一种不依赖钙离子的酸性α淀粉酶基因的克隆表达

Cloning and Expression of Ca-independent Acid α-amylase Gene

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a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。

关键词:酸性淀粉酶, 不依赖钙离子, 克隆表达, pKa

Abstract:

The a-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 optimal 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.

Key words:Acid amylase, Ca-independent, Cloning and expression, pKa