CN113481254A - 西他列汀中间体的制备方法 - Google Patents
西他列汀中间体的制备方法 Download PDFInfo
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- CN113481254A CN113481254A CN202110726892.3A CN202110726892A CN113481254A CN 113481254 A CN113481254 A CN 113481254A CN 202110726892 A CN202110726892 A CN 202110726892A CN 113481254 A CN113481254 A CN 113481254A
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- Prior art keywords
- ala
- gly
- val
- transaminase
- ile
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- 238000002360 preparation method Methods 0.000 title claims abstract description 47
- WJPYOCIWVYDFDT-UHFFFAOYSA-N ethyl 3-oxo-4-(2,4,5-trifluorophenyl)butanoate Chemical compound CCOC(=O)CC(=O)CC1=CC(F)=C(F)C=C1F WJPYOCIWVYDFDT-UHFFFAOYSA-N 0.000 title claims abstract description 36
- 238000006243 chemical reaction Methods 0.000 claims abstract description 133
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- 238000000034 method Methods 0.000 claims description 39
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- QAEDTLFWHIEVPK-UHFFFAOYSA-N 1-[3-(trifluoromethyl)-6,8-dihydro-5h-[1,2,4]triazolo[4,3-a]pyrazin-7-yl]-4-(2,4,5-trifluorophenyl)butane-1,3-dione Chemical compound C1=C(F)C(F)=CC(F)=C1CC(=O)CC(=O)N1CC2=NN=C(C(F)(F)F)N2CC1 QAEDTLFWHIEVPK-UHFFFAOYSA-N 0.000 abstract description 3
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- -1 2,4, 5-trifluorophenyl Chemical group 0.000 description 10
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- 102220064084 rs752330104 Human genes 0.000 description 9
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 8
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Abstract
本申请公开了一种西他列汀中间体的制备方法,以及经由该制备方法制得的西他列汀中间体,所述制备方法包括:在一个标准大气压下沸点不高于110℃的有机溶剂存在的前提下,将转氨酶与底物西他列汀前体酮(2Z)‑4‑氧代‑4‑[3‑(三氟甲基)‑5,6‑二氢‑[1,2,4]三唑并[4,3‑a]吡嗪‑7‑(8H)‑基]‑1‑(2,4,5‑三氟苯基)丁‑2‑酮转氨接触而发生酶催化反应,从而制得西他列汀中间体(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,其中,通过将低沸点有机溶剂与底物相混合获得的混合液以流加补料的方式加入至包含所述转氨酶的预混体系中,以及在预混体系中添加特定比例的酮还原酶和辅酶再生系统,提高底物转化率,底物转化率可达99%。
Description
技术领域
本申请涉及酶工程及生物制药领域,具体涉及一种西他列汀中间体的制备方法。
背景技术
西他列汀磷酸盐是一种治疗II型糖尿病的药物,商品名为捷诺维(Januvia),其活性成分为(3R)-3-氨基-1-[3-(三氟甲基)-5,6-二氢-1,2,4-三唑并[4,3-a]吡嗪-7(8H)-基]-4-(2,4,5-三氟苯基)丁-1-酮(西他列汀,Sitagliptin)。西他列汀是一种二肽基肽酶-4(DPP-4)抑制剂,主要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,具有较好的安全性和耐受性。西他列汀制备工艺中的关键步骤为手性氨基中间体的合成。
在现有技术中,西他列汀的手性氨基中间体的合成方法主要有化学合成工艺和生物催化合成工艺,其中,化学合成工艺虽然工艺成熟,但是具有工艺步骤繁多、所用试剂(如金属催化剂等)昂贵、部分试剂(如金属催化剂等)对环境和人体具有毒害作用、合成的手性氨基中间体纯度较低等缺点。生物催化合成工艺是利用酶作为催化剂来制备西他列汀,其中所用的酶大多为转氨酶(Transaminase,TA),生物催化合成工艺具有合成的手性氨基中间体立体选择性高、反应条件温和、对环境友好、纯化工序简单的优点。
目前公开的生物催化合成工艺仍具有一些不足之处,例如:由于底物水溶性差,所以需要在催化反应体系中加入有机溶剂以提高底物的溶解性,而有机溶剂的存在会加大后续产物分离纯化的难度。在专利申请WO2010099501中,采用生物催化合成工艺制备西他列汀的手性氨基中间体,在催化反应体系中添加了有机溶剂二甲基亚砜以提高底物的溶解性,由于二甲基亚砜沸点高,所以难以通过蒸馏的方式从反应产物中去除,使得反应产物在提纯过程中损失率较高,降低了产物的收率,导致制备成本的增加。因此,有必要对现有的生物催化合成工艺进行优化,以提供一种经济可靠、满足工业化生产需求的生物催化合成工艺来制备西他列汀的手性氨基中间体。
发明内容
针对现有技术的不足之处,本申请提供了一种西他列汀中间体的制备方法,以改善现有生物催化合成工艺制备西他列汀中间体所存在的产物分离提纯难度高、产物收率低、生产成本高等问题。
第一方面,本申请提供了一种西他列汀中间体的制备方法,所述制备方法包括步骤:
将作为底物的西他列汀前体酮与在一个标准大气压下沸点不高于110℃的有机溶剂相混合,以获得混合液;以及
将所述混合液与转氨酶进行转氨接触以发生酶催化反应,生成西他列汀中间体;
其中,所述底物为式(Ⅰ)所示的化合物:
所述西他列汀中间体为式(Ⅱ)所示的化合物:
进一步地,所述将所述混合液与转氨酶进行转氨接触的方式为:将所述混合液以流加补料的方式加入至包含所述转氨酶的预混体系中。
进一步地,所述混合液中所述底物的浓度为10克每升(g/L)至500克每升(g/L),流加速度为18毫升每小时(mL/h)至23毫升每小时(mL/h),流加时间为14小时(h)至17小时(h)。
进一步地,在所述酶催化反应的反应体系中除了包括所述混合液和所述转氨酶,还包括异丙胺和磷酸吡哆醛,其中,磷酸吡哆醛用作辅酶,异丙胺用作辅底物。
进一步地,所述有机溶剂包括甲醇、乙醇、异丙醇、四氢呋喃、乙酸乙酯、乙酸丙酯、乙酸丁酯、二甲基亚砜、二氯甲烷以及甲苯中的至少一种。
进一步地,所述有机溶剂为甲醇。
进一步地,在所述酶催化反应的反应体系中除了包括所述混合液和所述转氨酶,还包括酮还原酶和辅酶再生系统,所述辅酶再生系统用于提供循环再生的烟酰胺腺嘌呤二核苷酸或烟酰胺腺嘌呤二核苷酸磷酸。
进一步地,所述辅酶再生系统包括葡萄糖和葡萄糖脱氢酶,且所述转氨酶、所述葡萄糖脱氢酶和所述酮还原酶的质量比为6:(1~2):(1~2)。
进一步地,所述酶催化反应的反应条件为pH为7.5至11.0,温度为15℃至65℃,转速为250转每分钟(r/min)至500转每分钟(r/min)。
进一步地,所述转氨酶的氨基酸序列如SEQ ID NO:1所示,或者为与所述SEQ IDNO:1至少具有80%、至少具有85%、至少具有90%、至少具有95%、至少具有96%、至少具有97%、至少具有98%或至少具有99%相似性的氨基酸序列。
其中,所述转氨酶的添加方式可以是:直接将所述转氨酶的纯酶液或包含所述转氨酶的酶制剂添加于预混体系中;或者,将含有所述转氨酶的编码基因的转化体经发酵培养后获得的湿菌体添加于预混体系中;或者,将所述湿菌体经破碎处理后的细胞破碎液添加于预混体系中;或者,将所述细胞破碎液经分离纯化处理后的产物添加于预混体系中。
有益效果:本申请提供了一种西他列汀中间体的制备方法,所述制备方法包括:在低沸点有机溶剂存在的情况下,将转氨酶与底物西他列汀前体酮(2Z)-4-氧代-4-[3-(三氟甲基)-5,6-二氢-[1,2,4]三唑并[4,3-a]吡嗪-7-(8H)-基]-1-(2,4,5-三氟苯基)丁-2-酮转氨接触而发生酶催化反应,从而制得西他列汀中间体(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,具有简化产物分离纯化工序、提高产物收率和有机溶剂回收率、降低生产成本的优点。
经实验发现,采用甲醇作为有机溶剂,并且将甲醇与底物相混合获得的混合液以流加补料的方式加入至包含所述转氨酶的预混体系中的技术手段有利于提高底物转化率,底物转化率可达97%。
经实验发现,采用甲醇作为有机溶剂,并且在包含所述转氨酶的预混体系中添加酮还原酶和辅酶再生系统,以使转氨酶、酮还原酶以及辅酶再生系统按照特定的比例复配的技术方案有利于提高底物转化率,底物转化率至少为97%。此外,将甲醇与底物相混合获得的混合液以流加补料的方式加入至该预混体系中的技术手段可进一步提高底物转化率,底物转化率可达99%。
附图说明
下面结合附图,通过对本申请的具体实施方式详细描述,将使本申请的技术方案及其有益效果显而易见。
图1为实施例3中含有编号3的转氨酶的反应体系在初始反应时的HPLC图谱,其中,标号B位置处的波峰对应为底物峰。
图2为实施例3中含有编号3的转氨酶的反应体系在反应24h时的HPLC图谱,其中,标号A位置处的波峰对应为产物峰,标号B位置处的波峰对应为底物峰。
