Fermentation Optimization And Mutation Breeding For High-yield MTG-producing Strains Of Streptoverticillium Mobaraense

Transglutaminase (protein-glutamine-y-glutamytransferase, EC2.3.2.13, TG) is a kind of thiol enzyme that can catalyze protein cross-linking by introducing isopeptide bonds (ε-(γ-Glutamyl) lysine). These reactions can be used to change the function properties of proteins, improve the stability of the proteins and increase the nutrition value of proteins by introducing lysine. Therefore, TG is widely used in food, medicine and other industries. Microbial transglutaminase (MTG) has wider range of substrates and faster rate of cross-linking reaction, which make MTG a more extensively application.In this study, Streptoverticillium mobaraense was used to produce MTG. The microorganism metabolic characteristics, the establishment of high throughput screening method, the relationship between colony morphology and MTG activity, mutation breeding, effects of metal ions on MTG were investigated. Also, the optimal fermentation medium of MTG was studied by response surface method. The main results were as follows:(1) The growth of S. mobaraense could be divided into four periods. The first phase (0-6h) was lag phase, the second phase (6-18h) was logarithmic phase, the third phase (18-39h) was stationary phase and after39h was the forth phase named death phase. The value of pH in the fermentation process increased during0-6h, then significantly decreased during9-24h. From24-33h, the pH value was basically stable and then gradually rised after33h of fermentation. From lag phase to logarithmic phase, the amino nitrogen content continuously decreased and reached the minimum in20h. Then the value gradually increased. The consumption of glycerol was proportional to the fermentation time and the glycerol was exhausted at36h of fermentation. The strains didn’t produce MTG until25h of fermentation. MTG enzyme activity rapidly increased during28-40h and basically stable after40h.(2) The tube fermentation conditions were as follows:100μL spore suspension was put into test tube with3mL fermentation medium and cultured under the conditions of30℃,200r/min, measuring the MTG enzyme activity in48-60h. The96-well plate fermentation conditions were as follows:150μL fermentation medium was added into each hole. Single colonies were picked using sterilized toothpick and inoculated in the hole.The strains were cultured under the conditions of30℃,200r/min, measuring the MTG enzyme activity in72h. The process of high throughput screening method for high-yield of MTG strains was as follows:Strains were cultured using96-well plate fermentation for preliminary screening. Then the high-yield strains were selected by second screening using tube fermentation. Finally, the high-yield strains were confirmed by flask shaking fermentation.Experiments shows that this method could achieve a good effect.(3) The relationship between colony morphology and MTG activity of S. mobaraense was studied. The aim was to build a method to screen rapidly for high-yield of MTG strains according to the colony morphology. Original strains of S. mobaraense were isolated and purified by natural selection. Typical colony surfaces and edges characteristics were selected. Strains were cultivated in test tube and MTG activities were measured. The second screening of flask fermentation was used to verify its feasibility that high-yield of MTG strains according to the colony morphology. The results showed that the MTG activity of colonies with plump and full surfaces and rough edges were the highest; the MTG activity of colonies with thin-flat surfaces and neat edges were the lowest. According to the relativity, a high-yield of MTG strain was selected and MTG activity reached to6.33U/mL, which was20.1%higher than that of original strain. The method to screen rapidly for high-yield of MTG strains of S. mobaraense according to different colony morphology types was feasible.(4) The optimal conditions for ultraviolet mutagenesis of S. mobaraense were as follows:Filtered and nun-diluted spores were treated with UV for45-60s at a distance of30cm, and then dark treated for45min. After several rounds of ultraviolet mutagenesis, a high-yield of MTG strain was selected from5356strains and MTG activity reached to8.43U/mL, which was240%higher than that of original strain. The lethality rate of microwave mutation was proportional to microwave mutation time and the strains were almost all killed when mutated for90s. Through microwave, a high-yield of MTG strain was selected from608strains and MTG activity reached to5.42U/mL, which was59.4%higher than that of original strain. The lethality rate of LiCl mutation was proportional to the concentrations of LiCl and the optimal mutagenesis concentration was0.8%. The strains were all killed when the concentration of LiCl reached to1.2%.(5) The effects of Cu2+, Co2+, Mn2+, Zn2+, Ba2+, Fe2+on MTG and shake flask fermentation for MTG production of S. mobaraense were investigated. The restults showed that in the concentration range of0-1000mg/L, Cu2+, Co2+, Mn2+, Zn2+, Ba2+, Fe2+had no effect on MTG enzyme activity in low concentration range, while the inhibitory effect was enhanced with increasing concentration. The effect on MTG production in shake flask fermentation was not significant when Mn2+, Co2+, Ba2+, Fe2+at a concentration of1μg/mL were added in the medium. Zn2+could promote enzyme activity in low concentration range.(6) The optimal fermentation medium of MTG was studied by response surface method. The results showed that the effects of yeast extract and fish peptone on MTG activity were significant by Plackett-Burman design and analysis. And the optimum medium formulas were as follows:glycerol20g/L, yeast extract6.68g/L, fish peptone27.78g/L, MgSO4·7H2O2g/L, K2HPO4·3H2O2g/L, fermentation accelerator lOg/L.