CN110592035B - 一种羰基还原酶的突变体、重组表达载体及其在生产手性醇中的应用 - Google Patents
一种羰基还原酶的突变体、重组表达载体及其在生产手性醇中的应用 Download PDFInfo
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Abstract
本发明公开了一种羰基还原酶的突变体、重组表达载体及其在生产手性醇中的应用,突变体为在羰基还原酶EbSDR8氨基酸序列的基础上含有如下几个位点的单点突变或多点组合突变:94位甘氨酸、145位组氨酸、153位丝氨酸、188位酪氨酸、199位丝氨酸和202位酪氨酸;羰基还原酶的突变体或含有突变体的重组表达载体可在不添加任何辅酶的反应体系中高效催化高浓度的潜手性酮的不对称还原,生成高光学纯度手性醇(e.e.>99%);具有很好的工业化应用前景。
Description
技术领域
本发明涉及酶工程技术领域,特别是一种羰基还原酶的突变体、重组表达载体及其在生产手性醇中的应用。
背景技术
潜手性酮不对称还原得到的光学纯手性醇(R)-(3)-氯-(1)-苯丙醇(分子式为C9H11ClO,分子质量数为170.64,CAS-No.:100306-33-0)拥有三个活性基团(氯基、苯基、光学纯羟基),是合成医药、精细化学品的重要手性砌块。研究人员已开发出多种光学活性手性醇合成的方法,包括动力学拆分和不对称合成。其中,利用潜手性酮的不对称还原合成光学活性手性醇的途径,可实现100%的理论产率,是生产光学活性(R)-(3)-氯-(1)-苯丙醇的重要方法。化学家们已经发现手性金属衍生物可作为催化剂用于羰基的不对称还原,尽管该化学方法已被部分用于工业生产,然而该方法操作难度大,反应条件苛刻,且产物中可能会残留重金属,因此其应用受到限制。
生物催化法不仅反应条件温和、对环境友好,具有高度的区域选择性和立体选择性,而且避免了产物中重金属残留,恰好弥补了化学方法的不足之处,因此近几年来生物催化的羰基不对称还原反应在手性醇不对称合成中的应用越来越受到重视;市场需要一种通过蛋白质理性设计的方式,提供高催化活力的羰基还原酶EbSDR8的突变体及其工程菌等,为光学纯的手性醇合成提供强有力的生物催化剂;本发明解决这样的问题。
发明内容
为解决现有技术的不足,本发明的目的在于提供一种羰基还原酶的突变体、重组表达载体及其在生产手性醇中的应用,羰基还原酶的突变体催化活力高、对应选择性强、底物耐受性好。
为了实现上述目标,本发明采用如下的技术方案:
一种羰基还原酶的突变体,突变体为在羰基还原酶EbSDR8氨基酸序列的基础上含有如下几个位点的单点突变或多点组合突变:94位甘氨酸、145位组氨酸、153位丝氨酸、188位酪氨酸、199位丝氨酸和202位酪氨酸;
羰基还原酶EbSDR8氨基酸序列如SEQ ID No.2所示;
羰基还原酶EbSDR8核苷酸序列如SEQ ID No.1所示。
前述的一种羰基还原酶的突变体,突变体为在羰基还原酶EbSDR8氨基酸序列的基础上含有如下几个位点的单点突变或多点组合突变:153位丝氨酸、188位酪氨酸和202位酪氨酸。
前述的一种羰基还原酶的突变体,突变体为在羰基还原酶EbSDR8氨基酸序列的基础上在153位丝氨酸突变为缬氨酸;核苷酸序列如SEQ ID No.3所示。
前述的一种羰基还原酶的突变体,突变体为在羰基还原酶EbSDR8氨基酸序列的基础上在188位酪氨酸突变为丙氨酸,核苷酸序列如SEQ ID No.5所示。
前述的一种羰基还原酶的突变体,突变体为在羰基还原酶EbSDR8氨基酸序列的基础上在202位酪氨酸突变为甲硫氨酸,核苷酸序列如SEQ ID No.7所示。
前述的一种羰基还原酶的突变体,突变体的制备方法包括:培养重组表达转化体,诱导获得重组羰基还原酶突变体蛋白。
前述的一种羰基还原酶的重组表达载体,包括:羰基还原酶EbSDR8突变体的编码基因。
前述的一种羰基还原酶的重组表达载体,为连接EbSDR8突变体的编码基因的重组表达载体。
一种羰基还原酶的突变体、重组表达载体在生产手性醇中的应用,羰基还原酶EbSDR8的突变体为在羰基还原酶EbSDR8氨基酸序列的基础上含有如下几个位点的单点突变或多点组合突变:94位甘氨酸、145位组氨酸、153位丝氨酸、188位酪氨酸、199位丝氨酸和202位酪氨酸;
羰基还原酶EbSDR8氨基酸序列如SEQ ID No.2所示;
羰基还原酶的EbSDR8突变体以游离酶、固定化酶以及重组游离细胞的形式催化潜手性酮不对称还原光学活性手性醇。
前述的一种羰基还原酶的突变体、重组表达载体在生产手性醇中的应用,生产手性醇的具体方法为:以潜手性酮为底物,加入所述羰基还原酶EbSDR8的突变体或者重组表达载体,在20~50℃下,于pH 5.5~10.5的缓冲液构成的转化反应体系中反应,反应完全后,将反应液分离纯化得到相应产物(R)-(3)-氯-(1)-苯丙醇。
本发明的有益之处在于:
本发明所提供的羰基还原酶EbSDR8突变体与野生型相比较,具有更优良的催化活性,EbSDR8突变体催化合成手性醇的浓度在100~300g/L,转化率达70%~99.9%,产率70%~98%,光学纯度大于99%,催化剂易于制备、反应条件温和、底物适应性广、环境友好,且其重组细胞能够在不外加任何辅酶的含异丙醇反应体系中高效催化潜手性酮的不对称还原,具有很好的工业化应用开发前景。
附图说明
图1是本发明的短链脱氢酶EbSDR8及其突变体分离纯化后SDS-PAGE图。
具体实施方式
以下结合附图和具体实施例对本发明作具体的介绍。
一种羰基还原酶的突变体,突变体为在羰基还原酶EbSDR8氨基酸序列的基础上含有如下几个位点的单点突变或多点组合突变:145位组氨酸(His145)、153位丝氨酸(Ser153)、188位酪氨酸(Tyr188)、199位丝氨酸(Ser199)和202位酪氨酸(Tyr202);这几个氨基酸均处于底物结合位点附近,这些氨基酸的改变一方面可能改变底物结合的亲和性,使酶活得到提高,另一方面可以提高底物结合的方向性,使酶的特异性提高。
羰基还原酶EbSDR8的核苷酸序列如序列表中SEQ ID No.1所示,专利公开号CN105316250A。羰基还原酶EbSDR8的氨基酸序列如SEQ ID No.2所示。
一种羰基还原酶的突变体包括如下实施例:
实施例1,将如序列表中SEQ ID No.2所示氨基酸序列的第94位的甘氨酸替换为丙氨酸、第153位的丝氨酸替换为亮氨酸、第188位的酪氨酸替换为丙氨酸;
实施例2,将如序列表中SEQ ID No.2所示氨基酸序列的第94位的甘氨酸替换为丙氨酸、第188位的酪氨酸替换为丙氨酸,第153位的丝氨酸替换为异亮氨酸;