图3为实施例4中含有编号3的转氨酶的反应体系在反应0.5h时的HPLC图谱,其中,标号A位置处的波峰对应为产物峰,标号B位置处的波峰对应为底物峰。
图4为实施例4中含有编号3的转氨酶的反应体系在反应24h时的HPLC图谱,其中,标号A位置处的波峰对应为产物峰,标号B位置处的波峰对应为底物峰。
图5为实施例5中含有编号3的转氨酶的反应体系在初始反应时的HPLC图谱,其中,标号B位置处的波峰对应为底物峰。
图6为实施例5中含有编号3的转氨酶的反应体系在反应24h时的HPLC图谱,其中,标号A位置处的波峰对应为产物峰,标号B位置处的波峰对应为底物峰。
图7为实施例6中含有编号3的转氨酶的反应体系在反应0.5h时的HPLC图谱,其中,标号A位置处的波峰对应为产物峰,标号B位置处的波峰对应为底物峰。
图8为实施例6中含有编号3的转氨酶的反应体系在反应24h时的HPLC图谱,其中,标号A位置处的波峰对应为产物峰,标号B位置处的波峰对应为底物峰。
具体实施方式
下面将结合本申请实施例中的附图,对本申请实施例中的技术方案进行清楚、完整地描述。显然,所描述的实施例仅仅是本申请一部分实施例,而不是全部的实施例。基于本申请中的实施例,本领域技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本申请保护的范围。在本申请实施例中,未注明具体条件的实验方法,按照本领域常规方法进行,或者按照制造厂商所建议的条件进行。除非另行定义,本申请实施例中所使用的所有专业与科学用语与本领域技术人员所熟悉的意义相同。除非另有说明,文中涉及的试剂与材料均可商购获得,或本领域技术人员可依据公知常识自行制备。
如本申请所用,“包括”是指“包括但不限于”。
如本申请所用,“相似性”是指两个氨基酸序列或两个核苷酸序列之间的相关性。在本申请实施例中,至少具有80%以上相似性可以理解为80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%的序列相似性,对应相似性的数值为整数;也可以进一步理解为80.1%、81.2%、82.3%、83.4%、84.5%、85.6%、86.7%、87.8%、88.9%、89.8%、90.3%、91.7%、92.2%、93.5%、94.8%、95.9%、96.6%、97.5%、98.4%或99.9%,但小于100%的序列相似性,对应相似性的数值为小数。
如本申请所用,氨基酸由单字母或三字母代码表示,具有如下含义:A:Ala(丙氨酸);R:Arg(精氨酸);N:Asn(天冬酰胺);D:Asp(天冬氨酸);C:Cys(半胱氨酸);Q:Gln(谷氨酰胺);E:Glu(谷氨酸);G:Gly(甘氨酸);H:His(组氨酸);I:Ile(异亮氨酸);L:Leu(亮氨酸);K:Lys(赖氨酸);M:Met(甲硫氨酸);F:Phe(苯丙氨酸);P:Pro(脯氨酸);S:Ser(丝氨酸);T:Thr(苏氨酸);W:Trp(色氨酸);Y:Tyr(酪氨酸);V:Val(缬氨酸)。
如本申请所用,“点突变”是指特定位点氨基酸的取代、缺失或插入。在本申请的实施例中,点突变的方式为特定位点氨基酸的取代。对于氨基酸取代的点突变方式,命名方法为:原始氨基酸,原始氨基酸的位点,取代氨基酸,例如:T34A表示在SEQ ID NO:1所示氨基酸序列中第34位点处采用丙氨酸取代原始的苏氨酸,又如:V185R表示在SEQ ID NO:1所示氨基酸序列中第185位点处采用精氨酸取代原始的缬氨酸,又如:S181T、T237V和R259E表示在SEQ ID NO:1所示氨基酸序列中第181位点处采用苏氨酸取代原始的丝氨酸,且在SEQ IDNO:1所示氨基酸序列中237位点处采用缬氨酸取代原始的苏氨酸,且在SEQ ID NO:1所示氨基酸序列中259点处采用谷氨酸取代原始的精氨酸。
如本申请所用,“重组表达载体”是指一种DNA构建体,其含有与合适的控制序列可操作地连接的核酸分子,所述控制序列能够实现核酸分子在合适的表达系统中表达。在本申请实施例中,重组表达载体是指采用分子生物学技术将外源基因插入至载体上,由此形成的DNA构建体,示例pUC19-WT为重组表达载体。
本申请的各个实施例可以以一个范围的型式存在;应当理解,以一范围型式的描述仅仅是因为方便及简洁,不应理解为对本发明范围的硬性限制;因此,应当认为所述的范围描述已经具体公开所有可能的子范围以及该范围内的单一数值。例如,对于“所述有机溶剂的体积占所述反应体系总体积的1%至50%”的描述,应当认为从1%至50%的范围描述已经具体公开子范围,例如从1%到10%,从11%到20%,从21%到30%,从31%到40%,从41%到50%等,以及所数范围内的单一数字,例如1%、5%、10%、15%、20%、25%、30%、35%、40%、45%、50%等,此不管范围为何皆适用。此外,每当在本申请中指出数值范围,是指包括所指范围内的任何引用的数字(分数或整数)。
本申请实施例提供了一种西他列汀中间体的制备方法,以及经由该制备方法制得的西他列汀中间体,所述制备方法包括以下步骤:
Sa、将式(Ⅰ)所示的西他列汀前体酮(2Z)-4-氧代-4-[3-(三氟甲基)-5,6-二氢-[1,2,4]三唑并[4,3-a]吡嗪-7-(8H)-基]-1-(2,4,5-三氟苯基)丁-2-酮作为底物,与在一个标准大气压下沸点不高于110℃的有机溶剂相混合,以获得混合液;以及
Sb、将所述混合液与转氨酶进行转氨接触以发生酶催化反应,生成式(Ⅱ)所示的西他列汀中间体。
在一些实施例中,步骤Sb中将所述混合液与转氨酶进行转氨接触的方式为:全部的所述混合液直接与所述转氨酶相混合。作为示例,所述酶催化反应的反应体系包括所述混合液、转氨酶、异丙胺和磷酸吡哆醛,在整个反应体系中,底物的浓度为100mmol/L至800mmol/L,异丙胺的浓度为0.2mol/L至5mol/L,磷酸吡哆醛的浓度为5mmol/L至100mmol/L,有机溶剂的体积占整个反应体系总体积的1%至50%;反应条件为:pH为7.5至11.0,温度为15℃至65℃,转速为250r/min至500r/min。
在本申请的一些实施例中,步骤Sb中的转氨酶的氨基酸序列如SEQ ID NO:1所示,或者为与所述SEQ ID NO:1至少具有80%、至少具有85%、至少具有90%、至少具有95%、至少具有96%、至少具有97%、至少具有98%或至少具有99%相似性的氨基酸序列。
在本申请的一些实施例中,步骤Sb中的转氨酶的氨基酸序列是由SEQ ID NO:1所示的氨基酸序列发生一个或多个点突变得到的氨基酸序列。
在本申请的一些实施例中,发生所述点突变的氨基酸包括SEQ ID NO:1中的第34位氨基酸T、第77位氨基酸H、第80位氨基酸F、第153位氨基酸Y、第155位氨基酸I、第158位氨基酸L、第163位氨基酸P、第181位氨基酸S、第183位氨基酸A、第185位氨基酸V、第199位氨基酸V、第218位氨基酸S、第237位氨基酸T、第259位氨基酸R、第260位氨基酸D、第276位氨基酸R、第277位氨基酸G、第293位氨基酸V、第309位氨基酸W、第321位氨基酸M和第322位氨基酸D中的一个或多个。
在本申请的一些实施例中,发生所述点突变的方式为T34A、H77N、F80A、Y153T、I155V、L158Q、P163F、S181T、A183R、V185R、V199I、S218C、T237V、R259E、D260S、R276Y、G277D、V293F、W309V、M321N或D322V。
在本申请的一些实施例中,发生所述点突变的方式为以下任一所示组合:
(1)T34A和F80A;
(2)T34A、H77N和F80A;
(3)T34A、F80A和Y153T;
(4)Y153T、P163F、S181T和V185S;
(5)H77N、I155V、S181T和V185S;
(6)T34A、F80A、L158Q、P163F、S181T、A183R和V185S;
(7)P163F、S181T、A183R和V185S;
(8)S181T、T237V和R259E;
(9)V185S、A183R、S181T、T237V和R259E;
(10)F80A、L158Q、P163F、S181T、A183R、V185S和S218C;
(11)T34A、H77N、F80A和D260S;
(12)F80A、L158Q、P163F、S181T、R276Y和G277D;
(13)T34A、H77N、F80A、D260S、V293F、W306V和M321N;或
(14)T34A、F80A、L158Q、P163F、S181T、A183R、V185S、M321N和D322V。
在本申请的一些实施例中,步骤Sb中将所述混合液与转氨酶进行转氨接触的方式为:将所述混合液以流加补料的方式加入至包含所述转氨酶的预混体系中,该方式有利于提高底物转化率。作为示例,在所述预混体系中,异丙胺的浓度为0.2mol/L至5mol/L,磷酸吡哆醛的浓度为5mmol/L至100mmol/L;在所述混合液中,所述底物的浓度为10g/L至500g/L,流加速度为18g/h至23g/h,流加时间为14h至17h;反应条件为:pH为7.5至11.0,温度为15℃至65℃,转速为250r/min至500r/min。
在本申请的一些实施例中,所述有机溶剂包括甲醇、乙醇、异丙醇、四氢呋喃、乙酸乙酯、乙酸丙酯、乙酸丁酯、二甲基亚砜、二氯甲烷以及甲苯中的至少一种。
作为示例,所述有机溶剂为甲醇、乙醇、异丙醇、四氢呋喃、乙酸乙酯、乙酸丙酯、乙酸丁酯、二甲基亚砜、二氯甲烷或甲苯。
作为示例,所述有机溶剂包括以下任意一种组合剂:
(1)甲醇和乙醇复配获得的组合剂;
(2)甲醇和二甲基亚砜复配获得的组合剂;
(3)二甲基亚砜和乙醇复配获得的组合剂;
(4)甲醇和四氢呋喃复配获得的组合剂;
(5)甲醇、乙醇和异丙醇复配获得的组合剂;
(6)甲醇、乙醇和二甲基亚砜复配获得的组合剂;以及
(7)甲醇、异丙醇和二甲基亚砜复配获得的组合剂。
在本申请的一些实施例中,在所述酶催化反应的反应体系中除了包括所述混合液和所述转氨酶,还包括酮还原酶和辅酶再生系统,所述辅酶再生系统用于提供循环再生的烟酰胺腺嘌呤二核苷酸或烟酰胺腺嘌呤二核苷酸磷酸。
作为示例,所述辅酶再生系统包括葡萄糖和葡萄糖脱氢酶,且所述转氨酶、所述葡萄糖脱氢酶和所述酮还原酶的质量比为6:(1~2):(1~2)。反应体系的反应条件为:pH为7.5至11.0,温度为15℃至65℃,转速为250r/min至500r/min。
在本申请的一些实施例中,所述转氨酶的氨基酸序列如SEQ ID NO:1所示,或者为与所述SEQ ID NO:1至少具有80%、至少具有85%、至少具有90%、至少具有95%、至少具有96%、至少具有97%、至少具有98%或至少具有99%相似性的氨基酸序列。
