| Transgluaminase (EC 2.3.2.13, TGase) is a transferase that catalyzes the cross-linking between proteins or peptides, which can promote protein gelation and having a wide range of application in food, medicine, cosmetics and other fields. Microbial transglutaminase is a hotspot of current research, whereas it exists low activity and poor stability problems. In this paper, the optimal conditions for TGase fermentation with Streptomyces mobaraenis and its enzymatic characteristics were studied.The recombinant strains of Bacillus subtilis with high enzyme activity were obtained through TGase modifition by molecular biology techniques.The main results were as follows:Based on single factors and orthogonal experiment, the optimized medium of TGase from S. mobaraense was identified (g/L):glucose 40, peptone 40, ammonium nitratel 1, yeast extract 2, MgSO42, K2HPO42. Based on single factors and response surface test, the optimal conditions for TGase fermentation from S. mobaraense were determined:78 mL/250 mL of medium volume,10%(V/V) of inoculation and 150 r/min of rotation speed at 30 ℃ with initial pH 7.0 for 96 h cultivation.The crude enzyme was isolated and puried from the TGase supernatant of S. mobaraense by ammonium sulphate salting-out, ultrafiltration, dialysis, freeze drying and gelchromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed a single line and the relative molecular weight was about 37.2 kD. TGase was puried to 5.22 folds, the special activity was 5.73 U/mg with 53.97% enzyme recovery.The optimal temperature of TGase was 50 ℃ and it maintained more than 80% enzyme activity below 40 ℃, while there was almost no activity at 60 ℃. The optimal pH of TGase was 7.0 and the enzyme activity could remain more than 65% in the range of pH 5.0-9.0. The activity was found to decrease gradually at alkaline pH 8.0-10.0 while it droped rapidly at acidic pH 3.0-5.0. Metal ions such as Cu2+, Zn2+, Fe2+and Fe3+, Pb2+had strong inhibition to the enzyme activity, but Ca2+, Mn2+, Na+ had little effects. Antioxidants of EDTA, GSH, DTT were helpful to improve the TGase enzyme activity. When EDTA concentration was 1.0 mM, relative enzyme activity of TGase reached the maximum 133%; When the concentration of GSH and DTT were 5.0 mM, the relative enzyme activity were 137% and 128%, respectively.TGase gene was obtained from genomic DNA of S. mobaraense by PCR amplification. It was then cloned into secretory expression vector pWB980 to construct the recombinant plasmid which was transformed into B. Subtilis WB600. The heterologous expression was implemented. The enzyme activity of rocombinant protein was (3.33±0.21) U/mL after purifcation and zymogen activition. The special activity reached 11.36 U/mg. Using site-directed mutagenesis technology, the other two recombinant strains were constructed, one of which was mutated by changing three amino acid DSD before the N-terminus to M, the other was mutated serine at the 199th into alanine.They were named as B. subtilis WB600/pWB980-smtgM and B. subtilis WB600/pWB980-smtgS199A, respectively. After expression, purifcation and zymogen activition, the enzyme activity were (7.66±0.25) U/mL and (17.16±0.35) U/mL, the special activity were 13.38 U/mg and 26.27 U/mg respectively. Among the three kinds of recombinant protein (protein/smtg, protein/smtgM, protein/smtgS199A), the special activities were higher than the crude enzyme of original strain. Circular dichroism spectrum results showed that the secondary structure of recombinant proteins had been changed when compared to that of original one. The former were mainly a-Helix and β-turn, the latter was priority to a-Helix and β-Beta. The mount of β-turn from protein/smtgS199A was highest among them with 77.6%.The original protein had a higher β-Beta at 80.8%. |
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