CN115433721A - 一种羰基还原酶突变体及其应用 - Google Patents
一种羰基还原酶突变体及其应用 Download PDFInfo
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- CN115433721A CN115433721A CN202210728663.XA CN202210728663A CN115433721A CN 115433721 A CN115433721 A CN 115433721A CN 202210728663 A CN202210728663 A CN 202210728663A CN 115433721 A CN115433721 A CN 115433721A
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Abstract
本发明属于生物工程技术领域,具体涉及一种羰基还原酶突变体及其应用。本发明羰基还原酶突变体的野生菌来源于Rhodotorula toruloides,经定点突变获得与酶催化活性相关的核心氨基酸发生突变的突变体;在对应于SEQ ID NO.:1的氨基酸序列的第1至247位中仅存在下述之一的突变:T147A;G190A;E93S;L95A;D195S;E203A。本发明的羰基还原酶突变体实现在(2S,3R)‑2‑苯甲酰氨甲基‑3‑羟基丁酸甲酯合成中的应用,手性纯度显著提高,中控产品手性纯度达到98%以上。
Description
技术领域
本发明属于生物工程技术领域,特别涉及一种羰基还原酶突变体,还涉及上述突变体的应用。
背景技术
生物法催化羰基化合物的不对称还原是制备手性醇的重要方法,近年来已成为有机合成的研究热点。该反应由羰基还原酶催化,常用于合成一些高附加值的手性药物中间体,与传统的化学法相比,利用生物催化剂催化前手性羰基化合物的不对称还原具有显著优势。
(2S,3R)-2-苯甲酰氨甲基-3-羟基丁酸甲酯((2S,3R)-BHBM)是一种具有光学活性的β-羟基酯类物质,也是手性醇的一种,其化学结构式如式(II)所示。(2S,3R)-BHBM是合成(3R,4R)-3-[(R)-1-叔丁基二甲基硅氧乙基]-4-乙酰氧基-2-氮杂环丁酮(简称为4-AA)的关键起始原料,4-AA是一种重要的医药精细化学品,主要用于合成各类培南类的抗生素,如亚胺培南、比阿培南、美罗培南和法罗培南等。这些药物用途广泛,对革兰阴性和阳性菌、需氧菌、厌氧菌等均具有广谱强效抗菌作用,因而受到人们极大重视。目前,在(2S,3R)-BHBM的手性合成工艺方面,主要有两种方法。一种是化学合成法,该法需要贵重金属钌作为催化剂,且反应需要在高温高压条件下进行,对反应器的要求较高,限制了(2S,3R)-BHBM的大规模工业化生产。另一种是生物催化法,通过羰基还原酶不对称还原外消旋2-苯甲酰胺甲基-3-羰基丁酸甲酯(BOBM)制备(2S,3R)-BHBM。该方法可以有效解决传统化学法存在的不足,同时降低生产成本,提高生产效率,是(2S,3R)-BHBM工业化生产的发展趋势。目前,亟需提供一种可以高效催化该反应的羰基还原酶。
CN 112941043 A公开了一种羰基还原酶突变体及在制备手性β’-羟基-β-氨基酸酯中的应用。该发明提供了一种羰基还原酶突变体及其在(2S,3R)-2-苯甲酰氨甲基-3-羟基丁酸甲酯合成中的应用,具体地,所述羰基还原配的野生型来源于Sporobolomycessalmonicolor,经定点突变获得与酶催化活性相关的核心氨基酸发生突变的突变蛋白,其具有显著提高催化β’-羰基-β-(保护)氨基酸酯化合物的活性。
上述文献与本发明相比,存在以下局限:(1)缓冲体系不同:该专利中的酶要起到好的催化效果,需要在缓冲体系中添加甲醇、丙酮,二氯甲烷等助溶剂,而添加助溶剂后会导致后续提纯工艺繁琐且产生大量的有机废水,不利于环保;而本发明中的酶在仅用水作为反应介质的条件下即可达到良好的反应效果,环保的同时达到了很好的反应速率和产物纯度;(2)本发明的酶基因是通过基因挖矿技术获得的全新酶基因,与上述文献所用酶基因相比,相似度较低;(3)上述文献的羰基还原酶最适反应温度为28℃,而本发明的羰基还原酶突变体可以耐受更高的反应温度,更有利于工业化生产和储存。
发明内容
为了解决上述的技术问题,本发明提供了一种羰基还原酶突变体。本发明旨在挖掘高效的羰基还原酶突变体,并通过基因工程手段对其进行改造,获得性能明显提升的羰基还原酶突变体;改造后的羰基还原酶突变体合成手性(2S,3R)-BHBM的活力和立体选择性显著提高。
本发明羰基还原酶突变体的野生菌来源于Rhodotorula toruloides,经定点突变获得与酶催化活性相关的核心氨基酸发生突变的突变体;
本发明提供的羰基还原酶突变体,与SEQ ID NO:1所示的氨基酸序列具有95%以上的同一性,且所述突变体具有合成手性(2S,3R)-BHBM的能力,并且催化活性和立体选择性显著提高。
