CN113564136B - L-泛解酸内酯脱氢酶及其突变体、共表达工程菌及应用 - Google Patents
L-泛解酸内酯脱氢酶及其突变体、共表达工程菌及应用 Download PDFInfo
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Abstract
L‑泛解酸内酯脱氢酶及其突变体、共表达工程菌及应用。本发明涉及基因工程领域，具体涉及一种新的L‑泛解酸内酯脱氢酶(RhoLPLDH)及其突变体，编码基因，含有该突变体基因的重组载体以及共表达工程菌和应用。所述L‑泛解酸内酯脱氢酶氨基酸序列如SEQ ID NO:1所示，编码基因如SEQ ID NO:2所示。本发明提供了高催化活性的L‑泛解酸内酯脱氢酶RhoLPLDH及其突变体，RhoLPLDH突变体与分子伴侣pGro7共表达进一步提高了目的蛋白可溶性表达。其中突变体RhoLPLDH‑V241I‑pGro7在催化100mM底物时，产物浓度随时间的推移而逐渐升高，8h内可反应完成，底物转化率大于99％，没有中间产物酮泛解酸内酯的生成。突变体RhoLPLDH‑L254I‑pGro7在催化100mM底物时，产物浓度随时间的推移而逐渐升高，8h时底物转化率达到93％，16h监测时，底物转化率大于99％。
Description
(一)技术领域
本发明属于生物技术领域，具体涉及一种新的L-泛解酸内酯脱氢酶(RhoLPLDH)及其突变体，编码基因，含有该突变体基因的重组载体以及共表达工程菌和应用。
(二)背景技术
D-泛酸钙又称维生素B5，是辅酶A的组成部分，已经广泛应用于食品、饲料、医药、化工和化妆品等行业。D-(-)-泛解酸内酯，也称为(R)-泛解酸内酯，化学结构为D-(-)-泛解酸的γ-内酯，是合成D-(+)-泛酸的关键手性中间体。目前工业化合成D-泛解酸内酯采用化学法和水解酶拆分法相结合的技术路线，从异丁醛和甲醛为起始原料出发，化学法合成DL-泛解酸内酯；其中的D-泛解酸内酯可以被D-泛解酸内酯水解酶立体选择性水解生成D-泛解酸，再经内酯化生成D-泛解酸内酯，留下的L-泛解酸内酯经化学消旋化为DL-泛解酸内酯重新循环拆分。DL-泛解酸内酯的拆分是合成D-泛解酸内酯中的关键步骤。水解酶的手性拆分制备工艺需要L-泛解酸内酯的消旋，D-泛解酸和L-泛解酸内酯的分离以及D-泛解酸经酸化成环形成D-泛解酸内酯。水解酶催化的手性拆分法尽管工艺成熟，仍然存在过程复杂、能耗物耗较高、需要消耗较多的酸碱等问题。鉴于此，开发更为直接、高效、环保的D-泛解酸内酯不对称合成方法替代现有的手性拆分技术将具有重要的应用价值。D-泛解酸内酯可通过氧化还原法不对称合成，该方法可通过两条不同途径实现，第一条途径先以L-泛解酸内酯脱氢酶催化L-泛解酸内酯脱氢生成酮基泛解酸内酯，接着酮基泛解酸内酯自发水解形成酮基泛解酸，然后在D-酮基泛解酸还原酶的作用下生成D-泛解酸，接着D-泛解酸在酸的作用下闭环形成D-泛解酸内酯；更为简捷的第二条途径是以混旋的DL-泛解酸内酯为底物，利用立体选择性专一的L-泛解酸内酯脱氢酶催化L-泛解酸内酯脱氢生成酮基泛解酸内酯，接着酮基泛解酸内酯在D-酮基泛解酸内酯还原酶催化下不对称生成D-泛解酸内酯。与现有的水解酶催化通路相比，第二条途径工艺更为简单，混旋的底物经生物催化直接得到光学纯产物，不需要消旋步骤，也不需要内酯和酸的分离步骤；因此，第二条途径的氧化还原酶不对称合成D-泛解酸内酯的方法是一种非常有前景的生物水解酶法替代者。该途径中L-泛解酸内酯的脱氢是其关键步骤之一，由L-泛解酸内酯脱氢酶催化。目前已知的L-泛解酸内酯脱氢酶数量不多，缺乏催化性能优异的L-泛解酸内酯脱氢酶限制了氧化还原酶法在不对称合成D-泛解酸内酯中的应用。研究较多的L-泛解酸内酯脱氢酶包括源自红串红球菌的L-泛解酸内酯脱氢酶和源自星状诺卡氏菌的L-泛解酸内酯脱氢酶。源自红串红球菌的L-泛解酸内酯脱氢酶在大肠杆菌系统中可溶性较差，这一特性加大了多酶组合催化的难度。以红串红球菌L-泛解酸内酯脱氢酶基因在其原始宿主中增强表达的基因工程菌AKU2103为生物催化剂，催化0.768M的L-泛解酸内酯脱氢反应144h，反应的转化率为91.9％。考虑到L-泛解酸内酯脱氢产物为酮基泛解酸内酯，酮基泛解酸内酯容易自发水解为酮基泛解酸。在上述反应144h后，需进一步添加表达了酮基泛解酸还原酶的重组大肠杆菌为生物催化剂，将产生的酮基泛解酸在还原反应24h后全部转化为D-泛解酸。最终，D-泛解酸再经酸化处理生成D-泛解酸内酯(SiD,Urano N,Nozaki S,et al.L-Pantoyl lactone dehydrogenase fromRhodococcus erythropolis:genetic analyses and application to thestereospecific oxidation of L-pantoyl lactone.Applied Microbiology andBiotechnology,2012,95:431-440)。此外，源自星状诺卡氏菌的L-泛解酸内酯脱氢酶虽然有较详细地酶学性质研究(Kataoka M,Shimizu S,Yamada H.Purification andcharacterization of a novel FMN-dependent enzyme:membrane-bound L-(+)-pantoyllactone dehydrogenase from Nocardia asteroides.European Journal ofBiochemistry,1992,204,799-806)，其编码基因并未被鉴定，阻碍了其在生物催化中的进一步应用。
筛选高催化活性的L-泛解酸内酯脱氢酶是手性翻转合成D-泛解酸内酯的重要步骤。目前从Rhodococcus hoagii中筛选到一个L-泛解酸内酯脱氢酶(RhoLPLDH)，RhoLPLDH在多酶级联催化L-泛解酸内酯手性翻转合成D-泛解酸内酯中具有重要的应用前景。
(三)发明内容
本发明的目的是提供一种新的L-泛解酸内酯脱氢酶(RhoLPLDH)及其突变体，编码基因，含有该突变体基因的重组载体以及共表达工程菌和应用，以解决现有L-泛解酸内酯催化剂(菌体)用量大、催化活性不高等问题
本发明采用的技术方案是：
一种L-泛解酸内酯脱氢酶(RhoLPLDH)，其氨基酸序列如SEQ ID NO:1所示。所述L-泛解酸内酯脱氢酶源自白色红球菌，编码基因核苷酸序列为SEQ ID NO.2所示。
