Study On Identification And Mutafacient Selective Breeding Of Transglutaminase Producing Strain

Transglutaminase could improve the characteristics of protein foods by catalyzing protein cross-linking within or between protein molecules, and is very valuable for the food industry. To obtain higher transglutaminase producing strain, the transglutaminase -producing strain H197 from soil by the laboratory was treated by UV and gene cloning of transglutaminase from the strain H197 was tried. In addition, we also tried to conduct high transglutaminase-producing E. coli by genetic engineering.Strain H197, which could produce transglutamiase, was selected from the soil. In order to identify this strain, were studied the strain patterns, culture characteristics, physiological and biochemical characteristics, as well as 16SrDNA sequences. The results showed as following. First, the strain H197 had typical morphology of Streptomyces species. Second, the strain H197 has a similar characteristic to Streptomyces hygroscopicus in strain patterns, culture characteristics, physiological and biochemical characteristics and 16SrDNA sequences. According to the multi- classification principle and the phylogenetic analysis, the strain H197 was identified as Streptomyces hygroscopicus. In addition, sequence analysis showed that TG gene of H197 exhibited a higher similarity to that of Streptomyces hygroscopicus than to other Streptomyces strains.H197 (transglutaminase activity for 0.34 u/mL) was used as the original strain, after two ultraviolet repeat mutation, high TGase producting strains was selected by gel-producing and measuring the enzyme activity. Finally, a high TGase producting strain (transglutaminase activity for 1.25 u / mL) was selected, with which the activity ratio of MTG got 3.68 to fold that of H197.The genome DNA was extracted from the strain H197. The full-length of the transglutaminase gene was amplified and sequenced. The analysis of gene sequence revealed that the full-length sequence of transglutaminase gene was 1257 bp in length with 61.4% GC content, and encoded 418 amino acids with was molecular mass of 47 kD and a pI value of 7.08. BamH I and Hind III was selected for the restriction enzyme to produce the cohesive end. The full length of the transglutaminase gene with an additional restriction site was conducted by ligating the enzyme gene into the vector pET-28a, the recombinant plasmid pET28a+TG was transferred into E. coli strain DH5α. But sequencing verification is needed before further studies.