CN117126823B - 一种酮还原酶突变体及其应用 - Google Patents
一种酮还原酶突变体及其应用 Download PDFInfo
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- CN117126823B CN117126823B CN202311123361.0A CN202311123361A CN117126823B CN 117126823 B CN117126823 B CN 117126823B CN 202311123361 A CN202311123361 A CN 202311123361A CN 117126823 B CN117126823 B CN 117126823B
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Classifications
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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Abstract
本发明提供了一种酮还原酶突变体及其应用，所述的突变体在如SEQ ID NO.2所示氨基酸序列基础上经过氨基酸突变得到，所述突变为(1)或(2)：(1)Y199A、Y199C、M196W、A146F、A146L中任意一种；(2)Y199A、Y199C、M196W A146F、A146L中任意一种与N149L、N149I、I195L和I195V中任意一种的组合。本发明得到催化活性更高的突变体，转化率提高可达2.4倍，de值超过99.8％。本发明突变体在生物转化甾体类化合物制备24R‑羟基构型的胆甾醇中具有巨大的潜力。
Description
技术领域
本发明涉及基因工程和生物酶催化技术领域，具体涉及一种酮还原酶及其应用。
背景技术
酮还原酶普遍存在于自然界动、植物和微生物体内。其中微生物来源的酮还原酶最为广泛，目前已有至少200种来自微生物的酮还原酶被报道参与多种物质的还原反应。酮还原酶在手性醇的合成中具有重要地位，催化活性和立体选择性良好的酮还原酶能够有效地催化手性醇的不对称合成，极大地提高手性醇的合成效率，简化产物制备和分离流程，如阿托伐他汀、杜洛西汀、替格瑞洛等原料药或其中间体都已采用酮还原酶法进行制备，减少了很多金属手性催化剂或拆分剂的使用，酮还原酶已经成为医药化工领域应用最广泛的酶。酮还原酶是依赖NAD(H)或NADP(H)为辅酶，能够催化醛或酮可逆还原生成相应醇的一类氧化还原酶。酮还原酶共同的分子特征是利用NAD(H)或NADP(H)为辅酶，辅酶结合区域位于N-端Rossmann折叠中，保守位点为Gly富集序列G-x-x-x-G-x-G。催化结构域位于N-端中部，保守的催化三联体为Ser-Tyr-Lys。该家族的酶一般由单体、二聚体或四聚体组成，单亚基含有250个左右的氨基酸，在手性醇合成过程中起到了重要的作用。
角鲨胺(squalamine)，又名(3β,5α,7α)-3-[[3-((4-氨基丁基)氨基)丙基]氨基]胆甾烷-7,24-二醇-24-磺酸酯，最初是Zasloff及其合作者从白斑角鲨肝脏中提取获得了一种海洋氨基甾醇类化合物，角鲨胺具有很强的广谱抗菌作用和抗血管生成效应。
传统的角鲨胺合成工艺主要以3β-羟基-5-胆烯酸、鹅去氧胆酸、岩藻甾醇、脱氢表雄酮或醋酸娠烯醇酮等为原料。如以鹅去氧胆酸甲酯为原料通过化学合成制备角鲨胺需要经过九步反应(Dong-Hui Zhang,Feng Cai,Xiang-Dong Zhou,et al.Aconcise andstereoselective synthesis of squalamine.Organic Letters,2003,5(18):3257-3259.)，在这一合成过程中，其关键就是构建24位手性羟基中间体。这一反应路线需要将C-7位羟基用MOMO保护，这是一种具有高毒性和致癌性的试剂，C-24位经手性试剂化合物的控制对化合物进行亲核加成得到R构型的羟基。手性试剂和二异丙基锌均为价格昂贵的试剂，合成成本极其高，需要研究人员寻找更好的替代制备方法。
专利202310759919.8公开了采用化学-酶方法制备3α,7α,24R-三羟基-5α-胆甾醇的方法。该方法具有很强的先进性，但是该方法使用的酮还原酶KRED-101，KRED-171及其突变体中的一种或多种，转化效率仅可以达到64％。
发明内容
本发明针对上述技术难题，提供了一种酮还原酶突变体，利用酮还原酶(NaKRED)为模板进行生物酶催化剂的改进。该突变体具有更高的活性和选择性，能够结合NADH再生还原系统将甾体化合物(1a)选择性还原，得到24R-羟基构型的胆甾醇(1b)，并能够提高转化效率。
