| Transglutaminase (R-glutaminyl-peptide:amine-γ-glutayle-transferase, TGase, EC2.3.2.13) catalyze acyl transferring between ε-amino of Lys and y-acylamino of Gln, creating an ε-(γ-Gln)-Lys isopeptide bond, thus TGase is widely used in food industry, as well as in medical, cosmetic and other fields. Most TGase in the market is collected from streptoverticillium fermentation. However, industrial yield of microbial transglutaminase (MTG) is largely limited by the productivity of strains. Filamentous streptoverticillium forms mycelium pellets in liquid medium, leading liquid fermention process hard to control and therefore being detrimental the MTG yield. To improve the productivity of strains, high yielding strains were screened through traditional mutation method and newly built high-throughput screening method. Furthermore, promoters were searched to cope with pelleting and enhance MTG express. This research was focused on following aspects.To improve the efficiency of strains screening, two high-throughput screening systems based on cellulose acetate film (CAF) preliminary assay and 96-well micro-titer plate preliminary screening, respectively, were established in the first step. In CAF assay spores were germinated on Gaose’s medium for 3 days, and then cultured on solid fermentation plate for 4 days before color assay. In 96-well micro-titer plate assay, colonies were directly fermented for 3 days without seed culture. Colonies with a diameter of 2-3 mm were inoculated in 96-well micro-titer plate fermentation and cultured at 30℃ for about 72 h. The quantitative 96-well micro-titer plate assay was proved to be more accurate than the semi quantitative CAF assay. Candidate strains sifted from highthroug-put preliminary assay were further checked by stout tube fermentation assay and shaking flask validation.In this research,5 MTG high-yield S. mobaraense mutants were finally confirmed from over 14 000 one-or multi-time(s) UV treated strains in 14 months. The MTG activity reached as high as 6.2 U/mL,41% improved than its screened parent strain (4.4 U/mL),1.3 times higher than the original starting strain (2.7 U/mL). Both of the 2 high-throughput screening methods were proved to be very efficient and low-cost in this process.To find ways to reduce the effect of mycelium pellet on MTG yield,7 dispersants, including gelatin, agar, soluble starch, tamarind gum, carboxymethylcellulose sodium (CM), pectin and sucrose, and their impacts on metabolisms of S. mobaraense were discussed in this research. It’s showed that all the 7 addictives reinforced dispersed growth of this filamentous microorganism. The outer side mycellin became much longer and densed in addictive medium, aphotic zone of pellet under microscope became smaller. Among these dispersants,3 g/L pectin achieved the best effect on both dispersed growth and MTG yield. MTG producing peak in 3 g/L pectin medium improved by over 10% and moved 12 h forward.0.5 g/L CM followed for its very low action concentration and advanced MTG expressed time (12 h). Besides,100 g/L sucrose has significantly enhanced MTG productivity by 30%. Thus in an overall condideration, pectin is the most efficient dispersant in this research.Two previously reported efficient MTG fermentation promoters were examined too. It was proved that an addition of 5 g/L CTAB (cetyl trimethyl ammonium bromide) at the end of exponential growth did really advance MTG peak time for at least 20 h. But the other agent studied, polymyxin (both B and E), did not affect growth or MTG yield of S. mobaraense.Proceeding from strains enhancement and mycelial growth improvement, this study helped to crack the profound hard nuts of MTG production by strains breeding and seeking fermentation promoter, providing new thoughts for MTG research. |
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