CN111411095B - 一种重组(R)-ω-转氨酶及其突变体和应用 - Google Patents
一种重组(R)-ω-转氨酶及其突变体和应用 Download PDFInfo
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- CN111411095B CN111411095B CN202010277479.9A CN202010277479A CN111411095B CN 111411095 B CN111411095 B CN 111411095B CN 202010277479 A CN202010277479 A CN 202010277479A CN 111411095 B CN111411095 B CN 111411095B
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Abstract
本发明公开了一种新型重组(R)‑ω‑转氨酶及其突变体和应用，该新型重组(R)‑ω‑转氨酶的氨基酸序列如SEQ ID NO.1所示，突变体由SEQ ID NO.1所示氨基酸序列的第72位和第248位经单点或二点突变得到。本发明先通过基因重组技术获得高酶活的新型重组(R)‑ω‑转氨酶，再进一步通过定点突变获得新型重组(R)‑ω‑转氨酶突变体，该突变体不仅具有高酶活(1245.2U/g)、高立体选择特性(e.e.值99.9％)，而且能够高效催化前体酮类似物制备西他列汀中间体，转化率最高可达97.4％。
Description
技术领域
本发明涉及生物化学技术领域，尤其涉及一种重组(R)-ω-转氨酶及其突变体和应用。
背景技术
西他列汀，英文名称sitagliptin，全称(3R)-3-氨基-1-[3-(三氟甲基)-5,6,7,8-四氢-1,2,4-三唑并[4,3-a]吡嗪-7-基]-4-(2,4,5-三氟苯基)丁-1-酮，是由Merck和Codexis公司研制和开发的一种二肽基肽酶-4(DPP-4)抑制药，可通过保护内源性肠降血糖素和增强其作用而控制血糖水平，是一种有巨大潜力Ⅱ型糖尿病的治疗药物。
制备sitagliptin主要是不对称合成法，通常使用转氨酶为生物催化剂进行生物转化。转氨酶(transaminase，简称TA，EC 2.6.1.X)是辅酶型5’-磷酸吡哆醛(PLP)依赖型酶。在反应过程中PLP与5’-磷酸吡哆胺(PMP)会相互进行转换，同时催化氨基从合适的供体到羰基受体的可逆转移。TA可以被分为α-TA和ω-TA。其中，ω-TA可以催化任何结构的酮和胺，具有更高的应用价值。
催化制备sitagliptin的酶属于(R)-ω-TA。Codexis公司以Arthrobacter sp.来源的(R)-ω-ATA-117为研究对象，首先利用定点饱和突变对底物结合区域大口袋进行改造，获得突变体对1-[3-(三氟甲基)-5,6,7,8-四氢-1,2,4-三唑并[4,3-a]吡嗪-7-基]-1,3-丁二酮(简称：截短前体酮)展现了催化活性。然后对该突变体进行11轮分子改造，最终获得了催化1-[3-(三氟甲基)-5,6,7,8-四氢-1,2,4-三唑并[4,3-a]吡嗪-7-基]-4-(2,4,5-三氟苯基)-1,3-丁二酮(简称：前体酮底物)的高产突变体，催化活性较野生菌酶活提高了28000倍。(Savile C K,Janey J M,Mundorff E C,et al.Biocatalytic AsymmetricSynthesis of Chiral Amines from Ketones Applied to Sitagliptin Manufacture[J].Science，2010，329(5989):305-309.)。
DNA重组技术是将不同的遗传物质在体外进行剪切、拼接和组合，使遗传物质重新组合，然后通过载体转入微生物、植物和动物细胞内，进行无性繁殖，并使所需的基因在细胞内表达，产生人类所需的产物或是新生物的类型。利用分子生物学技术，可建立高效的新型转氨酶基因工程菌株，实现高活性、高稳定性、并且能满足工业化生产的转氨酶菌株。通过使用该方法能改进传统定点突变技术的不确定性，迅速获得高产工业菌株。重组DNA技术已经在细胞分化、生长发育、肿瘤发生等基础研究和工农业生产、医药保健等实际应用两方面取得了重要的突破。
发明内容
本发明提供了一种重组(R)-ω-转氨酶、重组(R)-ω-转氨酶突变体及其应用，尤其涉及生物催化前体酮类似物1-(吡咯烷-1-基)-4-(2,4,5-三氟苯基)-1,3-丁二酮合成西他列汀中间体(R)-3-氨基-1-(吡咯烷-1-基)-4-(2,4,5-三氟苯基)丁-1-酮中的应用，该新型重组(R)-ω-转氨酶和新型重组(R)-ω-转氨酶突变体不仅具有较高的酶活，而且能够高效催化前体酮类似物制备西他列汀中间体，尤其是新型重组(R)-ω-转氨酶突变体的转化率最高可达97.4％。
具体技术方案如下：
本发明提供了新型重组(R)-ω-转氨酶，该新型重组(R)-ω-转氨酶的氨基酸序列如SEQ ID NO.1所示。
本发明还提供了编码该新型重组(R)-ω-转氨酶的核苷酸序列，具体如SEQ IDNO.2所示。该新型重组(R)-ω-转氨酶是AtTA6的C端区域B1与CeTA5的N端区域A2重组获得。
本发明还提供了新型重组(R)-ω-转氨酶突变体，该突变体为下列之一：
(1)由SEQ ID NO.1所示氨基酸序列的第72位的天冬氨酸突变为苯丙氨酸、酪氨酸、丙氨酸或甘氨酸；
(2)由SEQ ID NO.1所示氨基酸序列的第72位的天冬氨酸突变为甘氨酸，且第248位丙氨酸突变为赖氨酸、苏氨酸、丝氨酸、组氨酸或酪氨酸；
(3)由SEQ ID NO.1所示氨基酸序列的第72位的天冬氨酸突变为甘氨酸，且第248位丙氨酸突变为丝氨酸。
本发明在获得新型重组(R)-ω-转氨酶的基础上进行的单点和二点突变，获得了以上具有较高酶活的新型重组(R)-ω-转氨酶突变体，这些突变体能够高效催化前体酮类似物1-(吡咯烷-1-基)-4-(2,4,5-三氟苯基)-1,3-丁二酮合成西他列汀中间体(R)-3-氨基-1-(吡咯烷-1-基)-4-(2,4,5-三氟苯基)丁-1-酮。
作为优选，该新型重组(R)-ω-转氨酶突变体的氨基酸序列如SEQ ID NO.3所示，核苷酸序列如SEQ ID NO.4所示。
本发明还提供了一种所述的新型重组(R)-ω-转氨酶或所述的新型重组(R)-ω-转氨酶突变体的编码基因。
本发明还提供了一种包含所述编码基因的重组载体。
本发明还提供了一种包含所述编码基因的基因工程菌。
本发明还提供了所述新型重组(R)-ω-转氨酶或所述新型重组(R)-ω-转氨酶突变体在生物催化前体酮类似物1-(吡咯烷-1-基)-4-(2,4,5-三氟苯基)-1,3-丁二酮合成西他列汀中间体(R)-3-氨基-1-(吡咯烷-1-基)-4-(2,4,5-三氟苯基)丁-1-酮。
本发明还提供了所述基因工程菌在生物催化前体酮类似物1-(吡咯烷-1-基)-4-(2,4,5-三氟苯基)-1,3-丁二酮合成西他列汀中间体(R)-3-氨基-1-(吡咯烷-1-基)-4-(2,4,5-三氟苯基)丁-1-酮。
本发明还提供了一种催化前体酮类似物1-(吡咯烷-1-基)-4-(2,4,5-三氟苯基)-1,3-丁二酮合成西他列汀中间体(R)-3-氨基-1-(吡咯烷-1-基)-4-(2,4,5-三氟苯基)丁-1-酮的方法，包括：以前体酮类似物1-(吡咯烷-1-基)-4-(2,4,5-三氟苯基)-1,3-丁二酮为底物，所述的新型重组(R)-ω-转氨酶、所述的新型重组(R)-ω-转氨酶突变体或所述的基因工程菌为生物催化剂，异丙胺为氨基供体，磷酸吡哆醛为辅酶，在缓冲溶液中进行生物催化合成反应，得到西他列汀中间体(R)-3-氨基-1-(吡咯烷-1-基)-4-(2,4,5-三氟苯基)丁-1-酮。
