CN114686537B - 一种多酶级联催化l-氨基酸生成n-甲基-l-氨基酸的方法及其应用 - Google Patents
一种多酶级联催化l-氨基酸生成n-甲基-l-氨基酸的方法及其应用 Download PDFInfo
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明公开了一种多酶级联催化L‑氨基酸生成N‑甲基‑L‑氨基酸的方法及其应用。该方法是以天然L‑氨基酸为底物，利用L‑氨基酸氧化酶、N‑甲基氨基酸脱氢酶和NADPH再生酶同步级联催化L‑氨基酸反应生成N‑甲基‑L‑氨基酸。本发明方法高效、成本低，底物范围广，可以适应L‑苯丙氨酸、L‑谷氨酸、L‑亮氨酸、L‑赖氨酸、L‑缬氨酸和L‑异亮氨酸等，将其转化为相应N‑甲基‑L‑氨基酸，产物转化率高，具有良好的工业应用前景。
Description
技术领域
本发明属于酶工程技术领域，特别涉及一种多酶级联催化L-氨基酸生成N-甲基-L-氨基酸的方法及其应用。
背景技术
N-α-烷基化是调节手性α-氨基酸活性的一种机制。N-甲基-α-氨基酸在制药和精细化工等领域应用广泛，是制备多种生物活性分子的重要组成部分，如抗生素万古霉素、免疫抑制剂环孢素、细胞抑制放线菌素和铁载体需氧蛋白等。因此，对N-甲基-L-氨基酸的合成方法进行研究具有重要价值。
与其他手性化合物相似，N-甲基-L-氨基酸的制备方法主要分为化学合成法和生物合成法。化学合成法依赖于N-烷基化过程或还原胺化过程。这些合成路线通常需要使用有毒试剂和危险溶剂，如自燃金属氢化物或有基因毒性的烷基化剂，反应过程中会产生大量污染环境的废物，有些反应还需要特殊的温度和压力环境，并且部分方法获得的产物纯度较低，需要复杂的后处理程序。
随着生物技术的发展，生物法制备N-甲基-L-氨基酸越来越受到人们的关注。生物法合成N-甲基-L-氨基酸具有绿色清洁、反应条件温和以及转化率高的优势。目前的合成方法包括利用N-甲基转移酶或几种脱氢酶。N-甲基转移酶(EC2.1.1)在自然界中负责许多氨基酸、肽和蛋白质的N-α-甲基化，产生单甲基化、二甲基化或三甲基化产物，缺点在于这种酶通常对底物具有高度选择性，底物范围较窄。一些脱氢酶能催化α-酮酸或亚胺与胺供体进行还原胺化反应产生N-甲基-L-氨基酸，包括Opines脱氢酶、N-甲基氨基酸脱氢酶、酮亚胺还原酶和亚胺还原酶等。该类反应需要特定的底物，有些底物溶解度较差、价格昂贵，不适用于工业化的生产。
发明内容
本发明的首要目的在于克服现有技术的缺点与不足，提供一种多酶级联催化L-氨基酸生成N-甲基-L-氨基酸的方法，该方法联合利用L-氨基酸氧化酶和N-甲基氨基酸脱氢酶，以天然L-氨基酸为底物生成相应N-甲基-L-氨基酸。
本发明的另一目的在于提供所述多酶级联催化L-氨基酸生成N-甲基-L-氨基酸的方法的应用。
本发明的目的通过下述技术方案实现：
一种多酶级联催化L-氨基酸生成N-甲基-L-氨基酸的方法，是以L-氨基酸为底物，利用L-氨基酸氧化酶、N-甲基氨基酸脱氢酶和NADPH再生酶同步级联催化L-氨基酸反应生成N-甲基-L-氨基酸。
所述的L-氨基酸包括L-苯丙氨酸、L-谷氨酸、L-亮氨酸、L-赖氨酸、L-缬氨酸和L-异亮氨酸中的至少一种；优选为L-苯丙氨酸、L-谷氨酸和L-亮氨酸中的至少一种。
所述的N-甲基-L-氨基酸为N-甲基-L-苯丙氨酸、N-甲基-L-谷氨酸、N-甲基-L-亮氨酸、高脯氨酸、N-甲基-L-缬氨酸和N-甲基-L-异亮氨酸中的至少一种；优选为N-甲基-L-苯丙氨酸、N-甲基-L-谷氨酸和N-甲基-L-亮氨酸中的至少一种。
所述的L-氨基酸氧化酶优选为祖先L-氨基酸氧化酶，其氨基酸序列如SEQ ID NO:1所示。
所述的N-甲基氨基酸脱氢酶优选为来源于恶臭假单胞菌的N-甲基氨基酸脱氢酶，其氨基酸序列在NCBI的登陆号为AB190215.1。
所述的NADPH再生酶优选为来源于伯克霍尔德菌的甲酸脱氢酶，其氨基酸序列在NCBI的登陆号为ACF35003.1。
所述的L-氨基酸氧化酶、N-甲基氨基酸脱氢酶和NADPH再生酶均可以通过常规方法获得，如通过原核表达，经蛋白纯化获得。
