Mutation Breeding Of Glucose Oxidase Producing Strain And Optimization Of Fermentation Conditions

Aspergillus niger PCTC was studied to improve glucose oxidase production usingatmospheric and room temperature plasma(ARTP) mutagenesis technique. Firstly, fortystrains showed greater color zone on a screening medium plate, containing o-dianisidine andhorse radish peroxidase. Then the most optimal mutant strain PCTC-8was cultured on theshake flask fermentation. The enzyme activity of PCTC-8reached14.36U·mL-1, which was117.25%higher than the original strain. Optimization of medium composition, fermentationconditions and the conditions of cell lysis were investigated. The findings are as follows:(1) Different methods of cell lysis to stimulate release of glucose oxidase in Aspergillusniger were compared. The optimal conditons of ultrasonic wall-breaking for dilution,ultrasonic power and reaction time was5times,35%, and25min, respectively. Under theabove conditions, the intracellular enzyme activity reached16.66U·mL-1. The optimalenzyme for hydrolyzing wall-breaking was helicase and the reaction time was3.5h. Underthis conditions, the intracellular enzyme activity achieved13.25U·mL-1. Combining thesetwo methods, the enzyme activity of enzymolysis after supersonic treatment was23.39U·mL-1, and the reaction time was1.5h. However, the enzyme activity of first, enzymolysisand then supersonic was19.13U·mL-1.(2) The optimization of medium and fermentation conditions: Single factor experimentand response surface methodology were used to optimize fermentation medium in Aspergillusniger PCTC-8. The optimized medium composition (g·L-1): sugar87.50, beef extract peptone3.15, ammonium nitrate1.88,(NH4)2HPO40.34, KH2PO40.25, twain-6030.47, corn flour12.50. The production of glucose oxidase increased5.09times from17.13U·mL-1to87.50U·mL-1. Based on the optimal fermentation medium, the fermentation conditions forinoculum size, seed age, rotation speed, fermentation time, and culture medium volume were10%,24h,225r·min-1,48h, and30mL, respectively. Under these conditions the glucoseoxidase activity of the producer was93.26U·mL-1.(3) The scale-up fermentation in7L tank and effect of adding time of twain-60toproduction of glucose oxidase was investigated. twain-60was added to broth at the period offermentation prophase, logarithmic phase and stationary phase and the dry cell weight andproduction of glucose oxidase reached21.92g·L-1,24.34g·L-1,28.92g·L-1and62.18U·mL-1,92.88U·mL-1,42.87U·mL-1, respectively.(4) The effect of temperature, pH on enzyme activity and tolerance was investigated. Theoptimal temperature of glucose oxidase was about40℃, and its stability was in the range of20℃to40℃. Placed one hour, the activity was kept above95%between20℃to40℃. The optimal pH of glucose oxidase was about6.0, and its stability was between3.0to7.0, duringwhich the activity was kept above80%after placed10hours.