进一步地，所述生物催化合成反应为35～50℃；所述缓冲溶液为三乙醇胺-HCl缓冲液，pH为8～9；所述底物的浓度为800～1200mM。
其中，对于本发明提供的氨基酸序列如SEQ ID NO.3所示的新型重组(R)-ω-转氨酶突变体而言，生物催化合成反应的温度在50℃时效果最佳，且当底物浓度为1000mM时，转化率最高，达到97.4％。
与现有技术相比，本发明具有以下有益效果：
本发明先通过基因重组技术获得高酶活的新型重组(R)-ω-转氨酶，再进一步通过定点突变获得新型重组(R)-ω-转氨酶突变体，该突变体不仅具有高酶活(1245.2U/g)、高立体选择特性(e.e.值99.9％)，而且能够高效催化前体酮类似物制备西他列汀中间体，转化率最高可达97.4％，所得的西他列汀中间体前体可进一步通过水解、氨基保护、缩合等步骤得到西他列汀。
具体实施方式
下面结合具体实施例对本发明作进一步描述，以下列举的仅是本发明的具体实施例，但本发明的保护范围不仅限于此。
实施例1：新型重组ω-TA的筛选、立体选择性确定与酶活精准测定
1、酶的来源与基因合成
运用基因挖掘技术，从NCBI据库中基因挖掘获得两株新酶，分别来自Sinirhodobacter hungdaonensis(ShTA，Genbank编号WP_128154090)和Actinobacteriasp.(AcTA，Genbank编号WP_030166319)。依据E.coli密码子偏好性进行密码子优化，以全基因合成的方法合成了两株酶的核苷酸序列。
在核苷酸序列末端加入6×His-tag标签，两端加入酶切位Xho I和Nco I，将该基因克隆至pET28b(+)对应的Xho I和Nco I位点，获得重组表达质粒pET28b/ShTA和pET28b/AcTA。
2、重组工程菌的诱导表达
LB液体培养基组成：蛋白胨10g/L，酵母粉5g/L，NaCl 10g/L，溶剂为水，pH值自然；LB固体培养基在LB液体培养基中添加20g/L琼脂；121℃高压灭菌20min；使用前添加终浓度50μg/mL卡那霉素。
将基因工程菌接种至含终浓度50μg/mL卡那霉素的LB液体培养基，在37℃、150r/min培养至OD600约0.6～0.8，获得种子液；将种子液以体积浓度2％接种量接种至新鲜的含有终浓度50μg/mL卡那霉素的LB培养基中，于37℃、150r/min培养OD600至0.6～0.8，再向培养液中加入终浓度为1mM的IPTG，于28℃下诱导表达10h后，4℃、8000r/min离心10min，弃去上清液，用0.85％的生理盐水清洗两遍湿菌体，并收集湿菌体备用。
3、重组工程菌超声破碎
采用超声破碎法对湿菌体进行破碎。取1g制备的湿菌体，用10mL Na2CO3/NaHCO3缓冲液(pH 7.5)悬浮，在39W条件下超声破碎，有效时间5min，离心收集破碎上清液。
4、确定新型重组ω-TA的立体选择性
取重组ShTA和AcTA破碎上清液进行如下反应。
反应体系：10mM的1-[3-(三氟甲基)-5,6,7,8-四氢-1,2,4-三唑并[4,3-a]吡嗪-7-基]-1,3-丁二酮(简称：截短前体酮)、52mM(R,S)-α-甲基苄胺、1mL破碎上清液(ShTA酶液或AcTA酶液)、2mM PLP，加入0.1M Na2CO3/NaHCO3缓冲液(pH 7.5)至总体积10mL。反应条件：于30℃反应2h，冰浴10min终止反应，8000r/min离心10min，获得反应上清液。反应过程如下：
反应上清液进行衍生化反应以确定ω-TA的立体选择性。
衍生化反应体系：8mL反应上清液、1mg 4-异丁基恶唑烷-2,5-二酮、加入0.45M硼酸盐缓冲液(pH 10.4)至总体系10mL。衍生化反应条件：室温下条件下反应2min，加入0.1mL1M的HCl终止反应，8000r/min离心10min，取上清液；采用高效液相色谱(HPLC)检测对应产物的构型。分析柱为Agilent C18柱(250×4.6mm，5μm)(安捷伦科技有限公司，美国)。Agilent 2414荧光检测器，Agilent 1525泵，Agilent 717进样器。与产物标样衍生化的出峰时间对比，根据产物构型来判断所筛ω-TA的立体选择性。如表1所示，ShTA的催化产物为R构型，说明ShTA属(R)-ω-TA。
表1：各转氨酶立体选择性的鉴定
5、ShTA重组工程菌对截短前体酮酶活的精准测定
采用超声破碎法对湿菌体进行破碎。取1g制备的湿菌体，用10mL三乙醇胺-HCl缓冲液(pH 7.5)悬浮，在39W条件下超声破碎，有效时间5min，离心收集破碎上清液用于下述反应。反应体系：30mM 1-[3-(三氟甲基)-5,6,7,8-四氢-1,2,4-三唑并[4,3-a]吡嗪-7-基]-1,3-丁二酮(简称：截短前体酮)、200μL破碎上清液(ShTA酶液)、50mM(R)-α-甲基苄胺、1mM PLP，加入三乙醇胺-HCl缓冲液(pH 8.0)至总体系5mL。反应条件：30℃条件下反应2h，加入6mM HCl终止反应，8000r/min离心10min，取反应上清液；采用HPLC检测产物的浓度，分析方法同实施例1的“确定新型ω-TA的立体选择性”。
酶活定义：30℃和pH 8.0下，每h内催化截短前体酮类似物生成1μmoL的R型产物所需酶量定义为一个酶活单位(U)。经酶活检测，ShTA的酶活为127.4U/g。
表2：重组酶的活力测定
实施例2：酶的重组与筛选
发明人所在课题组在前期获得了一种高效R选择性Capronia epimyces TA突变体(以下简称CeTA5)，其核苷酸序列为SEQ ID NO.5所示，编码酶的氨基酸序列如SEQ ID NO.6所示。CeTA5能够催化前体酮类似物1-(吡咯烷-1-基)-4-(2,4,5-三氟苯基)-1,3-丁二酮合成西他列汀中间体(R)-3-氨基-1-(吡咯烷-1-基)-4-(2,4,5-三氟苯基)丁-1-酮，对1-(吡咯烷-1-基)-4-(2,4,5-三氟苯基)-1,3-丁二酮(简称：前体酮类似物)的活性为650.3U/g，可催化700mM前体酮类似物，转化率94.6％。
此外，发明人所在课题组在前期还获得了一种高效R选择性Aspergillus terreusTA突变体(以下简称AtTA 6)，其氨基酸序列为SEQ ID NO.7所示，编码酶的核苷酸序列如SEQ ID NO.8所示。AtTA 6能够催化前体酮类似物1-(3-氧吡咯烷-1-基)-4-(2,4,5-三氟苯基)-1,3-丁二酮合成西他列汀中间体(R)-1-[3-氨基-4-(2,4,5-三氟苯基)丁酰基]吡咯-3-酮，对1-(3-氧吡咯烷-1-基)-4-(2,4,5-三氟苯基)-1,3-丁二酮的活性为753.2U/g，可催化800mM前体酮类似物，转化率95.4％。
本发明将AtTA6分别与CeTA5和ShTA进行DNA重组，得到新型重组酶，具体内容如下：
1、克隆AtTA6、CeTA5和ShTA的N端、C端氨基酸序列
(1)根据需要合成的AtTA6的核苷酸序列片段设计引物，利用快速PCR技术，以重组载体pET28b/AtTA6为模板，合成AtTA6的N端区域(简称A1)的DNA片段，引物为：
正向引物：CTCGAGATGGCTTCTATGGATAAAGTTTTT(下划线为酶切位点，如SEQ IDNO.9所示)；