所述的原核表达的宿主菌优选为大肠杆菌；更优选为大肠杆菌BL21(DE3)。
所述的蛋白纯化可采用Ni柱亲和层析法进行纯化。
所述的反应的体系为pH 7.5～9.0的Tris-HCl缓冲液体系或pH 9.5～10.0的Na2CO3-NaHCO3缓冲液体系；优选为pH 9.5～10.0的Na2CO3-NaHCO3缓冲液体系；更优选为pH9.5的Na2CO3-NaHCO3缓冲液体系。
所述的反应的体系为：0.01～1mg/mL L-氨基酸脱氨酶，1～5mg/mL N-甲基氨基酸脱氢酶，0.2～1mg/mL NADPH再生酶，10～100mM L-氨基酸，10～200mM胺供体，10～300mM甲酸盐，1～10mM NADP+，20～200U/mL过氧化氢酶，pH 7.5～10。
所述的甲酸盐优选为甲酸钠。
所述的甲酸盐与L-氨基酸的摩尔比为1～5:1；优选为3:1。
所述的胺供体为甲胺。
所述的胺供体与L-氨基酸的摩尔比为1～5:1；优选为2:1。
所述的NADP+与L-氨基酸的摩尔比可为1:1～10；优选为1:5～10；更优选为1:10。
所述的NADP+为氧化型辅酶II。
所述的反应的体系优选为：0.01mg/mL L-氨基酸脱氨酶，1mg/mL N-甲基氨基酸脱氢酶，0.4mg/mL NADPH再生酶，10～100mM L-氨基酸，20～200mM胺供体，30～300mM甲酸盐，5～10mM NADP+，50U/mL过氧化氢酶，pH 9.5。
所述的反应的温度为20～50℃；优选为37℃。
所述的反应的转速为100～200rpm；优选为160rpm。
所述的反应的时间为6～24h；优选为12～24h；更优选为12h。
所述的多酶级联催化L-氨基酸生成N-甲基-L-氨基酸的方法在制备N-甲基-L-氨基酸中的应用。
所述的N-甲基-L-氨基酸为N-甲基-L-苯丙氨酸、N-甲基-L-谷氨酸、N-甲基-L-亮氨酸、L-高脯氨酸、N-甲基-L-缬氨酸和N-甲基-L-异亮氨酸中的至少一种；优选为N-甲基-L-苯丙氨酸、N-甲基-L-谷氨酸和N-甲基-L-亮氨酸中的至少一种。
本发明所提供的催化L-氨基酸生成N-甲基-L-氨基酸方法中，存在辅因子再生系统，其中因子再生系统为：L-氨基酸氧化酶催化L-氨基酸氧化生成α-酮酸，N-甲基氨基酸脱氢酶催化α-酮酸还原为N-甲基-L-氨基酸同时NADPH被氧化成NADP+，NADPH再生酶将NADP+还原为NADPH，生成的NADPH重新参与到α-酮酸的还原胺化(图1)。
本发明相对于现有技术具有如下的优点及效果：
(1)本发明开发了一种能高效、低成本、体外制备N-甲基-L-氨基酸的新方法，该方法以廉价易得的天然L-氨基酸为底物，利用L-氨基酸氧化酶、N-甲基氨基酸脱氢酶和NADPH再生酶催化合成N-甲基-L-氨基酸；其中，底物范围较广(将底物L-氨基酸转化为相应N-甲基-L-氨基酸)，可以适应L-苯丙氨酸、L-谷氨酸、L-亮氨酸、L-赖氨酸、L-缬氨酸和L-异亮氨酸等，产率在47％～100％之间，具有较好的工业应用前景。
(2)本发明中多酶级联催化合成N-甲基-L-氨基酸的方法，是以L-氨基酸为底物，并对反应的各影响因素，包括反应温度、pH值、甲酸钠与甲胺浓度等进行了优化，提高了底物转化率高和产物(N-甲基-L-氨基酸)转化率。
附图说明
图1是L-氨基酸生成相应N-甲基-L-氨基酸的级联反应途径与NADPH再生途径偶联示意图(图中，R基团为：-CH2-C6H5、-(CH2)2-COOH、-C4H8、-CH2-CH(CH3)2或-CH-(CH3)2)。
图2是SDS-PAGE验证L-氨基酸氧化酶、N-甲基氨基酸脱氢酶和甲酸脱氢酶的表达结果图(M：DNAMarker；泳道1：AncLAAO；泳道2：NMAADH；泳道3：BsFDH)。
图3是反应温度和pH对级联反应催化L-苯丙氨酸生成N-甲基-L-苯丙氨酸的影响图；其中，A为反应温度对级联反应催化L-苯丙氨酸生成N-甲基-L-苯丙氨酸的影响；B为pH对级联反应催化L-苯丙氨酸生成N-甲基-L-苯丙氨酸的影响。
