Study On Biosynthesis Of Microbial Transglutaminase By Streptoverticillium

Transglutaminase (TGase) is a kind of thiol enzyme that catalyzes acryl-transfer reaction, which introduces inter- or intra-molecular covalent crosslinks in many proteins, changing the quality of food products. TGase has been widely used in food industry.The effects of soybean cake powder hydrolyzates as nitrogen source on TGase biosynthesis of Streptoviticillium SK4.001 were studied. The experiment results showed that trypsin could hydrolyze soybean cake powder effectively. The amount of hydrolyzates added into fermented culture was 100%. The best hydrolysis conditions were that: temperature 35℃, pH 8.5, the concentration of soybean cake 10%, and the amount of enzyme 1000U per gram of substrate. After 42h's fermentation, TGase's activity could reach 5.04U/mL when soybean cake powder hydrolyzates were used as nitrogen source. So it could replace fish peptone as well.TGase from Streptoviticillium SK4.001 exhibited optimum activity at 35℃, pH 7.0 for CBZ-Gln-Gly. TGase was stable at 20-40℃, pH 6.0-7.0. TGase was inhibited by some common metal ions, but was not inhibited by EDTA. Kinetic parameters were determined in the reaction mixtures containing variable mounts of the substrate N-CBZ-Gln-Gly (0-30 mmol/L). The value of Michaelis constant (Km) and maximum velocity (Vmax) determined from Lineweaver-Burk plots were 53.88 mmol/L and 0.98μmol/mL·min, respectively.The zymogen of TGase (pro-TGase) was found during cultivation of Streptoviticillium SK4.001. The pro-TGase was purified by ion-exchange chromatography (Fractogel EMD SO3-). The pro-TGase was hydrolyzed by dispase under suitable condition. The pro-TGase and TGase were both hydrolyzed by dispase when the concentration of protease or the time of enzymolysis increased. TGase's activity increased when CTAB was added into zymotic fluid fermented for 30h, but CTAB did not act on pro-TGase and the mechanism was not clear.The models of Streptoviticillium SK4.001's fermention were constructed using neural network to train experimental data,and the predicted value of the models was compared with the measured value.The result showed that theses models could accurately predict the time course of cell growth,glycerol consumption,and TGase's activity well during the fermentation.The maximum relative error of netwok's simulation reached 4.76% compared with 13.99% of netwok's prediction, and the models constructed could be put into practical use.