粘质沙雷氏菌PL-06磷脂酶A1在大肠杆菌中的分泌表达及其条件优化  

Secretive expression and optimized condition of phospholipase A1 gene from Serratia marcescens PL-06 in Escherichia coli

作　　者：陈环[1,2] 薛正莲[1,2] 王洲[1,2] 张爽[1,2] 苏燕南[1] 胡艳瑾 

机构地区：[1]安徽工程大学生物与化学工程学院,安徽芜湖241000 [2]微生物发酵安徽省工程技术研究中心,安徽芜湖241000

出　　处：《食品工业科技》2015年第16期193-197,202,共6页Science and Technology of Food Industry

基　　金：国家自然科学基金(C200504);安徽省自然科学基金(11040606M81)

摘　　要：克隆粘质沙雷氏菌PL-06磷脂酶A1基因pla A,与载体p ET-22b(+)连接构建重组质粒p ET-22b(+)-pla A,并将重组质粒导入受体菌E.coli BL21(DE3)中表达,构建得到重组菌AP22,IPTG诱导表达目的蛋白后将培养基蛋白进行SDS-PAGE分析显示:重组蛋白以可溶性蛋白的形式大量存在于发酵液中,分子质量约35 ku,与预期蛋白大小一致。以磷脂酶A1酶活为指标,在单因素实验的基础之上,通过正交实验得到摇瓶培养最佳条件为:氨苄青霉素终浓度30μg/m L,IPTG加量0.25 mmol/L,温度为34℃,OD600值为0.3,诱导时间4 h。在此条件下重组胞外磷脂酶A1酶活可高达8.6 U/m L,比优化前提高了43.3%。AThe phopholipase A1 gene from Serratia marcescens PL-06 named plaA was cloned,ligased with pET-22b （＋） to construct recombinant plasmid pET-22b （＋）-p/aA and then transferred to E.coil BL21 （DE3）. The engineered stains expressing phospholipase A1 was obtained,and named AP22. After subsequent indution by IPTG,Iots of approximately 35 ku protein was detected in the fermented liquid by SDS-PAGE. This protein was in the same size with the expected protein. Selectioning phospholipase A1 enzyme activity as reference,by single factor and orthogonal experiment method,optimized induction conditions for phophelipase A1 expression were obtained as follows.ampicillin concentration 30 μg/mL,IPTG concentration 0.25 mmol/mL, inducing temperature 34 ℃,strain density OD600 0.3 and induction time 4 h. Under the optimized conditions, maximum phospholipase A1 enzyme activity was observed to be 8.6 U/mL and increased by 43.3% than before.

关 键 词：磷脂酶A1 粘质沙雷氏菌 分泌表达 优化 

分 类 号：TS201.2[轻工技术与工程—食品科学]