Molecular modification and food-grade system construction for protein-glutaminase production in Bacillus subtilis

Protein-glutaminase (PG) is an enzyme used to enhance the functional properties of food proteins, such as emulsification, solubility and foaming. However, the use of PG in the food industry is limited because of its low catalytic activity and expression levels. In this study, a mutant library of PG was systematically constructed and screened through a structure-guided semi-rational design. This resulted in the identification of a mutant strain, A291S, which exhibited a 45.71% increase in activity. Moreover, a food-grade expression system for PG was established in Bacillus subtilis, demonstrating PG expression levels comparable to those obtained using plasmid-based expression systems that utilized antibiotics. The activity of PG remained above 99% even after the plates were incubated at ambient temperature for 5 days. However, the antibiotic-based expression system retained only 36% of PG activity. The efficacy of fed-batch fermentation for PG production was also evaluated and a maximum activity of 8.15 U/mL was obtained. These findings highlight the significance of PG modification and potential applications of the food-grade expression system in the food industry.