CN115011569B - 一种老黄酶NemR-PS突变体及其在制备(S)-香茅醇中的应用 - Google Patents
一种老黄酶NemR-PS突变体及其在制备(S)-香茅醇中的应用 Download PDFInfo
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- CN115011569B CN115011569B CN202210616904.1A CN202210616904A CN115011569B CN 115011569 B CN115011569 B CN 115011569B CN 202210616904 A CN202210616904 A CN 202210616904A CN 115011569 B CN115011569 B CN 115011569B
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Abstract
本发明公开了一种老黄酶NemR‑PS突变体及其在制备(S)‑香茅醇中的应用，所述老黄酶NemR‑PS突变体是将SEQ ID NO.2所示氨基酸序列中第275位、第351位氨基酸进行单突变或双突变获得的。通过构建共表达老黄酶NemR‑PS突变体、醇脱氢酶YsADH和葡萄糖脱氢酶BmGDHM6的基因工程菌，以该基因工程菌作为催化剂，以恒速流加方式加入底物，一锅法多酶级联催化底物(E/Z)‑柠檬醛的二步还原，产物(S)‑香茅醇累积浓度达500mM，产物e.e.值>99％，反应时间仅需12h，没有中间产物和副产物残留。
Description
(一)技术领域
本发明属于生物催化领域，特别涉及老黄酶NemR-PS突变体及其在多酶级联催化还原(E/Z)-柠檬醛合成(S)-香茅醇中的应用。
(二)背景技术
香茅醇是一种重要的香精原料，具有一个手性碳原子。自然界中的香茅醇含有两种对映体，(R)-香茅醇和(S)-香茅醇。较(R)-香茅醇而言，(S)-香茅醇的香气更为幽雅。(S)-香茅醇也是重要的手性中间体，可合成全顺式的玫瑰醚。(S)-香茅醇还具有多种生物活性，包括体外的抗细菌和抗真菌作用。
工业上香茅醇的合成主要以柠檬醛、香叶醇或橙花醇为底物，通过催化剂催化加氢制备香茅醇。化学法加氢选择性低，产生的副产物较多。以(E/Z)-柠檬醛为底物时，(E)-柠檬醛和(Z)-柠檬醛的还原产物往往光学性质是互补的，因而产物的光学纯化不高。与化学法相比，生物法还原柠檬醛生成(S)-香茅醇，具有优异的化学选择性和对映体选择性。天津大学元英进通过强化代谢途径构建了发酵产香茅醇的重组酿酒酵母，该酵母发酵116h香茅醇产量达到8.30g/L。2021年，Ying等人通过构建老黄酶、醇脱氢酶和葡萄糖脱氢酶的多酶级联体系实现了一锅法还原(E/Z)-柠檬醛生成(S)-香茅醇。在400mM柠檬醛底物浓度下，催化反应36h时转化率为>99.5％。
为了提高多酶级联合成(S)-香茅醇的催化效率，来源于斯氏普罗威登斯菌Providencia stuartii老黄酶NemR-PS进行了分子改造，获得了活力提高的突变体NemR-PS-D275G/F351A。构建共表达老黄酶突变体NemR-PS-D275G/F351A、醇脱氢酶YsADH和葡萄糖脱氢酶BmGDHM6基因工程菌E.coli BL21(DE3)/pACYCDuet1-YsADH-BmGDHM6/pET28a-NemR-PS-D275G/F351A，以该菌为整细胞催化剂，采用恒速流加方式加入底物和辅底物，多酶级联催化还原(E/Z)-柠檬醛生成(S)-香茅醇，在12h内可累积500mM的光学纯(S)-香茅醇。目前，未见对基于NemR-PS的275位天冬氨酸和351位苯丙氨酸的活力改造报道，也未见利用老黄酶突变体NemR-PS-D275G/F351A用于合成(S)-香茅醇的报道。与专利文献(申请号：2021108203589；一种三酶共表达重组菌及其在(S)-香茅醇合成中的应用)相比，老黄酶突变体的应用以及底物和辅底物加入方式的改变，产物浓度从400mM提高到了500mM，并且所需的反应时间从36h缩短到了12h，显著地提高了催化效率。
(三)发明内容
本发明目的是提供一种老黄酶NemR-PS突变体及其在合成(S)-香茅醇中的应用，通过对源自斯氏普罗威登斯菌的老黄酶NemR-PS的分子改造，成功获得了高酶活力且保持严格(S)-对映体选择性老黄酶NemR-PS突变体，特别是突变体NemR-PS-D275G/F351A。同时采用多酶级联催化还原(E/Z)-柠檬醛合成(S)-香茅醇，通过共表达老黄酶突变体、醇脱氢酶和葡萄糖脱氢酶基因工程菌的运用，结合底物和辅底物加入方式的改变，显著提高了多酶级联还原(E/Z)-柠檬醛合成(S)-香茅醇的催化效率。
本发明采用的技术方案是：
本发明提供一种老黄酶NemR-PS突变体，所述老黄酶突变体是将SEQ ID NO.2所示氨基酸序列中第275位、第351位氨基酸进行单突变或双突变获得的。
优选，所述老黄酶NemR-PS突变体是将SEQ ID NO.2所示氨基酸序列中第275位天冬氨酸突变替换为甘氨酸，同时第351位苯丙氨酸突变替换为丙氨酸获得的，记为NemR-PSD275G/F351A。所述老黄酶突变体NemR-PS D275G/F351A的氨基酸序列为SEQ ID NO.4所示，编码基因核苷酸序列为SEQ ID NO.3所示。
本发明还提供一种所述老黄酶突变体编码基因、编码基因构建的重组载体以及重组载体构建的重组基因工程菌。所述含老黄酶突变体编码基因的重组载体及基因工程菌由如下方法得到：将老黄酶突变体编码基因插入pET28a载体的Nco I和Xho I限制性酶切位点，得到重组载体。以重组载体为模板，利用带有突变碱基的引物经过反向PCR扩增全质粒，得到的PCR产物经DpnⅠ酶消化甲基化，酶切产物转化至大肠杆菌E.coli BL21(DE3)中，即可得到含老黄酶突变体基因的基因工程菌(优选E.coli BL21(DE3)/pET28a-NemR-PS-D275G/F351A，其中含重组老黄酶突变体编码基因的质粒命名为pET28a-NemR-PS-D275G/F351A)。
本发明还提供一种老黄酶NemR-PS突变体在还原(E/Z)-柠檬醛合成(S)-香茅醇中的应用，所述应用为：以共表达老黄酶NemR-PS突变体、醇脱氢酶和葡萄糖脱氢酶的基因工程菌(优选E.coli BL21(DE3)/pACYCDuet-1-YsADH-BmGDHM6/pET28a-NemR-PS-D275G/F351A)经诱导表达获得的湿菌体为催化剂，以(E/Z)-柠檬醛为底物，以葡萄糖为辅底物，加入助溶剂和辅酶NADP+，以pH5.5～8.0的缓冲液为反应介质构成转化体系，在20～45℃、200～700rpm条件下转化反应12～24h(优选30℃、500rpm、12h)，获得含(S)-香茅醇的反应液，反应液分离纯化，获得(S)-香茅醇；所述助溶剂为异丙醇；所述醇脱氢酶优选醇脱氢酶YsADH编码基因(核苷酸序列如SEQ ID NO.5所示，氨基酸序列如SEQ ID NO.6所示)；所述葡萄糖脱氢酶优选葡萄糖脱氢酶BmGDHM6编码基因(核苷酸序列如SEQ ID NO.7所示，氨基酸序列如SEQ ID NO.8所示)。
进一步，所述转化体系中，催化剂加入量以转化体系体积计为10～100g/L(优选100g/L)；底物加入终浓度以转化体系体积计为300～500mM(优选300mM)；所述葡萄糖加入终浓度与底物加入终浓度的比值为0.5：1～5：1(优选3:1)；所述NADP+加入终浓度以转化体系体积计为0～1.0mM(0是指不添加，优选0.4mM)；所述助溶剂体积加入量以转化体系体积计为0.5～8％(优选4％)。
进一步，所述湿菌体按如下方法制备：将共表达老黄酶突变体、醇脱氢酶和葡萄糖脱氢酶的基因工程菌接种至含100μg/mL卡那霉素的LB液体培养基中，37℃、200rpm培养12h，获得种子液，将种子液以体积浓度2％的接种量接种至新鲜的含100μg/mL卡那霉素的LB液体培养基中，37℃、200rpm培养至OD600为0.6～0.8，再加入终浓度为0.5mM的IPTG，24℃诱导16h，获得诱导培养液，再将诱导培养液于4℃和8000rpm下离心10min，弃去上清液；然后用pH 8.0、50mM Tris-HCl缓冲液重悬菌体，于4℃和8000rpm下离心10min，弃去上清液，收集湿菌体。
本发明所述共表达老黄酶NemR-PS突变体、醇脱氢酶和葡萄糖脱氢酶的基因工程菌按如下方法构建：将醇脱氢酶YsADH编码基因(核苷酸序列如SEQ ID NO.5所示，氨基酸序列如SEQ ID NO.6所示)插入pACYCDuet-1载体上的第一克隆位点Nco I和Hind III之间，将葡萄糖脱氢酶BmGDHM6编码基因(核苷酸序列如SEQ ID NO.7所示，氨基酸序列如SEQ IDNO.8所示)插入pACYCDuet-1载体上的第二克隆位点Nde I和Xho I之间，得到重组载体pACYCDuet-1-YsADH-BmGDHM6；将重组载体pACYCDuet-1-YsADH-BmGDHM6导入宿主细胞E.coli BL21(DE3)，得到重组菌E.coli BL21(DE3)/pACYCDuet-1-YsADH-BmGDHM6；将携带老黄酶突变体编码基因的重组载体(优选pET28a-NemR-PS-D275G/F351A)导入基因工程菌E.coli BL21(DE3)/pACYCDuet-1-YsADH-BmGDHM6感受态细胞中，得到所述的三酶共表达基因工程菌(优选E.coli BL21(DE3)/pACYCDuet-1-YsADH-BmGDHM6/pET28a-NemR-PS-D275G/F351A)。
进一步，在反应过程中底物和辅底物的加入方式可以是一次加入或是恒速流加方式加入。所述恒速流加方式按如下方法进行：底物柠檬醛和辅底物葡萄糖分别通过恒速微量泵注入到反应体系中，底物和辅底物恒速流加速度分别为0.5mmol/h和1.5mmol/h(辅底物和底物的摩尔比为3:1)，恒速流加时间为10h，底物累计添加量为500mM，辅底物葡萄糖累计添加量为1500mM。
进一步，反应过程中利用自动滴定系统维持pH恒定，滴定所用的碱液为1M NaOH溶液。
