Purification, Characteristics And Application Of A Novel Transglutaminase Derived From Streptomyces Hygroscopicus

Transglutaminase catalyzes an acyl transfer reaction between the γ-carboxyamide group of a peptidebound glutaminyl residue and a variety of primary amines. In this paper, the separation and purification of microbial TGase, derived from a novel Streptomyces hygroscopicus, was studied, including its enzymic properties, and using it in modified vital wheat gluten.In the manufacture process of MTG powder, application of ultrafiltration(UF ) separation and condensation MTG culture filtrate was mostly considered, comparing the separation effect of 3 type UF, which are helix ultrafiltraion membrane with a molecular weight cut-off of 10KD and 30KD and hollow fiber ultrafiltraion membrane with a molecular weight cut-off of 30KD, and the membrane pollution and washing, cleaning was also discussed. The cut off ratio of 3 type UF membrane was 100%, 97%, 100% respectively;and the recovery was 85%, 83.5%, 86% respectively;and the flow velocity was 0.025 , 0.043 , 0.066mL/min · cm~2, respectively. From the separation effcet, the hollow fiber membrane is the best, but its pollution was also biggest, which the loss of flow velocity was 70%. As the whole, the helix membrane with a molecular weight cut-off of 30KD, is the most appropriately. After ultrafiltraion, the condensated MTG was been sedimentated, and dried to MTG powder.After that, the separation and purification of MTG by CM-cellulose and Superdax-75 have been studied. In the CM-cellulose ion-exchange chromotography, the volume of MTG was 40mL, on the linear gradient elution method, MTG could be eluted by 300mL, 0~0.3mol/L phosphate buffer, at a flow tate of 0.4 mL/min. the prification fold was 13.6, while the specific activity and the recovery were 25U/mg and 63%, respectively. The MTG was seperated and purificated by Superdax-75 chromotography, the purification fold was 1.14 and the recovery was 65%. The TGase was pure by the examination of SDS-polyacrylamide electrophoresis (SDS-PAGE), and the molecular weight of this TGase was approximately 38.324KD by Gel-Pro analysis.In this studies of the enzymic properties of MTG, the MTG powder and the liquid MTG were stable in 4℃, remaining more than 90% MTG activity after 180 days;and 86% MTG activity after 14 days. The optimum temperature for MTGase activity was 47 ℃, it maintained high thermal stability between 20℃ and 40℃. The optimum pH for MTGase activity was 7.0;between 5 and 8, MTG maitained 90% activity and stability beteen pH6.0~ 8.0.In the study of MTG modified vital wheat gluten, the optimum reaction condition is: 30℃, pH 6.0, the concentration of gluten is 10%, and MTG 20U.Under this condition, the reaction velocity of MTG is the mast. The functional properties of modified gulten such as gel properties is sthengthened 1.7 fold;and the Solubility was add to 45mg/g from 30mg/g, the water-keeping and oil-absorbing property are also improved to 1.8g/g and 1.38g/g, respectively.