Study On The Fementation Condition And Application Of Glucose Oxidase Form Pichia Pastoris

Glucose oxidase (EC 1.1.3.4, GOD) is the redox enzyme which can catalyzes the oxidation of β-D-glucose to gluconic acid using molecular oxygen as an electron acceptor, with the production concomitant of hydrogen peroxide.Preservation in distilled water method for GOD producing strain P. pastoris p51, the technology of producing enzyme with fermentation process, the ingredient of flask medium application fo titration in the determination of glucosamine in broth and the small scale automatic fermentor have been studied systematically in this paper.(1) In shaking flasks condation:the optimal seed age is 20h when do with YPD mudium, phosphate hydrogen II ammonium was the best nitrogen source. The optimum medium for enzyme production was obtained according to orthogonal experiments and its formulates was glucose (20g/L),phosphate hydrogen II ammonium (5g/L), sulfuric acid calcium (0.45g/L), potassium(9.1g/L), magnesium sulfate(14.9g/L), PTM1 (3.3mL/L),KOH 4.13g/L,26.7mL/L phosphate.The maximum GOD activity (30U/mL) was abtanied when the fermentation was canducted at initial pH 6.0,31℃,250 r/min of rotation speed, volume 50 mL/500 mL, The largest concentration of methanol is 2.5% for 8d.(2) When the strain was preserved with methods of distilled water preservation and slant preservation for 180 days, strain survival rate reached 90%and 45%, respectively, which indicated that the effect of distilled water was better than the latter. Furthermore, the final enyzme activity reached 22.5±0.5U/mL and 23±0.4U/mL under methods of distilled water preservation and slant preservation in flask culture system. It showed that distilled water preservation did not reduce the yield of glucose oxidase.(3) The highest GOD activities in a 5-L bioreactor were 230 U/ml by P. pastoris p51 when the fementation was conducted at pH5.0,biomass before induction is 90g/L, methanol was fed at a constant rate of 8ml/h/L in the earlier age for 130h;The conditions of trinary fermentation were studied in a 10-L bioreactor,the maximum GOD activety (260U/mL) was abtainied in 111h.(4) GOD catalyzed by glucose into gluconic acid in fermentation broth samples, making it lose the ability of oxidation reagent Lin, Then use titration method for the determination of Glucosamine. The conversion formula of these two methods was: value of titrimetric method=(value of colorimetric methed+0.1454)× 1.2868+0.3793.Compare with colorimetric methed the titrimetric method is not response by the pigment in the fermentation broth.