重组枯草芽孢杆菌分泌表达腺苷酸脱氨酶

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。

Expression of Adenosine Deaminase in Recombinant Bacillus subtilis

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 pHY-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/mL 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.