实施例3,将如序列表中SEQ ID No.2所示氨基酸序列的第94位的甘氨酸替换为丙氨酸、第153位的丝氨酸替换为亮氨酸、第188位的酪氨酸替换为丙氨酸,第202位的酪氨酸替换为甲硫氨酸;
实施例4,将如序列表中SEQ ID No.2所示氨基酸序列的第94位的甘氨酸替换为丙氨酸、第153位的丝氨酸替换为亮氨酸、第188位的酪氨酸替换为丙氨酸,第145位的组氨酸替换为丙氨酸;
实施例5,将如序列表中SEQ ID No.2所示氨基酸序列的第94位的甘氨酸替换为丙氨酸、第153位的丝氨酸替换为亮氨酸、第188位的酪氨酸替换为丙氨酸,第145位的组氨酸替换为丙氨酸,第202位的酪氨酸替换为甲硫氨酸;
实施例6,将如序列表中SEQ ID No.2所示氨基酸序列的第94位的甘氨酸替换为丙氨酸、第153位的丝氨酸替换为异亮氨酸、第188位的酪氨酸替换为丙氨酸,第202位的酪氨酸替换为甲硫氨酸;
实施例7,将如序列表中SEQ ID No.2所示氨基酸序列的第94位的甘氨酸替换为丙氨酸、第188位的酪氨酸替换为丙氨酸,第153位的丝氨酸替换为异亮氨酸,第202位的酪氨酸替换为甲硫氨酸,第199位的丝氨酸替换为天冬氨酸;
任何对上述突变体氨基酸序列中氨基酸经过缺失、插入或替换一个或几个氨基酸且具有短链脱氢酶活性的,仍属于本发明的保护范围。
一种羰基还原酶EbSDR8突变体的编码基因,其中突变体G94A/S153L/Y188A核苷酸序列如序列表中SEQ ID NO.3所示,其编码的氨基酸序列如序列表SEQ ID NO.4所示;突变体G94A/S153I/Y188A核苷酸序列如序列表中SEQ ID NO.5所示,其编码的氨基酸序列如序列表SEQ ID NO.6所示;突变体G94A/S153L/Y188A/Y202M核苷酸序列如序列表中SEQ IDNO.7所示,其编码的氨基酸序列如序列表
SEQ ID NO.8所示;突变体G94A/S153L/Y188A/H145A核苷酸序列如序列表中SEQID NO.9所示,其编
码的氨基酸序列如序列表SEQ ID NO.10所示;突变体G94A/S153L/Y188A/H145A/Y202M核苷酸序列如
序列表中SEQ ID NO.11所示,其编码的氨基酸序列如序列表SEQ ID NO.12所示;突变体G94A/Y188A/S153I/Y202M核苷酸序列如序列表中SEQ ID NO.13所示,其编码的氨基酸序列如序列表SEQ ID NO.14所示;突变体G94A/Y188A/S153I/Y202M/S199D核苷酸序列如序列表中SEQ ID NO.15所示,其编码的氨基酸序列如序列表SEQ ID NO.16所示。
一种羰基还原酶EbSDR8突变体的重组表达载体。这些重组载体可通过本领域常规方法将本发明的羰基还原酶突变体核苷酸序列连接于各种载体上构建而成。所述载体可为本领域常规的各种载体,如各种质粒、噬菌体或病毒载体等,优选pET-30a。
作为一种重组表达载体的应用,可通过将本发明的重组表达载体转化至宿主微生物中获得基因工程菌。宿主微生物可为本领域常规的各种宿主微生物,只要满足重组表达载体可以稳定自我复制且所携带的本发明的羰基还原酶突变体基因可以有效表达。本发明优选大肠杆菌,更优选为大肠杆菌E.coli BL21(DE3)。
一种重组羰基还原酶突变体的制备方法,包括如下步骤:培养本发明的重组表达转化体,诱导获得重组羰基还原酶突变体蛋白。其中,所述的培养重组表达转化子所用的培养基可以是本领域可使转化子生长并产生本发明的羰基还原酶突变体蛋白的培养基,优选LB培养基:蛋白胨10g/L,酵母粉5g/L,氯化钠10g/L,pH 7.2。培养方法和培养条件没有特殊限制,只要使转化子能够生长并产生羰基还原酶突变体蛋白即可。优选下述方法:将本发明涉及的重组大肠杆菌接种至含卡那霉素的LB培养基中培养,当培养液的光密度OD600达到0.5~0.8时,在终浓度为0.1~1.0mM异丙基-β-D-硫代吡喃半乳糖苷(IPTG)的诱导下,即可高效表达本发明的重组羰基还原酶突变体蛋白。
得到催化生产手性醇的催化剂的制备方法如下所示:
(1)斜面培养:将含大肠杆菌羰基还原酶EbSDR8突变体编码基因的重组基因工程菌接种至含50μg/ml卡那霉素的斜面培养基,在37℃培养8-16h,获得斜面菌体;所述斜面培养基终浓度组成为:蛋白胨10g/L,酵母提取物5g/L,氯化钠10g/L,1.5%琼脂,溶剂为去离子水,pH7.2。使用前加入50μg/ml卡那霉素。
(2)种子培养:将斜面菌体接种至种子培养基,在37℃培养8~10h,获得种子液;所述种子培养基终浓度组成为:蛋白胨10g/L,酵母提取物5g/L,氯化钠10g/L,50μg/ml卡那霉素,溶剂为去离子水,pH7.2。
(3)发酵培养:将种子液以体积浓度10%的接种量接种至无菌的装有3L发酵培养基的机械搅拌通风通用式发酵罐中,37℃发酵培养14h后将已灭菌的终浓度为15g/L乳糖分批添加到发酵罐中于26℃下诱导培养。待培养12~24h后OD600达到100-150,放罐收集湿菌体。所述发酵培养基终浓度组成为:,酵母浸粉15g/L,NaCl 10g/L,甘油10g/L,(NH4)2SO42.5g/L,NaCl 3g,柠檬酸2.1g,葡萄糖2g,NaH2PO4·2H2O 2.24g/L,K2HPO4·3H2O 4g/L,MgSO4·7H2O 0.49g/L,FeSO4 0.3g,溶剂为去离子水。
本发明的应用:羰基还原酶的EbSDR8突变体或其基因工程菌可以游离酶、固定化酶以及重组游离细胞的形式催化潜手性酮不对称还原光学活性手性醇的应用。
应用方法为:以潜手性酮为底物,加入上述突变体或基因工程菌,在20~50℃下,于pH 5.5~10.5的缓冲液构成的转化反应体系中反应,反应条件可按本领域所用的常规条件进行选择;转化体系中底物初始浓度为10~2000mmol/L,反应体系中菌体的质量用量以菌体湿重计为10~500g/L;体系中采用的有机溶剂为甲醇、乙醇、异丙醇中的一种或多种,优选的为异丙醇;反应体系中异丙醇的浓度为10~100%;反应完全后,将反应液分离纯化得到相应产物(R)-(3)-氯-(1)-苯丙醇;反应液分离纯化的方法为:取上清液用等体积的乙酸乙酯萃取,有机层即为含相应手性醇的粗品,将粗品提纯即获得相应手性醇。粗品提纯的方法为本领域公知技术,通常为有机溶剂萃取、色谱分离和吸附分离等。
以下通过实验验证本发明的有益效果;
实验准备:
步骤一:突变体的构建;
