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Engineering strain for co-displaying glucose oxidase and catalase on spore surface and application of engineering strain

A glucose oxidase and surface co-display technology, applied in the biological field, can solve the problems of low enzyme activity, low yield, complex detection methods, etc., and achieve the effects of wide pH adaptation range, improved stability, and changes in enzymatic properties.

Active Publication Date: 2019-11-26
QILU UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Low yield, low enzyme activity, and complex detection methods are the limiting factors for the industrialization of GOD

Method used

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  • Engineering strain for co-displaying glucose oxidase and catalase on spore surface and application of engineering strain
  • Engineering strain for co-displaying glucose oxidase and catalase on spore surface and application of engineering strain
  • Engineering strain for co-displaying glucose oxidase and catalase on spore surface and application of engineering strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Construction of recombinant plasmids

[0058] (1) Cloning to obtain the spore capsid protein gene fragment

[0059] Using the Bacillus subtilis WB800n genome as a template, primers were designed to amplify the gene fragment of cotX spore capsid protein and the gene fragment of P43 promoter by PCR.

[0060] The PCR primers of the CotX protein gene sequence are as follows:

[0061] cotX-F: 5'-aaaactggtctgatcggatccGATCAGACAAAGACGGAAAAACG-3'

[0062] cotX-R: 5'-gtctgcatGAGGACAAGAGTGATAACTAGGATGG-3'

[0063] The PCR reaction system is as follows:

[0064] 2×Phanta Max Master Mix 25μL, 10μmol / L upstream primer cotX-F 2.5μL, 10μmol / L downstream primer cotC-X 2.5μL, template 2.5μL, use ddH 2 O supplemented to 50 μL;

[0065] The above-mentioned PCR reaction is carried out according to the following procedures:

[0066] Pre-denaturation at 95°C for 3min; denaturation at 95°C for 15s, annealing at 60°C for 15s, extension at 72°C for 15s, 30 cycles; extension at 7...

Embodiment 2

[0147] Example 2: Preparation of Bacillus subtilis WB800n electroporation competent cells

[0148] Pick a single colony of Bacillus subtilis WB800n on the surface of fresh LB solid medium and culture it overnight in 5 mL LB medium. Take 1 mL of the overnight culture and insert it into 50 mL of GM medium (LB+0.5M sorbitol), and culture it with shaking at 37°C until OD 600 is 1.0. The bacterial solution was placed in an ice-water bath for 10 minutes, centrifuged at 5000 rpm at 4°C for 8 minutes, and the bacterial cells were collected. The cells were resuspended with 20 mL of pre-cooled ETM medium (0.5M sorbitol+0.5M mannitol+10% glycerol), centrifuged at 5000rpm at 4°C for 8min, the supernatant was removed, and washed three times in this way. Resuspend the washed bacteria in 500 μL of ETM medium, aliquot them into EP tubes, and aliquot 60 μL in each tube.

Embodiment 3

[0149] Example 3: Transferring recombinant plasmid pDG1730-cotX-god-SpyTag into Bacillus subtilis WB800n

[0150] Add 5 μL of the recombinant plasmid pDG1730-cotX-god-SpyTag to 50 μL of competent cells WB800n, incubate on ice for 5 min, add to a pre-cooled electroporation cuvette (2mm), and perform electroporation at 2500V and 5ms. Immediately after the electric shock, add 1 mL of RM medium (LB + 0.5M sorbitol + 0.38M mannitol) preheated at 37°C to the electric transfer cup, revive and culture at 37°C for 3 hours, and spread on the LB plate containing spectinomycin . Inverted culture at 37°C, and strains resistant to spectinomycin were screened.

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Abstract

The invention relates to an engineering strain for co-displaying glucose oxidase and catalase on spore surface and application of the engineering strain. According to an engineering strain of bacillussubtilis, a prepared fusion gene fragment cotX-god-SpyTag is transformed into bacillus subtilis WB800n, recombinant bacillus subtilis WB800n-cotX-god-SpyTag is obtained, then a fusion gene fragment P43-cat-SpyCatcheris electro-transformed into recombinant bacillus subtilis WB800n-cotX-god-SpyTag competent cells; and namely the engineering strain is obtained. The experiment shows that the interaction peptide has no effect on the glucose oxidase and the catalase linked by SpyTag / SpyCatcher, and the enzymatic properties of the expressed glucose oxidase and the catalase are obviously changed, theadaptive range of the temperature and pH is wide, and the temperature and acid resistant properties are improved.

Description

technical field [0001] The invention relates to an engineering bacterial strain and application of co-displaying glucose oxidase and catalase on the surface of spores, and belongs to the technical field of biotechnology. Background technique [0002] Glucose oxidase (GOD) is an oxidoreductase that can specifically catalyze β-D-glucose to generate gluconic acid and hydrogen peroxide under aerobic conditions. GOD is widely distributed in animals, plants and microorganisms. Due to the rapid growth and reproduction of microorganisms and wide sources, they are the main source of GOD production. The main production strains are Aspergillus niger and Penicillium. At present, the application fields of GOD are continuously expanding, and the demand in domestic and foreign markets is increasing sharply. Low yield, low enzyme activity, and complex detection methods are the limiting factors for the industrialization of GOD. [0003] Catalase (CAT), an enzyme that catalyzes the decompo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/75C12P7/58C12R1/125
CPCC12N9/0006C12N9/0065C12N15/75C12P7/58C12Y101/03004C12Y111/01006
Inventor 王腾飞林平刘洪玲
Owner QILU UNIV OF TECH
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