本发明提供一种羰基还原酶突变体,在对应于SEQ ID NO.:1的氨基酸序列的第1至247位中仅存在第93,95,147,190,195,203在内的单个位点突变。
优选的,上述突变体在对应于SEQ ID NO.:1的氨基酸序列的第1至247位中仅存在下述之一的突变:T147A;G190A;E93S;L95A;D195S;E203A;其氨基酸序列为SEQ ID NO.3-8之一所示。
所述的SEQ ID NO:1所示的氨基酸序列的编码基因为SEQ ID NO:2所示的核苷酸序列。
本发明还提供上述突变体的编码基因,进一步提供含所述基因的表达载体、重组细胞。
本发明所述的羰基还原酶突变体的制备方法,包括如下步骤:
(1)合成SEQ ID NO:1所示的氨基酸序列的编码基因,得到SEQ ID NO:2所示的核苷酸序列;
(2)将SEQ ID NO:2所示的核苷酸序列进行定点突变,得到SEQ ID NO:3-8的编码基因;
(3)将SEQ ID NO:3-8的编码基因进行基因克隆,得到含有羰基还原酶突变体编码基因的质粒;
(4)将含有羰基还原酶突变体编码基因的质粒导入感受态细胞中,得到羰基还原酶突变体的表达菌株;
(5)羰基还原酶突变体的表达菌株进行诱导发酵,得到羰基还原酶突变体。
本发明还提供了利用羰基还原酶突变体制备的培南类药物中间体手性(2S,3R)-3-羰基-2(邻苯二甲酰亚胺)甲基丁酸甲酯化合物。
上述(2S,3R)-3-羰基-2(邻苯二甲酰亚胺)甲基丁酸甲酯化合物,是以表达所述羰基还原酶突变体的编码基因的工程菌经发酵培养获得的湿菌体为催化剂,以β-羰基-β-氨基酸酯化合物为底物,以水作为反应介质,在pH6.0-8.5,温度30-55℃条件下进行催化反应获得的。
优选的,上述底物的浓度为200-400g/L,菌体的浓度为10-20g/L,反应体系的pH为6.0-8.5,反应温度为30-55℃。
优选的,上述制备(2S,3R)-3-羰基-2(邻苯二甲酰亚胺)甲基丁酸甲酯化合物的应用中,在反应体系里加入NADP+、葡萄糖、葡萄糖脱氢酶,在100-200rpm的条件下反应,反应时间为8-20h。
优选的,上述制备(2S,3R)-3-羰基-2(邻苯二甲酰亚胺)甲基丁酸甲酯化合物的应用中,NADP+浓度为0.05-0.1g/L,葡萄糖浓度为120~180g/L,葡萄糖脱氢酶浓度为1-2.5g/L,在120-200rpm的条件下反应,反应时间为10-20h。
本发明中羰基还原酶突变体催化β-羰基-β-氨基酸酯化合物(BOBM)制备(2S,3R)-3-羰基-2(邻苯二甲酰亚胺)甲基丁酸甲酯(BHBM)的工艺路线如下:
本发明以BOBM为原料经过羰基还原酶突变体催化一步制得BHBM,可显著降低生产成本。
为了实现以BOBM为原料制备BHBM的目的,本发明开发了能够高效催化该反应的羰基还原酶突变体。本发明从一级结构至高级结构多角度多层系比较了Genbank中ID为PRQ77350.1的羰基还原酶(氨基酸序列如SEQ ID NO:1所示)与同源酶蛋白的异同,确定了影响羰基还原酶酶学性质的关键氨基酸位点为第93,95,147,190,195,203位氨基酸,然后通过密码子替换将这些位点进行如下突变:T147A;G190A;E93S;L95A;D195S;E203A,得到羰基还原酶突变体;通过构建突变体基因的6×His融合表达载体并导入基因工程菌E.coliBL21(DE3)中诱导表达,获得了突变体酶蛋白。
本发明提供了一种羰基还原酶突变体,所述突变体的蛋白为非天然蛋白,并具有高效催化BOBM制备BHBM的特点。本发明羰基还原酶突变体是在PRQ77350.1的羰基还原酶的第93,95,147,190,195,203位氨基酸发生突变得到的。
本发明的有益效果:
1.本发明羰基还原酶突变体及其在编码基因、表达载体、重组细胞的应用中均以水作为反应介质替代现有技术中的甲醇、丙乙醇等助溶剂体系,在保证产物纯度的同时,更具有环保性;
2.本发明的羰基还原酶突变体实现了以BOBM为原料生产BHBM的高效催化,显著降低了生产成本,产物纯度大幅度提高,产品手性纯度达到了98.11%;
3.本发明的羰基还原酶突变体可以耐受45-55℃的反应温度,具有更广的温度耐受范围,有利于工业化生产和储存。
附图说明
为了更清楚地说明本发明实施例中的技术方案,下面将对实施例描述中所需要使用的附图作简单地介绍。
图1是羰基还原酶突变体D195S在不同宿主中表达的SDS-PAGE电泳图;其中,BL21-CodonPlus(DE3)、BL21(DE3)、Rosetta(DE3)和OverexpressC43(DE3)为四种E.coli蛋白表达宿主,Marker为蛋白质分子量标准;
图2是利用羰基还原酶突变体制得的BHBM的HPLC检测图;
图3是BHBM标准品的HPLC检测图;
图4是利用羰基还原酶初始酶制得BHBM的HPLC检测图。
具体实施方式
为了能使本领域技术人员更好的理解本发明,现结合具体实施方式对本发明进行更进一步的阐述。以下实施例仅用于说明本发明而非用于限制本发明的范围。
实施例1
一、羰基还原酶突变体设计
