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Glucose dehydrogenase mutant and preparation method thereof

A glucose dehydrogenase and mutant technology, applied in the field of glucose dehydrogenase mutants and its preparation, can solve problems such as difficulties in expression and separation and purification, poor substrate specificity, and restrictions on the application of blood glucose measurement

Active Publication Date: 2019-11-12
遵义医科大学珠海校区
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Soluble expressed sPQQ-GDH has poor substrate specificity and a wide substrate spectrum; membrane-bound mPQQ-GDH mesangial protein is difficult to express, isolate and purify, which severely limits its use in blood glucose measurement Applications

Method used

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  • Glucose dehydrogenase mutant and preparation method thereof
  • Glucose dehydrogenase mutant and preparation method thereof
  • Glucose dehydrogenase mutant and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1. Construction of recombinant plasmid pPIC9K-fad-gdh

[0052] Using the restriction-free (RF) cloning method, 9K99a F and 9K99a R were used as primers, and fad-gdh (SEQ ID NO: 2) was used as a template for PCR amplification. Use the SanPrep Column PCR Product Purification Kit (Sangon Bioengineering (Shanghai) Co., Ltd.) to purify the PCR amplified product, use the purified product as a primer, use the pPIC9K plasmid as a template, and use Takara PrimeSTAR GXL DNA polymerase In the second PCR, the recombinant plasmid pPIC9K-fad-gdh was obtained.

[0053] Among them, the primer sequence is:

[0054] 9K99a F:

[0055] 5-CTCTCGAGAAAAGAGAGGCTGAAGCTTCACACAGGAAACAGACCATG-3' (SEQ ID NO: 3),

[0056] 9K99aR:

[0057] 5-GGAACAGTCATGTCTAAGGCGAATTACATCCGCCAAAACAGCCAAGC-3' (SEQ ID NO: 4).

Embodiment 2

[0058] Embodiment 2. Obtain the mutant construct of the Y406 site of fad-gdh

[0059] Using the random mutation primer Y406X-F / R at the Y406 site, the recombinant plasmid pPIC9K-fad-gdh prepared in Example 1 was used as a template to perform site-directed saturation mutation. The saturation mutant PCR products were transformed into XL10-GOLD competent cells for cloning. Plasmids were extracted using an extraction kit (Sangon Bioengineering (Shanghai) Co., Ltd.), and the plasmids were sequenced. From it, 19 kinds of plasmid pPIC9K-fad-gdh with mutation at Y406 site of FAD-GDH were screened.

[0060] Among them, the random mutation primers are:

[0061] Y406X-F:

[0062] 5'-TGCCGAAGTTTTAAAANNNCCAGGCTCCGCCAC-3' (SEQ ID NO: 5)

[0063] Y406X-R:

[0064] 5'-GTGGCGGAGCCTGGNNNGTTTAAAACTTCGGCA-3' (SEQ ID NO: 6).

Embodiment 3

[0065] Example 3. Expression of FAD-GDH in recombinant Pichia strains

[0066] The mutant plasmid prepared in Example 2 was linearized, and then the mutant plasmid was transformed into Pichia pastoris cells using a gene transfer instrument.

[0067] Spread the recombinant Pichia pastoris on the MD plate and incubate at 30°C for 2-3 days until white colonies grow. Use a toothpick to pick the white single colony on the MD plate, inoculate it on a YPD plate containing 0.5 mg / mL geneticin, and incubate at 30°C for 2 to 3 days until the white colony grows. Use a toothpick to pick up the white single colony grown on the YPD plate containing 0.5 mg / mL geneticin, inoculate it into the YPD plate containing 1.0 mg / mL geneticin, and culture it at 30°C for 2-3 days until the white colony is formed. Taking Y406E (the 406th tyrosine is replaced by glutamic acid) as an example, the result is as follows figure 1 As shown in A~1D, figure 1 The results of screening for multi-copy recombinant...

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Abstract

The invention belongs to the field of a biological technology, and particularly relates to a glucose dehydrogenase mutant and a preparation method thereof. Glucose dehydrogenase is from aspergillus terreus, and the mutant is obtained by replacing the 406th amino acid of the glucose dehydrogenase with other amino acids. Compared with the prepared FAD-GDH mutant with wild type FAD-GDH, the activityand the substrate selectivity are greatly improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a glucose dehydrogenase mutant and a preparation method thereof. Background technique [0002] Glucose dehydrogenase (GDH) is a class of enzymes that can catalyze the conversion of glucose into gluconolactone. In this reaction, glucose loses electrons and passes them to the coenzyme or prosthetic group of GDH. In the presence of other electron acceptors, electrons can continue to be transferred to achieve GDH regeneration. Therefore, the enzyme is widely used in blood glucose test strips, biofuel cells and electrochemical biosensors. As an important biological component of the above devices, GDH determines the performance of the devices to a large extent. Therefore, the acquisition of GDH with excellent enzymatic properties is very important for the development of related biomedical engineering equipment. [0003] Daily blood sugar testing is very important for diabet...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12N15/81C12Q1/26C12Q1/00H01M8/16
CPCC12N9/0006C12N15/815C12Q1/006C12Q1/26C12Y101/9901G01N2333/902H01M8/16Y02E60/50
Inventor 杨愈丰向红英郭坚刘迪嘉许佳伟彭莉萍
Owner 遵义医科大学珠海校区
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