CN111172124A - 一种羰基还原酶突变体及其在制备(r)-4-氯-3-羟基-丁酸酯中的应用 - Google Patents
一种羰基还原酶突变体及其在制备(r)-4-氯-3-羟基-丁酸酯中的应用 Download PDFInfo
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- CN111172124A CN111172124A CN202010121015.9A CN202010121015A CN111172124A CN 111172124 A CN111172124 A CN 111172124A CN 202010121015 A CN202010121015 A CN 202010121015A CN 111172124 A CN111172124 A CN 111172124A
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- carbonyl reductase
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Abstract
本发明属于生物制药和生物工程技术领域，具体为一种羰基还原酶突变体及其在制备(R)‑4‑氯‑3‑羟基‑丁酸酯中的应用。本发明的羰基还原酶突变体是对如SEQ ID NO.2序列的第85位苯丙氨酸替换为甲硫氨酸得到，或者在此基础上对85位之外的一个或几个位点经过氨基酸残基替换得到。本发明还涉及含有羰基还原酶突变体基因的重组表达载体，含有羰基还原酶突变体基因和葡萄糖脱氢酶基因的工程菌，以及该基因工程菌在不对称还原氯乙酰乙酸酯制备(R)‑4‑氯‑3‑羟基‑丁酸酯中的应用。本发明的羰基还原酶突变体与野生酶相比，对氯乙酰乙酸酯的还原能力明显提高，具有良好的工业应用前景。
Description
技术领域
本发明属于生物制药和生物工程技术领域，具体涉及羰基还原酶YOL151W突变体及其编码基因，涉及含有所述羰基还原酶突变体基因和葡萄糖脱氢酶基因的工程菌的制备方法，涉及该基因工程菌在不对称还原氯乙酰乙酸酯（I），制备(R)-4-氯-3-羟基-丁酸酯（II）中的应用。
背景技术
(R)-4-氯-3-羟基-丁酸酯是一类重要的手性化合物，其结构式如下述反应式中化合物（II）所示，式中R为C1-C8烷基或环烷基，单取代或多取代芳基或芳烷基。化合物（II）被广发应用于许多有价值的药物及活性分子的合成，例如减肥营养补充品L-肉碱[中国专利CN104726507、文献沈阳化工大学学报，2017，31，311-314]。生物不对称还原氯乙酰乙酸酯（I）是制备(R)-4-氯-3-羟基-丁酸酯（II）的一条简便、高效、高选择性、绿色、经济的途径。其中，Kim和Sakai等（文献J. Microbiol. Biotechnol., 2010, 20, 1300-1306;Tetrahedron, 2006, 62, 6143-6149.）报道采用来自于酿酒酵母的羰基还原酶YOL151W催化还原氯乙酰乙酸乙酯生成(R)-4-氯-3-羟基-丁酸乙酯，反应的对映选择性优异（97%ee）。但是，这些报道的底物浓度非常低（≤10g/L,质量百分浓度≤1%），限制了其工业应用前景。通过基于蛋白结构信息的定点突变改造YOL151W，获得对氯乙酰乙酸酯（I）还原能力提升的突变体酶，将其应用于(R)-4-氯-3-羟基-丁酸酯（II）的高底物浓度合成具有良好的工业应用价值。
发明内容
本发明所要解决的技术问题是，针对野生型羰基还原酶YOL151W催化还原氯乙酰乙酸酯（I）反应中底物浓度低的问题，提供一种催化活力高的YOL151W突变体蛋白质、编码该突变体蛋白质的核酸序列，还提供含有该核酸序列的重组表达载体，还提供含有所述羰基还原酶突变体基因和葡萄糖脱氢酶基因的工程菌的制备方法，以及其在不对称还原氯乙酰乙酸酯（I），制备(R)-4-氯-3-羟基-丁酸酯（II）中的应用。
本发明首先提供一种对氯乙酰乙酸酯（I）还原能力高（即催化活力高）的羰基还原酶YOL151W突变体；该YOL151W突变体，是对如SEQ ID NO.2（野生型羰基还原酶YOL151W基因）所示氨基酸序列的蛋白质进行如下突变得到：
（a）将第85位苯丙氨酸替换为甲硫氨酸，或者，
（b）在（a）氨基酸序列的基础上且保持酶催化活力的前提下，对85位之外的一个或几个位点经过氨基酸残基替换，获得的由YOL151W衍生的蛋白质。
