Glucose Oxidase Producing Strain Mutation Breeding And Optimization

Glucose Oxidase（GOD）is a kind of aerobic dehydrogenase which canspecifically catalyticβ-D-glucose to generate gluconic acid and hydrogen peroxide inaerobic condition. As biological catalyst, GOD shows widely value of applications inthe food industry, medical diagnostics and scientific fields and so on.Now the GOD had already been used as commodity for sale in our country,however its purity and vitality is lower and has less stability. So almost all thedemands depend on the imports, therefore to improve the purity and vitality of GODto come out the locational possess very important sense. In this study we selected outan overproducing strain by mutagenesis and then the enzyme activity was improvedthrough the optimization of fermentation conditions. Finally we prepared some basicresearches which concluded the preparation of protoplast and production of calciumgluconate utilizes this strain to carry out fermentation for carrying out the followingexperiments.The results of mutagenic breeding：The enzyme activity of the originalAspergillus strain stored in our lab is 1.21U/ml. Through the treatment of ultravioletand microwave mutation, we finally selected out mutant of C3 which possess higheryield of GOD and well stability. Also the GOD of C3 showed obviously higherenzyme activity which can achieved 5.32U/ml . It is more than 4 times the initialstrain.Optimal results of medium conditions: We carried out fermentation to productenzyme under the different content of organic nitrogen such as peptone, meal, urea,yeast extract and inorganic nitrogen such as （NH₄）₂SO₄, NaNO₃. The resultsdedicated when peptone at a concentration of 0.4% can cause the maximum impact ofenzyme production and the enzyme activity can achieve 6.21U/ml. Also it has a betteractivity effect when NaNO₃at a concentration of 0.6%, its maximum activity can reach 6.15U/ml. This study used different content of industrial glucose, sucrose,lactose, wheat bran, soluble starch as carbon source. The results dedicated that sucrosehas the greatest impact on enzyme production and the greatest activity can reached6.25U/ml. When we used starch as the carbon source, cell growth is very little and theactivity of glucose oxidase is very low. When we used wheat bran as the carbonsource, mycelium is wound in the bran which will not conducive to the preparation ofcrude enzyme. When we used lactose as the carbon source, the somatic has almost nogrowth, probably because lactose cannot be used by the bacteria. We can concludefrom the fermentation results, when concentration of the sucrose is12%, we have thebest enzyme producing. Experimental results show that enzyme activity can beachieved 7.20U/mL under the condition of sucrose 12.3%, peptone 0.41% and NaNO₃0.60%. It is more than 6 times the initial strain.The results of the optimization of fermentation conditions: Enzyme activityreached the maximum when the incubation time of 48 h, and then the activity beganto decrease. Reasons of the decline may be due to the rapid decline of pH forproducing large amounts of acid during the fermentation process and the increased ofhydrolysates, all of which make the production and activity of the enzyme reduced;The enzyme activity is the largest at the temperature of 30℃, cell growth is veryslow when the temperature is below 25℃and enzyme activity is lower; Whentemperature is higher than 30℃, its activity will decrease. Initial pH of about 6 forenzyme production effect the best, the pH value is too high or too low will affect theenzyme production and activity, and enzyme production fermentation process into alot of gluconic acid and other acidic substances, so that the enzyme activity rapidlydeclined when the pH dropped to below 3 in fermentation broth. When the content ofbroth was 30ml and the inoculated with a volume of 3%, its maximum activity canreach 8.70U/ml. It is more than 7 times the initial strain.The optimum temperature andpH for enzyme is 30℃and pH5.5. The pH stability is wide and the theraml stabilityis very good ,the glucose oxidase was inhibited strongly by Ag⁺、Hg²⁺.