Purification And Characteristics Of Transglutaminase Derived From Mutant Streptomyces Mobaraensis

Transglutaminase (TGase, EC2.3.2.13) which was widely existed in the human, animals, plants and microorganisms, was an acyl transfer reaction catalyzed transferase. Transglutaminase has been widely applied in various fields, the sales of MTG have nearly 200 million yuan in 2013 in China. However, MTG are mainly used in food, whose purification method was simple and performance was very unstable. Novel potential applications which cover the areas of biomedical engineering, material science, textiles and leather processing have emerged during the last decade. Although a lot of resercher have been purified MTG in different methods based on level of food, however, and the study of purification of pharmaceutical grade MTG has not yet in full swing.The fermentation metabolic curve of Streptomyces strains uv847 and mutant strain uv587 were compared in this paper, and transgiutaminase which was up to 99% HPLC purity and endotoxin content in line with the requirements of pharmacopoeia were acquired by cation exchange and hydrophobic chromatography. Enzymatic properties and the preliminary mature mechanism of MTG derived from two strains were studied. The main conclusions were shown as follows:1. The growth of two Streptomyces mobaraensis indicated the logarithmic growth phase(6-15h), the cells increased rapidly to 32g/L, the growth of bacteria was stabilize in stationary phase(18-50h). The pH has been decreased firstly (0-18h) and then increased after 18h. The amino nitrogen trend same as pH, glycerol as a carbon source was consumed almost entirely consumed after been 30h. Pro-MTG was secreted into the medium in logarithmic period, then the protease beginning to be secreted, zymogen was gradually cut to form active MTG in the stabilization period.The production of MTG from uv587 was twice the one from uv847. Protease rised quickly in logarithmic phase and stationary phase. LC/MS detected proteins were involved in the fermentation metabolism, protein synthesis, signal transduction, energy transportation and other related proteins.2. A safe efficient microbial transglutaminase purification method applied for industrial manufacture was introduced in order to improve the purity of transglutaminase and extend its applications for pharmaceuticals. Cultures was centrifuged at 10000 rpm 4℃ to remove the cells before adjusting conductivity to 4.1 mS/cm and pH to 5.5 for purification. The sample was purified by cation-exchange chromatography at a linear velocity of 60 cm/h in the first step.In this step SP Sepharose FF as a packing provided high selectivity and high loading for the target protein. After primary purification, the sample was further processed/isolated with hydrophobic chromatography packed with phenyl Sepharose 6 FF (high sub). This process purified yield was 69.7% and purification multiple was 21.7 times. Specific activity of MTG was 38 U/mg.SDS-PAGE pattern manifested a purity of over 95% and HPLC analysis indicated a purity of above 99% were achieved after this two purification step method. LPS of the purified sample was determined at 0.013 EU/ml with tachypleus amebocyte lysate, met the blood products requirement (<0.15 EU/ml) provided in Chinese Pharmacopoeia. Thus the method described/established in this study was efficient and practical in microbial transglutaminase purification.3. The enzymatic properties of MTG were studied after purification. The results showed that:The molecular of MTG derived from uv847 was 37781Da, isoelectric point of the enzyme was 8.1, the optimum temperature was 50℃, the thermal stability range was 0-40℃, the optimum pH was 6-8, pH stability range at 5-10. The activity of MTG was not inhibited by monovalent metal ion such as LiCl, NaCl, KCl and in the concentration of 5mM, MTG activity was enhance smaller; was inhibited by divalent metal ions ZnCl2, CuCl2 but not MgCl2, CaCl2, BaCl2; and was inhibited by trivalent metal ions FeCl3. The purified MTG was inhibited by NEM indicated TGase could possess a thiol group at the active site, EDTA and PMSF was not affected. The molecular of MTG derived from a strain uv587 was 37.814kDa, isoelectric point of the enzyme is 8.0, the optimum temperature was 50℃, the thermal stability range at 0-40℃, the optimum pH at 5-8, pH stability in the interval of 5-10. The effect of metal ions and inhibitors on MTG was similar with the one derived from uv847, MgCl2 was not effect on MTG from uv847 but inhibited the one a certain extent from uv587. The Vmax of MTG from uv587 was twice the one from uv847, which play stronger crosslinking effect in the adequate substrate.4. Pro-MTG derived from Streptoverticillium mobaraensis uv587 activited as the research object, the mature mechanism zymogen (pro-MTG) has carried on the preliminary explored. Pro-MTG was secreted into the bacterial fermentation, pro peptide can be cut by different protease in fermentation broth after removal of cell the fermentation.With 50mM NaAc-HAc pH6.0 buffer reconstitution initial ethanol precipitation fermentation, the rate of MTG formation will be higher than MTG activation did not do any processing of the fermentation broth. The speed of MTG activation was difference in broth at 12h,15h,18h, respectively.56h was need for culture at 12h to reach maximum activity, and only 8h for culture at 182h. The protease for MTG activation was Zn2+-dependent metalloprotease and not inhibited by NEM and PMSF. pH and temperature could affect the maturity and activity of MTG, the best mature conditions was at low temperature, pH6.0. Pro-MTG has no effect on the activity of MTG, while increased the stability of MTG.