Discovery,purification,enzymatic characterization,and recombinant expression of a novel transglutaminase
[Background]Transglutaminase EC 2.3.2.13(TGase)catalyzes cross-linking between the γ-carboxamido group of glutamine residues and the ε-amino group of lysine residues.This process leads to the formation of an isopeptide bond,which modulates the conformation and functions of proteins.TGases play a crucial role in food,pharmaceutical,textile,and leather processing industries.[Objective]To mine a high-performance TGase from natural Streptomyces mobaraensis and enhance the titer of this enzyme by recombinant expression in the industrial chassis.[Methods]The TGase production potential of S.mobaraensis(CGMCC 4.266)was evaluated by shake-flask fermentation.The TGase(TGe)was purified by Capto S cation exchange chromatography.The enzymatic properties including optimal pH,pH stability,optimal temperature,thermal stability,and cross-linking ability with casein were evaluated.We knocked out tg from industrial S.mobaraensis and obtained △tg,in which tge was introduced and expressed.[Results]TGe showcased the optimal pH 5.0,with high activity within the range of pH 4.0-10.0.This enzyme achieved the highest activity at approximately 50 ℃,which was comparable to that of commercially available TGases.TGe exhibited good stability within 4-40 ℃,and its activity surpassed those of commercial TGases at 40-65 ℃.In addition,TGe demonstrated higher cross-linking ability with casein at 50 ℃ than commercial TGases.The recombinant expression of tge in △tg increased the TGe titer(6.3 U/mL)by 162.5％compared with the wild-type strain,without compromising the catalytic activity of TGe.[Conclusion]High-performance TGases can be mined from natural S.mobaraensis.The heterologous expression of TGases in mature industrial strains gives a novel insight into enhancing the TGase titer of S.mobaraensis.
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