优选地，在（a）氨基酸序列的基础上，将第128位的苯丙氨酸、第132位的苯丙氨酸、第162位的缬氨酸分别或共同替换为其它任意氨基酸，获得由YOL151W衍生的且具有还原氯乙酰乙酸酯（I）的蛋白质。
优选的，所述羰基还原酶YOL151W突变体，为野生型羰基还原酶YOL151W的氨基酸残基发生如下突变所获得的突变体：野生型羰基还原酶YOL151W的氨基酸序列中第85位苯丙氨酸替换为甲硫氨酸得到突变体，命名为YOL151WF85M。
本发明中，野生型羰基还原酶YOL151W基因，其序列如SEQ ID NO.1所示。
突变体YOL151WF85M所示的氨基酸序列组成的蛋白质是由基于YOL151W结构信息的定点突变得到。在获得催化活力有明显提高的突变体YOL151WF85M的基础之上，我们将继续对YOL151WF85M进行改造，将酶催化活性中心的另外三个氨基酸残基（即第128位的苯丙氨酸、第132位的苯丙氨酸、第162位的缬氨酸）突变为其它的氨基酸残基得到突变体。这些突变体可以进一步强化该酶对氯乙酰乙酸酯（I）的还原能力。
所述突变体的获得方法具体为：以含有来源于酿酒酵母中的羰基还原酶野生酶YOL151W基因的重组载体pET28b-YOL151W为模板，利用含有F85M突变碱基信息的突变引物，通过PCR方法扩增，得到含有突变体基因YOL151WF85M的重组表达载体pET28b-YOL151WF85M；再以含有突变体基因YOL151WF85M的重组表达载体pET28b-YOL151WF85M为模板，利用含有其它突变位点碱基信息的突变引物，通过PCR方法扩增，得到含有其它相应突变体基因的重组表达载体。
本发明还包括一种分离的核酸，所述核酸编码前述任一羰基还原酶突变体。
本发明还包括含有所述核酸的重组表达载体。
本发明还包括既包含所述的重组表达载体，又包含葡萄糖脱氢酶重组表达载体的基因工程菌。
所述基因工程菌的制备方法较佳地为：将含上述突变信息的重组表达载体，例如pET28b-YOL151WF85M和含有葡萄糖脱氢酶基因的重组表达载体pACYC-GDH共同转化至宿主微生物中，即得到所述基因工程菌。其中所述宿主微生物较佳地为：大肠杆菌（E. coli），更佳的为大肠杆菌BL21(DE3)。将前述两个重组表达质粒共转化至E. coli BL21(DE3)中，即可得本发明优选的基因工程菌株，记为E. coli BL21(DE3)/pET28b-YOL151WF85M/pACYC-GDH。转化方法可选择本领域常规方法，如热激法、电转法等，较佳地选择热激法进行转化即可，热激条件较佳的是：42℃，热激45秒。
培养上述的基因工程菌E. coli BL21(DE3)/pET28b-YOL151WF85M/pACYC-GDH，从培养物中获得全细胞生物催化剂。方法较佳地为：将上述重组大肠杆菌接种至含有卡那霉素（25μg/mL）和氯霉素（12.5μg/mL）的LB培养基中，37℃，150-200rpm培养，培养液的吸光密度OD600达到0.5-1.0（优选0.6），加入0.05-1.0mM（优选0.1mM）异丙基-β-D-硫代半乳糖苷（IPTG）进行诱导，诱导温度为18℃，诱导时间18小时即可得到高效表达的重组基因工程菌全细胞生物催化剂。
本发明提供以上述基因工程菌为催化剂，用于还原氯乙酰乙酸酯（I）生成(R)-4-氯-3-羟基-丁酸酯（II）反应；反应体系包括：底物氯乙酰乙酸酯（I）、酶催化剂、葡萄糖、与水不互溶的有机溶剂，其中，酶催化剂为上述基因工程菌全细胞，或其对应的粗酶液或固定化酶；反应路线为：
其中，R为C1-C8烷基或环烷基，单取代或多取代芳基或芳烷基。
优选地，在起始反应体系中，底物的质量百分浓度为10%-30%（w/v），更优选的为15%-25%。所述工程菌的用量（按湿菌体计算）为底物质量的20%-150%，更优选的为50%-150%。
优选地，在起始反应体系中，葡萄糖的用量为底物摩尔当量的100%-400%，更有选的为150%-300%。
优选地，在反应体系中加入与水不互溶的有机溶剂，其百分浓度为5%-40%（v/v），更优选的为加入甲苯，其百分浓度为15%-30%（v/v）。
作为优选，所述反应的温度为20-50℃，更优选的为25-37℃。
作为优选，反应液的pH为6-10，更优选的为6.5-7.5。
上述反应结束后，反应液需经后处理才能获得成品，所述后处理为：加入乙酸乙酯搅拌4-10分钟后，将反应液在9000-10000 rpm下，离心15-30分钟。收集有机相，并用等体积乙酸乙酯将水相洗涤三次，9000-10000rpm下，离心15-30分钟后收集有机相。合并的有机相用无水硫酸钠干燥后，旋干溶剂得到产品。