进一步，所述反应液分离纯化方法为：反应液在12000rpm下离心2min，取上清，加入4倍反应液体积的乙酸乙酯，在200rpm和30℃条件下萃取1.5h，萃取结束后，在12000rpm下离心1min，取上层有机相；在上层有机相中加入无水硫酸钠去除水分后，减压蒸馏去除溶剂乙酸乙酯，得到产物(S)-香茅醇。
本发明利用利用共表达老黄酶突变体NemR-PS-D275G/F351A、醇脱氢酶和葡萄糖脱氢酶的基因工程菌作为整细胞催化剂，一锅法多酶级联催化(E/Z)-柠檬醛的两步还原生成(S)-香茅醇(图1)。
与现有技术相比，本发明的有益效果主要体现在：本发明筛选获得的所述的老黄酶突变体活力显著提高且保持严格的(S)-对映体选择性，特别是突变体NemR-PS D275G/F351A与野生型NemR-PS相比，催化效率提高了2.5倍。利用共表达老黄酶突变体NemR-PSD275G/F351A、醇脱氢酶和葡萄糖脱氢酶的基因工程菌催化(E/Z)-柠檬醛还原生成(S)-香茅醇，结合底物和辅底物加入方式的改变，有效地消除了副产物(橙花醇和香叶醇)和中间产物(S)-香茅醛，产物为光学纯的(S)-香茅醇，产物累积浓度高达500mM，所需反应时间仅为12h，反应时空得率提高了3.75倍。
(四)附图说明
图1为一锅法三酶级联催化(E/Z)-柠檬醛两步还原生成(S)-香茅醇的反应示意图。
图2为老黄酶NemR-PS及突变体纯酶液SDS-PAGE图；从左到右，泳道M，Blue plusII protein marker；泳道1，NemR-PS纯酶液，加粗条带为NemR-PS，其分子量大小为39kDa；泳道2，NemR-PS D275G/F351A纯酶液。
图3为实施例4上清液气相色谱图；(S)-香茅醛，20.091min；(R)-香茅醛，20.542min；橙花醇，27.196min；香茅醇，27.433min；香叶醇，28.433min；(E)-柠檬醛，28.796min；(Z)-柠檬醛，29.840min。
图4为实施例5老黄酶NemR-PS单点突变PCR产物的核酸电泳图，泳道M为2K PlusII DNA marker，单位为bp；泳道1-9对应的突变体依次为：F351A、D275A、F241A、W103A、Q242A、R143A、W276A、S272A和Y352A。
图5为实施例5老黄酶NemR-PS单点突变体粗酶液的SDS-PAGE图，泳道M为Blueplus II protein marker，单位为kDa；泳道1-9对应的突变体依次为：F351A、D275A、F241A、W103A、Q242A、R143A、W276A、S272A和Y352A。
图6为实施例6老黄酶NemR-PS的D275位点饱和突变PCR产物的核酸电泳图，泳道M为2K Plus II DNA marker，单位为bp；泳道1-20对应的突变体依次为：WT、D275A、D275G、D275T、D275R、D275I、D275P、D275Y、D275S、D275K、D275N、D275L、D275C、D275V、D275Q、D275E、D275F、D275W、D275H和D275M。
图7为实施例6老黄酶NemR-PS的D275位点饱和突变体粗酶液的SDS-PAGE图，泳道M为Blue plus II protein marker，单位为kDa；泳道1-20对应的突变体依次为：WT、D275A、D275T、D275R、D275I、D275P、D275Y、D275S、D275K、D275N、D275L、D275C、D275G、D275V、D275Q、D275E、D275W、D275F、D275H和D275M。
图8为实施例7老黄酶NemR-PS的F351位点迭代饱和突变PCR产物的核酸电泳图，泳道M为2K Plus II DNA marker，单位为bp；泳道1-19对应突变体依次为：F351A、F351V、F351W、F351M、F351C、F351P、F351Y、F351S、F351L、F351N、F351K、F351I、F351G、F351F、F351E、F351Q、F351T、F351H和F351R。
图9为实施例7老黄酶NemR-PS的F351位点迭代饱和突变体粗酶液的SDS-PAGE图，泳道M为Blue plus II protein marker，单位为kDa；泳道1-20对应的突变体依次为：M1(D275G/F351)、F351A、F351W、F351L、F351M、F351Q、F351Y、F351S、F351F、F351N、F351R、F351V、F351G、F351C、F351P、F351E、F351T、F351K、F351H、F351I。
图10为实施例9共表达老黄酶NemR-PS-D275G/F351A、醇脱氢酶YsADH和葡萄糖脱氢酶BmGDHM6基因工程菌所制得粗酶液的SDS-PAGE图。其中泳道M对应的是Protein Marker；泳道1是对照组，共表达老黄酶NemR-PS-D275G/F351A、醇脱氢酶YsADH和葡萄糖脱氢酶BmGDHM6基因工程菌未经诱导条件下制得的粗酶液；泳道2对应的是共表达老黄酶NemR-PS-D275G/F351A、醇脱氢酶YsADH和葡萄糖脱氢酶BmGDHM6基因工程菌经诱导培养所制得的粗酶液，老黄酶NemR-PS-D275G/F351A大小约39kDa、醇脱氢酶YsADH大小约37kDa和葡萄糖脱氢酶BmGDHM6大小约28kDa。
图11为实施例11一锅法催化(E/Z)-柠檬醛还原生成(S)-香茅醇的最适催化pH。
图12为实施例12一锅法催化(E/Z)-柠檬醛还原生成(S)-香茅醇的最适催化温度。
图13为实施例13一锅法催化(E/Z)-柠檬醛还原生成(S)-香茅醇的最适搅拌速度。
图14为实施例14一锅法催化(E/Z)-柠檬醛还原生成(S)-香茅醇的最适葡萄糖添加量。
图15为实施例15一锅法催化(E/Z)-柠檬醛还原生成(S)-香茅醇的最适助溶剂异丙醇添加量。
图16为实施例16一锅法催化(E/Z)-柠檬醛还原生成(S)-香茅醇的最适NADP+添加量。
图17为实施例17一锅法催化(E/Z)-柠檬醛还原生成(S)-香茅醇在400mM底物浓度下的反应进程；(S)-Citronellol代表(S)-香茅醇，(S)-Citronellal代表(S)-香茅醛，Nerol代表橙花醇，Geraniol代表香叶醇，Neral代表橙花醛，Geranial代表香叶醛。
图18为实施例18底物和辅底物恒速流加方式下一锅法催化(E/Z)-柠檬醛还原生成(S)-香茅醇的反应进程。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：
LB液体培养基组成：胰化蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，溶剂为水，用1M的NaOH调节pH值到7.0～7.5。在121℃高压灭菌20min，4℃储存。
实施例1：老黄酶NemR-PS编码基因、醇脱氢酶编码基因和葡萄糖脱氢酶编码基因的获取
利用已公开的来源于斯氏普罗威登斯菌(Providencia stuartii；Department ofInfectious Diseases,National Institute of Health Dr.Ricardo Jorge,Lisbon,Portugal)老黄酶NemR-PS编码基因(GenBank登录号为KNZ86848)，经过密码子优化后，人工合成(杭州擎科生物技术有限公司提供基因合成服务)该老黄酶NemR-PS编码基因，核苷酸序列和氨基酸序列分别如SEQ ID NO.1和SEQ ID NO.2所示。
SEQ ID NO.1
ATGAGCCAGAAAAAGCTGTTTACGCCGCTGAAAGTTGGTACCCTGACCGCACCGAATCGTATTTTTATGGCGCCGCTGACCCGCCTGCGTAGCATTGAACCGGGCGATATTCCGACGCCGCTGATGGGCGAATATTATCGCCAGCGTGCCACCGCAGGCCTGATTATTAGCGAAGCAACCCAGATTAGTGCACAGTCAAAAGGTTATGCAGGTGCACCAGGTCTGCATAGCGCAGAACAGATTGCAGCATGGAAAAAGATTACCTCAACCGTTCATGAAGCAGGTGGTCGTATTGCAGTACAGCTGTGGCATACGGGTCGTATTAGTCATGTTAGCCTGCAGCCGAACGGTCTGGCACCGGTTGCACCGTCAGCAATTTCAGCCGGCACACGTACCAGTCTGCGTGATGAAAATGGTCGTGCCATTCGTGTAGATACCAGCATGCCGCGTGCACTGGAAACCGAAGAAATTCCGGCAATTGTTAATGATTTTCGCCAGGCAGTTGCAAATGCCCGTGAAGCTGGTTTTGATATGGCTGAACTGCATGCAGCACATGGTTATCTGCTGCATCAGTTTCTGAGCCCGTCAGCAAATCATCGTACCGATCAGTATGGCGGTACCCGTGAAAATCGTGCACGTTTTCTGCTGGATGTTGTTGATGCAGTTTGTGCAGAATGGGGTAGCGAACATATTGGTATTCGTATTAGTCCGATTGGTACCTTTCAGAATACAGATAATGGTCCTAATGAAGTTGATGATGCTCTGTATCTGATTGAAGAACTGGATAAACGTCATATTGCATATCTGCATCTGAGCGAACCGGATTGGGCAGGTGGTCAGCCATATACCGATGATTTTCGTCAGAAAGTTCGTGAACGTTTTCATGGTGTTATTATTGGTGCAGGCGCATATACGACAGAAAAAGCAGAAAATCTGATTGAAAAAGGTCTGATTGATGCAGTTGCCTTTGGTCGTGATTTTATTGCGAATCCGGATCTGGTTGTGCGTCTGAAAAATAAAGCAGCACTGAACCCACAGCGTCCGGAAAGCTTTTATGGTGGTGGTGCCGAAGGTTATACCGATTATCCGAGCCTG.