一种L-泛解酸内酯脱氢酶突变体，由SEQ ID NO:1所示氨基酸经单点突变或者定点半饱和突变获得，突变的位点为254位，第241位，第272位或第308位。
优选的，所述突变体为下列之一：(1)SEQ ID NO:1所示氨基酸第254位亮氨酸突变为异亮氨酸(L254I)；(2)SEQ ID NO:1所示氨基酸第241位缬氨酸突变为异亮氨酸(V241I)；(3)SEQ ID NO:1所示氨基酸第254位亮氨酸突变为异亮氨酸，且第272位丝氨酸突变为天冬氨酸(L254I/S272D)；(4)SEQ ID NO:1所示氨基酸第254位亮氨酸突变为异亮氨酸，且第308位缬氨酸变为亮氨酸(L254I/V308L)。
本发明还涉及编码所述L-泛解酸内酯脱氢酶或其突变体的基因。本发明RhoLPLDH及突变体碱基序列全长均为1203bp，从第一个碱基起至第1203个碱基止，起始密码子为ATG，终止密码子为TAA。
本发明所述RhoLPLDH突变体的获取是采用定点突变技术与迭代饱和突变技术，使用该技术对RhoLPLDH的基因(SEQ ID NO.2)进行突变，将获得的突变质粒以热击方式转入E.coli BL21(DE3)感受态细胞，对获得菌株进行接种、转接、诱导、菌体回收，利用重悬菌液催化L-泛解酸内酯，具体方法如下：第一步将对照菌E.coli BL21(DE3)/pET28bpET28b(+)-RhoLPLDH活化并提取质粒pET28b(+)-RhoLPLDH，并保存于-20℃。第二步通过SWISS-MODEL进行同源建模，获得RhoLPLDH的三维结构；然后再通过HOTSPOT WIZARD预测RhoLPLDH的活性中心和相关的氨基酸。获得影响底物与RhoLPLDH结合的关键氨基酸位点Leu 254、Val241、Ser272、Val308。以pET28b(+)-RhoLPLDH为模板质粒，通过对Leu 254、Val 241进行定点半饱和突变，获得突变质粒，并转化，获得突变体文库。用氧化还原指示剂2,6-二氯酚靛酚(DCPIP)在脱氢酶的催化反应中获得H+，由氧化态的蓝色变为还原态的无色，氧化态在600nm波长下具有特征吸收峰的原理建立高通量筛选方法，利用高通量方法对RhoLPLDH定点半饱和突变文库进行优势突变菌株的筛选，得到优势体，再利用气相复筛获得优势突变L254I和V241I，获得RhoLPLDH突变体菌株E.coli BL21(DE3)/pET28b(+)-RhoLPLDH-L254I(记为RhoLPLDH-L254I)和E.coli BL21(DE3)/pET28b(+)-RhoLPLDH-V241I(记为RhoLPLDH-V241I)。再以突变菌株的重组质粒pET28b(+)-RhoLPLDH-L254I为模板，通过对Ser272和Val308分别进行定点半饱和突变，获得突变质粒，并转化。利用上述同样的高通量方法得到优势突变体，再利用气相复筛获得优势突变S272D和V308L，获得RhoLPLDH双突变菌株E.coli BL21(DE3)/pET28b(+)-RhoLPLDH-L254I/S272D(记为RhoLPLDH-L254I/S272D)和E.coli BL21(DE3)/pET28b(+)-RhoLPLDH-L254I/V308L(记为RhoLPLDH-L254I/V308L)。发现突变体菌株RhoLPLDH-L254I、RhoLPLDH-V241I、RhoLPLDH-L254I/S272D和RhoLPLDH-L254I/V308L相较于其出发菌株RhoLPLDH的比细胞活力分别提高了0.53倍、0.27倍、0.15倍和0.19倍。
本发明还涉及含有所述编码基因的表达载体。
本发明还涉及所述L-泛解酸内酯脱氢酶与分子伴侣pGro7构建的共表达工程菌。本发明所述RhoLPLDH突变体与分子伴侣pGro7共表达的工程菌是将RhoLPLDH突变体基因的重组质粒导入分子伴侣菌E.coli BL21(DE3)-pGro7制备的感受态细胞中，在卡那霉素和氯霉素的双抗性固体培养基中筛选获得。RhoLPLDH突变体与分子伴侣pGro7共表达工程菌经接种、转接、诱导和菌体回收后，湿菌体用于测试比细胞活力。发现共表达菌株RhoLPLDH-pGro7、RhoLPLDH-L254I-pGro7、RhoLPLDH-V241I-pGro7、RhoLPLDH-L254I/S272D-pGro7和RhoLPLDH-L254I/V308L-pGro7相较于出发菌株RhoLPLDH的比细胞活力分别提高了0.44、0.98、0.92、0.73和1.03倍。
所述共表达工程菌按如下方法获得：将L-泛解酸内酯脱氢酶编码基因的重组质粒导入分子伴侣菌E.coli BL21(DE3)-pGro7制备的感受态细胞中，在卡那霉素和氯霉素的双抗性固体培养基中筛选获得所述共表达工程菌。
本发明还涉及所述共表达工程菌在微生物催化制备D-泛解酸内酯中的应用。用RhoLPLDH及其突变体与分子伴侣pGro7重组菌作为生物催化剂，可用于催化L-泛解酸内酯生成酮泛解酸内酯，进一步制备D-泛解酸内酯。
具体的，所述的应用为：L-泛解酸内酯脱氢酶、共轭聚酮还原酶和葡萄糖脱氢酶三酶偶联催化L-泛解酸内酯构型反转制备D-泛解酸内酯。L-泛解酸内酯脱氢酶催化L-泛解酸内酯构型反转制备D-泛解酸内酯的反应示意图如图1所示，具体方法为：将含RhoLPLDH突变体和分子伴侣pGro7共表达的工程菌经诱导培养获得的湿菌体和含共轭聚酮还原酶的工程菌以及含葡萄糖脱氢酶基因的工程菌经诱导培养获得的湿菌体混合，以混合菌体为催化剂，以L-泛解酸内酯为底物，以葡萄糖为辅底物，以pH 7.0、50mM的PB缓冲液(0.05MNa2HPO4，0.05M NaH2PO4)为反应介质构成转化体系，在30～40℃、600～800rpm(优选30℃、800rpm)条件下进行反应，制得D-泛解酸内酯。
所述转化体系中，底物加入终浓度30～250mM(优选50～100mM)，葡萄糖加入终浓度45～375mM(优选75～150mM)，催化剂用量以菌体干重计(DCW细胞干重)为4～25g/L，所述混合菌体中RhoLPLDH突变体和分子伴侣pGro7共表达的工程菌经诱导培养获得的湿菌体与含共轭聚酮还原酶的工程菌以及含葡萄糖脱氢酶基因的工程菌经诱导培养获得的湿菌体以干重比1.0～20:2:1(w/w/w)，优选10:2:1混合。所述共轭聚酮还原酶基因为密码子优化后核苷酸序列，核苷酸序列为SEQ ID NO.3所示。共轭聚酮还原酶原始序列(GenBankNO.CAG61069.1)来自光滑念珠菌Candida glabrata。所述葡萄糖脱氢酶基因(GenBankNO.KM817194.1)来自Exiguobacterium sibirium DSM 17290，核苷酸序列为SEQ ID NO.4所示。