通过基因挖掘,从NCBI数据库中筛选获得了Genbank ID为PRQ77350.1的羰基还原酶基因,该基因的开放阅读框全长741bp,其编码的羰基还原酶由247个氨基酸组成,其氨基酸序列(247aa)为SEQ ID NO:1,编码该羰基还原酶的核苷酸序列为SEQ ID NO:2。
通过从一级结构至高级结构多角度多层系比较了Genbank中ID为PRQ77350.1的羰基还原酶(氨基酸序列如SEQ ID NO:1所示)与同源酶蛋白的异同,确定了影响羰基还原酶酶学性质的关键氨基酸位点为第93,95,147,190,195,203位氨基酸,然后通过密码子替换将这些位点进行如下突变:T147A;G190A;E93S;L95A;D195S;E203A,得到羰基还原酶突变体,其氨基酸序列如SEQ ID NO:4-8所示。
二、羰基还原酶突变体基因的获得
羰基还原酶突变体基因可以通过全基因合成方法或分子克隆方法获得。本实验采用全基因合成方法获得Genbank ID为PRQ77350.1的羰基还原酶基因,采用PCR法获得羰基还原酶突变体基因。
1、PRQ77350.1的羰基还原酶全基因合成
对Genbank ID为PRQ77350.1的羰基还原酶进行全基因合成,合成后的基因片段连入pUC57质粒中,委托生工生物工程(上海)股份有限公司合成上述基因。
2、羰基还原酶基因定点突变
(1)定点突变引物设计
对羰基还原酶突变体基因序列设计引物,引入定点突变。引物的核苷酸序列如表1。委托生工生物工程(上海)股份有限公司合成上述引物。
表1引物的核苷酸序列
(2)定点突变
以含有PRQ77350.1的羰基还原酶基因的pUC57质粒为模板,采用步骤(1)获得的上游引物和下游引物,按照下列PCR体系和程序扩增羰基还原酶突变体基因。PCR体系:KOD-Plus-0.5μL,质粒模板0.8μL,上游引物V-492-P-F(10μM)1μL,下游引物V-492-P-R(10μM)1μL,10×PCRBuffer2.5μL,dNTP(2mM)3μL,MgSO4(25mM)1.5μL,ddH2O 14.7μL。PCR程序如下:a.94℃预变性4min;b.94℃变性40sec,68℃退火并延伸7.5min;18个循环;c.68℃延伸20min。使用DpnI酶消化含有WP_016501746.1的羰基还原酶基因的模板质粒,37℃,消化30min。
(3)DH5α感受态细胞转化
将步骤(2)获得的含有羰基还原酶突变体基因的pUC57质粒转化到DH5α感受态细胞中。将DH5α感受态细胞放置于冰上,待细胞融化后加入10μL质粒溶液,冰上放置30min;42℃热激50s,然后冰上静置3min;加入600μL无菌LB液体培养基,37℃,200rpm摇床中培养1h;吸取200μL培养好的菌液,涂布于含有Kana抗性(50μg/mL)的LB平板培养基上,37℃倒置培养过夜。
(4)阳性克隆筛选
挑取LB平板上的单菌落,接种于含有Kana抗性(50μg/mL)LB液体培养基中,37℃,220rpm摇床过夜培养,用于质粒提取。使用OMEGA Plasmid Mini Kit I(货号:D6943)按照说明书提取质粒,并将提取后的质粒送金唯智(中国,苏州)公司测序,鉴定PRQ77350.1的羰基还原酶是否突变成功。
3、羰基还原酶突变体基因克隆
(1)基因克隆
以含有羰基还原酶突变体基因的pUC57质粒为模板,采用获得的上游引物和下游引物,按照下列PCR体系和程序扩增羰基还原酶突变体基因。PCR体系:PrimeSTAR MaxPremix(2×)25μL,质粒模板0.5μL,上游引物NcoI-F(10μM)2μL,下游引物XhoI-R(10μM)2μL,补ddH2O至总体积为50μL。PCR程序如下:a.98℃预变性2min;b.98℃变性10sec,65℃退火10sec,72℃延伸30sec;30个循环;c.72℃延伸3min。使用1.0%琼脂糖凝胶电泳检测PCR扩增产物,得到大小约为1600bp的目的条带。在紫外灯下切取目的条带,使用Omega GelExtraction Kit(货号:D2500),按照试剂盒说明书回收羰基还原酶突变体基因片段。
(3)表达载体构建
利用NcoI和Xho I限制性内切酶分别对羰基还原酶突变体基因和pET28(a)载体进行双酶切。酶切体系(基因):羰基还原酶突变体基因25μL,NcoI酶2μL,Xho I酶2μL,10×Buffer 5μL,补无菌双蒸水至体系为50μL。酶切体系(载体):pET28(a)载体2μL,NcoI酶0.5μL,XhoI酶0.5μL,10×Buffer1μL,补无菌双蒸水至体系为10μL。酶切条件:37℃酶切30min。然后使用T4 DNA连接酶连接经过双酶切后的羰基还原酶突变体基因和pET28(a)线性载体,16℃连接过夜,得到含有羰基还原酶突变体基因的pET28(a)质粒。
(4)DH5α感受态细胞转化
将步骤(3)获得的含有羰基还原酶突变体基因的pET28(a)质粒转化到DH5α感受态细胞中。将DH5α感受态细胞放置于冰上,待细胞融化后加入10μL质粒溶液,冰上放置30min;42℃热激50s,然后冰上静置3min;加入600μL无菌LB液体培养基,37℃,200rpm摇床中培养1h;吸取200μL培养好的菌液,涂布于含有Kana抗性(50μg/mL)的LB平板培养基上,37℃倒置培养过夜。