SEQ ID NO.2
MSQKKLFTPLKVGTLTAPNRIFMAPLTRLRSIEPGDIPTPLMGEYYRQRATAGLIISEATQISAQSKGYAGAPGLHSAEQIAAWKKITSTVHEAGGRIAVQLWHTGRISHVSLQPNGLAPVAPSAISAGTRTSLRDENGRAIRVDTSMPRALETEEIPAIVNDFRQAVANAREAGFDMAELHAAHGYLLHQFLSPSANHRTDQYGGTRENRARFLLDVVDAVCAEWGSEHIGIRISPIGTFQNTDNGPNEVDDALYLIEELDKRHIAYLHLSEPDWAGGQPYTDDFRQKVRERFHGVIIGAGAYTTEKAENLIEKGLIDAVAFGRDFIANPDLVVRLKNKAALNPQRPESFYGGGAEGYTDYPSL.
利用已公开的来源于约克氏菌(Yokenella sp.)CCTCC NO.M2013099(已在专利申请2013101888839中公开)的醇脱氢酶YsADH编码基因(GenBank登录号为KF887947)，经过密码子优化后，人工合成(苏州金唯智生物技术有限公司提供基因合成服务)该醇脱氢酶编码基因，核苷酸序列和氨基酸序列分别如SEQ ID NO.5和SEQ ID NO.6所示。
SEQ ID NO.5
ATGTCTATTATAAAAAGCTATGCCGCAAAAGAGGCGGGCAGCGAACTCGAACTTTACGAATATGATGCCGGTGAACTCAGGCCGGAAGATGTCGAGGTGCAGGTCGACTACTGCGGTATCTGCCATTCCGATCTTTCCATGATCGACAACGAATGGGGATTCTCTCAGTATCCGCTGGTTGCCGGGCATGAAGTGATTGGCCGCGTGGCGGCGCTCGGCAGTGCGGCGCAGGAAAAAGGGGTGAAAGTTGGTCAGCGCGTGGGCGTAGGCTGGACGGCGCGCAGCTGTGGGCATTGCGATGCATGTATCAGCGGTAATCAGATTAACTGCCTGGAAGGCGCCGTAGCCACCATTCTCAACCGTGGCGGTTTTGCCGAGAAACTGCGGGCAGACTGGCAGTGGGTGATCCCGCTTCCGGAGAGCATCGATATTGAGTCGGCAGGTCCTCTGTTATGCGGCGGTATTACGGTTTTTAAACCTCTGCTGATGCACCACATCACCGCGACCAGTCGCGTGGGGGTGATCGGCATCGGCGGTCTTGGGCACATTGCCATTAAACTGTTGCACGCAATGGGCTGTGAAGTGACCGCATTCAGCTCGAATCCGTCGAAAGAACAGGAAGTGCTGGCAATGGGGGCGGATAAAGTCGTGAACAGTCGCGATCCAGACGCGTTAAATGCGCTGGCAGGCCAGTTTGATCTCATTATCAACACCGTTAATGTCGACCTCGACTGGCAGCCCTACTTTGAAGCGCTGGCCTATGGCGGCCATTTCCACACCGTCGGCGCAGTGATGAAGCCGCTGCCGGTTCCGGCGTTTACATTGATTGCTGGCGATCGCAGCATCTCCGGCTCAGCAACCGGTACGCCCTATGAGCTGCGCAAATTGATGAAGTTTGCCGGGCGCAGCAAGGTCTCGCCGACGACAGAGCTGTTCCCAATGTCGCAAATCAACGAAGCCATCCAGCACGTTCGCGACGGCAAAGCGCGTTACCGCGTGGTACTGCAAGCCGACTTTAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGA.
SEQ ID NO.6
MSIIKSYAAKEAGSELELYEYDAGELRPEDVEVQVDYCGICHSDLSMIDNEWGFSQYPLVAGHEVIGRVAALGSAAQEKGVKVGQRVGVGWTARSCGHCDACISGNQINCLEGAVATILNRGGFAEKLRADWQWVIPLPESIDIESAGPLLCGGITVFKPLLMHHITATSRVGVIGIGGLGHIAIKLLHAMGCEVTAFSSNPSKEQEVLAMGADKVVNSRDPDALNALAGQFDLIINTVNVDLDWQPYFEALAYGGHFHTVGAVMKPLPVPAFTLIAGDRSISGSATGTPYELRKLMKFAGRSKVSPTTELFPMSQINEAIQHVRDGKARYRVVLQADF.
来源于巨大芽胞杆菌(Bacillus megaterium)的葡萄糖脱氢酶BmGDH的GenBank登录号为AAA22475，对葡萄糖脱氢酶BmGDH氨基酸序列进行Q252L/E170K/S100P/K166R/V72I/K137R多位点替换获得突变子BmGDHM6(已在专利申请2020103075429中公开)。突变子BmGDHM6编码基因经过密码子优化后人工合成(杭州擎科生物技术有限公司提供基因合成服务)，核苷酸序列和氨基酸序列分别如SEQ ID NO.7和SEQ ID NO.8所示。
SEQ ID NO.7
ATGTATAAAGATCTGGAAGGTAAAGTGGTGGTGATTACAGGTAGCAGCACGGGTCTGGGTAAAAGCATGGCAATTCGTTTTGCGACGGAAAAAGCGAAAGTTGTTGTGAATTATCGTAGCAAAGAAGATGAAGCAAATAGCGTGCTGGAAGAAATTAAAAAGGTGGGTGGTGAAGCAATCGCAGTTAAAGGTGATGTTACAGTGGAAAGCGATATTATTAATCTGGTTCAGAGCGCAATCAAAGAATTTGGTAAACTGGATGTTATGATCAACAATGCAGGTCTGGAAAATCCGGTTCCGAGTCATGAAATGAGCCTGAGCGATTGGAATAAAGTGATCGATACCAATCTGACCGGCGCATTTCTGGGTAGCCGTGAAGCAATTAAATATTTTGTTGAAAACGATATCCGTGGTACCGTTATTAATATGTCATCTGTTCATGAAAAAATTCCGTGGCCGCTGTTTGTTCATTATGCAGCAAGCAAAGGTGGTATGCGTCTGATGACCAAAACCCTGGCACTGGAATATGCACCGAAAGGTATTCGTGTTAATAATATTGGTCCGGGTGCAATTAATACCCCGATTAATGCAGAAAAATTTGCAGATCCGGAACAGCGTGCAGATGTTGAAAGCATGATTCCGATGGGTTATATTGGTGAACCGGAAGAAATTGCAGCAGTTGCAGCATGGCTGGCAAGCAGCGAAGCAAGCTATGTTACCGGTATTACCCTGTTTGCAGATGGTGGTATGACCCTGTATCCGAGCTTTCAGGCAGGTCGTGGTTAA.
SEQ ID NO.8
MYKDLEGKVVVITGSSTGLGKSMAIRFATEKAKVVVNYRSKEDEANSVLEEIKKVGGEAIAVKGDVTVESDIINLVQSAIKEFGKLDVMINNAGLENPVPSHEMSLSDWNKVIDTNLTGAFLGSREAIKYFVENDIRGTVINMSSVHEKIPWPLFVHYAASKGGMRLMTKTLALEYAPKGIRVNNIGPGAINTPINAEKFADPEQRADVESMIPMGYIGEPEEIAAVAAWLASSEASYVTGITLFADGGMTLYPSFQAGRG.