进一步，所述湿菌体按如下方法制备：将含RhoLPLDH突变体和分子伴侣pGro7共表达的工程菌接种到含终浓度50μg/mL卡那霉素和25μg/mL氯霉素的LB液体培养基中，37℃培养10h，获得种子液；将种子液以体积浓度1.0％的接种量接种到新鲜的含终浓度50μg/mL卡那霉素和25μg/mL氯霉素的LB液体培养基中，同时添加0.5g/L L-阿拉伯糖用于诱导分子伴侣蛋白，37℃、180rpm培养2h(OD600＝0.4-0.6)，培养液中加入终浓度为0.1mM异丙基硫代半乳糖苷(Isopropylβ-D-thiogalactoside，IPTG)，28℃培养12h后，4℃、8000rpm离心10min，获得含RhoLPLDH突变体蛋白和分子伴侣蛋白的湿菌体；所述含葡萄糖脱氢酶基因的工程菌经诱导培养获得的湿菌体制备方法中抗生素为50μg/mL卡那霉素，无需添加L-阿拉伯糖，诱导温度为28℃，其他同RhoLPLDH突变体和分子伴侣共表达的湿菌体；所述含共轭聚酮还原酶基因的工程菌经诱导培养获得的湿菌体制备方法中抗生素为25μg/mL氯霉素，无需添加L-阿拉伯糖，诱导温度为18℃，其他同RhoLPLDH突变体载体和分子伴侣共表达的湿菌体。
本发明RhoLPLDH突变体、共轭聚酮还原酶和葡萄糖脱氢酶基因工程菌的接种、转接、诱导、菌体回收，培养基可为本领域任何可使菌体生长并产生本发明的培养基，优选LB培养基：胰蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，蒸馏水溶解，调节pH 7.0。培养方法和培养条件没有特殊的限制，培养方法和条件可以根据宿主类型和培养方法等因素进行优化。
本发明的有益效果主要体现在：本发明提供了高催化活性的L-泛解酸内酯脱氢酶RhoLPLDH及其突变体，RhoLPLDH突变体与分子伴侣pGro7共表达进一步提高了目的蛋白可溶性表达。其中突变体RhoLPLDH-V241I-pGro7在催化100mM底物时，产物浓度随时间的推移而逐渐升高，8h内可反应完成，底物转化率大于99％，没有中间产物酮泛解酸内酯的生成。突变体RhoLPLDH-L254I-pGro7在催化100mM底物时，产物浓度随时间的推移而逐渐升高，8h时底物转化率达到93％，16h监测时，底物转化率大于99％。所有突变体及出发菌株在转化16h时，底物转化率均大于99％，从反应进程分析，突变体与出发菌株比较，反应速度进一步提高，为多酶级联催化L-泛解酸内酯手性翻转合成D-泛解酸内酯提供了基础。
(四)附图说明
图1为L-泛解酸内酯脱氢酶RhoLPLDH、共轭聚酮还原酶CglCPR和葡萄糖脱氢酶EsGDH三酶偶联催化L-泛解酸内酯构型反转制备D-泛解酸内酯的反应示意图。
图2为GC信号值(pA)与中间产物酮泛解酸内酯相应浓度(mM)的标准曲线。
图3为L-泛解酸内酯、酮泛解酸内酯和D-泛解酸内酯的气相色谱图。
图4为L-泛解酸内酯脱氢酶及突变体与分子伴侣共表达SDS-PAGE图。
图5为L-泛解酸内酯脱氢酶及突变体与分子伴侣共表达重组菌比细胞酶活。
图6为GC信号值(pA)与产物D-泛解酸内酯相应浓度(mM)的标准曲线。
图7为RhoLPLDH及突变体与分子伴侣共表达菌分别偶联CglCPR、EsGDH三酶催化50mM L-泛解酸内酯构型反转制备D-泛解酸内酯时间进程图。
图8为RhoLPLDH及突变体与分子伴侣共表达菌分别偶联CglCPR、EsGDH三酶催化100mM或250mM L-泛解酸内酯构型反转制备D-泛解酸内酯时间进程图。
(五)具体实施方式
下面结合具体实例对本发明做进一步详细地说明，但本发明并不限于以下实施例：
本发明中涉及到多种物质的添加量、含量及浓度，其中所述的百分含量，除特别说明外，皆指质量百分含量。
实施例1：L-泛解酸内酯脱氢酶突变体文库的构建及筛选
1、出发菌株：
以实验室保藏工程菌E.coli BL21(DE3)/pET28b(+)-RhoLPLDH为原始菌株，原始菌株构建过程如下：RhoLPLDH核酸序列(SEQ ID NO.2)连接到pET28b(+)的Nco I与Xho I酶切位点之间，形成重组质粒，重组质粒转化到E.coli BL21(DE3)感受态细胞中，在卡那霉素抗性固体LB培养基中筛选转化子，接种至卡那霉素抗性LB液体培养基中，经测序验证后保存在15％甘油中，-80℃保存。活化并提取质粒pET28b(+)-RhoLPLDH，其中L-泛解酸内酯脱氢酶RhoLPLDH氨基酸序列为SEQ ID NO.1所示，编码基因序列为SEQ ID NO.2所示。
2、单突变：
(1)突变文库的构建
RhoLPLDH突变体文库的制备通过定点突变来实现，以原始菌株中载体pET28b(+)-RhoLPLDH为模板，采用表1引物，进行聚合酶链式反应(PCR)。将经Clean-up纯化试剂盒(Axygen，USA)纯化所得的重组质粒转移到大肠杆菌BL21(DE3)感受态细胞中，并将克隆子接种至20mL LB平板培养基中，在37℃培养12～16h。
(2)初筛
随机挑选平板上的阳性克隆子及原始菌株，接种于96孔板中，加入1000μL LB培养基(含50μg/mL的卡那霉素)，在37℃，180rpm条件下培养10h，获得种子液。各转接50μL种子液于另一块新的96孔板(加有1000μL含50μg/mL的卡那霉素的LB培养基)中，37℃，180rpm振荡培养4h后，加入IPTG(终浓度0.10mM)，转至28℃培养12h。所得的细胞通过96孔板离心机，4000rpm，4℃下离心10min，得到突变体的湿菌体。
将含湿菌体的96孔板每个孔中加入300μL磷酸钠缓冲溶液(50mM pH 7.0)重悬细胞，再取100μL菌悬液加至96孔酶标板的对应位置，分别加入终浓度为100μM的2,6-二氯酚靛酚(DCPIP)，再分别加入终浓度为200μM L-泛解酸内酯起始反应，在酶标仪Kinetics(MDSpectraMax M5，USA)模式下，在温度30℃间隔30s测定5min内OD600吸光值的变化。相应地，突变体酶活越高，OD600的下降越多，从而筛选出突变文库内活力相对较高的突变体，用于进一步的复筛并进行测序验证。
