Site-directed Mutagenesis And Study On Enzymatic Properties Of Transglutaminase

Protein- glutamate-?- transglutaminase(EC2.3.2.13), named transglutaminase(referred as TGase orTG) catalyzed transglutaminase glutamine residue the ?-carboxamide transfering, and caused cross-linking recation between intermolecular and intramolecular, which could change the function of protein, improved the theoretical of food, increase its elasticity and nutrition. Therefore, TG had a great application values in food, pharmaceutical, cosmetics and other fields.TGase were widely distributed in various organisms. TG obtained from animal were complexity and expensive, which required Ca2+ to expose a cysteine residue in the active site domain. MTGase obtained from microorganism was wide variety of sources, short production period, not dependent on Ca2+, higher enzyme thermostability, stronger catalytic ability and more suitable for industrial production.In this article, the MTG DNA was cloned and sequenced from the strain Streptomyces H197 which had been kept in our laboratory. The results showed: the MTG gene had 1257 bp, encoded a protein of 418 amino acid, and had high GC content. The pI value was 7.08 and its molecular weight was 46.6 kd.To identify the enzyme active site, the mutant sites His(CAC) was insteaded of Lys(AAA) in active-site triad Cys151-Asp342-His361.The mutation plasmid pET-22b(+)-MTG-M was constructed by overlap extension PCR with the designed primers,and further transfered into E. coli BL21(DE3).Sequenced after colony PCR and digestion verification showed that the first C-1090 was mutated to A, 1092- C was mutated to A. It meant that His(CAC) was successfully mutated to Lys(AAA). Two recombinant strains was used for further study.They were the mutant-type pET-22b(+)-MTG-M E.coli BL21(DE3)(named as p-B-M-M)and the wild-type pET-22b(+)-MTG-E.coli BL21(DE3)(named as p-B-M).Crude protein concentration and enzyme activity of the two recombinant strains were measured after expression induced by IPTG.Protein concentration of the mutantwas 7.69g/L, and the crude enzyme activity was 0.437U/mL.Mean while protein concentration of the wild-type was 7.93g/L,and the crude enzyme activity was0.425U/mL. Both the protein concentration and crude enzyme activity from the two strains had no significant differences(T test P> 0.05).It meant that the mutation had not effect on protein expression and catalytic activity of MTG. The optimal induction time was 8-12 h, and the IPTG concentration for the best expression of MTG was 0.8g/L and the optimal induction temprature was 30?.The MTG specific activity of wild strain was increased from 0.056U/mg to3.28U/mg, after purified by Ni-NTA His, and that of the mutant was increased from 0.053U/mg to 3.38 U/ mg. In a very sour or very alkaline conditions, a lot of enzyme activity would rapied lost from wild,which could kept stabilization at pH 6-8 range,while the wild strain at pH5-9;less activity lossed at 20?-40? for 30 min,but the most lossed under 50 ?, the thermostability of the mutant reduced comparing with wild strain;most metal ions tested in the experiment had litte influence in the MTG activity of both the wild and the mutant.But Ni + and Cu2 +could inhibited MTG activity greatly.MTG stability of the mutant such as thermal stability, pH stability and stability of the metal ions were lower than that of the wild type.It suggested that the His enzyme active site Cys151-Asp342-His361 did not play a catalytic role in enzyme activity.But it had function in maintaining the stability of the active center, and ensured the catalytic process smoothly.