反向引物：ACCCCACTGCATATTTTTCACAGTCGGGTC(如SEQ ID NO.10所示)；
PCR反应体系：2×Phanta Max Buffer(含Mg2+)25μL，dNTPs 10mM，正向引物2μL，反向引物2μL，模板DNA 1μL，Phanta Max Super-Fidelity DNA Polymerase 50U，加入ddH2O至50μL。
PCR扩增条件：95℃5min；(95℃30s，55℃30s，72℃1min)共30循环；72℃5min。
根据需要合成的AtTA6的核苷酸序列片段设计引物，利用快速PCR技术，以重组载体pET28b/AtTA6为模板，合成AtTA6的C端区域(简称B1)的DNA片段，引物为：
正向引物：GATCTGGTGCGTGGTATGTTCGAAGCTGCA(如SEQ ID NO.11所示)；
反向引物：CCATGGTTAATGGTGATGGTGATGGTGGTT(下划线为酶切位点，如SEQ IDNO.12所示)；
PCR反应体系：2×Phanta Max Buffer(含Mg2+)25μL，dNTPs 10mM，正向引物2μL，反向引物2μL，模板DNA 1μL，Phanta Max Super-Fidelity DNA Polymerase 50U，加入ddH2O至50μL。
PCR扩增条件：95℃5min；(95℃30s，55℃30s，72℃1min)共30循环；72℃5min。
(2)根据需要合成的CeTA5的核苷酸序列片段设计引物，利用快速PCR技术，以重组载体pET28b/CeTA5为模板，合成CeTA5的N端区域(简称A2)DNA片段，引物为：
正向引物：CTCGAGATGGCTTCTATGGACAAAGTTTTC(下划线为酶切位点，如SEQ IDNO.13所示)；
反向引物：ACCACGCACCAGATCACCCCACTGGGTGTTTTTAACGGT(如SEQ ID NO.14所示)；
PCR反应体系：2应Phanta Max Buffer(含Mg2+)25μL，dNTPs 10mM，正向引物2μL，反向引物2μL，模板DNA 1μL，Phanta Max Super-Fidelity DNA Polymerase50U，加入ddH2O至50μL。
PCR扩增条件：95℃5min；(95℃30s，55℃30s，72℃1min)共30循环；72℃5min。
根据需要合成的CeTA5的核苷酸序列片段设计引物，利用快速PCR技术，以重组载体pET28b/CeTA5为模板，合成CeTA5的C端区域(简称B2)DNA片段，引物为：
正向引物：ACTGTGAAAAATATGCAGTGGGGTGACCTGACCCGTGCT(如SEQ ID NO.15所示)；
反向引物：CCATGGTTAGTGGTGGTGGTGGTGGTGTTT(下划线为酶切位点，如SEQ IDNO.16所示)；
PCR反应体系：2应Phanta Max Buffer(含Mg2+)25μL，dNTPs 10mM，正向引物2μL，反向引物2μL，模板DNA 1μL，Phanta Max Super-Fidelity DNA Polymerase 50U，加入ddH2O至50μL。
PCR扩增条件：95℃5min；(95℃30s，55℃30s，72℃1min)共30循环；72℃5min。
(3)根据需要合成的ShTA的核苷酸序列片段设计引物，利用快速PCR技术，以重组载体pET28b/ShTA为模板，合成ShTA的N端区域(简称A3)的DNA片段，引物为：
正向引物：CTCGAGATGACCATGAACCAGCTGACTATC(下划线为酶切位点，如SEQ IDNO.17所示)；
反向引物：ACCACGCACCAGATCACCCCACTGGTAGTTTTTGATCCA(如SEQ ID NO.18所示)；
PCR反应体系：2×Phanta Max Buffer(含Mg2+)25μL，dNTPs
10mM，正向引物2μL，反向引物2μL，模板DNA 1μL，Phanta Max Super-FidelityDNA Polymerase 50U，加入ddH2O至50μL。
PCR扩增条件：95℃5min；(95℃30s，55℃30s，72℃1min)共30循环；72℃5min。
根据需要合成的ShTA的核苷酸序列片段设计引物，利用快速PCR技术，以重组载体pET28b/ShTA为模板，合成ShTA的C端区域(简称B3)的DNA片段，引物为：
正向引物：ACTGTGAAAAATATGCAGTGGGGTGATCTGGTTCGTGCC(如SEQ ID NO.19所示)；
反向引物：CCATGGTTAATGGTGGTGATGATGGTGCAG(下划线为酶切位点，如SEQ IDNO.20所示)；
PCR反应体系：2×Phanta Max Buffer(含Mg2+)25μL，dNTPs 10mM，正向引物2μL，反向引物2μL，模板DNA 1μL，Phanta Max Super-Fidelity DNA Polymerase 50U，加入ddH2O至50μL。
PCR扩增条件：95℃5min；(95℃30s，55℃30s，72℃1min)共30循环；72℃5min。
2、构建重组酶基因工程菌
将AtTA6的N端区域A1分别与CeTA5、ShTA的C端区域B2、B3重组，获得2个新型重组酶；同时，再将AtTA6的C端区域B1分别与CeTA5、ShTA的N端区域A2、A3重组，获得2个新型重组酶。重组策略见表3，具体操作如下。
表3新型重组酶的重组策略
(1)以AtTA6的N端区域A1的正向引物为正向引物，以CeTA5、ShTA的C端区域B2、B3的反向引物为反向引物，利用Ex Taq酶扩增目的片段，分别进行AtTA6的N端活性区域A1与CeTA5的C端区域B2的基因片段融合PCR，AtTA6的N端区域A1与ShTA的C端区域B3的基因片段融合PCR。
AtTA6与CeTA5的片段融合PCR(A1与B2片段融合)反应体系为：10×Phanta MaxBuffer(含Mg2+)10μL，dNTPs 10mM，正向引物2μL，反向引物2μL，A1片段PCR产物为模板2μL，B2片段PCR产物为模板2μL，Ex Taq酶1μL，加入ddH2O至100μL。
AtTA6与ShTA的片段融合PCR(A1与B3片段融合)反应体系为：10×Phanta MaxBuffer(含Mg2+)10μL，dNTPs 10mM，正向引物2μL，反向引物2μL，A1片段PCR产物为模板2μL，B3片段PCR产物为模板2μL，Ex Taq酶1μL，加入ddH2O至100μL。
PCR扩增条件：94℃5min，(94℃1min，57℃30s，72℃1.5min)共30个循环，72℃10min。
(2)以AtTA6的C端区域B1的反向引物为反向引物，以CeTA5、ShTA的N端区域A2、A3的正向引物为正向引物，利用Ex Taq酶扩增目的片段，分别进行CeTA5的N端区域A2与AtTA6的C端区域B1的基因片段融合PCR，ShTA的N端区域A3与AtTA6的C端区域B1的基因片段融合PCR。
AtTA6与CeTA5的片段融合PCR(A2与B1片段融合)反应体系为：10×Phanta MaxBuffer(含Mg2+)10μL，dNTPs 10mM，正向引物2μL，反向引物2μL，A2片段PCR产物为模板2μL，B1片段PCR产物为模板2μL，Ex Taq酶1μL，加入ddH2O至100μL。