图4是甲酸钠以及甲胺与底物的初始摩尔比对级联反应催化L-苯丙氨酸生成N-甲基-L-苯丙氨酸的影响图；其中，A为甲酸钠与底物的初始摩尔比对级联反应催化L-苯丙氨酸生成N-甲基-L-苯丙氨酸的影响；B为甲胺与底物的初始摩尔比对级联反应催化L-苯丙氨酸生成N-甲基-L-苯丙氨酸的影响。
图5是级联反应催化L-氨基酸生成N-甲基-L-氨基酸的HPLC检测结果图；其中，(a)～(f)为标准品液相检测图谱和样品检测图谱(图中，A：N-甲基-L-苯丙氨酸和L-苯丙氨酸标准品液相检测图谱，B：样品L-苯丙氨酸检测图谱；C：N-甲基-L-谷氨酸和L-谷氨酸标准品液相检测图谱，D：样品L-谷氨酸检测图谱；E：N-甲基-L-亮氨酸和L-亮氨酸标准品液相检测图谱，F：样品L-亮氨酸检测图谱；G：L-高脯氨酸和L-赖氨酸标准品液相检测图谱，H：样品L-高脯氨酸检测图谱；I：N-甲基-L-缬氨酸和L-缬氨酸标准品液相检测图谱，J：样品L-缬氨酸检测图谱；K：N-甲基-L-异亮氨酸和L-异亮氨酸标准品液相检测图谱，L：样品L-异亮氨酸检测图谱)。
具体实施方式
下面结合实施例对本发明作进一步详细的描述，但本发明的实施方式不限于此。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。下列实施例中未注明具体实验条件的试验方法，通常按照常规实验条件。除非特别说明，本发明所用试剂和原材料均可通过市售获得。
实施例1：L-氨基酸生成N-甲基-L-氨基酸的相关酶的重组表达
(1)将祖先L-氨基酸氧化酶(AncLAAO，SEQ ID NO.1)(序列来自于文献DOI:10.1038/s42004-020-00432-8)编码基因、恶臭假单胞菌N-甲基氨基酸脱氢酶(NMAADH)(NCBI的登陆号：AB190215.1)编码基因和伯克霍尔德菌甲酸脱氢酶(BsFDH)(NCBI的登陆号：ACF35003.1)编码基因交由金斯瑞公司进行密码子优化与合成，分别插入到常规市售pET-28a载体的Xho I位点和Nde I位点之间，酶切分析鉴定，获得相应的重组质粒。
祖先L-氨基酸氧化酶(SEQ ID NO.1)：
MGTHYTFGKEITDKPLPTQVKVAIVGAGMSGLYSAWRLQQEANCQDLAIFERSNRTGGRLDSDLIEFKNLRSETPKTITVKEEQGGMRFLFDGMDDLMALFLKLNLQDDIVPFPMNSGGNNRLFFRGESFSVEDAQQDDYAIWSHLYNLDQSEQGVNPKDIVNVVFNRILEANPQFQQRPEVRGPEFWQAFRLECQWQGQTLNEWTLWDLYTDMGYSQECINMLYRVLGFNGTFLSQMNAGVAYQLLEDFPAGVQFKTFKDGFSTLPNKLVEEVGTDNIHLQTSIEEIDFAEESGLYSLHYSHTDEHGRVHKGQVKAEKVILGLPRLALEKLFVRSNAFNRLDKDRSEQLWNTLQSASNQPLLKINLYYDSAWWGRGTTGRPAVEFGPNFADLPTGSVYPFYAVNDELAAALMYQERSTNPSKAVQAKLDRIGNEKYERPAALTIYCDYLNINFWSNLQNIGETYHHPHQDDYVEDVPADIYPASTAVVEQATRFFKDIFNTHYVPEPILTSARIWEGSVRFDIPASRQFGFGVHQWAVGANDKEVMATLAEPLPNLFTCGEAFSDYQGWVEGALRSTDLALEKGFGLKPLSQVYFENTNISSSDAIKAVYEENSSKLINQYIETNFSANTAPIEKTADVDSVIGVNLSYFDTK。
(2)利用化学法将各重组质粒导入大肠杆菌BL21(DE3)感受态细胞，涂布于含有50ug/mL卡那霉素的LB固体培养基，37℃倒置过夜培养，挑取单克隆菌株接种到5～20mL LB液体培养基中，在37℃、180rpm振荡培养，以1％(v/v)的接种量分别转接到含有50μg/mL卡那霉素的100mL LB液体培养基中，37℃、180rpm培养2～3h至OD 600为0.6～0.8时，加入终浓度为0.1mM的异丙基-β-D-硫代半乳糖苷(IPTG)进行诱导，在16℃继续诱导20h，离心收集菌体(8000rpm，5min)，用pH 6.5～9.5的PB缓冲液或Tris-HCl缓冲液重悬菌体，进行超声破碎(350W，工作2s、间歇3s，20min)，破碎结束后离心(12000rpm，10min)收集可溶的上清表达组分(粗酶液)。
