Engineering a carboxypeptidase from Aspergillus oryzae M30011 to improve the terminal-specific enzymatic hydrolysis of aromatic amino acids

Abstract

Due to unique amino acid composition and anti-fatigue properties, high Fischer ratio oligopeptides have shown pharmacological effects on multiple diseases. Currently, preparing oligopeptides with high Fisher ratios by enzymic hydrolysis has many limitations such as complexity of optimization conditions of enzymes, time costing, and process inconvenience. Therefore, it is necessary to investigate the specificity mechanism of enzymatic digestion to obtain high Fischer ratio oligopeptides. In this study, serine carboxypeptidase (CPY) from the fungus Aspergillus Oryzae M30011 was cloned and expressed in Escherichia coli (E. coli). Based on prediction from docking by AutoDock Vina which clarified the binding mode of the CPY with synthetic peptides to improve its specificity to recognize aromatic amino acids, we deduced that the key substrate binding sites 271Tyr, 464Ile and 517Met in the hydrophobic cavity of CPY are associated with substrate specificity of the enzyme. Through a series of mutations, we found that the triple mutant Y271R/I464R/M517R had an activity of 1065 U/mg for the synthetic peptide Phe-Gly-Leu-Gly-Phe. In the combination of a specific chymotrypsin mutant P308R and CPY mutant M517R, the Fisher ratio of the oligopeptides from rice bran protein was 43.16, which was 54.09% higher than that of the non-specific enzyme combination.