实施例1:提供转氨酶催化反应所用的转氨酶
本申请实施例共提供了五种转氨酶(对应编号1至编号5),其中,编号1至编号4的转氨酶是由具有SEQ ID NO:1所示氨基酸序列的转氨酶发生一个或多个点突变获得,其中,具有SEQ ID NO:1所示氨基酸序列的转氨酶的编码基因的核苷酸序列如SEQ ID NO:2所示,编号1至编号5的转氨酶具体信息详见下表1:
表1编号1至编号5的转氨酶的突变方式及序列信息
1.1、构建含有转氨酶编码基因的基因工程菌
分别构建含有编号1至编号5转氨酶编码基因的基因工程菌,其中,构建含有编号1至编号4转氨酶编码基因的基因工程菌的步骤流程相同,下面以构建含有编号1的转氨酶编码基因的基因工程菌为例进行详细说明:
1.11、构建重组表达载体pUC19-WT
选择未插入外源基因的pUC-19质粒作为载体,pUC-19质粒上具有酶切位点BamH I和Nde I,pUC19-WT的构建包括如下步骤:
S1.11-a、人工合成如SEQ ID NO:2所示的核苷酸序列,然后采用BamH I和Nde I限制性内切酶对合成的核苷酸序列进行双酶切,1%琼脂糖凝胶电泳检测酶切完全后,胶回收目的基因片段,其中,双酶切后回收目的基因片段的操作根据胶回收试剂盒操作说明实施;
S1.11-b、采用BamH I和Nde I限制性内切酶对pUC-19质粒进行双酶切,1%琼脂糖凝胶电泳检测酶切完全后,胶回收载体骨架,其中,双酶切后回收载体骨架的操作根据胶回收试剂盒操作说明实施;
S1.11-c、将步骤S1.11-b获得的目的基因片段,与步骤S1.11-b获得的载体骨架相混合,在T4连接酶的作用下16℃连接过夜,然后将连接产物转化至DH5a感受态细胞内,挑取单克隆子测序验证,提取测序正确的重组质粒,获得包含编号1的转氨酶编码基因的重组表达载体,命名pUC19-WT,其中,连接体系为20μL,具体是:2μL的10×T4连接酶缓冲液(Buffer)、5μL的目的基因片段、5μL的载体骨架、2μL的T4连接酶以及6μL的双蒸水(ddH2O)。
1.12、构建含有编号1的转氨酶编码基因的基因工程菌
S1.12-a、采用定点突变策略,根据待突变的氨基酸位点利用Oligo7软件来设计点突变引物,通过在上下游突变引物的5’端以插入、替换或缺失碱基的方式引入突变,上游突变引物的核苷酸序列如SEQ ID NO.13所示,下游突变引物的核苷酸序列如SEQ ID NO.14所示;
S1.12-b、以1.11中构建的重组表达载体pUC19-WT为模板,以步骤S1.2.1中的上游突变引物和下游突变引物作为PCR扩增引物,采用KOD高保真酶试剂盒进行反向PCR,反向PCR的反应程序为:95℃预变性3min;98℃变性30s,55℃退火30s,68℃延伸3min,28个循环;72℃延伸5min,获得编号1的转氨酶编码基因的核苷酸序列;
S1.12-c、使用Dpn I限制性内切酶处理步骤S1.12-b中编号1的转氨酶编码基因的核苷酸序列,酶切后的产物经T4连接酶连接后转化大肠杆菌BL21(DE3)感受态,随后涂布含卡那霉素的LB抗性平板,置于37℃倒置培养18h,挑选单菌落转接含卡那霉素的LB液体培养基中,挑选培养液送样测序,将测序正确的克隆子保存备用,从而获得以大肠杆菌为宿主的含有编号1的转氨酶编码基因的基因工程菌。
1.13、构建含有编号5的转氨酶编码基因的基因工程菌
S1.13-a、人工合成如SEQ ID NO:12所示的核苷酸序列,然后参照步骤S1.11-a至S1.11-c构建获得包含编号5的转氨酶编码基因的重组表达载体pUC19-AF;
S1.13-b、使用Dpn I限制性内切酶处理重组表达载体pUC19-AF,酶切后的产物经T4连接酶连接后转化大肠杆菌BL21(DE3)感受态,随后涂布含卡那霉素的LB抗性平板,置于37℃倒置培养18h,挑选单菌落转接含卡那霉素的LB液体培养基中,挑选培养液送样测序,将测序正确的克隆子保存备用,从而获得以大肠杆菌为宿主的含有编号5的转氨酶编码基因的基因工程菌。
1.2、含有转氨酶编码基因的基因工程菌的诱导表达及后处理
含有编号1的转氨酶编码基因的基因工程菌至含有编号5的转氨酶编码基因的基因工程菌的诱导表达及后处理方法均相同,下面以对含有编号1的转氨酶编码基因的基因工程菌进行诱导表达及后处理为例进行详细说明。
将含有编号1的转氨酶编码基因的基因工程菌接种至含50μg/mL卡那霉素的LB液体培养基中,在37℃、180r/min的条件下培养至OD600为0.6~0.8,获得种子菌液。将种子菌液以1%的体积浓度接种至新鲜的含50μg/mL卡那霉素的自诱导培养基,置于30℃培养18h,获得培养液。将培养液在25℃、8000r/min的条件下离心10min,弃上清液以收集沉淀物,将沉淀物用pH为7.0的PB缓冲液清洗数遍,收集湿菌体备用。
其中,自诱导培养基的配制方法为:分别称取120g的酵母粉、60g胰蛋白胨、0.75g的硫酸镁(MgSO4)、16.5g的硫酸铵((NH4)2SO4)、32.5g的磷酸二氢钾(KH2PO4)、35.5g的磷酸氢二钠(Na2HPO4)、2.5g的葡萄糖以及10g的α-乳糖,然后将各个称取好的组分全部加入至磨粉机内,充分研磨至粉状,获得粉末状的自诱导培养基。将粉末状的自诱导培养基溶于1L去离子水,充分混匀后调节pH至7.0,然后121℃灭菌30min。
通过超纯水重悬制得的湿菌体,以制得菌体浓度为20%的菌液。采用超声波破碎法或高压均质破碎法处理菌液,破碎条件可依据实际需要自行选择。示例超声波破碎法的工作参数为:破碎1s;暂停2s;在180W的功率下,破碎10min。示例高压均质破碎法的工作参数为:在50HZ和800bar的条件下,破碎两次。
菌液破碎处理后,在4℃、12000r/min的条件下离心10~15min,以除去细胞碎片和大分子杂质,收集上清液保存于-20℃和4℃以备用,上清液即为编号1的转氨酶酶液。
同理,采用上述方法分别制得编号2至编号5的转氨酶酶液。
实施例2:选择转氨酶催化反应所用的有机溶剂
将实施例1制得的编号1至编号5的转氨酶酶液进行有机溶剂筛选实验。
2.1、有机溶剂单剂实验
有机溶剂单剂实验的方法流程如下:
S2.1.1、取100μL实施例1中制得的单种转氨酶酶液(编号1的转氨酶酶液、编号2的转氨酶酶液、编号3的转氨酶酶液、编号4的转氨酶酶液或编号5的转氨酶酶液)置于反应板上,然后向转氨酶酶液中加入1.5mL pH为8.5的储备混合物,其中,储备混合物包含0.2mol/L的三乙醇胺、2mmol/L的磷酸吡哆醛(PLP)、2mol/L的异丙胺(IPM)以及无菌水,获得预混体系;
S2.1.2、向步骤S2.1.1获得的预混体系中加入400μL的混合液,其中,混合液包含浓度为25g/L的底物化合物和有机溶剂,获得pH为8.5的反应体系,反应体系包含25g/L的底物化合物、1.5mol/L的IPM、2mmol/L的PLP以及20%(v/v)的有机溶剂,底物化合物为西他列汀前体酮(2Z)-4-氧代-4-[3-(三氟甲基)-5,6-二氢-[1,2,4]三唑并[4,3-a]吡嗪-7-(8H)-基]-1-(2,4,5-三氟苯基)丁-2-酮;
S2.1.3、将含有步骤S2.1.2的反应体系的反应板热密封,在45℃、200r/min的振荡条件下持续反应24h,待反应结束后,取样离心获得反应液,并利用高效液相色谱(HighPerformance Liquid Chromatography,HPLC)法对反应液进行检测分析。
在步骤S2.1.2中,有机溶剂分别选用二甲基亚砜(DMSO)、甲醇、乙醇、异丙醇、四氢呋喃、乙酸乙酯、乙酸丁酯、乙酸丙酯、二氯甲烷或甲苯进行实验。
在步骤S2.1.3中,HPLC的仪器型号为Agilent 1260(安捷伦),HPLC的工作条件如下:
(1)进样液的制备:取50μL的反应液,向其中加入950μL的无水乙腈,并充分混匀,然后在12000r/min离心1min,收集上清液作为进样液,每次进样5μL;
(2)色谱柱:DIKMA Diamond C18,4.6*150mm,5μm。
(3)流动相的制备:将无水乙腈和0.1%(体积百分数)的三氟乙酸按照30:70的体积比混合配制而成。
(4)流速:1mL/min。
(5)检测波长:268nm。
(6)柱温:30℃。
根据底物化合物的减少量计算转化率(%),转化率(%)的测定结果详见表2,转化率(%)的计算公式如下式(1):
在式(1)中,A1为反应后的底物化合物峰面积,A2为反应前的底物化合物峰面积。
实验结果如下表2所示:
表2有机溶剂单剂实验结果
由表1可知,相较于表1中其他的有机溶剂,选择甲醇作为反应体系中的有机溶剂,可使酶催化转氨反应的转化率具有明显的优势,底物转化率可达95%。其次,选择乙醇作为反应体系中的有机溶剂,可使酶催化转氨反应的底物转化率达到85%。选择甲苯作为反应体系中的有机溶剂,对应酶催化转氨反应的转化率具有明显的劣势,转化率为18%至30%。
2.2、有机溶剂组合剂实验
有机溶剂组合剂实验的方法流程如下:
S2.2.1、取200μL实施例1制得的单种转氨酶酶液置于反应板上,然后向酶液中加入3.0mL pH为8.5的储备混合物,其中,储备混合物包含0.2mol/L的三乙醇胺、2mmol/L的磷酸吡哆醛(PLP)、2mol/L的异丙胺(IPM)以及无菌水,获得预混体系;
S2.2.2、向步骤S2.2.1获得的预混体系中加入800μL的混合液,其中,混合液包含浓度为25g/L的底物化合物和有机溶剂,获得pH为8.5的反应体系,反应体系包含5g/L的底物化合物、1.5mol/L的IPM、2mmol/L的PLP以及20%(v/v)的有机溶剂,底物化合物为西他列汀前体酮1-(3-氧吡咯烷-1-基)-4-(,4,5-三氟苯基)丁二酮;
S2.2.3、将含有步骤S2.2.2的反应体系的反应板热密封,在45℃、200r/min的振荡条件下持续反应24h,取样离心获得反应液,并利用HPLC法对反应液进行检测分析。
在步骤S2.2.2中,有机溶剂为至少两种单剂的组合剂,具体组合情况详见表2。
在步骤S2.2.3中,HPLC的检测条件参照实施例1中步骤S2.1.3进行。
实验结果如下表3所示:
表3有机溶剂组合剂实验结果
由表1和表2可知,相较于表1中其他的单剂以及表2中所有的组合剂,选择甲醇作为反应体系中的有机溶剂,可使酶催化转氨反应的底物转化率具有明显的优势,底物转化率可达95%,即:甲醇与表1中除甲醇之外的一种或两种单剂以特定比例混合获得的组合剂,对应酶催化转氨反应的转化率均不如甲醇单剂。因此,选择甲醇单剂作为反应体系中的有机溶剂。实施例3:利用转氨酶催化制备西他列汀中间体(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
