Isolation Of Glucose Oxidase Producing Bacteria,Solid Fermentation Process And Enzymatic Properties

Glucose oxidase（glucose oxidase）system name isβ-D-glucose oxidoreductase（EC1.1.3.4）,referred to as GOD.Glucose oxidase can specifically catalyzeβ-D-glucose to gluconic acid under aerobic conditions.Glucose oxidase is widely used in flour processing,food preservation,animal feeding,animal disease prevention and control,medical diagnosis and beer industry.At present,the preparation of glucose oxidase mainly adopts Aspergillus niger,Penicillium punctatum,Penicillium notatum through liquid deep ventilation fermentation.Fieldure K.J chromogenic method was used to isolate and screen glucose oxidase producing strain from soil in this paper.The isolated strains were identified by morphological observation and ITS sequence analysis.The composition of solid fermentation medium and fermentation process conditions of glucose oxidase producing strain were studied by single factor test and response surface methodology,and the catalytic kinetics of glucose oxidase and other related enzymatic properties were studied.The main results were as follows:（1）The glucose oxidase producing strain,which named LJ-1,was isolated and purified from the collected soil samples by using Fiedure K.J color development method.Strain LJ-1was identified as Talaromyces pinophilus LJ-1 through morphological observation and phylogenetic tree construction based on ITS-r DNA sequence analysis.（2）Effects of glucose content,the type and amount of nitrogen source,KH₂PO₄ addition,Mg SO₄.7H₂O addition,Ca Cl₂ addition,Zn SO₄ addition and solid water ratio,fermentation temperature,initial p H value,inoculation amount,loading and fermentation time on glucose oxidase activity by solid fermentation of strain LJ-1 through single factor test.The composition of fermentation medium and fermentation conditions were optimized by response surface methodology based on the results of single factor experiment.The results showed that the suitable composition of solid fermentation medium were:wheat bran 100%,glucose14.245%and Na NO₃ 0.644%,KH₂PO₄ 0.2%,Mg SO₄·7H₂O 0.25%,Ca Cl₂0.197%,Zn SO₄0.25%,Tween80 1%;The optimum fermentation conditions were as follows:solid water ratio1:0.78,temperature 32℃,initial p H 6.68,solid loading 20g and inoculation amount 6%.Under these fermentation condition,the glucose oxidase activity reached 47.40U/g·dry basis.（3）The enzymatic properties of glucose oxidase of Talaromyces pinophilus LJ-1 were analyzed.The optimal reaction p H of glucose oxidase was 6.4,and maintained strong stability at Ph5.6-8.0;The optimal reaction temperature of glucose oxidase was 40℃,which was stable in the temperature range of 20-80℃.The concentration of 0.01mol/L metal ion Fe³⁺could promoted the enzyme activity significantly and Al³⁺promoted the enzyme activity slightly,while Zn²⁺inhibited the enzyme activity significantly,and Ca²⁺,K⁺,Mg²⁺,Na⁺had little effect on the enzyme activity.The Michaelis constant km and Vmax of glucose oxidase enzymatic reaction of strain LJ-1 were 118 mmol/L and 5.07 mol/（L·min）.