实施例2：单独表达老黄酶NemR-PS基因工程菌与共表达醇脱氢酶YsADH和葡萄糖脱氢酶BmGDHM6的基因工程菌构建
1、单独表达老黄酶NemR-PS基因工程菌的构建
将SEQ ID NO.1所示老黄酶NemR-PS编码基因插入pET28a载体的Nco I和Xho I限制性酶切位点，得到重组载体pET28a-NemR-PS；将重组载体pET28a-NemR-PS导入宿主细胞E.coli BL21(DE3)，得到基因工程菌E.coli BL21(DE3)/pET28a-NemR-PS。工程菌E.coliBL21(DE3)/pET28a-NemR-PS经提取质粒测序表明，老黄酶NemR-PS编码基因插入无误。
2、共表达醇脱氢酶YsADH和葡萄糖脱氢酶BmGDHM6的基因工程菌构建
将SEQ ID NO.5所示醇脱氢酶YsADH编码基因插入质粒pACYCDuet-1上的第一克隆位点Nco I和Hind III之间，将SEQ ID NO.7所示葡萄糖脱氢酶BmGDHM6编码基因插入pACYCDuet-1载体上的第二克隆位点Nde I和Xho I之间，得到重组质粒pACYCDuet-1-YsADH-BmGDHM6。将重组质粒pACYCDuet-1-YsADH-BmGDHM6导入宿主细胞E.coli BL21(DE3)，得到重组菌E.coli BL21(DE3)/pACYCDuet-1-YsADH-BmGDHM6。工程菌E.coli BL21(DE3)/pACYCDuet-1-YsADH-BmGDHM6经提取质粒测序表明，醇脱氢酶YsADH和葡萄糖脱氢酶BmGDHM6的编码基因插入无误。
实施例3：老黄酶NemR-PS的湿菌体制备、粗酶液的分离纯化及比酶活测定
1、湿菌体E.coli BL21(DE3)/pET-28a-NemR-PS的制备
将实施例2构建的基因工程菌E.coli BL21(DE3)/pET-28a-NemR-PS接种于含有终浓度100μg/mL卡那霉素的LB液体培养基中，在37℃、200rpm下培养过夜，然后以体积浓度2％的接种量转接于含有100μg/mL卡那霉素的LB液体培养基中，在37℃、200rpm培养至菌体浓度OD600至0.6～0.8，向培养物中加入终浓度0.5mM的IPTG，在24℃下诱导培养16h，获得诱导培养液。再将诱导培养液于4℃、8000rpm下离心10min，弃去上清液；然后用pH 8.0、50mMTris-HCl缓冲液重悬菌体，于4℃和8000rpm下离心10min，弃去上清液，收集老黄酶NemR-PS湿菌体作为生物催化剂，于-20℃条件下保存备用。同时，除了不添加IPTG，在其他条件相同下基因工程菌E.coli BL21(DE3)/pET-28a-NemR-PS经培养、离心、洗涤并收集菌体，用作为对照。
2、粗酶液的制备
称取步骤1得到的湿菌体1g，加入15mL的50mM Tris-HCl缓冲液(pH 8.0)充分重悬菌体后，然后将菌悬液在冰浴(0℃)、400W条件下超声破碎20min，超声过程工作2s，间歇4s，以相同条件重复破碎3次。破碎菌液在4℃和8000rpm下离心10min，所得到的上清液即为目的蛋白的粗酶液。将目的蛋白的粗酶液用截留分子量为10kDa的超滤管在4℃和5000rpm下离心30min进行浓缩。离心结束后，去除沉淀，得到的上层溶液即为浓缩后的目的蛋白的粗酶液，作为NemR-PS粗酶液进行下一步分离纯化。
3、目的蛋白的分离纯化
按照Ni-NTA金属螯合亲和层析(购自Bio-Rad公司，简称Ni2+柱，柱内径1.6cm，柱高15cm)使用说明，取步骤2NemR-PS粗酶液15mL上样至预平衡Ni2+柱中，再依次用含5mM咪唑、50mM咪唑、100mM咪唑、200mM咪唑、500mM咪唑的洗脱液(洗脱液组成：相应浓度的咪唑、300mM的氯化钠，溶剂为50mM的Tris-HCl缓冲液，pH 8.0)洗脱杂蛋白和目的蛋白，洗脱速度均为2.5mL/min，每个浓度洗脱液洗脱3个柱体积，收集含200mM咪唑的洗脱液对应的流出液，并用截留分子量为10kDa的超滤管在4℃和5000rpm下离心30min进行脱盐浓缩，取截留液即NemR-PS纯酶液，存于-20℃备用。
4、SDS-PAGE检测样品的制备
取20μL纯酶液，加入4μL 6x Protein Loading Buffer混匀后，煮沸10min。煮沸结束后，12000rpm离心1min，各取15μL上清液用于SDS-PAGE检测，蛋白质Marker为BluePlusProtein Marker(14-120kDa)。老黄酶NemR-PS纯酶液的纯度经SDS-PAGE凝胶电泳验证，SDS-PAGE电泳结果如图2中泳道1所示。老黄酶NemR-PS经SDS-PAGE电泳后均为单一条带，表明分离纯化后的老黄酶NemR-PS为电泳纯。老黄酶NemR-PS的理论大小分别为39.9kDa，而在SDS-PAGE电泳上的表观大小约为39kDa。
5、蛋白浓度测定
根据BCA法蛋白质浓度测定试剂盒(型号：E112-02，购自南京诺唯赞生物科技股份有限公司)绘制蛋白浓度标准曲线，测得的线性关系公式为Y＝0.0007930×X+0.1558，其中Y：是562nm处的吸光度值，X：为BSA溶液浓度(μg/mL)，标准偏差为R2＝0.9989。然后根据标准蛋白浓度曲线计算蛋白浓度，进而计算比酶活。每次做三组平行实验，计算平均值和标准误差。
经蛋白浓度测定，步骤2制备的老黄酶NemR-PS的粗酶液蛋白浓度为11.14mg/mL。
6、酶活测定
(1)老黄酶的比酶活测定
老黄酶NemR-PS的酶活力采用多功能酶标仪的单因素动力学方法测定在340nm处吸光值的变化来计算酶活。比酶活测定体系：100μg粗酶液(以粗酶液的蛋白浓度计)，10mM(E/Z)-柠檬醛，0.4mM NADPH，加50mM PIPES缓冲液(pH 7.0)补足200μL。酶活力单位(U)定义：在30℃下，每分钟氧化1μmol NADPH所需的酶量。每次做三组平行实验，计算平均值和标准误差。老黄酶NemR-PS体积酶活力及比活力计算公式如公式1和公式2：
①ΔA为1min之内吸光值的变化；
②V1、V2分别为反应液的总体积和添加的酶液体积，mL；
③6220为NAD(P)H在340nm下的摩尔消光系数；
④t为反应时间，1min；
⑤L为光程距离，为1cm
经比酶活测定，老黄酶NemR-PS粗酶液的体积酶活为3.85U/mL，比酶活为0.34U/mg。
实施例4：测定老黄酶NemR-PS对映体选择性的催化反应体系
1、湿菌体E.coli BL21(DE3)/pACYCDuet-1-YsADH-BmGDHM6的制备
将实施例2构建的基因工程菌E.coli BL21(DE3)/pACYCDuet-1-YsADH-BmGDHM6接种于含有终浓度100μg/mL氯霉素的LB液体培养基中，在37℃、200rpm下培养过夜，然后以体积浓度2％的接种量转接于含有100μg/mL氯霉素的LB液体培养基中，在37℃、200rpm条件下培养至菌体浓度OD600至0.6～0.8，向培养物中加入终浓度0.5mM的IPTG，在24℃下诱导培养16h，获得诱导培养液。再将诱导培养液于4℃、8000rpm下离心10min，弃去上清液；然后用pH8.0、50mM Tris-HCl缓冲液重悬菌体，于4℃和8000rpm下离心10min，弃去上清液，收集共表达湿菌体作为生物催化剂，于-20℃条件下保存备用。同样条件下，以不添加IPTG制备的生物催化剂作为对照。
2、反应体系
构建测定老黄酶NemR-PS对映体选择性的反应体系为5mL，各组分分别为：实施例3步骤1制备的老黄酶NemR-PS湿菌体和实施例4步骤1制备的共表达湿菌体各0.1g/mL、100mM柠檬醛、300mM D-葡萄糖、0.2mM NADP+以及50mM Tris-HCl pH 8.0缓冲液补充至5mL。30℃、200rpm振荡反应8h，同样条件下，以不加催化剂为空白对照。
反应结束后，取0.2mL反应液加入到离心管中，加入2倍体积的乙酸乙酯在30℃和200rpm下萃取2h，在高速离心机中以12000rpm离心2min，吸取上层有机相。向有机相中加适量无水硫酸钠进行除水处理，在高速离心机中以12000rpm离心1min，将上清液转移至气相瓶中用于气相色谱分析。利用气相色谱法检测样品中各成分((E)-柠檬醛、(Z)-柠檬醛、(S)-香茅醛、(R)-香茅醛、橙花醇、香叶醇、(S)-香茅醇和(R)-香茅醇)的含量。每次做三组平行实验，计算平均值和标准误差。
气相色谱条件：
气相色谱仪，SHIMADZU GC-2014；手性色谱柱，色谱柱：Agilent 6890N的手性柱BGB-174(30m×250μm×0.25μm)；检测器，FID，250℃；载气，N2；载气流量，3mL/min；分流比：1:19；进样量：1.0μL；进样口温度：250℃。
(E)-柠檬醛、(Z)-柠檬醛、(S)-香茅醛、(R)-香茅醛、橙花醇、香叶醇、(S)-香茅醇和(R)-香茅醇的气相色谱升温程序：90℃保持25min，20℃/min升到160℃保持2min，20℃/min升到180℃保持3min。结果如图3中所示，按出峰顺序，保留时间分别为：(S)-香茅醛，20.091min；(R)-香茅醛，20.542min；橙花醇，27.196min；香茅醇，27.433min；香叶醇，28.433min；(E)-柠檬醛，28.796min；(Z)-柠檬醛，29.840min。
上述色谱条件不能区分(S)-香茅醇和(R)-香茅醇，因而建立了一种专门分析(S)-香茅醇和(R)-香茅醇的气相色谱条件，如下：Agilent 6890N手性柱BGB-174(30m×250μm×0.25μm)；检测器FID，250℃；载气，N2；载气流量，3mL/min；分流比，1:19；进样量，1.0μL；进样口温度，250℃。升温程序：初始温度75℃，保持30min，以0.4℃/min升温至120℃，保持10min，随后以20℃/min升温至180℃，保持3min。(S)-香茅醇和(R)-香茅醇的保留时间分别为95.489min和96.249min。
实施例5：老黄酶NemR-PS的单点突变
以实施例2的质粒pET28a-NemR-PS为模板，设计点突变引物序列如表1，利用全质粒扩增PCR技术构建突变体质粒。
表1用于NemR-PS单点突变的引物序列
注：下划线部分为突变位点
PCR扩增体系见表2所示。
表2 PCR扩增反应体系
PCR反应进程为：95℃预变性5min；之后，以95℃变性15s，60℃复性15s，72℃保持1min20s为一个循环，重复这样循环30次；最后，72℃保持5min。PCR产物经0.8％琼脂糖凝胶电泳检测，从图4中可看到约6000bp处有明亮的条带，条带与质粒的理论值相符。
PCR产物经37℃酶切1h去除甲基化的模板，酶切体系如表3所示。
表3 PCR产物中甲基化模板的消化体系
DpnⅠ酶切后的PCR产物经纯化后，直接转化于宿主菌E.coli BL21(DE3)中，得到基因工程菌E.coli BL21(DE3)/pET-28a-NemR-PS-Mut(Mut表示引入的突变)。转化子经菌落PCR验证后接入含100μg/mL卡那霉素的LB液体培养基中，37℃培养12h，离心收集菌体，提取质粒，送去测序。分别获得W103A、R143A、F241A、Q242A、S272A、D275A、W276A、F351A和Y352A等9个单点突变体。
根据实施例3的诱导表达方法，突变体粗酶液的SDS-PAGE验证如图5所示，并测定粗酶液的比酶活，以未突变老黄酶酶活为100％，计算其他突变体粗酶液的相对活力。结果如下表4，D275A和F351A突变体比活力有显著提高，相对活力分别提高了46.50％和43.10％。
表4不同NemR-PS单点突变体粗酶液的相对活力
实施例6：老黄酶NemR-PS的D275位点的饱和突变
