脯氨酸氨肽酶枯草芽孢杆菌分泌表达及特性表征  

Secretory Expression and Characterization of Prolyl Aminopeptidase in Bacillus Subtilis

作　　者：汪克红 王开道 田亚平[1] WANG Kehong;WANG Kaidao;TIAN Yaping(Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学工业生物技术教育部重点实验室,江苏无锡214122

出　　处：《食品与生物技术学报》2018年第11期1135-1140,共6页Journal of Food Science and Biotechnology

基　　金：国家863计划项目(2011AA100905)

摘　　要：构建重组枯草芽孢杆菌大量分泌米曲霉脯氨酸氨肽酶,纯化后对其基本酶学性质进行表征。将脯氨酸氨肽酶cDNA序列从载体pMD19-pap上克隆后连接到载体pMA5上得到重组质粒,转化枯草芽孢杆菌WB600后进行分泌表达。通过在培养基中加入5%D-山梨醇和2 mmol/L CaCl2,胞外酶活由7.5 U/mL提高到36 U/mL。通过硫酸铵盐析、Hitrap Q和SuperdexTM75对重组脯氨酸氨肽酶进行了纯化,纯化后的比酶活为247.3 U/mg,纯化倍数达到8.8倍。该酶最适温度为50℃,最适pH为7.5,米氏常数Km和最大反应速率Vmax分别为0.171 mmol/L和55.99μmol/(L·min)。Construct recombinant Bacillus subtilis to over-expression Aspergillus oryzae prolyl minopeptidase,then purified and characterized its basic enzyme properties.The pap gene was cloned from pMD19-pap vector and ligated into pMA5 to generate recombinant pMA5-pap expression vector.Then the vector was transformed into Bacillus subtilis WB600 competent cells and secretory expressed.By adding 5%D-sorbitol and 2 mmol/L CaCl2 in TB medium,the enzyme activity in fermented supernatant increased from 7.5 to 36.0 U/mL.The recombinant prolyl aminopeptidase was purified by ammonium sulfate,Hitrap Q anion exchange chromatography and Superdex^TM 75 gel chromatography,the purification factor was 8.8 and the specific activity of the purified enzyme was 247.3 U/mg.The purified enzyme has the highest activity at pH 7.5 and 50℃,the Km and Vmax was estimated to be 0.171 mmol/L and 55.99μmol/(L·min)respectively.

关 键 词：脯氨酸氨肽酶 枯草芽孢杆菌 分泌表达 特性表征 

分 类 号：Q55[生物学—生物化学]