(1. 广西大学生命科学与技术学院 广西亚热带生物资源保护利用重点实验室 广西 南宁 530005)
(2. 广西科学院国家非粮生物质能源研究工程中心 广西 南宁 530007)
(3. 南京工业大学生物与制药工程学院 江苏 南京 210009)
摘 要: 以产酸性淀粉酶菌株Bacillus sp. CN7的基因组DNA为模板, PCR扩增到 淀粉酶成熟肽基因, 将该基因插入表达质粒pSE380中, 构建重组质粒pSE380-cn7a。将重组质粒导入到Escherichia coli JM109中, IPTG诱导表达。重组酶经Sephacryl S300、Ni-NTA纯化后测定其酶学性质。重组酶CN7A的最适温度为65°C, 最适pH为5.5?6.0, 对可溶性淀粉的Km值为3.784 g/L, 最大反应速度为101.2 mg/(L·min), 该酶的热稳定性不依赖钙离子
(1. Guangxi Key Laboratory of Subtropical Bio-resource Conservation and Utilization, College of Life Science and Technology, Guangxi University, Nanning, Guangxi 530005, China)
(2. National Non-grain Bio-energy Engineering Research Center, Guangxi Academy of Sciences, Nanning,Guangxi 530007, China)
(3. College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing,Jiangsu 210009, China)
Abstract: The -amylase gene cn7a was amplified by PCR from Bacillus sp. CN7 genome DNA. The recombinant plasmid pSE380-cn7a was constructed by inserting gene cn7a into expression vector pSE380 and then transformed into Escherichia coli JM109. The purified amylase CN7A showed an op-timal activity at pH 5.5?6.0 and 65°C, the Km value is 3.784 g/L taking soluble starch as substrate, and the maximum velocity was determined as 101.2 mg/(L·min). Ca ion was not required for the thermal stability of CN7A.
Keywords: Acid amylase, Ca-independent, Cloning and expression, pKa