以实施例2的质粒pET-28a-NemR-PS为模板，对275位设计饱和突变引物，引物序列如表5，利用全质粒扩增PCR技术构建突变体质粒。PCR产物经0.8％琼脂糖凝胶电泳检测，从图6中可看到约6000bp处有明亮的条带，条带与质粒的理论值相符。
表5 NemR-PS的D275位点饱和突变引物序列
注：下划线部分为突变位点
以实施例2方法制备得到工程菌E.coli BL21(DE3)/pET-28a-pnbA-BS-Mut(Mut表示在D275位点引入的突变)。转化子经菌落PCR验证后接入含100μg/mL卡那霉素的LB液体培养基中，37℃培养12h，离心收集菌体，提取质粒，送去测序。分别获得275位点的19个不同突变体。
根据实施例3的诱导表达方法，19个不同突变体粗酶液的SDS-PAGE验证如图7所示，并测定其比酶活，以未突变老黄酶酶活为100％，计算其他突变体粗酶液的相对活力。根据实施例4的方法，测定其对映体选择性。结果如下表6，275位点突变体D275G、D275A、D275V、D275L、D275I、D275M、D275F、D275P、D275Q、D275S、D275T、D275C的相对活力有提高，其中D275G最佳，相对酶活提高46.97％。此外，所有饱和突变子的(S)-对映体选择性均>99％。
表6不同NemR-PS的D275位点突变体粗酶液的相对活力和对映体选择性
实施例7：老黄酶NemR-PS的F351位点的迭代饱和突变
以实施例6的质粒pET-28a-NemR-PS-D275G为模板，对351位设计饱和突变引物序列如表7，利用全质粒扩增PCR技术构建突变体质粒。PCR产物经0.8％琼脂糖凝胶电泳检测，从图8中可看到约6000bp处有明亮的条带，条带与质粒的理论值相符。
表7 NemR-PS的F351位点迭代饱和突变引物序列
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注：下划线部分为突变位点
以实施例2方法制备得到工程菌E.coli BL21(DE3)/pET-28a-pnbA-BS-D275G/Mut(Mut表示在D351位点引入的突变)。转化子经菌落PCR验证后接入含100μg/mL卡那霉素的LB液体培养基中，37℃培养12h，离心收集菌体，提取质粒，送去测序。分别获得19个351位点迭代饱和体。
根据实施例3的诱导表达方法，19个突变体粗酶液的SDS-PAGE验证如图9所示，并测定粗酶液的比酶活，以未突变老黄酶酶活为100％，计算其他突变体粗酶液的相对活力。根据实施例4方法，测定其对映体选择性。结果如下表8，迭代突变体D275G/F351A、D275G/F351G、D275G/F351V、D275G/F351T、D275G/F351S、D275G/F351I、D275G/F351L、D275G/F351C、D275G/F351M、D275G/F351W、D275G/F351P、D275G/F351H、D275G/F351K、D275G/F351R等的相对活力有提高，其中D275G/F351A最佳，与野生型相比相对活力提高了132.08％。此外所有饱和突变子对映体选择性均>99％。
表8不同NemR-PS D275G/F351位点突变体粗酶液的相对活力和对映体选择性
注：M1为突变子D275G/F351
实施例8：表达老黄酶突变体D275G/F351A的基因工程菌、粗酶液的分离纯化及纯酶的动力学参数测定
按实施例2中的基因工程菌构建方法，获得基因工程菌E.coli BL21(DE3)/pET-28a-NemR-PS-D275G/F351A，经实施例3方法诱导表达得到表达突变体D275G/F351A的湿菌体，湿菌体经细胞破碎、离心获得粗酶液。粗酶液经实施例3方法分离纯化获得老黄酶突变体D275G/F351A纯酶液(图2中泳道2)。分别测定NemR-PS及突变体D275G/F351A的底物抑制动力学参数，突变体D275G/F351A的Ki为128.5mM，Km为0.35mM，Kcat为1.75s-1。突变体D275G/F351A的Kcat/Km达5.01s-1mM-1，约为老黄酶NemR-PS的2.5倍，表明老黄酶的活力改造显著地提高了催化效率。
表9 NemR-PS及突变体D275G/F351A的动力学参数
实施例9：共表达老黄酶突变体NemR-PS D275G/F351A、醇脱氢酶YsADH和葡萄糖脱氢酶BmGDHM6的基因工程菌
1、共表达老黄酶突变体NemR-PS D275G/F351A、醇脱氢酶YsADH和葡萄糖脱氢酶BmGDHM6的基因工程菌构建
取1mL实施例2方法构建的E.coli BL21(DE3)/pACYCDuet-1-YsADH-BmGDHM6接种于含有终浓度100μg/mL氯霉素的LB液体培养基中，在37℃、200rpm下培养过夜，然后将1mL种子液转接至50mL含有50μg/mL氯霉素的LB液体培养基中，在37℃和200rpm下培养至OD600至0.4，冰上冷却半小时，取菌液离心后弃去上清液，用冰上预冷的100mM氯化钙水溶液轻轻悬浮细胞，冰上放置30min，然后4℃和4000rpm下离心收集菌体，再用冰上预冷的100mM氯化钙水溶液轻轻悬浮细胞制成E.coli BL21(DE3)/pACYCDuet-1-YsADH-BmGDHM6感受态细胞。将实施例7制备的重组质粒pET28a-NemR-PS-D275G/F351A导入感受态细胞E.coli BL21(DE3)/pACYCDuet-1-YsADH-BmGDHM6中，获得基因工程菌E.coli BL21(DE3)/pACYCDuet-1-YsADH-BmGDHM6/pET28a-NemR-PS-D275G/F351A。经提取质粒测序表明，老黄酶突变体NemR-PS-D275G/F351A、醇脱氢酶YsADH和葡萄糖脱氢酶BmGDHM6的编码基因插入无误。
按实施例3的方法，获得基因工程菌E.coli BL21(DE3)/pACYCDuet1-YsADH-BmGDHM6/pET28a-NemR-PS-D275G/F351A的湿菌体及其粗酶液，同样条件下以未经诱导表达湿菌体所制得粗酶液为对照(图10中泳道1)。如图10中泳道2所示，诱导表达得到的粗酶液经SDS-PAGE电泳后显示三条加粗条带，NemR-PS D275G/F351A的蛋白大小为39kDa，YsADH的蛋白大小为37kDa，BmGDHM6的蛋白大小为28kDa。粗酶液蛋白浓度经测定为2.15mg/mL。
2、三酶共表达基因工程菌中老黄酶NemR-PS D275G/F351A、醇脱氢酶YsADH和葡萄糖脱氢酶BmGDHM6的粗酶液活力测定
老黄酶突变体NemR-PSD275G/F351A的酶活力依据实施例3的对应方法测定，其中底物浓度为20mM。
醇脱氢酶的酶活力采用酶标仪的单因素动力学方法测定在340nm处吸光值的变化来计算酶活。比酶活测定体系：100μg粗酶液(以粗酶液的蛋白浓度计)，20mM(S)-香茅醛，0.4mM NADPH，加50mM PIPES缓冲液(pH 7.0)补足200μL。酶活力单位(U)定义：在30℃下，每分钟氧化1μmol NADPH所需的酶量。每次做三组平行实验，计算平均值和标准误差。醇脱氢酶YsADH的体积酶活力及比活力计算公式依据实施例3中的公式1和公式2。
葡萄糖脱氢酶的酶活力采用酶标仪的单因素动力学方法测定在340nm处吸光值的变化来计算酶活。比酶活测定体系：100μg粗酶液(以粗酶液的蛋白浓度计)，20mM葡萄糖，0.4mM NADP+，加50mM PIPES缓冲液(pH 7.0)补足200μL。酶活力单位(U)定义：在30℃下，每分钟生成1μmol NADPH所需的酶量。每次做三组平行实验，计算平均值和标准误差。葡萄糖脱氢酶BmGDHM6的体积酶活力及比活力计算公式依据实施例3中的公式1和公式2。
经比酶活测定，三酶共表达基因工程菌中老黄酶NemR-PS D275G/F351A、醇脱氢酶YsADH和葡萄糖脱氢酶BmGDHM6的比酶活分别是0.79U/mg，0.79U/mg和0.36U/mg。
实施例10：一锅法催化(E/Z)-柠檬醛还原生成(S)-香茅醇的初始催化体系
以实施例9所述三酶共表达基因工程菌的湿菌体为催化剂，一锅法催化(E/Z)-柠檬醛还原生成(S)-香茅醇的初始反应体系与反应条件为：基因工程菌E.coli BL21(DE3)/pACYCDuet-1-YsADH-BmGDHM6/pET28a-NemR-PS-D275G/F351A湿菌体100g/L，300mM(E/Z)-柠檬醛，900mM葡萄糖，4％(v/v)助溶剂异丙醇，0.4mM NADP+，50mM PIPES缓冲液(pH 6.5)补充至10mL的反应总体系，葡萄糖与底物浓度摩尔比为3:1。转速400rpm，反应温度30℃。反应过程中利用自动滴定系统维持pH恒定，滴定所用的碱液为1M NaOH水溶液。反应时间为12h，反应结束后取1mL上清加入1mL乙酸乙酯萃取2h，12000rpm离心1min，取上层有机相。上层有机相经无水硫酸钠除水后，按实施例4气相色谱方法品进行分析。每次做三组平行实验，计算平均值和标准误差。在所述条件下，测得(S)-香茅醇转化率为86.01％。
实施例11：一锅法催化(E/Z)-柠檬醛还原生成(S)-香茅醇的最适催化pH
将实施例10中反应pH分别改为5.5、6.0、6.5、7.0、7.5和8.0，其它反应体系及反应条件设置同实施例10。每次做三组平行实验，计算平均值和标准误差。结果如图11所示，当pH为5.5时，(S)-香茅醇的转化率为49.24％；当pH为6.5时，转化率高达87.93％。当pH偏中性或弱碱性时，转化率降低，当pH 8.0时，产物转化率为59.02％。在pH 6.5时产物转化率最高，因而催化反应的最适pH为6.5。
实施例12：一锅法催化(E/Z)-柠檬醛还原生成(S)-香茅醇的最催化温度
将实施例10中温度分别改为20℃、25℃、30℃、35℃、40℃和45℃，其它反应体系及反应条件设置同实施例10。每次做三组平行实验，计算平均值和标准误差，结果如图12所示。在20℃时，(S)-香茅醇的转化率为47.84％；当催化温度为30℃时，转化率已达到87.93％。当温度升高至40和45℃时，转化率降至43.09％和34.47％。在30℃时转化率最高，因而确定催化反应的最适温度为30℃。
实施例13：一锅法催化(E/Z)-柠檬醛还原生成(S)-香茅醇的最适搅拌速度
将实施例10中转速分别为200rpm、300rpm、400rpm、500rpm、600rpm和700rpm。反应温度为30℃、pH为6.5，其它反应体系及反应条件设置同实施例10。每次做三组平行实验，计算平均值和标准误差。如图13所示，在200rpm时，(S)-香茅醇转化率为60.57％；当转速为500rpm时，转化率为92.33％。当搅拌速度继续提高至600rpm和700rpm时，转化率降至78.48％和58.86％。在500rpm转速时转化率最高，因而催化反应的最适转速为500rpm。
实施例14：一锅法催化(E/Z)-柠檬醛还原生成(S)-香茅醇的最适葡萄糖添加量
将实施例10中葡萄糖与底物浓度比例分别改为0.5:1、1:1、2:1、3:1、4:1和5:1。反应温度为30℃，pH为6.5，转速为500rpm，其它反应体系及反应条件设置同实施例10。每次做三组平行实验，计算平均值和标准误差。如图14所示，当添加比例为0.5:1时，(S)-香茅醇转化率为33.01％，当添加比例为3:1时，转化率为92.33％。继续提高葡萄糖添加浓度至4:1和5:1时，转化率下降至83.10％和70.63％。在葡萄糖与底物浓度比例为3:1时转化率最高，因而催化反应的最适葡萄糖添加量为与底物浓度比例3:1。
实施例15：一锅法催化(E/Z)-柠檬醛还原生成(S)-香茅醇的最适助溶剂添加量
