乳糖诱导重组大肠杆菌高效表达3-甾酮-Δ^(1)-脱氢酶的研究  

Lactose-induced Efficient Expression of 3-Ketosteroid-Δ^(1)-Dehydrogenase in Recombinant Escherichia coli

作　　者：胡青 吕德强 王烨 宋士奎 苏正定[1,2] HU Qing;LYU Deqiang;WANG Ye;SONG Shikui;SU Zhengding(College of Biology and Food,Hubei University of Technology,Wuhan 430068,China;Wuhan Amersino Biodevelop Inc.,Wuhan 430027,China)

机构地区：[1]湖北工业大学生物与食品学院,湖北武汉430068 [2]武汉艾默佳华生物科技有限公司,湖北武汉430027

出　　处：《食品与生物技术学报》2025年第1期125-131,共7页Journal of Food Science and Biotechnology

摘　　要：【目的】探索乳糖代替异丙基-β-D-硫代半乳糖苷(IPTG)诱导3-甾酮-Δ^(1)-脱氢酶(KstD)在大肠杆菌(Escherichia coli,E.coli)BL21(DE3)中高效表达的可行性。【方法】首先通过IPTG诱导获得大量高活性可溶性重组蛋白SZD-KstD,在摇瓶培养条件下对乳糖诱导表达参数进行了系统优化,随后在15 L发酵罐中开展了高密度发酵试验。重点考察了添加时间、乳糖诱导方式、诱导时间以及碳、氮源的补料工艺等关键工艺参数对重组蛋白SZD-KstD表达的影响。【结果】在终浓度为0.2 mmol/L的乳糖诱导下获得最大量的重组蛋白SZDKstD和菌体量,由于乳糖同时可作为碳源被利用,采用3次分批添加乳糖的效果优于一次性添加。在乳糖诱导体系中重组蛋白SZD-KstD表达量可达39.58%,与常规IPTG诱导效果无明显差异。【结论】乳糖作为廉价、高效的诱导剂可以代替高价格的IPTG用于KstD的规模化发酵,为生物酶法制备甾体药物提供了一个新思路,同时也为其他重组蛋白的生产提供了借鉴。[Objective]To investigate the feasibility of substituting lactose for isopropyl-β-D-1-thiogalactopyranoside(IPTG)in inducing efficient expression of 3-ketosteroid-Δ^(1)-dehydrogenase(KstD)in Escherichia coli(E.coli)BL21(DE3).[Method]Upon the established IPTG-induced protocol for obtaining high yields of soluble and catalytically active recombinant protein SZD-KstD,the lactose induction parameters under shake-flask culture conditions were optimized.Subsequently,high-density fermentation was conducted in a 15 L bioreactor.Critical process parameters influencing expression of the recombinant protein SZD-KstD were evaluated,including addition time,lactose induction method,induction duration,and carbon/nitrogen feeding protocols.[Result]The maximum amounts of SZD-KstD and bacteria were obtained under the induction with lactose at the final concentration of 0.2 mmol/L.Since lactose can be used as a carbon source at the same time,adding lactose in three batches outperformed adding it in one batch.Under lactose induction conditions,SZD-KstD accounted for 39.58%of total soluble protein content,which was not significantly different from that under IPTG induction.[Conclusion]Lactose,as a cheap and efficient inducer,can replace IPTG for the large-scale fermentation of KstD,which solves a major problem for the preparation of steroid drugs by bioenzyme method and provides a reference for the production of other recombinant proteins.

关 键 词：重组大肠杆菌 3-甾酮-Δ^(1)-脱氢酶 乳糖诱导 高密度发酵 

分 类 号：Q78[生物学—分子生物学]