Expression of a Salt-Tolerant Pseudolysin in Yeast for Efficient Protein Hydrolysis under High-Salt Conditions

Protease biocatalysis in a high-salt environment is very attractive for applications in the detergent industry, the production of diagnostic kits, and traditional food fermentation. However, high-salt conditions can reduce protease activity or even inactivate enzymes. Herein, in order to explore new protease sources, we expressed a salt-tolerant pseudolysin of Pseudomonas aeruginosa SWJSS3 isolated from deep-sea mud in Saccharomyces cerevisiae. After optimizing the concentration of ion cofactors in yeast peptone dextrose (YPD) medium, the proteolytic activity in the supernatant was 2.41 times more than that in the control group when supplemented with 5 mM CaCl₂ and 0.4 mM ZnCl₂. The extracellular proteolytic activity of pseudolysin reached 258.95 U/mL with optimized expression cassettes. In addition, the S. cerevisiae expression system increased the salt tolerance of pseudolysin to sodium chloride (NaCl)and sodium dodecyl sulfate (SDS) and the recombinant pseudolysin retained 15.19% activity when stored in 3 M NaCl for 7 days. The recombinant pseudolysin was able to efficiently degrade the β-conglycinin from low-denatured soy protein isolates and glycinin from high-denatured soy protein isolates under high temperatures (60 °C) and high-salt (3 M NaCl) conditions. Our study provides a salt-tolerant recombinant protease with promising applications in protein hydrolysis under high-salt conditions.