摘要
将去信号肽的耐酸性高温α-淀粉酶突变基因amyd克隆到大肠杆菌表达载体pET-30a上，实现重组质粒pET-amyd在大肠杆菌BL21（DE3）中的高效表达。经硫酸铵盐析、DEAE-Sepharose Fast Flow离子交换层析、Sephadex G-75凝胶层析，重组酶AMYD的比活达到354．6U·mg^-1、纯化倍数为83．83，获得凝胶电泳条带单一的蛋白样品，经SDS-PAGE检测，AMYD酶分子量为63．5kDa。重组酶AMYD的最适温度80℃、最适反应pH值为4．5，在温度低于90℃、反应pH值4．0～6．5的条件下，酶活较稳定。
In this research, the acid-resistant and heat-stable oramylase mutation gene （amyd） was cloned into expression vector pET-30a to constitute recombination vector pET-amyd which was transformed into BL21 （DE3）. Positive transformant was cultivated and IPTG was added into culture to induce the expression of the or amylase. The recombinant enzyme AMYD purification procedures include salting-out by ammonium sulfate, DEAE-Sepharose Fast Flow ion exchange and Sephadex G-75 gel filtration chromatography. By multi-step purification, the specific activity of AMYD was 354. 6 U · mg^-1 , it was purified to 83.83 folds and its recovery was 51.00%. Analyzed by SDS-PAGE, the recombinant enzyme AMYD had a molecular mass of 63.5 kDa. The optimum reaction temperature and pH value were 80℃ and 4.5, respectively. The AMYD was relatively stable when the temperature was below 90℃ and the pH value was in the range of 4.0-6.5.
出处
《化学与生物工程》
CAS
2007年第3期58-62,共5页
Chemistry & Bioengineering
基金
天津市科委科技发展计划攻关培育项目(06YFGPSH03500)
关键词
耐酸性高温α-淀粉酶突变基因
表达
纯化
酶学性质
acid-resistant and heat-stable or amylase mutation gene
expression
purification
characterization of the enzyme
