表5环1-四点突变的变异体Thr58Asn + Gly60Glu + Gly64Pro + Thr65GlnAsp59Glu + Gly60Ser + Gly64Pro + Thr65ProThr58Ser + Gly60Asn + Asn61Gln + Thr65ProAsp59Glu + Gly60Ser + Gly64Ser + Thr65GlyAsp59Glu + Gly60Asn + Asn61Ser + Gly62SerAsp59Glu + Gly60Asn + Asn61Ser + Gly64AsnThr58Gln + Asn61Gln + Gly62Glu + Gly64AsnThr58Asn + Asp59Glu + Gly60Gln + Thr65ProThr58Gly + Gly60Gln + Gly62Pro + Gly64GluGly60Asp + Asn61Asp + Gly62Pro + Gly64GlnThr58Pro + Gly60Glu + Asn61Asp + Gly64AsnThr58Asp + Asp59Glu + Gly62Asn + Thr65GlnThr58Asp + Asp59Glu + Gly60Ser + Gly64AsnThr58Glu + Asp59Glu + Gly62Asn + Thr65SerThr58Asp + Asp59Glu + Gly60Pro + Asn61SerThr58Asn + Gly60Gln + Asn61Glu + Gly62AspAsp59Glu + Gly60Glu + Asn61Glu + Gly62SerThr58Gln + Asp59Glu + Gly60Glu + Asn61GluThr58Pro + Asp59Glu + Gly60Glu + Asn61GluThr58Gln + Asp59Glu + Gly60Asp + Asn61AspThr58Pro + Gly60Glu + Asn61Asp + Gly62GluGly60Glu+Asn61Glu+Gly62Glu+Thr65GlyGly60Asp+Asn61Asp+Gly62Asp+Thr65GlnThr58Asp+Asp59Glu+Gly60Glu+Thr65SerAsp59Glu+Asn61Asp+Gly62Asp+Thr65GlnAsp59Glu+Asn61Asp+Gly62Glu+Thr65GlyAsp59Glu+Gly60Asp+Gly62Asp+Thr65GlnThr58Ser+Asp59Glu+Gly60Asp+Gly62Asp表6环2-单一突变的变异体Val 94AlaVal 94AsnVal 94AspVal 94CysVal 94GlnVal 94GluVal 94GlyVal 94HisVal 94MetVal 94ProVal 94SerVal 94ThrLeu 95AlaLeu 95AsnLeu 95AspLeu 95CysLeu 95GlnLeu 95GluLeu 95GlyLeu 95HisLeu 95IleLeu 95MetLeu 95ProLeu 95SerLeu 95ThrLeu 95ValAsn 96AspAsn 96GlnAsn 96GluAsn 96SerSer 97AspSer 97GluSer 98AspSer 98GluGly 99AsnGly 99AspGly 99GlnGly 99GluGly 99ProGly 99SerSer100AspSer100GluGly101AsnGly101AspGly101GlnGly101GluGly101ProGly101SerThr102AsnThr102AspThr102GlnThr102GluThr102GlyThr102ProThr102SerTyr103AlaTyr103AsnTyr103AspTyr103CysTyr103GlnTyr103GluTyr103GlyTyr103HisTyr103IleTyr103LeuTyr103MetTyr103ProTyr103SerTyr103ThrTyr103ValSer104AspSer104GluAla105AsnAla105AspAla105GlnAla105GluAla105GlyAla105HisAla105ProAla105SerAla105ThrIle106AlaIle106AsnIle106AspIle106CysIle106GlnIle106GluIle106GlyIle106HisIle106LeuIle106MetIle106ProIle106SerIle106ThrIle106Val表7环2-双点突变的变异体Val 94Gln+Ser100GluAsn 96Ser+Gly 99GlnVal 94Asp+Thr102GlnVal 94His+Ala105ProVal 94Pro+Ser 97GluAsn 96Ser+Ile106AspSer 97Glu+Gly101SerLeu 95Gly+Thr102GluSer100Glu+Ile106ThrAsn 96Ser+Gly 99SerAla105Ser+Ile106ProAsn 96Ser+Gly101SerThr102Asn+Ile106GlnVal 94His+Asn 96GlnLeu 95Cys+Ala105AspLeu 95Gly+Asn 96AspLeu 95Cys+Asn 96AspThr102Ser+Ile106CysAsn 96Gln+Thr102GlnGly101Ser+Tyr103GlyAsn 96Ser+Ile106ThrSer 98Asp+Gly 99GlnLeu 95Ser+Ser100GluLeu 95Met+Thr102GlyLeu 95His+Thr102GluAsn 96Asp+Tyr103HisTyr103Gln+Ile106AspSer104Asp+Ile106GlnThr102Asn+Tyr103AspVal 94Asn+Ser 97GluVal 94Gly+Gly101SerLeu 95Gln+Ile106AspAsn 96Ser+Ser104AspTyr103Glu+Ile106HisThr102Ser+Tyr103GluVal 94Cys+Ser104AspLeu 95Gln+Thr102AspAsn 96Glu+Gly101SerLeu 95Glu+Ala105SerVal 94Met+Leu 95ProVal 94Thr+Gly101GlnTyr103Gly+Ile106HisThr102Asn+Ile106GlySer104Asp+Ile106ThrGly 99Ser+Ser100AspVal 94Glu+Gly 99AsnGly 99Gln+Gly101SerVal 94Thr+Asn 96AspSer 97Glu+Ile106LeuThr102Asp+Tyr103MetSer 97Asp+Ala105HisAsn 96Gln+Ser100AspGly101Asn+Ile106GlnAsn 96Glu+Tyr103SerTyr103Thr+Ile106ProSer100Asp+Tyr103IleGly101Glu+TyrI03LeuSer100Asp+Thr102AsnVal 94His+Leu 95ThrSer104Asp+Ala105Asn表8环2-三点突变的变异体Val 94Gln+Leu 95Thr+Ser100GluSer 98Asp+Ala105Ser+Ile106AsnVal 94Gly+Gly 99Pro+Ile106ValVal 94Ser+Leu 95Gln+Ser104GluVal 94His+Tyr103Glu+Ile106GlyVal 94Pro+Asn 96Gln+Ile106CysVal 94Thr+Ser 98Asp+Ile106CysVal 94Met+Ser100Asp+Gly101SerVal 94Ala+Ser 97Asp+Gly 99GlnLeu 95Gly+Gly 99Pro+Ala105GlyVal 94Cys+Ser100Asp+Tyr103AlaGly101Asn+Thr102Asp+Ile106AlaGly 99Ser+Ser104Glu+Ile106ValAsn 96Gln+Ser100Glu+Tyr103ThrLeu 95Met+Thr102Pro+Ile106LeuAsn 96Glu+Thr102Ser+Ala105HisSer100Glu+Thr102Asn+Tyr103ValSer 98Glu+Gly 99Gln+Ile106MetAsn 96Asp+Gly 99Ser+Ala105ThrGly101Asp+Thr102Asn+Tyr103ThrVal 94Ala+Gly 99Gln+Tyr103HisThr102Gly+Tyr103Glu+Ala105GlySer 97Glu+Gly101Ser+Thr102GlnVal 94Pro+Gly 99Ser+Gly101AspSer 97Asp+Gly 99Ser+Ile106GlyVal 94Cys+Leu 95Val+Gly101GlnSer100Asp+Gly101Asn+Tyr103GlnThr102Asp+Tyr103Leu+Ile106ValVal 94Cys+Ser104Glu+Ala105AsnVal 94Ser+Thr102Asn+Ala105ThrSer 98Glu+Gly 99Asn+Tyr103ValLeu 95Val+Gly101Gln+Ala105AspSer 97Glu+Gly 99Pro+Thr102SerVal 94Ser+Gly101Glu+Ile106AlaGly 99Asn+Gly101Asp+Thr102GluGly 99AsP+Ser100Glu+Thr102AsnVal 94Thr+Gly 99Glu+Ser100AspGly 99Asp+Ser100Asp+Tyr103ProGly 99Glu+Ser100Glu+Gly101GlnAsn 96Glu+Ser 97Glu+Ala105AsnAsn 96Glu+Ser 97Asp+Gly101ProLeu 95Met+Asn 96Asp+Ser 97GluGly 99Ser+Ser104Asp+Ala105GluSer 97Asp+Ser 98Glu+Gly101SerAsn 96Asp+Ser 98Asp+Gly 99GluAsn 96Glu+Ser 97Glu+Gly 99GluLeu 95Asp+Ser100Glu+Ile106LeuAsn 96Asp+Ser100Glu+Gly101GluSer104Asp+Ala105Thr+Ile106GluAsn 96Glu+Ser 98Asp+Tyr103AlaLeu 95Asn+Ser 98Asp+Ser100GluLeu 95Gly+Ser 98Glu+Ser100GluSer 98Asp+Ser100Glu+Ala105SerSer 98Glu+Ser100Asp+Thr102AsnVal 94Asn+Ser 97Glu+Gly 99GluAsn 96Glu+Ser100Glu+Thr102ProVal 94Asp+Gly101Asp+Thr102ProGly101Glu+Thr102Asp+Ser104AspSer 98Glu+Ser100Asp+Gly101AspLeu 95Glu+Gly101Asp+Ile106Glu表9环2-四点突变的变异体Val 94Asr+Gly101Asp+Tyr103Ile+Ala105ThrVal 94Asn+Gly 99Ser+Ser100Glu+Thr102SerLeu 95Cys+Ser 98Asp+Gly101Pro+Ile106AlaLeu 95His+Gly101Ser+Tyr103Ile+Ala105SerVal 94Ser+Leu 95Val+Gly 99Pro+Ser100GluGly 99Ser+Ser100Glu+Tyr103Ile+Ala105SerVal 94Ala+Ser 97Glu+Thr102Ser+Ala105ProVal 94Thr+Tyr103Gly+Ser104Asp+Ala105GlnVal 94Ala+Leu 95Pro+Ser100Glu+Ile106SerLeu 95Asn+Ser 98Asp+Tyr103Gly+Ala105ProAsn 96Ser+Gly101Glu+Tyr103Leu+Ala105ProLeu 95Met+Ser 98Glu+Gly 99Asn+Tyr103ThrVal 94Ala+Leu 95Ala+Ser 97Asp+Thr102AsnGly 99Gln+Ser100Glu+Thr102Ser+Ile106LeuAsn 96Glu+Gly 99Gln+Ala105Pro+Ile106ThrVal 94Gly+Gly101Glu+Tyr103Gln+Ile106HisVal 94Thr+Ser 97Asp+Gly101Gln+Ala105ThrVal 94Gly+Leu 95Val+Asn 96Gln+Ser104GluLeu 95Gln+Gly 99Glu+Ser100Glu+Thr102ProLeu 95Met+Ser100Asp+Gly101Asp+Tyr103SerLeu 95Ile+Thr102Pro+Ser104Asp+Ala105AspSer 97Asp+Ser 98Asp+Tyr103Met+Ala105ProLeu 95Ser+Ser 97Asp+Ser 98Asp+Thr102SerSer 97Glu+Ser 98Glu+Ala105Pro+Ile106AsnSer 97Glu+Ser 98Glu+Gly101Ser+Ile106CysSer 97Asp+Ser 98Asp+Gly101Pro+Thr102ProLeu 95Glu+Ser100Glu+Gly101Asp+Thr102SerVal 94Met+Leu 95Glu+Asn 96Glu+Gly101AspLeu 95His+Gly 99Glu+Ser100Glu+Gly101GluAsn 96Glu+Gly 99Glu+Gly101Ser+Ile106AsnLeu 95Val+Asn 96Asp+Ser 98Glu+Tyr103GlnAsn 96Asp+Ser 98Glu+Gly 99Ser+Thr102AsnAsn 96Glu+Ser 98Glu+Gly101Ser+Thr102ProLeu 95Val+Asn 96Glu+Ser 98Asp+Ile106AlaVal 94Gly+Asn 96Glu+Ser 98Asp+Tyr103ThrSer 98Glu+Gly 99Gln+Ser100Asp+Gly101ProSer 98Glu+Gly 99Gln+Ser100Asp+Thr102GlnAsn 96Glu+Ser100Glu+Tyr103Gln+Ile106ProAsn 96Asp+Gly 99Asn+Ser100Glu+Tyr103IleLeu 95Glu+Asn 96Gln+Ser 98Glu+Gly 99GluSer 97Asp+Ser 98Glu+Ser100Asp+Ile106ValSer 97Asp+Ser 98Asp+Ser100Glu+Thr102SerSer 97Asp+Ser 98Asp+Ser100Glu+Gly101GlnLeu 95Thr+Ser 97Asp+Ser 98Glu+Ser100AspVal 94Met+Asn 96Glu+Gly 99Asp+Gly101AspSer 98Asp+Ser100Glu+Gly101Glu+Ala105SerVal 94Cys+Ser 98Asp+Ser100Asp+Gly101AspVal 94Glu+Ser100Glu+Gly101Glu+Tyr103IleLeu 95Glu+Ser 97Asp+Ser 98Asp+Ala105SerLeu 95Ala+Asn 96Asp+Gly101Asp+Thr102AspVal 94Cys+Gly 99Asp+Gly101Asp+Ile106ProLeu 95Ser+Ser100Glu+Gly101Gln+Thr102GluLeu 95Met+Ser100Asp+Thr102Glu+Ile106GlnAsn 96Ser+Gly101Asp+Tyr103Asn+Ile106AspLeu 95Cys+Gly101Glu+Ala105Pro+Ile106GluVal 94Asp+Gly 99Glu+Ser100Asp+Ala105SerSer 97Glu+Ser 98Glu+Gly101Asp+Tyr103SerSer 97Asp+Ser100Glu+Thr102Gln+Tyr103ThrSer 97Glu+Ser100Asp+Tyr103Ser+Ala105Gly表10环3-单一突变的变异体Leu125AlaLeu125AsnLeu125AspLeu125CysLeu125GlnLeu125GluLeu125GlyLeu125HisLeu125IleLeu125MetLeu125ProLeu125SerLeu125ThrLeu125ValGly126AsnGly126AspGly126GlnGly126GluGly126ProGly126SerGly127AsnGly127AspGly127GlnGly127GluGly127ProGly127SerPro128AsnPro128AspPro128GlnPro128GluPro128GlyPro128SerSer129AspSer129GluGly130AsnGly130AspGly130GlnGly130GluGly130ProGly130SerSer131AspSer131GluThr132AsnThr132AspThr132GlnThr132GluThr132GlyThr132ProThr132Ser表11环3-双点突变的变异体Leu125Gln+Ser129GluLeu125Pro+Gly126SerGly127Gln+Ser131AspGly130Ser+Thr132GlyLeu125Met+Gly130AspLeu125Pro+Pro128AspSer129Asp+Gly130AsnLeu125Asp+Pro128AsnGly127Ser+Ser131GluLeu125Ile+Gly130SerLeu125Thr+Thr132AspLeu125Thr+Thr132AsnLeu125Pro+Gly127GluLeu125Glu+Thr132AsnPro128Asn+Thr132ProLeu125Thr+Gly130AspGly127Pro+Thr132SerGly127Asn+Thr132AsnGly126Gln+Ser131AspLeu125Val+Thr132GlyGly126Gln+Gly130SerSer129Asp+Thr132GlyGly126Asp+Gly130ProLeu125Cys+Pro128GlnGly127Gln+Thr132GlyLeu125His+Gly126AspLeu125Val+Ser129GluGly126Ser+Pro128AspLeu125Val+Gly126GluGly127Glu+Pro128SerLeu125Asn+Thr132ProLeu125Met+Gly126AspLeu125Ser+Ser129GluLeu125Ser+Gly126AsnLeu125Val+Thr132GlnLeu125Val+Gly130AspSer129Glu+Thr132SerPro128Gly+Ser129AspGly126Pro+Thr132AspGly126Asp+Thr132SerPro128Asp+Thr132GlnGly126Pro+Ser129AspGly130Ser+Thr132GluGly127Glu+Pro128GlnGly126Asn+Gly130ProPro128Asn+Ser129GluGly130Asp+Thr132GlyGly126Gln+Ser131GluGly127Glu+Gly130SerGly126Asn+Ser129GluPro128Asp+Gly130AsnPro128Asp+Thr132GlyGly126Gln+Pro128AspSer131Glu+Thr132GlyGly130Glu+Thr132AsnLeu125Val+Pro128GlyLeu125Ile+Gly130ProPro128Asn+Ser131AspGly130Asn+Thr132AspGly127Ser+Pro128Glu 表12环3-三点突变的变异体Gly127Asp+Gly130Gln+Thr132SerPro128Asp+Gly130Pro+Thr132SerGly127Asn+Ser131Asp+Thr132ProLeu125Ser+Ser131Asp+Thr132ProLeu125Met+Pro128Glu+Thr132ProLeu125Asp+Gly126Ser+Thr132GlnGly127Asn+Pro128Glu+Gly130AsnGly127Ser+Pro128Gln+Ser129GluGly126Pro+Gly127Pro+Ser131GluLeu125Ile+Gly127Asp+Thr132GlnGly126Asn+Gly130Ser+Thr132ProGly126Pro+Gly127Gln+Thr132AspGly126Asp+Gly130Asn+Thr132GlnLeu125Ser+Pro128Gln+Thr132AsnGly126Pro+Gly127Pro+Pro128GluLeu125Met+Gly126Glu+Gly130GlnLeu125Met+Ser129Asp+Thr132GlnLeu125His+Ser129Asp+Gly130SerGly127Pro+Ser129Glu+Gly130SerPro128Gln+Gly130Asn+Thr132GlnGly126Asn+Gly127Pro+Gly130GlnLeu125Pro+Gly126Ser+Gly127GlnLeu125His+Gly127Ser+Ser131GluLeu125His+Gly127Asn+Thr132GluGly126Glu+Gly130Pro+Thr132GlnLeu125His+Gly127Gln+Gly130GluGly127Pro+Pro128Gly+Thr132AsnLeu125Gly+Pro128Gly+Gly130AspGly126Asn+Pro128Asn+Ser129GluGly126Pro+Gly130Ser+Thr132GlyLeu125Ser+Gly126Pro+Ser131GluLeu125Val+Gly126Glu+Thr132GlyPro128Asn+Ser131Asp+Thr132ProLeu125Gly+Pro128Asn+Thr132ProGly127Glu+Pro128Ser+Thr132ProGIy127Gln+Ser129Glu+Thr132GlnLeu125Ile+Gly127Pro+Gly130AspGly126Ser+Gly127Asp+Gly130AsnPro128Glu+Gly130Pro+Thr132GlnLeu125Val+Gly126Asp+Gly127AspPro128Asn+Gly130Asp+Ser131AspGly130Glu+Ser131Asp+Thr132GlnGly126Pro+Gly130Glu+Ser131AspGly127Glu+Pro128Glu+Thr132SerGly130Gln+Ser131Glu+Thr132AspLeu125Gln+Ser131Asp+Thr132AspGly130Ser+Ser131Glu+Thr132GluGly130Pro+Ser131Asp+Thr132AspGly127Pro+Ser131Glu+Thr132AspLeu125Asn+Ser131Asp+Thr132AspPro128Gln+Ser131Asp+Thr132GluPro128Gly+Ser131Asp+Thr132AspGly126Gln+Ser129Asp+Gly130AspLeu125Ile+Ser129Asp+Gly130GluGly127Glu+Pro128Asp+Ser129GluSer129Asp+Gly130Asp+Ser131AspSer129Glu+Gly130Asp+Ser131GluSer129Glu+Gly130Glu+Ser131AspGly126Glu+Gly127Glu+Pro128GluGly130Glu+Ser131Asp+Thr132Asp表13环3-四点突变的变异体Gly126Ser+Pro128Glu+Gly130Ser+Thr132SerLeu125Cys+Gly127Gln+Ser129Glu+Thr132ProLeu125Gln+Gly126Pro+Pro128Ser+Ser129GluLeu125Thr+Ser129Glu+Gly130Ser+Thr132ProGly127Gln+Pro128Gln+Ser129Glu+Gly130ProGly127Asn+Pro128Asn+Ser131Glu+Thr132GlyLeu125Met+Gly127Glu+Gly130Asn+Thr132GlyLeu125Met+Gly127Asn+Pro128Asn+Ser129GluLeu125Met+Gly126Ser+Gly127Gln+Ser131AspLeu125Asn+Gly126Asp+Gly127Gln+Thr132SerLeu125Gln+Gly127Gln+Gly130Glu+Thr132ProLeu125Ala+Gly130Asn+Ser131Asp+Thr132ProLeu125Met+Gly126Pro+Gly127Ser+Ser131AspLeu125Cys+Gly126Pro+Ser129Glu+Thr132AsnLeu125Gly+Pro128Gly+Ser131Asp+Thr132GlnLeu125Met+Gly130Asn+Ser131Asp+Thr132GlnLeu125Val+Gly126Pro+Gly130Gln+Thr132AspGly126Asn+Gly127Pro+Pro128Gln+Ser129GluLeu125Gln+Gly126Glu+Gly127Glu+Gly130AsnLeu125Val+Gly126Asp+Gly127Glu+Gly130ProLeu125Asp+Gly126Glu+Gly127Pro+Thr132ProLeu125Glu+Gly126Glu+Gly127Asn+Thr132SerLeu125Gln+Gly127Pro+Gly130Glu+Ser131AspLeu125Cys+Gly130Glu+Ser131Asp+Thr132GlnGly126Gln+Gly127Glu+Pro128Glu+Gly130AsnGly126Ser+Gly127Asp+Pro128Asp+Gly130SerLeu125Thr+Gly127Ser+Ser131Glu+Thr132AspPro128Asn+Gly130Asn+Ser131Asp+Thr132GluLeu125Met+Gly126Ser+Ser129Asp+Gly130AspGly126Gln+Pro128Gln+Ser129Glu+Gly130GluGly127Asn+Pro128Asn+Ser129Glu+Gly130GluGly127Ser+Ser129Glu+Gly130Asp+Thr132GlnGly127Asp+Pro128Glu+Ser129Asp+Gly130AsnGly127Asp+Pro128Glu+Ser129Asp+Thr132SerLeu125Gln+Ser129Glu+Gly130Asp+Ser131AspLeu125Cys+Ser129Glu+Gly130Asp+Ser131GluGly127Gln+Pro128Glu+Ser129Glu+Gly130AspLeu125Met+Gly126Asp+Gly127Glu+Pro128GluGly127Glu+Pro128Gln+Ser129Glu+Thr132SerLeu125Pro+Gly127Asp+Pro128Ser+Ser129AspGly126Pro+Gly127Glu+Pro128Gly+Ser129AspLeu125Pro+Gly127Asp+Ser129Glu+Gly130GluGly127Asp+Pro128Ser+Ser129Glu+Gly130AspGly127Asp+Ser129Glu+Gly130Glu+Thr132ProLeu125Ile+Gly127Glu+Ser129Asp+Gly130AspGly126Glu+Gly127Asp+Ser129Glu+Gly130ProGly126Asp+Gly127Glu+Ser129Asp+Thr132ProLeu125Met+Gly126Asp+Gly127Asp+Ser129AspGly126Ser+Ser129Asp+Ser131Glu+Thr132GlnGly127Gln+Pro128Asn+Ser129Asp+Ser131AspPro128Gln+Ser129Glu+Ser131Asp+Thr132SerGly126Pro+Ser129Asp+Ser131Glu+Thr132SerLeu125Ala+Ser129Glu+Gly130Pro+Ser131AspGly127Gln+Ser129Glu+Gly130Ser+Ser131AspGly127Ser+Ser129Glu+Ser131Glu+Thr132GlnGly127Gln+Pro128Gln+Ser129Glu+Ser131AspLeu125Ala+Gly126Ser+Ser129Asp+Ser131GluGly126Pro+Pro128Gln+Ser129Asp+Ser131AspGly126Ser+Pro128Ser+Ser129Asp+Ser131GluGly126Glu+Pro128Asp+Ser129Asp+Thr132Asn表14环4-单一突变的变异体Gly153AsnGly153AspGly153GlnGly153GluGly153ProGly153SerAsn154AspAsn154GlnAsn154GluAsn154SerSer155AspSer155GluGly156AsnGly156AspGly156GlnGly156GluGly156ProGly156SerSer157AspSer157GluSer158AspSer158GluGly159AsnGly159AspGly159GlnGly159GluGly159ProGly159SerSer160AspSer160GluGln161AsnGln161AspGln161GluGln161SerAsn162AspAsn162GlnAsn162GluAsn162SerThr163AsnThr163AspThr163GlnThr163GluThr163GlyThr163ProThr163SerIle164AlaIle164AsnIle164AspIle164CysIle164GlnIle164GluIle164GlyIle164HisIle164LeuIle164MetIle164ProIle164SerIle164ThrIle164ValGly165AsnGly165AspGly165GlnGly165GluGly165ProGly165SerTyr166AlaTyr166AsnTyr166AspTyr166CysTyr166GlnTyr166GluTyr166GlyTyr166HisTyr166IleTyr166LeuTyr166MetTyr166ProTyr166SerTyr166ThrTyr166Val表15环4-双点突变的变异体Asn154Ser+Ser155GluGly165Glu+Tyr166CysSer158Asp+Gly165ProSer157Asp+Thr163AsnGly156Pro+Thr163SerAsn154Glu+Ile164CysGly153Asn+Thr163AsnThr163Glu+Ile164GlyGly156Gln+Ile164ThrSer157Asp+Ile164HisSer158Glu+Tyr166HisGly153Pro+Tyr166AspGly153Pro+Gly165GlnSer157Glu+Gly159AsnAsn154Ser+Gly159ProGly156Glu+Asn162GlnGln161Asp+Ile164MetSer157Asp+Thr163SerSer157Glu+Tyr166IleGln161Glu+Ile164HisSer157Glu+Asn162GlnGly159Pro+Tyr166AspAsn162Asp+Ile164CysGln161Ser+Asn162AspAsn154Asp+Asn162GlnSer158Glu+Gly165AsnIle164Met+Tyr166ValGln161Asn+Gly165ProSer155Asp+Gly159ProSer160Asp+Asn162SerGly156Glu+Ile164CysThr163Asp+Gly165ProGly153Asn+Ser155GluSer158Asp+Thr163GlySer160Asp+Ile164MetGly159Asp+Thr163GlnSer155Glu+Tyr166CysGln161Ser+Tyr166GlySer160Asp+Ile164AlaSer157Asp+Gln161SerSer160Glu+Asn162GlnGly153Asn+Gly156GlnSer157Asp+Ile164ValAsn162Gln+Thr163GluSer155Asp+Gln161SerGly159Glu+Gly165GlnSer160Glu+Ile164HisGly153Ser+Ser160AspGly159Gln+Gly165GluGly153Glu+Gly159GlnGly156Glu+Thr163GlyGly156Ser+Tyr166AsnGly153Asp+Thr163AsnAsn154Ser+Ser157GluThr163Ser+Ile164ProSer155Glu+Asn162SerGly156Pro+Ile164GlyThr163Glu+Gly165AsnGly153Gln+Ser155GluIle164Ser+Tyr166Cys表16环4-三点突变的变异体Gly153Gln+Asn154Ser+Ser155GluSer155Asp+Thr163Pro+Tyr166GlnGly156Gln+Thr163Glu+Ile164GlyGly159Gln+Gln161Ser+Gly165SerAsn154Gln+Thr163Asp+Tyr166GlnAsn154Gln+Gly159Pro+Ile164ThrAsn162Gln+Ile164Gln+Gly165GluGly153Asp+Gln161Ser+Thr163ProGly153Gln+Asn154Gln+Tyr166IleAsn154Gln+Gln161Asn+Tyr166ProGly153Gln+Ser158Glu+Tyr166SerSer157Asp+Ile164Val+Tyr166AlaThr163Gly+Gly165Asp+Tyr166ProGIn161Glu+Asn162Gln+Tyr166ThrGly156Pro+Gln161Glu+Thr163SerSer158Glu+Gly159Pro+Gln161AsnSer158Glu+Gln161Asn+Asn162SerAsn154Glu+Gln161Ser+Gly165AsnGly153Asn+Asn154Gln+Ser158AspSer157Glu+Ile164Val+Tyr166ValSer155Asp+Gln161Asn+Tyr166IleGly153Glu+Gln161Asn+Gly165SerGly156Pro+Asn162Asp+Gly165ProGly156Asn+Asn162Asp+Ile164LeuAsn154Gln+Thr163Asn+Gly165SerSer158Asp+Gly159Asn+Gln161AsnSer155Glu+Gln161Asn+Tyr166LeuSer158Glu+Asn162Ser+Tyr166HisAsn154Asp+Thr163Gln+Ile164MetGly156Asp+Ser157Asp+Gly159SerGly159Asp+Ser160Glu+Thr163GlnSer158Asp+Gly159Asp+Tyr166CysAsn154Glu+Ser155Glu+Gly156SerSer155Asp+Gly156Asp+Asn162SerSer157Asp+Ser158Glu+Gly159ProSer160Asp+Gln161Asp+Ile164AlaGly159Gln+Ser160Asp+Gln161AspGly156Asp+Ser157Asp+Ser158AspGly159Asp+Gln161Glu+Gly165GlnGly153Pro+Gly159Glu+Gln161GluSer157Glu+Ser158Asp+Gln161GluSer157Glu+Gly159Glu+Gly165SerSer157Glu+Gly159Glu+Ser160GluSer158Asp+Ser160Asp+Ile164ThrSer158Asp+Ser160Glu+Tyr166ValAsn154Gln+Ser158Asp+Ser160AspSer158Glu+Ser160Asp+Tyr166HisGly153Asp+Asn154Glu+Gly165GluSer155Asp+Ser157Asp+Tyr166IleSer155Glu+Ser157Asp+Gly159ProGly153Asn+Ser157Glu+Thr163GluSer155Asp+Ser157Asp+Ser158GluSer157Asp+Gly159Asp+Gln161GluSer155Asp+Gly156Glu+Ser158AspAsn154Gln+Ser160Glu+Asn162AspSer158Glu+Ser160Glu+Asn162GluSer157Glu+Gln161Asp+Thr163AspAsn154Glu+Ser155Glu+Ser157GluSer157Asp+Ser160Glu+Tyr166GlyGly153Asn+Ser157Glu+Ser160Asp表17环4-四点突变的变异体Asn154Ser+Gly159Asp+Ile164Met+Gly165ProSer155Glu+Gly156Ser+Asn162Gln+Thr163AsnGly156Gln+Ser157Asp+Gly159Ser+Asn162SerSer155Asp+Gly159Pro+Gln161Ser+Ile164CysAsn154Gln+Gly156Asp+Thr163Gly+Tyr166MetAsn154Gln+Ser155Asp+Thr163Asn+Gly165SerGln161Ser+Asn162Gln+Thr163Gly+Tyr166GlySer155Asp+Gly159Pro+Gly165Pro+Tyr166GlnAsn154Ser+Ser155Asp+Thr163Gly+Ile164ValGly159Asp+GIn161Ser+Thr163Gln+Ile164ThrGly153Pro+Ser158Asp+Asn162Gln+Ile164CysGly153Pro+Gly156Asp+Ile164Ser+Tyr166LeuGly153Glu+Gly156Pro+Gln161Ser+Tyr166MetSer155Glu+Thr163Ser+Gly165Pro+Tyr166LeuSer157Asp+Thr163Gly+Ile164Gln+Tyr166GlyGly153Ser+Ser155Asp+Thr163Pro+Ile164ProAsn154Ser+Gly159Gln+Gln161Glu+Tyr166MetAsn154Ser+Ser160Asp+Ile164Gln+Gly165SerGly159Asn+Asn162Glu+Gly165Gln+Tyr166CysGly156Pro+Gly159Pro+Ile164Gln+Gly165GlnGly153Ser+Gly156Asn+Asn162Gln+Gly165AspSer158Asp+Gly159Glu+Gln161Ser+Ile164ThrGly153Gln+Asn154Glu+Ser155Glu+Tyr166GlyGly153Ser+Ser155Asp+Gly156Asp+Ile164GlnAsn154Ser+Ser155Glu+Gly156Asp+Tyr166ValSer155Glu+Gly156Glu+Thr163Asn+Ile164LeuSer157Glu+Ser158Asp+Gln161Asn+Ile164CysSer160Glu+Gln161Asp+Thr163Gln+Gly165ProSer157Asp+Ser158Glu+Gly159Asp+Ile164CysAsn154Gln+Ser160Glu+Gln161Glu+Asn162AspGly153Glu+Gly159Gln+Gln161Asn+Gly165GluAsn154Ser+Ser158Asp+Gly159Asn+Gln161GluAsn154Glu+Gly156Asp+Thr163Ser+Tyr166AsnSer158Glu+Ser160Asp+Gln161Asn+Thr163ProAsn154Glu+Ser155Asp+Gly156Gln+Thr163AspSer158Glu+Gln161Glu+Thr163Asp+Ile164HisSer155Glu+Ser157Asp+Asn162Ser+Gly165ProSer155Asp+Ser157Asp+Gly159Pro+Ile164CysGly159Glu+Ser160Asp+Asn162Glu+Ile164AsnSer157Glu+Gln161Asp+Thr163Asp+Gly165GlnAsn154Glu+Ser155Glu+Ser157Asp+Gly159ProAsn154Gln+Ser160Asp+Gln161Asp+Thr163AspSer160Asp+Gln161Asp+Asn162Gln+Thr163AspSer155Asp+Thr163Asp+Ile164Gly+Gly165AspGly156Ser+Ser158Asp+Thr163Asp+Ile164LeuSer155Glu+Ser158Glu+Thr163Glu+Ile164MetGly153Glu+Gly156Pro+Thr163Asp+Tyr166HisGly156Ser+Ser157Asp+Ser160Glu+Asn162AspGly153Asn+Ser157Glu+Asn162Asp+Ile164LeuGly153Glu+Ser155Glu+Ser157Asp+Ile164MetGly156Pro+Ser157Asp+Gly159Gln+Ser160GluGly153Pro+Ser155Glu+Gly156Glu+Gly165AspSer155Asp+Ser158Glu+Gln161Asn+Gly165ProSer155Asp+Ser158Glu+Gly159Gln+Ile164GlyGly153Asp+Ser157Glu+Gly159Pro+Thr163GluSer155Asp+Gly156Pro+Gln161Ser+Gly165AspGly156Glu+Ser157Glu+Ser160Asp+Ile164SerAsn154Ser+Gly156Asp+Ser158Asp+Ser160AspGly153Gln+Ser155Asp+Ser158Asp+Gly159GluAsn154Asp+Gly156Glu+Ser158Glu+Ile164Ala表18环5-单一突变的变异体Ala186AsnAla186AspAla186GlnAla186GluAla186GlyAla186HisAla186ProAla186SerAla186ThrSer187AspSer187GluPhe188AlaPhe188AsnPhe188AspPhe188CysPhe188GlnPhe188GluPhe188GlyPhe188HisPhe188IlePhe188LeuPhe188MetPhe188ProPhe188SerPhe188ThrPhe188TyrPhe188ValSer189AspSer189GluSer190AspSer190Glu表19环5-双点突变的变异体Phe188Met+Ser190GluAla186Ser+Ser187GluPhe188Ser+Ser190AspAla186His+Ser187GluSer187Asp+Phe188CysAla186Ser+Ser190GluAla186Pro+Ser190AspPhe188Ser+Ser190GluAla186Gln+Ser187GluPhe188Asn+Ser190AspAla186Glu+Phe188MetAla186Ser+Phe188AlaSer187Glu+Phe188ThrAla186Thr+Phe188AspSer187Asp+Phe188LeuAla186Gly+Phe188CysPhe188Val+Ser190GluAla186Asp+Phe188ProAla186Asn+Ser187AspPhe188Leu+Ser190GluSer187Asp+Phe188AsnAla186Ser+Ser187AspPhe188Ala+Ser190GluAla186Gly+Ser187GluAla186Glu+Phe188HisAla186Gly+Phe188GluSer187Glu+Phe188GlyAla186Glu+Phe188AsnSer187Glu+Phe188HisAla186Ser+Phe188AspAla186Gly+Ser187AspPhe188Ile+Ser190GluAla186Thr+Ser190GluAla186Gly+Ser190GluPhe188His+Ser190AspPhe188Leu+Ser190AspAla186Ser+Ser190AspSer187Glu+Phe188IleAla186Thr+Phe188HisPhe188Gln+Ser190GluAla186His+Ser187AspPhe188Tyr+Ser190GluAla186Pro+Phe188ThrAla186Gln+Ser187AspSer187Glu+Phe188AlaAla186Asp+Phe188IleAla186Ser+Phe188ProAla186Asn+Ser187GluPhe188His+Ser190GluAla186Glu+Phe188SerAla186Ser+Phe188LeuPhe188Pro+Ser190GluSer187Asp+Phe188ThrAla186His+Phe188AlaAla186Asn+Phe188AspPhe188Ile+Ser190AspAla186Thr+Phe188MetAla186Asp+Phe188HisSer187Asp+Phe188AlaAla186Pro+Ser190Glu表20环5-三点突变的变异体Ala186His+Phe188Ala+Ser190AspAla186Asn+Ser187Asp+Phe188AlaAla186Gly+Phe188Asn+Ser190AspAla186Pro+Phe188Met+Ser190AspAla186Gly+Phe188Leu+Ser190GluAla186Gln+Ser187Asp+Phe188IleAla186Thr+Ser187Glu+Phe188CysAla186Ser+Phe188Ala+Ser190GluAla186Gln+Ser187Asp+Phe188SerAla186Gln+Ser187Glu+Phe188IleAla186Ser+Phe188Gly+Ser190AspAla186Ser+Ser187Asp+Phe188MetAla186Gly+Ser187Glu+Phe188GlyAla186Gln+Phe188Pro+Ser190AspAla186Asn+Phe188Ala+Ser190GluAla186Ser+Phe188Pro+Ser190GluAla186Asn+Ser187Glu+Phe188ProAla186Gln+Phe188Gly+Ser190GluAla186Asn+Ser187Glu+Phe188IleAla186His+Ser187Glu+Phe188GlyAla186His+Phe188Cys+Ser190AspAla186Thr+Ser187Glu+Phe188ValAla186Gln+Ser187Glu+Phe188MetAla186His+Phe188Ala+Ser190GluAla186Gly+Phe188Met+Ser190GluAla186Ser+Ser187Glu+Phe188HisAla186Asn+Phe188Gly+Ser190GluAla186Asn+Phe188Pro+Ser190AspAla186Asn+Phe188Tyr+Ser190AspAla186Gly+Phe188Thr+Ser190GluAla186His+Ser187Asp+Phe188LeuAla186Ser+Phe188Pro+Ser190AspAla186Thr+Ser187Glu+Phe188ProAla186Pro+Phe188Pro+Ser190GluAla186Thr+Phe188Asn+Ser190AspAla186Pro+Ser187Glu+Phe188HisAla186Asp+Ser187Glu+Phe188MetAla186Asp+Ser187Glu+Phe188GlnAla186Asp+Ser187Glu+Phe188CysAla186Glu+Ser187Asp+Phe188ProAla186Asp+Ser187Glu+Phe188TyrAla186Glu+Ser187Asp+Phe188ThrAla186Asp+Ser187Asp+Phe188ValAla186Glu+Ser187Glu+Phe188AsnAla186Asp+Ser187Asp+Phe188HisAla186Asp+Ser187Glu+Phe188AlaAla186His+Ser187Asp+Phe188AspAla186Gly+Ser187Asp+Phe188AspAla186Gln+Ser187Glu+Phe188GluAla186Asn+Ser187Asp+Phe188GluAla186Pro+Ser187Glu+Phe188AspAla186Asp+Ser187Glu+Phe188AspAla186Glu+Ser187Glu+Phe188GluAla186Asp+Ser187Asp+Phe188GluAla186Thr+Phe188Asp+Ser190GluAla186Ser+Phe188Glu+Ser190GluAla186Thr+Phe188Glu+Ser190AspAla186Pro+Phe188Glu+Ser190GluSer187Glu+Phe188Glu+Ser190GluSer187Asp+Phe188Asp+Ser190Glu表21环5-四点突变的变异体Ala186Gly+Ser187Glu+Phe188Asp+Ser190GluAla186Gly+Ser187Asp+Phe188Asp+Ser190AspAla186Gln + Ser187Asp + Phe188Asp + Ser190AspAla186Gln + Ser187Glu + Phe188Asp + Ser190GluAla186Ser + Ser187Asp + Phe188Glu + Ser190GluAla186Pro + Ser187Glu + Phe188Asp + Ser190AspAla186Thr + Ser187Glu + Phe188Asp + Ser190AspAla186Thr + Ser187Glu + Phe188Asp + Ser190GluAla186His + Ser187Asp + Phe188Glu + Ser190AspAla186Asn + Ser187Glu + Phe188Asp + Ser190AspAla186Gly + Ser187Glu + Phe188Glu + Ser190AspAla186Gln + Ser187Asp + Phe188Glu + Ser190GluAla186Gln + Ser187Asp + Phe188Glu + Ser190AspAla186Gly + Ser187Glu + Phe188Asp + Ser190AspAla186Asp + Ser187Glu + Phe188Gly + Ser190AspAla186Asp + Ser187Asp + Phe188Gln + Ser190AspAla186Glu + Ser187Glu + Phe188Val + Ser190AspAla186Asp + Ser187Glu + Phe188Pro + Ser190GluAla186Glu + Ser187Asp + Phe188Met + Ser190GluAla186Glu + Ser187Asp + Phe188Ala + Ser190GluAla186Asp + Ser187Asp + Phe188His + Ser190AspAla186Glu + Ser187Glu + Phe188Met + Ser190GluAla186Asp + Ser187Asp + Phe188Ala + Ser190GluAla186Glu + Ser187Glu + Phe188Cys + Ser190AspAla186Glu + Ser187Glu + Phe188Thr + Ser190GluAla186Asp + Ser187Asp + Phe188His + Ser190GluAla186Asp + Ser187Asp + Phe188Gln + Ser190GluAla186Asp + Ser187Asp + Phe188Tyr + Ser190AspAla186Glu + Ser187Asp + Phe188Ser + Ser190AspAla186Glu + Ser187Asp + Phe188Gly + Ser190AspAla186Asp + Ser187Asp + Phe188Asn + Ser190AspAla186Glu + Ser187Glu + Phe188Cys + Ser190GluAla186Glu + Ser187Asp + Phe188Ile + Ser190GluAla186Glu + Ser187Glu + Phe188Val + Ser190GluAla186Asp + Ser187Asp + Phe188Cys + Ser190AspAla186Asp + Ser187Glu + Phe188Val + Ser190GluAla186Asp + Ser187Glu + Phe188Ser + Ser190AspAla186Asp + Ser187Asp + Phe188Ala + Ser190AspAla186Asp + Ser187Asp + Phe188Gly + Ser190GluAla186Asp + Ser187Asp + Phe188Thr + Ser190GluAla186Glu + Ser187Glu + Phe188His + Ser190GluAla186Glu + Ser187Asp + Phe188Pro + Ser190GluAla186Glu + Ser187Glu + Phe188Ile + Ser190GluAla186Glu + Ser187Glu + Phe188Pro + Ser190GluAla186Glu + Ser187Glu + Phe188Gly + Ser190AspAla186Glu + Ser187Glu + Phe188Pro + Ser190AspAla186Glu + Ser187Asp + Phe188Ser + Ser190GluAla186Asp + Ser187Asp + Phe188Tyr + Ser190GluAla186Asp + Ser187Glu + Phe188His + Ser190GluAla186Ser + Ser187Glu + Phe188Ser + Ser190GluAla186His + Ser187Glu + Phe188Ser + Ser190GluAla186Ser + Ser187Asp + Phe188Cys + Ser190GluAla186Ser + Ser187Glu + Phe188Asn + Ser190GluAla186Gln + Ser187Glu + Phe188Ala + Ser190AspAla186Gln + Ser187Asp + Phe188Met + Ser190GluAla186Asn + Ser187Glu + Phe188His + Ser190AspAla186Thr + Ser187Glu + Phe188Ala + Ser190GluAla186Pro + Ser187Asp + Phe188Cys + Ser190GluAla186Pro + Ser187Asp + Phe188Leu + Ser190AspAla186Asn + Ser187Asp + Phe188Gln + Ser190Glu表22环6-单一突变的变异体Ala199AsnAla199AspAla199GlnAla199GluAla199GlyAla199HisAla199ProAla199SerAla199ThrPro200AsnPro200AspPro200GlnPro200GluPro200GlyPro200SerGly201AsnGly201AspGly201GlnGly201GluGly201ProGly201SerVal202AlaVal202AsnVal202AspVal202CysVal202GlnVal202GluVal202GlyVal202HisVal202MetVal202ProVal202SerVa1202ThrSer203AspSer203GluVal204AlaVa1204AsnVal204AspVal204CysVal204GlnVal204GluVal204GlyVal204HisVal204MetVal204ProVal204SerVal204ThrTyr205AlaTyr205AsnTyr205AspTyr205CysTyr205GlnTyr205GluTyr205GlyTyr205HisTyr205IleTyr205LeuTyr205MetTyr205ProTyr205SerTyr205ThrTyr205ValSer206AspSer206GluThr207AsnThr207AspThr207GlnThr207GluThr207GlyThr207ProThr207SerTyr208AlaTyr208AsnTyr208AspTyr208CysTyr208GlnTyr208GluTyr208GlyTyr208HisTyr208IleTyr208LeuTyr208MetTyr208ProTyr208SerTyr208ThrTyr208ValPro209AsnPro209AspPro209GlnPro209GluPro209GlyPro209SerSer210AspSer210GluAsn211AspAsn211GlnAsn211GluAsn211SerThr212AsnThr212AspThr212GlnThr212GluThr212GlyThr212ProThr212SerTyr213AlaTyr213AsnTyr213AspTyr213CysTyr213GlnTyr213GluTyr213GlyTyr213HisTyr213IleTyr213LeuTyr213MetTyr213ProTyr213SerTyr213ThrTyr213ValThr214AsnThr214AspThr214GlnThr214GluThr214GlyThr214ProThr214SerSer215AspSer215GluLeu216AlaLeu216AsnLeu216AspLeu216CysLeu216GlnLeu216GluLeu216GlyLeu216HisLeu216IleLeu216MetLeu216ProLeu216SerLeu216ThrLeu216ValAsn217AspAsn217GlnAsn217GluAsn217SerGly218AsnGly218AspGly218GlnGly218GluGly218proGly218SerThr219AsnThr219AspThr219GlnThr219GluThr219GlyThr219ProThr219Ser表23环6-双点突变的变异体Val204Ser+Ser210GluPro200Gly+Thr214GlyThr212Asn+Thr219GlyThr207Ser+Asn211GluVal204Asn+Tyr208ThrSer215Asp+Asn217GlnPro200Gly+Ser215GluTyr208Gln+Tyr213GluPro200Gln+Leu216GluThr207Gln+Ser215AspTyr205Gly+Thr219GlyTyr208Gly+Asn211GluVal202Cys+Gly218AsnTyr205Ser+Tyr213LeuAla199Asn+Gly218ProThr207Gln+Thr214GluAsn211Gln+Ser215AspAla199Gly+Gly218GluPro200Asn+Thr214GluVal204Thr+Pro209AspPro209Gly+Tyr213CysThr214Gln+Asn217GluPro200Gln+Pro209GluAsn211Asp+Thr219GlySer203Asp+Val204GlnTyr205Val+Gly218GluTyr205Gln+Thr219GluSer203Asp+Leu216AlaSer210Asp+Gly218ProVal202Ala+Val204SerAla199Pro+Val204AlaTyr205Gln+Tyr213GlnTyr205Leu+Pro209AspVal204Ser+Tyr208MetThr212Asp+Tyr213MetVal202Ser+Asn211AspVal202Asp+Tyr213SerSer203Asp + Tyr205SerTyr213His + Thr219GluVal202Pro + Asn217GluVal202Ser + Ser210AspVal202Thr + Ser215AspTyr205Ser + Tyr208ProVal204Gly + Ser210GluThr212Glu + Thr219GlnTyr208Ala + Gly218GluPro200Gly + Tyr213AspThr207Pro + Asn211AspPro209Gly + Tyr213AlaVal204Met + Ser210AspThr214Pro + Asn217SerVal204Gln + Gly218ProTyr205Pro + Thr214GluAla199Gln + Ser215AspAla199Pro + Ser203AspGly201Pro + Thr212SerTyr205Pro + Tyr213HisVal204Gln + Thr207SerThr207Gly + Thr219AsnPro200Gln + Thr214ProVal202Cys + Ser215AspGly201Pro + Thr214SerVal204Ser + Pro209GluSer203Asp + Asn211GlnVal202Met + Thr212ProTyr205Gln + Thr207AsnAla199Gln + Tyr213MetGly201Ser + Ser210AspGly201Ser + Thr212AsnTyr208His + Pro209GlyPro209Gln + Ser215GluTyr208Pro + Leu216ThrTyr213Asp + Thr219GlnPro209Gln + Thr212GlyTyr208Met + Pro209GlyPro209Gly + Asn211AspAsn211Asp + Tyr213SerVal202Gln + Ser210GluSer203Glu + Thr207GlnPro200Gln + Val204ThrAsn211Asp + Thr214SerSer210Asp + Thr212GlnVal204Gln + Thr219GluVal202Gln + Tyr205HisThr214Glu + Gly218ProPro209Asn + Asn211SerAla199Gln + Asn217GluPro200Gly + Asn211SerGly201Pro + Tyr208ValVal202Asn + Tyr208CysVal202Gln + Ser215AspAla199Pro + Thr219SerGly218Pro + Thr219GluAla199Gln + Pro200SerThr207Pro + Asn217AspVal204Ser + Asn217GlnSer203Asp + Val204MetSer203Asp + Thr219ProThr212Pro + Thr219ProTyr205Ser + Ser210GluAla199His + Gly201GlnPro209Gly + Ser210GluTyr208Gln + Leu216AsnGly201Ser + Tyr205LeuVal202Met + Thr214GluTyr205Ser + Pro209AspPro209Ser + Thr212GluTyr205Gly + Asn217AspPro200Ser + Leu216SerAla199His + Tyr208HisAsn211Glu + Thr219AsnAla199Asn + Ser210GluTyr205Val + Tyr208HisThr207Ser + Asn211AspGly201Ser + Thr207SerThr207Asn + Tyr208ValTyr205Glu + Asn217GlnTyr208Pro + Thr212GluVal204Thr + Asn211GluTyr205Cys + Asn217AspLeu216Glu + Thr219SerTyr208Ala + Thr212ProAla199Ser + Ser203GluVal204His + Thr212SerVal204Gln + Gly218SerAla199Asn + Ser203GluPro200Gln + Val202MetAla199Asn + Ser203AspTyr205Pro + Leu216AspTyr205Gln + Ser210AspSer210Asp + Asn211GlnAla199Pro + Asn217AspAla199Gly + Asn211AspTyr205Ile + Leu216MetTyr205Gly + Thr214AsnPro209Glu + Thr219SerPro200Gln + Ser210GluAla199Gly + Pro200AsnTyr205Asp + Tyr213ValSer203Asp + Asn217GlnVal204Met + Thr219SerVal202Thr + Gly218SerPro200Gly + Tyr205CysThr207Gly+Thr219AspSer215Glu+Gly218ProGly201Pro+Tyr205MetTyr205Ser+Asn217AspVal202Ser+Pro209GlnPro200Gly+Gly218AsnTyr208Ser+Pro209AspPro209Gln+Ser215AspAla199Thr+Asn217GluThr214Asn+Leu216AspSer210Asp+Tyr213ValThr207Asn+Gly218AspThr212Pro+Thr219GluTyr213Asn+Leu216GluGly201Pro+Thr212AspGly201Gln+Asn211SerVal202Met+Leu216AlaSer215Asp+Leu216GlyGly201Gln+Leu216CysLeu216Gly+Thr219SerVal204Gln+Thr214GlnThr207Ser+Ser215GluThr207Gly+Asn217GluPro200Asn+Tyr213AlaThr212Asp+Thr219ProTyr205Glu+Leu216IleVal202Asp+Tyr208GlyTyr208Cys+Pro209AspThr207Asn+Tyr208IlePro200Gly+Ser210AspAsn211Glu+Thr212ProAla199His+Leu216SerVal204Thr+Asn211AspGly218Ser+Thr219GlySer203Glu+Val204AlaVal202Thr+Thr219GlyThr212Asp+Asn217Gln表24环6-三点突变的变异体Thr207Ser+Thr212Asn+Thr219GlyAla199Asn+Thr207Ser+Asn211GluPro200Gln+Leu216Glu+Gly218AsnTyr205Gly+Thr207Gln+Ser215AspTyr208Gly+Asn211Glu+Thr219GlyVal204Thr+Pro209Asp+Tyr213CysVal204Asn+Thr212Gly+Asn217GluPro200Ser+Tyr208Ser+Thr214GlyAla199Ser+Gly201Ser+Thr214SerAla199Gly+Val202Met+Ser210GluGly201Asn+Tyr205His+Thr214GlnAla199Thr + Asn211Asp + Gly218SerThr207Gln + Tyr213Gln + Leu216MetAla199Gly + Val202Cys + Ser210AspVal204Gln + Tyr213Ala + Thr214ProTyr213Leu + Asn217Ser + Thr219GluPro200Gly + Thr214Pro + Asn217AspPro200Gly + Leu216Val + Thr219GlnPro200Gly + Gly201Ser + Ser210AspGly201Gln + Val202His + Thr212GluTyr208His + Asn211Ser + Asn217AspThr207Ser + Ser210Asp + Gly218SerThr212Asn + Leu216Asn + Thr219GluVal202Pro + Val204Met + Pro209AspVal202Asn + Thr212Pro + Thr214GluTyr208Ile + Thr212Asp + Tyr213HisSer210Asp + Thr212Ser + Leu216AlaPro200Gln + Thr214Pro + Leu216HisTyr208Met + Pro209Asp + Leu216IleVal202Met + Leu216Gly + Thr219GlyPro200Ser + Tyr205Val + Leu216CysVal204Thr + Tyr205Glu + Asn217GlnThr212Glu + Leu216Ile + Asn217SerVal204Thr + Tyr208Pro + Asn211GluThr207Gly + Leu216Glu + Thr219SerSer203Glu + Tyr208Ala + Thr212ProAla199Ser + Val204His + Thr212SerAla199Asn + Val204Gln + Gly218SerPro200Gln + Thr214Pro + Gly218AspAla199Asn + Val202Met + Ser203AspPro200Gln + Ser210Asp + Asn211GlnTyr205Ile + Thr214Asn + Leu216MetPro200Gln + Pro209Glu + Thr219SerAla199Gly + Pro200Asn + Ser210GluVal204Met + Asn217Gln + Thr219SerVal202Thr + Thr214Asp + Gly218SerGly201Pro + Tyr205Met + Gly218ProVal202Ser + Tyr205Ser + Asn217AspPro200Gly + Pro209Gln + Gly218AsnAla199Thr + Thr214Asn + Asn217GluThr207Asn + Thr212Pro + Gly218AspGly201Pro + Asn211Ser + Thr212AspGly201Gln + Ser215Asp + Leu216GlyVal204Gln + Leu216Cys + Thr219SerThr207Ser + Thr214Gln + Ser215GluPro200Asn + Thr207Gly + Asn217GluThr212Asp + Tyr213Ala + Thr219ProThr207Asn + Tyr208Ile + Pro209GluAla199His + Asn211Glu + Thr212ProVal204Thr + Asn211Asp + Leu216SerSer203Glu + Val204Ala + Thr219GlyThr207Gly + Thr212Gly + Leu216HisGly201Asn + Thr207Gly + Tyr208GlyGly201Asn + Val202Glu + Thr207SerThr207Gln + Leu216Glu + Gly218SerTyr205His + Ser210Asp + Thr219ProPro200Gly + Tyr208Val + Ser210AspAla199Ser + Ser203Asp + Tyr208GlnGly201Ser + Asn211Glu + Tyr213ProAla199Gln + Tyr205His + Thr219GluPro200Ser + Thr207Pro + Thr214SerPro200Gln + Tyr205Met + Thr212ProAla199Gly + Val204His + Asn211GluThr207Asn + Thr212Ser + Thr219AsnTyr205Pro + Thr207Gln + Ser215GluVal204His + Gly218Glu + Thr219GlyTyr205Pro + Tyr208Ile + Ser215GluAla199Asn + Pro200Ser + Ser203GluTyr208Gln + Tyr213Met + Thr219GluVal202Glu + Val204Thr + Asn217SerVal204Gln + Pro209Gln + Thr214GlyThr214Gln + Ser215Asp + Leu216GlyVal204Thr + Tyr213Ile + Ser215GluVal204Gly + Asn211Gln + Ser215GluPro200Ser + Val204Gly + Tyr213IlePro200Gly + Thr212Asp + Leu216HisAsn211Glu + Tyr213Thr + Leu216ProLeu216Asn + Asn217Asp + Gly218SerGly201Gln + Pro209Asn + Ser215GluGly201Asn + Ser203Glu + Pro209AsnGly201Asn + Val202Ser + Leu216GlyAsn211Asp + Tyr213His + Thr219GlnThr207Gln + Asn217Asp + Gly218SerTyr208Gly + Ser210Glu + Thr214GlyThr207Gln + Tyr208Gln + Tyr213AspAla199Asn + Tyr205Ile + Gly218ProPro200Asn + Val202Gln + Val204SerVal204Asn + Ser215Glu + Leu216ProPro200Ser + Gly201Ser + Tyr208AlaTyr208Thr + Tyr213Val + Leu216AspThr207Gly + Thr212Ser + Tyr213AlaGly201Asn + Gly218Asp + Thr219AsnAla199Gly + Tyr205Cys + Tyr208ThrGly201Gln + Tyr213Cys + Thr214GlyAla199Gln + Pro200Ser + Thr214AspAla199Ser + Gly201Asn + Thr219ProVal204Gly + Leu216Val + Thr219GlnGly201Asn + Ser203Glu + Thr212GlyGly201Ser + Ser203Glu + Asn217GlnPro200Gly + Thr207Gln + Asn217AspAla199Thr + Tyr208Gln + Thr219ProThr207Ser + Thr212Ser + Gly218AspPro200Ser + Tyr208Ile + Asn217SerVal204Pro + Tyr205Asp + Thr214ProGly201Ser + Tyr205Glu + Thr207AsnPro200Ser + Thr207Gln + Asn217GlnGly201Asn + Val202Glu + Leu216CysAla199Ser + Pro209Ser + Gly218AspGly201Pro + Tyr205Met + Asn211AspGly201Ser + Thr214Pro + Ser215AspVal204Pro + Thr212Gly + Tyr213ValGly201Pro + Tyr208Ser + Thr212GluAla199Gln + Ser203Asp + Leu216SerVal202Gly + Asn211Glu + Tyr213LeuSer203Asp + Thr212Gly + Thr214GlnGly201Pro + Tyr205Gln + Thr207GlyGly201Asn + Tyr208Asn + Tyr213ProPro200Ser + Tyr213Asp + Gly218ProAla199Asn + Pro200Gly + Thr214SerSer210Glu + Tyr213Gln + Gly218AsnAla199Pro + Tyr213Thr + Ser215AspVal202Pro + Tyr208Cys + Asn211GlnAla199Gln + Ser203Glu + Asn211SerThr207Gln + Pro209Ser + Ser210GluPro200Ser + Val202Asn + Gly218SerTyr205Val + Tyr213Asn + Gly218GluVal202Glu + Val204His + Tyr205GlnAla199Gly + Thr207Pro + Asn211GluVal202Glu + Tyr208Met + Pro209GlyVal204Ser + Tyr208Ile + Leu216MetTyr205Thr + Tyr208Ser + Asn217SerThr207Ser + Pro209Ser + Leu216AsnGly201Asn + Pro209Asp + Tyr213MetGly201Asn + Pro209Gln + Thr214AsnPro200Asn + Val202Gln + Thr207GlnAla199Asn + Ser203Glu + Val204GlnSer203Asp + Pro209Gly + Leu216AlaAla199Thr + Pro200Asn + Ser210GluAla199Thr + Tyr205Leu + Asn217GluGly201Asn + Val202Met + Thr214AspThr212Gln + Thr214Glu + Thr219SerTyr205Glu + Tyr208Gly + Tyr213LeuPro200Ser + Asn217Glu + Gly218GlnSer210Glu + Thr214Pro + Leu216AsnTyr208Asn + Pro209Asn + Tyr213SerAla199Pro + Gly201Pro + Ser210AspGly201Gln + Thr207Gly + Gly218GluPro200Gln + Tyr208Gly + Ser215AspPro200Ser + Thr214Pro + Leu216GluAla199Gln + Asn217Asp + Gly218ProPro200Asn + Val202Asp + Pro209SerThr212Gly + Tyr213Asn + Ser215AspGly201Gln + Tyr205Met + Thr219GluPro200Gly + Tyr213Gly + Thr219GluPro200Gly + Ser203Glu + Thr214GlnTyr208Thr + Ser215Glu + Gly218SerSer210Asp + Asn211Ser + Leu216AsnAsn211Gln + Thr214Glu + Ser215AspPro209Glu + Ser210Asp + Leu216CysTyr208Pro + Asn217Asp + Gly218AspTyr213Cys+Ser215Asp+Leu216AspThr212Asp+Tyr213Asp+Gly218SerSer210Asp+Asn211Glu+Tyr213GlnVal204Cys+Ser210Glu+Asn211GluTyr205Glu+Tyr208Ala+Ser215GluVal202Glu+Ser203Glu+Asn217AspPro200Gln+Ser203Glu+Ser215AspAlal99Asn+Ser203Glu+Asn217GluSer203Asp+Tyr208Cys+Asn217GluTyr205Asp+Asn211Gln+Thr214Glu表25环6-四点突变的变异体Gly201Asn+Tyr205Gly+Thr207Gln+Tyr213IleVal202Cys+Tyr205Ser+Thr212Pro+Gly218AsnAlal99Ser+Tyr213His+Thr214Gln+Leu216AsnAlal99Ser+Pro200Ser+Tyr208Ser+Thr214SerAlal99Gly+Gly201Ser+Val202Met+Ser210GluGly201Asn+Tyr205His+Thr207Gln+Thr214GlnVal202Cys+Thr207Gln+Tyr213Gln+Leu216MetVal204Gln+Tyr20SAsp+Tyr213Ala+Thr214ProPro200Gly+Tyr213Leu+Thr214Pro+Asn217SerPro200Gly+Leu216Val+Asn217Asp+Thr219GlnAlal99Thr+Val204Ala+Asn217Gln+Gly218GlnGly201Gln+Val204Gln+Thr207Gln+Leu216Hispro200Asn+Val202Pro+Thr207Gly+Ser215AspAlal99Ser+Gly201Pro+Ser215Asp+Leu216SerAlal99Thr+Tyr205Gly+Ser215Glu+Asn217SerAlal99Gly+Thr207Gln+Tyr208Thr+Pro209GlnAla199His+Pro200Gly+Gly218Gln+Thr219GlnAlal99Gly+Thr207Asn+Asn211Ser+Thr212SerPro200Ser+Val202Gln+Tyr208Ile+Thr219ProPro200Gln+Tyr208Met+Pro209Asp+Leu216HisSer203Asp+Asn211Gln+Leu216Thr+Gly218GlnAla199Gly+Gly201Ser+Tyr205Asp+Leu216GlyAla199Gln+Gly201Asn+Tyr205Gln+Thr214AspPro200Ser+Gly201Pro+Val204Asn+Tyr208ProGly201Pro+Val202Gly+Ser210Asp+Leu216MetPro200Gly+Val202Cys+Ser203Asp+Leu216ProTyr205Gly+Thr207Pro+Asn211Gln+Asn217SerGly201Pro+Thr207Gln+Thr212Ser+Asn217AspPro200Gln+Tyr208Leu+Tyr213Met+Leu216GlnTyr205Met+Asn211Gln+Tyr213Met+Thr219AspVal202Met+Tyr205Leu+Tyr208Thr+Tyr213GlySer203Asp+Val204Ser+Tyr213Gly+Gly218ProGly201Gln+Val202Met+Ser215Asp+Leu216AlaVal204Gln+Thr214Gln+Leu216Cys+Thr219SerPro200Asn+Thr212Asp+Tyr213Ala+Thr219ProAlal99His+Val204Thr+Asn211Asp+Leu216SerVal202Thr+Ser203Glu+Val204Ala+Thr219AsnThr207Gly+Tyr208Gly+Thr212Gly+Leu216HisGly201Asn + Ser203Glu + Thr207Gly + Asn211GlnTyr205His + Thr207Gln + Leu216Gly + Gly218SerAla199Gln + Tyr205His + Tyr213Pro + Thr219GluAla199Gly + Tyr205Met + Asn211Glu + Thr212ProGly201Pro + Thr207Asn + Thr212Gln + Thr219AsnPro200Gln + Leu216Thr + Asn217Glu + Thr219ProGly201Ser + Tyr205Cys + Thr214Asn + Leu216ThrGly201Gln + Val204Pro + Asn211Asp + Gly218ProSer203Asp + Tyr205Val + Thr207Ser + Leu216CysVal202His + Pro209Gln + Ser215Asp + Leu216GlyPro200Asn + Gly201Gln + Val202Pro + Ser203GluPro200Ser + Gly201Gln + Val202His + Leu216ThrPro200Asn + Gly201Asn + Leu216Ala + Gly218ProAla199Pro + Val202Ser + Val204His + Thr214GluGly201Gln + Ser203Glu + Tyr213Ile + Gly218AsnAla199His + Ser203Asp + Val204Gly + Asn211GlnPro200Gln + Val202Ala + Val204Pro + Thr207ProGly201Asn + Thr212Ser + Tyr213Ala + Gly218AspGly201Gln + Tyr213Cys + Thr214Gly + Gly218GluAla199Gln + Pro200Ser + Gly201Asn + Thr214AspAla199Ser + Val204Gly + Leu216Val + Thr219ProPro200Gly + Val202His + Thr207Gln + Asn217AspAla199Thr + Tyr208Gln + Thr212Ser + Thr219ProPro200Ser + Thr207Ser + Tyr208Ile + Gly218AspVal204Pro + Tyr205Asp + Thr214Pro + Asn217SerGly201Pro + Tyr205Met + Asn211Asp + Thr214ProGly201Ser + Tyr213Val + Thr214Pro + Ser215AspGly201Pro + Tyr208Ser + Thr212Gly + Tyr213AlaVal202Asn + Asn211Glu + Tyr213Leu + Thr219ProGly201Pro + Tyr205Gln + Thr207Gly + Thr212GlyPro200Gln + Tyr208Cys + Ser215Glu + Thr219GlyGly201Asn + Tyr208Asn + Tyr213Pro + Ser215GluPro200Ser + Gly201Pro + Tyr213Asp + Gly218ProAla199Pro + Pro200Ser + Thr212Asp + Thr219SerPro200Asn + Ser203Asp + Thr207Gln + Thr212GlyVal204Cys + Pro209Ser + Ser215Asp + Gly218GlnVal202Gln + Val204Ser + Thr212Asp + Asn217GlnAla199Asn + Pro209Gly + Asn211Glu + Thr214ProAla199Asn + Val204Thr + Ser210Asp + Tyr213LeuPro200Asn + Val204Met + Thr207Gln + Tyr213AspVal204Gln + Pro209Gly + Thr212Gly + Tyr213CysPro209Asn + Tyr213Ile + Thr214Gly + Leu216MetPro200Gly + Ser203Asp + Leu216Cys + Asn217SerVal204Ser + Tyr208Ile + Asn211Asp + Leu216MetGly201Asn + Tyr205Ile + Pro209Gln + Leu216ValSer203Glu + Val204Gln + Tyr213Leu + Gly218AsnPro200Asn + Ser210Asp + Thr214Ser + Leu216CysAla199Thr + Pro200Asn + Ser210Glu + Leu216CysAla199Thr + Val202Met + Tyr205Leu + Thr214AspGly201Asn + Thr212Gln + Thr214Glu + Thr219SerGly201Pro + Tyr208Asn + Pro209Asn + Tyr213SerAla199Pro + Gly201Gln + Thr207Gly + Ser210AspAla199Gly + Pro200Gln + Tyr208Asn + Leu216ThrPro200Asn + Val202Asp + Pro209Ser + Gly218ProPro200Gly + Thr207Asn + Leu216Asn + Thr219AspAla199Ser + Pro200Gly + Val202Asp + Thr207SerVal204Ala + Thr207Asn + Ser210Glu + Tyr213MetPro200Gln + Gly201Ser + Tyr205Thr + Ser215GluAla199His + Gly201Asn + Tyr208Ala + Ser210AspPro200Gln + Val204Met + Tyr205Ala + Pro209GluVal202Ala + Tyr205Leu + Thr214Glu + Thr219GlnThr207Gln + Pro209Asn + Ser210Glu + Thr214ProGly201Asn + Ser203Asp + Tyr208Val + Asn211GlnAla199Ser + Pro200Gly + Leu216Val + Gly218ProGly201Gln + Asn211Gln + Thr212Glu + Leu216AlaVal202Cys + Thr207Asn + Tyr213Pro + Leu216AspAla199Gly + Val202His + Val204Ala + Ser215GluAla199Gln + Thr212Pro + Tyr213Glu + Leu216AlaPro200Gln + Val202Gln + Thr214Gln + Gly218GluGly201Gln + Thr207Pro + Tyr208Met + Thr219ProSer203Asp + Val204Gln + Tyr208Ser + Leu216ProAla199Asn + Ser203Glu + Val204Asn + Tyr208ProPro200Gly + Tyr213Met + Leu216Val + Gly218AsnTyr205Ile + Tyr213Ile + Thr214Asn + Thr219ProPro200Gln + Thr207Gly + Ser215Glu + Leu216MetAla199Ser + Tyr208Ala + Asn211Gln + Thr219AspGly201Pro + Val204Ser + Tyr205His + Thr219AspVal202Met + Thr207Pro + Thr212Gly + Asn217GluPro200Gln + Gly201Pro + Tyr208Thr + Ser215GluVal204Thr + Thr212Gly + Thr214Glu + Gly218SerPro200Gln + Tyr208Ala + Tyr213Glu + Asn217SerTyr213Met + Thr214Ser + Ser215Asp + Leu216ProThr207Ser + Pro209Asp + Thr212Gly + Thr219GlnThr207Gln + Asn211Gln + Thr214Glu + Ser215AspTyr205Ala + Tyr213Met + Thr214Glu + Ser215AspTyr205Met + Thr214Asp + Ser215Glu + Asn217GlnThr207Asn + Thr214Gly + Leu216Glu + Asn217AspVal202Thr + Val204His + Asn211Glu + Thr212AspPro200Gln + Asn211Asp + Thr212Glu + Thr214SerAsn211Glu + Thr212Glu + Tyr213Ile + Thr214AsnGly201Asn + Val202Glu + Ser203Glu + Thr212GlyThr212Asp + Tyr213Asp + Gly218Ser + Thr219GlyVal204Ala + Thr212Glu + Tyr213Glu + Thr214AsnPro200Gly + Ser210Asp + Asn211Glu + Thr212ProPro209Gln + Ser210Asp + Asn211Glu + Thr219ProPro200Ser + Val204Met + Ser210Asp + Asn211GluAla199Pro + Val202Ala + Ser210Glu + Asn211GluPro209Asp + Ser210Asp + Asn211Glu + Thr212ProVal202Asp + Ser203Asp + Tyr213Gln + Asn217GluSer203Asp + Ser215Asp + Leu216Glu + Thr219AsnPro200Ser + Ser203Glu + Leu216Glu + Asn217GluSer203Asp + Pro209Gly + Ser215Glu + Leu216AlaSer203Glu + Thr214Gln + Ser215Glu + Gly218SerGly201Asn + Ser203Asp + Tyr213Ala + Ser215GluSer203Glu + Thr207Ser + Tyr213Gly + Ser215AspAla199Gln + Ser203Glu + Thr214Pro + Asn217AspSer203Glu+Val204Asn+Thr212Gly+Asn217GluSer203Asp+Thr207Gln+Tyr208Cys+Asn217GluGly201Ser+Ser203Glu+Thr214Pro+Asn217AspAsn211Glu+Thr212Asp+Tyr213Glu+Thr219GlyAla199Pro+Ser203Asp+Val204Pro+Leu216GluAlal99Asn+Pro209Asp+Asn211Glu+Tyr213IlePro200Gln+Tyr205Leu+Pro209Glu+Thr212AspSer203Asp+Tyr205Glu+Tyr208Gly+Tyr213LeuAlal99Ser+Val202Glu+Leu216Cys+Gly218AspPro200Ser+Thr212Pro+Thr214Glu+Leu216GluAlal99Asn+Val202Pro+Ser215Glu+Asn217AspPro200Gly+Thr207Ser+Ser215Glu+Asn217GluGly201Asn+Val202Glu+Thr207Ser+Leu216GluAlal99Asn+Ser203Glu+Tyr205Glu+Asn217GluTyr205Glu+Thr214Pro+Ser215Asp+Asn217GluGly201Gln+Thr212Ser+Tyr213Glu+Ser215GluAlal99Ser+Tyr213Asp+Thr214Pro+Ser215GluAsn211Ser+Ser215Asp+Asn217Glu+Gly218AspSer203Asp+Thr212Gln+Ser215Asp+Gly218AspVal202Glu+Thr212Gly+Ser215Asp+Leu216GlnVal202Asp+Val204Cys+Tyr208Pro+Thr219GluSer203Asp+Pro209Gln+Tyr213Thr+Thr214AspVal202Asp+Tyr205Glu+Tyr208Gly+Leu216IleTyr208Gly+Thr214Asp+Asn217Glu+Gly218AspPro200Gln+Tyr208Gln+Tyr213Glu+Leu216GluGly201Gln+Tyr205Leu+Thr214Glu+Asn217AspSer203Asp+Val204Gly+Thr212Glu+Ser215AspGly201Gln+Val202Pro+Asn2l1Glu+Thr214GluThr207Gln+Pro209Ser+Asn211Asp+Thr214GluTyr205Ala+Ser210Glu+Thr212Asp+Ser215AspAlal99Gln+Thr214Glu+Leu216Glu+Thr219GluThr212Asp+Thr214Glu+Leu216Asn+Asn217GluGly201Asn+Asn211Glu+Thr214Asp+Leu216GluPro209Asp+Ser215Asp+Leu216Asp+Gly218GlnGly201Pro+Val202Cys+Ser203Asp+Thr219AspSer203Glu+Thr212Gln+Gly218Gln+Thr219Glu表26环6-五点取代的变异体Gly201Asn+Thr207Gln+Ser210Glu+Tyr213Ile+
Leu216IleVal202Cys+Tyr205Ser+Thr212Pro+Tyr213Leu+
Gly218AsnAlal99Ser+Pro200Ser+Tyr213His+Thr214Gln+
Leu216AsnAla199Ser+Gly201Ser+Tyr208Ser+Ser210Glu+
Thr214SerAla199Gly+Gly201Asn+Val202Met+Tyr205His+
Thr214GlnPro200Gly+Tyr213Leu+Thr214Pro+Asn217Ser+
Thr219GluAla199Thr + Tyr205Gln + Thr207Gln + Thr212Asp +
Gly218GlnVal204Gly + Tyr205Ala + Tyr208Gly + Asn211Gln +
Gly218AspAla199Ser + Gly201Pro + Tyr205Gly + Ser215Glu +
Leu216SerAla199His + Thr207Gln + Tyr208Thr + Gly218Gln +
Thr219GlnAla199Gly + Pro209Gly + Asn211Ser + Thr212Ser +
Tyr213SerPro200Ser + Val202Gln + Thr207Asn + Tyr208Ile +
Thr219ProPro200Ser + Gly201Ser + Tyr208Gln + Ser210Glu +
Asn217GlnAla199Gly + Gly201Ser + Val202Gly + Tyr205Asp +
Leu216GlyAla199Gln + Pro200Ser + Gly201Ser + Tyr205Gln +
Thr214AspAla199Pro + Gly201Pro + Val204Asn + Tyr208Pro +
Thr214GlyPro200Gly + Val202Gly + Ser203Asp + Asn211Ser +
Leu216ProTyr205Gly + Thr207Pro + Pro209Asp + Asn211Gln +
Asn217SerPro200Gln + Val204Ala + Tyr208Leu + Thr212Glu +
Tyr213MetTyr205Met + Asn211Gln + Tyr213Met + Thr214Asn +
Thr219AspSer203Asp + Val204Ser + Tyr213Gly + Thr214Asn +
Gly218ProGly201Gln + Val204Gln + Ser215Asp + Leu216Ala +
Thr219SerGly201Asn + Thr207Gly + Tyr208Gly + Thr212Gly +
Leu216HisGly201Asn + Tyr205His + Thr207Ser + Leu216Glu +
Gly218SerAla199Gln + Pro200Ser + Tyr205His + Thr214Ser +
Thr219GluPro200Gln + Gly201Gln + Leu216His + Asn217Asp +
Gly218GlnAla199Gly + Val204His + Tyr205Met + Asn211Glu +
Thr212ProVal204His + Tyr205Pro + Tyr208Ile + Gly218Glu +
Thr219GlyPro200Ser + Val202Gln + Val204Gly + Tyr213Ile +
Ser215GluAla199Ser + Pro200Asn + Asn211Glu + Tyr213Thr +
Leu216ProSer203Asp + Val204Gly + Asn211Gln + Tyr213Ile +
Gly218AsnAla199Asn + Pro200Gln + Val202Ala + Val204Pro +
Thr207ProAla199Thr + Gly201Gln + Val202Gly + Ser215Asp +
Leu216AlaThr212Gln + Tyr213Val + Leu216His + Gly218Gln +
Thr219GluAla199Gln + Pro200Ser + Gly201Asn + Tyr205Ala +
Thr214GlyGly201Asn + Val204Gly + Thr212Gly + Leu216Val +
Thr219ProPro200Gly + Val202His + Thr207Gln + Tyr208Gln +
Asn217AspAla199Thr + Thr207Ser + Thr212Ser + Gly218Asp +
Thr219ProPro200Ser + Val204Pro + Tyr208Ile + Thr214Pro +
Asn217SerPro200Gln + Val202Ser + Tyr205Ser + Asn211Ser +
Ser215AspVal202Thr + Thr212Gln + Ser215Asp + Asn217Ser +
Thr219AsnVal204Pro + Thr212Gly + Tyr213Val + Thr214Pro +
Ser215AspGly201Ser + Val204His + Tyr208Leu + Thr212Ser +
Gly218GluSer203Asp + Tyr205Gln + Thr207Gly + Thr212Gly +
Thr214GlnPro200Gln + Tyr208Cys + Thr212Gly + Ser215Glu +
Thr219GlyPro200Ser + Gly201Pro + Val202Ala + Tyr213Asp +
Gly218ProVal204Gly + Ser210Asp + Thr212Asn + Thr214Pro +
Thr219SerPro200Gly + Ser203Asp + Pro209Asn + Leu216Cys +
Asn217SerTyr205Thr + Thr207Ser + Tyr208Ser + Pro209Ser +
Asn217SerGly201Asn + Tyr205Ile + Pro209Gln + Thr214Asn +
Leu216ValAla199Asn + Ser203Glu + Val204Gln + Tyr213Leu +
Gly218AsnAla199Thr + Val202Met + Tyr205Leu + Asn211Ser +
Asn217GluAla199Pro + Gly201Pro + Pro209Asn + Ser210Asp +
Tyr213SerAla199Ser + Gly201Pro + Pro209Gly + Tyr213Pro +
Gly218ProPro200Ser + Pro209Gly + Thr212Pro + Thr214Pro +
Leu216GluGly201Ser + Thr207Gly + Ser210Asp + Asn211Ser +