实施例2：多酶级联催化L-氨基酸生成N-甲基-L-氨基酸相关酶的蛋白纯化
利用Ni柱亲和层析法将上述破碎后的可溶上清表达组分进行分离纯化，得到L-氨基酸氧化酶、N-甲基氨基酸脱氢酶和甲酸脱氢酶。具体操作为：用5～10倍柱体积的平衡缓冲液(pH 7.5 50mM Tris-HCl，50mM咪唑，500mM NaCl)平衡介质；将实施例1中破碎后得到的粗酶液用0.45μm滤头过滤；以1mL/min流速上样；上样结束后用10～20倍柱体积缓冲液(pH 7.5 50mM Tris-HCl，50mM咪唑，500mM NaCl)清洗杂蛋白；接着用5～10倍柱体积洗脱缓冲液(pH 7.5 50mM Tris-HCl，200mM咪唑，500mM NaCl)进行洗脱，收集洗脱液；将洗脱液用50mM pH 9.5的Na2CO3-NaHCO3缓冲液稀释、超滤浓缩(10KDa)，重复5～10次，除去咪唑；将超滤后得到的各目的蛋白采用Bradford法测定浓度，并分装保存于-80℃。各目的蛋白利用SDS-PAGE进行验证，结果如图2所示。
实施例3：反应温度和缓冲液pH值对催化合成N-甲基-L-苯丙氨酸的影响
探究反应温度和pH对多酶级联催化L-苯丙氨酸合成N-甲基-L-苯丙氨酸的影响，具体步骤如下：
(1)反应体系包含50mM L-苯丙氨酸(L-Phe)、100mM甲胺、100mM甲酸钠、5mM NADP+(氧化型辅酶II，购自上海麦克林生化科技有限公司)以及50U/mL过氧化氢酶(购自生工生物工程上海(股份)有限公司)，加入终浓度为0.1mg/mL的L-氨基酸脱氨酶、1mg/mL的N-甲基氨基酸脱氢酶、0.4mg/mL的甲酸脱氢酶进行反应，温度范围选择20～45℃(具体为：20℃、30℃、37℃、45℃)，缓冲液选择pH 7.5～9.0的Tris-HCl缓冲液(pH值为7.5、8.0、8.5、9.0)以及pH 9.5～10.0的Na2CO3-NaHCO3缓冲液(pH值为9.5、10.0)。
(2)①L-氨基酸氧化酶催化L-苯丙氨酸氧化生成苯丙酮酸(PPA)，PPA采用铁离子显色法进行检测。10mL铁离子显色剂的配制：FeCl3溶解于6mL二甲基亚砜(DMSO)，加入3.8mL去离子水进行稀释，再加入200μL冰醋酸使溶液呈酸性，FeCl3终浓度为100mM。取适量样品加入铁离子显色液中，混合均匀后于室温放置，颜色稳定后利用紫外分光光度计检测其在640nm波长处的吸光值。苯丙酮酸浓度的校准曲线：y＝0.1619x+0.0003361，x为苯丙酮酸浓度，mM。
②利用高效液相色谱法(HPLC)检测N-甲基-L-苯丙氨酸(N-Me-L-Phe)，检测条件：色谱柱为大赛璐CR(+)；流动相为pH 1.1～1.25高氯酸水溶液；检测波长210nm；柱温30℃；流量0.5mL/min；进样量20μL。利用该液相检测方法，测定多酶级联催化L-苯丙氨酸生成的N-甲基-L-苯丙氨酸的浓度。
结果如图3所示，反应温度在37～45℃时催化速率较高，考虑到温度对酶稳定性的影响，优选37℃为级联反应温度；当pH在9.5～10.0时产率达到最高，优选pH为9.5的Na2CO3-NaHCO3缓冲液作为反应溶液。
实施例4：甲酸钠和甲胺与底物的初始摩尔比对催化合成N-甲基-L-苯丙氨酸的影响
甲酸铵是辅酶循环的底物，甲胺作为还原胺化反应的胺供体，两者浓度均会对反应速率产生影响。以实施例3的反应体系，探究反应组分与底物的初始摩尔比对多酶级联催化L-苯丙氨酸合成N-甲基-L-苯丙氨酸的影响。甲胺或甲酸钠与底物(L-Phe)的摩尔浓度之比选择1:1，2:1，3:1，4:1，5:1，即体系中L-Phe浓度为50mM，分别加入50mM，100mM，150mM，200mM，250mM的甲酸钠或甲胺。再利用铁离子显色法检测苯丙酮酸(PPA)，高效液相色谱法检测N-甲基-L-苯丙氨酸(N-Me-L-Phe)，具体步骤同实施例3。
