D-对羟基苯甘氨酸合成酶重组菌的高密度发酵工艺  

High-density Fermentation Process of D-p-Hydroxyphenylglycine Synthase Recombinant Bacteria

作　　者：陈科奇 朱海 郑梦泽 李婷婷 史大华 CHEN Keqi;ZHU Hai;ZHENG Mengze;LI Tingting;SHI Dahua(School of Pharmacy,Jiangsu Ocean University,Lianyungang 222005,China;Jiangsu Key Laboratory of Marine Drug Active Molecular Screening,Jiangsu Ocean University,Lianyungang 222005,China;Jiangsu Key Laboratory of Marine Bioresources and Environment,Jiangsu Ocean University,Lianyungang 222005,China)

机构地区：[1]江苏海洋大学药学院,江苏连云港222005 [2]江苏海洋大学江苏省海洋药物活性分子筛选重点实验室,江苏连云港222005 [3]江苏海洋大学江苏省海洋生物资源与环境重点实验室,江苏连云港222005

出　　处：《江苏海洋大学学报（自然科学版）》2022年第1期57-65,共9页Journal of Jiangsu Ocean University:Natural Science Edition

基　　金：江苏省研究生科研与实践创新计划项目(SJCX20_1239)。

摘　　要：D-对羟基苯甘氨酸(D-HPG)是半合成青霉素的一种必需医药中间体。由于生物酶法属于环境友好的合成技术,酶法逐渐成为工业领域中化学合成法的理想替代方法。为了实现D-HPG酶法生产的工业规模化,首先构建了D-海因酶(DHD)和D-氨甲酰水解酶(DCB)的重组菌,并使目的蛋白可溶性表达。然后,基于单因素实验考察诱导剂浓度、温度、pH、碳源和氮源等条件对DHD与DCB重组菌酶活性的影响,进一步使用响应面法对限速酶DCB培养基成分进行优化。优化后最佳培养基配方为:淀粉和葡萄糖分别为DHD与DCB重组菌的碳源,大豆蛋白胨为氮源,添加阿拉伯糖为诱导剂。5 L发酵罐的发酵结果为:获得DHD菌体(53.4±0.75)g/L,DCB菌体(46.7±0.96)g/L。最后对双酶催化D-HPG条件进行优化,产率可达(92.43±3.76)%,为工业化生产D-对羟基苯甘氨酸提供了理论和技术支持。D-p-Hydroxyphenylglycine(D-HPG) is an essential pharmaceutical intermediate for semi-synthetic penicillin.Enzymatic synthesis has gradually become an ideal alternative to chemical synthesis in the industry because of the environmental friendliness of biological enzymatic synthesis.In order to realize the industrial scale of D-HPG enzymatic production,the recombinant bacteria of D-hydantoinase(DHD) and D-carbamyl hydrolase(DCB) was constructed and the soluble expression of target protein was carried out.Then,based on single factor experiments,effects of inducer concentrations,temperatures,pH,carbon sources and nitrogen sources on the enzyme activity of DHD and DCB recombinant bacteria were investigated.Furthermore,the response surface method was used to optimize the composition of the rate-limiting enzyme DCB medium.The optimal medium formula after optimization were as follows:starch and glucose as the carbon source of DHD and DCB recombinant bacteria,soy peptone as the nitrogen source,and arabinose as the inducer.(53.4±0.75)g/L of DHD cells and(46.7±0.96)g/L of DCB cells were obtained from 5 L fermentor.Finally,the conditions of D-HPG catalyzed by the two enzymes were optimized,and the molar yield was up to(92.43±3.76)%,which provided theoretical and technical support for the industrial production of D-p-hydroxyphenylglycine.

关 键 词：D-海因酶 D-氨甲酰水解酶 D-对羟基苯甘氨酸 高密度发酵 生物催化 

分 类 号：Q815[生物学—生物工程]