(3)GC复筛
对步骤(2)获得的突变体进行优势突变体的筛选，将优势突变体经摇瓶发酵获得湿菌体，湿菌体用于复筛反应，复筛条件如下：将获得的突变体湿菌体以干重2g/L的量加入pH 7.0的PB(50mM)中重悬，再加入终浓度4mM L-泛解酸内酯，在恒温震荡仪中30℃、1200rpm条件下进行反应30min，取反应液200μL加入50μL 6M盐酸(酸化)，加入200μL乙酸乙酯萃取3次，合并乙酸乙酯相。采用GC检测L-泛解酸内酯和酮泛解酸内酯的浓度和转化率。以产物酮泛解酸内酯的转化率为指标，筛选获得优势菌株。
GC信号值(pA)与中间产物酮泛解酸内酯相应浓度(mM)的标准曲线为y＝63.277x+1.2139，R2＝0.9994，标准曲线为图2所示。
转化率＝酮泛解酸内酯物质的量/(酮泛解酸内酯物质的量+L-泛解酸内酯物质的量)。
将获得优势菌株送杭州擎科生物技术有限公司测序，并保存在-80℃冰箱。最终筛选得到优势突变体为RhoLPLDH-L254I和RhoLPLDH-V241I。
3、双突变
以菌株E.coli BL21(DE3)/pET28b(+)-RhoLPLDH-L254I中载体pET28b(+)-RhoLPLDH-L254I为模板，设计定点半饱和突变引物，进行聚合酶链式反应(PCR)。按照高通量筛选、气相复筛的步骤在RhoLPLDH-L254I突变体的基础上进一步筛选优势突变。结果进一步筛选到双突变体RhoLPLDH-L254I/S272D和RhoLPLDH-L254I/V308L。最终获得RhoLPLDH突变体菌株E.coli BL21(DE3)/pET28b(+)-RhoLPLDH-L254I/S272D和BL21(DE3)/pET28b(+)-RhoLPLDH-L254I/V308L。
PCR反应体系(25μL)：1μL正向引物(100μM)，1μL反向引物(100μM)，12.5μL 2×Phanta缓冲液，0.5μL dNTP混合物(各10mM)，1μL质粒模板，0.5μL DNA聚合酶Phanta(诺唯赞，中国)和8.5μL超纯水。
根据Phanta Super-Fidelity DNA聚合酶手册设置的PCR程序如下：95℃预变性5min，然后30个循环(95℃变性15s，55℃退火15s，72℃延伸4min)，72℃终延伸10min，16℃保温。
气相检测条件：色谱柱BGB174(30m×0.25mm,0.25μm)，载气：氦气，流速：0.6mL/min，进样口、检测器温度：250℃；进样量：1μL；分流比：30:1；程序：170℃，8min。D-泛解酸内酯、L-泛解酸内酯和酮泛解酸内酯保留时间分别为：5.4min，5.6min和5.9min。D-泛解酸内酯、L-泛解酸内酯和酮泛解酸内酯气相色谱图如图3所示。
4、催化活性
分别将单突变株、双突变株和对照株菌液作为催化剂，以L-泛解酸内酯为底物，比较各突变体比细胞活力。反应体系选择为1mL，催化剂用量菌体干重2g/L，底物终浓度1mM，pH 7.0、50mM PB缓冲液为反应介质，30℃、1200rpm涡旋振荡反应30min，取反应液200μL加入50μL 6M盐酸(酸化)，加入200μL乙酸乙酯萃取3次，合并乙酸乙酯相。乙酸乙酯样品，采用实施例1所述GC检测L-泛解酸内酯、酮泛解酸内酯的浓度。
细胞酶活单位(U)定义为：在30℃、pH 7.0条件下，每分钟生成1μmol酮基泛解酸内酯所需的酶量定义为一个酶活单位U。比细胞酶活定义为每克菌体所具有的活力单位数，U/g。
各突变体比细胞酶活示于表2。
表1：L-泛解酸内酯脱氢酶定点半饱和突变引物设计
备注：下划线内容为定点半饱和突变的简并密码子。
表2：L-泛解酸内酯脱氢酶突变体比细胞酶活
实施例2：L-泛解酸内酯脱氢酶与分子伴侣共表达菌株的构建
将pET28b(+)-RhoLPLDH和pET28b(+)-RhoLPLDH-L254I、pET28b(+)-RhoLPLDH-V241I、pET28b(+)-RhoLPLDH-L254I/S272D、pET28b(+)-RhoLPLDH-L254I/V308L重组质粒分别通过热激转化到pGro7感受态细胞(分子伴侣菌E.coli BL21(DE3)-pGro7为实验室构建：将pGro7伴侣质粒导入E.coli BL21(DE3)获得。pGro7分子伴侣质粒购自Takara Bio.)中，在含终浓度25μg/mL氯霉素和50μg/mL卡那霉素双抗性的LB固体培养基中筛选L-泛解酸内酯脱氢酶与分子伴侣共表达的阳性克隆子，得到E.coli BL21(DE3)/pET28b(+)-RhoLPLDH-pGro7、E.coli BL21(DE3)/pET28b(+)-RhoLPLDH-L254I-pGro7、E.coli BL21(DE3)/pET28b(+)-RhoLPLDH-V241I-pGro7、E.coli BL21(DE3)/pET28b(+)-RhoLPLDH-L254I/S272D-pGro7和E.coli BL21(DE3)/pET28b(+)-RhoLPLDH-L254I/V308L-pGro7。
将RhoLPLDH及突变体与分子伴侣共表达菌摇瓶发酵后，收集菌体。菌体经超声破碎后，聚丙烯凝胶电泳(SDS-PAGE)验证L-泛解酸内酯脱氢酶与分子伴侣蛋白的表达，结果如图4所示。RhoLPLDH及突变体与分子伴侣共表达菌与未加分子伴侣的E.coli BL21(DE3)/pET28b(+)-RhoLPLDH出发菌株相比，可溶性表达的蛋白量有所提高。
实施例3：L-泛解酸内酯脱氢酶及其突变体、共轭聚酮还原酶和葡萄糖脱氢酶的诱导表达
1、葡萄糖脱氢酶基因工程菌：将来自E.sibirium DSM 17290葡萄糖脱氢酶基因EsGDH(GenBank NO.KM817194.1)插入到pET28b(+)的Nco I和Xho I两个酶切位点之间，构建重组表达载体；并将此表达载体转入E.coli BL21(DE3)，挑取单菌落接种至LB培养基，37℃培养12h，测序确定葡萄糖脱氢酶构建成功，制得E.coli BL21(DE3)/pET28b(+)-EsGDH。
2、共轭聚酮还原酶基因工程菌：将来自光滑念珠菌Candida glabrata(GenBankNO.CAG61069.1)共轭聚酮还原酶基因CglCPR密码子优化后插入到pACYCDuet的Nco I和XhoI两个酶切位点之间，构建重组表达载体；并将此表达载体转入E.coli BL21(DE3)，挑取单菌落接种至LB培养基，37℃培养12h，测序确定共轭聚酮还原酶基因构建成功，获得E.coliBL21(DE3)/pACYCDuet-CglCPR。
