Fermentative preparation and separation and purification method of heat-resistant and acid-resistant glucose oxidase

Abstract

The invention discloses a fermentation preparation method, and a separation and purification method of heat-resistant and acid-resistant glucose oxidase, and belongs to the technical field of microorganisms. The glucose oxidase prepared by fermentation has high heat resistance and acid resistance, and the defect of low enzyme activity caused by use in food and feed in a high temperature and acidic environment is overcome. The fermentation preparation method comprises the following specific steps: culturing a bacterial strain into a spore, cleaning the spore and then preparing a mono-spore suspension; introducing the mono-spore suspension into a seed culture medium for fermentation culture; introducing seed culture fluid into a shaking flask fermentation medium for fermentation culture, adding a fermentation supplementary nutrient solution into fermentation liquid; adding dextrin, soluble starch and potassium sorbate into the fermentation liquid, and uniformly stirring; freezing, drying and crushing the fermentation liquid to obtain the glucose oxidase. After crude enzyme fluid of the glucose oxidase obtained by the fermentation is subjected to super-heating treatment, ammonium sulfate-ethanol-compound precipitation, ion exchange chromatography and molecular sieve gel filtration chromatography, the glucose oxidase with high recovery rate and high purity is obtained.

Description

technical field [0001] The invention relates to the technical field of microorganisms, in particular to a fermentation preparation and separation and purification method of heat-resistant and acid-resistant glucose oxidase. Background technique [0002] Glucose oxidase is a very important enzyme in the field of enzyme technology. It can use molecular oxygen as an electron acceptor to specifically catalyze the oxidation of β-D-glucose into gluconic acid and hydrogen peroxide. Because of its close relationship with the vital substances glucose and oxygen, it has been widely used in scientific research, medicine and food industry production. In the food industry, glucose oxidase can be used to remove glucose in food or medicine, thereby preventing browning of food or obtaining oligosaccharides with higher purity; at the same time, glucose oxidase can be used as a relatively An ideal potassium bromate substitute, used as a safer flour improver; in the fermentation industry, the...

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Problems solved by technology

[0003] A patent application number 201410228211.0 published by the State Intellectual Property Office on August 6, 2014, titled: Preparation method of glucose oxidase preparation and its utilization, this patent proposes the following technical scheme: inoculate Penicillium inoculated into , nitrogen source, inorganic salt solution and trace elements of the fermentation medium in the fermenter are aerated and fermented to make a fermented liquid, and the fermented liquid is separated into solid and liquid to make a glucose oxidase preparation. The glucose oxidase prepared by this strain has the following technology Problem: First, the glucose oxidase produced by the fermentation of this strain has poor thermal stability, and is easily inactivated in an acidic environment, and does not have acid resistance; second, the enzyme of glucose oxidase produced by the fermentation of this strain Low activity and low production efficiency; these two problems greatly limit the scope of use of the glucose oxidase prepared by this strain, therefore, the glucose oxidase prepared by this preparation method is difficult to promote and use in the market

[0004] At present, there have been many research reports on glucose oxidase at home and abroad, most of which are derived from microorganisms. In microorganisms, there are generally more studies on the enzyme production of mesophilic bacteria, but relatively few studies on the production of thermostable glucose oxidase by thermophilic fungi.

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