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

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。

Abstract:

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.