3、诱导表达：将E.coli BL21(DE3)/pET28b(+)-EsGDH和E.coli BL21(DE3)/pACYCDuet-CglCPR分别接种到含有终浓度50μg/mL卡那霉素或终浓度25μg/mL氯霉素的10mL LB液体培养基中，37℃，180rpm条件下培养10h，获得种子液。将种子液以体积浓度1.0％(v/v)的接种量接种到新鲜的含有终浓度50μg/mL卡那霉素或终浓度25μg/mL氯霉素的100mL LB液体培养基摇瓶中，37℃、180rpm培养至OD600为0.6-0.8之间，再向培养液中加入终浓度为0.1mM IPTG，菌株E.coli BL21(DE3)/pACYCDuet-CglCPR置于18℃、其他菌株置于28℃培养12h后，4℃、8000rpm离心10min，获得相应的湿菌体细胞。
将实施例2出发菌株E.coli BL21(DE3)/pET28b(+)-RhoLPLDH-pGro7和突变菌株分别接种到含有终浓度50μg/mL卡那霉素和终浓度25μg/mL氯霉素二种抗生素的10mL LB液体培养基中，37℃，180rpm条件下培养10h，获得种子液。将种子液以体积浓度1.0％(v/v)的接种量接种到新鲜的含有终浓度50μg/mL卡那霉素和终浓度25μg/mL氯霉素以及终浓度0.5g/L阿拉伯糖的100mL LB液体培养基摇瓶中，37℃、180rpm培养至OD600为0.4-0.6之间，再向培养液中加入终浓度为0.1mM IPTG，置于28℃培养12h后，4℃、8000rpm离心10min，获得相应的湿菌体细胞。
以上获得的细胞产有相应的蛋白，可用于蛋白纯酶液的制备，也可用于粗酶液或全细胞催化L-泛解酸内酯构型反转制备D-泛解酸内酯。
4、催化活性
将步骤3诱导表达的L-泛解酸内酯脱氢酶和分子伴侣蛋白的湿菌体作为催化剂，以L-泛解酸内酯为底物，比较各突变体与分子伴侣共表达重组菌比细胞活力。反应体系选择为1mL，催化剂用量菌体干重2g/L，底物终浓度1mM，pH 7.0、50mM PB缓冲液为反应介质，30℃、1200rpm涡旋振荡反应30min，取反应液200μL加入50μL 6M盐酸(酸化)，加入200μL乙酸乙酯萃取3次，合并乙酸乙酯相。乙酸乙酯样品，采用实施例1所述GC检测L-泛解酸内酯、酮泛解酸内酯的浓度。
细胞酶活单位(U)定义为：在30℃、pH 7.0条件下，每分钟生成1μmol酮基泛解酸内酯所需的酶量定义为一个酶活单位U。比细胞酶活定义为每克菌体所具有的活力单位数，U/g。
各重组菌比细胞酶活示于图5。RhoLPLDH与分子伴侣共表达菌RhoLPLDH-pGro7相较于出发菌株RhoLPLDH的比细胞活力提高了0.44倍。表明分子伴侣pGro7提高RhoLPLDH的可溶性表达能够增强细胞的催化活力。突变体与分子伴侣共表达菌RhoLPLDH-L254I-pGro7、RhoLPLDH-V241I-pGro7、RhoLPLDH-L254I/S272D-pGro7和RhoLPLDH-L254I/V308L-pGro7比出发菌株RhoLPLDH的比细胞活力分别提高了0.98、0.92、0.73和1.03倍。
实施例4：原始及突变后的L-泛解酸内酯脱氢酶底物特异性考察
将实施例3基因工程菌E.coli BL21(DE3)/pET-28b(+)-RhoLPLDH-pGro7、E.coliBL21(DE3)/pET28b(+)-RhoLPLDH-L254I-pGro7、E.coli BL21(DE3)/pET28b(+)-RhoLPLDH-V241I-pGro7、E.coli BL21(DE3)/pET28b(+)-RhoLPLDH-L254I/S272D-pGro7和E.coliBL21(DE3)/pET28b(+)-RhoLPLDH-L254I/V308L-pGro7经诱导表达后所获得的湿菌体，用作为生物催化剂。分别以1mM的D-泛解酸内酯、L-泛解酸内酯、DL-泛解酸内酯为底物，利用全细胞催化考察RhoLPLDH及其突变体的底物特异性。L-泛解酸内酯脱氢酶催化L-泛解酸内酯脱氢的反应体系为1mL，分别含有：10mg/mL菌液，1mM底物和50mM磷酸盐缓冲液(pH 7.0)。在恒温振荡反应器中，30℃，1200rpm反应30min，取200μL反应液加入50μL 6M盐酸(酸化)，加入200μL乙酸乙酯充分萃取。萃取液离心后吸取上层有机相于离心管中，加入无水硫酸钠除水，下层水相再次加入200μL乙酸乙酯充分萃取。再离心取上层有机相合并至第一次萃取有机相中。取二次萃取乙酸乙酯有机相转移入气相样品瓶中用于气相色谱检测。底物特异性结果如表所示，源自Rhodococcus hoagii的L-泛解酸脱氢酶及其突变体不能催化D-泛解酸内酯，能催化L-泛解酸内酯和DL-泛解酸内酯。上述结果表明，源自Rhodococcus hoagii的L-泛解酸脱氢酶专一地作用于L-泛解酸内酯的脱氢反应。
表4：源自Rhodococcus hoagii的L-泛解酸内酯脱氢酶及其突变体的底物特异性
实施例5：RhoLPLDH及突变体催化50mM L-泛解酸内酯构型翻转制备D-泛解酸内酯
将实施例3方法制备的RhoLPLDH及突变体与分子伴侣共表达重组菌E.coli BL21(DE3)/pET-28b(+)-RhoLPLDH-pGro7、E.coli BL21(DE3)/pET28b(+)-RhoLPLDH-L254I-pGro7、E.coli BL21(DE3)/pET28b(+)-RhoLPLDH-V241I-pGro7、E.coli BL21(DE3)/pET28b(+)-RhoLPLDH-L254I/S272D-pGro7和E.coli BL21(DE3)/pET28b(+)-RhoLPLDH-L254I/V308L-pGro7湿菌体分别和共轭聚酮还原酶CglCPR、葡萄糖脱氢酶EsGDH湿菌体建立三酶偶联体系，一锅法催化L-泛解酸内酯构型翻转制备D-泛解酸内酯。
