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A kind of artificially modified glucose oxidase gene and its expression application

A glucose oxidase, artificial technology, applied in the direction of oxidoreductase, application, genetic engineering, etc., can solve the problem of insufficient comprehensiveness of enzyme molecular structure and catalytic mechanism

Active Publication Date: 2017-04-05
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the bottleneck of this method is that the current research on the molecular structure and catalytic mechanism of enzymes is not comprehensive enough, so it is still difficult to purposefully and rationally modify proteins

Method used

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  • A kind of artificially modified glucose oxidase gene and its expression application
  • A kind of artificially modified glucose oxidase gene and its expression application
  • A kind of artificially modified glucose oxidase gene and its expression application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] The amplification of embodiment 1 Aspergillus niger Z-25 glucose oxidase gene

[0053] 1.1 Strains and plasmids

[0054] Aspergillus niger Z-25 (Aspergillus.niger Z-25);

[0055] Escherichia coli BWTOP10 (Escherichia.coli BMTOP10) strain was purchased from Beijing Bomed Technology Development Co., Ltd.;

[0056] The synthesis of amplification primers was completed by BGI;

[0057] The sequencing vector pMD19-T was purchased from Takara Company;

[0058] 1.2 Amplification of the glucose oxidase gene

[0059] First, the genome of Aspergillus niger was extracted from the Aspergillus niger Z-25 mycelium used in the glucose oxidase fermentation experiment.

[0060] The genome of Aspergillus niger Z-25 was extracted by the improved benzyl chloride method.

[0061] (1) Suction filter the mycelial ball obtained in Chase's culture medium, and use filter paper to dry up the moisture in the culture medium as much as possible

[0062] (2) Grind the collected bacteria into pow...

Embodiment 2

[0077] Embodiment 2 Construction of glucose oxidase mutant gene library

[0078] 2.1 DNaseI randomly cuts the glucose oxidase gene

[0079] Firstly, use DNaseI to randomly cut the amplified glucose oxidase. DNaseI can cut any gene non-specifically. The system is as follows:

[0080] glucose oxidase gene 10μl DNaseI buffer 2μl DNaseI 0.8μl wxya 2 o

7.2μl condition 37 ℃ constant temperature 1h

[0081] 2.2 Glucose oxidase fragment reunion (DNA shuffling)

[0082] Take a certain amount of glucose oxidase fragments as a substrate, add enzyme and dNTP to carry out PCR without primers, this process is the process of DNA shuffling.

[0083] The specific system is as follows:

[0084] Tag Mix 10μl ddH2O 3μl gene fragment 7μl

[0085] The shuffling PCR program is:

[0086] 1 94℃ 3min 2 94℃ 30s

[0087] 3 56.5℃ 30s 4 72℃ 30s 5 Repeat steps 2-4 55 times 6...

Embodiment 3

[0101] Example 3 Introduction of glucose oxidase variant gene library into Pichia pastoris GS115 and its screening

[0102] 3.1 Strains and plasmids

[0103] Yeast expression strain Pichia GS115;

[0104] The shuttle expression vector pPIC9K was purchased from Invitrogen;

[0105] The sequencing vector pMD19-T was purchased from Takara Company.

[0106] 3.2 Culture medium and enzyme activity assay substrate

[0107] Refer to EasySelect for specific components of conventional medium YPD, yeast enrichment medium BMGY and yeast induction medium BMMY for yeast culture TM Pichia Expression Kit A Manual of Methods for Expression of Recombinant Proteins Using pPICZ and pPICZα in Pichia pastoris Catalog no. K1740-01.

[0108] The formulation of the 3L amplified fermentation medium for the screened high-enzyme-activity-expressing yeast refers to the composition of the medium involved in "Research on High-Density Culture Conditions for the Production of Recombinant Human Serum Albu...

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Abstract

The invention relates to an artificially modified glucose oxidase gene and expression application thereof, belonging to the fields of protein or enzyme preparation modification engineering and glucose oxidase production. The artificially modified glucose oxidase gene aims to further enhance enzyme activity on the basis of the existing glucose oxidase. An irrational means DNA (deoxyribonucleic acid) shuffling technique in protein or enzyme preparation modification engineering is utilized to modify a glucose oxidase gene derived from Aspergillus niger Z-25; and a gene segment subjected to DNaseI random cutting is subjected to the DNA shuffling technique to recombine the segment into a complete variant gene, and the complete variant gene is connected with a shuttling expression plasmid and integrated into the yeast genome. The strain with obviously higher enzyme activity is screened from the mutant library, and the variant sequence is disclosed as SEQ ID NO.2. In a 3L scale-up experiment of yeast, the end enzyme activity of the glucose oxidase reaches the higher level. Finally, in the aspect of enzyme activity and genes, the obtained high-enzyme-activity expression strain has favorable hereditary.

Description

technical field [0001] The present invention relates to irrational protein transformation means, especially to the transformation of glucose oxidase gene derived from Aspergillus niger by using DNA shuffling technology. The present invention also relates to the application of mutated glucose oxidase gene in secretion and production of glucose oxidase, which belongs to protein Or enzyme preparation engineering and the field of glucose oxidase production. Background technique [0002] Glucose Oxidase (Glucose Oxidase, E.C.1.1.3.4, referred to as GOD) can specifically catalyze β-D-glucose to generate gluconic acid and hydrogen peroxide under the condition of oxygen, and is widely distributed in animals, plants and microorganisms ( Pluschkell ​​S, Hellmuth K, Rinas U. Kinetics of glucose oxidase excretion by recombinant Aspergillus niger. Biotechnol Bioeng 1996; 51:215-20.). [0003] However, because microorganisms have the characteristics of fast growth and reproduction and wi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N9/04C12N1/19C12N15/81C12R1/84C12R1/19C12R1/685
Inventor 谭天伟张琛
Owner BEIJING UNIV OF CHEM TECH
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