Method for preparing recombinant-aspergillus niger glucose oxidase and application of recombinant-aspergillus niger glucose oxidase

A technology of glucose oxidase and recombinant vector, which is applied in the field of genetic engineering, can solve the problems of low expression level, difficult extraction and purification, cumbersome procedures, etc., and achieve the effect of high expression level

Active Publication Date: 2015-06-17
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 genera

Method used

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  • Method for preparing recombinant-aspergillus niger glucose oxidase and application of recombinant-aspergillus niger glucose oxidase
  • Method for preparing recombinant-aspergillus niger glucose oxidase and application of recombinant-aspergillus niger glucose oxidase
  • Method for preparing recombinant-aspergillus niger glucose oxidase and application of recombinant-aspergillus niger glucose oxidase

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Embodiment 1

[0089] Embodiment 1, preparation and identification of recombinant Aspergillus niger glucose oxidase

[0090] 1. Preparation of recombinant bacterial cells expressing recombinant Aspergillus niger glucose oxidase

[0091] The preparation of the recombinant cell expressing the recombinant Aspergillus niger glucose oxidase includes introducing the Agod gene into the recipient cell to obtain the recombinant cell producing the recombinant Aspergillus niger glucose oxidase. Wherein the coding sequence of the Agod gene is the DNA molecule at positions 4733-6520 of SEQ ID No.1, which is used to encode the recombinant Aspergillus niger glucose oxidase of SEQ ID No.5. The specific method is as follows:

[0092] 1. Construction of recombinant Aspergillus niger glucose oxidase expression vector

[0093] Prepare the Agod gene expression vector pPSK-AGOD-His6 shown in the nucleotide sequence of SEQ ID No.1, the recombinant Aspergillus niger glucose oxidase expressed in SEQ ID No.5 by the...

Embodiment 2

[0114] Example 2, Recombinant Aspergillus niger Glucose Oxidase Enzyme Activity Determination and Enzymatic Properties Research

[0115] 1. Determination of enzyme activity of recombinant Aspergillus niger glucose oxidase

[0116] 1. Determination of concentration of recombinant Aspergillus niger glucose oxidase

[0117] Dingguo Folin-phenol protein quantitative kit was used to measure the concentration of protein in the fermentation broth of Tu6△tku70::Agod prepared in Example 1 and the recombinant protein in the purified Tu6△tku70::Agod protein solution by the folin-phenol method The concentration of Aspergillus niger glucose oxidase Agod is done standard curve with bovine serum albumin (BSA), records respectively the mass concentration of the protein in the fermented liquid of Tu6△tku70::Agod is 7mg / mL, and the Tu6△tku70 of purification: : The mass concentration of the recombinant Aspergillus niger glucose oxidase Agod in the Agod protein solution is 2 mg / mL.

[0118] 2. ...

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Abstract

The invention discloses a method for preparing recombinant-aspergillus niger glucose oxidase and application of the recombinant-aspergillus niger glucose oxidase. Recombinant trichoderma reesei Tu6 delta tku70::Agod constructed by introducing an Agod gene into trichoderma reesei Tu6 delta tku70 can successfully express the recombinant-aspergillus niger glucose oxidase, the expression quantity is high, and the enzyme activity in fermentation broth can achieve 137 U/mL which is the highest level of the enzyme activity of shake flask fermentation in production of glucose oxidase strains at present. After the recombinant-aspergillus niger glucose oxidase in the fermentation broth is subjected to ni-sepharose purification, the detected enzyme activity can achieve 342 U/mL. The specific activity of the recombinant-aspergillus niger glucose oxidase is 155 U/mg protein. The recombinant-aspergillus niger glucose oxidase prepared by the method has good heat stability and good acid-alkali tolerance, the fermentation process is simple, transferring is not needed, raw materials are cheap and are easily obtained, and the cost is greatly reduced. The recombinant-aspergillus niger glucose oxidase can be widely applied to the fields 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...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/04C12N15/80C12N1/15C12R1/885
Inventor 董志扬马枝枝陈秀珍林洁黄振邦秦丽娜
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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