Construction Of High-yield Transglutaminase-producing Strain Of Streptomyces Mobaraense

Transglutaminase(EC 2.3.2.13,TGase)is the functional enzyme that can catalyze transfer reaction of acyl group between polypeptides and proteins,introducing the crosslinking among them,changing the functional properties and improving nutritional value of proteins.Because of its specific properties,TGase has been widely used in medical,food,textiles and cosmetics industry areas.In this study,in order to improve the production of TGase,we chosed the Streptomyces mobaraensis 11019 as host to express TGase gene by intergeneric conjugation between E.coli and S.mobaraensis and protoplast transformation.The main contents and results were described as follows:1.Applying S.mobaraensis 11019 genomic DNA as template,the TGase gene was amplified by PCR with the primers,which designed by the characteristic of the plasmid pL99.TGase gene fragment was engineered into plasmid pL99 to obtain expression vector pL99-T.TGase gene has the length of 1224 bp,has the G+C content of 65.22%and A+T content of 34.78%.TGase gene coding 407 amino acids,the first 26 amino acids show strong hydrophobic,and the first 30 amino acids is possible the signal peptide.Through the analysis of homologous modeling three-dimensional structure of TGase,we know TGase mainly consist of ?-helix and ?-fold,C64-D255-H274 is the activity center site.2.We studied several factors about the protoplasts preparation and regeneration of S.mobaraensis by losozymoe digestion to obtain the optimal conditions.The results showed the protoplasts preparation and regeneration rate could approach 97.47%and 13.27%respectively under the follow conditions:the secondary mycelium with the inoculation of 10%transferred to the R2YE liquit medium which containd 0.5%glycine,and cultured at 28? with 220 r/min for 28 h.collected the mycelium and lysed by 2 mg/mL lysozyme at 33? for 1.5 h,then spread protoplast on R2YE medium and cultured at 28? for generation.3.The expression vector pL99-T was transformed into S.mobaraensis to obtain TGase-overexpression strains by conjugation and protoplast transformation.We used the 24-well plate formentation combine 96-well plate detect the TGase activity in premary screening,and shaking flasks detect TGase activity in secondary screeing,and obtain the high-yield transglutaminase-producing strain SM-12.SM-12 produced 8.68 U/mL of the TGase,which was 6 times higher than that of original strain under the same condition.The SDS-PAGE analysis shows that TGase has been over-expressioned in SM-12.