以含有突变点的寡核苷酸片段为引物(表1),采用QuickChangeTM方法(Stratagene,La Jolla,CA)扩增含有羰基还原酶基因的pET-30a重组质粒。
表1突变体构建引物
备注:下划线标示为突变位点
PCR反应体系:5×PrimerSTAR buffer(Mg2+plus),5μL;dNTPs(各2.5mM),2.0μL;上游引物(10μM),1.0μL;下游引物(10μM),1.0μL;重组质粒模板,15ng;PrimerSTARpolymeraseTM HS(2.5U/μL),0.5μL;加ddH2O至总体积为25μL。
PCR程序:(1)98℃,1min;(2)98℃,10s;(3)55℃,10s;(4)72℃,7min。步骤(2)-(4)循环20次后冷却至4℃。
PCR产物经清洗后,利用特异性识别甲基化位点的限制性内切酶DpnI进行消化以降解模板质粒。酶切反应体系及条件:17.0μL经清洗处理的PCR产物,2.0μL10×缓冲液,1.0μL限制性内切酶DpnI,37℃保温1h。
将上述经酶切处理的PCR产物转化至大肠杆菌BL21(DE3)中,得到相应的重组大肠杆菌,涂布于含卡那霉素的平板,37℃下培养过夜,随机挑取克隆进行菌落PCR鉴定和测序验证,结果表明含有羰基还原酶突变体基因的重组表达载体成功转化至表达宿主E.coliBL21(DE3)中。最终获得突变体G94A/S153L/Y188A、G94A/Y188A/S153I、G94A/S153L/Y188A/Y202M、G94A/S153L/Y188A/H145A、G94A/S153L/Y188A/H145A/202M、G94A/Y188A/S153I/Y202M、G94A/Y188A/S153I/Y202M/S199D。核苷酸序列测序结果分别如序列表中SEQ IDNO.3、SEQ ID NO.5、SEQ ID NO.7、SEQ ID NO.9、SEQ ID NO.11、SEQ ID NO.13和SEQ IDNO.15所示,相应编码蛋白质氨基酸序列如序列表中SEQ ID NO.4、SEQ ID NO.6、SEQ IDNO.8、SEQ ID NO.10、SEQ ID NO.12、SEQ ID NO.14、SEQ ID NO.16所示。
步骤二:羰基还原酶突变体的诱导表达;
将步骤一构建的工程菌接种至50μg/mL卡那霉素的LB培养基中,37℃,220rpm培养过夜,再以1%接种量(v/v)接种至含50μg/mL卡那霉素的LB培养基中,37℃,220rpm培养至菌体浓度OD600至0.6左右,加入终浓度为0.1mM的异丙基硫代半乳糖苷(IPTG),25℃诱导培养10~16小时后,4℃、4000rpm离心10min收集菌体,于-80℃储藏备用。
步骤三:短链脱氢酶突变体的分离纯化;
步骤二收集的菌体细胞悬浮于10mL Na2HPO4-NaH2O4缓冲液(100mM,pH 8.0)中,振荡摇匀后置超声波下破碎(有效时间8min)。破碎液于12000rpm离心10min去除细胞碎片,收集上清液(粗酶液)用于酶的后续的分离纯化。纯化柱为Ni-NTA,装柱体积为5mL,先用上样平衡缓冲液(20mM磷酸钠,500mM NaCl和20mM咪唑,pH 7.4)平衡Ni-NTA柱,以5mL/min的速率上样粗酶液,用上样平衡缓冲液洗脱以除去未吸附的蛋白,最后用洗脱缓冲液(20mM磷酸钠,500mM NaCl和20mM咪唑,pH 7.4)洗脱收集目标蛋白。酶液用脱盐柱进行脱盐,脱盐缓冲液为Na2HPO4-NaH2PO4(100mM,pH 7.5)缓冲液,所得纯酶液于4℃贮存备用。纯化后的酶液用SDS-PAGE进行分析。
图1为羰基还原酶EbSDR8及其突变体分离纯化后SDS-PAGE图。其中Lane1,Marker;lane 2,EbSDR8;lane 3,G94A/S153L/Y188A;lane 4,G94A/Y188A/S153I;lane 5,G94A/S153L/Y188A/Y202M;lane 6,G94A/S153L/Y188A/H145A;lane 7,G94A/S153L/Y188A/H145A/Y202M;lane 8,G94A/Y188A/S153I/Y202M;lane 9,G94A/Y188A/S153I/Y202M/S199D。结果表明,经Ni-NTA亲和层析,得到电泳纯的重组羰基还原酶EbSDR8及其突变体。
步骤四:羰基还原酶突变体的发酵罐培养;
步骤一构建的工程菌接种至50μg/mL卡那霉素的LB培养基中,37℃,220rpm培养12h后,测OD,没有杂菌生长良好,接入一级种子液(3L),种子液中接种量2%,卡那霉素50mg/L加1‰,摇床37℃,220r/min培养12h,测OD,然后接入250L小罐。37℃,培养至测得OD达到15时,加入乳糖进行诱导12h后,利用管式离心机离心收集菌体备用。
步骤四中所用发酵培养基为本领域所熟知的可使突变体生长并产生本发明羰基还原酶突变体蛋白的培养基。
实验一:实验验证EbSDR8及其突变体的催化效率;
反应体系为(10.0ml):4.0mL异丙醇、6.0mL Na2HPO4-NaH2PO4(100mM,pH 7.5)缓冲液、适量纯酶。底物为(3)-氯-(1)-苯丙酮。野生型EbSDR8及其突变体催化相应底物效率和立体选择性如表2所示。
表2 EbSDR8及其突变体催化效率
实验二:羰基还原酶EbSDR8及其突变体的动力学参数;
在标准条件下,通过改变反应体系中底物的浓度进行酶活的测定,根据双倒数作图法计算相应的动力学常数。动力学常数计算中所用底物及其浓度如下:(3)-氯-(1)-苯丙酮(0~50.0mM),野生型EbSDR8及其突变体催化相应底物的表观动力学参数如表3所示。
表3 EbSDR8及其突变体不对称还原潜手性酮表观动力学参数
实验三:羰基还原酶EbSDR8及其突变体G94A/Y188A/S153I/Y202M/S199D转化(3)-氯-(1)-苯丙酮
反应体系(10.0mL):
0.5g实施例4中的湿菌体细胞,不同浓度的(3)-氯-(1)-苯丙酮,6.0mL Na2HPO4-NaH2PO4(100mM,pH7.5)缓冲液,4.0mL异丙醇,于37℃,220rpm条件下反应。野生型EbSDR8重组大肠杆菌全细胞催化明显低于突变体G94A/Y188A/S153I/Y202M/S199D,反应3h、6h、9h、12h后,EbSDR8重组大肠杆菌全细胞反应几乎无任何催化效果;突变体G94A/Y188A/S153I/Y202M/S199D重组大肠杆菌全细胞产率分别为60.36%、78.00%、81.14%、85.45%,且ee值高达99%;当底物浓度达到1000mM时,该G94A/Y188A/S153I/Y202M/S199D突变体全细胞仍能有效催化反应进行,反应24小时后,产率达90%,具体实验结果如表4所示。表明该全细胞生物催化剂具有广阔的工业化应用前景。
