【作者】 吴黎诚；

【导师】 刘子铎； 吴高兵；

【作者基本信息】 华中农业大学 ， 微生物学， 2022， 博士

【摘要】 随着国民生活水平提高和科技进步,液晶显示（Liquid crystal display,LCD）已成为国民日常生活中不可缺少的一部分。反式-2-（（4-正丙基）环己基）-1,3-丙二醇是合成反-1,3-二恶烷基类液晶材料的关键中间体。顺式-4-丙基环己醇可作为起始原料,用于合成反式-2-（（4-正丙基）环己基）-1,3-丙二醇。目前,国内外厂家采用化学合成法生产的顺式-4-丙基环己醇售价较高,阻碍了该材料的运用和液晶材料技术的发展。相对于传统化学制造,生物酶催化反应条件温和、催化效率高、绿色可持续,并且生物酶催化制备的顺式-4-丙基环己醇具有顺式值高、成本低等优点。而目前只有一篇采用野生菌转化制备顺式-4-丙基环己醇的报道,且转化率较低,难以产业化。因此,采用酶工程技术开发新的酶及酶催化工艺来制备顺式-4-丙基环己醇对于LCD工业具有重要的产业意义。首先,本研究从本实验室保存的醇脱氢酶（Alcohol dehydrogenase,ADH）重组菌资源库中筛选获得可高效催化4-丙基环己酮为顺式-4-丙基环己醇的开菲尔乳杆菌（Lactobacillus kefiri）来源的醇脱氢酶突变体LK-TADH,并利用Auto Dock4.2.6软件进行了分子对接分析;其次,为了解决双酶偶联工艺生产成本高的问题,本研究对LK-TADH进行了定点突变进化,筛选出以异丙醇为氢供体催化制备顺式-4-丙基环己醇的酶突变体LK-TADH-1（H40R/A94T/F147L/L199H/A202L）,并进行了分子对接分析,又进一步开展了发酵工艺研究和中试研究;然后,为从源头解决生产过程中游离酶使用成本高、产物提取收率低等问题,本研究进行了固定化酶催化制备顺式-4-丙基环己醇的工艺开发与中试研究,首次实现了利用固定化醇脱氢酶催化制备高顺式值、低成本的顺式-4-丙基环己醇产品;最后,本研究对LK-TADH-1固定化酶在其它手性医药中间体制造中的应用进行了拓展研究,提升了该固定化酶的产业化价值。主要研究结果如下:（1）从实验室保存的醇脱氢酶重组菌资源库中筛选获得可高效催化4-丙基环己酮为顺式-4-丙基环己醇的开菲尔乳杆菌（L.kefiri）来源的醇脱氢酶突变体LK-TADH（A94T/F147L/L199H/A202L）。利用LK-TADH与葡萄糖脱氢酶联用催化制备顺式-4-丙基环己醇。酶催化的最佳条件如下:温度35℃,p H值7.0-8.0,4-丙基环己酮浓度125 g/L,LK-TADH用酶量30 g/L,NAD⁺添加量0.1 g/L,葡萄糖脱氢酶用酶量10 g/L,葡萄糖与底物摩尔比1.2:1。在该条件下反应5 h后,4-丙基环己酮完全转化为顺式-4-丙基环己醇,提取收率为90.32%,产品的顺式值为99.5%,纯度为98.5%。分子对接结果表明,突变体中4-丙基环己酮与NADH之间的催化距离更近,4-丙基环己酮与LK-TADH的结合更加紧密。（2）为了解决双酶偶联工艺生产成本高等问题,理性设计突变位点,构建筛选出可利用异丙醇为氢供体催化4-丙基环己酮为顺式-4-丙基环己醇的突变体LK-TADH-1（H40R/A94T/F147L/L199H/A202L）。分子对接分析显示LK-TADH突变为LK-TADH-1后,酶与异丙醇之间的亲和力得到提升,异丙醇与NAD⁺催化原子距离缩短。进一步研究获得该酶的最佳催化条件如下:温度35℃,p H值7.0-8.0,底物与异丙醇摩尔比1:1.5,底物浓度150 g/L,LK-TADH-1酶用量20 g/L,NAD⁺添加量0.1 g/L。在该条件下反应6 h后,4-丙基环己酮完全转化为顺式-4-丙基环己醇,提取收率为92.5%,产品的顺式值为99.7%,纯度为98.8%。（3）对LK-TADH-1重组菌在10升罐中进行了发酵工艺优化,确定其最佳条件为:种子种龄OD₆₀₀值约4、接种量为5%、硫酸卡那霉素（Kana）终浓度75 mg/L、发酵p H 7.0、发酵溶解氧30%左右、补料速度80 m L/h、IPTG诱导浓度0.3 mmol/L、诱导时机为OD₆₀₀值约35、诱导温度26℃、结束发酵OD₆₀₀值约75。在此条件下,LK-TADH-1重组菌单位菌体比酶活最高为4664.8±84.2 U/g。LK-TADH-1重组菌在300 L发酵罐进行高密度发酵,单位菌体比酶活最高达4550 U/g左右。最后,利用LK-TADH-1重组菌破胞液（酶用量为10 g/L）在300 L催化罐中进行了顺式-4-丙基环己醇的中试生产,提取收率约为81.7%,产品纯度为98.7%。（4）开发了固定化酶催化制备顺式-4-丙基环己醇的工艺。确定了LK-TADH-1的最佳固定化条件为:固定化载体为环氧树脂LX-1000EP、固定化温度22℃、固定化p H8.0、菌体与载体比1.5:1,固定化时间24 h。该固定化酶的最佳酶催化条件为:4-丙基环己酮150 g/L,异丙醇与底物的摩尔比1.5:1,固定化酶加量25 g/L,NAD⁺添加量0.1 g/L,搅拌转速80 r/min,该固定化酶可反复使用25次以上。在300 L罐中进行了中试放大研究,反应8 h后,底物转化完全。提取收率约为96.2%,产品纯度为99.3%。（5）拓展了LK-TADH-1固定化酶在催化制备卢立康唑和替卡格雷关键手性中间体的应用。反应12 h后,250 g/L的2-氯-1-（2,4-二氯苯基）乙酮（CCPO）转化完全,提取收率约为94.2%,产品e.e.值为99.97%,产品纯度为99.5%;反应12 h后,250g/L的2-氯-1-（3,4-二氟苯基）乙酮（CFPO）转化完全,提取收率约为95.8%,产品e.e.值为99.91%,产品纯度为99.2%。更多还原

【Abstract】 With an improvement in living standards and the progress of technology,liquid crystal display（LCD）has become an indispensable part of our daily life.The wide use of electronic equipment has created a strong demand for LCDs.As a starting material,cis-4-propylcyclohexanol can be used to prepare trans-2-（4-alkylcyclohexyl）-1,3-propanediol.The price of cis-4-propylcyclohexanol produced by domestic and foreign manufacturers is high,which hinders the development of liquid crystal material technology.Therefore,it is of great significance for LCD industry to develop low cost and high quality cis-4-propylcyclohexanol production technology.Compared with traditional chemical