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Glucose dehydrogenase mutant with improved specific enzyme activity of catalytic xylose

A technology of glucose dehydrogenase and mutants, applied in the directions of oxidoreductase, enzymes, biochemical equipment and methods, etc., can solve the problems such as the ineffective utilization of xylose resources, and achieve the effect of reducing production costs

Active Publication Date: 2017-05-31
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method of the invention solves the problem that xylose resources cannot be effectively utilized, contributes to the rational utilization of abundant and cheap plant cellulose resources, and promotes sustainable economic development

Method used

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  • Glucose dehydrogenase mutant with improved specific enzyme activity of catalytic xylose
  • Glucose dehydrogenase mutant with improved specific enzyme activity of catalytic xylose
  • Glucose dehydrogenase mutant with improved specific enzyme activity of catalytic xylose

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Embodiment 1: Construction of recombinant bacteria expressing mutant A258F

[0028] 1. Acquisition of plasmid pET-GDH

[0029] The medium composition of recombinant E.coli BL21(DE3) / pET-GDH is: peptone 1%, yeast extract 0.5%, NaCl 1%.

[0030] The recombinant bacteria were inoculated in a test tube with a liquid volume of 5 mL of culture medium and cultured at 37° C. with shaking at 200 rpm for 8 h. After the cultivation, the cells were centrifuged at 12,000rpm for 1min and the cells were collected, and the plasmid pET-GDH was extracted using the plasmid extraction kit Mini-Plasmid RapidIsolation Kit (Beijing Biotech Biogene Technology Co., Ltd.); wherein the plasmid pET-GDH The amino acid sequence of glucose dehydrogenase GDH is shown in SEQ ID NO: 1 (the nucleotide sequence is shown in SEQ ID NO: 2).

[0031] 2. Construction of recombinant Escherichia coli E.coli BL21(DE3) / pET-GDH(A258F)

[0032] (1) Acquisition of A258F full-length gene:

[0033] Synthetic primer...

Embodiment 2

[0055] Embodiment 2: Induced expression culture of recombinant bacteria

[0056] LB medium: peptone 1%, yeast extract 0.5%, NaCl 1%, pH 7.0. When needed, add kanamycin sulfate (50 μg·mL) before use -1 ), add 1.5% agar powder to the solid medium.

[0057] Pick a single colony of positive clones and inoculate in 10mL containing 50μg·mL -1 In the LB liquid medium of kanamycin sulfate, culture overnight at 37° C. with shaking at 200 rpm. Take 10mL culture medium and transfer to 1L containing 50μg·mL -1 In the LB liquid medium of kanamycin sulfate, shake culture at 37°C and 200rpm to OD 600 about 1.0. Isopropyl-B-D-thiogalactopyranoside at a final concentration of 0.1 mM was added to the culture, and induction culture was carried out at 37° C. for 12 h.

Embodiment 3

[0058] Embodiment 3: Induced expression culture of recombinant bacteria

[0059] Induced expression: The composition of LB medium is the same as that in Example 2, and a single colony of positive clones is picked and inoculated in 10 mL containing 50 μg·mL -1 In the LB liquid medium of kanamycin sulfate, culture overnight at 37° C. with shaking at 200 rpm. Preserve two branches of glycerol bacteria (each containing 1mL bacterial solution, 15% glycerol), and transfer 1mL culture solution to 50mL containing 50μg·mL -1 In the LB liquid medium of kanamycin sulfate, shake culture at 37°C and 200rpm to OD 600 about 1.0. Isopropyl-B-D-thiogalactopyranoside at a final concentration of 0.1 mM was added to the culture, and induction culture was carried out at 37° C. for 12 h. Bacteria were collected, dissolved in 20mM phosphate buffer (pH 7.0), and ultrasonically disrupted for 20min with a working time of 1s and a resting time of 3s. The crushed bacteria were processed at 12,000 rpm...

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Abstract

The invention discloses a glucose dehydrogenase mutant with improved specific enzyme activity of catalytic xylose and belongs to the technical field of genetic engineering. A recombinant strain E.coli BL21(DE3) / pET-GDH (A258F) containing a target gene is successfully built. A mutate enzyme A258F is subjected to induced expression through the recombinant strain E.coli BL21(DE3) / pET-GDH (A258F); and a crude enzyme fluid is purified through His-Trap affinity chromatography to obtain a pure enzyme A258F. Through optimizing an enzyme activity determination condition, the specific enzyme activity of the A258F in a potassium phosphate buffer solution of pH 7.0 at 55 DEG C reaches 7.59U.mg<-1>. A novel substrate is provided for a coenzyme cycle of glucose dehydrogenase for asymmetric transformation reaction by the work; the problem that a xylose resource cannot be effectively utilized is solved; reduction of the production cost is facilitated; an excellent strain is provided for xylose utilization in industry; and a foundation is laid for transformation of the substrate specificity of the enzyme through a gene engineering method.

Description

technical field [0001] The invention relates to a glucose dehydrogenase mutant with improved specific enzyme activity for catalyzing xylose, which belongs to the technical field of genetic engineering. Background technique [0002] Glucose dehydrogenase can convert NAD(P) + As a cofactor to catalyze the production of glucono-1,5-lactone from glucose, it is widely used in detection kits and biosensors for blood glucose determination in industry. In addition, glucose dehydrogenase is widely used in the coenzyme cycle in the asymmetric reduction reaction catalyzed by oxidoreductase, such as (R)-carbonyl reductase coupled with glucose dehydrogenase to efficiently catalyze the preparation of 2-hydroxyacetophenone ( R) - phenyl glycol. At present, the main raw materials for glucose preparation are corn and sweet potato. However, the production cost of food crops is high. In order to save food and realize the sustainable development of our country, the use of renewable lignocell...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21
CPCC12N9/0006C12Y101/9901
Inventor 张荣珍徐岩
Owner JIANGNAN UNIV
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