本发明的技术方案如下：
本发明所提供的酮还原酶突变体可以在NADH再生系统下能够催化1a一步生成1b，酶促转化反应式如图1。
为达到发明的目的，本发明以已知的酮还原酶经过筛选，获得来自Novosphingobium aromaticivorans的具有立体选择的酮还原酶(NaKRED)为模板，通过研究酮还原酶的结合口袋，构建了突变体，能够实现1a生物酶催化转化生成1b，如SEQ IDNO.2所示的氨基酸序列经过一个或多个氨基酸位点突变且具有选择性还原24位羰基的活性。
一种酮还原酶突变体，所述的突变体在如SEQ ID NO.2所示氨基酸序列基础上经过氨基酸突变得到，所述突变为(1)或(2)：
(1)Y199A、Y199C、M196W、A146F、A146L中任意一种；
(2)Y199A、Y199C、M196W A146F、A146L中任意一种与N149L、N149I、I195L和I195V中任意一种的组合。
优选地，所述突变为如下任意一种组合：Y199A/N149L、Y199A/N149I、Y199A/I195L、Y199A/I195V。
一种DNA分子，编码上述酮还原酶突变体。
一种重组质粒，含有所述的DNA分子。
根据上述的重组质粒，其载体选自如下任意一种：
pET-21b(+)、pET-22b(+)、pET-3a(+)、pET-3d(+)、pET-11a(+)、pET-12a(+)、pET-14b、pET-15b(+)、pET-16b(+)、pET-17b(+)、pET-19b(+)、pET-20b(+)、pET-21a(+)、pET-23a(+)、pET-23b(+)、pET-24a(+)、pET-25b(+)、pET-26b(+)、pET-27b(+)、pET-28a(+)、pET-29a(+)、pET-30a(+)、pET-31b(+)、pET-32a(+)、pET-35b(+)、pET-38b(+)、pET-39b(+)、pET-40b(+)、pET-41a(+)、pET-41b(+)、pET-42a(+)、pET-43a(+)、pET-43b(+)、pET-44a(+)、pET-49b(+)、pQE2、pQE9、pQE30、pQE31、pQE32、pQE40、pQE70、pQE80、pRSET-A、pRSET-B、pRSET-C、pGEX-5X-1、pGEX-6p-1、pGEX-6p-2、pBV220、pBV221、pBV222、pTrc99A、pTwin1、pEZZ18、pKK232-8、p UC-18以及pUC-19。
一种重组菌，含有所述的重组质粒。
优选地，重组菌的宿主细胞为酵母细胞或大肠杆菌BL21(DE3)。
所述的酮还原酶突变体的应用，将酮还原酶突变体结合NADH再生还原系统用于催化还原反应制备手性甾体类化合物。
优选地，结合NADH再生系统，利用所述的酮还原酶突变体催化所述底物发生羰基不对称还原反应，得到24R-羟基构型的胆甾醇。
优选地，所述底物为具有如下结构式的任意一种：
优选地，所述NADH再生系统为：葡萄糖和葡萄糖脱氢酶；反应条件为：温度25±15℃，转速200±100rpm，时间12-24h。
与现有技术相比，本发明具有以下优点：
(1)本发明经过定点突变，改变酶与底物结合的活性口袋，得到催化活性更高的突变体。
(2)本发明提供的酮还原酶突变体以廉价易得的别鹅去氧胆酸(Allochenodeoxycholic Acid)为原料，通过两步反应合成1a底物，对24位羰基的选择性还原转化率相比于模板提高了2.4倍，可得到de超过99.8％的1b，该方法不仅反应条件温和，步骤少，而且可以充分减少副产物，绿色环保。因此，本发明提供的酮还原酶突变体在生物转化甾体类化合物制备24R-羟基构型的胆甾醇中具有巨大的潜力。
附图说明
图1为酮还原酶NaKRED突变体催化1a得到1b的反应式。
图2为模板NaKRED催化1a的HPLC图。
图3为模板NaKRED催化1a得到的产物R构型的1b的晶体结构。
图4为Y199A/N149L突变体催化1a的HPLC图。
具体实施方式
下面结合实施例进一步说明本发明，但不局限于本发明中的实施例。下述实验如无特别说明，均为常规方法，所用的实验材料若无特别说明，均可从商业公司购买得到。
实施例1模板pET28a-NaKRED的合成和重组表达
