灰产色链霉菌海藻糖合成酶的自诱导表达及两阶段温度控制发酵的优化  

Auto-induced expression of Streptomyces griseochromogenes trehalose synthase gene in E. coli and optimization of its two-stage temperature controlling fermentation

作　　者：杨鑫[1] 吴茜[1] 崔怀言 徐娴[1] 朱丽英[2] 江凌[1,3] 

机构地区：[1]南京工业大学生物与制药工程学院,江苏南京210009 [2]南京工业大学理学院,江苏南京210009 [3]南京工业大学食品与轻工学院,江苏南京210009

出　　处：《食品与发酵工业》2015年第5期14-18,共5页Food and Fermentation Industries

基　　金：江苏省自然科学基金项目(BK20131046;BK20130917);江苏省高校自然科学基金面上项目(14KJB530003);教育部高等学校博士学科点专项科研基金(20123221120011)

摘　　要：克隆灰产色链霉菌海藻糖合成酶基因(tres),导入大肠杆菌BL21(DE3)中高效表达,并对重组菌自诱导发酵条件进行优化。采用简并PCR的方法扩增灰产色链霉菌海藻糖合成酶基因(tres),将tres基因和表达载体p ET28a连接,构建重组质粒p ET28a-tres,将其转化到大肠杆菌BL21(DE3)中,筛选重组子进行自诱导表达。利用HPLC法测定酶活研究温度对自诱导发酵海藻糖合成酶催化酶活的影响。该研究成功构建了重组大肠杆菌BL21(DE3)。重组菌自诱导表达最优发酵条件:采用两阶段温度控制,37℃培养2.5 h,25℃继续培养14 h,并添加3%乙醇,酶活达到1.29×104U/m L,与传统IPTG恒温培养方式相比酶活提高了55.25%,海藻糖得率达71%。A novel trehalose synthase gene was cloned from Streptomyces griseochromogenes YX-1 and overexpressed in Escherichia coli BL21( DE3),and the auto-induced fermentative condition of recombinant bacteria was further optimized. Trehalose synthase gene was successfully amplified from S. griseochromogenes by degenerate PCR. The amplified fragment was linked to p ET28 a to construct p ET28a-tres which were then transformed into E. coli BL21( DE3). The recon was screened and gene was expressed by auto-induced fermentation. HPLC method was used to investigate the effect of auto-induced fermentative temperature on the trehalose synthase catalytic activity. The recombinant strains of bacteria E. coli BL21( DE3) were successfully constructed. The optimum fermentation conditions were as follows: cultivating at 37 ℃ for 2. 5 h firstly,then culturing in 25 ℃ for 14 h,and adding 3% ethanol simultaneously. The activity of the recombinant enzyme was 1. 29 × 104 U / m L,which was 55. 25% higher than that of strains traditionally induced with IPTG. The recombinant Tres could catalyze maltose-trehalose interconversion. The production of trehalose reached71%. The recombinant trehalose synthesized from auto-inducted and two-stage fermentative condition provided a theoretical basis for mass production as well as the high-efficiency expression of other recombinant proteins.

关 键 词：海藻糖合成酶 自诱导发酵 两阶段温度控制策略 

分 类 号：TQ920.6[轻工技术与工程—发酵工程]