Hydrolysis of myofibrillar proteins by protease AprA secreted from Pseudomonas fragi: Preference for degrading ala-linked peptide bonds

Abstract

Extracellular proteases of bacteria have attracted attention in recent years. Alkaline protease AprA secreted from Pseudomonas fragi has been shown to cause spoilage in chilled meat and to degrade myofibrillar proteins (MPs), but the spoilage mechanism was unknown. AprA possessed a high affinity for substrate proteins (K_m = 1.40 mg/mL, V_max = 11.20 mg/mL/min) and was controlled by metal ions and inhibitors. AprA exhibited strong hydrolytic activity on MPs, with alterations in secondary structure, tertiary structure and sulfhydryl content. Molecular docking and molecular dynamics revealed that AprA bound to actin and myosin heavy chain (MHC) through hydrogen bonds, hydrophobic interaction and salt bridges, respectively. AprA exhibited a broad spectrum of cleavage, with a relative preference for Ala-linked peptide bonds, according to peptide release kinetics. The above results reveal the mechanism of bacterial spoilage of chilled meat at low temperatures. Of course, this provides a theoretical basis for targeted control of the meat spoilage caused by AprA.