重组枯草芽孢杆菌分泌表达腺苷酸脱氨酶  

Expression of Adenosine Deaminase in Recombinant Bacillus subtilis

作　　者：郭自涛 张梁[1] 李由然[1] 李赢[1] 顾正华[1] 丁重阳[1] 石贵阳[1] 

机构地区：[1]粮食发酵工艺与技术国家工程实验室,江南大学生物工程学院,江苏无锡214122

出　　处：《食品科学》2015年第21期135-139,共5页Food Science

基　　金：国家高技术研究发展计划(863计划)项目(2011AA100905);教育部"新世纪优秀人才支持计划"项目(NCET-11-0665);江南大学食品科学与技术国家重点实验室自由探索课题(SKLF-ZZA-201201)

摘　　要：目的:利用基因重组技术,构建腺苷酸脱氨酶高效表达的重组菌株。方法:以前期构建的产腺苷酸脱氨酶重组大肠杆菌BL21(DE3)/pET28α-AMPD中来源于鼠灰链霉菌(Streptomyces murinus)目的基因为模板,设计特异性引物扩增腺苷酸脱氨酶基因,亚克隆至游离型表达载体pHY-WZX,转化枯草芽孢杆菌WB600。结果:成功筛选获得了一株产腺苷酸脱氨酶重组枯草芽孢杆菌WB600/pHY-AMPD,进一步在摇瓶中探究了发酵培养基成分对重组酶发酵的影响。获得了较优发酵培养基为:葡萄糖10 g/L、?酵母膏30 g/L、CaCl2 0.5 g/L、柠檬酸三钠1 g/L、NaH2PO4 10 g/L、K2HPO4 10 g/L、(NH4)2SO4 5 g/L,37℃、200 r/min发酵60 h摇瓶发酵液酶活力可达到(2 230±50)U/m L。结论:本研究实现了鼠灰链霉菌来源的腺苷酸脱氨酶基因在枯草芽孢杆菌中表达。Objective： To construct a recombinant strain to express adenosine deaminase（AMPD） efficiently. Methods： The AMPD gene from Streptomyces murinus was expressed in E. coli DE3/pET28α-AMPD. Specific primers were designed to amplify the AMPD gene. The PCR product was cloned into the plasmid p HY-WZX, and the recombinant plasmid was transformed into Bacillus subtilis WB600. Results： The recombinant Bacillus subtilis WB600/pHY-AMPD was successfully constructed to express AMPD. Furthermore, the fermentation medium was optimized to contain 10 g/L glucose, 30 g/L yeast extract, 0.5 g/L CaCl2, 1 g/L sodium citrate, 10 g/L NaH2PO4, 10 g/L K2HPO4, and 0.5 g/L（NH4）2SO4. The enzyme activity reached（2 230 ± 50） U/m L after shake flask cultivation at 37 ℃ for 60 h with a shaking speed of 200 r/min. Conclusion： The AMPD gene from Streptomyces murinus has been successfully expressed in Bacillus subtilis.

关 键 词：腺苷酸脱氨酶 鼠灰链霉菌 枯草芽孢杆菌 重组表达 

分 类 号：Q815[生物学—生物工程]