Pyranose oxidase and expression and purification method and application thereof

Abstract

The invention relates to a pyranose oxidase. A gene engineering technique is used for preparing a pyranose oxidase polypeptide chain, constructing recombinant plasmids and recombinant expression, purifying and determining activity of the pyranose oxidase, and a kit is configured to detect glucose content in human serum and comprises reagent I and reagent II. The pyranose oxidase has the advantages that gene engineering is used for recombinant expression of the pyranose oxidase and is high in yield, simple in technology, high in stability and low in cost; an amino acid sequence of the pyranose oxidase is analyzed through computer software, and the polypeptide chain high in homology is selected from different species of pyranose oxidases based on extensive literature research to express polypeptides with functionality, so that both activity of the pyranose oxidase and soluble expression of recombinant proteins are guaranteed, expression, purification and use are facilitated, and a process is simple; a pyranose oxidase method for detecting blood glucose is simple, stable, accurate and capable of resisting interference of VC, hemolysis, chyle and jaundice.

Description

technical field [0001] The invention relates to a pyranose oxidase and its expression and purification method and application. Background technique [0002] The methods for detecting glucose content in serum mainly include glucose oxidase method and hexokinase method. The glucose oxidase method uses glucose oxidase (GOD) to catalyze the first carbon atom of D-glucose to form gluconolactone, and the hexokinase method uses hexokinase (HK) to catalyze the phosphorylation reaction of glucose and ATP to generate Glucose-6-phosphate and ADP. This is the most widely used method for clinical determination of glucose content in serum. Pyranose oxidase (PROD, EC1.3.10) can catalyze the reaction of a class of carbohydrates on the second carbon atom to generate 2-keto derivatives and hydrogen peroxide. D-glucose is a highly specific substrate for pyranose oxidase with a small Km value (0.83mM-3.1mM), but the Km value of glucose oxidase is 33mM. Moreover, glucose oxidase and hexokina...

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Problems solved by technology

[0005] At present, pyranose oxidase is mainly obtained through the extraction of fungal microorganisms, but the yield is low and the purification process is complicated. Therefore, the construction of better PROD production strains by genetic engineering methods has always been highly valued.

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