广西科学2017，Vol.24Issue(1)：92-99,8.DOI:10.13656/j.cnki.gxkx.20170223.001
耐热α-淀粉酶的筛选、基因克隆和酶学性质分析
Screening of Thermostable α-Amylase Producing Strain and Cloning,Expression and Characterization of the Gene AmyGX
摘要
Abstract
[Objective]Screening thermostable α-amylase and expressed in Escherichia coli,and analyzing the recombinant enzyme characterization.[Methods]The strain Anoxybacillus sp.GXS-3 is isolated from soil samples collected from Tengchong hot spring,with M9 medium and soluble starch as the sole carbon source.According to the amino acid conserved sequence of amylase,degenerate primers are designed for PCR amplification.and then the target sequence is amplified and amplified to obtain the amylase gene AmyGX.AmyGX is ligated with the expression vector pQE30, introduced into E.coli M15, and the recombinantα-amylase is isolated and purified by NTA-affinity chromatography and its enzymological properties are analyzed.[Results]The gene of AmyGX is 1 515 bp in length and encoded a protein of 505 amino acids,of which,the first 23 amino acid residues are signal peptide sequences.The molecular mass of the recombinant AmyGX is 58.04 kDa.Assayed with soluble starch as substrate,this recombinant enzyme displays optimal activity at pH 8.0 and 60℃ with an apparent Km value of 3.14 mg/ml and Vmax of 0.19 U/mg respectively.The temperature at which 50% of the enzyme activity is lost after 30 min heat treatment (T3050) is 65.2℃.Zn2+、Cu2+、Co2+、Fe3+ 、Ba2+has significantly inhibitory effect on the activity of the recombinant enzyme,whereas Na+、K+could increase the activity and Mg2+、Ca2+ has slightly effect on it.[Conclusion]AmyGX is a moderate enzyme,it could be applied in the field of paper,detergent production and the removal of waste material in environment.关键词
Anoxybacillus sp.GXS-3菌株/重组淀粉酶AmyGX/克隆与表达/酶学性质Key words
Anoxybacillus sp.GXS-3/recombinant α-amylase AmyGX/cloning and expression/enzyme characterization分类
生物科学引用本文复制引用
廖思明,孙靓,王青艳,申乃坤,朱婧,黄桂媛,黄纪民,陈东,黄日波..耐热α-淀粉酶的筛选、基因克隆和酶学性质分析[J].广西科学,2017,24(1):92-99,8.基金项目
国家自然科学基金项目(31560251),广西自然科学基金项目(2015GXNSFAA139053),广西科学与技术开发计划项目(桂科合15104001-6)和广西科学院基本科研项目(15YJ22SW02)资助. （31560251）