表4实施例7中催化不同底物[(3)-氯-(1)-苯丙酮]浓度不对称还原结果
实验结果分析:本发明所提供的羰基还原酶EbSDR8突变体与野生型相比较,具有更优良的催化活性,EbSDR8突变体催化合成手性醇的浓度在100~300g/L,转化率达70%~99.9%,产率70%~98%,光学纯度大于99%,催化剂易于制备、反应条件温和、底物适应性广、环境友好,且其重组细胞能够在不外加任何辅酶的含异丙醇反应体系中高效催化潜手性酮的不对称还原,具有很好的工业化应用开发前景。
以上显示和描述了本发明的基本原理、主要特征和优点。本行业的技术人员应该了解,上述实施例不以任何形式限制本发明,凡采用等同替换或等效变换的方式所获得的技术方案,均落在本发明的保护范围内。
序列表
<110> 浙江大学
<120> 一种羰基还原酶的突变体、重组表达载体及其在生产手性醇中的应用
<141> 2019-08-29
<160> 30
<170> SIPOSequenceListing 1.0
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gattatctag aattattggt aacgaagcat ccaataggtc gtttaggaca gcctgaggaa 660
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Ala Ile Ala Pro Ala Gly Val Pro Thr Gly Cys Leu Thr Gly Leu Gly
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Ala Met Gly Leu Ala Gly Gly Gly Ser Ile Val Ala Met Ala Ser Ile
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His Gly Thr Val Ala Ala Pro Met Ile Ser Ala Thr Thr Ser Ala Leu
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tatggaaaat tggatattgc atgtaataat gccggaatag caggtccggc tgaattgaca 300
gaagattatc ctttagacgg ttggaaaaaa gtgattgata tcaacttcaa tggtgttttt 360
tatggatgta aatatcaatt gcaggcaatg gagaaaaatg gtggaggttc tattgtgaat 420
atggcctcaa tacacggtac agtagcggct cctatgttgt ctgcttatac ttctgcgaaa 480
catggtgttg tgggacttac aaaaaatatt ggagcagagt acggttcaaa aaatatccga 540
tgcaatgctg tgggacctgg tgcgcgcatg acaccattgt tgtcgaataa tttgagcgca 600
gatatgctag aattattggt aacgaagcat ccaataggtc gtttaggaca gcctgaggaa 660
gttgcagaat tagttttatt tctaagttct gataaagcgt cttttatgac aggaggttac 720
tatcttgtag atggaggata tacagcagtt taa 753
<210> 8
<211> 250
<212> PRT
<213> Artificial Sequence
<400> 8
Met Ser Ile Leu Leu Ala Leu Val Ala Ile Val Thr Gly Ala Ser Ser
1 5 10 15
Gly Ile Gly Leu Ala Val Ala Gly Leu Thr Ala Leu Gly Gly Ala Leu
20 25 30
Val Val Val Ser Ala Ile Ala Gly Gly Ala Gly Leu Gly Val Val Gly
35 40 45
Gly Ile Leu Leu Ala Gly Gly Gly Ala Ile Pro Pro Leu Ala Ala Thr
50 55 60
Ser Ser Pro Gly Gly Ala Gly Ala Leu Val Leu Leu Ala Val Gly Val
65 70 75 80
Thr Gly Leu Leu Ala Ile Ala Cys Ala Ala Ala Gly Ile Gly Ala Pro
85 90 95
Ala Gly Leu Thr Gly Ala Thr Pro Leu Ala Gly Thr Leu Leu Val Ile
100 105 110
Ala Ile Ala Pro Ala Gly Val Pro Thr Gly Cys Leu Thr Gly Leu Gly
115 120 125
Ala Met Gly Leu Ala Gly Gly Gly Ser Ile Val Ala Met Ala Ser Ile
130 135 140
His Gly Thr Val Ala Ala Pro Met Ser Ser Ala Thr Thr Ser Ala Leu
145 150 155 160
His Gly Val Val Gly Leu Thr Leu Ala Ile Gly Ala Gly Thr Gly Ser
165 170 175
Leu Ala Ile Ala Cys Ala Ala Val Gly Pro Gly Ala Ile Met Thr Pro
180 185 190
Leu Leu Ser Ala Ala Leu Ser Ala Ala Met Leu Gly Leu Leu Val Thr
195 200 205
Leu His Pro Ile Gly Ala Leu Gly Gly Pro Gly Gly Val Ala Gly Leu
210 215 220