本发明的羰基还原酶突变体与野生酶相比，对氯乙酰乙酸酯的还原能力明显提高。本发明获得的羰基还原酶突变体可用于高底物浓度、高立体选择性、绿色、经济合成(R)-4-氯-3-羟基-丁酸酯，具有良好的工业应用前景。
附图说明
图1为羰基还原酶突变体YOL151WF85M的SDS-PAGE电泳图。其中，M:蛋白质Marker;1:纯化得到的羰基还原酶突变体YOL151WF85M。
图2为本发明实施例3中纯化得到的(R)-4-氯-3-羟基-丁酸乙酯的衍生物的手性HPLC图。
具体实施方式
下面结合具体实施例对本发明做进一步详细地说明，但本发明并不限于以下实施例。
实施例1，定点突变和重组表达载体pET28b-YOL151WF85M的构建
定点突变采用TransStart FastPfu Fly DNA polymerase完成。首先设计含有突变点F85M的突变引物：
上游引物：GGCCTCTCCAATGTGCTTTGATATCACTGACAGT
下游引物：TATCAAAGCACATTGGAGAGGCCGTATGTAGAAC；
PCR反应体系（50μL）：野生型pET28b-YOL151W模板50ng，10μL5×TransStart®FastPfuFly Buffer，8μL dNTP（各2.5mM），一对突变引物各1μL（10μM），10μL5×PCR Stimulant，2.5个单位的TransStart FastPfu Fly DNA polymerase，加无菌蒸馏水至50μL。
PCR扩增程序：（1）98℃变性3min；（2）98℃变性20s，（3）65℃退火30s，（4）72℃延伸8min，步骤（2）-（4）共进行20个循环，最后72℃延伸10min，4℃保藏产物。
扩增得到的PCR产物经内切酶DpnI 37℃消化1小时候后转化E. coli DH5,涂布于含卡那霉素（50μg/mL）的LB固体培养基，于37℃培养过夜，挑选阳性克隆子，接种于含卡那霉素（50μg/mL）的LB培养基，培养约8小时，提取质粒，测序，序列正确的即为重组表达载体pET28b-YOL151WF85M。
实施例2，基因工程菌E.coli BL21(DE3)/pET28b-YOL151WF85M/pACYC-GDH的构建及诱导表达
用实施例1中构建的质粒pET28b-YOL151WF85M及实验室保存的pACYC-GDH来共同转化表达宿主E. coli BL21(DE3)，筛选得到阳性克隆子，命名此工程菌为E. coli BL21(DE3)/pET28b-YOL151WF85M/pACYC-GDH。将该工程菌接种到5mL含卡那霉素（25μg/mL）和氯霉素（12.5μg/mL）的LB液体培养基活化8h（37℃，200rpm）。取上述活化的培养物，以1/100的接种量转接到500mL含卡那霉素（25μg/mL）和氯霉素（12.5μg/mL）的LB液体培养基中培养（37℃，200rpm），当培养液的吸光密度OD600达到0.6，加入0.1mMIPTG进行诱导，诱导温度为18℃，诱导时间18小时。离心收集细胞（湿菌体），即为工程菌全细胞生物催化剂。
实施例3，工程菌E. coli BL21(DE3)/pET28b-YOL151WF85M/pACYC-GDH全细胞催化不对称合成(R)-4-氯-3-羟基-丁酸乙酯（百克级）
将氯乙酰乙酸乙酯（346.8g）加入到反应釜中，搅拌。加入甲苯（534mL），温度维持在30
℃。加入葡萄糖 （570.9g）,并搅拌5分钟。另一方面，把全细胞催化剂260g加入到1.2L
100mM磷酸盐缓冲液（pH6.7）中，并搅拌均匀，再把这个菌泥悬浊液加入到上述甲苯反应混
合物中。反应开始后时时监控pH，用2M K2CO3溶液维持pH在6.7。当GC-MS监测反应完全后，加
入300 mL乙酸乙酯，搅拌5分钟后，将反应液在9500rpm下离心20分钟。收集有机相，并用等
体积乙酸乙酯将水相萃取三次，9500rpm下离心20分钟后收集有机相。合并的有机相用无水
硫酸钠干燥，旋干溶剂得产物319.4g（91%收率）。旋光测定结果为[α]25 D =+21.5(c=5.0,
CHCl3), 文献值为[α]25 D =+22.3(c=5.0,CHCl3)[文献Org. Biomol. Chem., 2011, 9,
5463-5468]。通过将产物(R)-4-氯-3-羟基-丁酸乙酯衍生化成
，再用手
性HPLC测得ee值为97%，检测条件为: AS-H色谱柱，流动相为正己烷:异丙醇=85: 15的溶