(5)阳性克隆筛选
挑取LB平板上的单菌落,接种于含有Kana抗性(50μg/mL)LB液体培养基中,37℃,220rpm摇床过夜培养,用于质粒提取。使用OMEGA Plasmid Mini Kit I按照说明书提取质粒,并将提取后的质粒送金唯智(中国,苏州)公司测序,鉴定羰基还原酶突变体基因表达载体是否构建成功。
4、羰基还原酶突变体6×His融合蛋白的异源表达
(1)蛋白表达感受态细胞转化
从-80℃超低温冰箱中分别取出E.coli BL21(DE3)、E.coli Rosetta(DE3)和OverexpressC43(DE3)感受态细胞,冰上放置。待细胞融化后,分别向每种感受态细胞中加入1μL含有羰基还原酶突变体基因的pET28-(a)质粒,冰上放置30min;42℃热激50s,然后冰上放置3min;加入600μL无菌LB液体培养基,37℃,200rpm摇床培养1h;吸取200μL培养好的菌液,涂布于含有Kana抗性(50μg/mL)的LB平板培养基上,37℃倒置培养过夜。
(2)羰基还原酶突变体表达菌株保存
挑取LB平板上的单菌落,接种于含有Kana抗性(50μg/mL)LB液体培养基中,37℃,220rpm摇床过夜培养,用作羰基还原酶突变体的表达菌株。向菌液中加入终体积浓度为15%的无菌丙三醇,-80℃超低温冰箱中长期保存。
(3)蛋白表达
a.种子摇瓶培养:将100μL羰基还原酶突变体的表达菌株接种入50mL无菌TB液体培养基中,卡那霉素终浓度为50μg/mL,37℃,200rpm培养8h,得到羰基还原酶突变体的表达种子瓶菌液。b.发酵摇瓶培养:将10mL羰基还原酶突变体的表达种子瓶菌液接种入350mL无菌TB液体培养基中,卡那霉素终浓度为50μg/mL,37℃,200rpm;待OD600=0.6-0.8时,加入终浓度为0.3mM的无菌IPTG,28℃,200rpm诱导培养20h,得到酶液,进行后续的SDS-PAGE及酶活检测。c.酶活检测:通过检测340nm下NADPH吸光值的变化来计算突变体的催化活性,酶活检测底物为β’-羰基-β-(邻苯二甲酰亚胺)甲基丁酸甲酯,体系为底物25mM,10%DMF助溶,5mg/mL NADPH,缓冲液为200mM pH 6.8的磷酸缓冲液。检测的酶活力结果见表2。
实施例2
采用羰基还原酶突变体进行BHBM的合成。
反应体系采用pH7.0的磷酸缓冲液,反应过程使用Na2CO3调节pH为6.7-6.85,以β-羰基-β-氨基酸酯化合物为反应底物,浓度为300g/L;加入的葡萄糖浓度为120g/L,NADP+浓度为0.1g/L,葡萄糖脱氢酶浓度为1.5g/L,反应温度为45℃,实施例1中的羰基还原酶突变体(采用大肠杆菌BL21(DE3)制备的羰基还原酶突变体)用量为20g/L,以表达所述羰基还原酶突变体的编码基因的工程菌经发酵培养获得的湿菌体为催化剂,菌体浓度为20g/L;反应10h后将反应液过滤,滤饼使用乙酸乙酯萃取,将有机相旋蒸后获得产品。HPLC检测产品纯度,检测结果见表2。
突变体D195S反应后产品的HPLC结果见图2,BHBM的保留时间为10.287min,产品手性纯度98.11%。
对BHBM标准品进行HPLC检测,HPLC结果见图3,BHBM的保留时间为10.332min。通过将图2与图3进行对比,证明实施例2最终得到的产品是BHBM。
对比例1
本试验采用羰基还原酶初始酶进行BHBM的合成。
反应体系采用pH7.0的磷酸缓冲液,反应过程使用Na2CO3调节pH为6.7-6.85,以β-羰基-β-氨基酸酯化合物为反应底物,反应底物终浓度为300g/L;加入的葡萄糖浓度为150g/L,NADP+浓度为0.1g/L,葡萄糖脱氢酶浓度为2g/L,反应温度38℃,羰基还原酶初始酶(采用大肠杆菌BL21(DE3)制备的羰基还原酶初始酶)用量为20g/L,反应10h后将反应液过滤,滤饼使用乙酸乙酯萃取,将有机相旋蒸后获得产品。HPLC检测产品纯度,检测结果见表2。对其反应后产品的HPLC结果见图4,BHBM的保留时间为10.341min,产品手性纯度63.5%。
表2不同突变体的酶活及产物纯度对比
序列表
<110> 山东理工大学
<120> 一种羰基还原酶突变体及其应用
<141> 2022-06-24
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Ala Ser Asp Arg Thr Val Tyr Leu Val Thr Gly Gly Ser Arg Gly Leu
1 5 10 15
Gly Phe Gly Ile Val Thr Leu Leu Ala Gly Arg Ala Asn Thr Leu Val
20 25 30
Phe Ala Thr Ala Arg Asp Pro Ile Arg Ala Lys Asp Leu Gln Glu Leu
35 40 45