根据文献报道的能够遵循anti-prelog规则还原潜手性酮的酮还原酶NaKRED为模版(Wu K,Zheng K,Xiong L,et al.Efficient synthesis of an antiviral drugintermediate using an enhanced short-chain dehydrogenase in an aqueous-organic solvent system[J].Applied microbiology and biotechnology,2019,103(11):4417-4427.)，优化后的核苷酸序列如SEQ ID NO.1所示，氨基酸序列如SEQ ID NO.2所示。将优化后的核苷酸序列合成到pET28a载体上，然后转入大肠杆菌BL21(DE3)中。根据常规慢转方法恢复培养后取适量菌液涂布于含有50μg/mL卡那抗生素的LB平板，37℃倒置过夜培养。挑取平板上的单菌落进行活化，接种于25mL含有50μg/mL卡那抗生素的LB培养基中，37℃，220rpm至OD 0.6-0.8，加入诱导剂IPTG 0.2mM，在25℃，180rpm震荡培养20h。菌体在6500rpm，4℃，10min下收集，收集的菌体用反应缓冲液洗涤两次，菌体用超声破碎仪破碎，12000rpm离心15min得到上清进行活性检测并作为突变体活性筛选的对照。对于1mM的催化底物，酶的用量以湿菌体的重量计为50-60g/L缓冲液。
实施例2酮还原酶NaKRED突变体的构建
以pET28a-KRED整个质粒为模板，设计突变引物，需要突变的碱基放在引物中间。PCR体系为：模板1ng，上下游引物各1.5μL，2×PCR Buffer for KOD FX Neo 25μL，KOD FXNeo酶1μL，最后加灭菌蒸馏水将体积补至50μL。
PCR反应程序：(1)94℃预变性2min；(2)98℃变性10s；(3)根据突变引物设置退火温度，退火时间15s；(4)68℃延伸4min 20s，步骤(2)～(4)进行30个循环，最后68℃延伸10min，12℃保存PCR产物。
将上述PCR产物进行Dpn I酶消化，去除模板后转入大肠杆菌DH5α感受态细胞，恢复培养后的菌液涂布于含有50μg/mL卡那抗生素的LB培养基平板，37℃倒置培养过夜。挑取单克隆进行单菌落验证，选1-3个单菌落进行基因测序。采用上述同样的方法在单一突变体的基础上进行叠加突变，从而获得具有多突变点的突变体。
突变体的诱导表达如上述模板的诱导表达一致。
实施例3突变体的活性筛选
将得到的湿菌体按照1g(湿菌体):15mL(反应缓冲液)重悬，取900μL用于超声破碎，破1s，停2s，总时长10min，破碎液于12000rpm离心15min。收集上清加入1mM底物1a(用甲醇配制成100mM母液)，1mg/mL葡萄糖脱氢酶，100mM葡萄糖，1mM NAD+，终体积1mL。整个反应体系于25℃，220rpm反应12-24h。待反应结束，取等体积的乙酸乙酯萃取，萃取重复三次，最后合并有机相，用旋转蒸发旋干溶剂后用1mL甲醇重新溶解，用0.22μm的有机滤膜过滤制样，采用HPLC检测含量。
表1突变体的活性检测结果
以上结果是在同样反应条件下进行三次平行反应得到。转化率定义为:初始底物浓度-反应结束底物浓度/初始底物浓度％；选择性定义为：非对映选择性(de)定义为：[R-S]/[R+S]％。
针对以上活性检测结果，对Y199A/N149L突变体进行纯化。
实施例4Y199A/N149L突变体的菌种活化以及大量诱导表达
将保存在-80℃的Y199A/N149L甘油菌取10μL加入4mL含有50μg/mL的LB培养基中，37℃，220rpm，培养过夜。取2.5mL菌液加入250mL含有50μg/mL卡那抗生素的LB培养基中,37℃，220rpm，培养至OD 0.6-0.8，加入0.2mM IPTG，25℃，180rpm，诱导20h。培养液在6500rpm，4℃，10min进行收集，用纯化平衡缓冲液洗涤两次，将得到的湿菌体保存于-80℃备用。
将上述收集到的湿菌体按照1g(湿菌体):15mL(反应缓冲液)重悬，在冰浴的条件下超声破碎，开1s，关2s，总时长45min。细胞破碎液在12000rpm，4℃离心15min，上清再用0.22μm的滤膜过滤用于后续纯化。纯化方法为镍柱亲和层析方法：纯化柱为5mL预装柱，先用平衡液平衡整个纯化系统，再以1mL/min的流速上样，进一步用平衡液将未挂柱的杂蛋白去除，之后用洗脱缓冲液(500mM NaCl，500mM咪唑)进行梯度洗脱。目的蛋白大概是在150mM的咪唑浓度被洗脱下来，收集目的蛋白，用超滤管(截留50KDa)进行浓缩和脱盐至最后体积为1mL，最后将蛋白液分装保存于-80℃备用。
实施例5Y199A/N149L突变体的酶活检测
