A kind of preparation method and application of recombinant Aspergillus niger glucose oxidase

Abstract

The invention discloses a preparation method and application of recombinant Aspergillus niger glucose oxidase. The recombinant Trichoderma reesei Tu6△tku70::Agod constructed by introducing the Agod gene of the present invention into Trichoderma reesei Tu6△tku70 can successfully express recombinant Aspergillus niger glucose oxidase, and the expression level is relatively high. The activity can reach 137U / mL, which is the highest level of shake flask fermentation enzyme activity among glucose oxidase-producing strains. The enzyme activity measured after the recombinant Aspergillus niger glucose oxidase in the fermentation broth was purified by a nickel column can reach 342U / mL, and the specific activity of the recombinant Aspergillus niger glucose oxidase is 155U / mg protein. The recombinant Aspergillus niger glucose oxidase prepared by the method of the present invention has good thermal stability and good acid-base tolerance, simple fermentation process, no transfer, cheap and easy-to-obtain raw materials, greatly reduced cost, and can be widely used Applied in the field of food and medicine.

Description

technical field [0001] The invention relates to a preparation method and application of recombinant Aspergillus niger glucose oxidase in the field of genetic engineering. Background technique [0002] Glucose oxidase (Glucose oxidase, E.C.1.1.3.4) is a homodimeric glycoprotein composed of two identical polypeptide chains covalently bonded by disulfide bonds, and contains two non-covalently bonded flavin glands Purine dinucleotide (FAD) cofactor, an enzyme that uses molecular oxygen as an electron acceptor, can oxidize β-D-glucose to generate gluconic acid and hydrogen peroxide. [0003] Glucose oxidase has a wide application value in medical diagnosis, food processing, feed and textile industry due to its strong substrate specificity, high catalytic efficiency and no side effects. Glucose oxidase can specifically recognize glucose, so it is widely used in the detection of glucose content in clinics, such as the detection of urine sugar and the measurement of blood sugar, wh...

AI Technical Summary

Problems solved by technology

[0004] Glucose oxidase is widely distributed in animals, plants and microorganisms, but the content of glucose oxidase in animals and plants is low and the extraction and purification process is complicated; at present, glucose oxidase mainly comes from microorganisms, such as Aspergillus niger and Penicillium, but there are still low yields and the difficulty of extraction and purification

The expression level of glucose oxidase in other microbial expression hosts is generally not high (Table 1), although the expression level in Pichia pastoris and Saccharomyces cerevisiae is relatively high, but both need transfer and use A large amount of methanol is induced, the procedure is cumbersome, and the cost is high. More importantly, Pichia pastoris is not a safe microorganism (GRAS microorganism) certified by the U.S. Food and Drug Administration (FDA), and there are hidden dangers in biological safety. Suitable for use in food and medical fields

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