AtTA6与ShTA的片段融合PCR(A3与B1片段融合)反应体系为：10×Phanta MaxBuffer(含Mg2+)10μL，dNTPs 10mM，正向引物2μL，反向引物2μL，A3片段PCR产物为模板2μL，B1片段PCR产物为模板2μL，Ex Taq酶1μL，加入ddH2O至100μL。
PCR扩增条件：94℃5min，(94℃1min，57℃30s，72℃1.5min)共30个循环，72℃10min。
通过限制性核酸内切酶、DNA连接酶获得重组表达质粒pET28b/TAmutant-1、pET28b/TAmutant-2、pET28b/TAmutant-3和pET28b/TAmutant-4。
取8μL构建的重组质粒，加入100μL冰浴的感受态细胞悬液中，冰上静置30min，将转化产物于42℃热激90s，迅速置于冰上冷却2min。向Ep管中加入600μL的LB液体培养基，37℃，150r/min培养60min，12000r/min离心1min，弃掉600μL上清，将剩余的菌液悬浮后涂板，待菌液完全被培养基吸收后，37℃倒置培养12h，挑选转化子备用。
3、重组工程菌的诱导表达
同实施例1中的“重组工程菌的诱导表达”。
4、重组工程菌超声破碎
采用超声破碎法对湿菌体进行破碎。取1g制备的湿菌体(TAmutant-1、TAmutant-2、TAmutant-3和TAmutant-4)，用10mL三乙醇胺-HCl缓冲液(pH 7.5)悬浮，在39W条件下超声破碎，有效时间5min，离心收集破碎上清液。
5、重组工程菌对前体酮类似物酶活的精准测定
取各酶的破碎上清液用于下述反应，反应体系：20mM 1-(吡咯烷-1-基)-4-(2,4,5-三氟苯基)-1,3-丁二酮(简称：前体酮类似物)、400μL破碎上清液(酶液)、80mM异丙胺、1mM PLP，加入三乙醇胺-HCl缓冲液(pH 8.0)、25％(v/v)的DMSO至总体系10mL。反应条件：30℃下反应2h，加入6mM HCl终止反应，8000r/min离心10min，取反应上清液，采用HPLC检测产物(R)-3-氨基-1-(吡咯烷-1-基)-4-(2,4,5-三氟苯基)丁-1-酮(简称：sitagliptin中间体)的浓度。
反应过程如下：
分析方法：分析柱为Agilent C18柱(250×4.6mm，5μm)(安捷伦科技有限公司，美国)。Agilent 2414荧光检测器，Agilent 1525泵，Agilent 717进样器。流动相：乙腈与10mM乙酸铵的混合液(体积比55:45)，流速1.5mL/min，检测波长265nm。
酶活定义：30℃和pH 8.0下，每h将前体酮类似物催化生成1μmoL sitagliptin中间体所需酶量定义为一个酶活单位(U)。
经酶活检测(见表4)，TAmutant-3对前体酮类似物的酶活为817.3U/g，对比AtTA6展示了更高的催化活力，其他重组突变株对前体酮类似物活力较低或无活力。
表4：各重组突变株对前体酮类似物的酶活测定
6、重组工程菌立体选择性的确定
反应体系：20mM 1-(吡咯烷-1-基)-4-(2,4,5-三氟苯基)-1,3-丁二酮(简称：前体酮类似物)、200μL破碎上清液(酶液)、50mM(R)-α-甲基苄胺、1mM PLP，加入三乙醇胺-HCl缓冲液(pH 8.0)、25％(v/v)的DMSO至总体系5mL。
反应条件：30℃条件下反应2h，加入6mM HCl终止反应，8000r/min离心10min，取反应上清液；采用HPLC检测产物的构型。
分析方法：分析柱Daicel Chiralpak AD-H column(4.6×150mm,5μm)(大赛璐药物手性技术有限公司，上海，中国)。Ag1lent 2414荧光检测器，Agilent 1525泵，Agilent717进样器。流动相为乙醇与庚烷混合液(体积比60:40)，流速为0.8mL/min，柱温35℃。
由表5可以看出，TAmutant-1、TAmutant-2、TAmutant-3能够催化前体酮类似物生成(R)-sitagliptin中间体产物。
表5：重组突变株的立体选择性
实施例3：TAmutant-3单位点突变体的构建与筛选
1、突变体的构建
将突变库中的筛选到的(R)-ω-TA进行单点突变，根据TAmutant-3的核苷酸序列设计定点突变的引物，利用快速PCR技术，以重组载体pET28b/TAmutant-3为模板，对TAmutant-3氨基酸序列的72位引入单点突变，引物为：
正向引物：TCCGTCTGNNKGACCACATCTCTAACCTGG(下划线为突变碱基，如SEQ IDNO.21所示)；
反向引物：ATGTGGTCMNNCAGACGGAAGAACCTGCCG(下划线为突变碱基，如SEQ IDNO.22所示)；
PCR反应体系：2×Phanta Max Buffer(含Mg2+)25μL，dNTPs 10mM，正向引物2μL，反向引物2μL，模板DNA 1μL，Phanta Max Super-Fidelity DNA Polymerase 50U，加入ddH2O至50μL。
PCR扩增条件为：95℃5min；(95℃1min，58℃30s，72℃6min)30循环；72℃5min。
取5μL的PCR产物，加入100μL冰浴的感受态细胞悬液中，冰上静置30min，将转化产物于42℃热激90s，迅速置于冰上冷却2min。向Ep管中加入600μL的LB液体培养基，37℃，150r/min培养60min，12000r/min离心1min，弃掉600μL上清，将剩余的菌液悬浮后涂板，待菌液完全被培养基吸收后，37℃倒置培养12h，挑选阳性转化子备用。取单克隆于含50μg/mL卡那霉素的LB液体培养基中，37℃培养过夜。
2、高通量筛选阳性转化子
反应混合液组成：52mM邻苯二甲胺二盐酸、30mM前体酮类似物、1mM PLP、0.1MKOH，加入去离子水、25％(v/v)的DMSO至总反应体系配成1L。
在96孔聚苯乙烯微孔培养板中每孔加入100μL含有终浓度50μg/mL卡那霉素的LB培养液，接种不同的转化菌落，于37℃、150r/min培养OD600至0.6～0.8，再向培养液中加入终浓度为1mM的IPTG，于28℃下诱导表达10h后，4℃、8000r/min离心10min，弃去上清液。取265μL上述反应混合液加入含有菌体的的96孔板中，振荡器振荡混匀后在30℃、500r/min反应30min，冰浴3min停止反应。以重组菌E.coli BL21(DE3)/pET28b/TAmutant-3的反应为对照，取颜色比E.coli BL21(DE3)/pET28b/TAmutant-3的反应深的突变株进行酶活测定。
3、阳性转化子发酵产酶
同实施例1的“重组工程菌的诱导表达”。
4、单位点突变体的对前体酮类似物的酶活力检测
按实施例2的“重组工程菌对前体酮类似物酶活的精准测定”对各突变体进行活力测定，该实施例的结果为：运用高通量的方法，对获得的325株重组转化菌进行初筛，筛选出了4株酶活提高的突变株，在对其进行酶活测定，具体结果见表6。
经分析确定，其余311株重组酶保持不变或下降是第72位的天冬氨酸D变为了苯丙氨酸F、酪氨酸Y、丙氨酸A和甘氨酸G外的其他氨基酸。
表6：单点突变工程菌的酶活检测
将酶活提升最多的突变体pET28b/TAmutant-3-D72G标记为TAmutant-31，获得重组菌E.coli BL21(DE3)/pET28b/TAmutant-31。
实施例4：TAmutant-3二位点突变体的构建与筛选
