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

[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...

[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...

[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...