Heterologous expression and characterization of Bacillus velezensis SW5 serine protease involved in the hydrolysis of anchovy protein.

ABSTRACT

BACKGROUND: Bacillus velezensis SW5, with good enzyme production ability, was isolated and identified in our laboratory from fermented fish sauce. Its galactosidase has been expressed in Escherichia coli, which could hydrolyze lactose in milk. The present study aims to express a novel serine protease gene (SPr-SW5) of this strain by Bacillus subtilis WB800N, and applies the expressed enzyme in hydrolysis of anchovy to prepare antioxidant substances, aiming to alleviate the waste of low-value fish resources. RESULTS: SPr-SW5 with the open reading frame of 1353 bp encodes a serine protease (SPr-SW5) with 450 amino acids. The theoretical molecular weight and isoelectric point are 47.2 kDa and 5.22, respectively. The successful expression of SPr-SW5 in B. subtilis WB800N was confirmed by a skim milk plate test. Its optimal temperature and pH were 50 °C and 8.0, respectively. SPr-SW5 activity was increased by Ca2+ and Zn2+ , but inhibited by Fe3+ . Furthermore, SPr-SW5 was tolerant to 1% Tween-40 and Tween-80; however, its activity was strongly inhibited by 10 mm phenylmethylsulfonyl fluoride. Additionally, SPr-SW5 could be capable of hydrolyzing anchovy, the hydrolysate (AHP) at 10 g L-1 , with 2,2-diphenyl-1-picrylhydrazyl and hydroxyl (·OH) scavenging rates of 73.21% and 79.71%, displaying good antioxidant activity. CONCLUSION: The novel SPr-SW5 was successfully expressed in B. subtilis WB800N. It exhibited excellent temperature stability and good tolerance to several metal ions. In addition, the anchovy hydrolyzed by expressed SPr-SW5 has good antioxidant ability. Overall, this research lays a good foundation for SPr-SW5 with respect to exploration and application in the food industry as enzyme preparation. © 2023 Society of Chemical Industry.