Engineering a carboxypeptidase from Aspergillus niger M00988 by mutation to increase its ability in high Fischer ratio oligopeptide preparation

Abstract

High Fischer ratio oligopeptides have better conditioning effects on chronic diseases caused by long-term sub-health. At present, the enzymatic method for producing high Fischer ratio oligopeptides has a low yield, complicated purification, and a high cost. The use of exopeptidases with specific catalytic activity for aromatic amino acids in the preparation of high Fischer ratio oligopeptides is an important means to solve this problem. The carboxypeptidase from Aspergillus niger M00988 was cloned, which has good specificity for hydrophobic amino acids. Mutations at important substrate binding sites 135, 160, and 206 were performed to study important factors affecting the enzyme-specific recognition of aromatic groups. The results showed that the steric hindrance of amino acid residues at position 135 and the effects of positions 160 and 206 on the binding force of the enzyme to the substrate have important effects on the specific recognition of aromatic groups by the enzyme. Therefore, the S135 G, Y160S, and Y206S mutant enzymes have good application prospects in the preparation of high Fischer ratio oligopeptides with Chlorella powder. The obtained oligopeptides' Fischer ratio reached 31.45, 38.42, and 36.54, respectively. Compared with the original enzyme, the Fischer ratio increased by 2.58 %, 25.31 %, and 19.18 %, respectively.

Keywords: Aromatic amino acids; Binding sites; Enzyme specificity; High Fischer ratio oligopeptides; Site-directed mutations.