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

作　　者：廖思明[1,2] 孙靓[1,2] 王青艳[2] 申乃坤[2] 朱婧[2] 黄桂媛[2] 黄纪民[2] 陈东[2] 黄日波[1,2] 

机构地区：[1]广西大学生命科学与技术学院,广西南宁530004 [2]广西科学院国家非粮生物质能源工程技术研究中心非粮生物质酶解国家重点实验室广西生物炼制重点实验室,广西南宁530007

出　　处：《广西科学》2017年第1期92-99,共8页Guangxi Sciences

基　　金：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);广西科学院基本科研项目(15YJ22SW02)资助

摘　　要：【目的】筛选耐热α-淀粉酶并实现异源表达,同时分析其酶学性质特征。【方法】以M9培养基加上可溶性淀粉作为选择性分离培养基,从腾冲火山温泉土壤中筛选到产淀粉酶的菌株Anoxybacillus sp.GXS-3。根据淀粉酶氨基酸保守序列,设计引物进行PCR扩增,然后对目标序列进行步移扩增,获得淀粉酶基因AmyGX。将AmyGX与表达载体pQE30连接,导入大肠杆菌M15中表达,对重组酶进行分离纯化和酶学性质分析。【结果】AmyGX基因长1 515 bp,编码505个氨基酸残基,前23个氨基酸残基为信号肽序列;重组质粒pQE30-AmyGX编码的蛋白分子量为58.04kDa,对可溶性淀粉催化水解反应的最适温度为60℃,最适pH值为8.0,V_(max)、K_m值分别为0.19U/mg、3.14mg/mL,热半失活温度T_(50)^(30)值为65.2℃;Zn^(2+)、Cu^(2+)、Co^(2+)、Fe^(3+)、Ba^(2+)对该酶具有明显的抑制作用,Na^+、K^+对该酶有激活作用,Mg^(2+)、Ca^(2+)的影响则不明显。【结论】AmyGX是一种中等耐温碱性酶,在造纸、洗涤剂生产和有毒废弃物去除等方面具有潜在的应用前景。【Objective】Screening thermostable α-amylase and expressed in Escherichia coli,and analyzing the recombinant enzyme characterization.【Methods】The strain Anoxybacillus sp.GXS-3is 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 recombinant α-amylase is isolated and purified by NTA-affinity chromatography and its enzymological properties are analyzed.【Results】The gene of AmyGXis 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 pH8.0 and 60℃ with an apparent Km value of 3.14 mg/ml and Vmax of 0.19U/mg respectively.The temperature at which 50% of the enzyme activity is lost after 30 min heat treatment（T5030）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 

分 类 号：Q786[生物学—分子生物学]