将实施例10中助溶剂添加量分别改为0.5％、1％、2％、4％、6％和8％(v/v)。反应温度为30℃，pH为6.5，转速为500rpm，葡萄糖添加量为与底物浓度比例3:1，其它反应体系及反应条件设置同实施例10。每次做三组平行实验，计算平均值和标准误差。如图15所示，当助溶剂为0.5％(v/v)时，(S)-香茅醇转化率为25.24％；助溶剂浓度为4％(v/v)时，转化率为92.33％；助溶度为8％(v/v)时，转化率为72.39％。在添加助溶剂异丙醇体积浓度为4％(v/v)时转化率最高，因而催化反应最适助溶剂浓度为4％(v/v)。
实施例16：一锅法催化(E/Z)-柠檬醛还原生成(S)-香茅醇的最适NADP+浓度
将实施例10中辅酶NADP+的浓度分别为0mM、0.2mM、0.4mM、0.6mM、0.8mM和1.0mM。反应温度为30℃，pH为6.5，转速为500rpm，葡萄糖添加量为与底物浓度比例3:1，助溶剂添加量为4％(v/v)，其它反应体系及反应条件设置同实施例10。每次做三组平行实验，计算平均值和标准误差。如图16所示，当NADP+浓度在0～0.4mM范围内，NADP+浓度增加时，(S)-香茅醇转化率逐渐上升。当NADP+浓度为0.4mM时转化率为92.33％。NADP+浓度在0.4～1.0mM范围时，随着NADP+浓度增加转化率并没有显著增加，因此NADP+的最适添加量为0.4mM。
实施例17：一锅法催化400mM(E/Z)-柠檬醛还原生成(S)-香茅醇的反应进程
反应体系：基因工程菌E.coli BL21(DE3)/pACYCDuet-1-YsADH-BmGDHM6/pET28a-NemR-PS-D275G/F351A湿菌体100g/L，400mM(E/Z)-柠檬醛，1200mM葡萄糖(葡萄糖添加量为与底物浓度比例3:1)，4％(v/v)助溶剂异丙醇，0.4mM NADP+，50mM PIPES缓冲液(pH 6.5)补充至10mL的反应总体系。
反应条件：反应温度为30℃，pH为6.5，转速为500rpm，反应时长为24h，在反应前12h间隔2h取样，按实施例10方法检测。每次做三组平行实验，计算平均值和标准误差。如图17所示，反应12h内产物(S)-香茅醇逐渐累积，在12h时转化率为87.78％，在12～24h之间转化率没有显著变化。在24h时转化率为89.16％。同时残留副产物香叶醇9.53％及中间产物(S)-香茅醇0.31％。
实施例18：一锅法催化(E/Z)-柠檬醛还原生成(S)-香茅醇时底物流加反应进程
底物柠檬醛和辅底物葡萄糖按摩尔比3:1通过恒速微量泵注入到反应体系中，底物和辅底物对应的注入速度分别为0.5mmol/h和1.5mmol/h，恒速流加时间为10h，底物累计添加量为500mM，辅底物葡萄糖累计添加量为1500mM。其它反应体系及反应条件设置同实施例17。间隔2h取样，反应时长为12h。如图18所示，反应在流加底物时逐步积累产物(S)-香茅醇，反应过程中存在少量副产物香叶醇和中间产物(S)-香茅醛。在10h时，底物和辅底物流加停止，反应体系累积产物浓度达454.5mM，副产物香叶醇浓度为10.2mM，中间产物(S)-香茅醛浓度为35.3mM。继续反应2h至总反应时长为12h，产物(S)-香茅醇累计浓度达500mM且无副产物及中间产物。
从实施例11到实施例18中的所有催化反应，经对映体选择性测定，产物(S)-香茅醇e.e.值均>99％，表明所建立的(S)-香茅醇合成方法具有严格的(S)-对映体选择性。
序列表
<110> 浙江工业大学、浙江英沃迪生物科技有限公司
<120> 一种老黄酶NemR-PS突变体及其在制备(S)-香茅醇中的应用
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1095
<212> DNA
<213> 斯氏普罗威登斯菌(Providencia stuartii)
<400> 1
atgagccaga aaaagctgtt tacgccgctg aaagttggta ccctgaccgc accgaatcgt 60
atttttatgg cgccgctgac ccgcctgcgt agcattgaac cgggcgatat tccgacgccg 120
ctgatgggcg aatattatcg ccagcgtgcc accgcaggcc tgattattag cgaagcaacc 180
cagattagtg cacagtcaaa aggttatgca ggtgcaccag gtctgcatag cgcagaacag 240
attgcagcat ggaaaaagat tacctcaacc gttcatgaag caggtggtcg tattgcagta 300
cagctgtggc atacgggtcg tattagtcat gttagcctgc agccgaacgg tctggcaccg 360
gttgcaccgt cagcaatttc agccggcaca cgtaccagtc tgcgtgatga aaatggtcgt 420
gccattcgtg tagataccag catgccgcgt gcactggaaa ccgaagaaat tccggcaatt 480
gttaatgatt ttcgccaggc agttgcaaat gcccgtgaag ctggttttga tatggctgaa 540
ctgcatgcag cacatggtta tctgctgcat cagtttctga gcccgtcagc aaatcatcgt 600
accgatcagt atggcggtac ccgtgaaaat cgtgcacgtt ttctgctgga tgttgttgat 660
gcagtttgtg cagaatgggg tagcgaacat attggtattc gtattagtcc gattggtacc 720
tttcagaata cagataatgg tcctaatgaa gttgatgatg ctctgtatct gattgaagaa 780
ctggataaac gtcatattgc atatctgcat ctgagcgaac cggattgggc aggtggtcag 840
ccatataccg atgattttcg tcagaaagtt cgtgaacgtt ttcatggtgt tattattggt 900
gcaggcgcat atacgacaga aaaagcagaa aatctgattg aaaaaggtct gattgatgca 960
gttgcctttg gtcgtgattt tattgcgaat ccggatctgg ttgtgcgtct gaaaaataaa 1020
gcagcactga acccacagcg tccggaaagc ttttatggtg gtggtgccga aggttatacc 1080
gattatccga gcctg 1095
<210> 2
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<213> 斯氏普罗威登斯菌(Providencia stuartii)
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Ala Pro Asn Arg Ile Phe Met Ala Pro Leu Thr Arg Leu Arg Ser Ile
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Glu Pro Gly Asp Ile Pro Thr Pro Leu Met Gly Glu Tyr Tyr Arg Gln
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Arg Ala Thr Ala Gly Leu Ile Ile Ser Glu Ala Thr Gln Ile Ser Ala
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Gln Ser Lys Gly Tyr Ala Gly Ala Pro Gly Leu His Ser Ala Glu Gln
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Ile Ala Ala Trp Lys Lys Ile Thr Ser Thr Val His Glu Ala Gly Gly
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Arg Ile Ala Val Gln Leu Trp His Thr Gly Arg Ile Ser His Val Ser
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Gly Thr Arg Thr Ser Leu Arg Asp Glu Asn Gly Arg Ala Ile Arg Val
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Asp Thr Ser Met Pro Arg Ala Leu Glu Thr Glu Glu Ile Pro Ala Ile
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Val Asn Asp Phe Arg Gln Ala Val Ala Asn Ala Arg Glu Ala Gly Phe
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Asp Met Ala Glu Leu His Ala Ala His Gly Tyr Leu Leu His Gln Phe
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Leu Ser Pro Ser Ala Asn His Arg Thr Asp Gln Tyr Gly Gly Thr Arg
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Glu Asn Arg Ala Arg Phe Leu Leu Asp Val Val Asp Ala Val Cys Ala
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Glu Trp Gly Ser Glu His Ile Gly Ile Arg Ile Ser Pro Ile Gly Thr
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Phe Gln Asn Thr Asp Asn Gly Pro Asn Glu Val Asp Asp Ala Leu Tyr
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<210> 3
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<212> DNA
<213> 斯氏普罗威登斯菌(Providencia stuartii)
<400> 3
atgagccaga aaaagctgtt tacgccgctg aaagttggta ccctgaccgc accgaatcgt 60
atttttatgg cgccgctgac ccgcctgcgt agcattgaac cgggcgatat tccgacgccg 120
ctgatgggcg aatattatcg ccagcgtgcc accgcaggcc tgattattag cgaagcaacc 180
cagattagtg cacagtcaaa aggttatgca ggtgcaccag gtctgcatag cgcagaacag 240