本实施例提供了一种利用转氨酶催化西他列汀前体酮1-(3-氧吡咯烷-1-基)-4-(,4,5-三氟苯基)丁二酮生成西他列汀中间体(R)-1-[3氨基-4-(2,4,5-三氟苯基)丁酰基]吡咯-3-酮的制备方法,所述制备方法包括如下步骤:
S3.1、取一200mL的反应瓶,向反应瓶中加入18.8g的25%(质量百分比浓度,w/w)异丙胺水溶液、11.85g的无菌水以及39.35g的底物-甲醇溶液,控温至10℃以下,然后缓慢滴加30g的36%(质量百分比浓度,w/w)盐酸溶液,再升温至45℃,并用36%盐酸溶液或25%异丙胺水溶液调整pH至8.0,获得混合液;
S3.2、向步骤S3.1获得的混合液中加入247mg的PLP,以及10g的实施例1中制得的单种转氨酶酶液,获得反应体系;
S3.3、将步骤S3.2获得的反应体系于45℃、pH8.0以及400r/min的条件下反应24h,待反应结束后,取样离心获得反应液,并利用HPLC法对反应液进行检测分析;
S3.4、先调节步骤S3.3的反应液的pH至3.0,然后在35~45℃的条件下搅拌2h,然后过滤并收集滤液,再将滤液减压蒸馏以除去甲醇以获得粗提液,接着降温至室温,向粗提液中加入乙酸乙酯以萃取除杂并收集有机相,再将萃取后的水相调节pH至9.0,然后向调节pH后的水相中加入乙酸乙酯以萃取除杂并收集有机相,合并两次收集的有机相,无水硫酸镁干燥后减压浓缩以获得产物。
在步骤S3.1中,25%异丙胺水溶液的配制方法为:取一200mL的三口烧瓶,向其中加入150g的无菌水,然后控温至10℃以下,再缓慢加入50g的异丙胺,整个加料过程控温在20℃以内,加料结束后搅拌溶清,最后密封保存以备用。
在步骤S3.1中,底物-甲醇溶液的配制方法为:称取40g的底物,然后向称取好的底物中加入88.89g的甲醇,然后加热至30℃,搅拌溶清后密封保存以备用。
在步骤S3.3中,HPLC的检测条件参照实施例2中步骤S2.1.3进行。
在本实施例中,编号1至编号5的转氨酶的底物转化率详见下表4:
表4编号1至编号5的转氨酶的底物转化率
图1示出了含有编号3的转氨酶的反应体系在初始反应时的HPLC图谱,其中,在标号B位置处的波峰对应为底物峰,对应式(1)中的A2为1829464。图2示出了含有编号3的转氨酶的反应体系在反应24h时的HPLC图谱,在标号A位置处的波峰对应为产物峰,在标号B位置处的波峰对应为底物峰,对应式(1)中的A1为91473,按照式(1)计算获得转化率为95%。
实施例4:利用转氨酶催化制备西他列汀中间体(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
本实施例提供了一种利用转氨酶催化西他列汀前体酮1-(3-氧吡咯烷-1-基)-4-(,4,5-三氟苯基)丁二酮生成西他列汀中间体(R)-1-[3氨基-4-(2,4,5-三氟苯基)丁酰基]吡咯-3-酮的制备方法,所述制备方法包括如下步骤:
S4.1、取一2000mL的反应瓶,向反应瓶中加入90g的25%(质量百分比浓度,w/w)异丙胺水溶液和680g的无菌水,控温至10℃以下,然后缓慢滴加120g的36%(质量百分比浓度,w/w)盐酸溶液,再升温至50℃,并用25%异丙胺水溶液或36%盐酸溶液调整pH至8.5,获得混合液;
S4.2、向步骤S4.1获得的混合液中加入1.236g的PLP,以及30g的实施例1中制得的单种转氨酶酶液,获得预混体系;
S4.3、在50℃、pH8.5的条件下,按照21.5g/h的流速向步骤S4.2获得的预混体系中缓慢加入底物-甲醇溶液,流加时间为14h,获得反应体系;
S4.4、将步骤S4.3的反应体系置于50℃、pH8.5以及400r/min的条件下反应24h,待反应结束后,取样离心获得反应液,并利用HPLC法对反应液进行检测分析;
S4.5、参照步骤S3.4制得产物。
在步骤S4.1中,25%异丙胺水溶液的配制方法为:取一500mL的三口烧瓶,向其中加入300g的无菌水,然后控温至10℃以下,再缓慢加入100g的异丙胺,整个加料过程控温在20℃以内,加料结束后搅拌溶清,最后密封保存以备用。
在步骤S4.3中,底物-甲醇溶液的配制方法为:称取100g的底物,然后向称取好的底物中加入200g的甲醇,然后加热至30℃,搅拌溶清后密封保存以备用。
在本实施例中,编号1至编号5的转氨酶的底物转化率详见下表5:
表5编号1至编号5的转氨酶的底物转化率
图3和图4分别示出了含有编号3的转氨酶的反应体系在反应0.5h和24h时的HPLC图谱,其中,在标号A位置处的波峰对应为产物峰,在标号B位置处的波峰对应为底物峰,反应0.5h时的底物转化率已达到较高的水平,约为91%。
由实施例3和实施例4可知,在酶催化反应体系中,相较于一次性添加混合液(包含底物和在一个标准大气压下沸点不高于110℃的有机溶剂),所述混合液以流加的方式添加入酶催化反应体系中更有利于提升底物转化率,底物转化率例如可以提升2%。
实施例5:利用转氨酶催化制备西他列汀中间体(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
本实施例提供了一种利用转氨酶催化西他列汀前体酮1-(3-氧吡咯烷-1-基)-4-(,4,5-三氟苯基)丁二酮生成西他列汀中间体(R)-1-[3氨基-4-(2,4,5-三氟苯基)丁酰基]吡咯-3-酮的制备方法,所述制备方法包括如下步骤:
S5.1、取一200mL的反应瓶,向反应瓶中加入18.8g的25%(质量百分比浓度,w/w)异丙胺水溶液、11.85g的无菌水、2g的葡萄糖以及39.35g的底物-甲醇溶液,充分搅拌以使葡萄糖溶解后控温至10℃以下,然后缓慢滴加60g的36%(质量百分比浓度,w/w)盐酸溶液,再升温至37℃,并用36%盐酸溶液或25%异丙胺水溶液调整pH至9.0,获得混合液;
S5.2、向步骤S5.1获得的混合液中加入400mg的PLP、12g的实施例1中制得的单种转氨酶酶液、2g的酮还原酶(具有如SEQ ID NO.15所示的氨基酸序列)以及2g的葡萄糖脱氢酶(具有如SEQ ID NO.16所示的氨基酸序列),获得反应体系;
S5.3、将步骤S5.2获得的反应体系于37℃、pH9.0以及400r/min的条件下反应24h,待反应结束后,取样离心获得反应液,并利用HPLC法对反应液进行检测分析;
S5.4、参照步骤S3.4制得产物。
在步骤S5.1中,25%异丙胺水溶液和底物-甲醇溶液参照实施例3进行配制。
在步骤S5.3中,HPLC的检测条件参照实施例2中步骤S2.1.3进行。
在本实施例中,编号1至编号5的转氨酶的底物转化率详见下表6:
表6编号1至编号5的转氨酶的底物转化率
图5示出了含有编号3的转氨酶的反应体系在初始反应时的HPLC图谱,其中,在标号B位置处的波峰对应为底物峰,对应式(1)中的A2为1777077。图6示出了含有编号3的转氨酶的反应体系在反应24h时的HPLC图谱,其中,在标号A位置处的波峰对应为产物峰,在标号B位置处的波峰对应为底物峰对应式(1)中的A1为53312,按照式(1)计算获得转化率为97%。
由实施例3和实施例5可知,在酶催化反应体系中添加特定比例的酮还原酶和葡萄糖脱氢酶有利于提升底物转化率,底物转化率例如可以提升5%。
实施例6:利用转氨酶催化制备西他列汀中间体(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
本实施例提供了一种利用转氨酶催化西他列汀前体酮1-(3-氧吡咯烷-1-基)-4-(,4,5-三氟苯基)丁二酮生成西他列汀中间体(R)-1-[3氨基-4-(2,4,5-三氟苯基)丁酰基]吡咯-3-酮的制备方法,所述制备方法包括如下步骤:
S6.1、取一2000mL的反应瓶,向反应瓶中加入83.4g的25%(质量百分比浓度,w/w)异丙胺水溶液、697g的无菌水以及4g的葡萄糖,充分搅拌以使葡萄糖溶解后控温至10℃以下,然后缓慢滴加150g的36%(质量百分比浓度,w/w)盐酸溶液,再升温至45℃,并用25%异丙胺水溶液或36%盐酸溶液调整pH至9.5,获得混合液;
S6.2、向步骤S6.1获得的混合液中加入833.4mg的PLP,以及30g的实施例1中制得的单种转氨酶酶液、5g的酮还原酶(具有如SEQ ID NO.15所示的氨基酸序列)以及5g的葡萄糖脱氢酶(具有如SEQ ID NO.16所示的氨基酸序列),获得预混体系;
S6.3、在45℃、pH9.5的条件下,按照21.6g/h的流速向步骤S4.2获得的预混体系中缓慢加入底物-甲醇溶液,流加时间为14h,获得反应体系;
S6.4、将步骤S6.3的反应体系置于45℃、pH9.5以及400r/min的条件下反应24h,待反应结束后,取样离心获得反应液,并利用HPLC法对反应液进行检测分析;
S6.5、参照步骤S3.4制得产物。
在步骤S6.1和步骤S6.3中,25%异丙胺水溶液和底物-甲醇溶液参照实施例4进行配制。
在本实施例中,编号1至编号5的转氨酶的底物转化率、产物回收率和甲醇回收率详见下表7:
表7编号1至编号5的转氨酶的底物转化率、产物回收率和甲醇回收率
图7和图8分别示出了含有编号3的转氨酶的反应体系在反应0.5h和24h时的HPLC图谱,其中,在标号A位置处的波峰对应为产物峰,在标号B位置处的波峰对应为底物峰,反应0.5h时的底物转化率已达到较高的水平,约为91.7%。
由实施例5和实施例6可知,在酶催化反应体系中,相较于一次性添加混合液(包含底物和在一个标准大气压下沸点不高于110℃的有机溶剂),所述混合液以流加的方式添加入酶催化反应体系中更有利于提升底物转化率,底物转化率例如可以提升3%。
由实施例2至实施例6可知,在酶催化反应体系中添加特定比例的酮还原酶和葡萄糖脱氢酶,并且将甲醇与底物相混合制得的混合液以流加的方式添加入酶催化反应体系的制备方法最有利于提升底物转化率。
对比例1
本对比例提供了一种利用转氨酶催化西他列汀前体酮1-(3-氧吡咯烷-1-基)-4-(,4,5-三氟苯基)丁二酮生成西他列汀中间体(R)-1-[3氨基-4-(2,4,5-三氟苯基)丁酰基]吡咯-3-酮的制备方法,所述制备方法相较于实施例6中制备方法的区别之处仅在于:酶催化反应体系中未加入酮还原酶(具有如SEQ ID NO.15所示的氨基酸序列)。
在本对比例中,编号1至编号5的转氨酶的底物转化率详见下表8:
表8编号1至编号5的转氨酶的底物转化率
对比例2
本对比例提供了一种利用转氨酶催化西他列汀前体酮1-(3-氧吡咯烷-1-基)-4-(,4,5-三氟苯基)丁二酮生成西他列汀中间体(R)-1-[3氨基-4-(2,4,5-三氟苯基)丁酰基]吡咯-3-酮的制备方法,所述制备方法相较于实施例6中制备方法的区别之处仅在于:酶催化反应体系中未加入葡萄糖脱氢酶(具有如SEQ ID NO.14所示的氨基酸序列)。
在本对比例中,编号1至编号5的转氨酶的底物转化率详见下表9:
表9编号1至编号5的转氨酶的底物转化率
对比例3