Leu216IleAla199Ser + Gly201Gln + Val204Asn + Tyr205Leu +
Thr219AspThr207Pro + Tyr208Thr + Ser210Glu + Thr212Gly +
Asn217SerGly201Gln + Tyr205Asn + Thr207Asn + Pro209Asp +
Thr212GlyPro200Asn + Tyr205Glu + Tyr208Met + Tyr213Val +
Leu216IleTyr205Ala + Tyr213His + Thr214Ser + Asn217Gln +
Thr219GlnAla199Gln + Pro200Gly + Val202Met + Leu216Met +
Gly218ProPro200Ser + Val204Ala + Ser210Glu + Asn211Gln +
Gly218SerAla199Ser + Pro200Ser + Val202Asn + Leu216Val +
Gly218ProPro200Gln + Val202Gln + Tyr213Leu + Thr214Pro +
Gly218GluPro200Ser + Gly201Gln + Thr207Pro + Gly218Pro +
Thr219ProAla199Asn + Ser203Asp + Val204Asn + Tyr208Pro +
Leu216ProTyr205Ile + Tyr213Ile + Thr214Asn + Ser215Glu +
Thr219ProGly201Ser + Val204His + Asn211Gln + Thr212Gln +
Leu216MetAla199Ser + Gly201Pro + Asn211Glu + Gly218Gln +
Thr219SerAla199Ser + Thr207Ser + Thr214Gln + Ser215Glu +
Leu216AsnAla199His + Val204Cys + Asn211Ser + Thr214Gly +
Thr219GluAla199Asn + Val202His + Tyr208Asn + Leu216Thr +
Thr219GlySer203Glu + Thr207Pro + Tyr208Leu + Thr212Ser +
Asn217GlnPro200Ser + Val202Asp + Val204Asn + Tyr208Met +
Thr212GlnGly201Ser + Thr207Asn + Tyr208Leu + Ser210Asp +
Asn217SerVal202Asp + Val204Gln + Tyr208Cys + Pro209Gln +
Tyr213ValAla199Gly + Val202Thr + Thr207Gln + Thr212Glu +
Thr214GlnGly201Pro + Val202Cys + Ser210Asp + Thr212Pro +
Tyr213CysAla199Asn + Gly201Ser + Val204Met + Pro209Asn +
Thr214ProGly201Asn + Ser203Asp + Val204Thr + Thr207Ser +
Leu216AlaVal202Thr + Tyr205Gly + Thr207Gln + Asn211Asp +
Leu216AlaAla199Gln + Va1204Gly + Thr207Pro + Asn211Asp +
Thr214GlyGly201Pro + Val202Asn + Tyr205Ser + Asn211Glu +
Gly218AsnVal204Gly + Pro209Asn + Asn211Asp + Asn217Gln +
Thr219GlyVal202Gly + Val204Gly + Ser210Glu + Gly218Ser +
Thr219SerAla199Ser + Asn211Ser + Tyr213Met + Thr214Asp +
Thr219ProAla199Asn + Gly201Asn + Ser203Glu + Tyr213His +
Leu216IleAla199Gln + Pro200Asn + Thr207Ser + Pro209Ser +
Asn217SerTyr205Met + Thr214Asp + Ser215Glu + Leu216Cys +
Asn217GlnAla199Pro + Val202His + Tyr208Met + Thr214Glu +
Ser215AspPro200Asn + Pro209Glu + Ser210Asp + Thr214Ser +
Leu216CysVal204Ser + Tyr208Pro + Leu216Met + Asn217Asp +
Gly218AspTyr205Pro + Thr207Pro + Leu216Asn + Asn217Asp +
Gly218AspPro200Gly + Tyr205Asn + Tyr208Gly + Asn217Glu +
Gly218GluVal202Thr + Val204His + Pro209Gly + Asn211Glu +
Thr212AspGly201Gln + Val202Asn + Thr207Asn + Ser215Glu +
Leu216AspAla199His + Pro200Gly + Ser210Asp + Asn211Glu +
Thr212ProPro209Gln + Ser210Asp + Asn211Glu + Asn217Gln +
Thr219ProAla199Pro + Val202Ala + Ser210Glu + Asn211Asp +
Gly218AsnAla199Asn + Thr207Asn + Ser210Asp + Asn211Asp +
Thr212GlyVal204Ala + Tyr205Glu + Thr212Ser + Ser215Glu +
Asn217GlnGly201Pro + Val202Asp + Thr207Gln + Thr212Ser +
Asn217AspThr207Ser + Pro209Asp + Ser210Glu + Asn211Asp +
Tyr213AsnGly201Ser + Val202Glu + Ser203Glu + Thr214Pro +
Asn217AspSer203Glu + Thr207Asn + Thr214Gly + Leu216Glu +
Asn217AspGly201Ser + Ser203Asp + Tyr208Ile + Leu216Asp +
Asn217AspGly201Pro + Tyr208Gly + Leu216Asp + Asn217Glu +
Gly218AspSer203Glu + Val204Pro + Thr207Gly + Tyr213Leu +
Ser215AspGly201Asn + Ser203Asp + Tyr208Val + Asn211Gln +
Ser215AspAla199Asn + Pro200Ser + Ser203Glu + Tyr213Met +
Ser215GluAla199Ser + Ser203Glu + Asn211Gln + Ser215Glu +
Leu216CysSer203Glu + Val204Gln + Thr214Gln + Ser215Asp +
Leu216GlyAla199Pro + Gly201Pro + Ser203Asp + Tyr205Val +
Ser215GluAla199Thr + Ser203Asp + Val204Gln + Tyr213Gly +
Ser215AspVal202Cys + Ser203Asp + Tyr205Met + Tyr213Gln +
Asn217GluSer203Glu + Val204Asn + Thr212Gly + Asn217Glu +
Thr219GlnGly201Asn + Ser203Asp + Tyr208Val + Pro209Ser +
Asn217GluSer203Asp + Thr212Pro + Tyr213Gly + Asn217Glu +
Gly218SerVal204Thr + Asn211Asp + Thr212Asp + Tyr213Asp +
Leu216SerVal202Ala + Ser203Asp + Val204Gln + Tyr205Met +
Leu216GluSer203Glu + Val204Ser + Thr207Gly + Tyr208Val +
Leu216AspPro200Gln + Pro209Gln + Ser210Glu + Thr212Glu +
Thr214GlyPro209Glu + Asn211Asp + Asn217Gln + Gly218Gln +
Thr219ProPro200Ser + Tyr205Pro + Pro209Asp + Asn211Glu +
Tyr213IleAla199Asn + Ser203Asp + Tyr205Glu + Thr207Asn +
Leu216GluSer203Glu + Tyr205Glu + Tyr213Met + Leu216Ile +
Asn217GlnVal202Glu + Ser203Glu + Asn211Gln + Leu216Met +
Gly218GluAla199Ser + Val202Ser + Tyr205Asp + Tyr208Gly +
Tyr213AspPro200Ser + Thr207Pro + Thr212Asp + Thr214Asp +
Thr219SerPro200Gly + Thr207Ser + Thr214Gln + Ser215Glu +
Asn217GluPro200Gln + Thr207Gly + Ser215Asp + Leu216Gln +
Asn217AspAla199Asn + Tyr205Glu + Tyr208Ala + Ser215Glu +
Asn217GluGly201Pro + Val204Ser + Tyr205His + Asn217Glu +
Thr219AspAla199Ser + Tyr208Gln + Tyr213Asp + Thr214Pro +
Ser215GluThr207Asn + Tyr208Gln + Thr212Ser + Tyr213Glu +
Ser215GluGly201Gln + Tyr205Gly + Pro209Glu + Thr212Pro +
Tyr213GluSer203Asp + Pro209Ser + Thr212Gln + Ser215Asp +
Gly218AspSer203Asp + Pro209Gly + Ser215Glu + Leu216Ala +
Gly218AspPro200Ser + Ser203Asp + Thr207Gln + Leu216Gln +
Gly218GluPro200Asn + Gly201Pro + Ser203Glu + Thr207Gly +
Gly218GluVal202Asp + Val204Cys + Tyr208Ala + Gly218Gln +
Thr219GluVal202Glu + Val204Thr + Tyr208Gln + Asn217Ser +
Thr219GluPro200Ser + Gly201Pro + Val202Asp + Asn211Gln +
Thr219GluGly201Asn + Ser203Glu + Thr212Gln + Thr214Asp +
Thr219SerGly201Asn + Ser203Asp + Thr214Glu + Leu216Cys +
Thr219GlyGly201Asn + Val202Cys + Ser203Asp + Thr214Asp +
Thr219ProVal202Cys + Ser203Asp + Tyr208Thr + Thr214Asp +
Thr219ProVal204Ser + Tyr208Leu + Ser210Asp + Tyr213Asp +
Thr214AspAla199His + Ser203Asp + Tyr208Ser + Tyr213Glu +
Thr214AspVal204Cys + Ser210Asp + Asn211Ser + Tyr213Asp +
Thr219GlnAla199Asn + Ser210Glu + Thr212Asn + Tyr213Glu +
Thr214GlnSer203Asp + Tyr205Glu + Tyr208Gly + Tyr213Leu +
Gly218AspVal202Glu + Tyr205Glu + Pro209Asn + Leu216Thr +
Gly218GluTyr208Val + Ser215Asp + Asn217Ser + Gly218Asp +
Thr219GluTyr205Leu + Thr214Asp + Ser215Glu + Gly218Glu +
Thr219AsnAla199Pro + Pro209Asp + Thr212Asp + Ser215Asp +
Gly218SerSer203Glu + Thr212Glu + Tyr213Ser + Thr214Glu +
Leu216CysSer203Asp + Tyr208Cys + Thr212Asp + Tyr213Ile +
Thr214AspVal202Met + Val204Thr + Asn211Asp + Thr214Asp +
Ser215AspPro209Asn + Asn211Asp + Thr214Glu + Ser215Glu +
Leu216GlyPro200Asn + Tyr205Glu + Thr212Asp + Tyr213Ala +
Thr219ProPro200Asn + Ser203Asp + Tyr205His + Thr212Asp +
Ser215AspSer203Asp + Val204Thr + Thr212Glu + Tyr213Ile +
Ser215GluTyr205Gly + Thr207Gln + Tyr208Ile + Thr212Glu +
Ser215GluAla199Asn + Tyr205Asp + Ser210Glu + Thr212Glu +
Thr214GlyVal202Met- Tyr205Ala + Ser210Glu + Thr212Asp +
Ser215AspThr207Gln + Tyr208Leu + Asn211Glu - Tyr213Glu +
Leu216GluVal204Met + Pro209Ser + Asn211Asp + Thr214Glu +
Leu216AspPro200Ser - Ser203Glu + Tyr213Glu + Leu216Gly +
Asn217GluAla199Gly + Tyr213Pro + Thr214Asp + Asn217Asp +
Thr219AspAla199Gln + Tyr208Ala + Thr214Glu + Asn217Glu +
Thr219AspAla199His + Val202His + Ser203Glu + Thr207Gly +
Thr219AspGly201Ser + Val202Gly + Ser203Glu + Tyr205Thr +
Thr219AspGly201Pro + Pro209Glu + Ser210Asp + Ser215Glu +
Leu216MetTyr205Glu + Ser215Glu + Leu216Cys + Gly218Gln +
Thr219GluSer203Asp + Pro209Ser + Asn211Glu + Thr214Asp +
Leu216AsnAla199Thr + Ser203Glu + Thr207Gln + Asn211Asp +
Thr214GluGly201Ser + Ser203Asp + Tyr205Gly + Pro209Asp +
Ser215AspPro209Glu + Thr214Asn + Leu216Glu + Asn217Asp +
Thr219Asn表27环6-六点取代的变异体Gly201Asn + Tyr205Gly + Thr207Gln + Ser210Glu +Tyr213Ile + Thr219GlyPro200Asn + Gly201Ser + Tyr208Gly + Asn211Asp +Thr212Ser + Thr219AsnGly201Pro + Ser203Asp + Val204Gln + Tyr205Pro +Thr212Ser + Tyr213HisPro200Gln + Val204His + Thr207Ser + Thr214Pro +Ser215Asp + Thr219AsnAla199Pro + Pro200Ser + Gly201Pro + Val204Asn +Tyr208Pro + Thr214GlyPro200Gln + Val204Ala + Tyr208Leu + Thr212Glu +Tyr213Met + Leu216GlnAla199Pro + Val202Met + Ser203Glu + Tyr205Leu +Asn211Gln + Tyr213GlyGly201Gln + Val204Gln + Thr214Gln + Ser215Asp +Leu216Gly + Thr219SerVal204Thr + Tyr208Met + Pro209Glu + Tyr213Gly +Thr214Pro + Gly218SerSer203Asp + Val204Met + Thr207Gln + Pro209Gln +Asn217Ser + Thr219AsnPro200Ser + Val202Met + Val204Gly + Pro209Gln +Tyr213Ile + Ser215GluAla199His + Ser203Asp + Val204Gly + Thr207Gly +Asn211Gln + Thr214SerAla199Gln + Pro200Gln + Gly201Gln + Pro209Glu +Leu216Ile + Asn217SerPro200Gln + Tyr208Ile + Pro209Gln + Thr212Gly +Ser215Glu + Thr219GlyPro200Asn + Ser203Asp + Val204Cys + Thr207Gln +Pro209Ser + Thr214AsnAla199Asn + Val202Gln + Val204Ser + Pro209Gly +Thr212Asp + Asn217GlnPro200Asn + Val204Met + Thr207Gln + Pro209Gly +Thr212Gly + Tyr213AspPro200Gly + Val204Gln + Pro209Asn + Tyr213Ile +Thr214Gly + Leu216MetAla199Asn + Tyr205Thr + Tyr208Ser + Pro209Ser +Thr214Asp + Asn217SerTyr205Ser + Thr207Gln + Tyr208Cys + Pro209Asn +Thr212Asn + Leu216GluAla199Asn + Pro200Ser + Ser203Asp + Tyr205Cys +Tyr208Asn + Thr214GlnThr207Pro + Tyr213Ser + Thr214Asn + Asn217Ser +Gly218Ser + Thr219GluAla199Pro + Gly201Pro + Thr207Gly + Pro209Asn +Ser210Asp + Tyr213SerAla199Gln + Pro200Gly + Val202Met + Thr212Asp +Leu216Met + Gly218ProAla199Ser + Pro200Gly + Val202Asn + Asn211Gln +Leu216Val + Gly218ProAla199His + Pro200Gln + Val202Gln + Thr214Gln +Leu216Thr + Gly218GluPro200Ser + Gly201Gln + Thr207Pro + Tyr208Thr +Gly218Pro + Thr219ProPro200Gln + Val204Met + Thr207Gly + Ser215Glu +Leu216Met + Thr219ProThr207Ser + Pro209Asp + Thr212Gly + Thr214Ser +Leu216Pro + Thr219GlnPro200Asn + Gly201Ser + Thr207Asn + Tyr208Leu +Ser210Asp + Asn217SerAla199Asn + Val202Ser + Tyr205Cys + Thr207Gln +Tyr213Leu + Asn217SerVal202Ser + Pro209Gln + Tyr213Asp + Leu216Met +Asn217Ser + Thr219GlyGly201Gln + Val204Gly + Pro209Asn + Asn211Asp +Asn217Gln + Thr219GlyVal202Gly + Ser203Glu + Val204Gly + Tyr213Met +Gly218Ser + Thr219SerAla199Gln + Pro200Asn + Tyr205Thr + Thr207Ser +Thr214Gln + Asn217SerAla199Ser + Gly201Asn + Val202Met + Val204Asn + Asn211Gln + Gly218GluPro200Asn + Val202Pro + Val204Gln + Tyr208Pro +Gly218Gln + Thr219GluThr207Gly + Tyr208Ile + Pro209Gly + Tyr213Gly +Thr214Pro + Leu216AsnVal202Met + Ser203Asp + Val204Met + Thr212Asn +Thr214Asn + Leu216GlyAla199Ser + Val202Met + Val204Ala + Thr212Gly +Tyr213Thr + Thr214ProTyr208Val + Pro209Gln + Thr214Ser + Leu216Cys +Asn217Glu + Thr219AsnPro200Gln + Gly201Gln + Tyr205Ala + Pro209Gln +Leu216Pro + Thr219GluAla199Gly + Pro200Gln + Gly201Ser + Tyr208Gln +Ser215Asp + Gly218AsnAla199Asn + Pro200Asn + Val202Asn + Val204Gln +Thr214Gly + Asn217GluPro200Gly + Gly201Gln + Tyr213Gly + Thr214Asn +Leu216Gln + Thr219SerGly201Gln + Val202Gly + Thr207Gly + Asn211Gln +Thr214Gly + Ser215AspAla199Thr + Pro200Ser + Tyr208Ala + Thr212Ser +Ser215Asp + Asn217SerAla199His + Val202Asn + Ser203Glu + Val204His +Tyr213Ile + Asn217GlnGly201Asn + Val202Cys + Tyr205Cys + Tyr213Ile +Leu216Pro + Asn217AspAla199Thr + Gly201Ser + Val202Gly + Tyr205Pro +Leu216Thr + Gly218GluAla199Ser + Val204Cys + Tyr208Cys + Pro209Gly +Tyr213Ala + Leu216AspAla199Gly + Gly201Asn + Val204Met + Thr207Pro +Thr214Glu + Leu216MetPro200Gly + Tyr205Met + Tyr208Val + Thr212Gly +Tyr213Ser + Leu216ValGly201Pro + Tyr208Val + Pro209Glu + Asn211Gln +Tyr213Ala + Thr219GlyAla199Ser + Pro200Ser + Val202Thr + Tyr205Gln +Gly218Asp + Thr219ProAla199Gln + Ser203Glu + Val204Pro + Thr214Ser +Gly218Gln + Thr219ProAla199Thr + Pro200Gly + Gly201Gln + Thr212Ser +Leu216Val + Gly218SerPro200Ser + Val204Gln + Thr207Pro + Thr214Gly +Ser215Glu + Gly218GlnAla199Gly + Gly201Gln + Val204His + Tyr205Asp +Pro209Gln + Thr212SerPro200Gln + Tyr208Asn + Asn211Ser + Thr212Ser +Tyr213Thr + Leu216AspAla199His + Tyr208Leu + Thr212Pro + Ser215Glu +Leu216Ser + Gly218GlnGly201Pro + Val204Cys + Pro209Gln + Thr212Ser +Tyr213Cys + Thr214GlnVel204Ser + Tyr205Ser + Thr207Asn + Tyr208Cys +Ser215Asp + Asn217SerPro200Asn + Tyr208Als + Tyr213Pro + Thr214Glu +Ser215Asp + Thr219GlyVal202His + Tyr208Met + Pro209Asn + Thr212Asn +Thr214Glu + Ser215AspPro200Gln + Gly201Ser + Val202Pro + Val204Ser +Thr214Asp + Ser215GluAla199Gln + Gly201Ser + Val204Gly + Thr207Ser +Thr214Asp + Ser215GluVal202Asn + Tyr205Gly + Pro209Gln + Thr212Ser +Thr214Glu + Ser215GluPro200Asn + Pro209Glu + Ser210Asp + Asn211Ser +Thr214Ser + Leu216CysPro200Ser + Val204Pro + Tyr208Ile + Thr214Pro +Asn217Asp + Gly218AspGly201Gln + Tyr205Asn + Tyr208Gly + Thr212Gly +Asn217Glu + Gly218GluVal202His + Thr207Asn + Tyr208Gln + Tyr213Pro +Ser215Glu + Leu216AspAla199Asn + Gly201Ser + Val202Pro + Tyr205Thr +Ser215Asp + Leu216AspPro200Gly + Val202Ala + Val204Ala + Ser210Glu +Asn211Glu + Gly218SerVal204Asn + Pro209Ser + Ser210Asp + Asn211Asp +Tyr213Asn + Leu216MetAla199Asn + Val204Thr + Ser210Asp + Asn211Glu +Tyr213Leu + Thr214ProVal202Cys + Val204Ala + Tyr205Glu + Thr212Ser +Ser215Glu + Asn217GlnThr207Pro + Ser210Asp + Asn211Glu + Thr212Glu +Asn217Ser + Thr219SerPro200Asn + Val202Asp + Tyr205Ala + Pro209Ser +Asn217Asp + Gly218ProVal202Gln + Pro209Glu + Ser210Glu + Asn211Glu +Thr212Gln + Asn217GlnVal202Asp + Ser203Asp + Thr207Gln + Thr212Ser +Tyr213Gln + Asn217GluAla199Asn + Ser203Glu + Val204Asn + Tyr208Pro +Leu216Glu + Asn217AspVal204Thr + Thr212Gly + Thr214Glu + Ser215Asp +Leu216Glu + Gly218SerAla199Gln + Ser203Asp + Val204Cys + Thr207Gly +Ser215Glu + Leu216GlnAla199Pro + Pro200Gln + Ser203Asp + Asn211Ser +Ser215Asp + Leu216AsnSer203Glu + Tyr208Thr + Tyr213Cys + Thr214Gln +Ser215Glu + Gly218SerAla199Thr + Pro200Gln + Gly201Gln + Ser203Asp +Ser215Asp + Gly218SerPro200Gly + Val202Cys + Ser203Asp + Asn211Ser +Ser215Glu + Leu216ProAla199Gln + Ser203Glu + Tyr205Gly + Tyr213Met + Ser215Glu + Asn217SerPro200Ser + Gly201Asn + Ser203Asp + Tyr208Val +Pro209Ser + Asn217GluPro200Ser + Gly201Ser + Val202His + Ser203Asp +Asn217Glu + Gly218ProVal202Asn + Tyr205Glu - Thr207Gln + Tyr208Leu +Thr214Asp + Gly218ProSer203Asp + Val204Thr + Tyr205Glu + Thr212Ser +Tyr213Ile + Ser215GluAla199Asn + Pro200Ser + Tyr205Pro + Pro209Asp +Asn211Glu + Tyr213IleGly201Pro + Val202Cys + Val204Ser + Pro209Glu +Thr212Glu + Thr214SerAla199Asn + Val202Asp + Ser203Asp + Tyr208Cys +Ser215Asp + Leu216AsnAla199Asn + Ser203Glu + Tyr205Cys + Thr212Gly +Thr214Glu + Ser215AspAla199Thr + Pro200Ser + Tyr208Met + Thr214Ser +Ser215Glu + Asn217AspAla199Pro + Tyr208Gly + Ser215Asp + Leu216Val +Asn217Asp + Thr219SerPro200Asn + Pro209Gly + Tyr213Pro + Ser215Asp +Leu216Gly + Asn217AspPro200Ser + Gly201Pro + Va1202Ala + Asn211Glu +Tyr213Asp + Gly218ProVal202Met + Val204Ser + Tyr205His + Thr212Gly +Asn217Glu + Thr219AspTyr205Val + Tyr208Pro + Ser215Glu + Leu216Met +Asn217Asp + Gly218AspVal202Ser + Tyr208Ala + Asn211Ser + Thr214Glu +Leu216Asp + Asn217AspGly201Pro + Tyr208Gln + Thr212Asp + Tyr213Asp +Ser215Asp + Asn217SerAla199Gln + Gly201Gln + Tyr205Gly + Pro209Glu +Thr212Pro + Tyr213GluSer203Asp + Pro209Ser + Asn211Gln + Thr212Gln +Ser215Asp + Gly218AspSer203Asp + Pro209Gly + Tyr213Leu + Ser215Glu +Leu216Ala + Gly218AspVal202Glu + Val204Cys + Thr212Gly + Ser215Asp +Leu216Gln + Gly218GlnPro200Gly + Gly201Gln + Val202Glu + Val204Ala +Thr207Pro + Ser215AspAla199Gln + Pro200Ser + Gly201Ser + Ser203Asp +Gly218Glu + Thr219AspAla199Gln + Gly201Ser + Tyr205Gln + Ser210Asp +Thr212Glu + Thr214AspGly201Asn + Val202Pro + Ser210Glu + Thr212Glu +Tyr213Thr + Thr214GluPro200Gln + Val202Met + Ser203Asp + Val204Gly +Tyr208Ile + Gly218AspPro200Ser + Gly201Pro + Val202Asp + Tyr208Cys +Asn211Gln + Thr219GluSer203Asp + Tyr208Pro + Pro209Ser + Asn211Ser +Thr214Asp + Thr219ProAla199His + Gly201Asn + Ser203Glu + Tyr213His +Thr214Glu + Leu216IleSer203Glu + Tyr208Pro + Tyr213His + Thr214Asp +Leu216Cys + Asn217SerVal202Asp + Tyr205Met + Thr214Asp + Ser215Glu +Leu216Cys + Asn217GlnVal202Cys + Val204Ser + Tyr208Leu + Ser210Asp +Tyr213Asp + Thr214AspGly201Asn + Tyr205Gln + Pro209Asn + Ser210Glu+Tyr213Glu + Leu216AlaVal202Cys + Pro209Asp + Tyr213Asp + Thr214Asn +Ser215Glu + Asn217GlnAla199Gly + Gly201Gln + Ser215Asp + Leu216Asp +Gly218Asn + Thr219GluAla199His + Thr207Asn + Asn211Ser + Leu216Glu +Asn217Gln + Thr219AspVal202Met + Thr207Gln + Pro209Gly + Thr214Glu +Ser215Asp + Gly218AspPro200Gln + Val202Ala + Val204Pro + Thr207Gln +Pro209Glu + Thr214GluGly201Pro + Tyr208Gly + Thr214Asp + Leu216Cys +Asn217Glu + Gly218AspAla199Gln + Thr207Pro + Tyr208Cys + Pro209Gln +Tyr213Asp + Leu216AspSer203Asp + Thr214Gly + Ser215Glu + Leu216Ala +Asn217Ser + Thr219GluPro200Gln + Ser203Glu + Val204His + Thr212Asp +Thr214Asp + Gly218GlnAla199His + Gly201Ser + Ser203Glu + Tyr205Ser +Thr214Glu + Gly218GluGly201Pro + Ser203Glu + Thr207Gly + Tyr208Cys +Thr214Asp + Gly218GluSer203Glu + Tyr205Leu + Thr207Asn + Thr212Asp +Tyr213Met + Ser215GluAla199Pro + Ser203Asp + Pro209Ser + Thr212Glu +Ser215Asp + Gly218GlnSer203Glu + Tyr205His + Thr212Asp + Tyr213Met +Ser215Asp + Thr219SerSer203Asp + Val204Gly + Thr212Glu + Thr214Pro +Ser215Asp + Asn217SerGly201Pro + Tyr205Gln + Thr207Gly + Asn211Glu +Thr212Gly + Thr214GluPro200Asn + Val202Gln + Thr207Gln + Pro209Ser +Asn211Asp + Thr214GluGly201Gln + Thr207Gly + Pro209Gln + Thr212Glu +Ser215Glu + Gly218GlnVal202Met + Tyr205Ala + Ser210Glu + Thr212Asp +Ser215Asp + Leu216AlaGly201Gln + Pro209Gly + Asn211Asp + Tyr213Asp +Thr214Gly + Leu216AspAla199Gly + Val202Glu + Ser203Glu + Tyr213Glu + Thr214Pro + Thr219SerGly201Ser + Val202Cys + Ser203Asp + Tyr205Asp +Leu216Gly + Thr219AsPPro200Ser + Ser203Glu + Tyr213Glu + Leu216Gly +Asn217Glu + Thr219GlyTyr205Asn + Pro209Asn - Thr212Glu + Tyr213Met +Ser215Glu + Asn217AspAla199Gln + Tyr208Ala + Asn211Gln + Thr214Glu +Asn217Glu + Thr219AspGly201Pro + Val202Gly + Ser203Glu + Tyr205Thr +Tyr208Ala + Thr219AspPro200Asn + Gly201Pro + Ser203Asp + Thr207Gln +Gly218Ser + Thr219GluGly201Asn + Ser203Asp + Val204Gly + Tyr208Val +Asn211Gln + Thr219AspAla199His + Pro200Asn + Ser203Asp + Tyr205Thr +Pro209Asp + Thr214GluGly201Pro + Val202Gly + Pro209Glu + Ser210Asp +Ser215Glu + Leu216MetAla199Gly + Val202His + Val204Ala + Pro209Asp +Ser210Glu + Ser215GluGly201Asn + Val204Gln + Tyr208Leu + Thr214Glu +Ser215Glu + Thr219GluSer203Glu + Tyr205Gln + Tyr208Gly + Asn211Glu +Thr214Glu + Thr219SerGly201Ser + Ser203Asp + Tyr205Ala + Pro209Asp +Tyr213Met + Ser215AspVal202Asn + Pro209Asp + Asn211Glu + Thr212Ser +Tyr213Leu + Ser215GluAla199Pro + Val204Thr + Tyr205Glu + Ser210Glu +Asn211Asp + Tyr213LeuVal202Ala + Ser203Asp + Val204Gln + Tyr205Met +Thr212Glu + Leu216GluGly201Pro + Ser203Asp + Tyr208Thr + Thr214Asp +Asn217Gln + Thr219AspPro200Gln + Gly201Asn + Thr207Gln + Tyr213Glu +Asn217Asp + Gly218AspPro200Gln + Thr207Gly + Tyr208Ala + Thr212Glu +Tyr213Glu + Asn217GluThr207Ser + Tyr208Ser + Pro209Glu + Thr214Glu +Asn217Glu + Thr219AsnAla199Gly + Tyr205Pro + Pro209Glu + Thr214Glu +Leu216Asn + Asn217AspAla199His + Gly201Asn + Tyr205Pro + Pro209Glu +Ser215Glu + Asn217GluPro200Gly + Pro209Asp + Tyr213Gln + Ser215Glu +Leu216Ile + Asn217AspAla199His + Val204Ser + Thr207Gln + Pro209Asp +Ser210Asp + Thr219AspAla199Thr + Thr207Pro + Tyr208Val + Pro209Glu +Ser210Asp + Thr219AspTyr208His + Pro209Glu + Ser210Glu + Tyr213Val +Leu216Cys + Thr219AspTyr205Met + Thr207Gly + Ser210Asp + Thr212Ser +Asn217Glu + Gly218AspSer210Glu + Thr212Pro + Thr214Gln + Asn217Asp +Gly218Glu + Thr219SerPro200Gln + Val204Thr + Tyr208Asn + Asn211Asp +Ser215Glu + Leu216GluGly201Pro + Tyr208Gln + Asn211Glu + Thr212Asp +Asn217Glu + Thr219SerVal202Asp + Ser203Asp + Asn211Glu + Thr212Pro +Tyr213Leu + Thr214AsnVal202Glu + Ser203Asp + Tyr205Leu + Pro209Gly +Thr212Asp + Thr219SerAla199Gly + Pro200Asn + Tyr208Leu + Ser210Asp +Asn211Asp + Asn217AspPro200Ser + Gly201Ser + Val202Asp + Tyr208Gln +Ser210Glu + Asn211AspVal202Asp + Thr207Gln + Tyr208Thr + Ser210Glu +Thr214Gln + Asn217GluThr207Gln + Asn211Asp + Thr214Glu + Leu216His +Asn217Asp + Gly218GlnSer203Glu + Thr207Ser + Pro209Gly + Asn211Asp +Tyr213Gly + Ser215AspSer203Glu + Tyr205Val + Pro209Gly + Asn211Asp +Ser215Asp + Thr219Gly表28环6-七点取代突变变异体Ala199Ser + Pro200Ser + Val204Met + Tyr208Ser +Tyr213His + Thr214Gln + Leu216AlaAla199Ser + Gly201Ser + Val202Met + Thr207Gln +Ser210Glu + Thr214Ser + Asn217GlnGly201Gln + Val204Gly + Tyr205Ala + Thr207Gln +Tyr208Gly + Asn211Gln + Gly218AspAla199Gly + Pro200Gly + Thr207Gln + Tyr208Thr +Pro209Gln + Gly218Gln + Thr219GlnAla199Gly + Pro200Ser + Val202Gln + Thr207Asn +Asn211Ser + Thr212Ser + Thr219ProPro200Gln + Val204Ala + Tyr208Leu + Thr212Glu +Tyr213Met + Thr214Asn + Leu216GlnSer203Asp + Val204Met + Thr207Gln + Tyr208Met +Pro209Gln + Asn217Ser + Thr219AsnAla199Gln+ Pro200Ser + Gly201Gln + Tyr205Ala +Tyr213Cys + Thr214Gly + Gly218GluAla199Thr + Pro200Ser + Thr207Gln + Tyr208Gln +Thr212Ser + Gly218Asp + Thr219ProGly201Ser + Val204Pro + Tyr208Ser + Thr212Gly +Tyr213Val + Thr214Pro + Ser215AspPro200Gln + Tyr208Cys + Pro209Gln + Thr212Gly +Tyr213Pro + Ser215Glu + Thr219GlyVal204Met + Thr207Gln + Pro209Gly + Thr212Gly +Tyr213Cys + Thr214Gly + Leu216MetPro200Gly + Ser203Asp + Thr207Asn + Pro209Asn +Tyr213Ile + Leu216Cys + Asn217SerPro200Asn + Gly201Asn + Tyr205Ile + Pro209Asp +Tyr213Met + Thr214Asn + Leu216ValAla199Asn + Ser203Glu + Val204Met + Pro209Gly +Tyr213Leu + Leu216Ala + Gly218AsnAla199His + Thr207Pro + Tyr213Ser + Thr214Asn +Asn217Ser + Gly218Ser + Thr219GluAla199Ser + Gly201Pro + Val204Gln + Thr207Ser +Thr212Asn + Tyr213Pro + Gly218GluAla199Ser + Pro200Asn + Tyr205Met + Tyr208Met +Thr212Asn + Thr214Gly + Leu216MetGly201Gln + Tyr205Asn + Thr207Asn + Pro209Asp +Thr212Gly + Tyr213Val + Leu216IlePro200Asn + Tyr205Glu + Tyr208Met + Tyr213His +Thr214Ser + Asn217Gln + Thr219GlnAla199Gln + Pro200Gly + Val202Met + Tyr208Ile +Thr212Asp + Leu216Met + Gly218ProAla199Ser + Pro200Gly + Gly201Gln + Val202Asn +Asn211Gln + Leu216Val + Gly218ProAla199Asn + Val202Ser + Tyr205Cys + Thr207Gly +Tyr213Leu + Thr214Asp + Asn217SerGly201Pro + Val202Asn + Val204Asn + Tyr205Ser +Asn211Glu + Thr212Ser + Gly218AsnAla199Ser + Val204Cys + Thr207Ser + Asn211Ser +Thr214Gln + Ser215Glu + Asn217GlnPro200Ser + Val202Met + Tyr205Gly + Tyr208Pro +Ser210Glu + Thr212Ser + Tyr213GlyPro200Ser + Val202Gln + Ser203Glu + Tyr205Cys +Thr207Gln + Tyr208Ile + Asn211SerAla199Pro + Gly201Ser + Val204Ala + Thr207Ser +Pro209Gln + Tyr213Asp + Leu216HisGly201Ser + Ser203Asp + Val204Gln + Tyr208Thr +Asn211Gln + Leu216Ser + Thr219SerAla199Thr + Gly201Ser + Tyr205Pro + Tyr213Pro +Thr214Gly +Leu216Thr + Gly218GluPro200Asn + Gly201Gln + Tyr208Val + Thr214Gln +Ser215Asp + Gly218Ser + Thr219AsnPro200Gln + Val202Met + Val204Gly + Tyr208Gly +Asn211Ser + Tyr213Pro + Gly218AspAla199Asn + Pro200Gly + Tyr205Thr + Tyr208Val +Thr212Gly + Tyr213Ser + Leu216ValAla199Ser + Gly201Ser + Val202Gly + Tyr205Leu +Thr214Asn + Leu216Ala + Gly218GluAla199Ser + Gly201Gln + Val204Ser + Tyr208Asn +Pro209Ser + Thr214Asn + Ser215GluAla199Pro + Gly201Asn + Tyr208Cys + Tyr213Gly +Thr214Glu + Asn217Ser + Gly218GlnAla199Pro + Val202His + Tyr208Met + Pro209Asn +Thr212Asn + Thr214Glu + Ser215AspAla199His + Val202Gly + Val204Ser + Thr207Gln +Pro209Asp + Ser210Asp + Asn211SerAla199Gly + Pro209Glu + Ser210Glu + Thr212Gln + Tyr213Ala + Leu216Pro + Thr219AsnAla199His + Gly201Gln + Val202Met + Val204His +Thr207Gly + Pro209Glu + Ser210AspAla199His + Val202Gln + Thr207Gly + Thr214Gln +Leu216Thr + Asn217Asp + Gly218GluGly201Gln + Val204Thr + Tyr205Pro + Thr207Gly +Leu216Asn + Asn217Asp + Gly218AspGly201Ser + Tyr208Thr + Pro209Gln + Tyr213Ser +Asn217Asp + Gly218Glu + Thr219GlyGly201Ser + Val202Cys + Tyr205Val + Thr207Gln +Tyr213Ile + Leu216Asp + Asn217AspGly201Asn + Val202Glu + Ser203Glu + Val204Gly +Thr212Gly + Leu216Val + Thr219ProVal202Asp + Ser203Glu + Val204Asn + Thr207Pro +Tyr208Leu + Thr212Ser + Asn217GlnGly201Gln + Tyr208Ser + Pro209Asn + Asn211Gln +Tyr213Asp + Thr214Asp + Asn217GlnAla199Asn + Pro200Asn + Val204Thr + Ser210Asp +Asn211Glu + Tyr213Leu + Thr214ProGly201Ser + Val202Ala + Tyr208Met + Ser210Asp +Asn211Asp + Thr212Asp + Asn217SerAla199Ser + Pro200Gly + Gly201Asn + Val202Glu +Leu216Cys + Asn217Asp + Gly218AsnAla199Asn + Pro200Ser + Ser203Asp + Tyr205Cys +Tyr208Asn + Thr214Gln + Ser215GluAla199Gln + Val202Gly + Ser203Glu + Tyr205Gly +Tyr213Met +Ser215Glu + Asn217SerGly201Asn + Val202Gly + Ser203Asp + Tyr208Ile +Tyr213Pro + Ser215Glu + Asn217SerVal202Met + Ser203Glu + Thr207Asn + Thr214Gly +Ser215Asp + Leu216Glu + Asn217AspAla199Gln + Ser203Glu + Val204Cys + Asn211Ser +Thr214Gly + Asn217Glu + Thr219ProAla199Gln + Ser203Asp + Tyr205Thr + Thr207Ser +Thr212Asn + Asn217Asp + Gly218AsnPro200Ser + Val202Ser + Ser203Asp + Val204Gly +Thr212Asn + Tyr213Met + Asn217GluSer203Glu + Tyr205Met + Tyr208Thr + Pro209Gly +Asn217Glu + Gly218Asn + Thr219SerAla199Gln + Tyr205Leu + Tyr208Pro + Asn211Glu +Thr212Asp +Tyr213Asp + Thr214GlyGly201Pro + Val202Thr + Val204His + Tyr205Met +Asn211Glu + Thr212Asp + Tyr213AspGly201Pro + Ser203Glu + Tyr205Asp + Thr207Ser +Ser215Asp +Leu216His + Thr219SerAla199Asn + Val202Gln + Val204Ser + Pro209Gly +Ser210Glu + Thr212Asp + Asn217GlnPro200Gly + Gly201Pro + Tyr208Val + Pro209Glu +Asn211Asp+Thr212Gln + Thr219SerAla199Asn + Pro200Gln + Val202Ala + Val204Pro +Thr207Asn + Pro209Glu + Asn211GluAla199Asn + Ser203Asp + Thr207Gly + Asn211Ser +Ser215Glu + Asn217Asp + Gly218GlnPro200Gln + Ser203Glu + Val204Gly + Tyr213Pro +Ser215Glu + Asn217Glu + Thr219GlnPro200Gly + Val202Asp + Ser203Asp + Thr207Ser +Thr212Gly + Ser215Glu + Gly218SerVal202Glu + Ser203Asp + Tyr208Ser + Asn211Gln +Tyr213Ala + Ser215Asp + Thr219SerAla199Gln + Ser203Asp + Tyr205Met + Thr214Asp +Ser215Glu + Leu216Cys + Asn217GlnGly201Gln + Ser203Glu + Tyr205Glu + Pro209Gln +Asn211Ser + Thr214Glu + Leu216GluGly201Asn + Val202Cys + Ser203Asp + Tyr205Asp +Thr214Asp + Leu216Gly + Thr219ProAla199Gly + Val202Met + Ser203Glu + Val204Pro +Tyr205Glu + Thr207Ser + Thr214AspGly201Ser + Val204Met + Pro209Gly + Asn211Ser +Tyr213Cys + Thr214Glu + Leu216AspGly201Pro + Val202Asp + Thr207Gln + Thr212Ser +Asn217Asp + Gly218Asn + Thr219AspPro200Ser + Val204Gly + Tyr205Gly + Tyr208Cys +Thr214Ser + Ser215Glu + Asn217GluGly201Asn + Tyr205Ile + Tyr213Ile + Thr214Asn +Ser215Glu + Asn217Asp + Thr219ProPro200Ser + Val202Gln + Tyr208His + Thr212Pro +Thr214Ser + Ser215Glu + Asn217GluPro200Gly + Val204Gln + Thr207Ser + Tyr213Leu +Thr214Gln + Ser215Glu + Asn217GluThr207Gly + Ser210Asp + Asn211Asp + Thr212Gly +Tyr213Asp + Asn217Gln + Thr219GlnSer203Glu + Thr207Asn + Tyr208His + Asn211Gln +Thr214Asp + Leu216Glu + Asn217GluGly201Pro + Val202Met + Val204Ser + Tyr205His +Thr212Gly + Asn217Glu + Thr219AspAla199Asn + Val204Cys + Tyr208Cys + Tyr213Ala +Thr214Glu + Leu216Asp + Asn217GluPro200Gln + Thr207Gly + Tyr208Cys + Thr214Asp +Ser215Asp + Leu216Gln + Asn217AspAla199Gln + Val204His + Asn211Ser + Thr212Ser +Thr214Glu + Ser215Glu+ Asn217GluVal202Asp + Ser203Asp + Tyr205Ala + Thr207Gln +Tyr213Met + Thr214Glu+ Ser215AspAla199Gly + Pro200Gln + Val204Cys + Thr212Glu +Thr214Glu + Ser215Glu +Leu216GlnVal204Ala + Thr207Gly + Ser210Asp + Asn211Asp +Thr212Glu + Thr214Glu + Asn217SerGly201Pro + Pro209Ser + Ser215Glu + Leu216Glu +Asn217Asp + Gly218Ser + Thr219AspPro200Asn + Gly201Pro + Ser203Asp + Thr207Gln +Asn217Asp + Gly218Ser + Thr219GluAla199Pro + Val202Gly + Ser203Asp + Pro209Gln +Thr212Asn + Ser215Glu + Gly218AspAla199Gln + Pro200Ser + Gly201Ser + Tyr205Gln +Ser210Asp + Thr212Glu + Thr214AspAla199Pro + Pro200Ser + Thr207Gln + Ser210Glu + Thr212Asp + Thr214Asp + Thr219SerAla199His + Ser203Asp + Val204Gly + Thr207Gly +Asn211Gln + Thr214Ser + Gly218GluGly201Asn + Val202Asp + Val204Gly + Tyr208Val +Asn211Gln + Asn217Ser + Thr219AspVal202Asn + Tyr205Glu + Thr207Gln + Tyr208Leu +Thr214Asp + Asn217Asp + Gly218ProAla199Asn + Gly201Asn + Ser203Asp + Tyr208Asn +Thr214Glu + Leu216Cys + Thr219GlyVal202Cys + Ser203Asp + Val204Ser + Tyr208Thr +Thr214Asp + Asn217Gln + Thr219ProSer203Asp + Tyr208Pro + Pro209Ser + Asn211Ser +Tyr213Met + Thr214Asp + Thr219ProAla199Asn + Pro200Gln + Ser203Asp + Thr212Glu +Thr214Asp + Ser215Glu + Asn217SerPro200Asn + Gly201Asn + Val202Glu + Ser203Asp +Thr212Gly + Ser215Glu + Thr219AspAla199Gln + Gly201Gln + Pro209Gly + Ser210Glu +Thr212Pro + Tyr213Glu + Leu216AlaPro200Ser + Val204Ser + Thr207Asn + Tyr208Leu +Ser210Asp + Asn211Ser + Tyr213AspAla199His + Ser203Asp + Tyr205Asp + Thr207Asn +Tyr208Ile + Leu216Ser + Gly218AspAla199Gln + Gly201Pro + Val202Glu + Tyr205Glu +Pro209Gly + Thr214Asp + Leu216GlnGly201Asn + Val204Pro + Tyr208Cys + Ser210Glu +Asn211Glu + Thr212Pro + Thr214AspAla199His + Pro200Asn + Tyr208Val + Thr214Glu +Ser215Glu + Leu216Glu + Thr219AspPro200Gly + Val202Glu + Tyr205Pro + Tyr208Ala +Thr214Glu + Asn217Asp + Gly218AsnVal202Glu + Tyr205His + Pro209Gln + Thr214Glu +Ser215Asp + Asn217Ser + Gly218AspPro200Ser + Tyr205Leu + Thr212Gln + Thr214Asp +Ser215Glu + Gly218Glu + Thr219AsnSer203Asp + Val204Gly + Tyr205Ser + Thr212Glu +Thr214Pro + Ser215Asp + Asn217GluPro200Asn + Val204Gln + Pro209Glu + Ser210Glu +Asn211Glu + Thr212Asn + Ser215AspAla199Gln + Thr207Pro + Tyr208Cys + Pro209Gln +Tyr213Asp + Thr214Asn + Leu216AspAla199Gly + Pro209Glu + Ser210Asp + Thr214Glu +Ser215Glu + Gly218Pro + Thr219GlyAla199His + Thr207Ser + Tyr208Thr + Pro209Gly +Ser215Asp + Asn217Gln + Gly218GluAla199Ser + Ser203Glu + Tyr208Ala + Asn211Gln +Ser215Glu + Leu216Cys + Thr219AspGly201Pro + Val202Gly + Ser203Glu + Tyr205Thr +Tyr208Ala + Ser215Asp + Thr219AspPro200Gln + Val202His + Ser203Glu + Asn211Gln +Thr212Asp + Thr214Asp + Thr219GlyGly201Pro + Ser203Asp + Thr207Gln + Ser210Glu +Thr214Glu + Ser215Asp + Thr219SerAla1995er + Tyr205Gly + Thr207Ser + Ser210Asp +Tyr213Glu + Ser215Glu + Leu216GluVal204Thr + Thr207Pro + Ser210Glu + Asn211Glu +Thr212Glu + Gly218Asp + Thr219SerVal202Asp + Ser203Asp + Thr207Gly + Pro209Asn +Asn211Glu + Asn217Asp + Gly218SerPro200Ser + Ser203Asp + Val204Pro + Tyr208Ile +Thr214Asp + Asn217Ser + Gly218AspPro200Ser + Gly201Pro + Val202Gly + Ser203Glu +Tyr205Glu + Pro209Asp + Tyr213GluAla199Gly + Gly201Gln + Val202Ser + Asn211Asp +Ser215Glu + Leu216Asp + Asn217GluAla199Thr + Tyr205Gln + Thr207Gln + Pro209Gln +Thr212Asp + Ser215Glu + Gly218GlnAla199Gln + Val204Ser + Tyr205Glu + Thr207Asn +Ser210Glu + Thr212Glu + Asn217GlnTyr205Val + Tyr208Pro + Pro209Gln + Ser210Asp +Thr212Glu + Ser215Glu + Leu216GlyAla199Pro + Gly201Ser + Thr214Glu + Ser215Glu +Asn217Gln + Gly218Glu + Thr219AspSer203Glu + Ser210Asp + Asn211Gln + Thr212Glu +Tyr213Ser + Thr214Glu + Leu216CysAla199His + Pro200Gln + Pro209Asn + Tyr213Gln +Thr214Asp + Leu216Glu + Thr219AspAla199Asn + Ser203Asp + Tyr205Glu + Thr207Asn +Pro209Asn + Asn211Glu + Leu216GluAla199Thr + Gly201Pro + Ser210Glu + Thr214Asp +Ser215Asp + Asn217Ser + Thr219SerSer203Glu + Thr207Pro + Tyr208Ala + Ser210Asp +Ser215Glu + Asn217Asp + Thr219ProVal202Asp + Val204Cys + Tyr205Asp + Tyr208Ala +Pro209Gly + Gly218Gln + Thr219GluVal202Ala + Val204Thr + Tyr208Leu + Asn211Glu +Thr212Glu + Ser215Asp + Leu216GluPro200Asn + Val202Gln + Thr207Gln + Pro209Ser +Asn211Asp + Thr214Glu + Leu216AspAla199Asn + Val202Met + Thr207Asn + Pro209Glu +Tyr213GIu + Ser215Glu + Asn217AspAla199Gly + Val202Cys + Val204Gln + Tyr205Asp +Ser210Asp + Asn211Gln + Thr214GluAla199His + Tyr208Thr + Pro209Asp + Ser215Asp +Leu216Met + Asn217Asp + Gly218AspAla199Asn + Pro200Asn + Gly201Gln + Pro209Asp +Thr214Ser + Ser215Asp + Leu216AspAla199Asn + Pro200Gln + Gly201Ser + Val202Asp +Asn211Asp + Ser215Glu + Leu216AspAla199Thr + Val202Asp + Val204Cys + Ser210Asp +Tyr213Ile + Ser215Asp + Leu216AspGly201Ser + Val202Glu + Tyr205Gly + Tyr208Leu +Ser210Asp + Ser215Glu + Asn217AspGly201Gln + Thr207Ser + Tyr208His + Pro209Glu +Asn211Glu + Tyr213Asp + Asn217GluPro200Gly + Gly201Asn + Ser203Glu + Tyr208Cys + Thr212Asn + Asn217Gln + Thr219GluAla199Gly + Gly201Asn + Val202His + Val204Ala +Pro209Asp + Ser210Glu + Ser215GluGly201Asn + Val202Glu + Pro209Gly + Ser210Glu +Thr212Glu + Tyr213Thr + Thr214GluVal202Cys + Val204Ala + Tyr205Glu + Thr212Ser +Ser215Glu + Asn217Gln + Thr219AspGly201Ser + Ser203Glu + Ser210Glu + Asn211Ser +Thr212Glu + Ser215Glu + Leu216ProAla199His + Ser203Glu + Thr207Gln + Ser210Asp +Thr212Asp + Ser215Asp + Gly218GlnAla199Gln + Pro200Asn + Gly201Gln + Val204Asn +Thr214Asp + Ser215Glu + Thr219GluAla199Pro + Gly201Pro + Ser203Asp + Tyr205Val +Pro209Glu + Ser215Glu + Thr219ProPro200Gly + Ser203Asp + Thr207Gln + Asn211Glu +Tyr213Glu + Leu216Thr + Asn217GlnVal202Asp + Tyr205Glu + Tyr208Gly + Thr212Asp +Tyr213Ala + Leu216Ile + Thr219ProPro200Ser + Val202Glu + Tyr205Asp + Asn211Glu +Thr212Asp + Tyr213Cys + Thr219GlyVal204Pro + Tyr205Asp + Asn211Asp + Thr214Pro +Ser215Asp + Asn217Ser + Thr219ProGly201Asn + Ser203Asp + Pro209Gln + Tyr213Thr +Thr214Asp + Gly218Asn + Thr219GluPro200Gln + Val204Thr + Thr212Asn + Tyr213Glu +Thr214Pro + Asn217Asp + Gly218AspAla199Gln + Gly201Gln + Val204Asn + Tyr208Met +Ser210Glu + Thr214Glu + Asn217SerGly201Pro + Val204Thr + Tyr208Gly + Asn211Asp +Thr214Asp + Asn217Glu + Gly218AspPro200Asn + Pro209Glu + Ser210Asp + Asn211Ser +Thr214Ser + Leu216Cys + Thr219AspSer203Asp + Thr207Ser + Tyr208Asn + Pro209Asp +Ser210Asp + Thr214Gln + Asn217GlnVal204Ser + Tyr208Pro + Pro209Ser + Asn211Asp +Leu216Met + Asn217Asp + Gly218AspTyr205Ser + Tyr208Cys + Pro209Asn + Asn211Asp +Thr212Asr + Leu216Glu + Asn217AspPro200Gly + Ser210Asp + Thr212Pro + Tyr213Val +Asn2I7Ser + Gly218Asp + Thr219AspVal202Cys + Tyr205Ala + Thr207Asn + Asn211Asp +Tyr213Val + Ser215Asp + Leu216AspAla199Gln + Val202Glu + Ser203Asp + Val204Thr +Pro209Gln + Asn211Asp + Tyr213ProGly201Pro + Val202Glu + Ser203Glu + Val204Gly +Pro209Gln + Thr212Glu + Leu216AlaPro200Gln + Val202Asp + Ser203Asp + Tyr208Ala +Thr212Glu + Leu216Val + Gly218GlnAla199Thr + Gly201Gln + Val202Gly + Ser210Glu +Asn211Asp + Ser215Asp + Leu216AlaAla199Gly + Gly201Gln + Val204Asn + Ser210Asp +Asn211Asp + Ser215Glu + Thr219AsnAla199Asn + Val202Ala + Ser203Asp + Val204Gln +Ser210Glu + Thr212Glu + Leu216GluVal202Asp + Val204Asn + Pro209Ser + Ser210Asp +Asn211Asp + Tyr213Asn + Leu216MetVal202Pro + Ser203Asp + Val204Ser + Thr212Glu +Tyr213Asp + Thr214Asn + Gly218AspSer203Glu + Val204Pro + Tyr205Asp + Pro209Glu +Thr212Gln + Tyr213Asn + Gly218AspPro200Ser + Thr207Gly + Ser210Glu + Thr212Gly +Thr214Glu + Ser215Asp + Gly218GluVal202Met + Val204His + Ser210Glu + Thr212Asp +Tyr213Ile + Leu216Asp + Gly218GluSer203Glu + Tyr208Ser + Asn211Asp + Thr212Pro +Thr214Asn + Ser215Glu + Gly218ProAla199Thr + Gly201Pro + Ser203Asp + Tyr205Gly +Asn211Asp + Ser215Glu + Asn217SerPro200Gln + Gly201Asn + Ser203Glu + Pro209Asn +Asn211Asp + Thr212Gly + Ser215Glu表29多环双点突变变异体Leu 95Gly+Ser203GluThr 58Gln+Asp 59GluTyr103Cys+Leu216GlnVal202Gln+Thr212GlyGly126Gln+Gly156GluThr214Gly+Ser215GluAsn 96Gln+Ile106AlaGly101Asp+Tyr205ProAsn 61Ser+Gly165GlnGly 64Gln+Thr132GlyAsp 59Glu+Tyr205AsnSer100Glu+Tyr205HisGln161Glu+Tyr205ProThr 58Asp+Tyr205IleAsn 61Asp+Asn162SerGln161Glu+Pro209GlnGly 62Asp+Pro209AsnIle106Leu+Asn211AspTyr166Pro+Ser215GluIle106Leu+Pro209GluThr163Pro+Val202CysThr 65Pro+Leu216SerThr132Gly+Phe188ValGly156Glu+Ala186ThrPro128Ser+Ser160AspGly 62Gln+Gly165AspLeu 95Gln+Pro128GluSer 97Glu+Asn211SerLeu 95Asn+Phe188LeuLeu125Gln+Gly159GluSer187Glu + Tyr213AlaTyr205Cys + Asn211AspAsn162Gln + Tyr213ProSer158Asp + Leu216GlnGly156Asp + Val202AlaSer100Asp + Tyr205IleGly 99Ash + Thr214GluThr 58Asp + Gly130GlnGly156Glu + Tyr208ProAla105Pro + Leu216IleSer187Asp + Tyr205GlnLeu125Asn + Ser187GluThr 65Gln + Thr102GluTyr103Ile + Tyr205GluThr132Asp + Gly218GlnAsn211Ser + Thr212AspThr212Gln + Ser215GluThr132Pro + Tyr205GlyTyr103Glu + Thr132SerGly165Gln + Ser203GluGly 60Pro + Thr 65ProGly 99Asn + Gly153AsnPro128Asn + Val202HisAla105Glu + Gly130SerThr212Gly + Gly218GluPhe188Val + Tyr205SerAsn217Glu + Thr219GlyLeu 95Gln + Ser215AspVal 94Gln + Thr212GluAsn 61Ser + Ser203AspSer129Glu + Asn154GlnGly 62Glu + Thr219AsnSer 97Glu + Tyr213HisGly 99Asp + Ile164ThrGly101Pro + Ser215AspThr 58Pro + Tyr205GlnGly101Gln + Tyr208GlnAsn 61Gln + Thr212AspThr212Asp + Leu216IleAsn211Glu + Leu216HisGly153Glu + Thr163GlnThr132Asn + Tyr205AspAsn 61Ser + Ser203GluSer215Asp + Leu216SerGly 60Asp + Met198GlnSer158Glu + Tyr205MetPhe188Leu + Thr212GluGly130Pro + Ser190GluGly101Asp + Gly126SerAla105Gly + Ser157AspAsn 61Ser + Ser131AspAsn 61Ser + Phe188SerSer100Glu + Asn162GlnThr 58Gly + Pro128AspSer100Glu + Tyr205AlaGly127Glu + Tyr213ThrAsn154Glu + Leu216GlnSer 97Asp + Ala199ProSer203Glu + Leu216SerGly126Gln + Ser215GluGly 60Ser + Asn154GluThr 65Ser + Gly156AspPro128Ser + Thr163GlnSer215Asp + Leu216ValSer129Glu + Leu216AlaAsn211Asp + Tyr213LeuVal 94Ser + Phe188ValSer158Glu + Thr212GlnGly 99Pro + Asn154GlnVal202Ser + Tyr205GluTyr205Val + Asn217GluVal 94Gly + Ser215AspTyr205Glu + Tyr213ProAsn 96Ser + Gly127GluTyr205Thr + Thr214GlyGly 62Ser + Thr 65SerThr 65Glu + Pro128AsnSer215Asp + Leu216GlnAsp 59Glu + Gly 60ProVal 94Thr + Leu216MetSer 97Glu + Thr212GlnGly101Asp + Val202AsnSer129Asp + Val202GlyTyr103Ala + Thr132SerLeu 95Met + Leu216AspSer100Asp + Gly101ProSer100Asp + Thr219ProVal 94Asn + Ser215AspGly153Asn + Tyr205AspThr 58Glu + Ile106CysThr 65Glu + Tyr103ProVal 94Met + Ser 98GluSer210Glu + Gly218SerLeu125Pro + Ala199HisIle106Gly + Thr212GluTyr205Ile + Thr214ProGly130Glu + Tyr208HisSer187Asp + Leu216ValThr102Glu + Thr219GlnLeu125Ser + Gly153GluGly153Asn + Val202AspThr163Glu + Tyr205GlySer129Glu + Tyr166ProGly153Gln + Pro209GlnThr212Ser + Ser215GluThr163Asp + Tyr205HisAsn154Glu + Tyr166ThrAsn 96Ser + Ala105GlyGly101Ser + Phe188GlyGly153Glu + Gly218AsnSer100Asp + Thr212ProGly126Pro + Gly156AspLeu125Ser + Ser215GluAsp 59Glu + Thr 65GlyGly156Pro + Gly159AspVa1202Cys + Tyr213SerLeu 95Met + Ser131GluSer 97Asp + Gly 99SerAsn 61Gln + Leu 95AspGly126Asn + Tyr205GluGly159Pro + Gly218AsnLeu 95Thr + Leu216AlaAla105Glu + Leu216ThrThr102Glu + Gly130ProGly 64Gln + Ser215GluTyr103Asp + Gly126AsnGly101Glu + Gly127GlnThr132Asn + Ser160AspThr 58Pro + Gly153AsnSer203Glu + Thr214SerTyr103His + Ser129AspSer 98Asp + Pro209SerAsp 59Glu + Tyr205SerGly 60Gln + Ser215AspThr132Asp + Val202AsnGly218Ser + Thr219GlySer190Asp + Val202ThrAsp 59Glu + Gln161AsnLeu216Asn + Asn217AspVal 94Asp + Tyr205AsnTyr103Ser + Ser203AspGly 99Pro + Phe188GlnSer 97Asp + Leu216HisThr163Asp + Leu216AsnAsn 61Asp + Thr132SerLeu 95Asn + Ser215AspSer100Asp + Thr132GlyTyr166Thr + Asn211GlnThr207Pro + Leu216ThrIle106Ala + Gly165AspTyr205Asn + Ser215GluLeu 95Asn + Val202GlnSer160Asp + Phe188GlyGly 64Ser + Asn 96GlnGly101Asp + Asn154GlnGly 64Asn + Ser157GluPhe188Asp + Tyr213AsnSer215Glu + Leu216IleTyr103His + Ser187GluAsn 96Ser + Thr214AsnGly 62Asn + Ser 98AspIle106Ser + Ser157GluThr 65Glu + Thr212GlyGly127Ser + Ser215AspGly156Ser + Asn217GluSer 98Asp + Tyr103ThrGly127Asn + Gly165ProGly153Glu + Leu216MetSer129Asp + Gly165ProAsn 61Asp + Thr163GlnGly126Asn + Tyr205MetGly165Gln + Ser215GluThr 65Glu + Gly101ProVal 94Gln + Gly165GluTyr103Ile + Ser190GluAsn217Gln + Gly218SerSer 98Glu + Gly 99AsnThr102Asp + Thr212SerThr212Ser + Ser215AspGly130Glu + Pro209AsnPro128Glu + Gly165ProThr 58Asn + Leu125GluLeu125His + Thr163SerThr212Asp + Tyr213AlaThr 65Pro + Ser210GluVal 94Thr + Thr132GlyTyr205Cys + Thr214GlnGly127Gln + Ala186GlnGly127Glu + Tyr205CysSer 98Glu + Gly130SerGly127Glu + Phe188TyrPhe188Asp + Asn211SerGly 64Asn + Leu216AspSer203Glu + Thr212SerTyr166Val + Ser210AspGly165Glu + Tyr205MetAsn 96Gln + Thr132GluSer 98Glu + Gly101ProLeul25Cys + Ser215AspLeu125Cys + Gly126SerGly 99Gln + Ser210AspSer160Asp + Phe188LeuVal204Gln + Ser215GluAsn 61Gln + Leu216AspLeu125Gly + Ser155AspAla186Pro + Val204GlnIle106Met + Ser160AspLeu125Ile + Leu216SerGly 60Ser + Ser 97GluGly165Asp + Tyr213GlnPro128Glu + Asn154GlnGly 60Asp + Gly126ProSer203Glu + Leu216AlaGly126Glu + Val202GlyThr132Asp + Tyr213SerSer157Asp + Thr212SerGly130Ser + Ser203GluGly 99Asn + Gly130SerAsp 59Glu + Gly159AsnGly153Asn + Thr212AspGly153Asp + Tyr205SerSer100Glu + Tyr205ThrGly126Asn + Asn154GluSer190Asp + Tyr205ValThr102Glu + Tyr166ThrSer157Glu + Pro209GlySer100Asp + Tyr205LeuSer131Glu + Thr132AsnGly127Asp + Leu216ThrSer210Glu + Asn217GlnAsn 61Asp + Ile106GlnAla105Pro + Tyr205ProSer190Asp + Leu216CysSer203Glu + Tyr205ThrLeu 95Asp + Asn 96SerTyr205Ala + Thr212GluThr163Gln + Phe188GluTyr103Ala + Thr212AspSer155Glu + Gly218AsnVal 94Thr + Thr163SerThr102Glu + Asn217GlnGly1595er + Val202GluAsn154Ser + Tyr166AlaAsp 59Glu + Phe188GlyThr163Gln + Gly218SerGly153Asn + Tyr205ValSer129Glu + Leu216ProGly126Ser + Thr212GluSer203Asp + Leu216AsnGly126Glu + Thr212ProVal 94Pro + Ala186GluSer129Asp + Gly153AsnGly165Ser + Ser215AspGly 60Gln + Ser187GluSer203Asp + Leu216HisGly101Glu + Ile106MetVal 94Glu + Gly127GlnVal202Gly + Leu216HisAsp 59Glu + Thr132AsnVal202Asn + Tyr205ProLeu 95Ile + Ser215GluPro128Glu + Leu216ProSer 97Asp + Tyr213MetSer187Glu + Leu216CysAsn154Ser + Gly156GlnLeu125Gly + Gly126SerAsn154Ser + Leu216AsnGly126Asn + Val202AsnAsn 96Gln + Gly127SerGly 64Ser + Thr212GlySer160Asp + Asn211SerSer210Glu + Gly218GlnTyr205Ala + Thr212AspSer158Asp + Leu216SerThr212Gly + Ser215GluAla199Ser + Va1202AlaSer129Glu + Tyr205AsnAsn154Gln + Gly201GlnSer190Glu + Thr212SerThr 58Pro + Ser160AspGly127Glu + Gly130SerAsn154Glu + Thr212GlyGly153Ser + Gln161AsnTyr205Thr + Ser210GluLeu125Gly + Pro200GlnTyr103Val + Thr132GlyGly126Asp + Leu216AlaSer100Glu + Gly127AsnSer129Asp + Ala186SerSer160Glu + Tyr205SerThr163Glu + Ile164HisGly159Ser + Gln161GluAsp 59Glu + Gly159ProGly101Asp + Tyr166CysThr 58Asn + Ser129GluAsn 61Glu + Thr214SerGly127Ser + Ser203GluGly127Pro + Gly130AspGly127Glu + Val202GlyPhe188Met + Ser203GluSer 97Glu + Tyr166AsnGly 60Glu + Gly 99AsnAsn162Glu + Asn217SerThr102Asp + Leu216GlyGly 99Gln + Ala105ThrGly153Ser + Ser203GluTyr103Pro + Ser158GluSer187Glu + Tyr205ProSer160Asp + Val204ThrSer100Asp + Leu216HisGly101Asn + Ser203AspAsn 96Ser + Tyr205ProThr132Gly + Thr212ProIle106Pro + Val202CysGly 62Ser + Pro209GluPhe188Cys + Leu216AspLeu 95Cys + Ser104GluSer104Asp + Tyr205MetTyr205Glu + Thr214GlyGly153Glu + Leu216AsnVal 94Thr + Ser100GluThr 65Asp + Val202HisVal202Gln + Ser215AspVal 94Ser + Ala105GluThr 58Pro + Gly153AspAla105Asn + Ser203GluSer104Asp + Ala105GlyGly126Gln + Ile164ValGly153Ser + Ser215GluPro200Gly + Ser215Glu表33多环三点突变变异体Thr 58Gln + Leu 95Gly + Ser203GluAsp 59Glu + Tyr103Cys + Leu216GlnGly156Glu + Val202Gln + Thr212GlyGly126Gln + Thr214Gly + Ser215GluAsn 96Gln + Ile106Ala + Gly156GluAsn 61Ser + Gly1o1Asp + Tyr205ProGly 64Gln + Thr132Gly + Gly165GlnThr 65Pro + Thr163Pro + Val202CysThr132Gly + Phe188Val + Leu216SerLeu 95Asn + Ser 97Glu + Asn211SerAsn162Gln + Ser187Asp + Tyr213ProSer100Asp + Val202Ala + Tyr205IleThr 58Asp + Gly 99Asn + Gly130GlnGly156Glu + Tyr208Pro + Leu216IleThr 65Gln + Leu125Asn + Ser187GluAsn211Ser + Thr212Asp + Gly218GlnTyr205Gly + Thr212Gln + Ser215GluThr 58Glu + Gly 60Pro + Thr 65ProAsn 96Gln + Gly153Pro + Leu216ValGly 99Asn + Gly153Asn + Pro209GluAla105Glu + Pro128Asn + Val202HisTyr205Ser + Thr212Gly + Gly218GluGly 99Gln + Asn217Glu + Thr219GlyLeu 95Gln + Val202Thr + Ser215AspGly101Pro + Ile164Thr + Ser215AspThr 58Pro + Gly130Asp + Tyr205GlnGly101Gln + Ser190Glu + Tyr208GlnAsn 61Gln + Thr212Asp + Leu216IleAsn 61Ser + Thr132Asn + Tyr205AspGly130Pro + Phe188Leu + Thr212GluAsn 61Ser + Ala105Gly + Ser131AspAsn 61Ser + Asn162Gln + Phe188SerGly126Gln + Ser203Glu + Leu216SerThr 65Ser + Gly156Asp + Thr163GlnPro128Ser + Ser215Asp + Leu216ValVal 94Ser + Ser100Asp +P he188ValGly 99Pro + Asn154Gln + Ser203GluTyr166Asp + Tyr205Thr + Thr214GlyGly 62Ser + Thr 65Ser + Asn211GluThr 65Glu + Pro128Asn + Leu216GlnVal 94Thr + Thr212Gln + Leu216MetSer129Asp + Thr132Ser + Val202GlyLeu 95Met + Tyr103Ala + Leu216AspVal 94Met + Ser 93Glu + Tyr103ProLeu125Pro + Ala199His + Ser203GluIle106Gly + Tyr205Ile + Thr214ProSer129Glu + Tyr166Pro + Tyr205GlyPro209Gln + Thr212Ser + Ser215GluAsn154Glu + Tyr166Thr + Tyr205HisAsn 96Ser + Gly101Ser + Ala105GlyGly153Glu + Phe188Gly + Gly218AsnGly126Pro + Gly156Asp + Thr212ProGly156Pro + Gly159Asp + Val202CysLeu 95Met + Ser 97Asp + Gly 99SerGly126Asn + Gly159Pro + Gly218AsnThr 65Gln + Leu 95Thr + Leu216AlaTyr103Asp + Gly126Asn + Gly127GlnThr 58Pro + Ala105Glu + Gly153AsnGly 60Gln + Tyr205Ser + Ser215AspThr132Asp + Gly218Ser + Thr219GlyTyr103Ser + Phe188Gln + Ser203AspSer 97Asp + Gly 99Pro + Leu216HisAsn 61Asp + Thr132Ser + Leu216AsnLeu 95Asn + Thr132Gly + Ser215AspTyr166Thr + Asn211Gln + Leu216ThrIle106Ala + Gly165Asp + Thr207ProLeu 95Asn + Val202Gln + Ser215GluGly 64Ser + Gly101Asp + Asn154GlnThr 58Asn + Gly 64Asn + Ser157GluGly 62Asn + Asn 96Ser + Thr214AsnGly130Asp + Phe188Asn + Gly218ProGly 99Ser + Pro128Glu + Leu216GlyVal 94Ala + Pro209Ser + Ser215AspLeu 95Thr + Gly126Gln + Leu216GlyAsp 59Glu + Gly159Ser + Tyr205ProVal202Gln + Ser203Glu + Tyr205AsnThr 58Ser + Ser190Glu + Tyr205ProLeu 95Asn + Tyr205Ala + Pro209GluThr 58Asn + Gly153Pro + Ser210GluSer131Glu + Thr132Asn + Gly165AsnGly127Ser + Thr212Asn + Ser215AspThr 65Gly + Thr102Asp + Gly127AsnGly 60Asn + Tyr2AsThr + Asn217GlnAla199Ser + Val202Ala + Thr212GlyThr 58Pro + Gly130Ser + Ser160AspGly153Ser + Asn154Glu + Thr212GlyLeu125Gly + Tyr205Thr + Ser210GluTyr103Val + Thr132Gly + Pro200GlnSer100Glu + Gly127Asn + Leu216AlaSer160Glu + Ala186Ser + Tyr205SerGly159Ser + Thr163Glu + Ile164HisPhe188Met + Val202Gly + Ser203GluThr102Asp + Ala105Thr + Leu216GlyThr 58Asp + Asn 96Ser + Tyr205ProSer 97Asp + Thr132Gly + Thr212ProIle106Pro + Val202Cys + Ser215AspLeu 95Cys + Phe188Cys + Leu216AspThr 65Asp + Val 94Thr + Val202HisThr 58Pro + Ala105Asn + Gly153AspSer104Asp + Ala105Gly + Ile164ValGly126Gln + Ser203Asp + Leu216HisSer160Asp + Gly218Gln + Thr219AsnGly 62Ser + Ala105Glu + Pro128SerThr 58Gly + Ile106Gly + Leu216AspSer 97Asp + Thr163Gln + Ala186SerThr 65Glu + Gly126Gln + Leu216HisPhe188Ile + Ser203Glu + Leu216ThrThr132Gln + Gly165Asp + Thr212SerSer129Asp + Asn211Ser + Leu216ThrLeu125Ile + Asn154Ser + Leu216MetThr 65Pro + Gly101Pro + Thr212AspThr102Asn + Thr212Gly + Leu216AspThr 65Gln + Tyr205Gln + Leu216MetLeu125Asn + Pro209Gly + Thr214GluLeu125Pro + Val202His + Ser215AspSer160Glu + Gln161Asn + Tyr205ValGly126Ser + Thr212Glu + Gly218AsnVal 94Thr + Ser155Asp + Thr207GlySer157Glu + Tyr208Gln + Thr212GlnTyr205Met + Thr212Gln + Ser215GluPro128Gln + Thr132Pro + Tyr213AsnGly 60Ser + Gly156Asp + Pro209AsnAsn162Asp + Thr163Asn + Ala199GlyGly165Ser + Tyr205Thr + Ser215GluSer100Asp + Gly156Pro + Tyr205GlnAla105His + Ser157Glu + Ala186AsnAsn162Gln + Ser190Glu + Val202SerGly 99Gln + Gly126Gln + Gln161AspGly126Asp + Gly159Ser + Thr212GlyGly 62Glu + Asn154Gln + Leu216GlnAsn154Asp + Tyr205Ser + Tyr213MetAsn 61Asp + Leu 95Ser + Val204GlnThr102Gln + Gly159Gln + Ser190GluPro128Asn + Gly159Asn + Asn211SerGly159Ser + Thr163Gly + Ala199ThrAsn211Gln + Tyr213Gln + Ser215GluGly 99Ser + Leu125Gly + Ser215GluThr 65Gly + Ser190Glu + Thr219SerVal 94Met + Asn162Ser + Leu216MetLeu 95Ser + Tyr103His + Gly156GluThr 58Gly + Asn211Glu + Leu216AlaAla105Gly + Thr212Pro + Gly218GluSer158Asp + Gly159Ser + Tyr205ProThr 58Gln + Ser158Glu + Thr163GlyThr 65Pro + Leu125Met + Thr163GlyGly 62Asn + Gly126Pro + Ser210GluLeu 95Met + Thr132Asp + Asn211SerTyr103Ile + Gly159Asp + Val202GlyGly101Gln + Tyr166Pro + Asn211GlnPro128Gly + Thr132Pro + Thr214GlnPro200Ser + Thr212Asp + Leu216GlyThr212Asp + Leu216Met + Gly218GlnThr163Gln + Tyr205Cys + Thr212ProThr 58Gln + Ser 98Glu + Thr212ProThr102Pro + Ser155Asp + Tyr205AsnGly 62Ser + Gly 99Glu + Val202SerLeu 95Asp + Ile106Gln + Gly126AsnSer100Glu + Gly101Gln + Val202AlaThr 58Ser + Ser100Glu + Ile164LeuGly101Ser + Phe188Asp + Tyr213GlySer104Glu + Gly156Gln + Thr212GlyThr 65Asp + Val 94Gly + Tyr205MetSer203Glu + Thr212Ser + Tyr213ValThr 65Ser + Leu125Glu + Gln161SerThr 65Gly + Tyr205Asp + Tyr213ThrGly101Pro + Ser104Asp + Thr163GlnAla105Gly + Asn154Asp + Pro209GlyGly126Gln + Gln161Ser + Leu216AlaGly153Gln + Tyr166Cys + Gly218AspGly 99Gln + Val204Pro + Thr219GluThr 58Ser + Ser 97Asp + Thr212ProAsn162Ser + Met198Gly + Val202ThrVal 94Ser + Leu125Asp + Tyr205ThrSer157Asp + Tyr205Ser + Leu216ThrGly165Asn + Ser215Glu + Leu216ProGly156Asn + Thr163Glu + Ala199SerPro128Ser + Gly156Glu + Thr219GlyAsn 96Gln + Gly126Gln + Ser203GluGly159Asp + Gly165Pro + Tyr213IleThr 58Gln + Gly153Ser + Asn217GlnPro128Ser + Thr132Glu + Thr212GlyGly156Gln + Tyr205Pro + Ser215GluGly 62Ser + Tyr205Ile + Leu216AspLeu125Pro + Gly159Glu + Gly165ProThr 58Gln + Ser203Asp + Thr212SerThr102Gln + Val202His + Thr214SerGly 99Glu + Ile106Gly + Gly218AsnSer 97Asp + Leu125Val + Tyr213IleIle106Leu + Phe188Glu + Thr212AsnThr 58Pro + Gly153Pro + Asn162GlnGly127Ser + Pro128Gln + Thr212ProTyr103Pro + Ser203Asp + Tyr205IleThr 58Gln + Thr163Ser + Tyr205SerVal 94Cys + Ser215Glu + Gly218ProThr102Pro + Gly165Pro + Val202ThrThr 58Glu + Tyr103Asn + Pro200GlnPhe188Thr + Val202Asn + Asn217SerGly 99Asn + Tyr205Ser + Ser215GluTnr102Gly + Gly165Asp + Gly218GlnGly 60Asp + Ala186Pro + Tyr205AsnThr102Asn + Ile106Asn + Ser158GluGly 62Ser + Ser 97Glu + Gly218AsnIle106His + Ser129Glu + Thr212AsnGly126Glu + Pro128Asn + Va1202AsnAsp 59Glu + Gly 64Asn + Tyr213SerThr163Pro + Val204Thr + Tyr205CysSer129Glu + Asn162Ser + Leu216CysGly156Asn + Phe188Ala + Tyr205HisVal 94Ser + Gly101Ser + Leu125AspAsn 96Glu + Tyr166Ala + Leu216CysTyr103Gln + Phe188Met + Ser210GluGly101Ser + Thr212Pro + Thr219ProSer104Glu + Gly159Gln + Thr212ProGly 62Asp + Gly126Ser + Leu216ProGly101Asp + Gly218Pro + Thr219AsnSer157Glu + Tyr205Pro + Thr212SerPro209Glu + Ser210Glu + Gly218SerAsn154Glu + Ser155Glu + Gly218ProAsn211Glu + Thr212Asp + Leu216HisTyr205Gly + Ser215Glu + Leu216AspVal202Ser + Ser215Asp + Leu216AspThr 58Asp + Asp 59Glu + Thr212SerTyr205Glu + Tyr213Pro + Ser215AspGly156Ser + Tyr205Asp + Ser215GluGly 60Glu + Ser 97Glu + Gly 99AsnSer155Glu + Asn162Ser + Ser190GluLeu 95Asn + Ser203Asp + Ser215AspSer203Asp + Ser215Asp + Thr219GlnGly 62Asn + Ser203Glu + Ser215GluSer203Glu + Tyr205Pro + Ser215AspPhe188Asn + Ser203Asp + Ser215AspGly 62Asn + Ser203Glu + Ser215AspSer203Glu + Ser215Asp + Leu216SerGly156Gln + Ser203Glu + Ser215AspSer203Asp + Tyr205Ala + Asn217GluTyr205Ala + Ser210Glu + Thr212AspIle164Cys + Ser210Glu + Thr212AspSer158Asp + Gln161Glu + Tyr205ProAsp 59Glu + Asn 61Glu + Gly130SerGly 64Gln + Ser 98Glu + Ser100AspSer 97Glu + Gly 99Asp + Tyr213HisAsn154Gln + Ser157Glu + Ser190AspSer158Asp + Ser160Glu + Tyr205CysGly201Pro + 5er203Glu + Tyr205AspGly 99Gln + Ser129Asp + Tyr166GluSer160Asp + Asn162Glu + Phe188GlySer129Asp + Ser131Asp + Gly218AsnAsp 59Glu + Gly165Asn + Pro209GluVal 94Gly + Ser215Asp + Asn217GluVal202Glu + Tyr205Gln + Leu216GluGly156Glu + Ser158Glu + Tyr205MetAsn 61Asp + Asn 96Glu + Gly127SerGly159Asn + Phe188Asp + Gly218AspTyr103His + Asn162Glu + Ser190AspSer 97Asp + Ser100Glu + Gly201AsnSer157Glu + Ser160Glu + Tyr205ProGly153Asp + Val202Ser + Gly218AspSer157Glu + Gly159Glu + Ser190AspAsp 59Glu + Asn 96Glu + Tyr205SerGly153Ser + Asn154Asp + Gly165AspAsn154Glu + Gly165Asp + Asn211SerSer155Asp + Gly156Asp + Thr219AspSer210Asp + Tyr213Asp + Thr214GluAsn 61Glu + Ala186Gly + Pro209AspAsn154Asp + Ser157Glu + Thr214SerSer100Glu + Leu125Glu + Tyr213HisGly 60Glu + Leu 95Glu + Ile106LeuGly 99Glu + Gly101Glu + Tyr103GluThr 58Glu + Thr 65Glu + Ile106CysSer 97Glu + Gly101Asp + Val202AsnGly 62Asp + Ser210Asp + Leu216AlaAsn 61Asp + Ser100Glu + Asn162SerGly127Glu + Asn162Glu + Gly165GluIle106Asn + Gly156Glu + Gly159GluSer100Glu + Gly126Glu + Ala186GlnGly 62Asp + Phe188Leu + Thr212GluIle106Gln + Ser215Glu + Gly218GluTyr103His + Ser155Glu + Gly218GluAla186Glu + Val202Glu + Ser215AspGly127Glu + Asn154Glu + Tyr213ThrSer157Glu + Ser160Asp + Ser187AspGly 62Asp + Leu216Glu + Thr219GlnThr 58Asp + Ser210Asp + Thr212GluSer155Glu + Ser157Asp + Gly218AspPro128Asp + Asn162Glu + Asn217SerGly156Asp + Gly165Glu + Tyr205AsnSer158Glu + Phe188Asp + Val202MetThr102Glu + Ser104Asp + Leu125GluSer 98Asp + Ser100Asp + Ser104GluGly 62Asp + Pro209Asp + Asn211AspSer160Glu + Ser187Asp + Ser190AspGly159Asp + Val202Asp + Ser203AspPro128Glu + Thr212Asp + Tyr213AspLeu 95Glu + Tyr205Asp + Ser215AspAsp 59Glu + Tyr205Glu + Ser215GluGly 60Ser + Thr 65Glu + Leu216AspGly101Ser + Asn154Glu + Ser158GluPro128Asp + Ser203Asp + Ser215AspSer203Asp + Ser210Glu + Ser215GluSer104Glu + Ser203Asp + Asn217GluSer160Asp + Ser203Asp + Leu216AspSer203Glu + Ser210Asp + Leu216AspGly127Glu + Ser129Glu + Ser215GluPro128Glu + Ala186Glu + Phe188AspAsp 59Glu + Asn 61Glu + Ser203AspSer 97Asp + Gly 99Asp + Ser210AspAsn 96Glu + Ser100Asp + Thr212GluThr 65Asp + Thr163Gln + Ser215AspSer 98Asp + Thr102Glu + Leu216GlnThr132Glu + Ser157Asp + Ser190GluSer129Glu + Ser157Asp + Ser190GluSer190Glu + Ser203Glu + Tyr205GluAsn 61Asp + Ser 98Asp + Tyr205AspSer129Asp + Phe188Glu + Ser190AspSer100Glu + Ser104Glu + Tyr205MetGly127Glu + Ser190Asp + Tyr205LeuAsp 59Glu + Ser203Glu + Pro209AspGln161Glu + Ser215Glu + Asn217AspAsp 59Glu + Ser 97Glu + Ser157AspLeu125Glu + Gly153Glu + Ser203GluSer104Glu + Ser157Asp + Ser160AspAsp 59Glu + Asn 96Glu + Ser187GluSer129Asp + Thr132Glu + Asn162GluSer131Glu + Ser203Glu + Gly218GluGly156Asp + Ser203Asp + Thr214AspSer160Glu + Thr163Asp + Ser215AspThr 58Asn + Gly 99Glu + Ala105GluAsp 59Glu + Phe188Leu + Thr212AspSer104Asp + Ser131Glu + Tyr205GluGly 62Asp + Ser104Asp + Ser131AspSer104Glu + Ser131Glu + Gly156GluGly 60Asp + Gly101Asp + Met198GlnSer 98Glu + Ser215Glu + Gly218AspTyr103Asp + Ser129Asp + Tyr205GluSer100Asp + Ile106Asp + Ser215AspThr 65Asp + Ser100Glu + Ala105GluAsp 59Glu + Ser100Asp + Tyr205IleGly153Glu + Asn162Asp + Ser203GluSer 98Glu + Thr212Glu + Ser215GluSer131Asp + Thr212Glu + Ser215AspAla105Asp + Thr212Glu + Ser215AspSer158Glu + Thr212Asp + Ser215AspSer157Asp + Ser203Glu + Thr219AspGly 62Gln + Thr212Glu + Leu216AspSer 97Glu + Ser187Glu + Asn217AspSer155Glu + Asn217Asp + Gly218AsnGly159Glu + Ser190Glu + Asn211GluPro128Asp + Ser190Glu + Ser215AspAsp 59Glu + Gly156Asp + Ser160GluGly 60Asp + Ser100Glu + Ser215GluGly130Glu + Gly153Glu + Tyr205AsnThr102Glu + Gly153Glu + Asn217AspSer129Glu + Ser155Glu + Gln161AsnTyr205Glu + Asn211Glu + Leu216CysGly153Glu + Gln161Asp + Ala199AsnGly 62Asn + Gly153Glu + Val202AspSer129Asp + Asn162Glu + Asn211GluGly153Glu + Ser158Asp + Leu216AspSer157Glu + Leu216Ile + Thr219AspSer158Glu + Ser215Aso + Thr219Asp表34多环四点突变变异体Tyr103Cys + Val202Gln + Thr212Gly + Leu216GlnAsn 61Gln + Gly101Asp + Ile106Ala + Tyr205proAsn 61Ser + Gly 64Gln + Thr132Gly + Gly165GlnLeu 95Ile + Tyr205Glu + Tyr213Ala + Thr219AsnGly126Ser + Val202Ser + Tyr205Cys + Asn217GluAsn 96Ser + Asn154Glu + Tyr166Thr + Tyr205HisGly101Ser + Ala105Gly + Phe188Gly + Gly218AsnGly165Ser + Val202Gln + Tyr205Thr + Thr212SerGly126Asn + Ser190Glu + Pro209Asn + Leu216ValIle106Glu + Gly127Ser + Asn154Ser + Tyr205LeuThr 58Pro + Ser203Asp + Tyr213Met + Leu216ValGly 62Gln + Asn 96Glu + Ile164Asn + Leu216IleSer 98Glu + Tyr166Gln + Thr212Asn + Leu216ValGly156Asp + Tyr166Ala + Tyr208Pro + Leu216IleThr 58Pro + Ser 98Asp + Gly130Pro + Tyr205ValGly130Asp + Phe188Asn + Leu216Gly + Gly218ProLeu 95Thr + Gly126Gln + Pro209Ser + Leu216GlyVal202Gln + Ser203Glu + Tyr205Asn + Gly218AsnThr 58Asn + Leu 95Asn + Gly153Pro + Ser210GluGly127Ser + Ser131Glu + Thr132Asn + Gly165AsnThr 65Gly + Gly127Asn + Thr212Gln + Thr214GluAsn 96Gln + Gly126Asn + Gly127Ser + Val202AsnGly 64Ser + Ser160Asp + Asn211Ser + Thr212GlyAsp 59Glu + Ala199Ser + Val202Ala + Tyr205SerLeu125Gly + Thr132Gly + Pro200Gln + Tyr205ThrGly159Ser + Thr163Glu + Ile164His + Tyr205SerGly127Pro + Phe188Met + Val202Gly + Ser215GluGly101Gln + Asn211Gln + Thr212Pro + Ser215AspPro128Gly + Pro200Ser + Thr214Gln + Leu216GlyTyr205Cys + Thr212Pro + Leu216Met + Gly218GlnThr102Pro + Val202Ser + Tyr205Asn + Gly218AspLeu 95Asp + Ile106Gln + Gly126Asn + Val202AlaTyr103Gln + Ser203Asp + Thr212Asn + Leu216AsnPhe188Met + Ser210Glu + Thr212Pro + Thr219ProSer104Glu + Gly159Gln + Thr212Pro + Leu216ProGly 60Gln + Ala186Gly + Thr212Gly + Ser215AspThr102Glu + Thr132Pro + Leu216Pro + Asn217GlnLeu 95Ala + Phe188Thr + Tyr205Gln + Ser215AspThr 58Gly + Gly 60Asp + Val202Cys + Thr212ProGly156Asn + Val202Asp + Thr212Ser + Thr214GlyThr 58Pro + Gly165Glu + Tyr205Ser + Tyr213HisGly 99Asn + Tyr166Asn + Ser203Asp + Leu216HisGly 62Glu + Leu 95Met + Ala105Ser + Gly126SerIle106Gln + Ser187Glu + Tyr205Gly + Thr219ProGer160Glu + Tyr205Ser + Thr212Pro + Leu216ThrGly130Gln + Ser131Glu + Asn162Gln + Leu216AsnGly156Glu + Thr163Ser + Asn211Ser + Thr212AsnGly165Gln + Phe188Leu + Val202His + Thr212GluVal 94Cys + Pro128Asp + Val202Thr + Tyr205AlaLeu 95Thr + Gly156Ser + Thr214Glu + Leu216AlaGly 99Pro + Tyr103Glu + Gly165Pro + Leu216CysGly 64Asn + Thr 65Glu + Tyr166Ala + Phe188TyrTyr103Gln + Gly156Pro + Tyr166Asn + Ser215GluGly126Gln + Pro128Gln + Val202Thr + Thr212GluGly 64Ser + Val 94Met + Gly130Asp + Tyr213GlyGly 99Asn + Val202Pro + Thr212Asn + Ser215GluGly101Pro + Asn154Ser + Leu216His + Asn217GluTyr103Asp + Gly153Pro + Ala186Asn + Val202SerLeu125Thr + Gln161Asn + Thr212Gly + Leu216AsnGly 64Pro + Val 94His + Ser210Asp + Thr212GlyAsn 61Ser + Thr 65Gln + Ser190Asp + Val202GlyAla105Thr + Gly153Gln + Gly156Gln + Asn211GlnAsn 61Gln + Tyr166Ile + Ala186Asn + Ser215AspGly 60Gln + Tyr103Gly + Gly130Asn + Asn217AspLeu 95Pro + Tyr205Leu + Asn211Gln + Ser215GluThr102Asp + Gly126Gln + Val202Asn + Thr212GlyVal 94Thr + Gly153Pro + Ala186Pro + Tyr205GluSer104Asp + Ala105Asn + Thr163Ser + Tyr205AlaGly 64Ser + Val 94Asn + Gly130Asp + Leu216CysGly101Asn + Leu125Asn + Gly201Pro + Leu216SerGly159Pro + Gly165Pro + Ala186Thr + Thr212ProPro128Gln + Ser129Asp + Ala186Ser + Asn217SerGly 60Asn + Gly159Ser + Phe188Val + Ser203GluSer 98Glu + Gly 99Asn + Thr102Asn + Ala186ProThr 65Asn + Gly 99Gln + Thr212Ser + Leu216AspVal202Gln + Tyr205Thr + Tyr213Val + Ser215GluThr132Ser + Gly156Ser + Phe188Thr + Asn211GluLeu125His + Gly156Ser + Thr212Glu + Leu216AsnGly126Ser + Tyr205Asp + Thr214Gln + Leu216AsnAla186Gly + Ser215Glu + Gly218Pro + Thr219GlnThr 58Gly + Gly 60Asn + Leu125His + Leu216GlyAsp 59Glu + Val 94Gly + Tyr205Gln + Tyr213IleSer155Glu + Met198Thr + Val202Met + Tyr205ThrGly127Asn + Tyr166Pro + Phe188Leu + Thr214GluLeu 95Cys + Gly130Gln + Thr212Ser + Leu216ValAsp 59Glu + Thr 65Gly + Leu 95Gly + Leu216AsnSer131Glu + Gly201Ser + Val202Gln + Tyr205GlyLeu125Val + Tyr205Gln + Ser215Asp + Gly218GlnVal 94Gly + Thr102Ser + Ser203Glu + Leu216AsnAsp 59Glu + Gly 60Gln + Thr212Gly + Leu216CysAla186Gln + Gly201Gln + Val202Pro + Ser203GluThr 65Ser + Ser 98Asp + Gln161Ser + Tyr205CysTyr166Ala + Ser187Glu + Tyr205Gly + Leu216ValGly 99Asp + Met198Cys + Pro209Gly + Thr212AsnThr 65Pro + Ile106Gly + Tyr205Gln + Leu216GluLeu 95Ser + Thr102Asn + Leu216Asp + Asn217SerThr 58Gly + Gly127Ser + Tyr166Glu + Val202AsnVal 94Pro + Tyr213Thr + Thr214Glu + Asn217GlnSer 97Glu + Gln161Asn + Tyr166Gly + Val202SerTyr103Met + Thr132Gly + Tyr166Met + Ser210AspLeu 95Met + Gly156Asn + Val202Glu + Leu216GlnSer 97Asp + Met198Ala + Val204Thr + Tyr205ThrGly101Ser + Gly153Pro + Asn217Glu + Thr219ProIle106Met + Thr163Glu + Tyr166Ile + Thr212SerGly165Ser + Ser190Asp + Pro200Ser + Val202MetGly 62Ser + Gly153Asp + Thr163Ser + Gly201GlnGly101Gln + Thr102Ser + Gly153Ser + Val202AspVal 94Ala + Leu 95Gly + Leu125Thr + Gly130AspAsn 96Asp + Tyr205Ala + Thr212Pro + Thr219GlyGly153Gln + Tyr205Ile + Thr212Gly + Ser215GluIle106Cys + Asn154Glu + Tyr205Ala + Leu216AlaAsn 96Asp + Gly 99Pro + Tyr205Val + Asn217GlnAla105Thr + Leu125Thr + Val204Ala + Tyr205MetThr102Pro + Gly156Pro + Tyr208Val + Thr212AsnGly 60Ser + Phe188Ile + Thr212Ser + Leu216AspSer100Asp + Tyr103Gln + Gly159Gln + Leu216ValPro128Gly + Gly165Gln + Ser203Glu + Leu216SerSer129Glu + Thr132Gln + Met198Thr + Thr219GlnVal 94Pro + Gly 99Asn + Thr132Gly + Gly159AspSer155Asp + Gly165Ser + Tyr205His + Thr212GlyGly 99Asp + Phe188Val + Thr207Asn + Leu216ProThr102Gln + Gly126Asn + Gly159Asn + Leu216AspAsp 59Glu + Thr132Pro + Thr163Pro + Phe188ThrGly101Ser + Ala105Glu + Ala186Pro + Thr212GlySer129Asp + Gly153Asn + Gly159Pro + Val202MetGly 60Pro + Asn 96Asp + Gly126Gln + Tyr166AlaThr 65Gln + Gly 99Pro + Tyr205Ala + Ser215AspVal 94Gln + Gln161Asp + Tyr205Pro + Leu216MetAsp 59Glu + Gly 60Glu + Ala186Gly + Gly218AsnSer 98Asp + Gly 99Glu + Phe188Met + Tyr213AlaSer160Asp + Gln161Asp + Tyr205Ser + Thr219GlnAla105Gly + Ile106Cys + Tyr205Asp + Ser215AspPro200Asn + Tyr205Asp + Asn211Gln + Ser215GluTyr205Asp + Thr207Ser + Thr214Ser + Ser215AspAsn 61Asp + Thr 65Gln + Ser 97Asp + Tyr205AlaLeu125His + Ser203Glu + Ser215Glu + Leu216AspThr 65Asn + Ser203Glu + Tyr205Cys + Ser215AspGly 64Pro + Ala186Ser + Ser203Asp + Ser215AspPro128Ser + Ser203Asp + Tyr213Ala + Asn217GluVal202His + Tyr205Val + Ser210Glu + Thr212AspGly 99Asn + Gly130Ser + Ser210Glu + Thr212AspTyr205Ala + Ser210Glu + Thr212Asp + Gly218GlnPhe188Ala + Ser203Asp + Tyr205Asp + Thr212SerAsn154Glu + Ser155Asp + Thr163Asp + Tyr213ThrGly 99Gln + Ser129Asp + Tyr166Glu + Leu216ThrThr102Gly + Ala105Thr + Ser155Glu + Ser157AspGly127Pro + Ser157Glu + Gly159Asp + Gln161AspGly101Asn + Asn154Asp + Thr163Gly + Gly218GluPro128Ser + Ser158Asp + Thr163Asp + Asn217SerIle106Val + Thr214Asn + Ser215Asp + Asn217AspThr 58Gly + Asn154Asp + Ser157Asp + Ser190GluLeu125Asn + Gly153Glu + Thr163Asp + Thr214ProGly 64Pro + Ser157Glu + Gln161Glu + Tyr205CysSer155Asp + Ser187Asp + Ser190Glu + Thr212GlnVal 94Pro + Ser155Asp + Ser157Asp + Ser187GluGly126Gln + Ser203Asp + Ser215Glu + Gly218AspGly156Ser + Ser187Asp + Ser190Glu + Thr212SerAsn154Asp + Ser187Glu + Val202Asn + Leu216SerAsn162Asp + Gly165Asp + Thr212Gln + Leu216CysSer155Glu + Ser187Asp + Pro200Ser + Tyr213ValAsn 96Glu + Thr102Asp + Ala105Pro + Tyr213ValAsn154Ser + Gly156Glu + Asn162Asp + Thr212SerAsn 61Glu + Ser100Asp + Tyr205Val + Leu216GlyThr 65Gly + Asn154Glu + Ser157Asp + Asn162GluTyr205Leu + Thr212Glu + Ser215Glu + Leu216GluAsn 61Gln + Gly 62Glu + Ser 98Asp + Thr214GlnGly 62Glu + Ser 98Glu + Gly130Ser + Val202ProThr 65Glu + Tyr205Met + Thr212Asp + Thr214GlyAla105His + Phe188Asp + Val202Asp + Asn211SerAsp 59Glu + Pro209Ser + Asn211Asp + Thr212GluLeu 95Gln + Gly126Gln + Gly127Asp + Ser155AspGly 60Asp + Val 94His + Asn154Ser + Ser210GluThr 58Gly + Gly126Glu + Ser155Asp + Gly201ProGly156Asn + Tyr166Pro + Thr212Glu + Ser215GluThr212Asp + Ser215Glu + Leu216Ala + Thr219SerThr 58Asp + Gly101Ser + Ser210Asp + Thr212GluIle106Glu + Gly126Asp + Gly127Asn + Leu216ProAsn 61Ser + Gly 99Asp + Thr102Asp + Ser104AspSer210Asp + Thr212Asp + Tyr213Pro + Ser215GluGly127Pro + Pro128Ser + Ala186Glu + Ser203AspGly101Glu + Thr102Asp + Ser129Asp + Leu216AsnGly 62Glu + Leu 95Asp + Gly159Asn + Leu216GlyAsn 61Asp + Ser 97Asp + Ser210Glu + Thr212GlnSer203Glu + Tyr205Pro + Thr212Ser + Thr219GluThr 65Glu + Tyr103Pro + Ser215Asp + Asn217AspGln161Asp + Thr163Glu + Ser187Glu + Asn211GlnGly153Glu + Tyr166Gly + Ser187Glu + Leu216MetAsp 59Glu + Gly159Glu + Ser160Asp + Pro209GlnGly 60Asn + Ser129Glu + Ser158Asp + Gly159AspGly130Glu + Ser131Asp + Ser155Glu + Thr212AsnGly130Asp + Asn154Glu + Ser155Asp + Gln161AsnSer129Asp + Gly130Glu + Gly156Glu + Gln161SerGly 99Glu + Ser100Asp + Ser203Glu + Leu216SerPro128Asp + Ser129Asp + Val202Asn + Ser215GluTyr103Asp + Ser104Glu + Thr212Pro + Ser215GluThr 65Pro + Ser157Glu + Ser158Asp + Ser215AspThr 58Asn + Asn 96Asp + Ser 97Asp + Ser187AspSer104Asp + Ile164Gly + Ser215Asp + Leu216AspSer157Asp + Ser215Asp + Leu216Glu + Gly218GlnGln161Ser + Ser187Asp + Ser215Glu + Leu216GluThr 58Pro + Ser104Asp + Thr212Glu + Tyr213GluThr102Glu + Ser160Glu + Gln161Asp + Leu216ValAsn 61Glu + Gly 62Glu + Ser203Glu + Thr212GlyTyr205Val + Ser210Asp + Asn211Glu + Leu216GluAsp 59Glu + Gly126Ser + Tyr205Glu + Ser215GluPro128Asn + Ser129Asp + Tyr205Asp + Ser215AspGly101Glu + Tyr205Glu + Ser215Asp + Leu216HisSer155Glu + Ile164Ser + Tyr205Asp + Ser215AspTyr103Ala + Tyr166Glu + Tyr205Glu + Ser215GluGly 60Glu + Ser 97Glu + Gly 99Asn + Asn162GluSer187Glu + Ser203Glu + Tyr205Asp + Tyr213ProSer157Glu + Ser203Asp + Tyr208Pro + Ser215GluSer 98Glu + Ser203Glu + Tyr205Val + Ser215GluGly127Gln + Ser160Glu + Ser203Asp + Ser215GluVal 94Glu + Ser203Asp + Ser215Asp + Leu216ProSer100Asp + Ser203Glu + Ser215Glu + Leu216ThrSer157Glu + Phe188Thr + Ser203Asp + Ser215AspVal 94Gly + Leu 95Glu + Ser203Glu + Ser215AspPhe188Val + Ser190Asp + Ser203Asp + Ser215GluSer158Asp + Ser203Asp + Ser215Asp + Asn217SerSer157Asp + Ser203Asp + Ser215Asp + Thr219GlnGly126Pro + Gly153Glu + Ser203Glu + Ser215GlnSer131Glu + Gly165Asn + Ser203Glu + Ser215AspTyr103Glu + Gly153Asp + Ser190Glu + Tyr205ValGly159Glu + Ser203Glu + Leu216Gln + Asn217GluSer100Asp + Gly153Gln + Thr163Asp + Ser190AApThr102Asp + Gly127Glu + Ser155Asp + Tyr213ProAsn 61Glu + Asn154Glu + Ser190Asp + Val204MetAsn154Asp + Gln161Ser + Ser190Asp + Ser203GluThr 65Gly + Gly127Asp + Gly165Asp + Ser203AspAsn 96Asp + Gly 99Glu + Gly101Asn + Ser187GluThr102ASP + Ala105Glu + Gln161Ser + Pro209AspGly127Glu + Ser129Glu + Gly130Ser + Ser160AspThr102Glu + Ser104Glu + Pro200Asn + Thr212GluSer 98Asp + Ser100Glu + Pro128Gly + Gly159AspGly159Asp + Gln161Asp + Val204Gly + Ser215GluSer155Glu + Phe188Glu + Tyr205Asn + Ser215GluSer129Glu + Thr132Asn + Gly165Asp + Gly218AspSer100Glu + Gly153Asp + Gly165Gln + Thr219AspGly126Asp + Ser131Glu + Thr207Gly + Gly218GlnAsn 61Asp + Gly 99Glu + Tyr103Thr + Ser131AspGly 99Asp + 5er203Asp + Tyr205Asp + Thr212ProSer190Glu + Ser203Glu + Tyr205Asp + Leu216HisGly 60Pro + Ser157Asp + Ser203Glu + Tyr205AspSer157Glu + Thr163Glu + Pro209Ser + Ser215GluGly 60Asn + Ser157Asp + Thr163Glu + Asn211AspThr102Glu + Gly127Asn + Gly130Glu + Tyr205GlnAsn 61Glu + Gly153Ser + Pro209Asp + Ser215GluAsp 59Glu + Asn 61Gln + Leu2I6Asp + Gly218AspLeu125His + Ser129Asp + Ser131Asp + Val202GluIle106Cys + Tyr205Asp + Ser210Glu + Leu216AspSer 97Glu + Gly156Asp + Thr163Asp + Thr212GlnSer131Asp + Asn154Gln + Ser215Glu + Asn217AspSer104Glu + Thr212Ser + Ser215Asp + Asn217GluSer157Asp + Ser187Asp + Ser203Asp + Thr212GlyAsn 61Asp + Asn 96Asp + Ala105His + Asn162AspAsn 61Asp + Asn 96Glu + Gly127Ser + Gly130AspThr 58Gly + Ser158Glu + Asn211Asp + Tyr213AspSer158Asp + Asn162Asp + Thr212Asp + Leu216ThrGly 99Ser + Ser157Asp + Asn162Glu + Leu216AspGly159Gln + Asn162Glu + Ser190Asp + Ser215GluSer 97Asp + Ser100Asp + Gly126Asn + Ser160AspGly 60Glu + Ser 98Glu + Gly153Gln + Ala186AspSer157Asp + Ser160Asp + Thr212Asn + Ser215GluGly 62Pro + Ser131Glu + Gly165Glu + Thr219AspSer190Asp + Val202Glu + Ser215Glu + Leu216AlaAsn 96Glu + Gly153Asp + Gly156Asp + Gln161AsnTyr103Glu + Ser131Asp + Ser203Asp + Leu216CysGly130Glu + Ser157Asp + Ser187Asp + Tyr208HisAsn 61Asp + Thr163Gln + Ser203Asp + Thr214GluSer155Asp + Ser158Asp + Ser203Asp + Tyr205ThrSer155Asp + Gly156Ser + Ser215Glu + Thr219AspGly 60Gln + Thr 65Gln + Asn 96Asp + Leu125GluAsp 59Glu + Asn 96Glu + Ser203Glu + Thr214AsnAsp 59Glu + Asn 96Asp + Ser215Asp + Leu216IleGly 62Asp + Ser 97Glu + Ala105His + Ser131GluLeu125Asp + Gly165Asp + Ser203Asp + Tyr213GlnLeu 95Glu + Tyr166Asn + Ser203Glu + Thr214GluGly101Asp + Asn162Asp + Gly165Glu + Leu216AsnGly 99Glu + Tyr103Glu + Ser129Glu + Tyr205AsnAsp 59Glu + Asn 96Ser + Thr212Glu + Ser215AspAsn 61Glu + Ser100Asp + Ala186Gly + Thr212GluThr 65Gly + Ser 97Glu + Gly101Glu + Gly130GluAla186Glu + Phe188Tyr + Thr212Asp + Asn217AspAsn 61Gln + Gly156Glu + Ser215Glu + Thr219GluGly 99Gln + Ser129Glu + Ser155Glu + Gly218GluSer160Glu + Thr163Asp + Thr212Ser + Ser215AspAsn 96Asp + Thr102Asp + Ser157Glu + Ala186HisGly 99Glu + Ala105Asp + Thr214Asn + Leu216ValAsn162Gln + Ala186Asp + Tyr205Ser + Leu216AspSer 97Glu + Gly156Glu + Gly159Asp + Val202AsnIle106Cys + Ser129Glu + Ser155Glu + Ala186GluSer100Glu + Leu125Gly + Gly127Glu + Ser203AspThr 65Asp + Ser131Asp + Ile164Asn + Thr212AspSer 97Glu + Thr102Glu + Gln161Ser + Tyr205LeuAsp 59Glu + Gly 99Glu + Ser203Asp + Tyr205AsnAsp 59Glu + Gly 99Asp + Gly126Ser + Thr214GluSer160Asp + Pro209Asn + Ser215Asp + Gly218GluSer155Glu + Gly159Asn + Pro209Glu + Gly218GluSer155Asp + Asn211Asp + Tyr213Met + Gly218AspSer129Asp + Thr132Glu + Ser160Glu + Ala186SerVal 94Glu + Ser 97Asp + Gly101Pro + Ser203GluGly101Asp + Gly126Asp + Thr132Asp + Thr212AsnSer 98Glu + Gly127Glu + Thr163Asp + Thr212AsnGly 99Asp + Gly101Pro + Thr102Glu + Ser203GluGly159Asp + Thr212Asp + Ser215Asp + Asn217SerSer158Glu + Asn162Gln + Thr212Glu + Ser215Asp Val 94Pro + Ser155Asp + Thr212Glu + Ser215GluGly 99Gln + Ile106Asp + Thr212Glu + Ser215GluThr 58Asp + Tyr205Pro + Thr212Glu + Ser215AspAsp 59Glu + Ser 98Glu + Ser203Asp + Tyr205GlyAsp 59Glu + Ser100Asp + Ser104Asp + Ala105ProAsp 59Glu + Ser100Asp + Ser104Asp + Tyr205IlePro128Glu + Asn162Asp + Thr212Glu + Leu216CysAsn 96Gln + Thr102Asp + Ser129Glu + Asn162GluSer 97Glu + Gly130Asn + Ser155Asp + Ala186GluAla105Glu + Ser155Glu + Gln161Asp + Tyr205ThrGly 62Asp + Ser158Asp + Ser215Asp + Leu216AsnThr102Asp + Gly126Asp + Ser158Asp + Thr212SerThr 58Asn + Gly 64Pro + Thr 65Glu + Val 94AspSer190Glu + Ser203Glu + Tyr213Glu + Leu216HisGly159Asp + Ser203Asp + Tyr205Cys + Tyr213AspIle106Asp + Gly130Asp + Ser215Asp + Asn217GlnSer 98Glu + Thr102Asp + Ser203Asp + Thr214GlyGly 99Gln + Ser104Asp + Gly130Asp + Asn154GluSer104Glu + Gly126Ser + Gly130Asp + Asn217GluAsn 96Gln + Ser100Glu + Ser104Glu + Tyr205AspAsn 61Gln + Gly101Asp + Ser157Asp + Gly165GluThr102Ser + Val202Cys + Thr214Glu + Gly218GluAsn 61Gln + Ser104Glu + Gly127Glu + Gly165ProThr 65Gly + Tyr205Glu + Asn211Glu + Leu216CysGly 99Gln + Gly159Ser + Pro209Asp + Leu216AspAsp 59Glu + Ser158Asp + Tyr205Pro + Thr212GluAsp 59Glu + Ser131Glu + Thr212Glu + Leu216IleAla105Asp + Leu125Glu + Asn211Glu + Leu216IleThr 58Asp + Gly156Ser + Asn211Asp + Ser215GluSer157Glu + Gly159Ser + Gly165Pro + Thr219GluAsn162Asp + Tyr205Gln + Thr212Glu + Leu216AspSer100Glu + Met198Gln + Thr212Asp + Leu216GluGly 64Gln + Pro128Glu + Thr132Asp + Thr163GlyGly 62Gln + Pro128Asp + Thr132Asp + Leu216ThrGly101Asp + Ile106Met + Leu125Gly + Pro128GluThr 65Glu + Asn 96Glu + Gly156Asp + Leu216CysSer104Asp + Leu125Gln + Ser129Asp + Asn162GlnAsp 59Glu + Gly130Glu + Gly153Glu + Tyr205AsnSer129Asp + Ser155Asp + Tyr205Val + Leu216AspSer129Glu + Ser155Glu + Gln161Asn + Ser210GluGly 60Asp + Gly127Asp + Asn162Glu + Phe188AsnGly 62Gln + Ser 98Glu + Ser187Asp + Ser203GluSer104Asp + Gly130Gln + Ser187Asp + Ser203GluAsn 61Asp + Tyr103Ser + Thr212Asp + Thr219AspAsp 59Glu + Gly101Asp + Gly159Pro + Gln161GluGly 60Glu + Leu125Ser + Gly153Glu + Val202AspThr132Glu + Ser157Glu + Thr212Gly + Thr219AspGly101Glu + Leu125Cys + Gly130Glu + Thr219AspGly 60Asp + Pro128Glu + Ser157Asp + Tyr205CysPro128Glu + Asn154Gln + Ser157Asp + Ser203AspSer 97Asp + Ala105Asp + Ser203Asp + Thr212GlnSer100Asp + Ser158Glu + Tyr166Asp + Phe188ProTyr205Thr + Asn211Glu + Ser215Glu + Leu216Asn 表35多环五点突变变异体Tyr103Cys + Gly126Gln + Gly156Glu + Val202Gln +
Thr212GlyVal 94Met + Gly101Asn + Thr102Asn + Tyr205Leu +
Gly218AspSer 98Asp + Tyr103His + Leu125Cys + Gly159Asn +
Met198CysLeu 95Met + Gly101Pro + Tyr103Ala + Gly165Ser +
Leu216AspThr 65Asp + Val 94Thr + Thr132Gly + Asn154Ser +
Thr219GlyVal 94Thr + Thr132Gly + Ala186Gln + Tyr205Cys +
Thr214GlnThr163Gln + Tyr205Cys + Thr212Pro + Leu216Met +
Gly218GlnLeu 95Asp + Gly101Gln + Ile106Gln + Gly126Asn +
Val202AlaGly 99Glu + Gly126Ser + Tyr166Ala + Asn211Gln +
Leu216AsnGly 60Asn + Gly159Glu + Gln161Ser + Asn162Gln +