结果如图4所示，当甲酸钠与底物的摩尔浓度比在3:1时，产物得率得到显著提升，因此甲酸钠与底物的摩尔浓度比优选为3:1；随着甲胺浓度的增加，N-甲基-L-苯丙氨酸的产率随之增加，考虑经济性，选择甲胺与底物的摩尔浓度比为2:1。
实施例5：三酶偶合催化L-苯丙氨酸生成N-甲基-L-苯丙氨酸
将终浓度为0.1mg/mL L-氨基酸脱氨酶、1mg/mL N-甲基氨基酸脱氢酶、0.4mg/mL甲酸脱氢酶、100mM L-苯丙氨酸、200mM甲胺、300mM甲酸钠、10mM NADP+以及50U/mL过氧化氢酶在pH 9.5的Na2CO3-NaHCO3缓冲液中进行多酶级联催化反应，37℃、160rpm反应12h。
实施例6：三酶偶合催化L-谷氨酸生成N-甲基-L-谷氨酸
将终浓度为0.1mg/mL L-氨基酸脱氨酶、1mg/mL N-甲基氨基酸脱氢酶、0.4mg/mL甲酸脱氢酶、10mM L-谷氨酸、20mM甲胺、30mM甲酸钠、1mM NADP+以及50U/mL过氧化氢酶在pH 9.5的Na2CO3-NaHCO3缓冲液中进行多酶级联催化反应，37℃、160rpm反应12h。
实施例7：三酶偶合催化L-亮氨酸生成N-甲基-L-亮氨酸
将终浓度为0.1mg/mL L-氨基酸脱氨酶、1mg/mL N-甲基氨基酸脱氢酶、0.4mg/mL甲酸脱氢酶、10mM L-亮氨酸、20mM甲胺、30mM甲酸钠、1mM NADP+以及50U/mL过氧化氢酶在pH 9.5的Na2CO3-NaHCO3缓冲液中进行多酶级联催化反应，37℃、160rpm反应12h。
实施例8：三酶偶合催化L-赖氨酸生成L-高脯氨酸
将终浓度为0.1mg/mL L-氨基酸脱氨酶、1mg/mL N-甲基氨基酸脱氢酶、0.4mg/mL甲酸脱氢酶、10mM L-赖氨酸、20mM甲胺、30mM甲酸钠、1mM NADP+以及50U/mL过氧化氢酶在pH 9.5的Na2CO3-NaHCO3缓冲液中进行多酶级联催化反应，37℃、160rpm反应12h。
实施例9：三酶偶合催化L-缬氨酸生成N-甲基-L-缬氨酸
将终浓度为0.1mg/mL L-氨基酸脱氨酶、1mg/mL N-甲基氨基酸脱氢酶、0.4mg/mL甲酸脱氢酶、10mM L-缬氨酸、20mM甲胺、30mM甲酸钠、1mM NADP+以及50U/mL过氧化氢酶在pH 9.5的Na2CO3-NaHCO3缓冲液中进行多酶级联催化反应，37℃、160rpm反应12h。
实施例10：三酶偶合催化L-异亮氨酸生成N-甲基-L-异亮氨酸
将终浓度为10μg/mL L-氨基酸脱氨酶、1mg/mL N-甲基氨基酸脱氢酶、0.4mg/mL甲酸脱氢酶、10mM L-异亮氨酸、20mM甲胺、30mM甲酸钠、1mM NADP+以及50U/mL过氧化氢酶在pH 9.5的Na2CO3-NaHCO3缓冲液中进行多酶级联催化反应，37℃、160rpm反应12h。
实施例11：产物的检测方法
利用高效液相色谱法(HPLC)检测实施例5～10中的L-氨基酸以及N-甲基-L-氨基酸，检测条件：色谱柱为大赛璐CR(+)；流动相为pH 1.1～1.25高氯酸水溶液；检测波长210nm；柱温30℃；流量0.5mL/min；进样量20μL。利用该液相检测方法，测定多酶级联催化L-氨基酸生成相应N-甲基-L-氨基酸的转化率，检测结果如图5和表1所示。
表1 多酶级联催化多种L-氨基酸生成相应N-甲基-L-氨基酸的转化率结果
注：
“*”：底物转化率＝(C0-C底物)/C0×100％；
式中，C0表示反应起始底物的摩尔浓度，mM；C底物表示反应t时间后底物的摩尔浓度，mM；
“**”：产物转化率＝C产物/C理论×100％；
式中，C产物表示反应t时间后目标产物的摩尔浓度，mM；C理论表示目标产物的理论摩尔浓度，mM。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
序列表
<110> 华南理工大学