本对比例提供了一种利用转氨酶催化西他列汀前体酮1-(3-氧吡咯烷-1-基)-4-(,4,5-三氟苯基)丁二酮生成西他列汀中间体(R)-1-[3氨基-4-(2,4,5-三氟苯基)丁酰基]吡咯-3-酮的制备方法,所述制备方法相较于实施例6中制备方法的区别之处仅在于:酮还原酶(具有如SEQ ID NO.15所示的氨基酸序列)和葡萄糖脱氢酶(具有如SEQ ID NO.16所示的氨基酸序列)的添加量不相同。
在本对比例中,酮还原酶和葡萄糖脱氢酶的添加量均为30g,以使酶催化反应体系中转氨酶、葡萄糖脱氢酶和酮还原酶的质量比为1:1:1。
在本对比例中,编号1至编号5的转氨酶的底物转化率详见下表10:
表10编号1至编号5的转氨酶的底物转化率
对比例4
本对比例提供了一种利用转氨酶催化西他列汀前体酮1-(3-氧吡咯烷-1-基)-4-(,4,5-三氟苯基)丁二酮生成西他列汀中间体(R)-1-[3氨基-4-(2,4,5-三氟苯基)丁酰基]吡咯-3-酮的制备方法,所述制备方法相较于实施例6中制备方法的区别之处仅在于:酮还原酶(具有如SEQ ID NO.15所示的氨基酸序列)和葡萄糖脱氢酶(具有如SEQ ID NO.16所示的氨基酸序列)的添加量不相同。
在本对比例中,酮还原酶和葡萄糖脱氢酶的添加量均为2.5g,以使酶催化反应体系中转氨酶、葡萄糖脱氢酶和酮还原酶的质量比为8:1:1。
在本对比例中,编号1至编号5的转氨酶的底物转化率详见下表11:
表11编号1至编号5的转氨酶的底物转化率
由实施例6和对比例1至对比例2的结果可知,在酶催化反应体系中单独添加葡萄糖脱氢酶或酮还原酶均不能有效提升底物转化率,在酶催化反应体系中同时添加葡萄糖脱氢酶和酮还原酶具有协同增效的作用。
由实施例6、对比例3至对比例4的结果可知,在酶催化反应体系中,转氨酶、葡萄糖脱氢酶和酮还原酶的质量比范围优选为6:(1~2):(1~2)。在对比例3中,葡萄糖脱氢酶和酮还原酶的添加量过多,对应的底物转化率与实施例6差异不明显,但会浪费葡萄糖脱氢酶和酮还原酶,从而提高生产成本。在对比例4中,葡萄糖脱氢酶和酮还原酶的添加量过少,对应的底物转化率低于实施例6差,并且会浪费底物。
对比例5
本对比例提供了一种利用转氨酶催化西他列汀前体酮1-(3-氧吡咯烷-1-基)-4-(,4,5-三氟苯基)丁二酮生成西他列汀中间体(R)-1-[3氨基-4-(2,4,5-三氟苯基)丁酰基]吡咯-3-酮的制备方法,所述制备方法相较于实施例6中制备方法的区别之处仅在于:底物-甲醇溶液的流加速度不相同。
在本对比例中,底物-甲醇溶液的流加速度为15g/h。
在本对比例中,编号1至编号5的转氨酶的底物转化率、产物回收率和甲醇回收率详见下表12:
表12编号1至编号5的转氨酶的底物转化率、产物回收率和甲醇回收率
对比例6
本对比例提供了一种利用转氨酶催化西他列汀前体酮1-(3-氧吡咯烷-1-基)-4-(,4,5-三氟苯基)丁二酮生成西他列汀中间体(R)-1-[3氨基-4-(2,4,5-三氟苯基)丁酰基]吡咯-3-酮的制备方法,所述制备方法相较于实施例6中制备方法的区别之处仅在于:底物-甲醇溶液的流加速度不相同。
在本对比例中,底物-甲醇溶液的流加速度为30g/h。
在本对比例中,编号1至编号5的转氨酶的底物转化率、产物回收率和甲醇回收率详见下表13:
表13编号1至编号5的转氨酶的底物转化率、产物回收率和甲醇回收率
由实施例6、对比例5和对比例6可知,底物-甲醇溶液以21.6g/h的速度添加至酶催化反应体系中的流加方法更有利于提升底物转化率。
以上对本申请所提供的一种西他列汀中间体的制备方法及西他列汀中间体,进行了详细介绍。本文中应用了具体个例对本申请的原理及实施方式进行了阐述,以上实施例的说明只是用于帮助理解本申请的技术方案及其核心思想;本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本申请实施例的技术方案的范围。
序列表
<110> 台州酶易生物技术有限公司
<120> 西他列汀中间体的制备方法
<141> 2021-06-24
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Ala Ala Arg Asn Val Leu Pro Gly Ile Thr Arg Arg Thr Ala Leu Glu
245 250 255
Ile Ala Arg Asp Phe Gly Leu Gln Thr Val Ile Gly Asp Val Thr Pro
260 265 270
Glu Met Leu Arg Gly Ala Asp Glu Ile Phe Ala Ala Ser Thr Gly Gly
275 280 285
Gly Ile Thr Pro Val Val Ala Leu Asp Gly Ala Pro Val Gly Ala Gly
290 295 300
Val Pro Gly Asp Trp Thr Arg Lys Ile Arg Thr Arg Tyr Trp Gln Met
305 310 315 320
Met Asp Glu Pro Ser Asp Leu Ile Glu Pro Val Arg Tyr Ile
325 330
<210> 4
<211> 1005
<212> DNA
<213> 人工序列
<400> 4
atgaaccaac tgactatcct ggaagctggt ctggatgaaa tcatctgtga aactgttcca 60
ggtgaagcta tccaatactc tcgttactct ctggatcgta ctaacccact ggctggtggt 120
tgtgcttgga tcgaaggtgc tttcgttcca gctgctgctg ctcgtatctc tatcttcgat 180
gctggtttct acacttctga tgctacttac actactgctc acgtttggca cggtaacttc 240
ttccgtctgg aagatcacgt tgaacgtttc ctggctggtg ctgaaaagat ccgtctgcca 300
atgccagcta ctaaggctga aatcatggat ctgatgcgtg gttgtgttgc tcgttctggt 360
ctgcgtgaag ctgttgttac tgttactgtt actcgtggtt acggtcgtaa gccattcgaa 420
aagactctgg aagctctgga atctcaactg tacgttactg ctatcccgta cctgtgggtt 480
ttcagcttta tccgtcaaat cgaaggtatc gatgctgtta tcgctcaatc tgttcgtcgt 540
actccagcta actctatgga cccatggatc aagaactacc aatggggtga tctggttcgt 600
gctatcttcg aagctcaaga acgtggtgct cgtactgctt tcctgctgga ttctgatggt 660
ttcgttactg aaggtccagg tttcaacgtt ctgatggtta aggatggtac tgttttcact 720
gctgctcgta acgttctgcc aggtatcact cgtcgtactg ctctggaaat cgctcgtgat 780
ttcggtctgc aaactgttat cggtgatgtt actccagaaa tgctgcgtgg tgctgatgaa 840
atcttcgctg cttctactgg tggtggtatc actccggttg ttgctctgga tggtgctcca 900
gttggtgctg gtgttccagg tgattggact cgtaagatcc gtactcgtta ctggcaaatg 960
atggatgaac catctgatct gatcgaacca gttcgttaca tctaa 1005
<210> 5
<211> 334
<212> PRT
<213> 人工序列
<400> 5
Met Asn Gln Leu Thr Ile Leu Glu Ala Gly Leu Asp Glu Ile Ile Cys
1 5 10 15
Glu Thr Val Pro Gly Glu Ala Ile Gln Tyr Ser Arg Tyr Ser Leu Asp
20 25 30
Arg Ala Asn Pro Leu Ala Gly Gly Cys Ala Trp Ile Glu Gly Ala Phe
35 40 45
Val Pro Ala Ala Ala Ala Arg Ile Ser Ile Phe Asp Ala Gly Phe Tyr
50 55 60
Thr Ser Asp Ala Thr Tyr Thr Thr Ala His Val Trp His Gly Asn Ala
65 70 75 80
Phe Arg Leu Glu Asp His Val Glu Arg Phe Leu Ala Gly Ala Glu Lys
85 90 95
Ile Arg Leu Pro Met Pro Ala Thr Lys Ala Glu Ile Met Asp Leu Met
100 105 110
Arg Gly Cys Val Ala Arg Ser Gly Leu Arg Glu Ala Val Val Thr Val
115 120 125
Thr Val Thr Arg Gly Tyr Gly Arg Lys Pro Phe Glu Lys Thr Leu Glu
130 135 140
Ala Leu Glu Ser Gln Leu Tyr Val Tyr Ala Ile Pro Tyr Gln Trp Val
145 150 155 160
Phe Ser Phe Ile Arg Gln Ile Glu Gly Ile Asp Ala Val Ile Ala Gln
165 170 175
Ser Val Arg Arg Thr Pro Arg Asn Ser Met Asp Pro Trp Ile Lys Asn
180 185 190
Tyr Gln Trp Gly Asp Leu Val Arg Ala Ile Phe Glu Ala Gln Glu Arg
195 200 205
Gly Ala Arg Thr Ala Phe Leu Leu Asp Ser Asp Gly Phe Val Thr Glu
210 215 220
Gly Pro Gly Phe Asn Val Leu Met Val Lys Asp Gly Thr Val Phe Thr