Val Leu Pro Leu Ser Ser Ala Leu Ala Ser Pro Met Thr Gly Gly Thr
225 230 235 240
Thr Leu Val Ala Gly Gly Thr Thr Ala Val
245 250
<210> 9
<211> 753
<212> DNA
<213> Artificial Sequence
<400> 9
atgtcaatat taaaagataa ggtagctatt gtgacaggag caagttccgg aataggtaaa 60
gctgttgcag aattgtatgc aaaagaaggt gcaaaagttg ttgtttctga tatcgatgaa 120
gaaagaggaa aagaagttgt agaacagatt aaaaaaaatg gaggagaagc catctttttc 180
aaagcggata catcatctcc cgaagagaat gaagcgttgg taaaaaaagc agttgaagtg 240
tatggaaaat tggatattgc atgtaataat gccggaatag caggtccggc tgaattgaca 300
gaagattatc ctttagacgg ttggaaaaaa gtgattgata tcaacttcaa tggtgttttt 360
tatggatgta aatatcaatt gcaggcaatg gagaaaaatg gtggaggttc tattgtgaat 420
atggcctcaa tagcgggtac agtagcggct cctatgttgt ctgcttatac ttctgcgaaa 480
catggtgttg tgggacttac aaaaaatatt ggagcagagt acggttcaaa aaatatccga 540
tgcaatgctg tgggacctgg tgcgcgcatg acaccattgt tgtcgaataa tttgagcgca 600
gattatctag aattattggt aacgaagcat ccaataggtc gtttaggaca gcctgaggaa 660
gttgcagaat tagttttatt tctaagttct gataaagcgt cttttatgac aggaggttac 720
tatcttgtag atggaggata tacagcagtt taa 753
<210> 10
<211> 250
<212> PRT
<213> Artificial Sequence
<400> 10
Met Ser Ile Leu Leu Ala Leu Val Ala Ile Val Thr Gly Ala Ser Ser
1 5 10 15
Gly Ile Gly Leu Ala Val Ala Gly Leu Thr Ala Leu Gly Gly Ala Leu
20 25 30
Val Val Val Ser Ala Ile Ala Gly Gly Ala Gly Leu Gly Val Val Gly
35 40 45
Gly Ile Leu Leu Ala Gly Gly Gly Ala Ile Pro Pro Leu Ala Ala Thr
50 55 60
Ser Ser Pro Gly Gly Ala Gly Ala Leu Val Leu Leu Ala Val Gly Val
65 70 75 80
Thr Gly Leu Leu Ala Ile Ala Cys Ala Ala Ala Gly Ile Gly Ala Pro
85 90 95
Ala Gly Leu Thr Gly Ala Thr Pro Leu Ala Gly Thr Leu Leu Val Ile
100 105 110
Ala Ile Ala Pro Ala Gly Val Pro Thr Gly Cys Leu Thr Gly Leu Gly
115 120 125
Ala Met Gly Leu Ala Gly Gly Gly Ser Ile Val Ala Met Ala Ser Ile
130 135 140
Ala Gly Thr Val Ala Ala Pro Met Ser Ser Ala Thr Thr Ser Ala Leu
145 150 155 160
His Gly Val Val Gly Leu Thr Leu Ala Ile Gly Ala Gly Thr Gly Ser
165 170 175
Leu Ala Ile Ala Cys Ala Ala Val Gly Pro Gly Ala Ile Met Thr Pro
180 185 190
Leu Leu Ser Ala Ala Leu Ser Ala Ala Thr Leu Gly Leu Leu Val Thr
195 200 205
Leu His Pro Ile Gly Ala Leu Gly Gly Pro Gly Gly Val Ala Gly Leu
210 215 220
Val Leu Pro Leu Ser Ser Ala Leu Ala Ser Pro Met Thr Gly Gly Thr
225 230 235 240
Thr Leu Val Ala Gly Gly Thr Thr Ala Val
245 250
<210> 11
<211> 753
<212> DNA
<213> Artificial Sequence
<400> 11
atgtcaatat taaaagataa ggtagctatt gtgacaggag caagttccgg aataggtaaa 60
gctgttgcag aattgtatgc aaaagaaggt gcaaaagttg ttgtttctga tatcgatgaa 120
gaaagaggaa aagaagttgt agaacagatt aaaaaaaatg gaggagaagc catctttttc 180
aaagcggata catcatctcc cgaagagaat gaagcgttgg taaaaaaagc agttgaagtg 240
tatggaaaat tggatattgc atgtaataat gccggaatag caggtccggc tgaattgaca 300
gaagattatc ctttagacgg ttggaaaaaa gtgattgata tcaacttcaa tggtgttttt 360