Leu216SerAla186His + Val202His + Thr212Pro + Ser215Glu +
Leu216ThrGly 99Gln + Leu125Ile + Gly127Ser + Tyr205Val +
Thr212AspThr 58Gly + Val 94Gly + Tyr166Cys + Ser203Asp +
Tyr205HisVal 94Asp + Gly 99Asn + Gln161Ser + Gly201Ser +
Thr212AsnGly126Asn + Gly127Pro + Gly153Asp + Gly159Pro +
Ile164AlaAsn 61Glu + Thr102Asn + Gly165Gln + Phel88Leu +
Val202HisVal 94Cys + Gly156Ser + Val202Thr + Tyr205Ala +
Leu216AlaTyr103Gln + Gly126Gln + Gly156Pro + Tyr166Asn +
Ser215GluGly 99Asn + Val202Pro + Thr212Asn + Tyr213Gly +
Ser215GluGly101Asp + Leu125Thr + Gln161Asn + Thr212Gly +
Leu216AsnAsn 61Ser + Gly153Gln + Gly156Gln + Val202Gly +
Asn211GlnAsn 61Gln + Ala105Thr + Tyr166Ile + Ala186Asn +
Ser215AspAla105Gln + Leu125Gly + Thr163Ser + Val202Gln +
Asn217AspPro200Ser + Val202Ser + Ser203Glu + Thr207Gly +
Thr214GlyVal 94Asn + Gly130Pro + Tyr205Ser + Ser215Asp +
Thr219ProThr163Asn + Ala199Gln + Tyr205Asn + Thr212Ser +
Ser215GluLeu 95Met + Gly156Asn + Val202Cys + Leu216Gln +
Gly218GluVal 94His + Gly 99Glu + Asn211Ser + Tyr213His +
Leu216MetGly 64Ser + Val 94Gln + Ser157Asp + Tyr205Ser +
Thr207ProIle106Ala + Gly165Asn + Ser190Glu + Ala199Asn +
Tyr205AsnAsp 59Glu + Gly159Ser + Gly165Asn + Phe188Thr +
Asn217GlnGly130Pro + Ser155Glu + Gly159Asn + Tyr205Pro +
Thr212SerTyr103Gln + Ser155Glu + Val202Asn + Thr212Ser +
Thr219AsnGly 60Pro + Val 94Ser + Ser100Glu + Tyr103Leu +
Tyr205SerGly 60Gln + Leu 95Val + Pro128Gly + Ser158Asp +
Tyr205ThrThr132Gln + Asn162Ser + Gly165Asp + Tyr205Gly +
Tyr208CysGly 62Asn + Val 94Thr + Ala105His + Pro128Glu +
Tyr205HisThr 58Pro + Gly156Pro + Val202Pro + Thr212Glu +
Leu216ThrLeu 95Met + Gly 99Gln + Tyr103Cys + Asn154Gln +
Thr212GlnSer 98Glu + Tyr205Asn + Pro209Gln + Tyr213His +
Thr219SerGly 62Ser + Val 94His + Tyr103Gln + Ser187Glu +
Phe188TyrGly 62Gln + Asn 96Ser + Gly165Glu + Val202Gly +
Tyr205ThrGly 60Pro + Ile106Thr + Ser160Glu + Thr214Gln +
Leu216IleSer100Asp + Ala105Thr + Tyr205Ile + Thr214Gly +
Leu216CysLeu 95Pro + Ala105Asn + Phe188Leu + Thr212Pro +
Leu216ValAsp 59Glu + Gly 60Ser + Gly127Pro + Tyr205Thr +
Leu216AlaAla105Asn + Ser131Asp + Thr163Pro + Leu216His +
Gly218AsnLeu 95Cys + Tyr103Met + Ile106Cys + Ala186Asp +
Tyr205MetLeu125Cys + Tyr205His + Leu216Asn + Asn217Ser +
Thr219AsnAsn 96Ser + Gly153Glu + Asn154Gln + Ala186Gln +
Thr212AsnGly101Gln + Ala105Pro + Gly130Asp + Asn154Gln +
Gly156AsnThr 58Gly + Gly159Pro + Thr163Ser + Tyr205Gln +
Thr212AsnTyr205Pro + Thr212Asn + Tyr213Gln + Ser215Asp +
Leu216GlnGly130Pro + Gly153Pro + Gln161Glu + Tyr205Met +
Thr212GlnAla105Gly + Ser157Asp + Tyr166Asn + Val202Gln +
Thr212SerThr 58Gln + Asn 96Ser + Ser203Asp + Thr207Gly +
Leu216SerSer158Glu + Val202Ser + Thr212Asn + Thr214Gln +
Thr219AsnAsn 61Ser + Ser100Glu + Tyr205Leu + Thr212Ser +
Asn217SerGly 60Gln + Gly 99Glu + Thr212Ser + Tyr213Gln +
Leu216ThrLeu125Asn + Ser129Asp + Asn162Ser + Ala186His +
Tyr205GlnGly 64Gln + Leu 95Glu + Ala199Asn + Val204His +
Leu216MetLeu 95His + Gly101Pro + Val204His + Tyr205Asn +
Leu216GlnThr163Ser + Gly165Asn + Pro209Gly + Tyr213His +
Leu216AspGly127Gln + Ser131Asp + Thr132Pro + Tyr205Cys +
Leu216AsnAsn 61Gln + Gly101Pro + Ser190Asp + Tyr205Pro +
Thr214GlyGly126Asn + Pro200Gly + Val204Gly + Tyr205Asp +
Thr212AsnAsn 61Gln + Val 94Gln + Gly126Asp + Tyr205Val +
Thr212GlnGly 60Gln + Ile106Pro + Ser131Glu + Ile164Ala +
Val202ProGly101Ser + Tyr103Glu + Ser104Asp + Gly165Asn +
Pro209GlnThr 58Glu + Asp 59Glu + Tyr166His + Val202Cys +
Thr219AsnGly126Pro + Ala186His + Tyr205Asp + Tyr213Val +
Ser215AspGly 60Asp + Ser 97Glu + Ala105Pro + Leu125Cys +
Gly153ProAsn154Ser + Pro209Ser + Thr214Glu + Ser215Asp +
Leu216GluThr163Pro + Ser203Glu + Val204Thr + Tyr205Cys +
Ser215AspGly 62Ser + Met198Gln + Ser203Glu + Ser215Asp +
Leu216HisGly156Asn + Ser203Asp + Tyr205Met + Ser215Asp +
Gly218ProGly 60Gln + Pro128Ser + Ser203Glu + Ser215Glu +
Leu216ProTyr166Cys - Tyr205Glu + Pro209Asn + Ser215Glu +
Leu216AspTyr103Leu + Leu125Gly + Gly153Glu + Ser190Glu +
Leu216IleGly 99Pro + Ile106Thr + Gly153Asp + Ser155Asp +
Ser190AspThr 65Pro + Leu125His + Ser210Glu + Thr212Asp +
Tyr213AlaGly 64Ser + Thr 65Ser + Gly 99Asn + Pro209Glu +
Asn211GluLeu 95Asn + Gly101Asn + Asn154Asp + Gly156Glu +
Tyr166GlnSer155Glu + Thr163Asp + Tyr205Cys + Thr212Pro +
Leu216ValGly159Pro + Thr163Ser + Ser203Asp + Tyr205Asp +
Thr212SerThr102Gln + Gly126Asp + Gly153Asp + Phe188Thr +
Tyr205HisAsn 61Ser + Val202Glu + Ser203Glu + Tyr205Asp +
Thr212ProThr 58Gln + Thr102Pro + Tyr213Cys + Ser215Glu +
Asn217AspGly 60Asp + Gly 62Glu + Thr 65Glu + Gly126Asn +
Leu216ThrAla105Gly + Gly126Pro + Gly153Glu + Leu216Thr +
Gly218AspTyr103Gln + Ala105Thr + Val202Asp + Tyr205His +
Ser215GluVal202Glu + Tyr205Thr + Pro209Asn + Ser215Asp +
Leu216MetVal 94Asn + Ala105Asn + Ile106Val + Ser203Glu +
Gly218AspAsn 96Ser + Ser203Asp + Tyr205Ile + Thr212Gln +
Gly218AspAsn 61Asp + Gly 62Gln + Thr 65Asp + Gly126Asn +
Gly159ProGlyI53Gln + Ala186Glu + Tyr205Thr + Leu216Thr +
Asn217AspLeu125Asp + Gly126Asp + Ala186Ser + Gly218Glu +
Thr219ProThr 58Glu + Thr 65Glu + Val 94Gln + Gly101Pro +
Gly165GlnGly153Glu + Ser158Glu + Thr163Glu + Gly165Pro +
Leu216MetGly159Asp + Thr163Glu + Tyr166Thr + Ala186His +
Pro200SerLeu125Thr + Gly130Ser + Asn154Asp + Ser157Asp +
Ala186GluAsp 59Glu + Asn 96Glu + Gly101Glu + Pro128Ser +
Thr212AsnAsp 59Glu + Asn 96Asp + Gly101Asp + Tyr166Asn +
Thr219AsnGly 99Glu + Gly101Glu + Gly127Glu + Tyr166Leu +
Asn217GlnAla186Gln + Ser187Asp + Leu216Pro + Gly218Glu +
Thr219GlnThr 58Pro + Leu 95Ile + Gly126Glu + Gly153Glu +
Phe188AspThr 58Glu + Asn 96Glu + Gly 99Asp + Phe188Met +
Gly218GlnGly 62Glu + Asn162Ser + Val202Met + Thr212Asp +
Leu216MetGly 62Pro + Gly 99Gln + Ser104Glu + Ser131Asp +
Tyr213ThrGly 62Glu + Gly130Ser + Tyr205Glu + Ser215Glu +
Leu216AsnGly126Gln + Gly127Glu + Asn154Glu + Tyr213Pro +
Leu216HisSer129Glu + Asn154Glu + Asn162Ser + Gly165Asp +
Leu216CysThr 58Gly + Leu125Asn + Gly156Asp + Gln161Glu +
Tyr205GlyGly126Ser + Gly153Gln + Gly156Glu + Gln161Asp +
Tyr205ProThr163Gln + Val202His + Tyr205Asp + Pro209Gly +
Thr212AspGly 62Gln + Val202Met + Asn211Asp + Thr212Asn +
Thr214GluThr102Gln + Gly126Pro + Thr212Asp + Ser215Glu +
Leu216AsnGly101Ser + Gln161Asn + Thr212Glu + Ser215Asp +
Leu216GlnLeu125Gln + Ala186Glu + Val202Cys + Ser203Asp +
Ser215AspSer129Glu + Ser155Glu + Thr163Asp + Ala186Ser +
Tyr208SerThr 58Ash + Leu125Glu + Pro128Glu + Thr163Ser +
Gly165ProAsn 61Glu + Gly 62Asp + Gly130Ser + Asn211Asp +
Thr212GlyThr 65Gln + Asn154Asp + Ser215Glu + Leu216His +
Asn217GluAsp 59Glu + Ser 97Glu + Thr102Gln + Ser210Asp +
Leu216ThrGly153Glu + Ile164Thr + Val202Gln + Ser203Glu +
Asn217GluVal 94His + Leu125Cys + Gly126Ser + Ser155Glu +
Gln161GluSer187Glu + Tyr205Thr + Thr212Asn + Tyr213Gly +
Asn217GluGly 99Ser + Gly130Glu + Asn162Asp + Thr163Asp +
Tyr205AsnPro128Gln + Gly156Asp + Ala186Glu + Ser203Glu +
Tyr213GlnGly 62Asp + Gly156Asn + Ala186Pro + Thr212Glu +
Leu216AspGly126Ser + Tyr166Gly + Ser203Asp + Pro209Asp +
Ser215AspTyr103Val + Pro128Asp + Gly153Asn + Asn162Ser +
Ser190AspGly130Pro + Gly156Asn + Gln161Asp + Thr163Glu +
Ser187GluLeu125Pro + Ser203Asp + Pro209Gln + Thr212Glu +
Leu216AspLeu 95Asp + Gly126Glu + Pro128Gly + Gly159Gln +
Ala186HisGly 62Ser + Ser190Asp + Thr214Gly + Ser215Asp +
Gly218AspGly 64Asn + Tyr103Ile + Leu125Asp + Ser203Asp +
Asn217GluGly126Ser + Gln161Asp + Asn162Ser + Thr214Glu +
Ser215GluIle106Leu + Gly126Asp + Gly127Glu + Gly130Gln +
Val202GluAsp 59Glu + Thr102Ser + Ala105Gly + Gly159Glu +
Ser160GluLeu 95Asp + Asn 96Asp + Tyr166Leu + Val204Ala +
Gly218GluGly130Glu + Ser131Asp + Gly153Gln + Ser158Glu +
Ala199ProGly 60Glu + Asn 61Asp + Gln161Asn + Pro209Gln +
Ser215GluThr 65Asn + Gly 99Gln + Asn154Asp + Pro209Asp +
Ser210GluAsn154Glu + Ser155Asp + Tyr213Thr + Ser215Glu +
Gly218ProSer100Asp + Asn154Glu + Ser155Asp + Pro209Ser +
Thr214GlyGly 62Asn + Gly153Asp + Asn211Glu + Thr212Asp +
Gly218GlnTyr103His + Ser129Glu + Tyr205Pro + Asn211Asp +
Thr212GluSer129Asp + Asn162Gln + Tyr205Leu + Asn211Asp +
Thr212AspSer100Glu + Gly153Pro + Thr163Pro + Val202Glu +
Ser203GluThr 58Asp + Thr 65Pro + Val202Glu + Ser203Asp +
Leu216AsnThr 65Pro + Gly126Asn + Ser157Glu + Ser158Asp +
Ser215AspGly101Asn + Ser157Asp + Ser158Glu + Ser215Glu +
Leu216IleAsn 96Glu + Ser 97Glu + Gly159Asn + Phe188His +
Ser210GluThr 65Asn + Ser 98Asp + Gly159Asn + Ser215Asp +
Leu216AspAsn 96Glu + Pro128Ser + Tyr205Cys + Ser215Glu +
Leu216GluIle106Gly + Ser155Glu + Thr212Asn + Ser215Glu +
Leu216AspLeu125Glu + Thr163Asn + Val202Thr + Ser215Asp +
Leu216GluGly101Gln + Gly130Glu + Tyr205Ala + Ser215Glu +
Leu216GluPro128Gly + Ser210Asp + Thr212Ser + Ser215Glu +
Leu216GluSer104Asp + Ala105Glu + Met198Pro + Ser203Glu +
Leu216SerSer 97Glu + Ser 98Glu + Ala105Gly + Gly153Asp +
Asn217GlnGly153Gln + Ser210Asp + Asn211Asp + Thr212Ser +
Ser215AspThr 65Glu + Leu125Cys + Gly126Ser + Val202Glu +
Ser215AspThr102Gln + Gly165Asn + Ser203Glu + Tyr205Glu +
Pro209AspLeu 95Glu + Asn 96Gln + Tyr205Glu + Pro209Ser +
Ser215GluGly126Asn + Tyr205Asp + Ser210Asp + Ser215Glu +
Leu216MetGly 62Gln + Leu 95Pro + Ser158Asp + Tyr205Asp +
Ser215GluAsn 61Glu + Gly101Glu + Gly126Ser + Leu216Met +
Thr219SerGly156Asp + Ala186Gln + Ser190Glu + Ser203Glu +
Thr212ProGly 60Glu + Ser 97Glu + Gly 99Asn + Asn162Glu +
Asn217SerThr 58Pro + Ser160Asp + Ser190Asp + Val202Asn +
Thr212AsnGln161Asn + Ser203Asp + Asn211Glu + Tyr213Cys +
Ser215GluPro128Glu + Gly165Gln + Ala186His + Ser203Glu +
Ser215GluGly101Pro + Thr132Ser + Ser158Glu + Ser203Glu +
Ser215GluPro128Ser + Ala186Gly + Ser203Glu + Ser210Glu +
Ser215AspAsn 61Gln + Ser158Asp + Ala186Ser + Ser203Asp +
Ser215GluLeu 95Val + Ser100Asp + Tyr166Met + Ser203Glu +
Ser215AspGly153Glu + Ala186Gln + Ser203Glu + Ser215Asp +
Thr219GlySer160Asp + Ser203Glu + Val204Gln + Thr212Pro +
Ser215GluThr 58Pro + Leu 95His + Ser155Asp + Ser203Asp +
Ser215AspAsn154Gln + Ser157Asp + Ser203Glu + Thr212Ser +
Ser215GluLeu 95Gln + Ser100Asp + Ser203Glu + Tyr205His +
Ser215GluAsn154Asp + Tyr166His + Ser203Asp + Tyr205Ala +
Ser215GluSer 98Asp + Ser203Glu + Asn211Ser + Ser215Asp +
Leu216SerAsn162Glu + Ser203Asp + Tyr208Asn + Thr212Ser +
Ser215AspGln161Asp + Thr163Gln + Ser203Glu + Ser215Glu +
Leu216ThrLeu 95Cys + Ser129Glu + Ser203Asp + Asn211Gln +
Ser215AspThr132Asn + Asn154Glu + Ser203Asp + Thr212Asn +
Ser215AspPro128Gln + Ser131Asp + Gly156Asn + Ser203Glu +
Ser215AspThr 58Glu + Asn 61Ser + Gly 99Pro + Ser203Glu +
Ser215AspSer100Asp + Gly153Ser + Ser203Asp + Tyr205Val +
Ser215GluAla105Glu + Ser203Asp + Thr212Asn + Ser215Glu +
Gly218AsnThr102Asn + Ser104Asp + Gly127Asn + Ser203Asp +
Ser215AspGly 62Asp + Leu125Thr + Gly153Glu + Gly165Ser +
Ser190GluGly153Glu + Phe188His + Ser190Glu + Ser203Glu +
Leu216GlyThr 58Asp + Phe188Asn + Ser203Glu + Tyr205Leu +
Asn217AspThr 65Gly + Thr102Ser + Gly126Asp + Thr212Ser +
Gly218AspAsp 59Glu + Thr163Glu + Ser190Glu + Thr212Gln +
Leu216SerTyr166Val + Ser203Glu + Ser210Asp + Thr212Ser +
Leu216AspGly159Asp + Ser203Glu + Thr207Pro + Thr212Asn +
Leu216AspSer157Asp + Asn162Gln + Gly165Gln + Ser203Glu +
Leu216AspLeu 95Thr + Ser131Glu + Val202Ser + Ser203Asp +
Leu216GluSer100Asp + Gly127Asn + Ser203Asp + Tyr208Val +
Leu216AspThr102Gly + Ser129Glu + Ser203Asp + Tyr205Ala +
Leu216AspThr 65Gln + Ser 98Asp + Thr102Pro + Gly126Asp +
Gly165AspThr 65Asn + Gly127Glu + Ser129Asp + Tyr205Ile +
Ser215GluGly 60Asn + Ser203Glu + Tyr205Val + Ser210Asp +
Thr212GluVal 94Cys + Ser 98Glu + Pro209Ser + Ser210Asp +
Thr212GluGly 60Gln + Tyr103Asn + Pro128Glu + Ser157Asp +
Gly159AspSer 98Glu + Ser100Asp + Ser203Glu + Thr212Gln +
Leu216ThrGly 60Glu + Thr 65Asp + Thr132Gln + Asn154Ser +
Leu216AspAsn 96Asp + Ser100Asp + Thr102Pro + Tyr205Asp +
Thr212GlnThr102Gly + Phe188His + Gly201Asn + Tyr205Glu +
Gly218AspAsn154Gln + Ser155Asp + Tyr205Gln + Pro209Glu +
Thr212AspTyr103Val + Gly153Asn + Pro209Asp + Thr212Asp +
Gly218GluThr 65Glu + Ile106Met + Leu125His + Tyr205Leu +
Ser215AspLeu125Met + Gly156Asp + Tyr166Ser + Ser187Glu +
Ser215AspThr 58Glu + Gly 60Glu + Ser131Glu + Tyr205Val +
Gly218ProThr 58Asp + Gly 60Asp + Asn 61Ser + Asn162Gln +
Ser203AspSer157Glu + Ser203Asp + Tyr205Asp + Thr212Ser +
Leu216CysThr 58Asn + Thr102Gln + Ser155Glu + Ser157Asp +
Thr212GluAla105Gln + Ser155Asp + Ser157Asp + Ser203Asp +
Thr212GlyThr163Glu + Val202Asp + Tyr205Cys + Leu216Pro +
Gly218GluThr 58Gln + Asn 61Glu + Ile106Thr + Pro209Asp +
Ser215AspGly 62Glu + Ser 97Glu + Asn154Ser + Tyr205Asp +
Tyr213ValLeu125Gln + Gly130Asp + Leu216Glu + Gly218Asp +
Thr219GlyGly126Ser + Ser131Glu + Tyr205Ala + Leu216Asp +
Gly218GluIle106Ala + Ser160Glu + Asn162Glu + Ala186Gly +
Ser203AspAsp 59Glu + Thr 65Asp + Asn162Asp + Leu216Ile +
Asn217SerAsn 96Gln + Thr102Pro + Gly127Glu + Ala186Pro +
Ser190AspGly 99Asn + Leu125Thr + Ser157Glu + Gly165Glu +
Thr212GlnVal 94His + Gly126Asn + Ser157Glu + Gly165Glu +
Tyr205ValAsp 59Glu + Asn 61Ser + Val202Asn + Thr207Pro +
Asn211AspAsp 59Glu + Tyr103Ala + Ser155Glu + Pro209Glu +
Thr212AsnGly101Gln + Gly127Glu + Gly153Ser + Asn154Glu +
Ser158AspSer 97Glu + Thr212Ser + Ser215Asp + Leu216Cys +
Asn217GluGly 99Gln + Ser157Glu + Ser215Asp + Leu216Ser +
Asn217AspSer131Glu + Gly159Asn + Tyr205Met + Ser215Glu +
Asn217GluVal 94Ala + Gly101Asp+ Gly156Gln + Ser215Asp +
Asn217AspAsn 61Asp + Asn 96Glu + Pro209Ser + Ser215Glu +
Leu216AlaAsn 61Glu + Asn 96Glu + Gly127Ser + Ser203Glu +
Thr214SerGly 99Pro + Ser100Asp + Thr102Glu + Ala105Gly +
Asn211GluSer155Glu + Ala186Pro + Tyr205Asp + Leu216Cys +
Gly218GluPhe188Asp + Ser203Asp + Tyr213Ser + Thr214Glu +
Gly218AsnSer129Asp + Thr163Gly + Gly165Glu + Ser203Asp +
Tyr205IleSer104Asp + Ser158Glu + Asn162Glu + Val202Gln +
Leu216ValSer 98Asp + Ser158Asp + Asn162Glu + Leu216Ile +
Thr219GlyPro128Gly + Ser158Asp + Asn162Glu + Val202Gln +
Asn217GluThr102Glu + Ala105Asn + Ser129Asp + Tyr208Thr +
Leu216GlnThr 65Asn + Ser157Asp + Asn162Glu + Ser203Asp +
Tyr205GlyAla105Gly + Ser157Asp + Ser160Glu + Ala186His +
Ser210AspSer 98Glu + Gly153Asp + Val202Ser + Tyr205Asn +
Gly218AspGly 62Asp + Gly 64Asn + Gly 99Pro + Gly127Asp +
Gly153GluThr 58Ser + Val 94Met + Ser104Glu + Gly153Asp +
Tyr166AspAsn 61Asp + Thr 65Gly + Ala186Gln + Ser210Asp +
Thr214AspGly127Asn + Val202Asp + Ser210Glu + Ser215Glu +
Asn217SerGly 62Asn + Gly101Asp + Tyr103Met + Ser104Glu +
Ser203GluGly159Asp + Asn162Glu + Ile164Ala + Thr212Asn +
Ser215AspSer 98Asp + Gly101Glu + Asn154Gln + Tyr205Gln +
Ser210AspLeu 95Glu + Tyr166Asn + Phe188Met + Ser203Glu +
Thr214GluGly101Pro + Ser129Glu + Ser203Asp + Thr214Glu +
Gly218GlnAsn 96Gln + Asn162Glu + Gly165Glu + Val202Asp +
Tyr205MetThr 65Gln + Val 94Asp + Ser104Glu + Leu125Asn +
Thr212AspThr102Pro + Ser155Asp + Ser187Glu + Tyr205Val +
Ser215GluAsn 96Glu + Thr102Gln + Ala186Asp + Phe188Gln +
Ser190AspThr 58Glu + Asn 61Glu + Gly 64Asn + Ala105Pro +
Asn217GluVal 94Ala + Gly126Asp + Asn154Glu + Tyr166Pro +
Thr212GluThr 58Asn + Asn 61Ser + Gly130Glu + Phe188Glu +
Asn217AspThr 58Asp + Ser 97Asp + Ile106Leu + Gly153Gln +
Gly156AspThr 58Asp + Ser 97Glu + Thr102Pro + Ala105Ser +
Ser158AspTyr103Glu + Gly127Glu + Asn154Gln + Ser157Asp +
Thr163SerAsn 96Gln + Pro128Glu + Gly130Gln + Ser131Glu +
Ser157AspGly127Glu + Gly153Asn + Pro209Asp + Thr214Glu +
Thr219SerSer100Asp + Ile106Met + Pro128Asn + Ser190Asp +
Gly218GluThr 58Asp + Asn 96Asp + Gly127Asp + Tyr166Ala +
Ala186SerAsn 96Glu + Thr163Pro + Phe188Asp + Gly201Gln +
Val202GluSer 98Glu + Gly127Asp + Ser131Glu + Val202Ala +
Thr214SerGly 99Asn + Gly127Glu + Ser131Glu + Gly156Gln +
Asn211GluVal 94Asp + Pro128Asp + Ser131Asp + Tyr205Thr +
Leu216SerGly101Glu + Gly126Ser + Gly127Asp + Tyr205His +
Ser215AspSer160Asp + Tyr205Glu + Asn211Gln + Thr212Asp +
Thr219GlyGly 62Asp + Gly127Gln + Gly153Pro + Ser187Glu +
Leu216GluGly 60Asp + Gly127Asp + Gly159Pro + Ser210Asp +
Gly218AsnAsn 61Ser + Gly 99Asp + Thr102Asp + Ser203Asp +
Thr212GlnGly126Pro + Ser160Asp + Tyr205Met + Thr212Glu +
Ser215AspThr 65Ser + Gly127Asp + Thr132Ser + Thr212Asp +
Ser215AspAsn154Ser + Ser187Glu + Thr212Glu + Tyr213Gln +
Ser215GluSer129Asp + Gly153Glu + Ser158Glu + Gly165Asn +
Leu216ThrIle106His + Leu125Pro + Ser129Glu + Ala186Glu +
Ser203GluGly 60Pro + Gly 62Gln + Tyr205Thr + Ser215Glu +
Thr219GluAsp 59Glu + Val 94Pro + Gly130Gln + Ser187Asp +
Thr219AspGly153Ser + Ser155Glu + Ser160Asp + Asn162Ser +
Thr212ProSer155Asp + Gln161Glu + Ala186Thr + Thr214Gly +
Ser215GluSer104Asp + Gly130Asn + Ser187Glu + Thr212Gln +
Asn217GluAla105Asn + Ser203Glu + Thr212Glu + Asn217Ser +
Thr219AspLeu125His + Gly165Gln + Phe188Asp + Ser203Glu +
Tyr205LeuAsn 96Asp + Ser104Asp + Gly201Pro + Val202His +
Leu216SerGly 62Asp + Tyr103Val + Ser190Glu + Asn211Glu +
Thr219SerGly 62Asp + Ser190Glu + Val202Ser + Asn211Glu +
Thr214GlnSer100Asp + Pro128Asp + Tyr205Ile + Thr212Gln +
Leu216HisThr132Gly + Gly156Asn + Gly159Glu + Ser190Asp +
Ser203GluAsn 96Ser + Ser 98Asp + Gly159Asp + Ser190Asp +
Leu216MetVal 94Asp + Ser 98Glu + Thr132Asn + Phe188Cys +
Thr219AspGly101Ser + Tyr103Glu + Gly153Glu + Tyr166His +
Ser187AspGly 99Ser + Ser160Asp + Ser190Glu + Val202Glu +
Tyr205SerAsn 61Asp + Gly 99Gln + Ala186Glu + Thr212Gln +
Leu216AspAla186His + Phe188Gln + Ser190Glu + Pro200Ser +
Asn217AspAsn 61Ser + Thr 65Asp + Ser129Glu + Tyr205Glu +
Tyr213GlyAsn 61Asp + Gly 62Ser + Ser203Asp + Asn211Gln +
Tyr213GluThr102Asn + Gly130Asp + Tyr166Ser + Tyr205Asp +
Gly218AspAsn 61Glu + Asn154Gln + Gln161Asn + Tyr205Asp +
Gly218GluGly 99Glu + Gly127Asp + Thr163Pro + Tyr213Ile +
Asn217GluGly101Gln + Gly126Asp + Ser131Asp + Gly156Asn +
Ser203GluThr102Asp + Gly130Glu + Pro209Asn + Thr212Ser +
Ser215AspThr 58Ser + Ser100Glu + Ser104Glu + Ile164Leu +
Ser215GluAsp 59Glu + Ile106Val + Gly127Glu + Tyr166Asn +
Ser190GluGly 99Ser + Pro128Asp + Gly156Asp + Tyr205Cys +
Asn217AspThr 65Asp + Leu 95Glu + Gly 99Pro + Ser158Asp +
Tyr205GlnSer 97Asp + Ala105His + Ser155Glu + Tyr205Asn +
Pro209GluGly 62Asn + Thr 65Asp + Ser 97Glu + Ser131Glu +
Leu216GlyGly130Pro + Gly153Glu + Asn154Gln + Gln161Asp +
Pro209AsnGly159Ser + Tyr166Glu + Tyr205Asp + Thr212Pro +
Thr219GluGly 62Asp + Leu 95Cys + Ser100Glu + Thr163Pro +
Ser187AspThr 58Gly + Gly159Glu + Ser187Asp + Ser203Glu +
Thr219AsnThr 58Pro + Tyr166Asp + Val202Met + Pro209Asp +
Ser215AspLeu 95Asp + Thr102Pro + Ala105Gly + Gly127Glu +
Leu216GluPro128Glu + Gln161Asp + Phe188Gln + Ser203Glu +
Asn217GlnLeu125Pro + Pro128Glu + Thr132Asn + Gln161Glu +
Leu216AspSer 97Glu + Thr102Asp + Val202Thr + Ser215Glu +
Gly218SerAsp 59Glu + Ser100Asp + Phe188Gln + Thr212Ser +
Ser215GluGly101Gln + Thr102Asn + Gly130Glu + Thr163Glu +
Ser210AspAsn 61Glu + Gly101Asn + Ser129Glu + Tyr205Thr +
Leu216GluLeu 95Glu + Ile164Val + Thr212Glu + Tyr213Gly +
Leu216AspThr 58Gln + Gly126Glu + Gly156Gln + Thr212Asp +
Leu216AspLeu125Met + Gly126Ser + Ser131Asp + Thr212Asp +
Leu216AspGly 60Asn + Asn 96Gln + Ser104Glu + Thr212Asp +
Leu216GluThr 65Asp + Gly126Gln + Ser158Asp + Gly165Asp +
Leu216ValSer158Asp + Gly165Glu + Ser203Asp + Leu216Ser +
Gly218GlnGly101Asp + Leu125Gly + Pro128Glu + Ala186Gly +
Thr219GlnThr 65Glu + Asn 96Glu + Tyr103Val + Ser155Glu +
Met198AlaThr 65Asp + Asn 96Glu + Leu125His + Asn162Glu +
Tyr166HisAsn 61Ser + Ser100Asp + Phe188Asp + Ser203Glu +
Gly218SerSer104Glu + Gly156Gln + Phe188Asp + Ser203Glu +
Thr212GlyGly 99Asp - Tyr205Asp + Pro209Asp + Thr212Pro +
Tyr213MetThr102Asp + Asn154Ser + Tyr166Glu + Ser203Asp +
Gly218SerGly101Glu + Ser129Asp + Ser157Asp + Thr214Ser +
Leu216AsnAsp 59Glu + Gly101Asp + Gly127Ser + Gly159Pro +
Gln161GluGly 60Asn + Gly127Asp + Thr132Asp + Asn162Gln +
Ser203GluThr 58Asn + Gly 60Asp + Gly130Glu + Val202Ser +
Thr212GluSer100Asp + Tyr166Asp + Ala186His + Tyr205His +
Leu216ThrSer 97Asp + Gly101Ser + Ala105Glu + Tyr205Met +
Ser215GluSer 97Glu + Ala105Glu + Leu125Ala + Ser158Asp +
Tyr205CysSer100Glu + Ile106His + Gly127Ser + Ser158Glu +
Tyr166GluThr102Asn + Gly130Ser + Ser131Asp + Asn211Asp +
Ser215GluGly127Gln + Ser131Glu + Asn211Asp + Tyr213His +
Ser215GluSer129Glu + Ser158Glu + Val202Gly + Ser215Asp +
Leu216GlnLeu125pro + Pro128Ser + Ser157Glu + Ser203Glu +
Gly218SerThr 65Gln + Gln161Glu + Ser210Glu + Thr212Gln +
Ser215GluVal 94Met + Gly101Glu + Ala105Asn + Ala186His +