225 230 235 240
Ala Ala Arg Asn Val Leu Pro Gly Ile Thr Arg Arg Thr Ala Leu Glu
245 250 255
Ile Ala Arg Asp Phe Gly Leu Gln Thr Val Ile Gly Asp Val Thr Pro
260 265 270
Glu Met Leu Arg Gly Ala Asp Glu Ile Phe Ala Ala Ser Thr Gly Gly
275 280 285
Gly Ile Thr Pro Val Val Ala Leu Asp Gly Ala Pro Val Gly Ala Gly
290 295 300
Val Pro Gly Asp Trp Thr Arg Lys Ile Arg Thr Arg Tyr Trp Gln Met
305 310 315 320
Met Asp Glu Pro Ser Asp Leu Ile Glu Pro Val Arg Tyr Ile
325 330
<210> 6
<211> 1005
<212> DNA
<213> 人工序列
<400> 6
atgaaccaac tgactatcct ggaagctggt ctggatgaaa tcatctgtga aactgttcca 60
ggtgaagcta tccaatactc tcgttactct ctggatcgtg ctaacccact ggctggtggt 120
tgtgcttgga tcgaaggtgc tttcgttcca gctgctgctg ctcgtatctc tatcttcgat 180
gctggtttct acacttctga tgctacttac actactgctc acgtttggca cggtaacgct 240
ttccgtctgg aagatcacgt tgaacgtttc ctggctggtg ctgaaaagat ccgtctgcca 300
atgccagcta ctaaggctga aatcatggat ctgatgcgtg gttgtgttgc tcgttctggt 360
ctgcgtgaag ctgttgttac tgttactgtt actcgtggtt acggtcgtaa gccattcgaa 420
aagactctgg aagctctgga atctcaactg tacgtttacg ctatcccgta ccaatgggtt 480
ttcagcttta tccgtcaaat cgaaggtatc gatgctgtta tcgctcaatc tgttcgtcgt 540
actccaagaa actctatgga cccatggatc aagaactacc aatggggtga tctggttcgt 600
gctatcttcg aagctcaaga acgtggtgct cgtactgctt tcctgctgga ttctgatggt 660
ttcgttactg aaggtccagg tttcaacgtt ctgatggtta aggatggtac tgttttcact 720
gctgctcgta acgttctgcc aggtatcact cgtcgtactg ctctggaaat cgctcgtgat 780
ttcggtctgc aaactgttat cggtgatgtt actccagaaa tgctgcgtgg tgctgatgaa 840
atcttcgctg cttctactgg tggtggtatc actccggttg ttgctctgga tggtgctcca 900
gttggtgctg gtgttccagg tgattggact cgtaagatcc gtactcgtta ctggcaaatg 960
atggatgaac catctgatct gatcgaacca gttcgttaca tctaa 1005
<210> 7
<211> 334
<212> PRT
<213> 人工序列
<400> 7
Met Asn Gln Leu Thr Ile Leu Glu Ala Gly Leu Asp Glu Ile Ile Cys
1 5 10 15
Glu Thr Val Pro Gly Glu Ala Ile Gln Tyr Ser Arg Tyr Ser Leu Asp
20 25 30
Arg Thr Asn Pro Leu Ala Gly Gly Cys Ala Trp Ile Glu Gly Ala Phe
35 40 45
Val Pro Ala Ala Ala Ala Arg Ile Ser Ile Phe Asp Ala Gly Phe Tyr
50 55 60
Thr Ser Asp Ala Thr Tyr Thr Thr Ala His Val Trp His Gly Asn Ala
65 70 75 80
Phe Arg Leu Glu Asp His Val Glu Arg Phe Leu Ala Gly Ala Glu Lys
85 90 95
Ile Arg Leu Pro Met Pro Ala Thr Lys Ala Glu Ile Met Asp Leu Met
100 105 110
Arg Gly Cys Val Ala Arg Ser Gly Leu Arg Glu Ala Val Val Thr Val
115 120 125
Thr Val Thr Arg Gly Tyr Gly Arg Lys Pro Phe Glu Lys Thr Leu Glu
130 135 140
Ala Leu Glu Ser Gln Leu Tyr Val Tyr Ala Ile Pro Tyr Gln Trp Val
145 150 155 160
Phe Ser Phe Ile Arg Gln Ile Glu Gly Ile Asp Ala Val Ile Ala Gln
165 170 175
Ser Val Arg Arg Thr Pro Ala Asn Val Met Asp Pro Trp Ile Lys Asn
180 185 190
Tyr Gln Trp Gly Asp Leu Val Arg Ala Ile Phe Glu Ala Gln Glu Arg
195 200 205
Gly Ala Arg Thr Ala Phe Leu Leu Asp Ser Asp Gly Phe Val Thr Glu
210 215 220
Gly Pro Gly Phe Asn Val Leu Met Val Lys Asp Gly Thr Val Phe Thr
225 230 235 240
Ala Ala Arg Asn Val Leu Pro Gly Ile Thr Arg Arg Thr Ala Leu Glu
245 250 255
Ile Ala Arg Asp Phe Gly Leu Gln Thr Val Ile Gly Asp Val Thr Pro
260 265 270
Glu Met Leu Tyr Asp Ala Asp Glu Ile Phe Ala Ala Ser Thr Gly Gly
275 280 285
Gly Ile Thr Pro Val Val Ala Leu Asp Gly Ala Pro Val Gly Ala Gly
290 295 300
Val Pro Gly Asp Trp Thr Arg Lys Ile Arg Thr Arg Tyr Trp Gln Met
305 310 315 320
Met Asp Glu Pro Ser Asp Leu Ile Glu Pro Val Arg Tyr Ile
325 330
<210> 8
<211> 1005
<212> DNA
<213> 人工序列
<400> 8
atgaaccaac tgactatcct ggaagctggt ctggatgaaa tcatctgtga aactgttcca 60
ggtgaagcta tccaatactc tcgttactct ctggatcgta ctaacccact ggctggtggt 120
tgtgcttgga tcgaaggtgc tttcgttcca gctgctgctg ctcgtatctc tatcttcgat 180
gctggtttct acacttctga tgctacttac actactgctc acgtttggca cggtaacgct 240
ttccgtctgg aagatcacgt tgaacgtttc ctggctggtg ctgaaaagat ccgtctgcca 300
atgccagcta ctaaggctga aatcatggat ctgatgcgtg gttgtgttgc tcgttctggt 360
ctgcgtgaag ctgttgttac tgttactgtt actcgtggtt acggtcgtaa gccattcgaa 420
aagactctgg aagctctgga atctcaactg tacgtttacg ctatcccgta ccaatgggtt 480
ttcagcttta tccgtcaaat cgaaggtatc gatgctgtta tcgctcaatc tgttcgtcgt 540
actccagcta acgttatgga cccatggatc aagaactacc aatggggtga tctggttcgt 600
gctatcttcg aagctcaaga acgtggtgct cgtactgctt tcctgctgga ttctgatggt 660
ttcgttactg aaggtccagg tttcaacgtt ctgatggtta aggatggtac tgttttcact 720
gctgctcgta acgttctgcc aggtatcact cgtcgtactg ctctggaaat cgctcgtgat 780
ttcggtctgc aaactgttat cggtgatgtt actccagaaa tgctgtatga tgctgatgaa 840
atcttcgctg cttctactgg tggtggtatc actccggttg ttgctctgga tggtgctcca 900
gttggtgctg gtgttccagg tgattggact cgtaagatcc gtactcgtta ctggcaaatg 960
atggatgaac catctgatct gatcgaacca gttcgttaca tctaa 1005
<210> 9
<211> 334
<212> PRT
<213> 人工序列
<400> 9
Met Asn Gln Leu Thr Ile Leu Glu Ala Gly Leu Asp Glu Ile Ile Cys
