Mutation Breeding Of Streptoverticillium Mobaraense For Potent Transglutaminase-production

Transglutaminase (TGase) can catalyze the cross-linking within or between protein molecules, the connection between the protein and the hydrolysis of Glutamine residue in protein molecules. The enzyme has been widely used in food industry, pharmacy and so on. At present, commercialized transglutaminase is produced by microbial fermentation mostly. But the research and development of microbial transglutaminase (MTG) in China is behind international advanced level. Especially the TGase activity of industrial strains is relatively low, which limit the further development of the enzyme in food industry. Based on the Streptoverticillium mobaraense in our laboratory, in order to improve the TGase activity, we investigated establishment of the mutagenesis method, establishment of the screening method, identification of high yield strains and the effects of common salts on fermentation.The main results were as follows:(1) The optimal ultraviolet mutagenesis conditions were as follows:the power of uv lamp was15w, the distance was30cm, the irradiation time was60s or80s. The important influential factors for the nitrosoguanidine(NTG) mutation were the pH of buffer solution, the concentration of NTG and the mutagenesis time. Based on the orthogonal test of NTG mutation, the influence of the above three factors on the fatality rate in turn is:the pH of buffer solution>the concentration of NTG>the mutagenesis time, and the influence of the above three factors on the positive mutation frequency in turn is:the mutagenesis time> the concentration of NTG>the pH of buffer solution. Treatment of spore with NTG in the presence of50μg/mL chloramphenicol caused enhancement of mutagenesis. With the addition of chloramphenicol, the positive mutation rate increased from5.3%to13.3%.(2) We established preliminary screening with96-well plates, then screening with test tube and screening with shake flask. Appropriate fermentation conditions of each screening technique have been determined. With the colony morphology observation, and the use of2-Deoxy-D-Glucose plate, the screening efficiency can be improved. The correlation of screening by96-well plates and test tube was not ideal, which proved that the accuracy of screening by96-well plates is not high. Because this method can reduce the workload and improve screening efficiency, so screening by96-well plates as preliminary screening method was appropriate. Screening by test tube and screening by shake flask had a high correlation, which proved that the accuracy of screening by test tube was ideal.(3) We selected tow high yield strains (20121227NTG-1and20121130NTG-31). Compared with original strain, the enzyme production time of the two high yield strains was later, but the the highest enzyme activity of the two high yield strains improved a lot. Furthermore, we also studied the enzymatic and molecular property of the TGase produced by the two high yield strains. The results showed that the TGase produced by the two high yield strains was the same as TGase produced by original strain. So the TGase produced by the two high yield strains was safe and effective.(4) The effects of three inorganic salts (NaCl, KC1and MgCl2) on the production of transglutaminase (TGase) were tested in this study. It indicated that enzyme activity increased in the presence of NaCl or MgCl2compared with control. The optimum addition of NaCl was0.5%, and the optimum addition of MgCl2was1%. With0.5%NaCl or1%MgCl2, the TGase activity increased observably, and the fermentative period shortened about15hours. Based on the SDS-PAGE and Western Blot results, we found that NaCl and MgCl2had a significant effect on the TGase production by promoting the secretion of proenzyme (pro-TGase) and the maturation of TGase.