Ala Val Lys His Ser Asn Val Ile Pro Val Gln Leu Glu Leu Thr Ser
50 55 60
Glu Ala Ser Val Glu Ala Leu Ala Arg Val Ile Asn Glu Lys Ala Gly
65 70 75 80
Arg Leu Asp Val Ala Ile Asn Asn Ala Ala Asn Cys Glu Gly Leu Ala
85 90 95
Thr Ala Leu Thr Thr Ser Ile Ser Asp Phe Arg Asp Lys Leu Glu Ser
100 105 110
Asn Ala Val Ala Pro Leu Leu Leu Phe Gln His Leu Tyr Pro Leu Leu
115 120 125
Met Lys Ser Lys Gln Arg Gln Phe Val Gly Ile Ser Thr Ala Gly Ala
130 135 140
Ser Leu Thr Leu Val Pro His Ile Ser Tyr Pro Ile Leu Gly Tyr Ala
145 150 155 160
Ala Ser Lys Thr Ala Met Asn Met Val Tyr Thr Lys Ile Ala Ala Glu
165 170 175
His Ala Gly Asp Glu Phe Leu Ser Tyr Val Val His Pro Ala Leu Val
180 185 190
Lys Thr Asp Ser Ala Ala Ser Ala Ile Gln Glu Leu Gly Leu Asp Glu
195 200 205
Ser Glu Ala Leu Ser Pro Met Ala Ser Ala Ala Gly Val Leu Lys Val
210 215 220
Val Ala Ala Ala Arg Arg Glu Thr His Ser Gly Arg Phe Trp Asp Tyr
225 230 235 240
Glu Gly Lys Glu Val Pro
245
<210> 5
<211> 246
<212> PRT
<213> 人工序列(Artificial)
<400> 5
Ala Ser Asp Arg Thr Val Tyr Leu Val Thr Gly Gly Ser Arg Gly Leu
1 5 10 15
Gly Phe Gly Ile Val Thr Leu Leu Ala Gly Arg Ala Asn Thr Leu Val
20 25 30
Phe Ala Thr Ala Arg Asp Pro Ile Arg Ala Lys Asp Leu Gln Glu Leu
35 40 45
Ala Val Lys His Ser Asn Val Ile Pro Val Gln Leu Glu Leu Thr Ser
50 55 60
Glu Ala Ser Val Glu Ala Leu Ala Arg Val Ile Asn Glu Lys Ala Gly
65 70 75 80
Arg Leu Asp Val Ala Ile Asn Asn Ala Ala Asn Cys Ser Gly Leu Ala
85 90 95
Thr Ala Leu Thr Thr Ser Ile Ser Asp Phe Arg Asp Lys Leu Glu Ser
100 105 110
Asn Ala Val Ala Pro Leu Leu Leu Phe Gln His Leu Tyr Pro Leu Leu
115 120 125
Met Lys Ser Lys Gln Arg Gln Phe Val Gly Ile Ser Thr Ala Gly Ala
130 135 140
Ser Leu Thr Leu Val Pro His Ile Ser Tyr Pro Ile Leu Gly Tyr Ala
145 150 155 160
Ala Ser Lys Thr Ala Met Asn Met Val Tyr Thr Lys Ile Ala Ala Glu
165 170 175
His Ala Gly Asp Glu Phe Leu Ser Tyr Val Val His Pro Gly Leu Val
180 185 190
Lys Thr Asp Ser Ala Ala Ser Ala Ile Gln Glu Leu Gly Leu Asp Glu
195 200 205
Ser Glu Ala Leu Ser Pro Met Ala Ser Ala Ala Gly Val Leu Lys Val
210 215 220
Val Ala Ala Ala Arg Arg Glu Thr His Ser Gly Arg Phe Trp Asp Tyr
225 230 235 240
Glu Gly Lys Glu Val Pro
245
<210> 6
<211> 246