液，流速0.5mL/min，柱温24℃，波长215nm（附图二）。1H NMR (CDCl3, 400 MHz):δ/ppm
4.31-4.25 (m, 1H), 4.18 (q,J = 7.2 Hz, 2H), 3.63-3.58 (m, 2H), 3.15 (s, 1H),
2.68-2.61 (m, 2H), 1.30 (t, J = 7.2 Hz, 3H). 13C NMR (CDCl3, 100 MHz): δ/ppm
172.0, 67.8, 60.6, 48.2, 38.7, 14.0。
实施例4，工程菌E. coli BL21(DE3)/pET28b-YOL151WF85M/pACYC-GDH全细胞催化不对称合成(R)-4-氯-3-羟基-丁酸甲酯（百克级）
将氯乙酰乙酸甲酯（115.6g）加入到反应釜中，搅拌。加入甲苯（115mL），温度维持在30
℃。加入葡萄糖 （253.7g）,并搅拌5分钟。另一方面，把全细胞催化剂115.6g加入到463mL
100mM磷酸盐缓冲液（pH6.7）中，并搅拌均匀，再把这个菌泥悬浊液加入到上述甲苯反应混
合物中。反应开始后时时监控pH，用2M K2CO3溶液维持pH在6.7。当GC-MS监测反应完全后，加
入100 mL乙酸乙酯，搅拌5分钟后，将反应液在9500rpm下离心20分钟。收集有机相，并用等
体积乙酸乙酯将水相萃取三次，9500rpm下离心20分钟后收集有机相。合并的有机相用无水
硫酸钠干燥，旋干溶剂得产物103.1g（88%收率）。通过将产物(R)-4-氯-3-羟基-丁酸甲酯衍
生化成
，再用手性HPLC测得ee值为98%，检测条件为: AS-H色谱柱，流动相
为正己烷:异丙醇=85: 15的溶液，流速0.5mL/min，柱温24℃，波长215nm。1H NMR (CDCl3,
400 MHz):δ/ppm 4.31-4.24 (m, 1H), 3.73 (s, 3H), 3.64-3.57 (m, 2H), 3.06 (s,
1H), 2.68-2.61 (m, 2H). 13C NMR (CDCl3, 100 MHz):δ/ppm 172.1, 67.7, 51.9,
48.3, 38.3。
实施例5，工程菌E. coli BL21(DE3)/pET28b-YOL151WF85M/pACYC-GDH全细胞催化不对称合成(R)-4-氯-3-羟基-丁酸叔丁酯（百克级）
将氯乙酰乙酸乙酯（173.4g）加入到反应釜中，搅拌。加入甲苯（534mL），温度维持在30
℃。加入葡萄糖 （325.1g）,并搅拌5分钟。另一方面，把全细胞催化剂346.8g加入到1.2L
100mM磷酸盐缓冲液（pH6.7）中，并搅拌均匀，再把这个菌泥悬浊液加入到上述甲苯反应混
合物中。反应开始后时时监控pH，用2M K2CO3溶液维持pH在6.7。当GC-MS监测反应完全后，加
入600 mL乙酸乙酯，搅拌5分钟后，将反应液在9500rpm下离心20分钟。收集有机相，并用等
体积乙酸乙酯将水相萃取三次，9500rpm下离心20分钟后收集有机相。合并的有机相用无水
硫酸钠干燥，旋干溶剂得产物157.7g（90%收率）。通过将产物(R)-4-氯-3-羟基-丁酸叔丁酯
衍生化成
，再用手性HPLC测得ee值为97%，检测条件为: AS-H色谱柱，流动
相为正己烷:异丙醇=90: 10的溶液，流速0.5mL/min，柱温24℃，波长215nm。1H NMR (CDCl3,
400 MHz): δ/ppm 4.30-4.23 (m, 1H), 3.66-3.58 (m, 2H), 3.08 (s, 1H), 2.68-2.61
(m, 2H), 1.29 (s, 9H). 13C NMR (CDCl3, 100 MHz): δ/ppm 173.0, 82.1, 69.5,
51.5, 38.6, 28.8。
以上所述仅为本发明的较佳实施例，只为说明本发明的技术构思和特点，其目的在于让熟悉此项技术的人士能够了解本发明的内容并据以实施，并不能以此限制本发明的保护范围。凡根据本发明精神实质所作的任何修改、等同替换、改进等，都应涵盖在本发明的保护范围之内。
序列表
<110> 复旦大学
<120> 一种羰基还原酶突变体及其在制备(R)-4-氯-3-羟基-丁酸酯中的应用
<130> 001
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1029
