Insight into the cross-linking preferences and characteristics of the transglutaminase from Bacillus subtilis by in vitro RNA display

Transglutaminase (TGase) has high value to the field of food processing due to their protein cross-linking capacity. Compared with the widely used TGase from Streptococcus mobaraensis (MTG), Bacillus subtilis-derived TGase (BTG) exhibits higher thermal stability and broader range of pH, and is thus better suitable to food processing conditions. However, BTG's cross-linking preferences and characteristics are still unclear, restricting the application in food process. In this study, a modified in vitro RNA display was used to efficiently screen BTG's peptide substrates with a high library capacity. The results showed that BTG preferred to cross-link the Q sites around which were hydrophobic residues in the substrate sequences. LC-MS analysis validated that the top five screened peptides indeed showed high cross-linking rates by BTG. Moreover, the molecular dynamics simulation indicated that their binding affinity with BTG was mainly due to van der Waals interaction, which explained the cross-linking preference of BTG on the substrate. This study provided knowledge about BTG's cross-linking preference and characteristics, which will benefit the comprehensive understanding of BTG and promote its rational application in food industry.

Automated summary

In this study, BTG's peptide substrates were efficiently screened using a modified in vitro RNA display technique. It was found that BTG preferred to cross-link the Q sites around which were hydrophobic residues in the substrate sequences. Experimental validation and molecular dynamics simulation indicated that van der Waals interactions played a key role in the binding affinity between BTG and the substrate, explaining BTG's cross-linking preference.