attgcagcat ggaaaaagat tacctcaacc gttcatgaag caggtggtcg tattgcagta 300
cagctgtggc atacgggtcg tattagtcat gttagcctgc agccgaacgg tctggcaccg 360
gttgcaccgt cagcaatttc agccggcaca cgtaccagtc tgcgtgatga aaatggtcgt 420
gccattcgtg tagataccag catgccgcgt gcactggaaa ccgaagaaat tccggcaatt 480
gttaatgatt ttcgccaggc agttgcaaat gcccgtgaag ctggttttga tatggctgaa 540
ctgcatgcag cacatggtta tctgctgcat cagtttctga gcccgtcagc aaatcatcgt 600
accgatcagt atggcggtac ccgtgaaaat cgtgcacgtt ttctgctgga tgttgttgat 660
gcagtttgtg cagaatgggg tagcgaacat attggtattc gtattagtcc gattggtacc 720
tttcagaata cagataatgg tcctaatgaa gttgatgatg ctctgtatct gattgaagaa 780
ctggataaac gtcatattgc atatctgcat ctgagcgaac cgggttgggc aggtggtcag 840
ccatataccg atgattttcg tcagaaagtt cgtgaacgtt ttcatggtgt tattattggt 900
gcaggcgcat atacgacaga aaaagcagaa aatctgattg aaaaaggtct gattgatgca 960
gttgcctttg gtcgtgattt tattgcgaat ccggatctgg ttgtgcgtct gaaaaataaa 1020
gcagcactga acccacagcg tccggaaagc gcttatggtg gtggtgccga aggttatacc 1080
gattatccga gcctg 1095
<210> 4
<211> 365
<212> PRT
<213> 斯氏普罗威登斯菌(Providencia stuartii)
<400> 4
Met Ser Gln Lys Lys Leu Phe Thr Pro Leu Lys Val Gly Thr Leu Thr
1 5 10 15
Ala Pro Asn Arg Ile Phe Met Ala Pro Leu Thr Arg Leu Arg Ser Ile
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Glu Pro Gly Asp Ile Pro Thr Pro Leu Met Gly Glu Tyr Tyr Arg Gln
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Arg Ala Thr Ala Gly Leu Ile Ile Ser Glu Ala Thr Gln Ile Ser Ala
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Gln Ser Lys Gly Tyr Ala Gly Ala Pro Gly Leu His Ser Ala Glu Gln
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Ile Ala Ala Trp Lys Lys Ile Thr Ser Thr Val His Glu Ala Gly Gly
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Arg Ile Ala Val Gln Leu Trp His Thr Gly Arg Ile Ser His Val Ser
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Leu Gln Pro Asn Gly Leu Ala Pro Val Ala Pro Ser Ala Ile Ser Ala
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Gly Thr Arg Thr Ser Leu Arg Asp Glu Asn Gly Arg Ala Ile Arg Val
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Asp Thr Ser Met Pro Arg Ala Leu Glu Thr Glu Glu Ile Pro Ala Ile
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Val Asn Asp Phe Arg Gln Ala Val Ala Asn Ala Arg Glu Ala Gly Phe
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Asp Met Ala Glu Leu His Ala Ala His Gly Tyr Leu Leu His Gln Phe
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Leu Ser Pro Ser Ala Asn His Arg Thr Asp Gln Tyr Gly Gly Thr Arg
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Glu Asn Arg Ala Arg Phe Leu Leu Asp Val Val Asp Ala Val Cys Ala
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Glu Trp Gly Ser Glu His Ile Gly Ile Arg Ile Ser Pro Ile Gly Thr
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Phe Gln Asn Thr Asp Asn Gly Pro Asn Glu Val Asp Asp Ala Leu Tyr
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Leu Ile Glu Glu Leu Asp Lys Arg His Ile Ala Tyr Leu His Leu Ser
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Glu Pro Gly Trp Ala Gly Gly Gln Pro Tyr Thr Asp Asp Phe Arg Gln
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Lys Val Arg Glu Arg Phe His Gly Val Ile Ile Gly Ala Gly Ala Tyr
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Thr Thr Glu Lys Ala Glu Asn Leu Ile Glu Lys Gly Leu Ile Asp Ala
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Val Ala Phe Gly Arg Asp Phe Ile Ala Asn Pro Asp Leu Val Val Arg
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Leu Lys Asn Lys Ala Ala Leu Asn Pro Gln Arg Pro Glu Ser Ala Tyr
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Gly Gly Gly Ala Glu Gly Tyr Thr Asp Tyr Pro Ser Leu
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<210> 5
<211> 1059
<212> DNA
<213> 约克氏菌(Yokenella sp.)
<400> 5
atgtctatta taaaaagcta tgccgcaaaa gaggcgggca gcgaactcga actttacgaa 60
tatgatgccg gtgaactcag gccggaagat gtcgaggtgc aggtcgacta ctgcggtatc 120
tgccattccg atctttccat gatcgacaac gaatggggat tctctcagta tccgctggtt 180
gccgggcatg aagtgattgg ccgcgtggcg gcgctcggca gtgcggcgca ggaaaaaggg 240
gtgaaagttg gtcagcgcgt gggcgtaggc tggacggcgc gcagctgtgg gcattgcgat 300
gcatgtatca gcggtaatca gattaactgc ctggaaggcg ccgtagccac cattctcaac 360
cgtggcggtt ttgccgagaa actgcgggca gactggcagt gggtgatccc gcttccggag 420
agcatcgata ttgagtcggc aggtcctctg ttatgcggcg gtattacggt ttttaaacct 480
ctgctgatgc accacatcac cgcgaccagt cgcgtggggg tgatcggcat cggcggtctt 540
gggcacattg ccattaaact gttgcacgca atgggctgtg aagtgaccgc attcagctcg 600
aatccgtcga aagaacagga agtgctggca atgggggcgg ataaagtcgt gaacagtcgc 660
gatccagacg cgttaaatgc gctggcaggc cagtttgatc tcattatcaa caccgttaat 720
gtcgacctcg actggcagcc ctactttgaa gcgctggcct atggcggcca tttccacacc 780
gtcggcgcag tgatgaagcc gctgccggtt ccggcgttta cattgattgc tggcgatcgc 840
agcatctccg gctcagcaac cggtacgccc tatgagctgc gcaaattgat gaagtttgcc 900
gggcgcagca aggtctcgcc gacgacagag ctgttcccaa tgtcgcaaat caacgaagcc 960
atccagcacg ttcgcgacgg caaagcgcgt taccgcgtgg tactgcaagc cgactttaag 1020
cttgcggccg cactcgagca ccaccaccac caccactga 1059
<210> 6
<211> 339
<212> PRT
<213> 约克氏菌(Yokenella sp.)
