摘要
利用反向-巢式PCR(IN-PCR)从富集土壤宏基因组DNA中克隆到一种α-淀粉酶基因的全序列,其基因登录号为GU045523,测序分析显示与来自Bacillussp.KR-8104耐酸淀粉酶不完整基因同源性为99%。将获得的α-淀粉酶成熟肽基因与表达载体pSE380连接,导入Escherichia coliJM109中,IPTG诱导表达。粗酶液经Ni-NTA、SephacrylS-200纯化后测定酶学性质:重组酶GXAA的最适作用pH为7.0,最适作用温度为75℃,对可溶性淀粉的Km值为11.6g/L。构建突变子E27G、A450T、E27G-A450T,其酶学性质与原始酶没有显著差别。
Inverse-nest PCR was applied to clone the full sequence of some α-amylase from enrichment soil metagenomic DNA.The successfully achieved gene（GU045523）showed high homology （Identity：99%）with the partial coding sequence of acid-stable amylase from Bacillus sp.KR-8104.The putative mature peptide gene was inserted into pSE380,a recombinant plasmid pSE380-gxaa was constructed and transformed into Escherichia coli JM109.The purified amylase GXAA showed an optimal activity at pH 7.0 and 75℃,the Km value is 11.6g/L taking soluble starch as substrate.Mutants E27G,A450T and E27G-A450T were constructed,the property showed no remarkable difference from the wild type.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2010年第3期56-60,共5页
China Biotechnology
基金
国家科技支撑计划课题(2007BAD75B05)
国际科技合作项目(2008DFA30710)资助项目