tatggatgta aatatcaatt gcaggcaatg gagaaaaatg gtggaggttc tattgtgaat 420
atggcctcaa tagcgggtac agtagcggct cctatgttgt ctgcttatac ttctgcgaaa 480
catggtgttg tgggacttac aaaaaatatt ggagcagagt acggttcaaa aaatatccga 540
tgcaatgctg tgggacctgg tgcgcgcatg acaccattgt tgtcgaataa tttgagcgca 600
gatatgctag aattattggt aacgaagcat ccaataggtc gtttaggaca gcctgaggaa 660
gttgcagaat tagttttatt tctaagttct gataaagcgt cttttatgac aggaggttac 720
tatcttgtag atggaggata tacagcagtt taa 753
<210> 12
<211> 250
<212> PRT
<213> Artificial Sequence
<400> 12
Met Ser Ile Leu Leu Ala Leu Val Ala Ile Val Thr Gly Ala Ser Ser
1 5 10 15
Gly Ile Gly Leu Ala Val Ala Gly Leu Thr Ala Leu Gly Gly Ala Leu
20 25 30
Val Val Val Ser Ala Ile Ala Gly Gly Ala Gly Leu Gly Val Val Gly
35 40 45
Gly Ile Leu Leu Ala Gly Gly Gly Ala Ile Pro Pro Leu Ala Ala Thr
50 55 60
Ser Ser Pro Gly Gly Ala Gly Ala Leu Val Leu Leu Ala Val Gly Val
65 70 75 80
Thr Gly Leu Leu Ala Ile Ala Cys Ala Ala Ala Gly Ile Gly Ala Pro
85 90 95
Ala Gly Leu Thr Gly Ala Thr Pro Leu Ala Gly Thr Leu Leu Val Ile
100 105 110
Ala Ile Ala Pro Ala Gly Val Pro Thr Gly Cys Leu Thr Gly Leu Gly
115 120 125
Ala Met Gly Leu Ala Gly Gly Gly Ser Ile Val Ala Met Ala Ser Ile
130 135 140
Ala Gly Thr Val Ala Ala Pro Met Ser Ser Ala Thr Thr Ser Ala Leu
145 150 155 160
His Gly Val Val Gly Leu Thr Leu Ala Ile Gly Ala Gly Thr Gly Ser
165 170 175
Leu Ala Ile Ala Cys Ala Ala Val Gly Pro Gly Ala Ile Met Thr Pro
180 185 190
Leu Leu Ser Ala Ala Leu Ser Ala Ala Met Leu Gly Leu Leu Val Thr
195 200 205
Leu His Pro Ile Gly Ala Leu Gly Gly Pro Gly Gly Val Ala Gly Leu
210 215 220
Val Leu Pro Leu Ser Ser Ala Leu Ala Ser Pro Met Thr Gly Gly Thr
225 230 235 240
Thr Leu Val Ala Gly Gly Thr Thr Ala Val
245 250
<210> 13
<211> 753
<212> DNA
<213> Artificial Sequence
<400> 13
atgtcaatat taaaagataa ggtagctatt gtgacaggag caagttccgg aataggtaaa 60
gctgttgcag aattgtatgc aaaagaaggt gcaaaagttg ttgtttctga tatcgatgaa 120
gaaagaggaa aagaagttgt agaacagatt aaaaaaaatg gaggagaagc catctttttc 180
aaagcggata catcatctcc cgaagagaat gaagcgttgg taaaaaaagc agttgaagtg 240
tatggaaaat tggatattgc atgtaataat gccggaatag caggtccggc tgaattgaca 300
gaagattatc ctttagacgg ttggaaaaaa gtgattgata tcaacttcaa tggtgttttt 360
tatggatgta aatatcaatt gcaggcaatg gagaaaaatg gtggaggttc tattgtgaat 420
atggcctcaa tacacggtac agtagcggct cctatgatct ctgcttatac ttctgcgaaa 480
catggtgttg tgggacttac aaaaaatatt ggagcagagt acggttcaaa aaatatccga 540
tgcaatgctg tgggacctgg tgcgcgcatg acaccattgt tgtcgaataa tttgagcgca 600
gatatgctag aattattggt aacgaagcat ccaataggtc gtttaggaca gcctgaggaa 660
gttgcagaat tagttttatt tctaagttct gataaagcgt cttttatgac aggaggttac 720
tatcttgtag atggaggata tacagcagtt taa 753
<210> 14
<211> 250
<212> PRT
<213> Artificial Sequence
<400> 14
Met Ser Ile Leu Leu Ala Leu Val Ala Ile Val Thr Gly Ala Ser Ser
1 5 10 15
Gly Ile Gly Leu Ala Val Ala Gly Leu Thr Ala Leu Gly Gly Ala Leu