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgtcagttt tcgtttcagg tgctaacggg ttcattgccc aacacattgt cgatctcctg 60
ttgaaggaag actataaggt catcggttct gccagaagtc aagaaaaggc cgagaattta 120
acggaggcct ttggtaacaa cccaaaattc tccatggaag ttgtcccaga catatctaag 180
ctggacgcat ttgaccatgt tttccaaaag cacggcaagg atatcaagat agttctacat 240
acggcctctc cattctgctt tgatatcact gacagtgaac gcgatttatt aattcctgct 300
gtgaacggtg ttaagggaat tctccactca attaaaaaat acgccgctga ttctgtagaa 360
cgtgtagttc tcacctcttc ttatgcagct gtgttcgata tggcaaaaga aaacgataag 420
tctttaacat ttaacgaaga atcctggaac ccagctacct gggagagttg ccaaagtgac 480
ccagttaacg cctactgtgg ttctaagaag tttgctgaaa aagcagcttg ggaatttcta 540
gaggagaata gagactctgt aaaattcgaa ttaactgccg ttaacccagt ttacgttttt 600
ggtccgcaaa tgtttgacaa agatgtgaaa aaacacttga acacatcttg cgaactcgtc 660
aacagcttga tgcatttatc accagaggac aagataccgg aactatttgg tggatacatt 720
gatgttcgtg atgttgcaaa ggctcattta gttgccttcc aaaagaggga aacaattggt 780
caaagactaa tcgtatcgga ggccagattt actatgcagg atgttctcga tatccttaac 840
gaagacttcc ctgttctaaa aggcaatatt ccagtgggga aaccaggttc tggtgctacc 900
cataacaccc ttggtgctac tcttgataat aaaaagagta agaaattgtt aggtttcaag 960
ttcaggaact tgaaagagac cattgacgac actgcctccc aaattttaaa atttgagggc 1020
agaatataa 1029
<210> 2
<211> 342
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Ser Val Phe Val Ser Gly Ala Asn Gly Phe Ile Ala Gln His Ile
1 5 10 15
Val Asp Leu Leu Leu Lys Glu Asp Tyr Lys Val Ile Gly Ser Ala Arg
20 25 30
Ser Gln Glu Lys Ala Glu Asn Leu Thr Glu Ala Phe Gly Asn Asn Pro
35 40 45
Lys Phe Ser Met Glu Val Val Pro Asp Ile Ser Lys Leu Asp Ala Phe
50 55 60
Asp His Val Phe Gln Lys His Gly Lys Asp Ile Lys Ile Val Leu His
65 70 75 80
Thr Ala Ser Pro Phe Cys Phe Asp Ile Thr Asp Ser Glu Arg Asp Leu
85 90 95
Leu Ile Pro Ala Val Asn Gly Val Lys Gly Ile Leu His Ser Ile Lys
100 105 110
Lys Tyr Ala Ala Asp Ser Val Glu Arg Val Val Leu Thr Ser Ser Tyr
115 120 125
Ala Ala Val Phe Asp Met Ala Lys Glu Asn Asp Lys Ser Leu Thr Phe
