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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