20 25 30
Val Val Val Ser Ala Ile Ala Gly Gly Ala Gly Leu Gly Val Val Gly
35 40 45
Gly Ile Leu Leu Ala Gly Gly Gly Ala Ile Pro Pro Leu Ala Ala Thr
50 55 60
Ser Ser Pro Gly Gly Ala Gly Ala Leu Val Leu Leu Ala Val Gly Val
65 70 75 80
Thr Gly Leu Leu Ala Ile Ala Cys Ala Ala Ala Gly Ile Gly Ala Pro
85 90 95
Ala Gly Leu Thr Gly Ala Thr Pro Leu Ala Gly Thr Leu Leu Val Ile
100 105 110
Ala Ile Ala Pro Ala Gly Val Pro Thr Gly Cys Leu Thr Gly Leu Gly
115 120 125
Ala Met Gly Leu Ala Gly Gly Gly Ser Ile Val Ala Met Ala Ser Ile
130 135 140
His Gly Thr Val Ala Ala Pro Met Ile Ser Ala Thr Thr Ser Ala Leu
145 150 155 160
His Gly Val Val Gly Leu Thr Leu Ala Ile Gly Ala Gly Thr Gly Ser
165 170 175
Leu Ala Ile Ala Cys Ala Ala Val Gly Pro Gly Ala Ile Met Thr Pro
180 185 190
Leu Leu Ser Ala Ala Leu Ser Ala Ala Met Leu Gly Leu Leu Val Thr
195 200 205
Leu His Pro Ile Gly Ala Leu Gly Gly Pro Gly Gly Val Ala Gly Leu
210 215 220
Val Leu Pro Leu Ser Ser Ala Leu Ala Ser Pro Met Thr Gly Gly Thr
225 230 235 240
Thr Leu Val Ala Gly Gly Thr Thr Ala Val
245 250
<210> 15
<211> 753
<212> DNA
<213> Artificial Sequence
<400> 15
atgtcaatat taaaagataa ggtagctatt gtgacaggag caagttccgg aataggtaaa 60
gctgttgcag aattgtatgc aaaagaaggt gcaaaagttg ttgtttctga tatcgatgaa 120
gaaagaggaa aagaagttgt agaacagatt aaaaaaaatg gaggagaagc catctttttc 180
aaagcggata catcatctcc cgaagagaat gaagcgttgg taaaaaaagc agttgaagtg 240
tatggaaaat tggatattgc atgtaataat gccggaatag caggtccggc tgaattgaca 300
gaagattatc ctttagacgg ttggaaaaaa gtgattgata tcaacttcaa tggtgttttt 360
tatggatgta aatatcaatt gcaggcaatg gagaaaaatg gtggaggttc tattgtgaat 420
atggcctcaa tacacggtac agtagcggct cctatgatct ctgcttatac ttctgcgaaa 480
catggtgttg tgggacttac aaaaaatatt ggagcagagt acggttcaaa aaatatccga 540
tgcaatgctg tgggacctgg tgcgcgcatg acaccattgt tgtcgaataa tttggacgca 600
gatatgctag aattattggt aacgaagcat ccaataggtc gtttaggaca gcctgaggaa 660
gttgcagaat tagttttatt tctaagttct gataaagcgt cttttatgac aggaggttac 720
tatcttgtag atggaggata tacagcagtt taa 753
<210> 16
<211> 250
<212> PRT
<213> Artificial Sequence
<400> 16
Met Ser Ile Leu Leu Ala Leu Val Ala Ile Val Thr Gly Ala Ser Ser
1 5 10 15
Gly Ile Gly Leu Ala Val Ala Gly Leu Thr Ala Leu Gly Gly Ala Leu
20 25 30
Val Val Val Ser Ala Ile Ala Gly Gly Ala Gly Leu Gly Val Val Gly
35 40 45
Gly Ile Leu Leu Ala Gly Gly Gly Ala Ile Pro Pro Leu Ala Ala Thr
50 55 60
Ser Ser Pro Gly Gly Ala Gly Ala Leu Val Leu Leu Ala Val Gly Val
65 70 75 80
Thr Gly Leu Leu Ala Ile Ala Cys Ala Ala Ala Gly Ile Gly Ala Pro
85 90 95
Ala Gly Leu Thr Gly Ala Thr Pro Leu Ala Gly Thr Leu Leu Val Ile
100 105 110
Ala Ile Ala Pro Ala Gly Val Pro Thr Gly Cys Leu Thr Gly Leu Gly
115 120 125
Ala Met Gly Leu Ala Gly Gly Gly Ser Ile Val Ala Met Ala Ser Ile
130 135 140
His Gly Thr Val Ala Ala Pro Met Ile Ser Ala Thr Thr Ser Ala Leu
145 150 155 160