<400> 6
Met Ser Ile Ile Lys Ser Tyr Ala Ala Lys Glu Ala Gly Ser Glu Leu
1 5 10 15
Glu Leu Tyr Glu Tyr Asp Ala Gly Glu Leu Arg Pro Glu Asp Val Glu
20 25 30
Val Gln Val Asp Tyr Cys Gly Ile Cys His Ser Asp Leu Ser Met Ile
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Asp Asn Glu Trp Gly Phe Ser Gln Tyr Pro Leu Val Ala Gly His Glu
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Val Ile Gly Arg Val Ala Ala Leu Gly Ser Ala Ala Gln Glu Lys Gly
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Val Lys Val Gly Gln Arg Val Gly Val Gly Trp Thr Ala Arg Ser Cys
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Gly His Cys Asp Ala Cys Ile Ser Gly Asn Gln Ile Asn Cys Leu Glu
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Gly Ala Val Ala Thr Ile Leu Asn Arg Gly Gly Phe Ala Glu Lys Leu
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Arg Ala Asp Trp Gln Trp Val Ile Pro Leu Pro Glu Ser Ile Asp Ile
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Glu Ser Ala Gly Pro Leu Leu Cys Gly Gly Ile Thr Val Phe Lys Pro
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Leu Leu Met His His Ile Thr Ala Thr Ser Arg Val Gly Val Ile Gly
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Ile Gly Gly Leu Gly His Ile Ala Ile Lys Leu Leu His Ala Met Gly
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Cys Glu Val Thr Ala Phe Ser Ser Asn Pro Ser Lys Glu Gln Glu Val
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Leu Ala Met Gly Ala Asp Lys Val Val Asn Ser Arg Asp Pro Asp Ala
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Leu Asn Ala Leu Ala Gly Gln Phe Asp Leu Ile Ile Asn Thr Val Asn
225 230 235 240
Val Asp Leu Asp Trp Gln Pro Tyr Phe Glu Ala Leu Ala Tyr Gly Gly
245 250 255
His Phe His Thr Val Gly Ala Val Met Lys Pro Leu Pro Val Pro Ala
260 265 270
Phe Thr Leu Ile Ala Gly Asp Arg Ser Ile Ser Gly Ser Ala Thr Gly
275 280 285
Thr Pro Tyr Glu Leu Arg Lys Leu Met Lys Phe Ala Gly Arg Ser Lys
290 295 300
Val Ser Pro Thr Thr Glu Leu Phe Pro Met Ser Gln Ile Asn Glu Ala
305 310 315 320
Ile Gln His Val Arg Asp Gly Lys Ala Arg Tyr Arg Val Val Leu Gln
325 330 335
Ala Asp Phe
<210> 7
<211> 786
<212> DNA
<213> 巨大芽胞杆菌(Bacillus megaterium)
<400> 7
atgtataaag atctggaagg taaagtggtg gtgattacag gtagcagcac gggtctgggt 60
aaaagcatgg caattcgttt tgcgacggaa aaagcgaaag ttgttgtgaa ttatcgtagc 120
aaagaagatg aagcaaatag cgtgctggaa gaaattaaaa aggtgggtgg tgaagcaatc 180
gcagttaaag gtgatgttac agtggaaagc gatattatta atctggttca gagcgcaatc 240
aaagaatttg gtaaactgga tgttatgatc aacaatgcag gtctggaaaa tccggttccg 300
agtcatgaaa tgagcctgag cgattggaat aaagtgatcg ataccaatct gaccggcgca 360
tttctgggta gccgtgaagc aattaaatat tttgttgaaa acgatatccg tggtaccgtt 420
attaatatgt catctgttca tgaaaaaatt ccgtggccgc tgtttgttca ttatgcagca 480
agcaaaggtg gtatgcgtct gatgaccaaa accctggcac tggaatatgc accgaaaggt 540
attcgtgtta ataatattgg tccgggtgca attaataccc cgattaatgc agaaaaattt 600
gcagatccgg aacagcgtgc agatgttgaa agcatgattc cgatgggtta tattggtgaa 660
ccggaagaaa ttgcagcagt tgcagcatgg ctggcaagca gcgaagcaag ctatgttacc 720
ggtattaccc tgtttgcaga tggtggtatg accctgtatc cgagctttca ggcaggtcgt 780
ggttaa 786
<210> 8
<211> 261
<212> PRT
<213> 巨大芽胞杆菌(Bacillus megaterium)
<400> 8
Met Tyr Lys Asp Leu Glu Gly Lys Val Val Val Ile Thr Gly Ser Ser
1 5 10 15
Thr Gly Leu Gly Lys Ser Met Ala Ile Arg Phe Ala Thr Glu 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 Ile 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 Pro Ser His Glu Met Ser Leu Ser Asp Trp Asn 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 Arg 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 Arg Leu Met Thr Lys 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 Arg Ala Asp
195 200 205
Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile
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 Leu Tyr Pro Ser Phe
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Gln Ala Gly Arg Gly
260
Claims (9)
1.一种老黄酶NemR-PS突变体，其特征在于，所述老黄酶NemR-PS突变体是将SEQ IDNO.2所示氨基酸序列中第275位天冬氨酸突变替换为甘氨酸，同时第351位苯丙氨酸突变替换为丙氨酸获得的。
2.权利要求1所述老黄酶NemR-PS突变体的编码基因。
3.含权利要求2所述老黄酶NemR-PS突变体编码基因的重组基因工程菌。
4.权利要求1所述老黄酶NemR-PS突变体在制备(S)-香茅醇中的应用。
5.如权利要求4所述的应用，其特征在于，所述应用为：以共表达老黄酶NemR-PS突变体、醇脱氢酶和葡萄糖脱氢酶的基因工程菌经诱导表达获得的湿菌体为催化剂，以(E/Z)-柠檬醛为底物，以葡萄糖为辅底物，加入助溶剂和辅酶NADP+，以pH 5.5～8.0的缓冲液为反应介质构成转化体系，在20～45℃、200～700rpm条件下转化反应12～24h，获得含(S)-香茅醇的反应液，反应液分离纯化，获得(S)-香茅醇；所述助溶剂为异丙醇。
6.如权利要求5所述的应用，其特征在于，所述转化体系中，催化剂加入量以转化体系体积计为10～100g/L；底物加入终浓度以转化体系体积计为300～500mM；所述葡萄糖加入终浓度与底物加入终浓度的比值为0.5：1～5：1；所述NADP+加入终浓度以转化体系体积计为0～1.0mM；所述助溶剂体积加入量以转化体系体积计为0.5～8％。
7.如权利要求5所述的应用，其特征在于，所述湿菌体按如下方法制备：将共表达老黄酶NemR-PS突变体、醇脱氢酶和葡萄糖脱氢酶的工程菌接种至含100μg/mL卡那霉素的LB液体培养基中，37℃、200rpm培养12h，获得种子液，将种子液以体积浓度2％的接种量接种至新鲜的含100μg/mL卡那霉素的LB液体培养基中，37℃、200rpm培养至OD600为0.6～0.8，再加入终浓度为0.5mM的IPTG，24℃诱导16h，获得诱导培养液，再将诱导培养液于4℃和8000rpm下离心10min，弃去上清液；然后用pH 8.0、50mM Tris-HCl缓冲液重悬菌体，于4℃和8000rpm下离心10min，弃去上清液，收集湿菌体；所述醇脱氢酶核苷酸序列如SEQ ID NO.5所示；所述葡萄糖脱氢酶核苷酸序列如SEQ ID NO.7所示。
8.如权利要求5所述的应用，其特征在于，在反应过程中，底物一次加入或者恒速流加方式加入；所述底物恒速流加按如下方法进行：底物和辅底物分别通过恒速微量泵注入到反应体系中，底物和辅底物恒速流加速度分别为0.5mmol/h和1.5mmol/h。
9.如权利要求5所述的应用，其特征在于，所述共表达老黄酶NemR-PS突变体、醇脱氢酶和葡萄糖脱氢酶的基因工程菌按如下方法构建：将SEQ ID NO.5所示醇脱氢酶YsADH编码基因插入pACYCDuet-1载体上的第一克隆位点Nco I和Hind III之间，将SEQ ID NO.7所示葡萄糖脱氢酶BmGDHM6编码基因插入pACYCDuet-1载体上的第二克隆位点Nde I和Xho I之间，得到重组载体pACYCDuet-1-YsADH-BmGDHM6；将重组载体pACYCDuet-1-YsADH-BmGDHM6导入宿主细胞E.coli BL21(DE3)，得到重组菌E.coli BL21(DE3)/pACYCDuet-1-YsADH-BmGDHM6；将携带老黄酶NemR-PS突变体编码基因的重组载体导入基因工程菌E.coli BL21(DE3)/pACYCDuet-1-YsADH-BmGDHM6感受态细胞中，得到共表达基因工程菌。
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