Optimization And Purification Of Glucose Oxidase In Aspergillus Niger Z-25

Glucose oxidase has attracted considerable research interest in recent years because of its potential application in food industry and as a tool enzyme.In food industry,the enzyme,it can ues as potassium bromate substitute.And it can immobilize on many kinds films to made glucose biosensors that use to detect glucose rapidly.In this paper, through optimization of fermentation,we got higher yield of glucose oxidase;through purification of the enzyme,we got electrophretic pure glucose oxidase on PAGE.In the research,we also studied the characterize of the enzyme.The results were as follows:The effect of various on producing of glucose oxidase by Aspergillusn niger Z-25 was studied.On the base of single factor experiments,the best conditions for liquid fermentation was found to be:glucose 10%,(NH4) 2_SO₄ 0.5%,proteose peptone 1.5%,tween-80 3%.The effcct of inoculation stage,liquid volume in flask and inoculum were studied on the growth of Aspergillus niger Z-25,and the optimal fermentation condition was obtained as follow:7days,50mL and 2.5%respectiviely. After the optimization test,the enzyme activity increased by 19.6 times,and compared with the same midium,the enzyme activity increased by 2.4 times.To study further the properties of glucose oxidase from Aspergillus niger Z-25, purification of glucose oxidase was investigated.The glucose oxidase,which showed as a single protein band on PAGE,was achieved by using standard purification techniques,such as Sephadex G-100 chromatography and DEAE -Sephacel ion exchange chromatography.And an unusual method called tween 80 - ammonium sulphate extraction system.After three-step treatment,596.59-fold purification was obtained with 3%activity recovery and 525- U/mg specific activities. The properties of glucose oxidase which had been purificated were studied and the result were as follow:the enzyme is a homodimer made up of two identical subunits of molecular weight approximately 80kDa each;the optimal temperature and pH for the action of the enzyme were at 40℃and 5.5,respectively;The enzyme was fully stable at pH 3-5 and the temperature up to 50℃;The activity was stimulated by Ag⁺, but inhibited strongly by Cu²⁺ and Pb²⁺.The Km of the glucose oxidase was 0.01mmol·L^-1,and the Vmax was 25.12μmol·min^-1·mL^-1.