His Gly Val Val Gly Leu Thr Leu Ala Ile Gly Ala Gly Thr Gly Ser
165 170 175
Leu Ala Ile Ala Cys Ala Ala Val Gly Pro Gly Ala Ile Met Thr Pro
180 185 190
Leu Leu Ser Ala Ala Leu Ala Ala Ala Met Leu Gly Leu Leu Val Thr
195 200 205
Leu His Pro Ile Gly Ala Leu Gly Gly Pro Gly Gly Val Ala Gly Leu
210 215 220
Val Leu Pro Leu Ser Ser Ala Leu Ala Ser Pro Met Thr Gly Gly Thr
225 230 235 240
Thr Leu Val Ala Gly Gly Thr Thr Ala Val
245 250
<210> 17
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 17
ggaatagcag gtccggctga attga 25
<210> 18
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 18
cggacctgct attccggcat tattac 26
<210> 19
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 19
gcctcaatag ccggtacagt agcggc 26
<210> 20
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 20
gtaccggcta ttgaggccat attcacaata g 31
<210> 21
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 21
cctatgttgt ctgcttatac ttctg 25
<210> 22
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 22
gcagacaaca taggagccgc tac 23
<210> 23
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 23
cctatgatct ctgcttatac ttctg 25
<210> 24
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 24
gcagagatca taggagccgc tac 23
<210> 25
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 25
gacctggtgc tatcatgaca ccattgttgt c 31
<210> 26
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 26
gtcatgatag caccaggtcc cacagcattg c 31
<210> 27
<211> 33
<212> DNA
<213> Artificial Sequence
<400> 27
tttggacgca gattatctag aattattggt aac 33
<210> 28
<211> 34
<212> DNA
<213> Artificial Sequence
<400> 28
gataatctgc gtccaaatta ttcgacaaca atgg 34
<210> 29
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 29
gcagatatgc tagaattatt ggtaa 25
<210> 30
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 30
aataattcta gcatatctgc gctca 25
Claims (1)
1.一种羰基还原酶突变体,其特征在于,在SEQ ID NO:2所示氨基酸序列的基础上,发生G94A/Y188A/S153I突变的突变体多肽具有羰基还原酶的活性。
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Citations (4)
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CN102618513A (zh) * | 2012-05-04 | 2012-08-01 | 华东理工大学 | 一种羰基还原酶、基因和突变体及在不对称还原羰基化合物中的应用 |
CN104099305A (zh) * | 2014-07-18 | 2014-10-15 | 华东理工大学 | 一种羰基还原酶突变体及其基因和应用 |
CN107586763A (zh) * | 2017-11-02 | 2018-01-16 | 杭州馨海生物科技有限公司 | 羰基还原酶突变体、载体、工程菌及其应用 |
CN109837254A (zh) * | 2019-03-28 | 2019-06-04 | 中国科学院成都生物研究所 | 一种热稳定性提高的羰基还原酶突变体 |
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CN102618513A (zh) * | 2012-05-04 | 2012-08-01 | 华东理工大学 | 一种羰基还原酶、基因和突变体及在不对称还原羰基化合物中的应用 |
CN104099305A (zh) * | 2014-07-18 | 2014-10-15 | 华东理工大学 | 一种羰基还原酶突变体及其基因和应用 |
CN107586763A (zh) * | 2017-11-02 | 2018-01-16 | 杭州馨海生物科技有限公司 | 羰基还原酶突变体、载体、工程菌及其应用 |
CN109837254A (zh) * | 2019-03-28 | 2019-06-04 | 中国科学院成都生物研究所 | 一种热稳定性提高的羰基还原酶突变体 |
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