1 5 10 15
Glu Thr Val Pro Gly Glu Ala Ile Gln Tyr Ser Arg Tyr Ser Leu Asp
20 25 30
Arg Ala Asn Pro Leu Ala Gly Gly Cys Ala Trp Ile Glu Gly Ala Phe
35 40 45
Val Pro Ala Ala Ala Ala Arg Ile Ser Ile Phe Asp Ala Gly Phe Tyr
50 55 60
Thr Ser Asp Ala Thr Tyr Thr Thr Ala His Val Trp Asn Gly Asn Ala
65 70 75 80
Phe Arg Leu Glu Asp His Val Glu Arg Phe Leu Ala Gly Ala Glu Lys
85 90 95
Ile Arg Leu Pro Met Pro Ala Thr Lys Ala Glu Ile Met Asp Leu Met
100 105 110
Arg Gly Cys Val Ala Arg Ser Gly Leu Arg Glu Ala Val Val Thr Val
115 120 125
Thr Val Thr Arg Gly Tyr Gly Arg Lys Pro Phe Glu Lys Thr Leu Glu
130 135 140
Ala Leu Glu Ser Gln Leu Tyr Val Tyr Ala Ile Pro Tyr Leu Trp Val
145 150 155 160
Phe Ser Pro Ile Arg Gln Ile Glu Gly Ile Asp Ala Val Ile Ala Gln
165 170 175
Ser Val Arg Arg Ser Pro Ala Asn Val Met Asp Pro Trp Ile Lys Asn
180 185 190
Tyr Gln Trp Gly Asp Leu Val Arg Ala Ile Phe Glu Ala Gln Glu Arg
195 200 205
Gly Ala Arg Thr Ala Phe Leu Leu Asp Ser Asp Gly Phe Val Thr Glu
210 215 220
Gly Pro Gly Phe Asn Val Leu Met Val Lys Asp Gly Thr Val Phe Thr
225 230 235 240
Ala Ala Arg Asn Val Leu Pro Gly Ile Thr Arg Arg Thr Ala Leu Glu
245 250 255
Ile Ala Arg Ser Phe Gly Leu Gln Thr Val Ile Gly Asp Val Thr Pro
260 265 270
Glu Met Leu Arg Gly Ala Asp Glu Ile Phe Ala Ala Ser Thr Gly Gly
275 280 285
Gly Ile Thr Pro Phe Val Ala Leu Asp Gly Ala Pro Val Gly Ala Gly
290 295 300
Val Pro Gly Asp Val Thr Arg Lys Ile Arg Thr Arg Tyr Trp Gln Met
305 310 315 320
Asn Asp Glu Pro Ser Asp Leu Ile Glu Pro Val Arg Tyr Ile
325 330
<210> 10
<211> 1005
<212> DNA
<213> 人工序列
<400> 10
atgaaccaac tgactatcct ggaagctggt ctggatgaaa tcatctgtga aactgttcca 60
ggtgaagcta tccaatactc tcgttactct ctggatcgtg ctaacccact ggctggtggt 120
tgtgcttgga tcgaaggtgc tttcgttcca gctgctgctg ctcgtatctc tatcttcgat 180
gctggtttct acacttctga tgctacttac actactgctc acgtttggaa tggtaacgct 240
ttccgtctgg aagatcacgt tgaacgtttc ctggctggtg ctgaaaagat ccgtctgcca 300
atgccagcta ctaaggctga aatcatggat ctgatgcgtg gttgtgttgc tcgttctggt 360
ctgcgtgaag ctgttgttac tgttactgtt actcgtggtt acggtcgtaa gccattcgaa 420
aagactctgg aagctctgga atctcaactg tacgtttacg ctatcccgta cctgtgggtt 480
ttcagcccaa tccgtcaaat cgaaggtatc gatgctgtta tcgctcaatc tgttcgtcgt 540
tctccagcta acgttatgga cccatggatc aagaactacc aatggggtga tctggttcgt 600
gctatcttcg aagctcaaga acgtggtgct cgtactgctt tcctgctgga ttctgatggt 660
ttcgttactg aaggtccagg tttcaacgtt ctgatggtta aggatggtac tgttttcact 720
gctgctcgta acgttctgcc aggtatcact cgtcgtactg ctctggaaat cgctcgttct 780
ttcggtctgc aaactgttat cggtgatgtt actccagaaa tgctgcgtgg tgctgatgaa 840
atcttcgctg cttctactgg tggtggtatc actccgtttg ttgctctgga tggtgctcca 900
gttggtgctg gtgttgttgg tgattggact cgtaagatcc gtactcgtta ctggcaaatg 960
aatgatgaac catctgatct gatcgaacca gttcgttaca tctaa 1005
<210> 11
<211> 323
<212> PRT
<213> 人工序列
<400> 11
Met Ala Ser Met Asp Lys Val Phe Ser Gly Tyr Tyr Ala Arg Gln Lys
1 5 10 15
Leu Leu Glu Arg Ser Asp Asn Pro Phe Ser Lys Gly Ile Ala Tyr Val
20 25 30
Glu Gly Lys Leu Val Leu Pro Ser Asp Ala Arg Ile Pro Leu Leu Asp
35 40 45
Glu Gly Phe Met His Ser Asp Leu Thr Tyr Asp Val Ile Ser Val Trp
50 55 60
Asp Gly Arg Phe Phe Arg Leu Asp Asp His Leu Gln Arg Ile Leu Glu
65 70 75 80
Ser Cys Asp Lys Met Arg Leu Lys Phe Pro Leu Ala Leu Ser Ser Val
85 90 95
Lys Asn Ile Leu Ala Glu Met Val Ala Lys Ser Gly Ile Arg Asp Ala
100 105 110
Phe Val Glu Val Ile Val Thr Arg Gly Leu Thr Gly Val Arg Gly Ser
115 120 125
Lys Pro Glu Asp Leu Tyr Asn Asn Asn Ile Tyr Leu Leu Val Leu Pro
130 135 140
Tyr Ile Trp Val Met Ala Pro Glu Asn Gln Leu His Gly Gly Glu Ala
145 150 155 160
Ile Ile Thr Arg Thr Val Arg Arg Thr Pro Pro Gly Ala Phe Asp Pro
165 170 175
Thr Ile Lys Asn Leu Gln Trp Gly Asp Leu Thr Lys Gly Leu Phe Glu
180 185 190
Ala Met Asp Arg Gly Ala Thr Tyr Pro Phe Leu Thr Asp Gly Asp Thr
195 200 205
Asn Leu Thr Glu Gly Ser Gly Phe Asn Ile Val Leu Val Lys Asn Gly
210 215 220
Ile Ile Tyr Thr Pro Asp Arg Gly Val Leu Arg Gly Ile Thr Arg Lys
225 230 235 240
Ser Val Ile Asp Val Ala Arg Ala Asn Ser Ile Asp Ile Arg Leu Glu
245 250 255
Val Val Pro Val Glu Gln Ala Tyr His Ser Asp Glu Ile Phe Met Cys
260 265 270
Thr Thr Ala Gly Gly Ile Met Pro Ile Thr Leu Leu Asp Gly Gln Pro
275 280 285
Val Asn Asp Gly Gln Val Gly Pro Ile Thr Lys Lys Ile Trp Asp Gly
290 295 300
Tyr Trp Glu Met His Tyr Asn Pro Ala Tyr Ser Phe Pro Val Asp Tyr
305 310 315 320
Gly Ser Gly
<210> 12
<211> 969
<212> DNA
<213> 人工序列
<400> 12
atggcttcga tggacaaagt cttctcaggt tactacgccc gtcaaaaact gctggaacgc 60
tcagataatc cgttctcaaa aggtattgcc tatgtcgaag gtaaactggt gctgccgagt 120
gatgcgcgca ttccgctgct ggacgaaggc tttatgcata gtgatctgac ctacgacgtt 180