根据实施例3构建的突变体TAmutant-31序列设计定点突变的突变引物，利用快速PCR技术，以重组载体pET28b/TAmutant-31为模板，对TAmutant-31氨基酸序列248位引入单突变，引物为：
正向引物：CTGCGGAANNKTTCGGTATCGAAGTTCGTG(下划线为突变碱基，如SEQ IDNO.23所示)；
反向引物：ATACCGAAMNNTTCCGCAGCGTTGATCACA(下划线为突变碱基，如SEQ IDNO.24所示)；
PCR反应体系方法同实施例4的“突变体的构建”。
PCR扩增条件为：95℃5min；(95℃1min，58℃30s，72℃6min)30循环；72℃5min。
PCR产物转化E.coli BL21(DE3)感受态细胞，挑单克隆于含50μg/mL卡那霉素的LB液体培养基中，37℃培养过夜。利用上述提到的高通量的筛选方法对突变体进行初筛，方法同实施4的“高通量筛选阳性转化子”，以重组菌E.coli BL21(DE3)/pET28b/TAmutant-31的反应为对照，取颜色比E.coli BL21(DE3)/pET28b/TAmutant-31的反应深的突变株进行酶活测定。
对初筛的阳性突变株进行酶活的检测。该实施例的结果为：对获得的375株重组转化菌初筛，筛选出了5株酶活提高的突变株，再对其进行酶活检测，具体结果见表7。
经分析确定，其余370株重组菌酶或保持不变或下降是第248位丙氨酸A突变为了赖氨酸K、苏氨酸T、丝氨酸S、组氨酸H和酪氨酸Y外的其他氨基酸。
表7：二点突变重组菌的酶活测定
将酶活提高最显著的突变体pET28b/TAmutant-31-A248S记为TAmutant-32，获得重组菌E.coli BL21(DE3)/pET28b/TAmutant-32。
实施例5重组大肠杆菌发酵产酶
分别将重组菌E.coli BL21(DE3)/pET28b/TAmutant-3、E.coli BL21(DE3)/pET28b/TAmutant-31、E.coli BL21(DE3)/pET28b/TAmutant-32接种至含终浓度50μg/mL卡那霉素的LB液体培养基，在37℃，150r/min培养OD600约为0.6～0.8，获得种子液；将种子液以2％(v/v)接种量接种至新鲜的含有终浓度50μg/mL卡那霉素的LB液体培养基中，与37℃，150r/min培养OD600至0.6～0.8，再向培养液中加入终浓度为1mM的IPTG，于28℃下诱导表达12h后，4℃，8000r/min离心10min，弃去上清液，用0.85％的生理盐水清洗两遍湿菌体，备用。
实施例6重组菌全细胞催化前体酮类似物不对称合成sitagliptn中间体
按实施例5的方法，获得重组菌E.coli BL21(DE3)/pET28b/TAmutant-3、E.coliBL21(DE3)/pET28b/TAmutant-31、E.coli BL21(DE3)/pET28b/TAmutant-32湿菌体作为生物催化剂，反应体系如下：不同浓度的前体酮类似物(见表8)、1.4M异丙胺、10mM磷酸吡哆醛(PLP)、5g重组菌全细胞，加入三乙醇胺-HCl缓冲液(pH 9.0)、50％(v/v)的DMSO至总体系100mL。反应条件：50℃和400r/min条件下反应30h，加入6mM HCl终止反应，8000r/min离心10min，取上清采用HPLC检测sitagliptin中间体的浓度和e.e.值，并计算转化率。
由表8可知，E.coli BL21(DE3)pET28b/TAmutant-32在底物浓度为1000mM时，转化30h，转化率97.4％。E.coli BL21(DE3)pET28b/TAmutant-32获得的转化效果不仅优于本发明其他突变体的转化效果，而且其底物浓度和转化率明显优于已报道的技术水平。
表8：不同底物浓度下生成sitagliptin中间体的比较
实施例7：化学法合成sitagliptin
在1.3L蒸馏水中，加入150g的(R)-3-氨基-1-(吡咯烷-1-基)-4-(2,4,5-三氟苯基)丁-1-酮(简称：sitagliptin中间体，实施例6的反应产物)、40g NaOH，补加蒸馏水至总体系为1.5L。升温至55℃，反应3h。结束后降温至30℃，加入140g二碳酸二叔丁酯，反应6h，结束后通入盐酸调节pH至1.5～2.0，结晶过滤，蒸馏水水洗，加入2L二氯甲烷、150g二氯亚砜，25℃下反应2h，结束后加入140g二乙胺、185.4g 3-(三氟甲基)-5,6,7,8-四氢-[1,2,4]三唑并[4,3-a]吡嗪盐酸盐，反应6.5h，结束后添加2L三氟乙酸，反应4h，结束后添加等体积蒸馏水，分层，留取有机层，水洗两次后浓缩结晶获得sitagliptin干品201.2g，HPLC检测产率为98.1％，收率为96.2％。
序列表
<110> 浙江工业大学
浙江永太科技股份有限公司
浙江永太药业有限公司
<120> 一种新型重组(R)-ω-转氨酶及其突变体和应用
<160> 24
<170> SIPOSequenceListing 1.0
<210> 1
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ccgttcctga ccgacggcga cgctcatctg accgagggct ccgctttcaa tatcgttctg 660
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275 280 285
Val Asn Gly Gly Gln Ile Gly Pro Ile Thr Lys Lys Ile Trp Asp Gly
290 295 300
Tyr Trp Ala Met His Tyr Asp Ala Ala Tyr Ser Phe Glu Ile Asp Tyr
305 310 315 320
Asn Glu Arg Asn His His His His His His
325 330
<210> 4
<211> 1008
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atggcttcta tggacaaagt tttcgctggt taccagtctc gtctgcgtgt tctggaagct 60
tctaccaacc cgctggctca gggtgttgct tggatcgaag gtgaactggt tccgctgtct 120
caggctcgta tcccgctgat ggaccagggt ttcctgcact ctgacctgac ctacgacgtt 180
ccagctgtgt gggacggcag gttcttccgt ctgggtgacc acatctctaa cctggaaaaa 240
tcttgctcta aactgcgtct gaaactgccg ctgccgcgtg acgaagttaa acgtgttctg 300
gttgacatgg ttgctcgttc tggtatacgt gacgcgttcg ttgaactgat agttacccgt 360
ggtctgaccg gtgttcgtgg tgctggtatc ccggaagacc tggttaacaa cctgttcatg 420
ttcctgcagc cgtacctgtg ggttatgccg ccggaaaccc agctggttgg tggttctgct 480