<120> 一种多酶级联催化L-氨基酸生成N-甲基-L-氨基酸的方法及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 654
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Gly Thr His Tyr Thr Phe Gly Lys Glu Ile Thr Asp Lys Pro Leu
1 5 10 15
Pro Thr Gln Val Lys Val Ala Ile Val Gly Ala Gly Met Ser Gly Leu
20 25 30
Tyr Ser Ala Trp Arg Leu Gln Gln Glu Ala Asn Cys Gln Asp Leu Ala
35 40 45
Ile Phe Glu Arg Ser Asn Arg Thr Gly Gly Arg Leu Asp Ser Asp Leu
50 55 60
Ile Glu Phe Lys Asn Leu Arg Ser Glu Thr Pro Lys Thr Ile Thr Val
65 70 75 80
Lys Glu Glu Gln Gly Gly Met Arg Phe Leu Phe Asp Gly Met Asp Asp
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Leu Met Ala Leu Phe Leu Lys Leu Asn Leu Gln Asp Asp Ile Val Pro
100 105 110
Phe Pro Met Asn Ser Gly Gly Asn Asn Arg Leu Phe Phe Arg Gly Glu
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Ser Phe Ser Val Glu Asp Ala Gln Gln Asp Asp Tyr Ala Ile Trp Ser
130 135 140
His Leu Tyr Asn Leu Asp Gln Ser Glu Gln Gly Val Asn Pro Lys Asp
145 150 155 160
Ile Val Asn Val Val Phe Asn Arg Ile Leu Glu Ala Asn Pro Gln Phe
165 170 175
Gln Gln Arg Pro Glu Val Arg Gly Pro Glu Phe Trp Gln Ala Phe Arg
180 185 190
Leu Glu Cys Gln Trp Gln Gly Gln Thr Leu Asn Glu Trp Thr Leu Trp
195 200 205
Asp Leu Tyr Thr Asp Met Gly Tyr Ser Gln Glu Cys Ile Asn Met Leu
210 215 220
Tyr Arg Val Leu Gly Phe Asn Gly Thr Phe Leu Ser Gln Met Asn Ala
225 230 235 240
Gly Val Ala Tyr Gln Leu Leu Glu Asp Phe Pro Ala Gly Val Gln Phe
245 250 255
Lys Thr Phe Lys Asp Gly Phe Ser Thr Leu Pro Asn Lys Leu Val Glu
260 265 270
Glu Val Gly Thr Asp Asn Ile His Leu Gln Thr Ser Ile Glu Glu Ile
275 280 285
Asp Phe Ala Glu Glu Ser Gly Leu Tyr Ser Leu His Tyr Ser His Thr
290 295 300
Asp Glu His Gly Arg Val His Lys Gly Gln Val Lys Ala Glu Lys Val
305 310 315 320
Ile Leu Gly Leu Pro Arg Leu Ala Leu Glu Lys Leu Phe Val Arg Ser