manufacturing,cis-4-propylcyclohexanol catalyzed by biological enzymes has the advantages of high cis-value,low price,mild reaction conditions,high catalytic efficiency,green and sustainable.At present,there wss only one report that adopted the transformation process of wild bacteria,and its conversion rate was low,which was difficult to be industrialized.Therefore,it is of great industrial significance to develop a new recombinant enzyme route to prepare cis-4-propyl cyclohexanol.Firstly,the mutant of Lactobacillus kefiri alcohol dehydrogenase（LK-ADH）,LK-TADH（A94T/F147L/L199H/A202L）,was screened to efficiently catalyze4-propylcyclohexanone to cis-4-propylcyclohexanol from the ADH library that previously constructed in our laboratory,and then the small amount preparation of cis-4-propylcyclohexanol was carried out by using this enzyme coupled with glucose dehydrogenase（GDH）.Secondly,in order to solve the problem of high purification cost in the downstream of the existing process,LK-TADH was mutated and modified in this study,in order to construct the LK-TADH-1（H40R/A94T/F147L/L199H/A202L）catalyzing the preparation of cis-4-propylcyclohexanol with isopropanol as hydrogen donor,and the fermentation process and pilot study were also carried out.Then,in order to solve the problems of high cost of free enzyme and low extraction yield of this product,the process and pilot study of preparation of cis-4-propylcyclohexanol catalyzed by immobilized enzyme were carried out.Finally,this study expanded the application of LK-TADH-1 immobilized enzyme in the manufacture of other chiral compounds,and improved the industrial value of the immobilized enzyme.The main research results are as follows:（1）Firstly,LK-TADH（A94T/F147L/L199H/A202L）which could catalyze4-propylcyclohexanone to cis-4-propylcyclohexanol was screened from the laboratory enzyme library.Cis-4-propylcyclohexanol was prepared by combining LK-TADH-1 with GDH.The optimal conditions for catalysis were as follows:the catalytic temperature was35oC,the p H value of the reaction system was maintained between 7.0 and 8.0,the concentration of 4-propylcyclohexanone was 125 g/L,the amount of recombinant LK-TADH-1 cells was 30 g/L,the optimal amount of NAD⁺was 0.1 g/L,the amount of recombinant GDH cells was 10 g/L and the molar ratio of glucose to substrate was 1.2:1.Under these conditions,4-propylcyclohexanone could be completely converted to cis-4-propylcyclohexanol with the yield of 90.32%,the cis value of the product was99.5%,and with a purity of 98.5%.The results of molecular docking showed that the catalytic distance between 4-propylcyclohexanone and NADH in the mutant was closer,and the binding between 4-propylcyclohexanone and LK-TADH was closer.