gttatcaccc gtaccgttcg tcgtaccccg ccgggttcta tggacccgac cgttaaaaac 540
acccagtggg gtgatctggt gcgtggtatg ttcgaagctg cagaccgtgg tgcgacgtac 600
ccgttcctga ccgacggcga cgctcatctg accgagggct ccgctttcaa tatcgttctg 660
gttaaagatg gcgttctgta cactccggat cgtggtgtgg aacagggcgt tacccgcaaa 720
tctgtgatca acgctgcgga atcattcggt atcgaagttc gtgttgaatt tgtgccggtc 780
gaactggcat atcgttgcga cgagatcttc atgtgcacca ctgccggcgg cattatgccg 840
attactaccc tggatggcat gccggtcaat ggcggccaga tcggtccaat taccaagaaa 900
atttgggatg gttactgggc catgcattac gacgctgctt attcttttga aatcgactac 960
aacgagcgta accaccatca ccatcaccat taacaccatc accattaa 1008
<210> 5
<211> 341
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Met Ala Ser Met Asp Lys Val Phe Ala Gly Tyr Gln Ser Arg Leu Arg
1 5 10 15
Val Leu Glu Ala Ser Thr Asn Pro Leu Ala Gln Gly Val Ala Trp Ile
20 25 30
Glu Gly Glu Leu Val Pro Leu Ser Gln Ala Arg Ile Pro Leu Met Asp
35 40 45
Gln Gly Phe Leu His Ser Asp Leu Thr Tyr Asp Val Pro Ala Val Trp
50 55 60
Asp Gly Arg Phe Phe Arg Leu Asp Asp His Ile Ser Asn Leu Glu Lys
65 70 75 80
Ser Cys Ser Lys Leu Arg Leu Lys Leu Pro Leu Pro Arg Asp Glu Val
85 90 95
Lys Arg Val Leu Val Asp Met Val Ala Arg Ser Gly Ile Arg Asp Ala
100 105 110
Phe Val Glu Leu Ile Val Thr Arg Gly Leu Thr Gly Val Arg Gly Ala
115 120 125
Gly Ile Pro Glu Asp Leu Val Asn Asn Leu Phe Met Phe Leu Gln Pro
130 135 140
Tyr Leu Trp Val Met Pro Pro Glu Thr Gln Leu Val Gly Gly Ser Ala
145 150 155 160
Val Ile Thr Arg Thr Val Arg Arg Thr Pro Pro Gly Ser Met Asp Pro
165 170 175
Thr Val Lys Asn Thr Gln Trp Gly Asp Leu Thr Arg Ala Leu Leu Glu
180 185 190
Ala Ser Asp Arg Gly Ala Ser Tyr Pro Phe Leu Thr Asp Gly Asp Ala
195 200 205
Asn Ile Thr Glu Gly Ser Gly Tyr Asn Ile Val Leu Ile Lys Asp Gly
210 215 220
Ala Ile His Thr Pro Asp Arg Gly Val Leu Glu Gly Val Thr Arg Lys
225 230 235 240
Thr Val Phe Asp Ile Ala Lys Ala Asn Gly Phe Glu Val Arg Leu Glu
245 250 255
Val Val Pro Val Glu Leu Ala Tyr Arg Ala Asp Glu Ile Phe Met Cys
260 265 270
Tyr Thr Ala Gly Gly Ile Met Pro Ile Thr Ser Leu Asp Gly Gln Pro
275 280 285
Val Asn Gly Gly Gln Ile Gly Pro Ile Thr Lys Lys Ile Trp Asp Asp
290 295 300
Tyr Trp Ala Leu His Tyr Asp Pro Ala Phe Ser Phe Glu Ile Lys Tyr
305 310 315 320
Asp Glu Ala Gly Ala Ser Thr Asn Gly Val Asn Gly Val His Lys His
325 330 335
His His His His His
340
<210> 6
<211> 1026
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atggcttcta tggacaaagt tttcgctggt taccagtctc gtctgcgtgt tctggaagct 60
tctaccaacc cgctggctca gggtgttgct tggatcgaag gtgaactggt tccgctgtct 120
caggctcgta tcccgctgat ggaccagggt ttcctgcact ctgacctgac ctacgacgtt 180
ccagctgtgt gggacggcag gttcttccgt ctggacgacc acatctctaa cctggaaaaa 240
tcttgctcta aactgcgtct gaaactgccg ctgccgcgtg acgaagttaa acgtgttctg 300
gttgacatgg ttgctcgttc tggtatacgt gacgcgttcg ttgaactgat agttacccgt 360
ggtctgaccg gtgttcgtgg tgctggtatc ccggaagacc tggttaacaa cctgttcatg 420
ttcctgcagc cgtacctgtg ggttatgccg ccggaaaccc agctggttgg tggttctgct 480
gttatcaccc gtaccgttcg tcgtaccccg ccgggttcta tggacccgac cgttaaaaac 540
acccagtggg gtgacctgac ccgtgctctg ctggaagctt ctgaccgtgg tgcttcttac 600
ccgttcctga ccgacggtga cgctaacatc accgaaggtt ctggttacaa catcgttctg 660
atcaaagacg gtgctatcca caccccggac cgtggtgttc tggaaggtgt aacccgtaaa 720