因为酮还原酶是NADH依赖性氧化还原酶，NADH在340nm处具有最大紫外吸收，而NAD+在此处没有最大吸收，所以通过检测反应在340nm的吸收值变化来评价突变体的活性。具体方法为：于250μL反应体系中(100mM PBS Buffer,pH 7.2)中加入0.5μM的纯酶，0.5mM的1a，1mM的NADH，监测反应5min内的吸光值变化。反应结束后立即用等体积的乙酸乙酯萃取，萃取重复三次，合并有机相后用氮吹仪将溶剂挥发，最后用等体积的甲醇进行溶解，用0.22μm的滤膜过滤制样，采用HPLC检测产物的形成。
表2 Y199A/N149L突变体的酶活结果
以上所述，仅为本发明相对较好的具体实施方式，但不仅仅限于此方式，凡在本发明申请专利范围之内的轻易能想到的改进都在本发明的保护范围之内。
Claims (7)
1.一种酮还原酶突变体，其特征在于，所述的突变体在如SEQ ID NO. 2所示氨基酸序列基础上经过氨基酸突变得到，所述突变为（1）或（2）：
（1）Y199A、Y199C、M196W、A146F中任意一种；
（2）Y199A/N149L、Y199A/N149I、Y199A/I195L、Y199A/I195V中任意一种。
2.一种DNA分子，其特征在于，编码权利要求1所述的酮还原酶突变体。
3.一种重组质粒，其特征在于，含有权利要求2所述的DNA分子。
4.一种重组菌，其特征在于，含有权利要求3所述的重组质粒。
5.根据权利要求4所述的重组菌，其特征在于，宿主细胞为酵母细胞或大肠杆菌BL21(DE3)。
6.权利要求1所述酮还原酶突变体的应用，其特征在于，结合NADH再生系统，利用所述的酮还原酶突变体催化底物发生羰基不对称还原反应，得到24R-羟基构型的胆甾醇，所述底物具有如下结构：
。
7.根据权利要求6所述的应用，其特征在于，所述NADH再生系统为：葡萄糖和葡萄糖脱氢酶；反应条件为：温度25±15℃，转速200±100rpm，时间12-24h。
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Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102482648A (zh) * | 2009-06-22 | 2012-05-30 | 科德克希思公司 | 酮还原酶介导的产生α氯代醇的立体选择性途径 |
| CN103289970A (zh) * | 2013-06-18 | 2013-09-11 | 杭州师范大学 | 酮还原酶lek、编码基因、突变体及应用 |
| CN103898072A (zh) * | 2014-02-26 | 2014-07-02 | 杭州师范大学 | 一种酮还原酶突变体及其应用 |
| CN105671010A (zh) * | 2016-03-04 | 2016-06-15 | 浙江工业大学 | 一种醛酮还原酶突变体、基因、工程菌及其应用 |
| WO2016095223A1 (zh) * | 2014-12-19 | 2016-06-23 | 凯莱英医药集团（天津）股份有限公司 | 双羰基还原酶突变体及其应用 |
| CN108048417A (zh) * | 2018-01-22 | 2018-05-18 | 吉林凯莱英医药化学有限公司 | 酮还原酶突变体及其应用 |
| CN109957584A (zh) * | 2017-12-25 | 2019-07-02 | 尚科生物医药（上海）有限公司 | 一种用于制备（r）-(4-氯苯基)-2-吡啶甲醇的生物催化方法 |
| CN111394324A (zh) * | 2020-06-08 | 2020-07-10 | 凯莱英生命科学技术(天津)有限公司 | 酮还原酶突变体及其应用 |
| CN113801859A (zh) * | 2021-09-29 | 2021-12-17 | 山东寰酶生物制药有限公司 | 一种用于制备手性醇化合物的羰基还原酶突变体及其用途 |
| WO2023011627A1 (zh) * | 2021-08-05 | 2023-02-09 | 上海医药工业研究院有限公司 | 一种羰基还原酶突变体及其应用 |
| CN115747183A (zh) * | 2022-12-23 | 2023-03-07 | 奥锐特药业(天津)有限公司 | 一种酮还原酶突变体及其应用 |
| CN116496997A (zh) * | 2022-01-19 | 2023-07-28 | 南京药石科技股份有限公司 | 羰基还原酶突变体及其应用 |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8357523B2 (en) * | 2009-06-16 | 2013-01-22 | Codexis, Inc. | Beta-glucosidase variant enzymes and related polynucleotides |
