芽胞杆菌XM-1酸性α-淀粉酶基因的克隆及原核表达  

Cloning of Acid alpha-Amylase Gene in Bacillus Subtilis XM-1 and Its Prokaryotic Expression

作　　者：胡元森[1] 蒋炳伸[2] 潘涛[1] 李翠香[1] 

机构地区：[1]河南工业大学生物工程学院,郑州450001 [2]黄淮学院农林科学系,河南驻马店463000

出　　处：《中国食品学报》2012年第2期41-45,共5页Journal of Chinese Institute Of Food Science and Technology

基　　金：河南省教育厅自然科学基金项目(2009B210002)

摘　　要：为使枯草芽胞杆菌XM-1菌株酸性α-淀粉酶基因高通量表达,提高酶产量,利用PCR方法从基因组DNA中扩增出该酶基因As-Amy,并构建pET30a(+)/As-Amy原核表达载体,在大肠杆菌BL21中成功表达。测序结果表明As-Amy基因编码框全长为1434bp(Genbank登录号GQ153530),编码477个氨基酸,预测蛋白质分子质量为61ku。序列比对分析表明,As-Amy与多个枯草芽胞杆菌α-淀粉酶基因相似性在98%以上,所编码氨基酸与枯草芽胞杆菌(Bacillus subtilis natto)IAM1212同源性最高。SDS-PAGE分析显示,As-Amy基因在大肠杆菌BL21中获得表达,酶活力较原菌株XM-1提高了2.7倍,表达蛋白分子质量大小与预测值相符。Abstract In this paper, the acid alpha-amylase （As-Amy） gene from genomic DNA of Bacillus subtilis XM-1 was amplified using PCR approach,and constructed the prokaryotic expression vector pET30a （＋）/As-Amy which successfully expressed in BL21. The sequencing result showed that the full length of As-Amy gene coding region was 1 43g（Genbank accession no.GQ153530） and encoded 477 amino acid residues whose predicted molecular weight was 61 ku. The homo- geneous analysis demonstrated that the deduced amino acid of As-Amy gene shared the highest homolgy of 98% with Bacillus subtilis （natto） IAM1212, and the As-Amy ORF sequences were more than 98% resemble to that of a few B. subtilis. The SDS-PAGE analysis indicated that As-Amy was expressed in Escherichia coli BL21, enabled enzyme activi- ty of the recombinant strain was 2.7 times than that of the original strain XM-1. The expressed protein was found to be about 60 ku, which was accordant to the predicted one.

关 键 词：酸性Α-淀粉酶 基因克隆 原核表达 

分 类 号：TS201.25[轻工技术与工程—食品科学]