accgtgttcg acatcgcaaa agctaacggt ttcgaagttc gtctggaagt tgttccggtt 780
gaactggctt accgtgctga cgaaatcttc atgtgctaca ccgctggtgg tatcatgccg 840
atcacctctc tggacggtca gccggttaac ggtggtcaga tcggtccgat caccaaaaaa 900
atctgggacg actactgggc tctgcactac gacccggctt tctctttcga aatcaaatac 960
gacgaagctg gtgcttctac caacggtgtt aacggtgttc acaaacacca ccaccaccac 1020
cactaa 1026
<210> 7
<211> 331
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Met Ala Ser Met Asp Lys Val Phe Ala Gly Tyr Ala Ala Arg Gln Ala
1 5 10 15
Ile Leu Glu Ser Thr Glu Thr Thr Asn Pro Phe Ala Lys Gly Ile Ala
20 25 30
Trp Val Glu Gly Glu Leu Val Pro Leu Ala Glu Ala Arg Ile Pro Leu
35 40 45
Leu Asp Ser Gly Phe Met His Ser Asp Leu Thr Tyr Asp Val Pro Ser
50 55 60
Val Trp Asp Gly Arg Phe Phe Arg Leu Asp Asp His Ile Thr Ala Leu
65 70 75 80
Glu Ala Ser Cys Thr Lys Leu Arg Leu Arg Leu Pro Leu Pro Arg Asp
85 90 95
Gln Val Lys Gln Ile Leu Val Glu Met Val Ala Lys Ser Gly Ile Arg
100 105 110
Asp Ala Phe Val Glu Leu Ile Val Thr Arg Gly Leu Lys Gly Val Arg
115 120 125
Gly Thr Arg Pro Glu Asp Ile Val Asn Asn Leu Tyr Met Phe Val Gln
130 135 140
Pro Tyr Val Trp Asp Met Glu Pro Asp Met Gln Arg Val Gly Gly Ser
145 150 155 160
Ala Val Val Ala Arg Thr Val Arg Arg Val Pro Pro Gly Ala Ile Asp
165 170 175
Pro Thr Val Lys Asn Met Gln Trp Gly Asp Leu Val Arg Gly Met Phe
180 185 190
Glu Ala Ala Asp Arg Gly Ala Thr Tyr Pro Phe Leu Thr Asp Gly Asp
195 200 205
Ala His Leu Thr Glu Gly Ser Ala Phe Asn Ile Val Leu Val Lys Asp
210 215 220
Gly Val Leu Tyr Thr Pro Asp Arg Gly Val Glu Gln Gly Val Thr Arg
225 230 235 240
Lys Ser Val Ile Asn Ala Ala Glu Ala Phe Gly Ile Glu Val Arg Val
245 250 255
Glu Phe Val Pro Val Glu Leu Ala Tyr Arg Cys Asp Glu Ile Phe Met
260 265 270
Cys Thr Thr Ala Gly Gly Ile Met Pro Ile Thr Thr Leu Asp Gly Met
275 280 285
Pro Val Asn Gly Gly Gln Ile Gly Pro Ile Thr Lys Lys Ile Trp Asp
290 295 300
Gly Tyr Trp Ala Met His Tyr Asp Ala Ala Tyr Ser Phe Glu Ile Asp
305 310 315 320
Tyr Asn Glu Arg Asn His His His His His His
325 330
<210> 8
<211> 996
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atggcttcta tggataaagt ttttgcgggt tacgctgctc gtcaagcaat tctggaatct 60
accgaaacca ccaacccgtt cgcgaaaggt atcgcctggg ttgagggcga actggtacct 120
ctggctgaag ctcgtatccc gctgctggac tccggtttca tgcactctga cctgacctac 180
gacgttccga gcgtgtggga cggtcgtttc ttccgtctgg atgaccacat tacggcactg 240
gaggctagct gcaccaaact gcgcctgcgt ctgccgctgc cgcgtgatca ggttaaacaa 300
atcctggttg aaatggttgc gaagagcggc atccgtgacg ccttcgttga gctgatcgtg 360
acccgtggcc tgaagggtgt gcgtggcact cgtccggaag acatcgttaa taacctgtac 420
atgttcgtcc agccgtatgt gtgggacatg gaaccggata tgcagcgtgt aggtggctct 480
gctgtcgttg ctcgtaccgt acgccgcgta ccgccgggtg cgattgaccc gactgtgaaa 540
aatatgcagt ggggtgatct ggtgcgtggt atgttcgaag ctgcagaccg tggtgcgacg 600
tacccgttcc tgaccgacgg cgacgctcat ctgaccgagg gctccgcttt caatatcgtt 660
ctggttaaag atggcgttct gtacactccg gatcgtggtg tggaacaggg cgttacccgc 720
aaatctgtga tcaacgctgc ggaagcgttc ggtatcgaag ttcgtgttga atttgtgccg 780
gtcgaactgg catatcgttg cgacgagatc ttcatgtgca ccactgccgg cggcattatg 840
ccgattacta ccctggatgg catgccggtc aatggcggcc agatcggtcc aattaccaag 900
aaaatttggg atggttactg ggccatgcat tacgacgctg cttattcttt tgaaatcgac 960
