In vitro activation of recombinant pro-protein-glutaminases from Bacteroides helcogenes and Flavobacterium sp.

Abstract

Protein-glutaminases (PGs) are extracellular enzymes and catalyze the deamidation of protein-bound glutamine residues in other proteins. This often increases the respective protein's solubility. Therefore, PGs are interesting for the food industry since protein-rich foods are trending. Microbial PGs are intracellularly produced in their inactive zymogenic pre-pro-form and subsequently activated after the secretion process, whereby the pre-pro-peptide sequence is cleaved off extracellularly by an endogenous peptidase. However, a former study showed that a recombinantly produced pro-PG from Bacteroides helcogenes (PGB) was surprisingly active, although the pro-peptide was still attached. In this study, the specific in vitro cleavage of the PGB pro-peptide by HRV-3C peptidase was investigated and led to a 16-fold activity increase compared to the non-cleaved pro-PGB. Alternatively, trypsin was used for activation (17.5-fold activity increase) and mass spectrometry analysis revealed that the pro-peptide was indeed cleaved in proximity to the putative native cleavage site. In addition, another formerly unknown PG from Flavobacterium sp. 316 (PGF) was recombinantly produced as pro-PGF in E. coli, partially purified, and biochemically characterized. Pro-PGF was almost inactive, but was activated similarly to pro-PGB through in vitro pro-peptide hydrolysis with trypsin. The mature PGF showed maximal specific activity of 123.7 nkat mg⁻¹ at 35 °C and pH 9.