Active expression of a charge-engineered protein glutaminase FBPG in Komagataella phaffii.

FBPG is a novelly reported protein glutaminase derived from the marine bacterium Flavobacterium, exhibiting unique catalytic properties in the deamidation of plant proteins. However, its industrial application has been hindered by low specific activity, lack of an efficient expression system, and expression of its proenzyme form. To end those shortcomings, we optimized charge distribution near the active site of FBPG by site-directed mutation. A mutant A223H/R225K achieved a specific activity of 15.18 U/mg, a 9.43-fold increase over the wild type. We then evaluated different promoters for expressing mutant A223H/R225K in Komagataella phaffii, with PDH showing the highest expression. To obtain active enzyme, a Kex2 cleavage site was introduced between the propeptide and the mature peptide, where optimizing the P1' site (Asn) markedly enhanced active FBPG yield. We found that overexpression of Sko1 increased FBPG production by 60 % and achieved full-active enzyme expression in a 7 L bioreactor with a yield of 2610 U/L. This study significantly improved the catalytic efficiency and production yield of active FBPG without additional enzymatic digestion, laying a crucial foundation for its application in the food industry.