易错PCR技术改造β-环糊精葡萄糖基转移酶的催化特性

Modification of the Catalytic Properties of β-cyclodextrin Glucosyltransferase through Error-prone PCR

摘要:

β-环糊精是一种环状多糖，可用于改变某些大分子化合物的理化性质，在食品、生物及医药等领域都具有很高的工业应用前景。通过利用β-环糊精葡萄糖基转移酶（β-CGTase），使其作用于淀粉等含有葡萄糖基的多糖化合物，可转化生成β-环糊精。该研究采用易错PCR技术对来源于Paenibacillus campinasensis的β-CGTase进行定向进化，得到酶活力高的突变体，并对突变体进行镍柱亲和纯化与酶学性质分析。实验最终获得了一株突变体Q280L，其酶活力与原始-CGTase相比提高了42.10%，对β-环糊精的转化率提高了7.60%，最适反应pH值和稳定性均有所变化，底物亲和力提高46.13%。经序列比对及结构分析，发现突变体Q280L与野生β-CGTase相比，第280位氨基酸残基种类以及周围氢键发生变化。该试验结果表明，基于易错PCR技术对β-CGTase基因进行定向进化，可提高酶活力和改善酶学性质，为实现β-环糊精的工业生产提供参考意义。

Abstract:

β-cyclodextrin is a cyclic polysaccharide that can be used to alter the physical and chemical properties of certain macromolecular compounds. It has remarkable industrial application prospects in the fields of food, biology, and medicine. β-cyclodextrin is obtained by converting polysaccharide compounds containing glucose groups, such as starch, by the action of β-cyclodextrin glucosyltransferase (β-CGTase). In the present study, β-CGTase derived from Paenibacillus campinasensis was subjected to directed evolution through error-prone PCR to obtain a mutant with potent enzymatic activity. The mutants were subjected to nickel column affinity purification and analyzed for their enzymatic properties. Finally, the mutant Q280L was obtained. Compared with that of the wildtype β-CGTase, the enzymatic activity of Q280L was increased by 42.10%, conversion rate of β-cyclodextrin was increased by 7.6%, its optimal reaction pH and pH stability were shifted, and its substrate affinity was increased by 46.13%. Sequence alignment and structural analysis revealed that compared with that in wildtype β-CGTase, the amino acid residue at position 280 and the hydrogen bonding interaction between this and its surrounding residues were altered in Q280L. These findings indicate that directed evolution of β-CGTase based on error-prone PCR could improve enzymatic activity and properties, thereby providing a reference for the industrial production of β-cyclodextrin.