Thermococcus siculi HJ21 α-淀粉酶Asp186定点突变对酶活性的影响  

Effect of Site-Directed Mutagenesis of Asp186 on Activity of Thermococcus siculi HJ21 α-Amylase

作　　者：吕明生[1] 杨帆[1,2] 胡建恩[2] 房耀维[1] 焦豫良[1] 王淑军[1] 刘姝[1] 

机构地区：[1]淮海工学院海洋学院,江苏连云港222005 [2]大连海洋大学食品工程学院,辽宁大连116023

出　　处：《食品科学》2012年第11期200-204,共5页Food Science

基　　金：江苏省高校自然科学研究重大项目(09KJA170001);江苏省科技支撑计划项目(BE2008340);江苏省高校自然科学基础研究项目(08KJB550001)

摘　　要：为确定超嗜热古菌Thermococcus siculi HJ21 α-淀粉酶(TSA)中特定氨基酸及所在位点与酶活性间的关系,以含TSA基因的pEt-28a-His6质粒为模板,利用定点突变技术,将第186位点的天冬氨酸(Asp)突变为天冬酰胺(Asn),突变子转化至E.coli BL21(DE3)并经IPTG诱导表达,测定突变前后的酶活性,定点突变后的TSA无酶活性;采用DNAStar和Deepview蛋白分析软件,预测突变前后TSA蛋白的二级结构和三级结构,结果表明Thermococcus siculi HJ21 α-淀粉酶的186位点对维持TSA的酶活性具有重要意义,它的突变导致酶蛋白二级结构组件及氢键网络的改变。In order to determine the relationship between amino acid residue at the special site and the activity of Thermococcus.siculi HJ21 α-amylase,pEt-28a-His6 plasmid with TSA gene was used as a template for the site mutagenesis from Asp186 to Asn186.The activity of α-amylase with and without mutation was measured.According to the difference between per-mutant and mutant cDNA sequences,the secondary structure and three-dimensional structure of TSA protein were also predicted using DNAStar and Deepview protein analysis software.The results showed that Asp186 was of critical significance for maintaining the activity of T.siculi HJ21 α-amylase.The site-directed mutagenesis of T.siculi HJ21 α-amylase at position 186 changed the properties of secondary structure as well as hydrogen-bonding networks,thus resulting in the loss of its activity.

关 键 词：Α-淀粉酶 定点突变 TSA基因 1 86位点 酶活性 

分 类 号：Q71[生物学—分子生物学]