Li_2025_Appl.Biochem.Biotechnol__2

Cutinase (EC 3.1.1.74), a serine esterase that degrades a variety of ester substrates into fatty acids, has significant potential for various applications. Bacillus subtilis is a food-safe strain known for its exceptional protein secretion capabilities. This study aims to enhance the expression of cutinase from Humicola insolens (HIC) in B. subtilis. Specifically, B. subtilis WS9 was selected as the expression host, with pHY300PLK as the expression vector, achieving an initial enzymatic activity of 35.00 U.mL(-1) in shake flask fermentation. Subsequently, the effect of different promoters on HIC expression was investigated, and the optimal promoter, P(amyQ'), was screened, achieving an enzyme activity of 75.85 U.mL(-1). Then, by co-expressing the chaperone protein PrsA and the intracellular chaperone proteins DnaK and GroE, the enzyme activity increased to 121.43 U.mL(-1). Based on that, we optimized the fermentation medium and conditions for cutinase production, and the highest activity achieved was 232.31 U.mL(-1), which was 6.63 times higher than the original level. The recombinant strain was further subjected to high-density fermentation in a 3-L fermenter. After 96 h, the enzymatic activity reached 1548.53 U.mL(-1), which was 6.67 times higher than that achieved in the shake flask. Notably, the cutinase expressed by B. subtilis exhibited comparable efficacy to that expressed by Pichia pastoris in the application of tomato preserves. This study establishes a robust framework for industrial-scale HIC production, advancing its application in food technology.