Targeted reduction of bitterness in infant extensively hydrolyzed formula by engineering a chymotrypsin from Metarhizium.

This study investigated the reduction of bitterness in extensively hydrolyzed formula (EHF) for milk-allergic infants by engineering chymotrypsin CHY1 through molecular simulation and mutagenesis. Molecular docking identified key mutation sites (Ser201, Pro309, Gly345) for enhancing binding to bitter peptides. Virtual mutation analysis predicted stable interactions. Mutant enzymes were expressed in Pichia pastoris GS115, with expression levels increased to 86.24-112.17 mg/L. The P309W mutant exhibited more than twice higher activity than CHY1. Optimized enzyme combinations, particularly P309F with flavor protease, were applied to milk protein hydrolysis. Results demonstrated significant reductions in hydrophobic amino acid content and bitterness, alongside improved peptide distribution below 500 Da. Sensory evaluation and electronic tongue confirmed near-zero bitterness, while antigenicity was substantially reduced. These enhancements were attributed to the mutant's efficient cleavage and synergistic exopeptidase action. The findings provide a strategic basis for industrial low-bitterness EHF production using microbial chymotrypsin.