Breeding And Lab-scale Fermentation Production Process Of Transglutaminase From StreptomycesMobaraensis

Transglutaminases (TGase, EC 2.3.2.13) are a class of transferases that exist in animals and microorganisms. These enzymes catalyze transfer reactions between the y-carboxylamide groups of glutamine residues in various proteins, peptides, and primary amines, and have been used to improve the texture, elasticity, appearance, taste, flavor, and shelf-life of food. The industrial breeding process of a new kind of microbial transglutaminase (MTG) production strain was showed in this research. On the basis of high-yield strain S. mobaraensis ANNN2-19-38, an optimized fermentation medium was also showed, as well as fermentation technology and purifying process of the new kind of MTG (called MTG-TX2).An efficient and stable mutagenesis method was established by evaluating lethality and mutation rate of physical and chemical mutagenic agents. NTG (nitrosoguanidine) mutagenesis was considered as the best mutagenic agents by mutanting strains at the concentration of 2 mg·mL-1 and pH 8.5 for 30 min. A high-yield strain named S. mobaraensis ANNN2-19-38 was obtained by 4 times mutating S. mobaraensis HS47 by ARTP(atmospheric and room temperature plasma) and NTG mutagenesis. The output of the new mutant strain was 105% higher than the wild strain to reach at 4.3 U·mL-1.Using glycerol as carbon source and peptone from fish as nitrogen source at the same time could provide the highest yield of S. mobaraensisANNN2-19-38 in this research. But if the cost of carbon source occupied 12.1% to 55.2% of total cost, glucose was the cheapest carbon source. Furthermore if the cost of carbon source occupied less than 12.1% of total cost, glycerol was the best carbon source. On the other hand, peptone from fish was the cheapest nitrogen source if the cost occupied less than 35.6% of total cost, nor was ammonium chloride. Because the percentage cost of carbon source is easily reaching to 12.1%, glucose might be a great carbon source in the fermentation medium.On the basis of determination on the physiological status of S. mobaraensis ANNN2-19-38, an optimized fermentation process evolving changes of pH, temperature and feed strategy was showed in this report. The optimized process includes the following changes. Mg3(PO4)2 was removed, instead of adding 50 mM MgCl2 at the beginning of fermentation, then pH was stabled at 6.0-6.5 by adding 50 mM Na3PO4. The temperature was set at 35℃ from 0-14 h and at 25℃ after 14 h. Feeding carbon source was used from 20 h to 22 h at the rate of using carbon source. The optimized process enhanced 11.6-23.3% of MTG output at 10 L bench scale fermentation. And also, the Optimized process reduced 61.5-71.5% of phosphorus concentration in fermentation broth.Two purification methods was suggested to adapt high recovery or high purity by this research. Methods adapting high purity included adjusting pH to 8.5, then precipitated by 50% ethanol and finally purified by cation ion-exchange chromatography, the purity was more than 95% detected by SDS-PAGE. Methods adapting high recovery included ultrafitration and cation ion-exchange chromatography, the recovery reached to 70.96%.MTG-TX2 was found more stable than Chinese commercial MTG at pH and thermolstablity. Furthermore, MTG-TX2 performed better at low temperature which suggested it has better application in the future. Yogurt within MTG-TX2 was much firmer and better at storage.