将实施例3方法制备的RhoLPLDH与分子伴侣共表达重组菌湿菌体和共轭聚酮还原酶CglCPR、葡萄糖脱氢酶EsGDH湿菌体以干重比20：2：1(w/w/w)混合成混合菌体，在5mL反应体系中，先将混合菌体用pH 7.0、50mM的PB缓冲液重悬，转化体系中混合菌体加入干重量为23g DCW/L，底物L-泛解酸内酯的投料量为50mM，葡萄糖浓度为75mM，以pH 7.0、50mM的PB缓冲液为反应介质构成转化体系，在30℃、800rpm条件下反应，1M NaOH调节反应pH，维持pH7.0-7.5。采用实施例1方法检测，RhoLPLDH-L254I-pGro7 6h能将底物完全转化成产物D-泛解酸内酯，底物转化率大于99％。所述RhoLPLDH及突变体与分子伴侣共表达菌24h转化率均大于99％，反应过程中未检测到中间产物酮泛解酸内酯的生成。
GC信号值(pA)与产物D-泛解酸内酯相应浓度(mM)的标准曲线为y＝83.584x-0.0204，R2＝0.9998，标准曲线为图6所示。
反应过程转化率见于图7。与RhoLPLDH-pGro7催化效果相比，突变体分子伴侣菌反应速率更快。RhoLPLDH-pGro7在6h转化率为86％，其他突变体分子伴侣菌6h转化率均高于86％。RhoLPLDH-L254I-pGro7 6h转化率接近100％。
实施例6：RhoLPLDH及突变体催化100mM L-泛解酸内酯构型翻转制备D-泛解酸内酯
将实施例3方法制备的RhoLPLDH及突变体与分子伴侣共表达重组菌E.coli BL21(DE3)/pET-28b(+)-RhoLPLDH-pGro7、E.coli BL21(DE3)/pET28b(+)-RhoLPLDH-L254I-pGro7、E.coli BL21(DE3)/pET28b(+)-RhoLPLDH-V241I-pGro7、E.coli BL21(DE3)/pET28b(+)-RhoLPLDH-L254I/S272D-pGro7和E.coli BL21(DE3)/pET28b(+)-RhoLPLDH-L254I/V308L-pGro7湿菌体分别和共轭聚酮还原酶CglCPR、葡萄糖脱氢酶EsGDH湿菌体建立三酶偶联体系，一锅法催化L-泛解酸内酯构型翻转制备D-泛解酸内酯。
将实施例3方法制备的RhoLPLDH与分子伴侣共表达重组菌湿菌体和共轭聚酮还原酶CglCPR、葡萄糖脱氢酶EsGDH湿菌体以干重比20：2：2(w/w/w)混合成混合菌体，在5mL反应体系中，先将混合菌体用pH 7.0、50mM的PB缓冲液重悬，转化体系中混合菌体加入干重量为24g DCW/L，底物L-泛解酸内酯的投料量为100mM，葡萄糖浓度为150mM，以pH 7.0、50mM的PB缓冲液为反应介质构成转化体系，在30℃、800rpm条件下反应，1M NaOH调节反应pH，维持pH7.0-7.5。采用实施例1方法检测，RhoLPLDH-V241I8h能将底物完全转化成产物D-泛解酸内酯，底物转化率大于99％。所述RhoLPLDH突变体及出发菌株16h转化率均大于99％，反应过程中未检测到中间产物酮泛解酸内酯的生成。反应过程转化率见于图8。与RhoLPLDH-pGro7催化效果相比，突变体分子伴侣菌反应速率更快。RhoLPLDH-V241I-pGro7 8h转化率大于99％，RhoLPLDH-L254I-pGro7 8h转化率为93％。RhoLPLDH-pGro7在8h转化率为73％。
实施例7：RhoLPLDH催化250mM L-泛解酸内酯构型翻转制备D-泛解酸内酯
将实施例3方法制备的RhoLPLDH与分子伴侣共表达重组菌E.coli BL21(DE3)/pET-28b(+)-RhoLPLDH-pGro7湿菌体和共轭聚酮还原酶CglCPR、葡萄糖脱氢酶EsGDH湿菌体建立三酶偶联体系，一锅法催化L-泛解酸内酯构型翻转制备D-泛解酸内酯。
将实施例3方法制备的RhoLPLDH与分子伴侣共表达重组菌湿菌体和共轭聚酮还原酶CglCPR、葡萄糖脱氢酶EsGDH湿菌体以干重比20：2：2(w/w/w)混合成混合菌体，在5mL反应体系中，先将混合菌体用pH 7.0、50mM的PB缓冲液重悬，转化体系中混合菌体加入干重量为24g DCW/L，底物L-泛解酸内酯的投料量为250mM，葡萄糖浓度为375mM，以pH 7.0、50mM的PB缓冲液为反应介质构成转化体系，在30℃、800rpm条件下反应，1M NaOH调节反应pH，维持pH7.0～7.5。采用实施例1方法检测，RhoLPLDH催化24h时底物转化率为71％。反应过程中未检测到中间产物酮泛解酸内酯的生成。反应过程转化率见于图8。
序列表
<110> 浙江工业大学
<120> L-泛解酸内酯脱氢酶及其突变体、共表达工程菌及应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 400
<212> PRT
<213> Rhodococcus hoagii
<400> 1
Met Ala Lys Asn Thr Trp Phe Glu Thr Val Ala Glu Ala Gln Arg Arg
1 5 10 15
Ala Lys Lys Arg Leu Pro Lys Ser Val Tyr Ala Ala Leu Val Ala Gly
20 25 30
Ser Glu Lys Gly Leu Thr Val Asp Asp Asn Ile Ala Ala Phe Ser Glu
35 40 45
Leu Gly Phe Ala Pro His Val Ala Gly Leu Ser Gly Glu Arg Asp Leu
50 55 60
Ser Thr Thr Val Met Gly Gln Pro Ile Ser Met Pro Val Met Ile Ser
65 70 75 80
Pro Thr Gly Val Gln Ala Val His Pro Asp Gly Glu Val Ala Val Ala
85 90 95
Arg Ala Ala Ala Ala Arg Gly Thr Ala Ile Gly Leu Ser Ser Phe Ala
100 105 110
Ser Lys Ser Ile Glu Glu Val Ala Ala Ala Asn Pro Gln Thr Phe Phe
115 120 125
Gln Met Tyr Trp Val Gly Asp Arg Asp Thr Leu Leu Gln Arg Met Glu
130 135 140
Arg Ala Arg Ala Ala Gly Ala Thr Gly Leu Ile Ile Thr Leu Asp Trp
145 150 155 160
