Studies On Optimization Of Fermentation Medium, Purification And Enzyme Preparation Of Glucose Oxidase

Glucose oxidase can oxidize β-D-glucose to produce gluconic acid and utilize molecularoxygen as electron acceptor with simultaneous production of hydrogen peroxide, so it had a widerange of applications in food, medicine, feed and chemical industry. This paper studied theoptimal fermentation medium and fermentation condition by Aspergillus niger, purification ofglucose oxidase and dry of glucose oxidase.First, in order to improve the production of glucose oxidase by Aspergillus niger M2632of ourlaboratory, the effects of carbon source nitrogen source and emulsifier of fermentation mediumwere explored in single factor experiments, results indicated that: the effect of glucose and（NH₄）₂HPO₄on enzyme was remarkable, the concentration level of carbon source, nitrogen source,tween, KH₂PO₄, MgSO₄·7H₂O and CaCO₃were studied though uniform design methodology,results indicated that: the optimum fermentation medium for production of glucose oxidase iscomposed of162.4g/L of glucose,1.2g/L of （NH₄）₂HPO₄,5.7g/L of KH₂PO₄,6.3g/L ofMgSO₄·7H₂O,56.9g/L of CaCO₃. The condition of fermentation was studied by single factorexperiments, results indicated that: culture temperature was32℃, medium pH was7.0, inoculumwas6%, rotation speed was200r/min, strain age was18h. The glucose oxidase was produced byabove condition in a5L fermenter, results indicated that the fermentation time of48h was to obtainthe maximum enzyme activity.The method of purification about glucose oxidase by aspergillus niger was studied. Theoptimal conditions were obtained: sedimentation of30%～80%about ammonium sulfate, dialysis,ion exchange chromatography of DEAE-Sephrose and gel chromatography of Superdex-G200, theenzyme activity was increased by30.22times, the recovery of enzyme activity was31.58%. Theresults showed that the enzyme was a single band after SDS-PAGE and electrophoretichomogeneity was obtained. The molecular weight of enzyme was measured and was approximately154.2kD. It contained two identical subunits. The properties of glucose oxidase were studied andthe results were as follow: the optimum temperature of reaction was40℃, the range of thermalstability was20℃to45℃, the optimum pH of reaction was5.5, the range of pH stability was4.5to7.0, the activity was stimulated by Na⁺, Zn²⁺, Ca²⁺and restrained by Hg⁺, Ag⁺, the activity wasstimulated by5mmol/L Mg²⁺and restrained by high concentrations of Mg²⁺.The effects of three additional agent （soluble starch, maltodextrin and β-cyclodextrins） andfour protectives（sucrose, trehalose, NaCl and PEG1000） on the stability of glucose oxidase duringspray drying were discussed. The condition of spray drying was studied by single factor experiments. On these basis, the condition of glucose oxidase preparations was studied and resultsindicated that: maltodextrin and trehalose were the best protectives, the optimimal concentrationlevel were15%and0.3%, the effect of the temperature of inlet was170℃, the temperature ofoutlet was80℃and speed of injection was8mL/min.