325 330 335
Asn Ala Phe Asn Arg Leu Asp Lys Asp Arg Ser Glu Gln Leu Trp Asn
340 345 350
Thr Leu Gln Ser Ala Ser Asn Gln Pro Leu Leu Lys Ile Asn Leu Tyr
355 360 365
Tyr Asp Ser Ala Trp Trp Gly Arg Gly Thr Thr Gly Arg Pro Ala Val
370 375 380
Glu Phe Gly Pro Asn Phe Ala Asp Leu Pro Thr Gly Ser Val Tyr Pro
385 390 395 400
Phe Tyr Ala Val Asn Asp Glu Leu Ala Ala Ala Leu Met Tyr Gln Glu
405 410 415
Arg Ser Thr Asn Pro Ser Lys Ala Val Gln Ala Lys Leu Asp Arg Ile
420 425 430
Gly Asn Glu Lys Tyr Glu Arg Pro Ala Ala Leu Thr Ile Tyr Cys Asp
435 440 445
Tyr Leu Asn Ile Asn Phe Trp Ser Asn Leu Gln Asn Ile Gly Glu Thr
450 455 460
Tyr His His Pro His Gln Asp Asp Tyr Val Glu Asp Val Pro Ala Asp
465 470 475 480
Ile Tyr Pro Ala Ser Thr Ala Val Val Glu Gln Ala Thr Arg Phe Phe
485 490 495
Lys Asp Ile Phe Asn Thr His Tyr Val Pro Glu Pro Ile Leu Thr Ser
500 505 510
Ala Arg Ile Trp Glu Gly Ser Val Arg Phe Asp Ile Pro Ala Ser Arg
515 520 525
Gln Phe Gly Phe Gly Val His Gln Trp Ala Val Gly Ala Asn Asp Lys
530 535 540
Glu Val Met Ala Thr Leu Ala Glu Pro Leu Pro Asn Leu Phe Thr Cys
545 550 555 560
Gly Glu Ala Phe Ser Asp Tyr Gln Gly Trp Val Glu Gly Ala Leu Arg
565 570 575
Ser Thr Asp Leu Ala Leu Glu Lys Gly Phe Gly Leu Lys Pro Leu Ser
580 585 590
Gln Val Tyr Phe Glu Asn Thr Asn Ile Ser Ser Ser Asp Ala Ile Lys
595 600 605
Ala Val Tyr Glu Glu Asn Ser Ser Lys Leu Ile Asn Gln Tyr Ile Glu
610 615 620
Thr Asn Phe Ser Ala Asn Thr Ala Pro Ile Glu Lys Thr Ala Asp Val
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Asp Ser Val Ile Gly Val Asn Leu Ser Tyr Phe Asp Thr Lys
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Claims (4)
1.一种多酶级联催化L-氨基酸生成N-甲基-L-氨基酸的方法，其特征在于：以L-氨基酸为底物，利用L-氨基酸氧化酶、N-甲基氨基酸脱氢酶和NADPH再生酶同步级联催化L-氨基酸反应生成N-甲基-L-氨基酸；