<212> PRT
<213> 人工序列(Artificial)
<400> 6
Ala Ser Asp Arg Thr Val Tyr Leu Val Thr Gly Gly Ser Arg Gly Leu
1 5 10 15
Gly Phe Gly Ile Val Thr Leu Leu Ala Gly Arg Ala Asn Thr Leu Val
20 25 30
Phe Ala Thr Ala Arg Asp Pro Ile Arg Ala Lys Asp Leu Gln Glu Leu
35 40 45
Ala Val Lys His Ser Asn Val Ile Pro Val Gln Leu Glu Leu Thr Ser
50 55 60
Glu Ala Ser Val Glu Ala Leu Ala Arg Val Ile Asn Glu Lys Ala Gly
65 70 75 80
Arg Leu Asp Val Ala Ile Asn Asn Ala Ala Asn Cys Glu Gly Ala Ala
85 90 95
Thr Ala Leu Thr Thr Ser Ile Ser Asp Phe Arg Asp Lys Leu Glu Ser
100 105 110
Asn Ala Val Ala Pro Leu Leu Leu Phe Gln His Leu Tyr Pro Leu Leu
115 120 125
Met Lys Ser Lys Gln Arg Gln Phe Val Gly Ile Ser Thr Ala Gly Ala
130 135 140
Ser Leu Thr Leu Val Pro His Ile Ser Tyr Pro Ile Leu Gly Tyr Ala
145 150 155 160
Ala Ser Lys Thr Ala Met Asn Met Val Tyr Thr Lys Ile Ala Ala Glu
165 170 175
His Ala Gly Asp Glu Phe Leu Ser Tyr Val Val His Pro Gly Leu Val
180 185 190
Lys Thr Asp Ser Ala Ala Ser Ala Ile Gln Glu Leu Gly Leu Asp Glu
195 200 205
Ser Glu Ala Leu Ser Pro Met Ala Ser Ala Ala Gly Val Leu Lys Val
210 215 220
Val Ala Ala Ala Arg Arg Glu Thr His Ser Gly Arg Phe Trp Asp Tyr
225 230 235 240
Glu Gly Lys Glu Val Pro
245
<210> 7
<211> 246
<212> PRT
<213> 人工序列(Artificial)
<400> 7
Ala Ser Asp Arg Thr Val Tyr Leu Val Thr Gly Gly Ser Arg Gly Leu
1 5 10 15
Gly Phe Gly Ile Val Thr Leu Leu Ala Gly Arg Ala Asn Thr Leu Val
20 25 30
Phe Ala Thr Ala Arg Asp Pro Ile Arg Ala Lys Asp Leu Gln Glu Leu
35 40 45
Ala Val Lys His Ser Asn Val Ile Pro Val Gln Leu Glu Leu Thr Ser
50 55 60
Glu Ala Ser Val Glu Ala Leu Ala Arg Val Ile Asn Glu Lys Ala Gly
65 70 75 80
Arg Leu Asp Val Ala Ile Asn Asn Ala Ala Asn Cys Glu Gly Leu Ala
85 90 95
Thr Ala Leu Thr Thr Ser Ile Ser Asp Phe Arg Asp Lys Leu Glu Ser
100 105 110
Asn Ala Val Ala Pro Leu Leu Leu Phe Gln His Leu Tyr Pro Leu Leu
115 120 125
Met Lys Ser Lys Gln Arg Gln Phe Val Gly Ile Ser Thr Ala Gly Ala
130 135 140
Ser Leu Thr Leu Val Pro His Ile Ser Tyr Pro Ile Leu Gly Tyr Ala
145 150 155 160
Ala Ser Lys Thr Ala Met Asn Met Val Tyr Thr Lys Ile Ala Ala Glu
165 170 175
His Ala Gly Asp Glu Phe Leu Ser Tyr Val Val His Pro Gly Leu Val
180 185 190