| EP3744836A4 (en) * | 2018-01-22 | 2021-03-10 | Jilin Asymchem Laboratories Co., Ltd. | MUTANT KETOREDUCTASE AND RELATED APPLICATION |
-
2023
- 2023-09-01 CN CN202311123361.0A patent/CN117126823B/zh active Active
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102482648A (zh) * | 2009-06-22 | 2012-05-30 | 科德克希思公司 | 酮还原酶介导的产生α氯代醇的立体选择性途径 |
| CN103289970A (zh) * | 2013-06-18 | 2013-09-11 | 杭州师范大学 | 酮还原酶lek、编码基因、突变体及应用 |
| CN103898072A (zh) * | 2014-02-26 | 2014-07-02 | 杭州师范大学 | 一种酮还原酶突变体及其应用 |
| WO2016095223A1 (zh) * | 2014-12-19 | 2016-06-23 | 凯莱英医药集团（天津）股份有限公司 | 双羰基还原酶突变体及其应用 |
| CN105671010A (zh) * | 2016-03-04 | 2016-06-15 | 浙江工业大学 | 一种醛酮还原酶突变体、基因、工程菌及其应用 |
| CN109957584A (zh) * | 2017-12-25 | 2019-07-02 | 尚科生物医药（上海）有限公司 | 一种用于制备（r）-(4-氯苯基)-2-吡啶甲醇的生物催化方法 |
| CN108048417A (zh) * | 2018-01-22 | 2018-05-18 | 吉林凯莱英医药化学有限公司 | 酮还原酶突变体及其应用 |
| CN111394324A (zh) * | 2020-06-08 | 2020-07-10 | 凯莱英生命科学技术(天津)有限公司 | 酮还原酶突变体及其应用 |
| WO2023011627A1 (zh) * | 2021-08-05 | 2023-02-09 | 上海医药工业研究院有限公司 | 一种羰基还原酶突变体及其应用 |
| CN113801859A (zh) * | 2021-09-29 | 2021-12-17 | 山东寰酶生物制药有限公司 | 一种用于制备手性醇化合物的羰基还原酶突变体及其用途 |
| CN116496997A (zh) * | 2022-01-19 | 2023-07-28 | 南京药石科技股份有限公司 | 羰基还原酶突变体及其应用 |
| CN115747183A (zh) * | 2022-12-23 | 2023-03-07 | 奥锐特药业(天津)有限公司 | 一种酮还原酶突变体及其应用 |
Non-Patent Citations (4)
| Title |
|---|
| Development of an Engineered Ketoreductase with Simultaneously Improved Thermostability and Activity for Making a Bulky Atorvastatin Precursor;Xu-Min Gong;CS Catal;147–153 * |
| Rapid asymmetric reduction of ethyl 4-chloro-3-oxobutanoate using a thermostabilized mutant of ketoreductase ChKRED20;Zhao FJ;Appl Microbiol Biotechnol;3567-3575 * |
| Single mutations of ketoreductase ChKRED20 enhance the bioreductive production of (1S)-2-chloro-1-(3, 4-difluorophenyl) ethanol.Feng-Jiao Zhao.2017,1945–1952. * |
| 分子改造羰基还原酶CpCR提高其催化合成2-苯乙醇能力;陈孟军;食品与发酵工业;30-37 * |
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