内切多聚半乳糖醛酸酶是果胶酶中研究最为广泛的酶之一,可以特异性水解多聚半乳糖醛酸链的α-1,4-糖苷键,生成不同聚合度的寡聚半乳糖醛酸,对提高果胶的附加值具有重要意义。目前关于内切多聚半乳糖醛酸酶的研究仍然较少,且其催化活力和热稳定性不佳是限制其工业化应用的主要因素。课题组前期研究获得了青霉Penicillium arizonens来源的内切多聚半乳糖醛酸酶基因pePGB,并在毕赤酵母中实现了异源表达。由于其催化活力仅处于同类酶的中等水平,该研究主要针对其催化活力进行了改造。首先对其底物结合口袋亚位点的氨基酸进行了丙氨酸突变,基于突变结果及序列比对,选择了相应的突变热点进行理性设计,筛选获得了3个酶活力提高的突变体V248L、V248R和G111R,有序叠加后获得最佳突变体G111R/V248R,比活力和催化效率(kcat/Km)分别为野生型的1.84倍和2.06倍。随后通过同源建模及分子对接分析,248位的缬氨酸突变为精氨酸后,与底物形成了两个氢键,增强了酶与底物相互作用;而G111R突变未直接对酶与底物的相互作用造成影响,但可能与248位的精氨酸存在协同作用,共同增加了底物口袋的正电性,从而提高了与负电荷底物的结合力,进而提高了酶的催化活力。该研究为该酶后续在果汁加工等行业的应用奠定了重要基础,也为理性设计提高酶催化活力提供了参考价值。
Endo-polygalacturonase is one of the most widely studied enzymes in pectinases, which specifically hydrolyzes the α-1,4-glycosidic bonds of polygalacturonic acid chains, generating oligogalacturonic acids of different polymerization degrees.This enzyme holds significant importance in enhancing the added value of pectin.Currently, research on this enzyme remains limited, and its poor catalytic activity and thermal stability are major factors limiting its industrial application.In previous work, a novel endo-polygalacturonase gene from Penicillium arizonense (pePGB) was obtained and heterologously expressed in Komagataella phaffii.However, its catalytic activity was only at a moderate level compared to similar enzymes.Therefore, this study focused on enhancing its catalytic activity.Initially, alanine scanning of amino acids in the substrate binding pocket subsites was conducted.Based on the scanning results and sequence alignment, specific mutation hotspots were chosen for rational design.Three mutants with increased enzymatic activity, V248L, V248R, and G111R, were screened.The beneficial mutations were combined to obtain an optimal mutant, G111R/V248R, which showed 1.84-fold and 2.06-fold improvements in specific activity and catalytic efficiency (kcat/Km), respectively.Homology modelling and molecular docking analysis revealed that the mutation of valine to arginine at position 248 formed two hydrogen bonds with the substrate, enhancing the enzyme-substrate interaction.While the mutation at position 111 did not directly affect the enzyme-substrate interaction.It likely synergized with the arginine at position 248 and collectively increased the positive charge of the substrate pocket, resulting in an increased binding affinity with negatively charged substrates, consequently enhancing the catalytic activity.This study lays an important foundation for the application of this enzyme in industries such as juice processing and provides valuable insights for rational design aimed at enhancing enzyme catalytic activity.
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