Geobacillus sp.CHB1环糊精葡萄糖基转移酶淀粉结合位点N623饱和突变对催化效率与产物特异性的影响  

Saturated Mutation Effects on Catalytic Efficiency and Product Specificity of Starch Binding Site N623 of Cyclodextrin Glucanotransferase from Geobacillus sp. CHB1

作　　者：郭永华[1,2] 陈济琛[1] 蔡海松[1] 陈龙军[1] 林新坚[1,2] 

机构地区：[1]福建省农业科学院土壤肥料研究所,福州350003 [2]福建农林大学生命科学学院,福州350002

出　　处：《中国生物工程杂志》2016年第11期30-38,共9页China Biotechnology

基　　金：国家公益性农业科研专项(201303094-05);福建省自然科学基金项目(2014J01105);福建省属公益类科研院所基本科研专项(2014R1022-3);福建财政社会公益研究(2060302)资助项目

摘　　要：为了提高来源于Geobacillus sp.CHB1环糊精葡萄糖基转移酶(CGTase)的催化效率和产物特异性,对其氨基酸序列和模拟结构进行了分析,确定其淀粉结合位点2处第623位氨基酸残基的构象可能影响其催化效率。运用重叠PCR技术,在其淀粉结合位点2处第623位(N623)进行定点饱和突变,构建19种不同氨基酸残基突变体。将突变基因与p ET-28a(+)-omp A载体连接并在大肠杆菌BL21(DE3)中表达。以可溶性淀粉为底物进行酶催化实验,HPLC分析反应产物中的环糊精含量变化。结果表明,相对于野生型CGTase,突变酶N623T的催化效率明显提高,总环化活力提高了58.6%,α-环化活力提高了64%,β-环化活力提高了80.5%,而γ-环化活力降低了35.3%。产物特异性方面,相比野生型CGTase,突变酶N623T的淀粉总转化率从11.3%提高至39.7%,提高了251.3%,其中α-环糊精、γ-环糊精所占比例缩减为32.8%和7.7%,β-环糊精提高至59.5%。分析其可能机制为:与野生型CGTase相比,突变体N623T中苏氨酸残基代替了天冬酰胺,造成淀粉结合位点2处的构象发生了变化,该构象优化底物作用方向有利于反应的进行,从而提高了酶的催化效率。In order to improve catalytic efficiency and product specificity of cyclodextrin glucanotransferase（ CGTase） from Geobacillus sp. CHB1,acid sequences and simulation structure model were analyzed,found out that the 623 th amino acid residues of starch binding sites II probably affected its catalytic efficiency. Using overlapping PCR method,19 kinds of mtuants on the 623 th amino acid residues（ N623） of starch binding sites II of CGTase were built. The mutant CGTase genes were respectively linked with p ET-28a（ ＋）-omp A and expressed in Escherichia coli BL21（ DE3）. The recombinant pure enzyme was used to transform soluble starch into cyclodextrins（ CDs）. HPLC analysis results show that,compared to wild-type CGTase,mutant N623 T increases catalytic efficiency of CGTase,the total cyclization activity increased 58. 6%,α- cyclization activity increased 64%,β- cyclization activity increased 80. 5%,while γ- cyclization activity was reduced by 35. 3%.In terms of product specificity,compared to wild-type CGTase,the total starch conversion rate by mutant N623 T increased from 11. 3% to 39. 7%,of which α- cyclodextrin,γ- cyclodextrin proportion reduced to 32. 8% and7. 7%,β- cyclodextrin increased to 59. 5%. The possible mechanism was that,compared to wild-type CGTase,mutant N623 T. Threonine residue in place of asparagine caused conformation of starch binding site II was changed,the conformation optimized substrate acting direction,in favor of the reaction is carried out,thereby improving the catalytic efficiency of the enzyme.

关 键 词：环糊精葡萄糖基转移酶 饱和突变 催化效率 产物特异性 

分 类 号：Q812[生物学—生物工程]