Discovery, purification, enzymatic characterization, and recombinant expression of a novel transglutaminase

Abstract:

[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. mobaraensisand obtained Δtg, 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 Δtg 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.