（2）Secondly,In order to solve the problem of high production cost of double enzyme coupling process,mutation sites were designed rationally,and a mutant LK-TADH-1（H40R/A94T/F147L/L199H/A202L）that can catalyze 4-propylcyclohexanone to cis-4-propylcyclohexanol using isopropanol as hydrogen donor was screened.Molecular docking analysis showed that after LK-TADH mutation to LK-TADH-1,the affinity between the enzyme and isopropanol was improved to a certain extent.In addition,after mutation,the distance between isopropanol and NAD+catalytic atoms is greatly shortened.The optimal catalytic conditions of this recombinant enzymes were as follows:The catalytic temperature was 35oC,the p H value of the reaction system was maintained between 7.0 and 8.0,the molar ratio of substrate to isopropanol was 1:1.5,the substrate concentration was 150 g/L,the dosage of recombinant LK-TADH-1 cells was 20 g/L,and the addition amount of NAD⁺was 0.1 g/L.Under these conditions,4-propylcyclohexanone could be completely converted to cis-4-propylcyclohexanol with a final yield of 92.5%,a cis value of 99.7%,and with a purity of 98.5%after 6 h reaction.（3）Then,the expression conditions of recombinant LK-TADH-1 in a 10 L fermenter were optimized,and the best expression conditions were determined as follows:The OD₆₀₀of secondary seeds was 4,the inoculum size was 5%,the final concentration of kanamycin was 75 mg/L,the fermentation p H was 7.0,the fermentation dissolved oxygen（DO）was 30%,the feeding speed was 80 m L/h,the IPTG induction concentration was0.3 mmol/L,the OD₆₀₀of the induction was 35,the induction temperature was 26oC,and the final OD₆₀₀was 75.Under these conditions,the maximum LK-TADH-1 activity was achieved to 4664.8±84.2 U/g.Subsequently,the recombinant LK-TADH-1 was cultured at high density in a 300 L fermenter,and the maximum enzyme activity reached about4550 U/g.Finally,the pilot production of cis-4-propylcyclohexanol was carried out by using free enzyme in a 300 L catalytic tank.After substrate conversion（150 g/L）,the final yield was about 81.7%,and with a purity of 98.8%.（4）Subsequently,the preparation process of cis-4-propylcyclohexanol by immobilized enzyme was developed for the first time and the pilot study was carried out.The optimal immobilization conditions of LK-TADH-1 were determined as follows:the optimal immobilization medium was LX-1000EP,the immobilization temperature was 22oC,the immobilization p H was 8.0,the cell dosage to support ratio was 1.5:1,and the immobilization time was 24 h.The optimal catalytic conditions of the immobilized enzyme were as follows:the 4-propylcyclohexanone concentration was 150 g/L,the substrate/isopropanol molar ratio was 1:1.5,the 2.5%immobilized enzyme addition was25 g/L,the NAD⁺dosage was 0.1 g/L,the rotation speed was 80 r/min,and the immobilized enzyme could be used repeatedly for 25 times.Finally,a pilot scale up study was carried out in a 300 L reactor,and the substrate conversion was complete after 8 h of reaction.Through separation and extraction,the yield of about 96.2%,and with a purity of 99.3%.（5）Finally,the application of LK-TADH-1 immobilized enzyme was expanded to prepare the key chiral intermediates of luliconazole and ticagrelor.After 12 h of reaction,2-chloro-1-（2,4-dichlorophenyl）ketone（CCPO）was completely converted,with a yield of about 94.2%,e.e.value of 99.97%,and with a purity of 99.5%.After 12 h of reaction,2-chloro-1-（3,4-difluorophenyl）ethanone（CFPO）was completely transformed,with a yield of about 95.8%,e.e.value of 99.91%,and with a purity of 99.2%.更多还原