tacaacgagc gtaaccacca tcaccatcac cattaa 996
<210> 9
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ctcgagatgg cttctatgga taaagttttt 30
<210> 10
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
accccactgc atatttttca cagtcgggtc 30
<210> 11
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
gatctggtgc gtggtatgtt cgaagctgca 30
<210> 12
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
ccatggttaa tggtgatggt gatggtggtt 30
<210> 13
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
ctcgagatgg cttctatgga caaagttttc 30
<210> 14
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
accacgcacc agatcacccc actgggtgtt tttaacggt 39
<210> 15
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
actgtgaaaa atatgcagtg gggtgacctg acccgtgct 39
<210> 16
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
ccatggttag tggtggtggt ggtggtgttt 30
<210> 17
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
ctcgagatga ccatgaacca gctgactatc 30
<210> 18
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
accacgcacc agatcacccc actggtagtt tttgatcca 39
<210> 19
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
actgtgaaaa atatgcagtg gggtgatctg gttcgtgcc 39
<210> 20
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
ccatggttaa tggtggtgat gatggtgcag 30
<210> 21
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (9)..(10)
<223> n is a, c, g, or t
<400> 21
tccgtctgnn kgaccacatc tctaacctgg 30
<210> 22
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (10)..(11)
<223> n is a, c, g, or t
<400> 22
atgtggtcmn ncagacggaa gaacctgccg 30
<210> 23
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (9)..(10)
<223> n is a, c, g, or t
<400> 23
ctgcggaann kttcggtatc gaagttcgtg 30
<210> 24
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (10)..(11)
<223> n is a, c, g, or t
<400> 24
ataccgaamn nttccgcagc gttgatcaca 30
Claims (10)
1.重组(R)-ω-转氨酶，其特征在于，该重组(R)-ω-转氨酶的氨基酸序列如SEQ IDNO.1所示。
2.重组(R)-ω-转氨酶突变体，其特征在于，该突变体为下列之一：
(1)由SEQ ID NO.1所示氨基酸序列的第72位的天冬氨酸突变为苯丙氨酸、酪氨酸、丙氨酸或甘氨酸；
(2)由SEQ ID NO.1所示氨基酸序列的第72位的天冬氨酸突变为甘氨酸，且第248位丙氨酸突变为赖氨酸、苏氨酸、丝氨酸、组氨酸或酪氨酸；
(3)由SEQ ID NO.1所示氨基酸序列的第72位的天冬氨酸突变为甘氨酸，且第248位丙氨酸突变为丝氨酸。
3.如权利要求2所述的重组(R)-ω-转氨酶突变体，其特征在于，该突变体的氨基酸序列如SEQ ID NO.3所示，核苷酸序列如SEQ ID NO.4所示。
4.一种如权利要求1所述的重组(R)-ω-转氨酶或如权利要求2所述的重组(R)-ω-转氨酶突变体的编码基因。
5.一种包含权利要求4所述编码基因的重组载体。
6.一种包含权利要求4所述编码基因的基因工程菌。
7.如权利要求1所述重组(R)-ω-转氨酶或权利要求2所述的重组(R)-ω-转氨酶突变体在生物催化前体酮类似物1-(吡咯烷-1-基)-4-(2,4,5-三氟苯基)-1,3-丁二酮合成西他列汀中间体(R)-3-氨基-1-(吡咯烷-1-基)-4-(2,4,5-三氟苯基)丁-1-酮中的应用。
8.如权利要求6所述基因工程菌在生物催化前体酮类似物1-(吡咯烷-1-基)-4-(2,4,5-三氟苯基)-1,3-丁二酮合成西他列汀中间体(R)-3-氨基-1-(吡咯烷-1-基)-4-(2,4,5-三氟苯基)丁-1-酮中的应用。
9.一种催化前体酮类似物1-(吡咯烷-1-基)-4-(2,4,5-三氟苯基)-1,3-丁二酮合成西他列汀中间体(R)-3-氨基-1-(吡咯烷-1-基)-4-(2,4,5-三氟苯基)丁-1-酮的方法，其特征在于，包括：以前体酮类似物1-(吡咯烷-1-基)-4-(2,4,5-三氟苯基)-1,3-丁二酮为底物，权利要求1所述的重组(R)-ω-转氨酶、权利要求2所述的重组(R)-ω-转氨酶突变体或权利要求6所述的基因工程菌为生物催化剂，异丙胺为氨基供体，磷酸吡哆醛为辅酶，在缓冲溶液中进行生物催化合成反应，得到西他列汀中间体(R)-3-氨基-1-(吡咯烷-1-基)-4-(2,4,5-三氟苯基)丁-1-酮。
10.如权利要求9所述的方法，其特征在于，所述生物催化合成反应为35～50℃；所述缓冲溶液为三乙醇胺-HCl缓冲液，pH为8～9；所述底物的浓度为800～1200mM。
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Non-Patent Citations (2)
| Title |
|---|
| ω-转氨酶不对称合成手性胺及非天然氨基酸的研究进展;程峰;《生物加工过程》;20180531;第16卷(第3期);第1-11页 * |
| ω-转氨酶分子改造研究进展;高新星;《生物工程学报》;20180725;第34卷(第7期);第1057-1068页 * |
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