Studies On The Pilot-scale Fermentative Production, Purification And Application Of Microbila Transglutaminase

Streptomyces sp. WJS-825,which was preserved in our laboratory produced Transglutaminase.In this paper,Studies on the fermentation conditions,purification ,characterization and application of TGase was carried out.The main reports were as following:By single factor design and five factors quadratic orthogonal rotary regression experiment design, a mathematical model of the effect of the process conditions targeted on microbial transglutaminase(MTG) activity and dry cell weight was established.The optimical Media composed of 2.1%multi-peptone, 1.5%soluble starch,0.1%MgSO4.7H2O,0.2%K2HPO4,0.3%Na2HPO4,0.3%polyglyceralaether,initial pH was adjusted to 6.96. The seeds of Streptomyces sp.WJS-825 pre-treated for 48h was inoculated into 40mL media in 250mL flask,shaking in 30.8℃ at 200r/min for 72h,with 10% inoculation volume.In 5-200L fenmenter controlled by one on-line supervision computer,besides above all conditions the appropriate fermentation conditions also include:0.8-1.1v/vm aeration rate, 300-400r/min agitation speed: and adopt a stepwise controlled strategy:0-20h(32℃,pH7.2);20-72h(30.8℃, pH6.98).Applying these optimical conditions,the batch fermentation with the Streptomyces sp.WJS-825 went on smoothly and obtained repeatable MTG productions with stable enzyme activitives higher than 3000U/L.By ammonium sulphate fractional precipitation,CMC cation exchange chromatography and sephedex-75 gel filtration ,TGase was purified 158 fold from the extract content of Streptomyces sp.WJS-825.The extract recover rate of the enzyme activity was 78%,while the purification recover rate was 32%.SDS-PAGE showed the molecular weight of the enzyme was about 40KDa.Some properties of the enzyme were undertaken,the results showed:TGase had higest enzyme activity at 45℃ with pH6.8 and was stable under 50℃ within pH4.5-7.5.TGase was inhibited by Fe2+,Mg2+,Zn2+,while Ca2+ had no effect to its activity.Modification effect of TGase on casein from goat milk was investigated. The experiment results indicated:TGase could catalyze cross-linking reaction among casein molecules and as a result polymer with high molecule mass formed.The approapriate cross-linking reaction conditions include:50℃,pH6.0-8.0, 15u/g protein enzyme dosage.Under these conditions the reaction could be completed within 120 minutes.The properties of casein solution modified by TGase had been changed:the viscidity and emulsification of casein solution modified were increased while the vesicant of it was changed.