Ser Phe Ser Asn Gly Arg Asp Trp Gly Ser Pro Ser Ile Pro Glu Lys
165 170 175
Met Asp Leu Lys Ala Met Phe Gln Phe Ala Pro Glu Gly Ile Thr Arg
180 185 190
Pro Lys Trp Leu Trp Glu Phe Ala Lys Thr Gly Lys Val Pro Asp Leu
195 200 205
Thr Thr Pro Asn Leu Ala Ala Pro Gly Gln Gln Pro Pro Thr Phe Phe
210 215 220
Gly Ala Tyr Gly Gln Trp Met Gly Thr Pro Leu Pro Thr Trp Glu Asp
225 230 235 240
Val Ala Trp Leu Arg Glu Gln Trp Gly Gly Pro Phe Met Leu Lys Gly
245 250 255
Val Met Arg Val Asp Asp Ala Lys Arg Ala Leu Asp Ala Gly Cys Ser
260 265 270
Ala Ile Ser Val Ser Asn His Gly Gly Asn Asn Leu Asp Gly Thr Pro
275 280 285
Ala Pro Ile Arg Ala Leu Pro Ala Ile Ala Glu Ala Val Gly Asp Gln
290 295 300
Leu Glu Val Val Leu Asp Gly Gly Ile Arg Arg Gly Ser Asp Val Val
305 310 315 320
Lys Ala Leu Ala Leu Gly Ala Arg Ala Val Met Ile Gly Arg Ala Tyr
325 330 335
Leu Trp Gly Leu Ser Ala Asn Gly Gln Ala Gly Val Glu Asn Val Leu
340 345 350
Asp Ile Leu Arg Gly Gly Ile Asp Ser Ala Val Leu Gly Leu Gly His
355 360 365
Lys Ser Ile His Asp Leu Ser Pro Asn Asp Leu Val Val Pro Glu Gly
370 375 380
Phe Arg Arg Asp Leu Gly Val Gly Leu Glu His His His His His His
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<210> 2
<211> 1203
<212> DNA
<213> Rhodococcus hoagii
<400> 2
atggccaaaa atacctggtt tgaaaccgtt gccgaagcac agcgccgtgc caaaaaacgt 60
ctgccgaaaa gtgtttatgc agcactggtg gccggtagtg aaaaaggtct gaccgttgat 120
gataatattg cagcctttag tgaactgggc tttgccccgc atgtggcagg tctgagtggt 180
gaacgtgatc tgagtaccac cgtgatgggc cagccgatta gcatgccggt tatgattagt 240
ccgaccggtg tgcaggccgt gcatccggat ggtgaagttg cagttgcacg cgccgcagca 300
gcccgcggta cagcaattgg tctgagcagt tttgcaagta aaagtattga agaagtggca 360
gcagccaatc cgcagacctt tttccagatg tattgggttg gtgaccgcga taccctgctg 420
cagcgtatgg aacgtgcacg cgccgccggt gccaccggtt taattattac cctggattgg 480
agctttagta atggtcgcga ttggggcagc ccgagtattc cggaaaaaat ggatctgaaa 540
gccatgtttc agtttgcacc ggaaggtatt acccgtccga aatggctgtg ggaatttgcc 600
aaaaccggca aagttccgga tctgaccacc ccgaatctgg cagcaccggg ccagcagccg 660
ccgacctttt tcggcgccta tggccagtgg atgggtaccc cgctgccgac ctgggaagat 720
gttgcctggc tgcgtgaaca gtggggcggt ccgtttatgc tgaaaggcgt gatgcgtgtt 780
gatgatgcca aacgtgccct ggatgccggt tgcagcgcaa ttagtgttag caatcatggc 840
ggcaataatc tggatggtac cccggccccg attcgcgcac tgcctgctat tgcagaagcc 900
gttggcgatc agctggaagt ggtgctggat ggtggtattc gtcgtggcag tgatgttgtt 960
aaagccctgg cactgggcgc acgtgccgtg atgattggtc gtgcatatct gtggggtctg 1020
agcgccaatg gtcaggcagg cgtggaaaat gtgctggata ttctgcgtgg tggtattgat 1080
agtgcagtgc tgggtctggg tcataaaagt attcatgatc tgagcccgaa tgatctggtt 1140
gttccggaag gttttcgtcg tgatctgggc gtgggtctcg agcaccacca ccaccaccac 1200
taa 1203
<210> 3
<211> 954
<212> DNA
<213> 未知(Unknown)
<400> 3
atggttaagc aggaattttt caagctgaat aatggccatg aaatgccggg tgtggcaatt 60