Lys Thr Ser Ser Ala Ala Ser Ala Ile Gln Glu Leu Gly Leu Asp Glu
195 200 205
Ser Glu Ala Leu Ser Pro Met Ala Ser Ala Ala Gly Val Leu Lys Val
210 215 220
Val Ala Ala Ala Arg Arg Glu Thr His Ser Gly Arg Phe Trp Asp Tyr
225 230 235 240
Glu Gly Lys Glu Val Pro
245
<210> 8
<211> 246
<212> PRT
<213> 人工序列(Artificial)
<400> 8
Ala Ser Asp Arg Thr Val Tyr Leu Val Thr Gly Gly Ser Arg Gly Leu
1 5 10 15
Gly Phe Gly Ile Val Thr Leu Leu Ala Gly Arg Ala Asn Thr Leu Val
20 25 30
Phe Ala Thr Ala Arg Asp Pro Ile Arg Ala Lys Asp Leu Gln Glu Leu
35 40 45
Ala Val Lys His Ser Asn Val Ile Pro Val Gln Leu Glu Leu Thr Ser
50 55 60
Glu Ala Ser Val Glu Ala Leu Ala Arg Val Ile Asn Glu Lys Ala Gly
65 70 75 80
Arg Leu Asp Val Ala Ile Asn Asn Ala Ala Asn Cys Glu Gly Leu Ala
85 90 95
Thr Ala Leu Thr Thr Ser Ile Ser Asp Phe Arg Asp Lys Leu Glu Ser
100 105 110
Asn Ala Val Ala Pro Leu Leu Leu Phe Gln His Leu Tyr Pro Leu Leu
115 120 125
Met Lys Ser Lys Gln Arg Gln Phe Val Gly Ile Ser Thr Ala Gly Ala
130 135 140
Ser Leu Thr Leu Val Pro His Ile Ser Tyr Pro Ile Leu Gly Tyr Ala
145 150 155 160
Ala Ser Lys Thr Ala Met Asn Met Val Tyr Thr Lys Ile Ala Ala Glu
165 170 175
His Ala Gly Asp Glu Phe Leu Ser Tyr Val Val His Pro Gly Leu Val
180 185 190
Lys Thr Asp Ser Ala Ala Ser Ala Ile Gln Ala Leu Gly Leu Asp Glu
195 200 205
Ser Glu Ala Leu Ser Pro Met Ala Ser Ala Ala Gly Val Leu Lys Val
210 215 220
Val Ala Ala Ala Arg Arg Glu Thr His Ser Gly Arg Phe Trp Asp Tyr
225 230 235 240
Glu Gly Lys Glu Val Pro
245
Claims (10)
1.一种羰基还原酶突变体,其特征在于,在对应于SEQ ID NO.:1的氨基酸序列的第1至247位中仅存在第93,95,147,190,195,203在内的单个位点突变。
2.如权利要求1所述的一种羰基还原酶突变体,其特征在于,在对应于SEQ ID NO.:1的氨基酸序列的第1至247位中仅存在下述之一的突变:T147A;G190A;E93S;L95A;D195S;E203A。
3.如权利要求1或2所述的羰基还原酶突变体的编码基因。
4.如权利要求1或2所述的羰基还原酶突变体的编码基因的表达载体。
5.如权利要求1或2所述的羰基还原酶突变体的编码基因的重组细胞。
6.如权利要求1或2所述的羰基还原酶突变体在制备培南类药物中间体手性(2S,3R)-2-苯甲酰氨甲基-3-羟基丁酸甲酯化合物中的应用。
7.如权利要求6所述的应用,其特征在于,是以表达所述羰基还原酶突变体的编码基因的工程菌经发酵培养获得的湿菌体为催化剂,以β-羰基-β-氨基酸酯化合物为底物,以水作为反应介质,在pH6.0-8.5,温度30-55℃条件下进行催化反应,得到(2S,3R)-2-苯甲酰氨甲基-3-羟基丁酸甲酯。
8.如权利要求7所述的应用,其特征在于,所述的底物的浓度为200-400g/L,菌体的浓度为10-20g/L,反应体系的pH为6.0-8.5,反应温度为30-55℃。
9.如权利要求8所述的应用,其特征在于,在反应体系用加入NADP+、葡萄糖、葡萄糖脱氢酶,在100-200rpm的条件下反应,反应时间为8-20h。
