Mutation and Fermentation Conditions Optimization for the Production of Transglutaminase with Streptoverticillium Mobaraense
|Keywords||transglutaminase Streptoverticillium mobaraense atmospheric pressure glow discharge plasma fermentation optimization feeding strategy|
Transglutaminases are a family of enzymes (EC 188.8.131.52) that catalyse the formation of covalent cross-links between a free amine group and the y-carboxamide group of protein-bound or peptide-bound glutamine. It has gained interest due to its attractive potential applications in food sector, immobilization of enzymes, biomedical engineering, textiles and leather processing.This study is mainly aimed at enhancing the production of TGase by Streptoverticillium mobaraense 03-10. Firstly we mutagenized and selected the TGase producing strain and then optimized fermentation conditions. The main results are listed as following:(1) The atmospheric pressure glow discharge (APGD) plasma jet driven by a radio frequency (RF) power was used to treat the spores of S.mobaraense 03-10 for the selection of TGase producer. The mutant with high TGase production was quickly screened according to the formation of color on the double-layered plate and the different appearances of colonies. The total mutation rate was over 42.8% and the positive mutant rate was 20.6%. The obtained mutant G2-1 has good genetic and morphology stability and TGase activity reached 2.73 U/mL, which was 82% higher than that of original strain.(2) The effects of environmental conditions on fermentation by the mutant strain S. mobaraense 03-10 G2-1 were optimized in flask culture. The proper culture conditions in seed medium were as the following:the carbon source was 25 g/L soluble starch, and inoculation spores was 106per mL, and the seed age was 20 h, and the inoculum size was 8%. The optimal fermentation conditions were 2 g/L CaCl2 and 30 g/L glycerol as single carbon source. The TGase activity in flask reached 3.71 U/mL, which was improved by 40%.(3) The effects of different mixture carbon sources on fermentation were investigated. The optimal mixture carbon source were glycerol and corn starch in flask culture. Then compared the fermentation situations in different agitator speeds and different ratios of carbon sources in a 3 L stirred fermentor, the best results were that the agitator speed was 500 r/min, and the ratio was 10:20. The maximum activity was 3.54 U/mL.(4) Influence of different pH and feeding strategies on fermentation in a 3 L stirred fermentor by the screened mutant were investigated. The optimal pH control strategy in fementation processing was as the following:the initial pH7.4 and using NH3·H2O to control pH≥6.5. Based on the optimal pH control strategy, the effect of different initial glycerol concentrations and glycerol feeding rates were studied. A three-stage feeding-shift strategy was proposed:feeding rate 1.67 g/L/h during 10～20 h,0.83 g/L/h during 20～30 h,0.41 g/L/h during 30～40 h. By applying this feeding-shift strategy in fermentation, the maximal activity and productivity were significantly improved and reached 3.69 U/mL and 76.88 U/(L-h) respectively, compared with the results of before optimization(2.07 U/mL and 42.71 U/(L-h)).