gtgggcaccg gcaccaaatg gcataaagtt aatgaaaccg atgaaaattt cagccagacc 120
ctggtggatc agctgaaata tgccctgagc ctgccgggcg tggttcatct ggatgccgcc 180
gaattttata tgacctatcg cgaagttggt cgcgccctgg ccgaaaccag caaaccgcgc 240
gatgaaattt ttattaccga taaatactgg accctgagta aagtgaccga aaatccgatt 300
gttggtctgg aaaccggcct gaaacgcctg ggtctggaat atgttgatct gtatctgctg 360
catagcccgt ttattagcaa agaaaccaat ggctttagtc tggaagaagc atggggtatg 420
atggaagaac tgtatcatag cggtaaagca aaaaatattg gcgtgagcaa ttttgccaaa 480
gaagatctgg aacgtgtgct gaaagtttgc aaagttaaac cgcaggtgaa tcagattgaa 540
ttcaatgcct ttctgcagaa tcagaccccg ggtatctata atttttgcaa acagaatgac 600
atccagctgg ccgcatatag cccgctgggt ccgctgcaga aaaaaccggc cgatggtaat 660
agccagccgt tttatagtta tattaacaaa ctggcccagc attataataa gaccccgggt 720
caggtgctgc tgcgctgggt taccaaacgt ggtgttgttg cagtgaccac cagcgaaaag 780
aaagaacgca ttaagcaggc acaggaaatt tttgaatttg atctgaaaga cgacgaagtg 840
accgaaatta ccaaactggg tctggatcat gaaccgctgc gcctgtattg gcatgatcag 900
tataataagt ataacagtga gagccagaaa gcacatcatc atcatcacca ttaa 954
<210> 4
<211> 789
<212> DNA
<213> 未知(Exiguobacterium sibirium)
<400> 4
atgggttata attctctgaa aggcaaagtc gcgattgtta ctggtggtag catgggcatt 60
ggcgaagcga tcatccgtcg ctatgcagaa gaaggcatgc gcgttgttat caactatcgt 120
agccatccgg aggaagccaa aaagatcgcc gaagatatta aacaggcagg tggtgaagcc 180
ctgaccgtcc agggtgacgt ttctaaagag gaagacatga tcaacctggt gaaacagact 240
gttgatcact tcggtcagct ggacgtcttt gtgaacaacg ctggcgttga gatgccttct 300
ccgtcccacg aaatgtccct ggaagactgg cagaaagtga tcgatgttaa tctgacgggt 360
gcgttcctgg gcgctcgtga agctctgaaa tacttcgttg aacataacgt gaaaggcaac 420
attatcaata tgtctagcgt ccacgaaatc atcccgtggc ctactttcgt acattacgct 480
gcttctaagg gtggcgttaa actgatgacc cagactctgg ctatggaata tgcaccgaaa 540
ggtatccgca ttaacgctat cggtccaggc gcgatcaaca ctccaattaa tgcagaaaaa 600
ttcgaggatc cgaaacagcg tgcagacgtg gaaagcatga tcccgatggg caacatcggc 660
aagccagagg agatttccgc tgtcgcggca tggctggctt ctgacgaagc gtcttacgtt 720
accggcatca ccctgttcgc agatggtggc atgaccctgt acccgagctt tcaggctggc 780
cgtggttga 789
Claims (7)
1.一种L-泛解酸内酯脱氢酶突变体，由SEQ ID NO:1所示氨基酸经单点突变或者定点半饱和突变获得，突变的位点为254位，第241位，第272位或第308位，所述突变体为下列之一：（1）SEQ ID NO:1所示氨基酸第254位亮氨酸突变为异亮氨酸；（2）SEQ ID NO:1所示氨基酸第241位缬氨酸突变为异亮氨酸；（3）SEQ ID NO:1所示氨基酸第254位亮氨酸突变为异亮氨酸，且第272位丝氨酸突变为天冬氨酸；（4）SEQ ID NO:1所示氨基酸第254位亮氨酸突变为异亮氨酸，且第308位缬氨酸变为亮氨酸。
2.编码权利要求1所述L-泛解酸内酯脱氢酶突变体的基因。
3.含有权利要求2所述编码基因的表达载体。
4.权利要求1所述L-泛解酸内酯脱氢酶突变体与分子伴侣pGro7构建的共表达工程菌。
5.如权利要求4所述的共表达工程菌，其特征在于所述共表达工程菌按如下方法获得：将L-泛解酸内酯脱氢酶突变体编码基因的重组质粒导入分子伴侣菌E. coli BL21(DE3)-pGro7制备的感受态细胞中，在卡那霉素和氯霉素的双抗性固体培养基中筛选获得所述共表达工程菌。
6.权利要求4所述共表达工程菌在微生物催化制备D-泛解酸内酯中的应用。
7.如权利要求6所述的应用，其特征在于所述的应用为：L-泛解酸内酯脱氢酶、共轭聚酮还原酶和葡萄糖脱氢酶三酶偶联催化L-泛解酸内酯构型反转制备D-泛解酸内酯；所述共轭聚酮还原酶的核苷酸序列如SEQ ID NO.3所示，所述葡萄糖脱氢酶的核苷酸序列如SEQID NO.4所示。
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