130 135 140
Asn Glu Glu Ser Trp Asn Pro Ala Thr Trp Glu Ser Cys Gln Ser Asp
145 150 155 160
Pro Val Asn Ala Tyr Cys Gly Ser Lys Lys Phe Ala Glu Lys Ala Ala
165 170 175
Trp Glu Phe Leu Glu Glu Asn Arg Asp Ser Val Lys Phe Glu Leu Thr
180 185 190
Ala Val Asn Pro Val Tyr Val Phe Gly Pro Gln Met Phe Asp Lys Asp
195 200 205
Val Lys Lys His Leu Asn Thr Ser Cys Glu Leu Val Asn Ser Leu Met
210 215 220
His Leu Ser Pro Glu Asp Lys Ile Pro Glu Leu Phe Gly Gly Tyr Ile
225 230 235 240
Asp Val Arg Asp Val Ala Lys Ala His Leu Val Ala Phe Gln Lys Arg
245 250 255
Glu Thr Ile Gly Gln Arg Leu Ile Val Ser Glu Ala Arg Phe Thr Met
260 265 270
Gln Asp Val Leu Asp Ile Leu Asn Glu Asp Phe Pro Val Leu Lys Gly
275 280 285
Asn Ile Pro Val Gly Lys Pro Gly Ser Gly Ala Thr His Asn Thr Leu
290 295 300
Gly Ala Thr Leu Asp Asn Lys Lys Ser Lys Lys Leu Leu Gly Phe Lys
305 310 315 320
Phe Arg Asn Leu Lys Glu Thr Ile Asp Asp Thr Ala Ser Gln Ile Leu
325 330 335
Lys Phe Glu Gly Arg Ile
340
Claims (9)
1. 一种羰基还原酶突变体；其特征在于，是对如SEQ ID NO.2所示氨基酸序列的蛋白质进行如下突变得到：
（a）将第85位苯丙氨酸替换为甲硫氨酸，或者
（b）在（a）氨基酸序列的基础上且保持酶催化活力的前提下，对85位之外的一个或几个位点经过氨基酸残基替换。
2.根据权利要求1所述的羰基还原酶YOL151W突变体，其特征在于，所述（b）中所述位点为的第128位的苯丙氨酸、第132位的苯丙氨酸、第162位的缬氨酸，分别或共同替换为其它任意氨基酸。
3.一种分离的核酸，其特征在于，所述核酸编码如权利要求1或2中所述的任一羰基还原酶突变体。
4.一种包含如权利要求3所述的核酸的重组表达载体。
5.一种既包含如权利要求4所述的重组表达载体又包含葡萄糖脱氢酶重组表达载体的基因工程菌。
6.一种如权利要求1或2所述的羰基还原酶突变体在还原氯乙酰乙酸酯（I）生成(R)-4-氯-3-羟基-丁酸酯（II）反应中的应用，其特征在于，反应体系包括：底物氯乙酰乙酸酯（I）、酶催化剂、葡萄糖、与水不互溶的有机溶剂，其中，酶催化剂为如权利要求5所述的基因工程菌全细胞，或其对应的粗酶液或固定化酶；反应路线为：
其中，R为C1-C8烷基或环烷基，单取代或多取代芳基或芳烷基。
7.根据权利要求6所述的应用，其特征在于，在起始反应体系中，底物的质量百分浓度为10%-30%（w/v）；所述工程菌的用量按湿菌体计算为底物质量的20%-150%；葡萄糖的用量为底物摩尔当量的100%-400%；在反应体系中的与水不互溶的有机溶剂，其百分浓度为5%-40%（v/v）。
8.根据权利要求6所述的应用，其特征在于，在起始反应体系中，反应的温度为20-50℃，反应体系的pH为6-10。
9. 根据权利要求6所述的应用，其特征在于，上述反应结束后，反应液经后处理得到成品，所述后处理为：加入乙酸乙酯搅拌4-10分钟后，将反应液在9000-10000 rpm下，离心15-30分钟；收集有机相，并用等体积乙酸乙酯将水相洗涤三次，9000-10000rpm下，离心15-30分钟后收集有机相；合并的有机相用无水硫酸钠干燥后，旋干溶剂。
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