食品工业科技2025，Vol.46Issue(11)：185-193,9.DOI:10.13386/j.issn1002-0306.2024100117
新型α-葡萄糖苷酶Aga432分子改造及酶学性质分析
Molecular Modification and Enzymatic Properties of the Novelα-Glucosidase Aga432
摘要
Abstract
This study aimed to clone the novel α-glucosidase gene Aga432 from Paenibacillus sp.and enhance its catalytic activity through site-directed mutagenesis.A gene fragment encoding α-glucosidase was successfully amplified from the genomic DNA of Paenibacillus sp.,comprehensive sequence analysis was performed,and homology modeling and molecular docking were employed to construct gene-engineered strains.Eight positive mutant strains were identified,among which the enzymatic properties of recombinant Aga432 and the highest relative activity mutant AT-2 were characterized.Additionally,the dispersing effects of recombinant α-glucosidases Aga432 and AT-2 on biofilms were explored,and their toxicity to mouse embryo fibroblasts was evaluated.The results revealed that the specific activity of Aga432 was 45.05 U/mg,while the mutant AT-2 exhibited a significantly enhanced specific activity of 84.09 U/mg.Although the optimal reaction temperature and pH for AT-2 were essentially unaltered relative to Aga432,its thermal stability was significantly enhanced,and it exhibited heightened stability under acidic conditions.The Km of mutant AT-2 was 2.18 times that of Aga432,the Vmax was 3.19 times,the Kcat was 2.33 times,and the Kcat/Km was 1.07 times that of Aga432.In vitro cellular assays indicated that Aga432 and AT-2 at concentrations of 15.0～30.0 μg/mL were non-toxic and exhibited good cell compatibility.Biofilm dispersal assays demonstrated that both recombinant α-glucosidases at concentrations ranging from 10.0 to 50.0 μg/mL significantly dispersed bacterial biofilms(P＜0.0001).The thermostability of α-glucosidase Aga432 was successfully enhanced through molecular modification in this study,laying a foundation for the development of novel α-glucosidases and providing a reference for future targeted modification research.关键词
α-葡萄糖苷酶/同源建模/分子对接/酶学性质/生物膜分散作用Key words
α-glucosidase/homology modeling/molecular docking/enzymatic properties/biofilm dispersing effect分类
生物科学引用本文复制引用
路涵,方婷,牛晓旭,翁晓敏,严芬..新型α-葡萄糖苷酶Aga432分子改造及酶学性质分析[J].食品工业科技,2025,46(11):185-193,9.基金项目
中央引导地方科技发展专项(2022L3020). （2022L3020）
