亚位点7处突变对碱性芽胞杆菌CGT酶产物特异性的影响  

Effect of mutating subsite ?7 on product specificity of cyclodextrin glucanotransferase from alkalophilic Bacillus clarkii

作　　者：杨冬[1,2] 田靖斐[1,2] 陈晟[1,2] 吴敬[1,2] 

机构地区：[1]江南大学食品科学与技术国家重点实验室,江苏无锡214122 [2]江南大学生物工程学院工业生物技术教育部重点实验室,江苏无锡214122

出　　处：《生物工程学报》2012年第2期191-202,共12页Chinese Journal of Biotechnology

基　　金：中央高校基本科研业务费专项资金(No.JUSRP20917);食品科学与技术国家重点实验室科研基金(No.SKLF-MB-200802)资助~~

摘　　要：为了研究来源于碱性芽胞杆菌的γ-环糊精葡萄糖基转移酶(CGT酶)具有较高产物特异性的作用机理,对其氨基酸序列和模拟结构进行了分析,确定其亚位点7处氨基酸的缺失可能影响其产物特异性。运用重叠PCR的方法,在其亚位点7处添加缺失的6个氨基酸,造成插入突变。将突变基因与pET-20b(+)连接并在大肠杆菌BL21(DE3)中表达。以可溶性淀粉为底物进行酶转化,HPLC分析转化产物中的环糊精含量。结果表明,相对于野生型γ-CGT酶,突变酶转化生成的3种环糊精中,γ-环糊精所占的比例从76.0%降至12.5%,α-、β-环糊精分别从8.7%和15.2%提高至37.5%和50%。分析其可能机理为:与α-、β-CGT酶相比,野生型γ-CGT酶的亚位点7处缺失6个氨基酸,该构象为葡萄糖的结合提供了更大的空间,从而更适合γ-环糊精的生成;而在其亚位点7处插入6个氨基酸,造成插入突变后,葡萄糖链结合的空间变小,这种构象不利于γ-环糊精的生成。To investigate the mechanism of high product specificity of γ-clodextrin glucanotransferase（CGTase） from alkalophilic Bacillus clarkii 7364,we aligned protein sequence and structure model,found out that loss of 6 amino acids at subsite ？7 probably affected its product specificity.Using overlapping PCR method,we inserted 6 amino acids into subsite ？7 of CGTase.The mutant CGTase gene was ligated with pET-20b（＋） and expressed in Escherichia coli BL21（DE3）.The extracellular recombinant enzyme was used to transform soluble starch into cyclodextrins（CDs）.HPLC analysis results show that,compared to wild CGTase,the γ-CDs produced by mutant enzyme decreased from 76.0% to 12.5%,whereas the ratio of α-and β-CDs increased from 8.7% and 15.2% to 37.5% and 50%.The possible mechanism was that,compared to α-,β-CGTase,wild γ-CGTase lacks 6 amino acids in its subsite ？7.This conformation provided more space for glucose combination and was thus advantageous for forming γ-CD.When the 6 amino acids were inserted into the subsite ？7 of wild γ-CGTase,the space to bind with glucose reduced and consequently resulted in less γ-CD production.

关 键 词：环糊精 环糊精葡萄糖基转移酶 产物特异性 突变 

分 类 号：Q55[生物学—生物化学]