葡萄糖3-脱氢酶工程菌的构建、表达及其合成3-酮对硝基苯井冈霉胺  

Construction and expression of a glucoside 3-dehydrogenase engineering strain for N-p-nitrophenyl-3-ketovalidamine biosynthesis

作　　者：张建芬[1] 柯薇[1] 陈虹[1] ZHANG Jianfeng;KE Wei;CHEN Hong(College of Biology and Environmental Engineering,Zhejiang Shuren University,Hangzhou 310015,China)

机构地区：[1]浙江树人大学生物与环境工程学院,浙江杭州310015

出　　处：《生物加工过程》2019年第1期94-98,共5页Chinese Journal of Bioprocess Engineering

基　　金：国家自然科学基金青年基金(21102131);浙江省自然科学基金(LY18C010002)

摘　　要：采用基因挖掘技术,从NCBI数据库发现来源于Glaciecola polaris的葡萄糖3-脱氢酶基因,该基因经密码子优化后合成,构建p ET28b-GpG3DH重组表达载体,转入大肠杆菌E. coli BL21中进行重组表达,结果显示:GpG3DH实现了部分可溶表达,其分子量为5. 5×104。随后,优化了GpG3DH的表达条件,结果发现,重组菌在TB培养基中37℃培养至OD600为0. 8时,加入IPTG至终浓度为0. 5 mmol/L,16℃诱导表达10 h,GpG3DH酶活为2. 83×10-2U/m L。进一步将重组菌用于3-酮对硝基苯井冈霉胺的细胞转化法合成,细胞用10 mmol/L的EDTA处理,在p H为7. 5、25℃的条件下转化32 h,3-酮对硝基苯井冈霉胺的产率(Y3-KpNPV)为0. 54。In this study,a glucoside 3-dehydrogenase(G3DH)gene from Glaciecola polaris was found from NCBI databases by gene mining technique.The gene was optimized and synthesized,and a pET28b-G p G3DH recombinant expression vector was constructed,then it was transferred to E.coli BL21.GpG3DH realized partial soluble expression,and its relative molecular weight was 5.5×10^4.The expression conditions of GpG3DH were optimized.The recombinant strain was cultured with TB medium at 37℃until OD600 was 0.8.Then IPTG was added with a final concentration of 0.5 mmol/L.The recombinant strain was induced at 16℃for 10 h.G p G3DH activity was 2.83×10^-2 U/mL.Furthermore,the recombinant strain was used to synthesize N-p-nitrophenyl-3-ketovalidamine.The cells were treated with 10 mmol/L EDTA,Under the conditions of pH 7.5,25℃for 32 h,0.54 of the N-p-nitrophenyl-3-ketovalidamine yield was achieved.

关 键 词：葡萄糖3脱氢酶 基因挖掘 3-酮对硝基苯井冈霉胺 细胞转化 

分 类 号：Q78[生物学—分子生物学]