Activation Mechanism Of Transglutaminase From Streptomyces Hygroscopicus

Transglutaminase (TGase; EC 2.3.2.13) is a functional enzyme, which could introduce covalent cross-links between proteins as well as amines and peptides by catalyzing acyl transfer reactions. It has great wide applications in the fields of food processing, biomedical engineering, material science, textiles. It has been reported that Streptomyces TGase is secreted as zymogen (Pro-TGase) and activated by protease, but the mechanism of TGase activation was still unclear. To further explore this mechanism, the purification and activation process of TGase from S. hygroscopicus were studied in this paper.1. S. hygroscopicus was cultured in fermentation liquid added Cu2+, Mn2+, Zn2+, Fe2+ and Co2+ of different concentration respectively. Through the analysis of TGase activity, the activity of TGase could be promote by Cu2+, Mn2+, Zn2+, and the optimal was Cu2+in the concentration of 0.05 mmol/L. After cultured 60 h, Pro-TGase could entirely transformed into mature TGase in optimized culture.2. Two active TGases (TGase A and TGase B) and Pro-TGase were effectively purified from Streptomyces hygroscopicus fermentation liquid by ammonium sulfate precipitation, Hitrap Q HP and Superdex 7510/300GL columns. This is the first report for the purification of TGase and Pro-TGase using anion chromatography.3. The two TGases exhibit different hydrophobicities, molecular weights, and second structures by analysis using Hiprep 1610 phenyl FF,LC-MS and CD spectra. They also have different Vmax, Km, and kcat values.4. The N-terminal amino acid sequence of purified two TGase were determined as DAADER and N-terminal amino acid sequence of Pro-TGase was ASGGDG. According to the amino acid sequence alignment, the N-terminal sequences of Pro-TGase from S. hygroscopicus exhibited significant sequence similarity to those from S. cinnamoneus, S. fradiae and S. netropsis, but the N-terminal sequence of TGase from Streptomyces hygroscopicus exhibited low sequence similarity to them. This is the first report of the N-terminal amino acid sequence of TGase from Streptomyces hygroscopicus.5. The results of interaction between Pro-TGase and TGase indicated that Pro-TGase could be activated by one type of mature TGase (TGase B), while the activity of both TGases was inhibited by the heat-treated Pro-TGase.6. The activation process was studied using the fermentation liquid in different time. The results indicate that Pro-TGase could be activated by some enzyme to TGase B, then TGase B activate the Pro-TGase to TGase A.