10.如权利要求9所述的应用,其特征在于,NADP+浓度为0.05-0.1g/L,葡萄糖浓度为120~180g/L,葡萄糖脱氢酶浓度为1-2.5g/L。
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CN117230031A (zh) * | 2023-11-03 | 2023-12-15 | 山东理工大学 | 羰基还原酶突变体及其应用 |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6218156B1 (en) * | 1997-02-07 | 2001-04-17 | Kaneka Corporation | Gene encoding carbonyl reductase, and methods for its use |
WO2010025607A1 (zh) * | 2008-09-02 | 2010-03-11 | 南京工业大学 | 一种采用羰酰还原酶生产(s)-4-氯-3-羟基丁酸乙酯的方法 |
CN104846025A (zh) * | 2015-03-31 | 2015-08-19 | 浙江大学 | 一种制备(2s,3r)-2-苯甲酰氨甲基-3-羟基丁酸甲酯的方法 |
CN112941043A (zh) * | 2021-05-17 | 2021-06-11 | 中国科学院天津工业生物技术研究所 | 羰基还原酶突变体及在制备手性β’-羟基-β-氨基酸酯中的应用 |
CN113322291A (zh) * | 2020-02-28 | 2021-08-31 | 湖北美天生物科技股份有限公司 | 一种手性氨基醇类化合物的合成方法 |
CN113462666A (zh) * | 2021-08-18 | 2021-10-01 | 杭州文德阶生物科技有限公司 | 羰基还原酶突变体及其构建方法和应用 |
CN113652407A (zh) * | 2021-07-09 | 2021-11-16 | 浙江工业大学 | 羰基还原酶突变体及其不对称合成双手性化合物的应用 |
-
2022
- 2022-06-24 CN CN202210728663.XA patent/CN115433721B/zh active Active
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6218156B1 (en) * | 1997-02-07 | 2001-04-17 | Kaneka Corporation | Gene encoding carbonyl reductase, and methods for its use |
WO2010025607A1 (zh) * | 2008-09-02 | 2010-03-11 | 南京工业大学 | 一种采用羰酰还原酶生产(s)-4-氯-3-羟基丁酸乙酯的方法 |
CN104846025A (zh) * | 2015-03-31 | 2015-08-19 | 浙江大学 | 一种制备(2s,3r)-2-苯甲酰氨甲基-3-羟基丁酸甲酯的方法 |
CN113322291A (zh) * | 2020-02-28 | 2021-08-31 | 湖北美天生物科技股份有限公司 | 一种手性氨基醇类化合物的合成方法 |
CN112941043A (zh) * | 2021-05-17 | 2021-06-11 | 中国科学院天津工业生物技术研究所 | 羰基还原酶突变体及在制备手性β’-羟基-β-氨基酸酯中的应用 |
CN113652407A (zh) * | 2021-07-09 | 2021-11-16 | 浙江工业大学 | 羰基还原酶突变体及其不对称合成双手性化合物的应用 |
CN113462666A (zh) * | 2021-08-18 | 2021-10-01 | 杭州文德阶生物科技有限公司 | 羰基还原酶突变体及其构建方法和应用 |
Non-Patent Citations (1)
Title |
---|
LI J ET AL: "Structure-Guided Directed Evolution of a Carbonyl Reductase Enables the Stereoselective Synthesis of (2S, 3S)-2, 2-Disubstituted-3-hydroxycyclopentanones via Desymmetric Reduction.", 《 EPUB》, vol. 22, no. 9 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN117230031A (zh) * | 2023-11-03 | 2023-12-15 | 山东理工大学 | 羰基还原酶突变体及其应用 |
CN117230031B (zh) * | 2023-11-03 | 2024-01-26 | 山东理工大学 | 羰基还原酶突变体及其应用 |
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