Thr212Asp表36多环六点突变变异体Asn 61Ser + Gly 64Gln + Gly101Asp + Thr132Gly +Gly165Gln + Tyr205ProAsn 96Ser + Gly101Ser + Ala105Gly + Asn154Glu +Tyr166Thr + Tyr205HisVal 94Ala + Leu 95Thr + Gly126Gln + Pro209Ser +Ser215Asp + Leu216GlyThr 65Gln + Leu125Asn + Asn162Asp + Tyr205Gln +Pro209Gly + Leu216MetThr163Gln + Tyr205Cys + Thr212Pro + Leu216Met +Asn217Gln + Gly218GlnGly 60Gln + Val 94Cys + Asn 96Ser + Gly127Pro +Gly159Asp + Asn162SerGly 99Ser - Gly126Asn + Gly127Pro + Gly153Asp +3Ly159Prp - Ile164AlaTal-94Cys - Pro126Asp - Gly156Ser + Val202Thr +Tyr205Ala + Leu216AlaAsn 61Gln - Ala105Thr + Gly153Gln + Gly156Gln +Tyr166Ile + Asn211GlnGly 60Asn + Ser104Asp + Ala105Asn + Gln161Ser +Thr163Ser + Tyr205AlaGly 64Ser + Val 94Asn + Gly101Asn + Leu125Asn +Gly130Asp + Leu216CysGly159Pro + Gly165Pro + Ala186Thr + Gly201Pro +Thr212Pro + Leu216SerGly 99Asn + Gly159Pro + Ser203Glu + Tyr205Leu +Asn211Gln + Thr212SerThr 58Gln + Leu125Pro + Thr132Asp + Gly153Asn +Tyr213Ala + Leu216GlyIle106Ala + Asn154Glu + Asn162Gln + Met198Thr +Ala199Asn + Tyr205AsnAsp 59Glu + Gly159Ser + Gly165Asn + Phe198Thr +Pro200Gln + Asn217GlnGly 99Ser + Gly126Asn + Thr132Gly + Ser187Asp +Val202Ser + Tyr205HisTyr103Gln + Gly130Pro + Ser155Glu + Gly159Asn +Tyr205Pro + Thr212SerGly 60Pro + Val 94Ser + Ser100Glu + Tyr103Leu +Tyr205Ser + Thr219ProThr 58Gly + Leu125Ala + Val202Cys + Tyr205Asn +Ser215Asp + Leu216AlaVal 94Thr + Ser104Glu + Gly130Pro + Thr212Pro +Leu216Asn + Gly218SerSer 97Glu + Thr132Gly + Pro200Gly + Tyr205Ala +Asn211Ser + Tyr213GlnGly 62Asn + Leu 95Asp + Tyr166Val + Tyr205Cys +Asn211Ser + Leu216ValGly156Asn + Gly165Pro + Gly201Asn + Tyr213Ala +Thr214Gln + Asn217GluGIy 62Ser + Ser 98Asp + Gly126Ser + Tyr205Pro +Thr212Ser + Tyr213ThrAla105Gln + Gly127Ser + Gln161Asp + Thr163Gly +Tyr205Cys + Leu216ProVal 94Ala + Gly130Gln + Ala186His + Ser203Asp +Tyr205Cys + Thr214GlnThr 65Gln + Leu125Gly + Ser187Asp + Phe188Gln +Val202Asn + Thr212AsnThr 58Ser + Ile106Pro + Thr132Gly + Gln161Asn +Tyr205Asn + Thr212AspSer100Asp + Ala105Thr + Leu125Pro + Tyr205Ile +Thr214Gly + Leu216CysThr102Ser + Gly127Pro + Pro128Gly + Ser203Glu +Tyr208His + Tyr213ThrThr102Ser + Gln161Glu + Asn162Asp + Thr163Gln +Ala186His + Phe188IleGly 62Pro + Pro128Asp + Ser129Asp + Gly156Asn + Tyr213Cys + Gly218AsnThr 58Gln + Gly127Pro + Ala186Gln + Val202Asp +Ser203Asp + Leu216ValGly156Ser + Phe188Gly + Ser203Glu + Tyr213Gln +Ser215Asp + Leu216AspGly101Gln + Ala105His + Asn162Gln + Ala186Gly +Ser203Glu + Ser215GluThr 58Pro + Thr163Asn + Ser203Glu + Tyr205Ile +Ser215Glu + Leu216GlnThr 65Asn + Gly127Glu + Gly156Asn + Tyr166Glu +Phe188Met + Thr212ProLeu 95Asp + Ser100Glu + Gly101Gln + Ile106Gln +Ile164Leu + Val202AlaTyr103Pro + Gly127Glu + Ser129Asp + Gly130Glu +Asn162Gln + Leu216HisGly 60Gln + Gly165Ser + Ser203Glu + Tyr205Asp +Thr214Glu + Ser215AspLeu 95Asn + Leu125Pro + Ser203Asp + Tyr205Thr +Thr214Glu + Ser215AspGly 62Asn + Tyr103Ile + Leu125Cys + Gly126Glu +Gly153Asp + Tyr166CysGly 99Ser + Pro128Glu + Gly130Asp + Phe188Asn +Leu216Gly + Gly218ProAsp 59Glu + Gly 60Asp + Thr 65Asp + Asn162Ser +Thr163Gln + Tyr166GlnPhe188Glu + Tyr205Thr + Thr212Asn + Tyr213Gly +Asn217Glu + Gly218AspGly 62Gln + Ser203Glu + Tyr205Ser + Thr212Ser +Ser215Asp + Gly218AspSer187Glu + Ser190Asp + Tyr205Val + Pro209Ser +Thr214Ser + Thr219SerThr 58Ser + Asp 59Glu + Asn 96Asp + Asn154Ser +Ala186His +Leu216SerThr 65Gly + Thr102Gly + Leu125Asp + Gly165Asp +Ala199Ser + Val202ThrLeu125Asn + Ser158Glu + Gly159Asp + Ser190Glu +Thr212Gly + Leu216ValThr 58Pro + Asn 61Asp + Val 94Glu + Asn 96Asp +Gly126Ser +Gly165ProAla186Glu + Val202Glu + Tyr205Ala + Tyr213Val +Ser215Asp + Asn217GluTyr103Val + Ser203Glu + Tyr205Asp + Thr212Glu +Ser215Asp + Leu216IleGly 62Ser + Ile106Leu + Pro128Ser + Asn154Asp +Ser157Glu + Thr214SerVal 94Asp + Gly 99Asp + Pro128Gln + Ala199Gln +Tyr205Gln + Leu216IleAsn 61Gln + Thr102Gln + Gly126Asp + Ser155Glu +Ser190Asp + Val202SerLeu125Asp + Gly126Asp + Ala186Ser + Tyr205Gln +Gly218Glu + Thr219ProAsn 61Glu + Ser100Asp + Thr102Asn + Gly165Gln +Phe188Leu + Val202HisTyr103Gly + Tyr205Leu + Thr212Glu + Ser215Glu +Leu216Glu + Asn217AspGly 99Pro + Ser104Glu + Ile106Thr + Ser131Asp +Gly201Gln - Thr214SerLeu 95Ile + Gly126Glu + Gly153Glu + Ser187Asp +Phe166Asp - Asn211GlnGly 62Asn + Leu 95Ser + Gly153Ser + Tyr205Asp +Thr212Glu + Leu216AspAla105Gly + Ile106Cys + Ser155Asp + Tyr205Asp +Ser215Asp + Leu216AspTyr103Ala + Ser155Glu + Tyr166Pro + Ser203Glu +Tyr205Glu + Ser215GluPro128Gly + Asn162Glu + Gly165Asn + Ser203Glu +Tyr205Glu + Ser215GluGly 99Pro + Leu125Gln + Gly130Asn + Ala186Asp +Phe188Ser - Ser203GluIle106Asn + Gly156Glu + Gly159Asn + Phe188Asp +Ser215Glu + Gly218AspGly156Pro + Asn162Asp + Ser203Glu + Tyr205Gln +Ser215Asp + Asn217GluGly153Asn + Ser155Glu + Ser157Asp + Ser190Glu +Thr212Asp + Leu216IleLeu 95Glu + Gly 99Glu + Leu125Glu + Pro128Gly +Asn154Glu + Thr219GlyGly 60Asp + Ile106Met + Asn162Ser + Ser203Glu +Tyr205Asp + Leu216AspSer 97Asp + Asn162Ser + Thr163Gln + Ser203Glu +Tyr205Asp + Leu216AspLeu125Ile + Gly159Glu + Ser203Glu + Thr214Glu +Ser215Glu + Leu216GlnGly156Ser + Ser158Glu + Tyr205Asp + Tyr213Glu +Ser215Asp + Leu216CysGly 62Gln + Asn154Asp + Ser155Asp + Ser160Asp +Gly165Glu + Tyr205MetGly 64Pro + Ala186Glu + Ser187Asp + Ser203Glu +Tyr205His + Ser215GluTyr103Asp + Tyr166Ser + Ala186Asn + Val202Glu +Ser203Asp + Tyr205GluLeu 95Gln + Gly126Gln + Gly127Asp + Pro128Ser +Ser155Asp + Ser158GluAsp 59Glu + Ile106His + Tyr205Ala + Tyr213Glu +Ser215Glu + Leu216AspSer158Asp + Ser160Glu + Thr163Asp + Pro209Gln +Thr212Ser + Ser215GluGly 62Asp + Tyr103Met + Val202Ser + Asn211Glu +Thr214Gln + Leu216AlaAsp 59Glu + Ser 97Asp + Ser 98Asp + Gly153Ser +Gln161Asn + Ser203GluLeu125Asn + Gly153Glu + Ser157Asp + Thr163Glu +Val202Asp + Thr214ProLeu 95Glu + Ser100Asp + Leu125Asp + Pro128Asn +Ser131Asp + Tyr205GlyGly126Gln + Ser158Glu + Ser203Asp + Ser215Glu + Leu216Thr + Gly218AspGly101Ser + Pro128Asp + Ser158Glu + Gly159Asp +Ala166Gly + Phe188ValSer167Glu + Ser203Asp + Tyr205Cys + Pro209Gln +Ser215Asp + Leu216SerGly 62Ser + Ser187Glu + Met198Gln + Ser203Glu +Ser215Asp + Leu216HisThr132Asn + Gly165Pro + Ser187Glu + Ser203Glu +Tyr205His + Leu216AspAsn 96Ser + Ser131Glu + Gly159Asp + Ser160Asp +Thr214Gln + Leu216GlyGly130Glu + Ser158Asp + Gly159Glu + Thr163Gln +Tyr205Thr + Thr207AsnGly130Asp + Ser131Glu + Gly165Pro + Ser190Glu +Val202Gly + Asn217SerLeu 95Thr + Gly130Glu + Ser131Glu + Tyr205Ala +Ser210Glu + Gly218GlnThr 58Gly + Ala105Glu + Ile106Gly + Tyr205Val +Ser215Glu + Leu216AspThr 58Asp + Asp 59Glu + Leu125Asn + Ser203Glu +Tyr205Cys + Thr212SerThr 58Glu + Asp 59Glu + Tyr103Asn + Pro209Gln +Thr212Asn + Ser215GluThr 65Ser + Ser 97Asp + Ser 98Glu + Ser104Glu +Thr163Gln + Leu216SerSer 97Glu + Ser 98Glu + Gly101Gln + Thr212Asn +Leu216His + Gly218GluGly101Asp + Thr102Glu + Ile106Val + Thr163Pro +Tyr205Glu + Ser215AspGly 60Pro + Ser129Glu + Val202Glu + Thr214Asp +Ser215Asp + Leu216IleGly 62Asp + Gly 99Glu + Tyr205Asn + Asn211Asp +Tyr213Met + Thr214AspThr 58Gly + Gly127Glu + Ser131Glu + Ser155Asp +Gly165Pro + Tyr205CysGly159Asp + Tyr166Gly + Tyr205Asp + Tyr213Asn +Ser215Asp + Leu216ThrGly 62Glu + Gly101Ser + Thr132Gly + Ser203Glu +Asn211Asp + Tyr213GluAsn 96Asp + Ser 97Asp + Leu125Pro + Gly127Gln +Ser203Asp + Ser215AspAsp 59Glu + Val 94Glu + Ser 98Asp + Ser104Glu +Leu125Asn + Tyr213GlySer155Asp + Gly156Gln + Ser190Asp + Tyr205Glu +Thr212Ser + Leu216AlaVal 94Met + Gly 99Pro + Ile106Thr + Ser155Asp +Ser190Asp + Thr212GluGln161Ser + Thr163Glu + Val202Ala + Ser203Glu +Asn211Ser + Ser215AspGly156Glu + Ala186Asn + Ser203Asp + Thr212Gln +Ser215Glu + Leu216GlnSer 98Glu + Ser203Asp + Val204Cys + Ser215Asp +Asn217Ser + Thr219GlnAsn 61Gln + Thr 65Ser + Ser131Asp + Ser203Asp +Ser215Glu + Leu216CysIle106Val + Thr132Ser + Ser158Glu + Ser203Glu +Thr214Ser + Ser215GluVal 94Thr + Gly153Glu + Ala186Gln + Ser203Glu +Ser215Asp + Thr219GlyVal 94Met + Thr102Asp + Pro128Glu + Ser131Asp +Gly165Asn + Thr219SerSer155Asp + Gln161Asp + Serl90Glu + Ser203Glu +Thr212Ser + Leu216CysAsn 61Glu + Ser100Asp + Thr132Pro + Ala186Gly +Ser210Glu + Thr212GluAla105Glu + Thr132Ser + Gly201Ser + Ser203Glu +Thr212Pro + Asn217AspThr 58Gly + Gly 62Pro + Ala105Asp + Ser203Asp +Tyr208Ile + Leu216GluAsn 96Asp + Gly 99Asp + Ile106Gly + Asn154Gln +Gln161Asn + Thr214GluAsn 96Glu + Gly 99Glu + Thr102Asn + Gly127Ser +Ser203Glu + Asn217GlnAsp 59Glu + Gly127Asp + Ser129Asp + Tyr213His +Leu216Val + Thr219GlyThr 65Gln + Tyr205Ser + Pro209Gln + Ser210Glu +Thr212Glu + Thr219AspThr 58Asp + Asn 96Ser + Ser158Glu + Gln161Asp +Gly201Gln + Tyr205ValAsp 59Glu + Asn 61Glu + Tyr166His + Phe188Val +Thr212Gly + Ser215AspThr102Glu + Gly126Pro + Gly127Asn + Val202Asn +Pro209Asp + Asn211GluAsn 61Gln + Gly159Gln + Ser187Asp + Pro209Asp +Asn211Asp + Gly218SerSer 97Asp + Ser 98Glu + Gly156Asp + Ser158Glu +Thr163Gln + Pro209GlnGly 60Gln + Gly130Asn + Ser155Glu + Phe188Glu +Tyr205Asn + Ser215GluVal 94Asp + Ile106Glu + Pro128Asp + Ser131Asp +Tyr205Thr + Leu216SerGly 60Pro + Ser 98Asp + Ser100Glu + Pro128Gly +Ser157Asp + Gly159AspThr 58Asn + Gly 99Asp + Thr102Gln + Pro209Asp +Thr212Glu + Tyr213MetThr 65Glu + Tyr103Gln + Gly156Pro + Tyr166Asn +Phe188Tyr + Ser215GluVal 94Asp + Gly101Glu + Ile164Gly + Ala186Pro +Ser203Asp + Leu216ProGly 60Asn + Asn 61Glu + Gly153Ser + Pro209Asp +Asn211Asp + Ser215GluThr132Gly + Ser157Glu + Ser187Asp + Ser203Asp +Tyr205Met + Ser215GluAsn 61Asp + Asn 96Glu + Gly156Asn + Ser203Glu +Thr212Gln + Ser215GluGly 99Pro + Ser158Glu + Ser160Glu + Tyr205His + Thr212Ser + Ser215GluGly101Pro + Ser157Glu + Ser203Asp + Tyr205Asp +Thr212Ser + Leu216CysVal 94Ala + Pro128Ser + Gly156Gln + Ser190Asp +Ser203Asp + Tyr205GluIle106Met + Leu125Ile + Ser160Asp + Ser203Asp +Tyr205Glu + Leu216SerLeu 95Glu + Ser 97Glu + Thr102Glu + Gly159Asp +Tyr205Leu + Leu216AlaLeu125Cys + Gly126Ser + Gly159Asp + Gln161Glu +Ser215Glu + Asn217GluSer100Glu + Thr102Gly + Ala105Thr + Ser155Glu +Ser157Asp + Asn162SerSer 98Glu + Gly127Asn + Ser157Asp + Gln161Asn +Asn162Asp + Gly165GluAsn 61Gln + Ser100Asp + Ser104Glu + Ile106Asp +Ser215Asp + Leu216AlaSerl31Glu + Gly159Asn + Val202Cys + Ser215Asp +Leu216Asp + Thr219GluLeu125Cys + Gln161Asp + Asn162Glu + Ser203Glu +Leu216Thr + Gly218GluGly101Asp + Ala105Asp + Gly126Asn + Gln161Ser +Ala186Pro + Thr214GluSer100Glu + Thr102Glu + Tyr103Thr + Gly126Asp +Ser160Glu + Leu216MetGly127Gln + Asn154Ser + Ser157Glu + Thr163Glu +Ser215Glu + Asn217GluLeu 95Ile + Asn 96Glu + Thr102Gly + Tyr103Glu +Gly126Glu + Gly165GluSer129Asp + Ser131Asp + Thr132Asn + Gly159Ser +Tyr213Gln + Thr214GluGly101Pro + Gly126Glu + Ser129Asp + Tyr205Glu +Ser215Glu + Leu216GlyLeu 95Met + Pro128Gly + Ser129Glu + Gly153Glu +Thr163Gly + Leu216SerGly 60Glu + Val202Met + Tyr205Ile + Thr212Glu +Ser215Glu + Leu216AspThr 58Ser + Ser158Asp + Gln161Asn + Thr163Asp +Leu216Asp + Gly218AspIle106Ser + Ser158Asp + Ser187Asp + Ser190Asp +Tyr205Met + Ser215AspSer187Glu + Val202Gln + Tyr205Asn + Thr214Glu +Leu216Glu + Gly218SerAsp 59Glu + Ser100Asp + Gly126Pro + Gly127Asp +Tyr166Glu + Thr212AsnAsp 59Glu + Gly 62Pro + Gly127Asn + Ala186Pro +Val202Glu + Pro209AspAsp 59Glu + Asn 96Glu + Gly101Glu + Thr102Gln +Pro128Ser + Leu216GluGly159Asp + Asn162Asp + Ala186Gln + Val202Gln +Ser203Glu + Ser215AspGly159Ser + Ala186Asn + Ser210Asp + Tyr213Pro +Ser215Asp + Asn217AspSerl04Glu + Ser131Asp + Thr132Gln + Asn162Ser +Gly165Asp + Tyr208CysGly156Glu + Ser158Asp + Thr163Gly + Ser203Asp +Thr212Asn + Leu216SerGly 64Pro + Thr 65Glu + Ala105Asp + Val202Thr +Ser215Asp + Leu216GluThr 58Pro + Gly 99Ser + Ser100Asp + Gly101Ser +Thr102Asp + Asn211GluGly 62Glu + Asn 96Ser + Ser100Asp + Gly101Ser +Ser210Asp + Tyr213AlaGly 60Pro + Gln161Asp + Gly165Asp + Phe188Val +Ser190Glu + Ser210GluGly153Gln + Ser155Asp + Gly159Asn + Pro209Glu +Thr212Asp + Ser215AspThr 58Gln + Thr102Glu + Ser129Glu + Gly130Asp +Tyr205Ala + Ser215AspTyr103Ser + Asn154Asp + Ser187Glu + Val202Asn +Ser203Glu + Leu216SerLeu125Pro + Pro128Ser + Ser157Glu + Gln161Glu +Ser203Glu + Gly218SerSer 97Glu + Gly 99Asp + Ala105Ser + Leu125Asp +Gly165Gln + Tyr205AspThr 65Asp + Leu 95Cys + Ser 97Asp + Asn162Ser +Thr212Glu + Ser215AspSer 97Glu + Ser100Glu + Gly156Asn + Ser157Glu +Ser160Glu + Tyr205ProLeu 95Asp + Ala105Asp + Ser157Glu + Val202His +Tyr205Ile +Leu216MetLeu 95His + Gly156Pro + Ser157Asp + Asn162Glu +Ser187Glu + Leu216AspSer100Glu + Leu125Gly + Gly127Glu + Ser203Asp +Tyr208Val + Leu216AspAsn154Ser + Gly165Asp + Ser190Glu + Thr212Gly +Ser215Asp + Gly218ProThr 58Gln + Ser 98Glu + Gly153Asp + Val202Ser +Tyr205Asn + Gly218AspAsn 61Gln + Thr132Gly + Gly156Glu + Ser158Glu +Ser215Asp + Thr219GluVal 94Glu + Ser 97Glu + Ala105Asp + Gly165Pro +Leu216Met + Thr219AspVal 94Glu + Ser 97Glu + Ala105Asp + Asn154Gln +Gln161Asp + Ala186AsnGly130Gln + Ser155Glu + Ser158Glu + Ala186Gln +Ser203Asp + Leu216CysAsn 61Gln + Ser160Asp + Ser203Glu + Val204Gln +Thr212Glu + Ser215GluThr102Glu + Ala105Asp + Ser131Glu + Ser203Glu +Tyr205Ala + Thr214AsnThr 58Pro + Ser155Glu + Gly159Asn + Pro209Glu +Thr212Glu + Gly218GluAsp 59Glu + Gly 99Asp + Gly126Ser + Met198Thr +Thr212Asp + Gly218ProThr132Ser + Asn154Ser + Ser157Asp + Val202Glu + Ser215Glu + Leu216GlyVal 94Asp + Gly 99Asn + Ser100Asp + Gly130Ser +Asn154Gln - Ser215AspPro128Glu + Gly156Ser + Ser160Glu + Ser187Asp +Ser190Glu + Thr212SerAsn 61Gln + Ser 98Glu + Ile106Cys + Ser203Asp +Tyr205Gly + Gly218AspAla105His + Ser158Glu + Ser187Glu + Ser203Glu +Tyr205Leu + Ser215GluAsn 96Ser + Gly 99Gln + Gly153Glu + Ala186Glu +Tyr205Glu + Ser215GluAsp 59Glu + Ser 98Glu + Gly153Glu + Ser155Glu +Phe188Pro + Leu216HisAsn162Asp + Gly165Asp + Val204Gln + Tyr205Thr +Ser210Asp + Thr212ProGly101Pro + Gly156Asp + Tyr166Asp + Ser190Glu +Tyr205Asp + Thr212AsnGly126Glu + Thr132Asp + Ser155Asp + Ser157Asp +Val202Gly + Tyr213SerGly 60Asp + Asn 61Asp + Asn154Glu + Ser158Glu +Gly165Gln + Tyr213GlnGly 62Glu + Leu125His + Ser131Asp + Tyr166Leu +Ser203Glu + Ser215AspAsp 59Glu + Val 94Asp + Thr102Gly + Ser203Glu +Tyr205Asp + Thr219GlySer104Glu + Tyr166Cys + Val202Thr + Thr212Asp +Ser215Asp + Asn217GluSer100Asp + Gly126Pro + Asn154Asp + Gly201Ser +Ser203Glu + Thr219AspGly101Asp + Pro128Asp + Ser155Asp + Ser187Asp +Thr207Gly + Thr212GlnIle106Ser + Ser160Glu + Ser190Asp + Ser203Asp +Ser215Glu + Leu216ValGly 99Gln + Gly153Asp + Ser160Asp + Phe188Leu +Ser190Asp + Ser210AspSer158Asp + Gly165Asn + Ser190Glu + Val202His +Tyr205Asp + Tyr213LeuThr 65Asp + Leu 95Thr + Ser131Glu + Val202Ser +Ser203Asp + Leu216GluAsn 61Glu + Gly165Ser + Met198Gln + Val202Glu +Tyr205Glu + Leu216AsnVal 94Ala + Gly126Asp + Asn154Glu + Tyr166Pro +Val202Gln + Thr212GluSer 97Glu + Leu125Glu + Ser155Glu + Gly159Asn +Tyr166Asp + Leu216ValAsn 96Glu + Ser 97Asp + Thr102Gln + Leu125Asp +Leu216Glu + Thr219GlnTyr103Cys + Ser160Glu + Thr163Asp + Tyr208Ser +Leu216Met + Gly218GluVal 94Cys + Leu125Glu + Ser155Asp + Gly156Asp +Gly201Gln + Ser215GluAsn 61Ser + Thr 65Glu + Val 94Asp + Tyr103Pro +Ser215Asp + Asn217AspGly 99Gln + Gly127Asp + Thr132Pro + Ser155Glu +Ser187Glu + Ser203AspSer 98Glu + Ser129Glu + Gly153Glu + Ser190Asp +Tyr213Val + Thr214ProAsn 61Ser + Gly130Glu + Phe188Glu + Tyr205Asp +Thr212Pro + Asn217AspSer158Glu + Gly159Glu + Val202Ser + Tyr205Met +Pro209Glu + Ser215GluAsn 61Asp + Gly 99Asn + Gly101Asp + Ser203Asp +Tyr205Asp + Thr212ProSer 98Asp + Ser100Asp + Ser104Glu + Gly153Glu +Phe188Gly + Gly218AsnLeu 95Val + Gly 99Asp + Tyr103Val + Ala105Asp +Tyr205Asp + Ser215AspAsp 59Glu + Asn 61Asp + Ser160Glu + Pro200Asn +Asn211Ser + Thr212AspGly 62Glu + Thr 65Gly + Ser 97Asp + Thr102Pro +Ser131Glu + Leu216AspGly126Pro + Pro128Glu + Gly156Glu + Ser158Asp +Tyr205Asn + Asn217AspAsp 59Glu + Gly130Asn + Ser155Asp + Ser210Asp +Tyr213Gly + Gly218AsnAsn 96Gln + Gly 99Glu + Ser100Asp + Pro128Asp +Tyr205Cys + Asn217GluPro128Gly + Ser129Asp + Asn162Glu + Gly165Asn +Ser203Asp + Ser215AspAsn 96Ser + Ser100Glu + Tyr103Glu + Tyr205His +Ser215Glu + Leu216ProAsn 96Glu + Ser 98Glu + Ala186Asp + Val202Asn +Tyr213Met + Leu216GluGly 99Asp + Ser158Asp + Asn162Asp + Thr163Asn +Ser187Glu + Ala199GlyLeu 95Ser + Ser 97Asp + Ser 98Glu + Gly165Glu +Leu216Ile + Gly218GluGly101Asp + Leu125Asp + Gln161Asn + Tyr205Asp +Thr212Gly + Leu216AsnSer160Asp + Val202His + Ser203Asp + Thr212Asp +Tyr213Val + Leu216AspLeu 95Val + Tyr103Glu + Ala105Glu + Thr163Ser +Thr212Asp + Leu216GluSer129Asp + Gly130Ser + Ser157Asp + Gly165Asp +Tyr205Glu + Leu216GlnThr132Asn + Gly156Glu + Tyr166Thr + Ser203Glu +Thr212Glu + Asn217GluAsn 61Gln + Ser 97Glu + Ser187Glu + Ser203Glu +Pro209Gln + Ser215GluGly101Gln + Ala105Asp + Ser187Glu + Ser203Asp +Ser215Glu + Leu216HisGly 99Asn + Ser104Asp + Thr132Gly + Ser187Asp +Ser203Glu + Ser215AspGly 62Asp + Asn 96Gln + Ser104Glu + Tyr205Gln +Thr212Asp + Tyr213ValVal 94Gly + Asn 96Asp + Ala105Glu + Asn162Asp + Asn211Gln + Thr212GlnSer 97Glu + Ser187Asp + Ser203Glu + Tyr213Gln +Leu216Glu + Asn217GlnGly 64Pro + Ser104Glu + Gly126Glu + Tyr166Ser +Tyr205Glu + Ser215GluGly130Asn + Thr132Asp + Ser187Glu + Pro209Gln +Thr214Glu + Ser215GluGly126Glu + Ser160Asp + Tyr166Met + Val202Thr +Thr214Asp + Ser215GluSer 98Asp + Gly101Ser + Gly130Asp + Ser158Glu +Gly159Asp + Leu216AlaThr 58Glu + Tyr103Pro + Gly130Glu + Ser131Asp +Ser158Glu + Ala199ProGly 60Asp + Asn 61Asp + Ser131Asp + Gly153Ser +Thr163Glu + Val202CysGly 99Asp + Pro128Asn + Asn162Glu + Thr212Ser +Leu216Glu + Asn217GluGly 60Ser + Ser 98Asp + Asn154Asp + Ser155Glu +Gly159Pro + Asn211GluTyr103Pro + Ser104Glu + Leu125Cys + Val202Glu +Ser203Glu + Pro209GluGly101Gln + Ser104Asp + Leu125Val + Val202Asp +Ser203Asp + Thr212AspThr 65Asn + Ser 98Asp + Gly159Asn + Gly165Asp +Ser215Asp + Leu216AspGly127Glu + Gly165Pro + Tyr205Cys + Ser210Asp +Ser215Glu + Leu216GluVal 94Ser + Ser 98Glu + Gly 99Asp + Gly156Asp +Phe188Leu + Ser203GluGly 62Asp + Gly101Glu + Ser160Glu + Gln161Asp +Tyr205Met + Thr212ProSer 97Asp + Ser 98Glu + Ala105His + Ser157Glu +Thr212Ser + Ser215AspGly 62Gln + Ser 97Glu + Ser 98Asp + Gly126Glu +Gly130Pro + Thr212GluVal 94Glu + Ser 97Asp + Leu125Asn + Thr163Ser +Ser210Asp + Leu216IleLeu 95Glu + Asn 96Gln + Gly159Glu + Tyr205Glu +Pro209Ser + Ser215GluSer129Glu + Asn154Ser + Thr212Glu + Ser215Asp +Gly218Asp + Thr219SerAsp 59Glu + Gly127Gln + Val202Glu + Asn211Glu +Thr212Ser + Ser215AspSer104Asp + Ala105Pro + Ser187Glu + Ser203Glu +Tyr205Asp + Tyr213ProThr 58Pro + Asn 96Asp + Gly127Glu + Ser155Glu +Asn162Ser + Thr212SerThr 58Gly + Leu125Asn + Gly156Asp + Gln161Glu +Tyr205Gly + Ser215AspGly 60Ser + Gly156Asp + Pro209Ser + Thr214Asp +Leu216Thr + Thr219AspLeu125His + Gly127Gln + Ser131Glu + Ser203Asp +Asn211Asp + Ser215GluThr 58Glu + Pro128Glu + Gly165Gln + Ala186His +Ser203Glu + Ser215GluSer131Asp + Ser158Glu + Ser203Asp + Tyr205His +Ser215Asp + Leu216GlnThr 65Asn + Gly130Asp + Ser158Asp + Ser203Asp +Pro209Ser + Ser215GluGly 60Gln + Thr102Glu + Ser157Asp + Ser203Asp +Ser215Asp + Thr219GlnGly 64Gln + Gly127Asp + Ser160Glu + Ser203Asp +Ser215Glu + Gly218GlnIle106Leu + Thr132Asp + Thr163Asp + Ser203Glu +Ser215Asp + Leu216IleGly126Ser + Ser160Asp + Thr212Glu + Ser215Glu +Leu216Val + Thr219SerGly 60Pro + Gly101Asn + Ser158Glu + Tyr205Ile +Thr212Asp + Ser215GluGly 62Glu + Gly 99Ser + Val202Ser + Tyr205Asp +Thr212Asn + Thr214SerVal 94His + Thr132Glu + Gly165Asp + Tyr166Cys +Ser203Asp + Leu216GluGly 62Asn + Ser131Asp + Ser157Glu + Gln161Asn +Ser203Glu + Leu216GluGly 60Pro + Val 94Asp + Ala105Glu + Gly127Pro +Gln161Asp + Ser210AspGly 62Gln + Ser 97Asp + Asn154Asp + Ser190Asp +Ser203Glu + Thr212SerSer 97Glu + Asn154Glu + Gly159Asn + Ser190Asp +Tyr205Thr + Asn211GluThr 65Glu + Ser 98Asp + Thr102Pro + Gly126Asp +Gly165Asp + Gly218GlnAsn 96Glu + Tyr103Asp + Gly159Asp + Asn162Asp +Tyr208Leu + Thr212ProGly 62Asp + Ser104Glu + Ile106Asp + Ser157Asp +Gly218Ser + Thr219ProAsn 96Glu + Gly 99Asp + Ser160Asp + Gly201Ser +Asn211Gln + Ser215GluIle106Pro + Gly153Asp + Gly165Glu + Tyr166Ser +Ser203Asp + Thr212AspSer 97Asp + Gly 99Asn + Ser158Asp + Ser210Glu +Thr212Glu + Leu216ThrAsp 59Glu + Ser 98Asp + Ser160Glu + Gly201Ser +Thr212Gly + Tyr213AsnVal 94Ser + Ser 97Asp + Ser187Glu + Ser190Asp +Pro209Asp + Leu216AsnGly101Gln + Gly126Asp + Ser131Asp + Asn154Glu +Gly156Asn + Ser203AspGly 64Ser + Ser 98Glu + Ser100Glu + Ala186Glu +Thr212Glu +Leu216ProSer 97Asp + Gly 99Asp + Val202Asp + Thr207Asn +Asn211Glu + Leu216MetThr 65Pro + Thr132Gly + Ala186Asp + Val202Ala +Ser203Glu + Thr212AspIle106Asp + Gly127Ser + Asn154Glu + Gly156Glu + Thr214Pro + Ser215GluThr 58Asp + Ser129Glu + Thr132Asn + Gly165Asp +Leu216Val + Gly218AspThr 58Pro + Gly 60Glu + Gly 62Glu + Thr102Asp +Ser158Glu + Leu216AlaPro128Asp + Asn154Glu + Phe188Val + Val202Cys +Ser203Asp + Tyr205AsnGly101Gln + Ala105His + Ser155Asp + Tyr166Val +Ala186Asp + Pro209GluLeu 95Val + Ser155Glu + Tyr166Ala + Ala186Glu +Ser210Asp + Leu216MetAsn 96Asp + Gly101Glu + Asn162Glu + Val202Thr +Ser210Asp + Leu216AsnSer129Glu + Ser157Asp + Phe188Ala + Ser190Glu +Val202Ser + Tyr205GluSer131Asp + Ser158Asp + Ser160Asp + Tyr205Ile +Ser215Asp + Leu216MetGly 62Glu + Leu 95Glu + Tyr103Pro + Ala186His +Ser203Glu + Thr207GlySer100Glu + Leu125Ala + Ser157Glu + Ser203Asp +Tyr205Asp + Leu216CysThr102Gln + Ser104Asp + Gly153Pro + Ser158Glu +Ser203Glu + Tyr205AspAsn 61Glu + Asn 96Gln + Ser 98Asp + Ser158Asp +Thr212Asn + Thr219AspVal 94Thr + Thr132Asp + Ser155Asp + Ser157Glu +Thr207Gly + Ser215AspPro128Gly + Gly130Pro + Ser158Glu + Ser187Glu +Phe188Cys + Ser203AspThr 65Glu + Gly130Pro + Gly159Glu + Gly165Asp +Phe188Met + Pro209AspThr163Glu + Val202Asp + Tyr205Cys + Thr212Asp +Leu216Pro + Gly218GluSer 97Glu + Gly101Asn + Gly130Glu + Asn154Asp +Thr163Gly + Gly218GluThr 65Gly + Ala105Glu + Ser155Asp + Gln161Glu +Phe188Val + Asn217GlnThr 65Gln + Tyr103Asp + Pro128Glu + Ser155Asp +Ser215Asp + Leu216SerThr132Glu + Ala186Gln + Ser187Glu + Val202Pro +Thr207Gly + Asn217AspGly 60Glu + Ser129Glu + Ser131Glu + Gly165Asn +Ser203Glu + Pro209GlnGly101Pro + Pro128Asp + Gly130Asp + Ser203Glu +Tyr205Ile + Thr212AspVal 94Glu + Asn 96Glu + Asn154Asp + Tyr205Ala +Thr212Glu + Leu216CysSer 97Glu + Thr102Ser + Ser157Asp + Ser215Asp +Leu216Ser + Asn217GluSer 98Asp + Gly156Glu + Ser158Glu + Gln161Ser +Ser203Asp + Gly218AsnGly130Ser + Gly156Asp + Ser158Glu + Ser210Asp +Thr212Gln + Ser215AspSer100Glu + Thr102Asp + Gly126Pro + Gly127Ser +Ser187Glu + Ser215GluAsn 96Glu + Gly127Ser + Pro128Gln + Ser190Asp +Tyr213Asp + Ser215GluPro128Glu + Gly153Ser + Ser158Glu + Gly165Ser +Ala186Gly + Ser187GluThr 65Glu + Gly153Asp + Thr163Asp + Gly165Asn +Met198His + Ser203GluLeu125Gly + Ser129Glu + Thr163Glu + Ala186His +Ser203Glu + Tyr205LeuThr 58Glu + Asn 61Ser + Thr 65Gly + Ser158Asp +Asn162Asp + Ser203GluThr 65Asn + Val 94Thr + Gly126Glu + Ser157Asp +Asn162Glu + Leu216AspAsp 59Glu + Ser 97Glu + Pro128Asn + Thr132Glu +Ser157Asp + Tyr205GlyThr 58Glu + Gly 60Gln + Leu 95Glu + Val202Gln +Tyr205Gln + Ser215AspAsp 59Glu + Ser157Asp + Asn162Asp + Tyr166Asn +Leu216Ile + Asn217GluSer157Glu + Asn162Glu + Phe188Gly + Ser203Asp +Asn211Asp + Thr212SerSer157Glu + Asn162Asp + Ser210Asp + Thr212Ser +Tyr213Leu + Leu216AspGly 60Pro + Ser 97Glu + Ser100Asp + Ala105His +Ser203Glu + Asn211AspAsn 61Asp + Leu125His + Gly156Asn + Pro200Gln +Thr212Glu + Leu216AspThr 65Asp + Ser 97Asp + Leu125Asn + Ser157Glu +Thr163Gly + Thr212AspGly 62Glu + Thr 65Asn + Asn162Glu + Thr163Ser +Asn211Asp + Ser215GluVal 94Glu + Asn 96Ser + Gly101Pro + Thr102Asp +Ser155Glu + Thr212AspGly 60Glu + Ser 98Glu + Gly 99Gln + Ser104Glu +Ala105Thr + Thr214AspSer155Asp + Ser187Asp + Ala199Gln + Ser203Asp +Tyr205Val + Thr212GluSer104Glu + Ile106Cys + Thr163Ser + Gly165Glu +Ser190Glu + Thr212AspVal 94Thr + Ile106Asp + Gly130Asn + Ser131Asp +Ser203Glu + Tyr205ProSer 98Glu + Pro128Glu + Ser155Asp + Ser160Glu +Ala186Ser + Pro209AsnThr 58Glu + Gly159Ser + Tyr166Glu + Ser190Glu +Thr212Pro + Leu216SerLeu 95Glu + Leu125His + Gly126Asp + Tyr205Gln +Ser215Asp + Thr219AsnAsp 59Glu + Gly 99Asp + Ala105Glu + Ala186Pro +Ser187Asp + Thr212ProAsn 96Gln + Ser 98Glu + Gly101Pro + Thr132Glu +Val202Asp + Ser215AspGly 99Asp + Pro128Asp + Thr132Asn + Val202Asp + Ser215Asp + Leu216HisVal 94Gln + Gly101Asp + Ser104Asp + Thr163Pro +Ser187Asp + Ser215AspLeu 95Met + Gly101Asp + Ser104Glu + Gly153Glu +Tyr205Ala + Ser215GluSly101Glu - Ser104Glu + Ser203Asp + Tyr205Ile +Ser210Glu + Thr212ProVal 94Glu + Ser100Glu + Ile106Met + Gly130Glu +Val202Glu + Thr214AsnSer 98Asp + Ile106Asp + Asn154Asp + Gly165Asp +Val202Met + Leu216GlnLeu 95Met + Thr102Ser + Gly127Asn + Ser190Glu +Val202Gly + Asn217AspGly 62Pro + Ile106Gln + Gly126Asp + Ser129Glu +Ser160Asp + Ser215AspGly 62Asp + Asn154Glu + Ser160Glu + Ala186Thr +Ser167Asp + Leu216ThrGly 62Ser + Thr 65Ser + Ser155Asp + Gly159Glu +Ser203Glu + Thr219Gln
II.清洗组合物
在本发明另一实施方案中，在用于清洗各种需要除去蛋白质污物表面的组合物中包括一种或多种有效量的酶变异体。这种清洗组合物包括清洗硬表面的洗涤剂组合物，形式不限(如液体和颗粒状)；用于清洗织物的洗涤剂组合物，形式不限(如颗粒、液体和条块制剂)；洗碟剂组合物(形式不限)；口腔清洁组合物，形式不限(如牙粉、牙膏和漱口制品)；假牙清洗组合物，形式不限(如液体、片剂)；和隐形眼镜清洁组合物，形式不限(如液体、片剂)。
清洗组合物除包括上述枯草杆菌蛋白酶DY变异体外，还包括一种或多种与蛋白酶相容的清洗组合物材料。这里使用的“清洗组合物材料”是指被选择用在所需特殊类型的清洗组合物和产品型式(如液体、颗粒、条块、雾状、棒状、膏、胶)中的任意一种液体、固体或气体材料，这些材料也与用于组合物的枯草杆菌蛋白酶DY变异体相容。考虑所要清洗的表面材料、使用中清洗条件(如在洗涤剂使用的整个过程中)所需的组合物形式很容易作出清洗组合物材料的特定选择。这里使用的术语“相容的”是指清洗组合物材料不会降低枯草杆菌蛋白酶DY变异体的蛋白水解活性至该蛋白酶失去正常使用条件中所期望的效果的程度。后面将详细例举特定的清洗组合物材料。
这里所用的“有效量的酶变异体”是指在特定清洗组合物中获得所需酶活性而需要的酶变异体的量。本领域普通技术人员很容易确定这种有效量，其基于多种因素，如所用的特殊的酶变异体、清洗应用范围、清洗组合物的特定组成以及需要的是液体还是干燥的(如颗粒、条块)组合物等等。优选清洗组合物包括约0.0001％至约10％的一种或多种本发明的酶变异体，更优选约0.001％至约1％，更优选约0.01％至约0.1％。下面更为详细地讨论了使用酶变异体的各种清洗组合物的一些例子。除非另有说明，这里所用的所有的份、百分含量和比例均指重量。
这里所用的“非织物清洗组合物”包括硬表面清洗组合物、洗碟剂组合物、口腔清洁组合物、假牙清洗组合物和隐性眼镜清洗组合物。
A.用于硬表面、碟子和织物的清洗组合物
本发明的酶变异体可用于需要大量泡沫并能很好地去除不溶性底物的各种洗涤剂组合物中。因此，该酶变异体可与各种常规成分一起使用，提供全配制的硬表面洗涤剂、洗碟剂组合物、织物洗涤组合物等等。这种组合物可呈液体、颗粒、条块等形式，可被配制成现代的“浓缩”洗涤剂，含有多达30％-60％(重量百分含量)的表面活性剂。
这里的清洗组合物可选择性地但优选含各种阴离子、非离子、两性离子等表面活性剂。这种表面活性剂通常含量约为组合物的5％-35％。
这里所用的表面活性剂非限制性例子包括常规的C11-C18烷基苯磺酸盐及伯烷基和无规则烷基硫酸盐、C10-C18仲(2，3)烷基硫酸盐、分子式为CH3(CH2)X(CHOSO3)-M+)CH3和CH3(CH2)y(CHOSO3)-M+)CH2CH3，其中x和(y+1)是至少为大约7的整数，优选至少为大约9，M是水溶性阳离子，特别是钠；C10-C18的烷基烷氧基硫酸盐(特别是EO1-5乙氧基硫酸盐)，C10-C18的烷基烷氧基羧酸盐(特别是EO1-5乙氧基羧酸盐)，C10-C18烷基多苷、及其相应的硫酸化多苷、C12-C18α-磺酸化脂肪酸酯、C12-C18烷基和烷基苯酚烷氧基化物(特别是乙氧基化物和混合的乙氧基/丙氧基)、C12-C18甜菜碱和磺基甜菜碱(“sultaines”)、C10-C18氧化胺等等。此处优选烷基烷氧基硫酸盐(AES)和烷基烷氧基羧酸盐(AEC)。(根据配方设计的需要，还优选这种表面活性剂与前述氧化胺和/或甜菜碱或磺基甜菜碱表面活性剂结合使用。)其他常规使用的表面活性剂列于标准书籍中。特别有用的表面活性剂包括由Connor等人的于1993年3月16日公开的美国专利US5194639中所述的C10-C18N-甲基萄萄糖酰胺，这里引为参考。
此处的组合物可包括洗涤剂清洗组合物中有用的其他多种成分，包括其他活性成分、载体、水溶助长剂、加工助剂、染料或颜料、液体配方所需溶剂等。如果需要额外增加泡沫，可在组合物中掺入象C10-C16烷氧酰胺这样的增泡剂，通常含量约为1％至10％。C10-C14的单乙醇和二乙醇酰胺代表了一类这种典型的增泡剂。将这种增泡剂与多泡的辅助表面活性剂如上述的氧化胺、甜菜碱和磺基甜菜碱一起使用也是有利的。如果需要，可加入如MgCl2、MgSO4等这样的可溶性镁盐，通常加入量约为0.1％-2％，以提供更多的泡沫。
这里的液体洗涤剂组合物可包含作为载体的水及其他溶剂。低分子量的伯醇或仲醇例如甲醇、乙醇、丙醇和异丙醇是合适的。优选一元醇用于使表面活性剂增溶，但也可使用那些约含2-6个碳原子和2-6个羟基的多元醇(如1，3-丙二醇、乙二醇、甘油和1，2-丙二醇)。组合物可含有约5％-90％的，通常为约10％-50％的这种载体。
优选配制这里的洗涤剂组合物使其用于水溶液清洗操作中时，洗涤水的pH在约6.8-11.0。通常在此范围内配制成品。将pH值控制于推荐使用范围的技术包括使用缓冲剂、碱、酸等等，这是本领域技术人员所熟知的技术。
当配制本发明的硬表面清洗组合物和织物清洗组合物时，配方师可希望使用约5％-50％(重量百分比)的各种助洗剂。典型的助洗剂包括1-10微米的沸石、聚羧酸盐如柠檬酸盐和氧联二琥珀酸盐、层状硅酸盐、磷酸盐等等。其他常规助洗剂列于标准配方中。