atctccgtct gggacggccg tttctttcgc ctggatgacc acctgcagcg cattctggaa 240
tcatgcgata aaatgcgtct gaaatttccg ctggcactga gctctgtcaa aaatatcctg 300
gcagaaatgg tggctaaaag cggcattcgt gacgctttcg tcgaagtgat cgttacccgc 360
ggcctgacgg gtgttcgtgg ctctaaaccg gaagatctgt ataacaataa catttacctg 420
ctggtgctgc cgtatatctg ggttatggca ccggaaaatc agctgcatgg cggtgaagct 480
attatcaccc gtacggtgcg tcgcaccccg ccgggtgcct ttgatccgac gatcaaaaac 540
ctgcaatggg gtgacctgac caaaggcctg tttgaagcga tggatcgtgg tgccacctat 600
ccgttcctga cggatggcga caccaatctg acggaaggca gcggtttcaa tattgtcctg 660
gtgaaaaacg gtattatcta caccccggat cgtggtgttc tgcgcggcat tacgcgtaaa 720
tcagtgatcg atgttgcgcg cgccaactcg attgacatcc gtctggaagt ggttccggtg 780
gaacaagcgt accactccga tgaaattttc atgtgtacca cggccggcgg tattatgccg 840
atcaccctgc tggatggtca gccggttaac gacggtcaag tcggcccgat taccaagaaa 900
atttgggatg gctattggga aatgcactac aacccggctt attcgtttcc ggtggattat 960
ggcagcggt 969
<210> 13
<211> 31
<212> DNA
<213> 人工序列
<400> 13
gctaacccac tggctggtgg ttgtgcttgg a 31
<210> 14
<211> 31
<212> DNA
<213> 人工序列
<400> 14
acgatccaga gagtaacgag agtattggat a 31
<210> 15
<211> 283
<212> PRT
<213> 人工序列
<400> 15
Met Ala Lys Asn Phe Ser Asn Val Glu Tyr Pro Ala Pro Pro Pro Ala
1 5 10 15
His Thr Lys Asn Glu Ser Leu Gln Val Leu Asp Leu Phe Lys Leu Asn
20 25 30
Gly Lys Val Ala Ser Ile Thr Gly Ser Ser Ser Gly Ile Gly Tyr Ala
35 40 45
Leu Ala Glu Ala Phe Ala Gln Val Gly Ala Asp Val Ala Ile Trp Tyr
50 55 60
Asn Ser His Asp Ala Thr Gly Lys Ala Glu Ala Leu Ala Lys Lys Tyr
65 70 75 80
Gly Val Lys Val Lys Ala Tyr Lys Ala Asn Val Ser Ser Ser Asp Ala
85 90 95
Val Lys Gln Thr Ile Glu Gln Gln Ile Lys Asp Phe Gly His Leu Asp
100 105 110
Ile Val Val Ala Asn Ala Gly Ile Pro Trp Thr Lys Gly Ala Tyr Ile
115 120 125
Asp Gln Asp Asp Asp Lys His Phe Asp Gln Val Val Asp Val Asp Leu
130 135 140
Lys Gly Val Gly Tyr Val Ala Lys His Ala Gly Arg His Phe Arg Glu
145 150 155 160
Arg Phe Glu Lys Glu Gly Lys Lys Gly Ala Leu Val Phe Thr Ala Ser
165 170 175
Met Ser Gly His Ile Val Asn Val Pro Gln Phe Gln Ala Thr Tyr Asn
180 185 190
Ala Ala Lys Ala Gly Val Arg His Phe Ala Lys Ser Leu Ala Val Glu
195 200 205
Phe Ala Pro Phe Ala Arg Val Asn Ser Val Ser Pro Gly Tyr Ile Asn
210 215 220
Thr Glu Ile Ser Asp Phe Val Pro Gln Glu Thr Gln Asn Lys Trp Trp
225 230 235 240
Ser Leu Val Pro Leu Gly Arg Gly Gly Glu Thr Ala Glu Leu Val Gly
245 250 255
Ala Tyr Leu Phe Leu Ala Ser Asp Ala Gly Ser Tyr Ala Thr Gly Thr
260 265 270
Asp Ile Ile Val Asp Gly Gly Tyr Thr Leu Pro
275 280
<210> 16
<211> 261
<212> PRT
<213> 人工序列
<400> 16
Met Tyr Pro Asp Leu Lys Gly Lys Val Val Val Ile Thr Gly Ser Ser
1 5 10 15
Thr Gly Leu Gly Lys Ala Met Ala Ile Arg Phe Ala Thr Ala Lys Ala
20 25 30
Lys Val Val Val Asn Tyr Arg Ser Lys Glu Asp Glu Ala Asn Ser Val
35 40 45
Leu Glu Glu Ile Lys Lys Val Gly Gly Glu Ala Ile Ala Val Lys Gly
50 55 60
Asp Val Thr Val Glu Ser Asp Val Ile Asn Leu Val Gln Ser Ala Ile
65 70 75 80
Lys Glu Phe Gly Lys Leu Asp Val Met Ile Asn Asn Ala Gly Leu Glu
85 90 95
Asn Pro Val Ser Ser His Glu Met Ser Leu Ser Asp Trp Ile Lys Val
100 105 110
Ile Asp Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile
115 120 125
Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Thr Val Ile Asn Met Ser
130 135 140
Ser Val His Glu Lys Ile Pro Trp Pro Leu Phe Val His Tyr Ala Ala
145 150 155 160
Ser Lys Gly Gly Met Lys Leu Met Thr Glu Thr Leu Ala Leu Glu Tyr
165 170 175
Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn
180 185 190
Thr Pro Ile Asn Ala Glu Lys Phe Ala Asp Pro Glu Gln Ser Ala Asp
195 200 205
Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Thr
210 215 220
Ala Ala Val Ala Ala Trp Leu Ala Ser Ser Glu Ala Ser Tyr Val Thr
225 230 235 240
Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Gln Tyr Pro Ser Phe
245 250 255
Gln Ala Gly Arg Gly
260
Claims (10)
2.根据权利要求1所述的制备方法,其特征在于,所述将所述混合液与转氨酶进行转氨接触的方式为:将所述混合液以流加补料的方式加入至包含所述转氨酶的预混体系中。
3.根据权利要求2所述的制备方法,其特征在于,所述混合液中所述底物的浓度为10克每升至500克每升,流加速度为18毫升每小时至23毫升每小时,流加时间为14小时至17小时。
4.根据权利要求1所述的制备方法,其特征在于,在所述酶催化反应的反应体系中除了包括所述混合液和所述转氨酶,还包括异丙胺和磷酸吡哆醛。
5.根据权利要求1所述的制备方法,其特征在于,所述有机溶剂包括甲醇、乙醇、异丙醇、四氢呋喃、乙酸乙酯、乙酸丙酯、乙酸丁酯、二甲基亚砜、二氯甲烷以及甲苯中的至少一种。
6.根据权利要求5所述的制备方法,其特征在于,所述有机溶剂为甲醇。
7.根据权利要求1至6任一项中所述的制备方法,其特征在于,在所述酶催化反应的反应体系中除了包括所述混合液和所述转氨酶,还包括酮还原酶和辅酶再生系统,所述辅酶再生系统用于提供循环再生的烟酰胺腺嘌呤二核苷酸或烟酰胺腺嘌呤二核苷酸磷酸。
8.根据权利要求7所述的制备方法,其特征在于,所述辅酶再生系统包括葡萄糖和葡萄糖脱氢酶,且所述转氨酶、所述葡萄糖脱氢酶和所述酮还原酶的质量比为6:(1~2):(1~2)。
9.根据权利要求1至6和8任一项中所述的制备方法,其特征在于,所述酶催化反应的反应条件为:pH为7.5至11.0,温度为15℃至65℃,转速为250转每分钟至500转每分钟。
10.根据权利要求1至6和8任一项中所述的制备方法,其特征在于,所述转氨酶的氨基酸序列如SEQ ID NO:1所示,或者为与所述SEQ ID NO:1至少具有80%、至少具有85%、至少具有90%、至少具有95%、至少具有96%、至少具有97%、至少具有98%或至少具有99%相似性的氨基酸序列。
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