| TGase is a common food additive, which is mainly collected from Streptomyces mobaraense fermentation. At present, the industrial yield of TGase by Streptomyces mobaraense fermentation is still low in domestic. Above all, this research is trying to improve the TGase yield of Streptomyces mobaraense by breeding strains and optimizing the fermentation conditions and seeking fermentation promoter.Three high-throughput screening methods are established, which are 96 well shallow plate screening,96 well deep plate screening and colony blotting screening method. As the standard enzyme label plate, each well in the 96 well shallow plate can hold 250μL liquid. Colony strain is cultured in 96 well shallow plate, which contains 150μL fermented liquid culture in each hole and being tested the enzyme activity after 96 h; And each well in the 96 well deep plate can hold 1.2mL liquid. Colony strain which contain 150μL or 200μL fermented liquid culture in each hole and being tested the enzyme activity after 96 h; when it comes to Colony blotting screening method, filter paper or the cellulose membrane is needed. After being cultured for 3 day, spores in NA agar plate then been transferred to fermentation medium plate cultured for another 4 days, then been reacted by liquid quantitative for coloration. Screening with 96 well plate was more accurate than the colony blotting screening, but the cycle time by colony blotting screening is shorter.Streptomyces mobaraense were induced by two physical mutagenesis method of UV and ARTP (atmospheric pressure plasma mutation at room temperature) and three kinds of chemical mutagenesis method:nitrosoguanidine (NTG), sodium nitrite (NIT), two acid ethyl ester (DES). Moreover, at the same time the method of UV-DES composite mutagenesis is studied as well. With the method of high throughput screening, and the optimum conditions of each mutation,38000 strains of mutant culture are screened and 1522 strains of mutant culture are rescreened and 447 strains of high yield strains are verified. And the test is found that the TGase enzyme activity of the highest yield strain reached 9U/mL, which is increased by 91% than the wild type strain. In order to avoid the degradation of the strains, the natural breeding is used to the high yield strains during the breeding process. Five different kinds of mutagens lead to 18 kinds of mutation combinations. The different combination schemes are evaluated under the standard of the improvement of the yield. After the comparation of the 3 high-yielding strains and the original strains with the fermentation and metabolism characteristics, the results showed that the growth of the high yield strains appears a clear delay. In addition, the high yield strains has a significant difference on the metabolism of the glycerol and amino acid.By optiming the carbon and nitrogen sources of the high yield strains. The result turn out that the best carbon source of strain 20141026GSCN52 is glucose with 1.75% concentration. A research regarding the effects of Chitin on streptomyces mobaranse also has been done,and it is found that 0.05% chitin be play an significantly role in promoting Streptomyces mobaraense’s fermentation. It can be explained as that Chitin might pressure the growth process of Streptomyces mobaraense, and then enhance the secretion of secondary metabolites. In addition, Chitin might also be used as a dispersing agent to reduce the density of mycelial aggregates, which enhance the mycelia pellets’ permeability. Thereby it is benefit for strains growth and secretion of Pro-TGase to the extracellular. Consequently the TGase yield of Streptomyces mobaraense is improved.Mutation breeding, optimizing the culture of Streptomyces mobaraense in fermentation and adding the chitin as a fermentation accelerant all elevate the ability of Streptomyces mobaraense producing MTG in fermentation to different extent, so the three methods play a critical and practical role in fermentation industry. |
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