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Glucose dehydrogenase mutant, preparation method thereof and application

A technique for glucose dehydrogenase and mutants, which is applied in the field of glucose dehydrogenase mutants and its preparation, and can solve problems such as poor stability, difficult to reuse, and expensive coenzymes

Active Publication Date: 2017-05-31
JIANGSU ALPHA PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because coenzymes are expensive, poor in stability, and difficult to reuse, the industrial application of oxidoreductases is severely limited. It is extremely important to build an efficient and economical coenzyme regeneration system to relieve the restriction of coenzyme content on the reaction and reduce production costs.

Method used

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

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] The establishment of embodiment 1 genetically engineered bacteria

[0025] According to the glucose dehydrogenase gene GDH (GenBank: KU682779.1) included in NCBI, the glucose dehydrogenase gene fragment was artificially synthesized, and the gene fragment was used as a template to amplify and expand the fragment by PCR (adding NdeI and BamHI on both sides of the fragment) endonuclease fragment) its nucleotide sequence is shown in SEQ ID NO.3. And the gene was inserted into the pET-28a plasmid by using the NdeI and BamHI endonuclease sites, and the ligated vector was transferred into Escherichia coli BL21 (DE3) to establish the glucose dehydrogenase genetically engineered bacteria. The primers for PCR amplification of the glucose dehydrogenase gene are: the upstream primer is F1:5'-GGGCCATATGATGGACATGT ATCCGGATT-3' (SEQ ID NO.4), and the downstream primer is R1:5'-GCGGGGATCCTTAGCGGCC TGCCTGGAA T-3' (SEQ ID NO.5).

Embodiment 2

[0026] Embodiment 2 Obtaining of glucose dehydrogenase mutant gene

[0027] In this study, glucose dehydrogenase was protein engineered using an error-prone PCR method.

[0028] The 50 μl PCR reaction system is: 5 μl of 10× amplification buffer, 4 μl of each of the four dNTP mixtures (2.5 mmol / L), 50 pmol of each primer, 1.5 μg of template DNA, 0.5 μL of Taq DNA polymerase, Mg 2+ 2mmol / L, add double distilled water to 50μl.

[0029] The PCR amplification program was: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, and 30 cycles; further extension at 72°C for 5 min, cooling to 4°C, and ending the reaction.

[0030] experiment process

[0031] Chemically synthesize the glucose dehydrogenase gene and insert the gene into the pET-28a plasmid using NdeI and BamHI endonuclease sites as a template for gene mutation; error-prone PCR amplifies the glucose dehydrogenase gene, and the amplified gene fragments a...

Embodiment 3

[0034] The shaking flask culture of embodiment 3 recombinant escherichia coli

[0035] The recombinant Escherichia coli obtained in Example 1 and Example 2 were inoculated into LB medium (10 g / L of peptone, 5 g / L of yeast extract, 10 g / L of NaCl) containing 5 mL of kanamycin (50 μg / mL) respectively. , pH7.0), at 37°C, shake culture at 200rpm for 4-8 hours. Transfer 1 mL of bacterial culture solution to 50 mL of LB liquid medium (containing 50 μg / mL of kanamycin), shake culture at 37°C and 200 rpm until the OD600 is 0.6-0.8, and then add the inducer IPTG to the final concentration After inducing expression at 22-26°C and 200rpm for 8-12 hours, the cells were collected by centrifugation (8000rpm, 15min, 4°C) and washed twice with phosphate buffer (pH7.5, 10mmol / L). Disperse in the same pre-cooled buffer and perform sonication in an ice-water bath. Centrifuge (10000rpm, 15min, 4°C), discard the cell fragments, and obtain glucose dehydrogenase and its mutant crude enzyme solutio...

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Abstract

The invention discloses a glucose dehydrogenase mutant, a preparation method thereof and an application. A nucleotide sequence of a glucose dehydrogenase mutant gene is as shown in SEQ ID NO.1, and an amino acid sequence of the glucose dehydrogenase mutant is as shown in SEQ ID NO.2. The gene is led into Escherichia coli to obtain gene engineering bacteria with the gene, the gene engineering bacteria are cultured, and a fermentation process is optimized, so that recombinant glucose dehydrogenase is prepared. The glucose dehydrogenase mutant is good in catalytic activity, pH (potential of hydrogen) and heat stability and organic solvent tolerance and can be used for regeneration of coenzyme NADH and NADPH in oxidation-reduction reactions.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a glucose dehydrogenase mutant and its preparation method and application. Background technique [0002] Oxidoreductases are widely used to catalyze the preparation of pharmaceutical intermediates such as chiral alcohols and hydroxy acids, and most redox reactions require the participation of the coenzyme NAD(P)H. Because coenzymes are expensive, poor in stability, and difficult to reuse, the industrial application of oxidoreductases is severely limited. It is extremely important to build an efficient and economical coenzyme regeneration system to relieve the restriction of coenzyme content on the reaction and reduce production costs. [0003] Coenzyme regeneration methods can be divided into whole cell method, photochemical method, chemical method, electrochemical method and enzymatic method. Enzymatic regeneration of reduced coenzyme is the most popular meth...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12R1/19
CPCC12N9/0006C12N15/70C12N2800/101C12Y101/01047
Inventor 石利平陈峻青蔡进漆志文张维冰叶银梅徐春涛何义陈晓佩刘思琪
Owner JIANGSU ALPHA PHARM CO LTD
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