机构地区: 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
出 处: 《中国生物工程杂志》 2010年第3期56-60,共5页
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.
领 域: [生物学]