与上述情况相似，配方师可希望在这种组合物中使用各种其他酶，如纤维素酶、脂酶、淀粉酶和蛋白酶，通常重量百分含量约为0.001％-1％。在洗衣洗涤剂领域各种洗涤和保护织物的酶是熟知的。
可在这种组合物中使用各种漂白化合物，如过碳酸盐、过硼酸盐等，通常重量百分含量在约1％-15％。如果需要，这种组合物中还可含有如四乙酰乙二胺、壬酰氧基苯磺酸盐等这样的漂白活化剂，它们在本领域均是熟知的。用量通常约为1％-10％(重量百分比)。
各种污垢解脱剂特别是阴离子低聚酯型的污垢解脱剂、各种螯合剂特别是氨基膦酸盐和乙二胺二琥珀酸盐、各种粘土类去污垢剂特别是乙氧基化的四亚乙基五胺、各种分散剂特别是聚丙烯酸盐和聚天冬氨酸盐、各种增白剂特别是阴离子增白剂、各种抑泡剂特别是硅氧烷和仲醇、各种织物柔软剂特别是绿土粘土等等均可用于这种组合物中，重量百分含量约为1％-35％。标准配方书和公开专利中有这些常规材料的多种、详细说明。
在清洗组合物中还可使用酶稳定剂。这种酶稳定剂包括丙二醇(优选从约1％至约10％)、甲酸钠(优选约0.1％至约1％)及甲酸钙(优选约0.1％至约1％)。
1.硬表面清洗组合物
这里使用的“硬表面清洗组合物”是指用于清洗如地板、墙壁、浴室瓷砖等这类硬表面的液体和颗粒状洗涤剂。本发明的硬表面清洗组合物包括有效量的一种或多种本发明的酶变异体，优选组合物中活性酶的重量百分比为约0.001％至约10％，更优选约0.01％至约5％，更优选约0.05％至约1％。除包含一种或多种酶变异体外，通常这种硬表面清洗组合物还包括一种表面活性剂和一种水溶性螯合助洗剂。但是，在某些象窗户喷雾清洗剂这种特定的产品中，有时不使用表面活性剂，因为它们会在玻璃表面产生一层薄膜状/条纹残留物。
当存在表面活性剂成分时，它在本发明组合物中所占比例可少至0.1％，但通常组合物中含有约0.25％至约10％的表面活性剂，更优选约1％至5％。
通常该组合物含有约0.5％至约50％的洗涤助洗剂，优选约1％至10％。
优选pH应在约8至12的范围内。如果需要调节pH的话，可使用常规的pH调节剂，如氢氧化钠、碳酸钠或盐酸。
在组合物中可包括溶剂。有用的溶剂包括但不限于二醇醚，如二甘醇一己基醚，二甘醇一丁基醚、乙二醇一丁基醚、乙二醇一己基醚、丙二醇一丁基醚、二丙二醇一丁基醚，和二醇如2，2，4 三甲基-1，3-戊二醇和2-乙基-1，3-己二醇。使用时，通常这种溶剂的含量为约0.5％至约15％，优选约3％至约11％。
此外，可在本发明组合物中使用高挥发性溶剂，如异丙醇或乙醇，以便在本发明组合物完全应用于表面后不漂洗表面时易于组合物的快速挥发。当使用高挥发性溶剂时其在组合物中的含量约2％至约12％。
本发明的硬表面清洗组合物的实例描述在下面的实施例中。
实施例7-12
硬表面清洗液体组合物
实施例号组分 7 8 9 10 11 12Phe188Asp 0.05 0.50 0.02 0.03 0.10 0.03Val202Ala - - - - 0.20 0.02Na2DIDA*EDTA** - - 2.90 2.90 - -柠檬酸钠 - - - - 2.90 2.90C12烷基苯磺酸钠 1.95 - 1.95 - 1.95 -C12烷基硫酸钠 - 2.20 - 2.20 - 2.20C12乙氧基硫酸钠 - 2.20 - 2.20 - 2.20C12二甲胺氧化物 - 0.50 - 0.50 - 0.50枯烯磺酸钠 1.30 - 1.30 - 1.30 -己基卡必醇*** 6.30 6.30 6.30 6.30 6.30 6.30水**** 100％
*N-二甘醇-N，N-亚氨基二乙酸二钠
**乙二胺二乙酸四钠盐
***二甘醇一己醚
****所有配方均调节至pH7
在实施例7-10中，用表2-36中列举的其他枯草杆菌蛋白酶DY变异体代替Phe188Asp，得到基本相似的结果。
在实施例11-12中，用表2-36中列举的其他枯草杆菌蛋白酶DY变异体的任意一种组合代替Phe188Asp和Val202Ala，得到基本相似的结果。
实施例13-18
清洗硬表面和家庭除霉喷雾组合物
实施例号组分 13 14 15 16 17 18Asn 96Asp+Tyr103His 0.50 0.05 0.60 0.30 0.20 0.30Pro200Gly+Gly201Ser - - - - 0.30 0.10+Ser210Asp辛基硫酸钠 2.00 2.00 2.00 2.00 2.00 2.00十二烷基硫酸钠 4.00 4.00 4.00 4.00 4.00 4.00氢氧化钠 0.80 0.80 0.80 0.80 0.80 0.80硅酸盐(Na) 0.04 0.04 0.04 0.04 0.04 0.04香料 0.35 0.35 0.35 0.35 0.35 0.35水 平衡至100％
产品pH约为7。
在实施例13-16中，用表2-36中列举的其他枯草杆菌蛋白酶DY变异体代替Asn96Asp+Tyr103His，得到基本相似的结果。
在实施例17-18中，用表2-36中列举的其他枯草杆菌蛋白酶DY变异体的任意一种组合代替Asn 96Asp+Tyr103His和Pro200Gly+Gly201Ser+Ser210Asp，得到基本相似的结果。
2.洗碟剂组合物
在本发明另一实例中，洗碟剂组合物包括一种或多种本发明的酶变异体。这里使用的“洗碟剂组合物”是指洗涤盘碟的所有形式组合物，包括颗粒剂和液体形式，但不限于此。本发明洗碟剂组合物的实例用下面的实施例说明。
实施例19-24
洗碟组合物
实施例号组分 19 20 21 22 23 24Gly127Asn 0.05 0.50 0.02 0.40 0.10 0.03Gly201Asn+Tyr205Gly - - - - 0.40 0.02+Thr207Gln+Tyr213IleC12-C14 N-甲基-葡糖
酰胺 0.90 0.90 0.90 0.90 0.90 0.90C12乙氧基(1)硫酸盐 12.00 12.00 12.00 12.00 12.00 12.002-甲基十一烷酸 4.50 4.50 4.50 4.50 4.50 4.50C12乙氧基(2)羧酸盐 4.50 4.50 4.50 4.50 4.50 4.50C12醇乙氧基化物(4) 3.00 3.00 3.00 3.00 3.00 3.00C12氧化胺 3.00 3.00 3.00 3.00 3.00 3.00枯烯磺酸钠 2.00 2.00 2.00 2.00 2.00 2.00乙醇 4.00 4.00 4.00 4.00 4.00 4.00Mg++(为MgCl2) 0.20 0.20 0.20 0.20 0.20 0.20Ca++(为CaCl2) 0.40 0.40 0.40 0.40 0.40 0.40水 平衡至 100％
产品pH被调节至7。
在实施例19-22中，用表2-36中列举的其他枯草杆菌蛋白酶DY变异体代替Gly127Asn，得到基本相似的结果。
在实施例23-24中，用表2-36中列举的其他枯草杆菌蛋白酶DY变异体的任意一种组合代替Gly127Asn和Gly201Asn+Thr207Gln+Tyr205Gln+Tyr213Ile，得到基本相似的结果。
3.织物清洗组合物
在本发明另一实例中，织物清洗组合物包括一种或多种本发明的酶变异体。这里使用的“织物清洗组合物”是指洗涤织物的所有形式的洗涤剂组合物，包括颗粒剂、液体和条块形式，但不限于此。优选织物清洗组合物呈液体形式。
a.颗粒状织物清洗组合物
本发明的颗粒状织物清洗组合物包括有效量的一种或多种本发明的酶变异体，优选组合物中活性酶的重量百分比从约0.001％至约10％，更优选从约0.005％至约5％，更优选从约0.01％至约1％。除包含一种或多种酶变异体外，通常这种颗粒状织物清洗组合物还包括至少一种表面活性剂、一种或多种助洗剂，且在某些情况下包括一种漂白剂。
本发明颗粒状织物清洗组合物的实例描述于下面的实施例中。
实施例25-28
颗粒状织物清洗组合物
实施例号组分 25 26 27 28Gly156Glu+Asn162Gln 0.10 0.20 0.03 0.05Leu95Glu - - 0.02 0.05C13直链烷基苯磺酸盐 22.00 22.00 22.00 22.00磷酸盐(为三磷酸钠) 23.00 23.00 23.00 23.00碳酸钠 23.00 23.00 23.00 23.00硅酸钠 14.00 14.00 14.00 14.00沸石 8.20 8.20 8.20 8.20螯合剂(二亚乙基三胺五乙酸) 0.40 0.40 0.40 0.40硫酸钠 5.50 5.50 5.50 5.50水 平衡至100％
在实施例25-26中，用表2-36中列举的其他枯草杆菌蛋白酶DY变异体代替Gly156Glu+Asn162Gln，得到基本相似的结果。
在实施例27-28中，用表2-36中列举的其他枯草杆菌蛋白酶DY变异体的任意一种组合代替Gly156Glu+Asn162Gln和Leu95Glu，得到基本相似的结果。
实施例29-32
颗粒状织物清洗组合物
实施例号组分 29 30 31 32Gln161Glu 0.10 0.20 0.03 0.05Ala186Ser+Phe188Ala+Ser190Glu - - 0.02 0.05C12烷基苯磺酸钠 12.00 12.00 12.00 12.00沸石A(1-10微米) 26.00 26.00 26.00 26.002-丁基辛酸 4.00 4.00 4.00 4.00C12-C14仲(2，3)烷基硫酸钠 5.00 5.00 5.00 5.00柠檬酸钠 5.00 5.00 5.00 5.00荧光增白剂 0.10 0.10 0.10 0.10硫酸钠 17.00 17.00 17 00 17.00
在实施例29-30中，用表2-36中列举的其他枯草杆菌蛋白酶DY变异体代替Gln161Glu，得到基本相似的结果。
在实施例31-32中，用表2-36中列举的其他枯草杆菌蛋白酶DY变异体的任意一种组合代替Gln161Glu和Ala186Ser+Phe188Ala+Ser190Glu，得到基本相似的结果。
实施例33-36
颗粒状织物清洗组合物
实施例号组分 33 34 35 36Asn 96Glrn+Ile106Ala 0.10 0.20 0.03 0.05Gly 60Asn+Asn 61Glu+Gly153Ser - - 0.02 0.05+Pro209Asp+Asn211Asp+Ser215GluC13直链烷基苯磺酸盐 22.00 22.00 22.00 22.00磷酸盐(为三聚磷酸钠) 23.00 23.00 23.00 23.00碳酸钠 23.00 23.00 23.00 23.00硅酸钠 14.00 14.00 14.00 14.00沸石 8.20 8.20 8.20 8.20螯合剂(二亚乙基三胺五乙酸) 0.40 0.40 0.40 0.40硫酸钠 5.50 5.50 5.50 5.50水 平衡至 100％
在实施例33-34中，用表2-36中列举的其他枯草杆菌蛋白酶DY变异体代替Asn96Gln+Ile106Ala，得到基本相似的结果。
在实施例35-36中，用表2-36中列举的其他枯草杆菌蛋白酶DY变异体的任意一种组合代替Asn96Gln+Ile106Ala和Gly60Asn+Asn61Glu+Gly153Ser+Pro209Asp+Asn211Asp+Ser215Glu，得到基本相似的结果。
实施例37-40
颗粒状织物清洗组合物
实施例号组分 37 38 39 40Gly60Asn 0.10 0.20 0.03 0.05Ala186Asn+Ser187Asp+Phe188Gln - - 0.02 0.05
+Ser190GluC12十二烷基苯磺酸盐 12.00 12.00 12.00 12.00沸石A(1-10微米) 26.00 26.00 26.00 26.002-丁基辛酸 4.00 4.00 4.00 4.00C12-C14仲(2，3)烷基硫酸钠 5.00 5.00 5.00 5.00柠檬酸钠 5.00 5.00 5.00 5.00荧光增白剂 0.10 0.10 0.10 0.10硫酸钠 17.00 17.00 17.00 17.00水及次要组分 平衡至100％
在实施例37-38中，用表2-36中列举的其他枯草杆菌蛋白酶DY变异体代替Gly60Asn，得到基本相似的结果。
在实施例39-40中，用表2-36中列举的其他枯草杆菌蛋白酶DY变异体的任意一种组合代替Gly60Asn和Ala186Asn+Ser187Asp+Phe188Gln+Ser190Glu，得到基本相似的结果。
实施例41-42
颗粒状织物清洗组合物
实施例号组分 41 42直链烷基苯磺酸盐 11.4 10.70牛油烷基硫酸盐 1.80 2.40C14-15烷基硫酸盐 3.00 3.107倍乙氧基化C14-15烷醇 4.00 4.0011倍乙氧基化牛油醇 1.80 1.80分散剂 0.07 0.1硅氧烷流体 0.80 0.80柠檬酸三钠 14.00 15.00柠檬酸 3.00 2.50沸石 32.50 32.10马来酸丙烯酸共聚物 5.00 5.00二亚乙基三胺五亚甲基膦酸 1.00 0.20Ser215Asp 0.30 0.30脂酶 0.36 0.40淀粉酶 0.30 0.30硅酸钠 2.00 2.50硫酸钠 3.50 5.20聚乙烯吡咯烷酮 0.30 0.50过硼酸盐 0.5 1苯酚磺酸盐 0.1 0.2过氧化物酶 0.1 0.1次要组分 至100 至100
实施例43-44
颗粒状织物清洗组合物
实施例号组分 43 44C12直链烷基苯磺酸钠 6.5 8.0硫酸钠 15.0 18.0沸石A 26.0 22.0次氮基三乙酸钠 5.0 5.0聚乙烯吡咯烷酮 0.5 0.7四乙酰乙二胺 3.0 3.0硼酸 4.0 -过硼酸 0.5 1苯酚磺酸盐 0.1 0.2Val202Met+Thr212Pro 0.4 0.4填料(如硅酸盐、碳酸盐、香料、水) 至100 至100
实施例45
浓缩的颗粒状织物清洗组合物组分 重量％烷基硫酸盐 8.0烷基乙氧基硫酸盐 2.03和7倍乙氧基化的C25和C45烷醇混合物 6.0多羟基脂肪酸酰胺 2.5沸石 17.0多层硅酸盐/柠檬酸盐 16.0碳酸盐 7.0马来酸丙烯酸共聚物 5.0污垢解脱聚合物 0.4羧甲基纤维素 0.4聚(4-乙烯吡啶)-N-氧化物 0.1乙烯咪唑和乙烯吡咯烷酮共聚物 0.1PEG2000 0.2Thr 65Glu+Ile106Met+Leu125His+Tyr205Leu 0.5+Ser215Asp脂酶 0.2纤维素酶 0.2四乙酰乙二胺 6.0过碳酸盐 22.0乙二胺二琥珀酸 0.3抑泡剂 3.54，4′-二(2-吗啉代-4-苯胺基-S-三嗪-6- 0.25基氨基)茋-2，2′-二磺酸钠4，4′-二(2-磺基styril)联苯二钠 0.05水、香料及次要组分 至100
实施例46
颗粒状织物清洗组合物组分 重量％直链烷基苯磺酸盐 7.6C16-C18烷基磺酸盐 1.37倍乙氧基化的C14-15烷醇 4.0椰子油-烷基-二甲基羟乙基氯化铵 1.4分散剂 0.07硅氧烷流体 0.8柠檬酸三钠 5.0沸石4A 15.0马来酸丙烯酸共聚物 4.0二亚乙基三胺五亚甲基膦酸 0.4过硼酸盐 15.0四乙酰乙二胺 5.0绿土粘土 10.0聚氧乙烯(MW300,000) 0.3Thr207Asn+Tyr208Gln+Thr212Ser+Tyr213Glu 0.4+Ser215Glu脂酶 0.2淀粉酶 0.3纤维素酶 0.2硅酸钠 3.0碳酸钠 10.0羧甲基纤维素 0.2增白剂 0.2水、香料和次要组分 至100
实施例47
颗粒状织物清洗组合物组分 重量％直链烷基苯磺酸盐 6.92牛油烷基硫酸盐 2.057倍乙氧基化的C14-15烷醇 4.43倍乙氧基化的C12-15烷基乙氧基硫酸盐 0.16沸石 20.2柠檬酸盐 5.5碳酸盐 15.4硅酸盐 3.0马来酸丙烯酸共聚物 4.0羧甲基纤维素 0.31污垢解脱聚合物 0.30Gly 60Asp+Asn 61Asp+Gly 62Asp+Thr 65Gln 0.2脂酶 0.36纤维素酶 0.13四水合过硼酸盐 11.64一水合过硼酸盐 8.7四乙酰乙二胺 5.0二亚乙基三胺五甲基膦酸 0.38硫酸镁 0.40增白剂 0.19香料，硅氧烷，抑泡剂 0.85次要组分 至100
b.液体织物清洗组合物
本发明的液体织物清洗组合物包括有效量的一种或多种本发明的酶变异体，优选组合物中活性酶的重量百分比为约0.005％至约5％，更优选约0.01％至约1％。通常这种液体织物清洗组合物还包括一种阴离子表面活性剂、一种脂肪酸、一种水溶性助洗剂和水。
本发明的液体织物清洗组合物的实例描述于下面的实施例中。
实施例48-52
液体织物清洗组合物
实施例号
48 49 50 51 52Val 94Gly+Gly 99Pro+lle106Val 0.05 0.03 0.30 0.03 0.10Gly218Pro - - - 0.01 0.20C12-C14烷基硫酸钠 20.00 20.00 20.00 20.00 20.002-丁基辛酸 5.00 5.00 5.00 5.00 5.00柠檬酸钠 1.00 1.00 1.00 1.00 1.00C10烷醇乙氧基化物(3) 13.00 13.00 13.00 13.00 13.00单乙醇 2.50 2.50 2.50 2.50 2.50水/丙二醇/乙醇(100∶1∶1) 平衡至100％
在实施例48-50中，用表2-36中列举的其他枯草杆菌蛋白酶DY变异体代替Val94Gly+Gly99Pro+Ile106Val，得到基本相似的结果。
在实施例51-52中，用表2-36中列举的其他枯草杆菌蛋白酶DY变异体代替Val94Gly+Gly99Pro+Ile106Val和Gly218Pro，得到基本相似的结果。
实施例53-57
液体织物清洗组合物
实施例号组分 53 54 55 56 57Gly156Ser 0.05 0.03 0.30 0.03 0.10Ser187Glu - - - 0.01 0.20C12-C14烷基硫酸钠 20.00 20.00 20.00 20.00 20.002-丁基辛酸 5.00 5.00 5.00 5.00 5.00柠檬酸钠 1.00 1.00 1.00 1.00 1.00C10烷醇乙氧基化物(3) 13.00 13.00 13.00 13.00 13.00单乙醇胺 2.50 2.50 2.50 2.50 2.50水/丙二醇/乙醇(100∶1∶1) 平衡至100％
在实施例53-55中，用表2-36中列举的其他枯草杆菌蛋白酶DY变异体代替Gly156Ser，得到基本相似的结果。
在实施例56-57中，用表2-36中列举的其他枯草杆菌蛋白酶DY变异体的任意组合代替Gly156Ser和Ser187Glu，得到基本相似的结果。
实施例58-59
液体织物清洗组合物
实施例号组分 58 59C12-14链烯基琥珀酸 3.0 8.0柠檬酸一水合物 10.0 15.0C12-15烷基硫酸钠 8.0 8.02倍乙氧基化的C12-15烷醇硫酸钠 - 3.07倍乙氧基化C12-15烷醇 - 8.05倍乙氧基化C12-15烷醇 8.0 -二亚乙基三胺五(亚甲基膦酸) 0.2 -油酸 1.8 -乙醇 4.0 4.0丙二醇 2.0 2.0Gly101Glu+Ser104Glu+Ser203Asp+Tyr205Ile 0.2 0.2
+Ser210Glu+Thr212Pro聚乙烯吡咯烷酮 1.0 2.0抑泡剂 0.15 0.15NaOH 至pH7.5过硼酸盐 0.5 1苯酚磺酸盐 0.1 0.2过氧化物酶 0.4 0.1水及次要组分 至100份
在实施例58和59中，用表2-36中列举的其他枯草杆菌蛋白酶DY变异体代替Gly101Glu+Ser104Glu+Ser203Asp+Tyr205Ile+Ser210Glu+Thr212Pro，得到基本相似的结果。
实施例60-62
液体织物清洗组合物
实施例号组分 60 61 62柠檬酸 7.10 3.00 3.00脂肪酸 2.00 - 2.00乙醇 1.93 3.20 3.20硼酸 2.22 3.50 3.50单乙醇胺 0.71 1.09 1.091，2-丙二醇 7.89 8.00 8.00枯烯磺酸钠 1.80 3.00 3.00甲酸钠 0.08 0.08 0.08NaOH 6.70 3.80 3.80硅烷消泡剂 1.16 1.18 1.18Asn 61Glu+Gly 64Ser+Thr 65Gly 0.0145 - -Ser129Asp+Gly130Asn - 0.0145 -Leu216Asn - - 0.0145脂酶 0.200 0.200 0.200纤维素酶 - 7.50 7.50污垢解脱聚合物 0.29 0.15 0.15消泡剂 0.06 0.085 0.085增白剂36 0.095 - -增白剂3 - 0.05 0.05C12烷基苯磺酸 9.86 - -C12-15烷基聚乙氧基化(2.5)硫酸盐 13.80 18.00 18.00C12葡糖酰胺 - 5.00 5.00C12-13烷基聚乙氧基化物(9) 2.00 2.00 2.00水、香料和次要组分 平衡至 100％
c.条块状织物清洗组合物
适用于手洗脏衣物的本发明条块状织物清洗组合物包括有效量的一种或多种本发明的酶变异体，优选占组合物的重量百分比为约0.001％至约10％，更优选约0.01％至约1％。
本发明条块状织物清洗组合物的实例描述于下面的实施例中。
实施例63-66
条块状织物清洗组合物
实施例号组分 63 64 65 66Val202Ser+Asn211Asp 0.3 - 0.1 0.02Gly156Gln+lle164Thr - - 0.4 0.03C12-C16烷基硫酸钠 20.0 20.0 20.0 20.00C12-C14N-甲基葡酰胺 5.0 5.0 5.0 5.00C11-C13烷基苯磺酸钠 10.0 10.0 10.0 10.00碳酸钠 25.0 25.0 25.0 25.00焦磷酸钠 7.0 7.0 7.0 7.00三聚磷酸钠 7.0 7.0 7.0 7.00沸石A(0.1-10μ) 5.0 5.0 5.0 5.00羧甲基纤维素 0.2 0.2 0.2 0.20聚丙烯酸盐(MW1400) 0.2 0.2 0.2 0.20椰子油单乙醇酰胺 5.0 5.0 5.0 5.00增白剂、香料 0.2 0.2 0.2 0.20CaSO4 1.0 1.0 1.0 1.00MgSO4 1.0 1.0 1.0 1.00水 4.0 4.0 4.0 4.00填料* 平衡至100％
*可选自适宜的材料如CaCO3、滑石、粘土、硅酸盐等等。
在实施例63-64中，用表2-36中列举的其他枯草杆菌蛋白酶DY变异体代替Val202Ser+Asn211Asp，得到基本相似的结果。
在实施例65-66中，用表2-36中列举的其他枯草杆菌蛋白酶DY变异体的任何一种组合代替Val202Ser+Asn211Asp和Gly156Gln+Ile164Thr，得到基本相似的结果。
实施例67-70
条块状织物清洗组合物
实施例号组分 67 68 69 70Val204Thr+Pro209Asp+Tyr213Cys 0.3 - 0.1 0.02Gly126Gln+Thr214Gly+Ser215Glu - 0.3 0.4 0.03C12-C16烷基硫酸钠 20.0 20.0 20.0 20.00C12-C14N-甲基葡酰胺 5.0 5.0 5.0 5.00C11-C13烷基苯磺酸钠 10.0 10.0 10.0 10.00碳酸钠 25.0 25.0 25.0 25.00焦磷酸钠 7.0 7.0 7.0 7.00三聚磷酸钠 7.0 7.0 7.0 7.00沸石A(0.1-10μ) 5.0 5.0 5.0 5.00羧甲基纤维素 0.2 0.2 0.2 0.20聚丙烯酸盐(MW1400) 0.2 0.2 0.2 0.20椰子油单乙醇酰胺 5.0 5.0 5.0 5.00增白剂，香料 0.2 0.2 0.2 0.20CaSO4 1.0 1.0 1.0 1.00MgSO4 1.0 1.0 1.0 1.00水 4.0 4.0 4.0 4.00填料* 平衡至100％
*可选自适宜的材料如CaCO3、滑石、粘土、硅酸盐等等。
在实施例67中，用表2-36中列举的其他枯草杆菌蛋白酶DY变异体代替Val204Thr+Pro209Asp+Tyr213Cys，得到基本相似的结果。
在实施例68中，用表2-36中列举的其他枯草杆菌蛋白酶DY变异体代替Gly126Gln+Thr214Gly+Ser215Glu，得到基本相似的结果。
在实施例69-70中，用表2-36中列举的其他枯草杆菌蛋白酶DY变异体的任何一种组合代替Val204Thr+Pro209AsP+Tyr213Cys和Gly126Gln+Thr214Gly+Ser215Glu，得到基本相似的结果。
B.其他清洗组合物
除上述硬表面清洗组合物、洗碟剂组合物和织物清洗组合物外，本发明的一种或多种酶变异体还可搀入需要水解不溶性的底物的各种其他清洗组合物中。这类其它组合物包括口腔清洁组合物、假牙清洗组合物和隐性眼镜清洗组合物。
1.口腔清洁组合物
在本发明另一实施方案中，在用于去除牙齿或假牙上的蛋白质污物的组合物中可含有药物上可接受量的本发明的一种或多种酶变异体。这里所用的“口腔清洁组合物”是指牙粉、牙膏、牙胶、牙粉、漱口剂、口腔喷雾剂、口胶、口香糖、锭剂、sachets、片剂、生物胶、预防膏、牙齿治疗液等等。优选口腔清洁组合物包括占组合物重量百分比约0.0001％至约20％的一种或多种本发明的酶变异体，更优选约0.001％至约10％，更优选约0.01％至约5％，和药物可接受的载体。这里所用的“药物可接受”是指药物、药剂或惰性成分适于与人或低级动物的组织接触而不会有不当的毒性、不相容性、不稳定性、刺激性、过敏反应等等，具有相称的合理的利/弊之比。
通常口腔清洁组合物中的口腔清洁成分药物上可接受的口腔清洁载体成分占该组合物的重量百分比为约50％至99.99％，优选约65％至99.99％，更优选约65％至99％。
可包含在本发明口腔清洁组合物中的药物上可接受的载体成分和可选择性成分是本领域技术人员所熟知的。各种组合物类型、用于口腔清洁组合物中的载体成分和可选择成分描述于Seibel的1992年3月17日公开的美国专利5096700、Sampathkumar的1991年7月2日公开的美国专利5028414、Benedict，Bush和Sunbergd的1991年7月2日公开的美国专利5028415中，这些均在此引作参考文献。
本发明口腔清洁组合物的实例描述于下面的实施例中。
实施例71-74
牙粉组合物
实施例号组分 71 72 73 74Thr214Asp 2.000 3.500 1.500 2.000山梨醇(70％水溶液) 35.000 35.000 35.000 35.000PEG-6* 1.000 1.000 1.000 1.000二氧化硅牙用磨料** 20.000 20.000 20.000 20.000氟化钠 0.243 0.243 0.243 0.243二氧化钛 0.500 0.500 0.500 0.500糖精钠 0.286 0.286 0.286 0.286烷基硫酸钠(27.9％水溶液) 4.000 4.000 4.000 4.000香料 1.040 1.040 1.040 1.040羧乙烯聚合物*** 0.300 0.300 0.300 0.300角叉胶**** 0.800 0.800 0.800 0.800水 平衡至100％
*PEG-6＝分子量为600的聚乙二醇
**由J.M.Huber提供的鉴定为Zeodent119的沉淀硅石
***由B.F.Goodrich化学公司提供的Carbopol
****由Hercules化学公司提供的Lota Carrageenan
在实施例71-74中，用表2-36中列举的其他枯草杆菌蛋白酶DY变异体代替Thr214Asp，得到基本相似的结果。
实施例75-78
漱口剂组合物
实施例号组分 75 76 77 78Thr132Glu 3.00 7.50 1.00 5.00SDA40烷醇 8.00 8.00 8.00 8.00香料 0.08 0.08 0.08 0.08乳化剂 0.08 0.08 0.08 0.08氟化钠 0.05 0.05 0.05 0.05甘油 10.00 10.00 10.00 10.00甜味剂 0.02 0.02 0.02 0.02苯甲酸 0.05 0.05 0.05 0.05氢氧化钠 0.20 0.20 0.20 0.20颜料 0.04 0.04 0.04 0.04水 平衡至100％
在实施例75-78中，用表2-36中列举的其他枯草杆菌蛋白酶DY变异体代替Thr132Glu，得到基本相似的结果。
实施例79-82
锭剂组合物
实施例号组分 79 80 81 82Ser 97Glu+Ile106Leu 0.01 0.03 0.10 0.02山梨醇 17.50 17.50 17.50 17.50甘露糖醇 17.50 17.50 17.50 17.50淀粉 13.60 13.60 13.60 13.60甜味剂 1.20 1.20 1.20 1.20香料 11.70 11 70 11.70 11.70色料 0.10 0.10 0.10 0.10玉米糖浆 平衡至100％
在实施例79-82中，用表2-36中列举的其他枯草杆菌蛋白酶DY变异体代替Ser97Glu+Ile106Leu，得到基本相似的结果。
实施例83-88
口香糖组合物
实施例号组分 83 84 85 86Leu125Ala+Ser129Glu+Gly130Pro 0.03 0.02 0.10 0.05+Ser131Asp山梨醇结晶 38.44 38.40 38.40 38.40Paloja-T胶基* 20.00 20.00 20.00 20.00山梨醇(70％水溶液) 22.00 22.00 22.00 22.00甘露糖醇 10.00 10.00 10.00 10.00甘油 7.56 7.56 7.56 7.56香料 1.00 1.00 1.00 1.00
*由L.A.Dreyfus Company提供
在实施例83-86中，用表2-36中列举的其他枯草杆菌蛋白酶DY变异体代替Leu125Ala+Ser129Glu+Gly130Pro+Ser131Asp，得到基本相似的结果。
2.假牙清洗组合物
在本发明另一实施方案中，在口腔外清洗假牙的假牙清洗组合物包含一种或多种本发明的酶变异体。这种假牙清洗组合物包括有效量的一种或多种酶变异体，优选占组合物重量百分比约0.0001％至约50％的一种或多种酶变异体，更优选约0.001％至约35％，更优选0.01％至约20％，和假牙清洗载体。各种假牙清洗组合物形式如起泡片等是本领域所熟知的(参见Young的美国专利5055305，这里引作参考文献)，常常适于掺入一种或多种酶变异体除去假牙上的蛋白质污物。
本发明假牙清洗组合物的实例描述于下面的实施例中。
实施例87-90
双层起泡假牙清洗片
实施例号组分 87 88 89 90酸层Thr212Pro+Thr219Pro 1.0 1.5 0.01 0.05酒石酸 24.0 24.0 24.00 24.00碳酸钠 4.0 4.0 4.00 4.00氨基磺酸 10.0 10.0 10.00 10.00PEG20,000 4.0 4.0 4.00 4.00碳酸氢钠 24.5 24.5 24.50 24.50过硫酸钾 15.0 15.0 15.00 15.00酸式焦磷酸钠 7.0 7.0 7.00 7.00焦化的二氧化硅 2.0 2.0 2.00 2.00TAED* 7.0 7.0 7.00 7.00蓖麻油基磺基琥珀酸盐 0.5 0.5 0.50 0.50香料 1.0 1.0 1.00 1.00碱层一水合过硼酸钠 32.0 32.0 32.00 32.00碳酸氢钠 19.0 19.0 19.00 19.00EDTA 3.0 3.0 3.00 3.00三聚磷酸钠 12.0 12.0 12.00 12.00PEG20,000 2.0 2.0 2.00 2.00过硫酸钾 26.0 26.0 26.00 26.00碳酸钠 2.0 2.0 2.00 2.00焦化的二氧化硅 2.0 2.0 2.00 2.00颜料/香料 2.0 2.0 2.00 2.00
*四乙酰乙二胺
在实施例87-90中，用表2-36中列举的其他枯草杆菌蛋白酶DY变异体代替Thr212Pro+Thr219Pro，得到基本相似的结果。
3.隐性眼镜清洗组合物
在本发明另一实施方案中，隐性眼镜清洗组合物包含一种或多种本发明的酶变异体。这种隐性眼镜清洗组合物包括有效量的一种或多种酶变异体，优选占组合物重量百分比约0.01％至约50％的一种或多种酶变异体，更优选约0.01％至约20％，更优选约1％至约5％，和隐性眼镜清洗载体。各种隐性眼镜清洗组合物形式如片剂、液体等是本领域所熟知的(参见Davies，Meaken和Rees的公开于1989年9月5日的美国专利4863627，Huth，Lam和Kirai的再公开于1988年5月24日的美国再颁专利32672，Schaferd的公开于1986年9月2日的美国专利4609493，Ogunbiyi和Smith公开于1987年9月1日的美国专利4690793，Ogunbiyi，Riedhammer和Smith公开于1986年9月30日的美国专利4614549，Ogata公开于1981年8月25日的美国专利4285738，这些在此均引作参考文献)，且常常适于与本发明一种或多种酶变异体结合起来除去隐性眼镜上的蛋白质污物。
本发明的隐性眼镜清洗组合物的实例描述于下面的实施例中。
实施例91-94
隐形眼镜酶制清洗液
实施例号组分 91 92 93 94Gly156Glu+Thr163Ser+Asn211Ser 0.01 0.5 0.1 2.0+Thr212Asn葡萄糖 50.00 50.0 50.0 50.0非离子表面活性剂(聚氧乙烯- 2.00 2.0 2.0 2.0聚氧丙烯共聚物)阴离子表面活性剂(聚氧乙烯- 1.00 1.0 1.0 1.0烷基苯基醚硫酸酯钠)氯化钠 1.00 1.0 1.0 1.0硼砂 0.30 0.3 0.3 0.3水 平衡至100％
在实施例91-94中，用表2-36中列举的其他枯草杆菌蛋白酶DY变异体代替Gly156Glu+Thr163Ser+Asn211Ser+Thr212Asn，得到基本相似的结果。
尽管本发明仅叙述了具体实施方案，但很显然本领域的技术人员可以在不超出本发明的精神和范围的情况下对本发明作各种改变和修改。本发明意在后附的权利要求中包括在本发明范围之内的所有这些改变。
序列表(1)一般信息(i)申请人：BRODE，PHILIP F.
BARNETT，BOBBY L.
RUBINGH，DONN N.(ii)发明名称：具有降低的吸附作用和增强的水解作用的枯草杆菌
蛋白酶DY变异体(iii)序列数：1(iv)通讯地址：
(A)收件人：THE PROCTER&GAMBLE COMPANY
(B)街道：11810 East Miami River Road
(C)城市：Ross
(D)州：OH
(E)国家：USA
(F)邮政编码：45061(v)计算机可读形式：
(A)介质类型：软盘
(B)计算机：IBM PC兼容机
(C)操作系统：PC-DOS/MS-DOS
(D)软件：PatentIn Release#1.0，版本1.30(vi)本申请数据：
(A)申请号：
(B)申请日：
(C)分类号：(viii)代理人/代理机构信息：
(A)姓名：HAKE，RICHARD A.
(B)注册号：37,343
(C)参考/备案号：5606(iX)通讯信息：
(A)电话：(513)627-0087
(B)传真：(513)627-0260(2)序列信息：SEQ ID NO：1：(i)序列特征：
(A)长度：274氨基酸
(B)类型：氨基酸
(C)链：单链
(D)拓扑结构：线性(Xi)序列描述：SEQ ID NO：1：Ala Gln Thr Val Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp Lys Val1 5 10 15Gln Ala Gln Gly Tyr Lys Gly Ala Asn Val Lys Val Gly Ile Ile Asp
20 25 30Thr Gly Ile Ala Ala Ser His Thr Asp Leu Lys Val Val Gly Gly Ala
35 40 45Ser Phe Val Ser Gly Glu Ser Tyr Asn Thr Asp Gly Asn Gly His Gly
50 55 60Thr His Val Ala Gly Thr Val Ala Ala Leu Asp Asn Thr Thr Gly Val65 70 75 80Leu Gly Val Ala Pro Asn Val Ser Leu Tyr Ala Ile Lys Val Leu Asn
85 90 95Ser Ser Gly Ser Gly Thr Tyr Ser Ala Ile Val Ser Gly Ile Glu Trp
l00 105 110Ala Thr Gln Asn Gly Leu Asp Val Ile Asn Met Ser Leu Gly Gly Pro
115 120 125Ser Gly Ser Thr Ala Leu Lys Gln Ala Val Asp Lys Ala Tyr Ala Ser
130 135 140Gly Ile Val Val Val Ala Ala Ala Gly Asn Ser Gly Ser Ser Gly Ser145 150 155 160Gln Asn Thr Ile Gly Tyr Pro Ala Lys Tyr Asp Ser Val Ile Ala Val
165 170 175Gly Ala Val Asp Ser Asn Lys Asn Arg Ala Ser Phe Ser Ser Val Gly
180 185 190Ala Glu Leu Glu Val Met Ala Pro Gly Val Ser Val Tyr Ser Thr Tyr
195 200 205Pro Ser Asn Thr Tyr Thr Ser Leu Asn Gly Thr Ser Met Ala Ser Pro
210 215 220His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys Tyr Pro Thr Leu225 230 235 240Ser Ala Ser Gln Val Arg Asn Arg Leu Ser Ser Thr Ala Thr Asn Leu
245 250 255Gly Asp Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Glu Ala Ala
260 265 270Ala Gln
Claims (9)
1.一种对野生型枯草杆菌蛋白酶的氨基酸序列进行了改良的枯草杆菌蛋白酶DY变异体，该野生型氨基酸序列包括第一环区、第二环区、第三环区、第四环区、第五环区和第六环区；其中改良的氨基酸序列是在一个或多个环区中一个或多个位置上有取代；其中：
A、当取代发生在第一环区时，在58、59、60、61、62、64或65位的一个或多个位置上发生取代，其中
c.取代发生在第60位时，取代氨基酸为Asn、Gln、Pro或Ser；
f.取代发生在第64位时，取代氨基酸为Asn、Asp、Gln、Glu、Pro或Ser；以及
g.取代发生在第65位时，取代氨基酸为Asn、Asp、Gln、Glu、Gly、Pro或Ser；
B、当取代发生在第二环区时，在94、95、96、97、98、99、100、101、102、103、104、105或106位的一个或多个位置上发生取代，其中
a.取代发生在第94位时，取代氨基酸为Ala、Cys、Gln、Gly、His、Met、Pro或Ser；
b.取代发生在第95位时，取代氨基酸为Ala、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Met、Pro、Ser、Thr或Val
c.取代发生在第96位时，取代氨基酸为Gln或Ser；
f.取代发生在第99位时，取代氨基酸为Asn、Gln、Pro或Ser；
h.取代发生在第101位时，取代氨基酸为Asn、Asp、Gln、Glu、Pro或Ser；
i.取代发生在第102位时，取代氨基酸为Asn、Gln、Gly、Pro或Ser；
j.取代发生在第103位时，取代氨基酸为Ala、Asn、Cys、Gln、Gly、His、Ile、Leu、Met、Pro或Ser；
l.取代发生在第105位时，取代氨基酸为Asn、Gln、Gly、His、Pro、Ser或Thr；和
m.取代发生在第106位时，取代氨基酸为Ala、Cys、Gln、Gly、His、Met、Pro、Ser或Val；
C、当取代发生在第三环区时，在125、126、127、128、129、130、131或132位的一个或多个位置上发生取代，其中
a.取代发生在第125位时，取代氨基酸为Ala、Asn、Cys、Gln、Gly、His、Met、Pro、Ser、Thr或Val；
b.取代发生在第126位时，取代氨基酸为Asn、Asp、Gln、Glu、Pro或Ser
d.取代发生在第128位时，取代氨基酸为Asn、Gln或Ser；
g.取代发生在第131位时，取代氨基酸为Asp或Glu；和
h.取代发生在第132位时，取代氨基酸为Asn、Gln、Gly、Pro或Ser；
D、当取代发生在第四环区时，在153、154、155、156、157、158、159、160、161、162、163、164、165或166位的一个或多个位置上发生取代，其中
b.取代发生在第154位时，取代氨基酸为Gln或Ser
d.取代发生在第156位时，取代氨基酸为Asn、Asp、Gln、Glu、Pro或Ser；和
n.取代发生在第166位时，取代氨基酸为Ala、Gly、His、Ile、Leu、Met或Pro；
E、当取代发生在第五环区时，在186、187、188、189或190位的一个或多个位置上发生取代，其中
c.取代发生在第188位时，取代氨基酸为Ala、Asn、Cys、Gln、Gly、His、Ile、Leu、Met、Pro、Ser、Thr、Tyr或Val
F、当取代发生在第六环区时，在199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218或219位的一个或多个位置上发生取代，其中
a.取代发生在第199位时，取代氨基酸为Asn、Asp、Gln、Glu、Gly、His、Pro、Ser或Thr；
b.取代发生在第200位时，取代氨基酸为Asn、Asp、Gln、Glu、Gly或Ser；
c.取代发生在第201位时，取代氨基酸为Asn、Asp、Gln、Glu、Pro或Ser；
d.取代发生在第202位时，取代氨基酸为Ala、Cys、Gln、Gly、His、Met、Pro或Ser；
f.取代发生在第204位时，取代氨基酸为Ala、Cys、Gln、Gly、His、Met、Pro或Ser；
g.取代发生在第205位时，取代氨基酸为Ala、Asn、Gln、Gly、His、Ile、Leu、Met、Pro或Ser；
h.取代发生在第206位时，取代氨基酸为Asp或Glu；
i.取代发生在第207位时，取代氨基酸为Asn、Gln、Gly、Pro或Ser；
j.取代发生在第208位时，取代氨基酸为Ala、Asn、Cys、Gln、Gly、His、Ile、Met、Pro或Ser；
k.取代发生在第209位时，取代氨基酸为Asn、Gln、Gly或Ser；、
m.取代发生在第211位时，取代氨基酸为Gln或Ser；
n.取代发生在第212位时，取代氨基酸为Asn、Gln、Gly、Pro或Ser；
o.取代发生在第213位时，取代氨基酸为Ala、Asn、Cys、Gln、Gly、His、Ile、Leu、Met或Pro；
p.取代发生在第214位时，取代氨基酸为Asn、Gln、Gly、Pro或Ser；
r.取代发生在第216位时，取代氨基酸为Ala、Asn、Cvs、Gln、Gly、His、Ile、Met、Pro、Ser、Thr或Val；和
u.取代发生在第219位时，取代氨基酸为Asn、Asp、Gln、Glu、Gly、Pro或Ser；
由此与野生型枯草杆菌蛋白酶DY相比，本枯草杆菌蛋白酶DY变异体对不溶性底物具有降低的吸附作用和增强的水解作用。
2.权利要求1的枯草杆菌蛋白酶DY变异体，其中在第一环区发生一个或多个取代。
3.权利要求1的枯草杆菌蛋白酶DY变异体，其中在第二环区发生一个或多个取代。
4.权利要求1的枯草杆菌蛋白酶DY变异体，其中在第三环区发生一个或多个取代。
5.权利要求1的枯草杆菌蛋白酶DY变异体，其中在第四环区发生一个或多个取代。
6.权利要求1的枯草杆菌蛋白酶DY变异体，其中在第五环区发生一个或多个取代。
7.权利要求1的枯草杆菌蛋白酶DY变异体，其中在第六环区发生一个或多个取代。
8.一种选自硬表面清洗组合物、洗碟剂组合物、口腔清洁组合物、假牙清洗组合物、隐性眼镜组合物和织物清洗组合物的清洗组合物，其特征在于该清洗组合物包含权利要求1-7之一的枯草杆菌蛋白酶DY变异体和一种清洗组合物载体；该清洗组合物优选是硬表面清洗组合物或织物清洗组合物；优选该组合物包含至少约为组合物重量5％的表面活性剂和至少约为组合物重量5％的助洗剂；优选该组合物还包含选自溶剂、缓冲剂、酶、污垢解脱剂、粘土类去污垢剂、分散剂、增白剂、抑泡剂、织物柔软剂、增泡剂、酶稳定剂、漂白剂、染料、香料及其混合物的清洗组合物材料。
9.一种编码权利要求1-7之任一枯草杆菌蛋白酶DY变异体的DNA序列。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/401,575 US6475765B1 (en) | 1995-03-09 | 1995-03-09 | Subtilisin DY variants having decreased adsorption and increased hydrolysis |
| US08/401,575 | 1995-03-09 |
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| CN1183810A true CN1183810A (zh) | 1998-06-03 |
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| CN96193794A Pending CN1183810A (zh) | 1995-03-09 | 1996-03-06 | 具有降低吸附增强水解作用的枯草杆菌蛋白酶dy变异体 |
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| US (1) | US6475765B1 (zh) |
| EP (1) | EP0815243A2 (zh) |
| JP (1) | JPH11501815A (zh) |
| CN (1) | CN1183810A (zh) |
| AU (1) | AU5182796A (zh) |
| BR (1) | BR9607758A (zh) |
| CA (1) | CA2214579C (zh) |
| CZ (1) | CZ279897A3 (zh) |
| HU (1) | HUP9800767A3 (zh) |
| IL (1) | IL117351A0 (zh) |
| IN (1) | IN191825B (zh) |
| MA (1) | MA23821A1 (zh) |
| MX (1) | MX9706847A (zh) |
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| ZA (1) | ZA961821B (zh) |
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| CA2173106C (en) * | 1993-10-14 | 2000-02-15 | Michael Eugene Burns | Bleaching compositions comprising protease enzymes |
| ES2364776T3 (es) * | 1994-02-24 | 2011-09-14 | HENKEL AG & CO. KGAA | Enzimas mejoradas y detergentes que las contienen. |
| US6599730B1 (en) | 1994-05-02 | 2003-07-29 | Procter & Gamble Company | Subtilisin 309 variants having decreased adsorption and increased hydrolysis |
| US5780285A (en) * | 1995-03-03 | 1998-07-14 | Genentech, Inc. | Subtilisin variants capable of cleaving substrates containing dibasic residues |
-
1995
- 1995-03-09 US US08/401,575 patent/US6475765B1/en not_active Expired - Fee Related
-
1996
- 1996-03-04 IL IL11735196A patent/IL117351A0/xx unknown
- 1996-03-06 JP JP8527691A patent/JPH11501815A/ja active Pending
- 1996-03-06 ZA ZA961821A patent/ZA961821B/xx unknown
- 1996-03-06 MX MX9706847A patent/MX9706847A/es unknown
- 1996-03-06 WO PCT/US1996/003006 patent/WO1996028557A2/en not_active Application Discontinuation
- 1996-03-06 HU HU9800767A patent/HUP9800767A3/hu unknown
- 1996-03-06 BR BR9607758A patent/BR9607758A/pt not_active IP Right Cessation
- 1996-03-06 IN IN456DE1996 patent/IN191825B/en unknown
- 1996-03-06 EP EP96908661A patent/EP0815243A2/en not_active Withdrawn
- 1996-03-06 CA CA002214579A patent/CA2214579C/en not_active Expired - Fee Related
- 1996-03-06 AU AU51827/96A patent/AU5182796A/en not_active Abandoned
- 1996-03-06 CN CN96193794A patent/CN1183810A/zh active Pending
- 1996-03-06 CZ CZ972798A patent/CZ279897A3/cs unknown
- 1996-03-07 MA MA24178A patent/MA23821A1/fr unknown
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100462434C (zh) * | 2001-10-31 | 2009-02-18 | 汉高两合股份公司 | 新型碱性蛋白酶-变体和含有该种新型碱性蛋白酶-变体的洗涤剂 |
| CN109061813A (zh) * | 2018-08-29 | 2018-12-21 | 青岛海信宽带多媒体技术有限公司 | 一种光模块 |
| CN112852788A (zh) * | 2019-11-26 | 2021-05-28 | 江南大学 | 一种碱性底物选择性提高的枯草蛋白酶e突变体及其应用 |
| CN112852788B (zh) * | 2019-11-26 | 2022-10-11 | 江南大学 | 一种碱性底物选择性提高的枯草蛋白酶e突变体及其应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| CA2214579A1 (en) | 1996-09-19 |
| HUP9800767A3 (en) | 2001-01-29 |
| CZ279897A3 (cs) | 1998-01-14 |
| MX9706847A (es) | 1997-11-29 |
| BR9607758A (pt) | 1999-05-04 |
| IL117351A0 (en) | 1996-07-23 |
| MA23821A1 (fr) | 1996-10-01 |
| AU5182796A (en) | 1996-10-02 |
| ZA961821B (en) | 1996-09-10 |
| US6475765B1 (en) | 2002-11-05 |
| HUP9800767A2 (hu) | 1998-07-28 |
| WO1996028557A3 (en) | 1997-08-07 |
| WO1996028557A2 (en) | 1996-09-19 |
| JPH11501815A (ja) | 1999-02-16 |
| CA2214579C (en) | 2001-10-30 |
| EP0815243A2 (en) | 1998-01-07 |
| IN191825B (zh) | 2004-01-10 |
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