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

作　　者：郑婉 刘振杨 郑金珠 武国干[2] 吴华伟[1] ZHENG Wan;LIU Zhenyang;ZHENG Jinzhu;WU Guogan;WU Huawei(College of Life Sciences,Yangtze University,Jingzhou 434025,China;Shanghai Academy of Agricultural Sciences,Shanghai 200000,China)

机构地区：[1]长江大学生命科学学院,湖北荆州434025 [2]上海市农业科学院,上海200000

出　　处：《现代食品科技》2023年第5期25-31,共7页Modern Food Science and Technology

基　　金：国家自然科学基金项目(21576110);江苏省益生制剂重点建设实验室开放课题(JSYSZJ2017001)。

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β-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。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 ofβ-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 wildtypeβ-CGTase,the enzymatic activity of Q280L was increased by 42.10%,conversion rate ofβ-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 wildtypeβ-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 ofβ-CGTase based on error-prone PCR could improve enzymatic activity and properties,thereby providing a reference for the industrial production ofβ-cyclodextrin.

关 键 词：Paenibacillus campinasensis β-CGTase 定向进化 易错PCR 酶学性质 

分 类 号：TS201.25[轻工技术与工程—食品科学]