所述的L-氨基酸氧化酶为祖先L-氨基酸氧化酶，其氨基酸序列如SEQ ID NO:1所示；
所述的N-甲基氨基酸脱氢酶为来源于恶臭假单胞菌的N-甲基氨基酸脱氢酶，其氨基酸序列在NCBI的登陆号为AB190215.1；
所述的NADPH再生酶为来源于伯克霍尔德菌的甲酸脱氢酶，其氨基酸序列在NCBI的登陆号为ACF35003.1；
所述的L-氨基酸为L-苯丙氨酸、L-谷氨酸和L-亮氨酸中的至少一种；
所述的N-甲基-L-氨基酸为N-甲基-L-苯丙氨酸、N-甲基-L-谷氨酸和N-甲基-L-亮氨酸中的至少一种；
所述的反应的体系为：0.1mg/mL L-氨基酸脱氨酶，1 mg/mL N-甲基氨基酸脱氢酶，0.4mg/mL NADPH再生酶，10~100 mM L-氨基酸，20~200 mM胺供体，30~300 mM甲酸盐，5~10 mMNADP+，50 U/mL过氧化氢酶，pH 9.5；
所述的甲酸盐与L-氨基酸的摩尔浓度比为3:1；
所述的胺供体与L-氨基酸的摩尔浓度比为2:1；
所述的NADP+与L-氨基酸的摩尔浓度比为1:5～10；
所述的甲酸盐为甲酸钠；
所述的胺供体为甲胺；
所述的反应的温度为37～45℃。
2.根据权利要求1所述的方法，其特征在于：
所述的NADP+为氧化型辅酶II。
3.根据权利要求1所述的方法，其特征在于：
所述的反应的转速为100～200 rpm；
所述的反应的时间为6~24h。
4.权利要求1～3任一项所述的多酶级联催化L-氨基酸生成N-甲基-L-氨基酸的方法在制备N-甲基-L-氨基酸中的应用，其特征在于：
所述的N-甲基-L-氨基酸为N-甲基-L-苯丙氨酸、N-甲基-L-谷氨酸和N-甲基-L-亮氨酸中的至少一种。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1639328A (zh) * | 2002-02-28 | 2005-07-13 | 三菱化学株式会社 | 新型脱氢酶和编码该脱氢酶的基因 |
| CN104428313A (zh) * | 2012-05-11 | 2015-03-18 | 科德克希思公司 | 工程化亚胺还原酶以及用于酮和胺化合物的还原胺化的方法 |
| WO2020196619A1 (ja) * | 2019-03-28 | 2020-10-01 | 静岡県公立大学法人 | 新規なｌ－アミノ酸オキシダーゼ及びｄ－アミノ酸又はその誘導体の製造方法 |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1639328A (zh) * | 2002-02-28 | 2005-07-13 | 三菱化学株式会社 | 新型脱氢酶和编码该脱氢酶的基因 |
| CN104428313A (zh) * | 2012-05-11 | 2015-03-18 | 科德克希思公司 | 工程化亚胺还原酶以及用于酮和胺化合物的还原胺化的方法 |
| WO2020196619A1 (ja) * | 2019-03-28 | 2020-10-01 | 静岡県公立大学法人 | 新規なｌ－アミノ酸オキシダーゼ及びｄ－アミノ酸又はその誘導体の製造方法 |
Non-Patent Citations (2)
| Title |
|---|
| ACCESSION No.:ACF35003.1，formate dehydrogenase [Burkholderia stabilis];Hatrongjit,R,et al;《GenBank》;Features和Origin部分 * |
| Ancestral L-amino acid oxidases for deracemization and stereoinversion of amino acids;Shogo Nakano,et al.;《Communications chemistry》;第3卷(第1期);摘要、第2页右栏第2段至第9页左栏第2段、补充材料、图1 * |
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