Recombinant Fungal Aspartic Endopeptidases: Insights into Protein Hydrolysis and Combined Effect with Pepsin for Animal Feed Application

Abstract

Protein hydrolysis under acidic conditions can improve the product quality, nutrient availability, and cost efficiency, particularly when neutral or alkaline enzymes are ineffective. Six fungal aspartic endopeptidases (FAPs) were recombinantly expressed as active enzymes in Komagataella phaffi, with peak activity between 30–50 °C and pH 3.0–4.0. Despite FAP1 yielding a higher degree of hydrolysis for soy protein isolate (SPI) than FAP4, mass spectrometry analysis revealed similar cleavage preferences for the two peptidases. FAP1 and FAP4 experienced competitive product inhibition (K_i: 2.8 mg mL^–1, K_m: 3.2 mg mL^–1 for FAP1 and K_i: 9.67 mg mL^–1, K_m: 6.58 mg mL^–1 for FAP4). These findings suggest that K_i and K_m values, when studied in isolation, do not always predict a peptidase’s hydrolytic efficacy. Among the FAPs, FAP6 notably increased soluble protein content in animal feed by ∼3-fold. FAP1, when combined with pepsin, had a positive effect on the hydrolysis of SPI. These results underscore the potential of FAPs to hydrolyze proteins─specifically, animal feed proteins─in acidic environments.