Recombinant bacillus subtilis, construction method and applications thereof

A Bacillus subtilis, construction method technology, applied in the direction of recombinant DNA technology, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of Bacillus dissolved oxygen, etc., to achieve increased biomass, low production cost, and reduced production cost effect

Inactive Publication Date: 2013-12-04
EAST CHINA NORMAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problem of dissolved oxygen of Bacillus in the process of fermenting γ-polyglutamic acid, the present invention provides a recombinant Bacillus subtilis, which is a γ-polyglutamic acid producing bacterium capable of expressing Vitiligo hyaline hemoglobin

Method used

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  • Recombinant bacillus subtilis, construction method and applications thereof
  • Recombinant bacillus subtilis, construction method and applications thereof
  • Recombinant bacillus subtilis, construction method and applications thereof

Examples

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Effect test

Embodiment 1

[0024] Example 1 Construction of a recombinant Bacillus subtilis expressing Vitreum hemoglobin: a genetically engineered bacterium containing Vitreum hemoglobin.

[0025] The first step base sequence synthesis

[0026] The Vitiligo hyaline hemoglobin gene is transformed according to the preferred codons of Bacillus subtilis gene translation, and the transformation method is as follows: From the NCBI database, the vgb sequence of the Vitiligo hyaline hemoglobin gene (as shown in SEQ ID: 2), according to the Bacillus subtilis Favored codon tables (eg figure 1 Shown) Optimizing and transforming the hemoglobin gene vgb of Vitiligo hyaline, among which 78 bases are changed, the change rate is 17.69%, and the G+C content of vgb gene is reduced from 45.3% to 31.1%, thus improving the hemoglobin gene of Vitria hyaline Expression efficiency of vgb in Bacillus subtilis.

[0027] In the above method, a professional biotechnology company is entrusted to chemically synthesize the optimi...

Embodiment 2

[0044] Example 2 Production of γ-polyglutamic acid by recombinant Bacillus subtilis expressing Vitella hyaline hemoglobin

[0045] The first step is to ferment γ-polyglutamic acid

[0046] The successfully constructed Bacillus subtilis expressing Vitella hyaline hemoglobin was added to the fermentation medium (maltose 50g / L, yeast extract 10g / L, sodium glutamate 30g / L, sodium chloride 10g / L, potassium dihydrogen phosphate 5g / L, magnesium sulfate 0.5g / L), 37°C, 210r / min, cultured for 48 hours, to produce and accumulate γ-polyglutamic acid.

[0047] The second step is to collect γ-polyglutamic acid

[0048] Use hydrochloric acid to adjust the fermentation culture solution to make the pH value 3.0, centrifuge the fermentation culture solution at 16,000r / min for 20 min, remove the bacteria in the fermentation solution, and obtain the supernatant, adjust the pH value of the supernatant to 7.0 , slowly add ethanol three times its volume into the solution, and use a glass rod to s...

Embodiment 3

[0052] Example 3 Preparation of small molecular weight γ-polyglutamic acid

[0053] Use the γ-polyglutamic acid prepared above as the parent material, weigh its powder, prepare a 1% concentration of γ-polyglutamic acid solution with distilled water, adjust its pH to 4.0, and place it at a constant temperature of 80°C In the water bath, degrade for 0.5h, 1.0h, 1.5h, 2.0h, 3.0h, 5.0h respectively, then cool it in an ice bath, adjust its pH to 7.0, and then identify it by horizontal 0.8% agarose gel electrophoresis The molecular weight of the degradation product, the experimental results are as follows figure 2 shown. figure 2 Among them, 1-3 means: 14.5 kD, 20.5 kD, 64.0 kD γ-polyglutamic acid standard; 4 means: macromolecular γ-polyglutamic acid control; 5-10 means: macromolecular γ-polyglutamic acid The samples were degraded by acid for 0.5 h, 1.0 h, 1.5 h, 2.0 h, 3.0 h, and 5.0 h, respectively.

[0054] The results showed that by adjusting the different degradation condi...

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Abstract

The invention relates to a recombinant bacillus subtilis, a construction method and applications thereof. The invention discloses a bacillus subtilis containing vitreoscilla hemoglobin gene vgb, and the bacillus subtilis can generate gamma-polyglutamic acid. The invention also discloses a construction method that recombinant plasmid containing the vitreoscilla hemoglobin gene vgb sections is transferred into the bacillus subtilis to obtain the recombinant bacillus subtilis, which contains the vitreoscilla hemoglobin gene vgb and is capable of expressing gamma-polyglutamic acid. The recombinant bacillus subtilis can achieve the goal of high yield production of gamma-polyglutamic acid under the conditions of high viscosity culture medium and low dissolved oxygen, solves the problem of requirement on high dissolved oxygen in mass production of gamma-polyglutamic acid, and thus the production cost is effectively reduced.

Description

technical field [0001] The invention relates to a recombinant bacillus subtilis, its construction method and the application of using the bacterium to produce gamma-polyglutamic acid, which belongs to the technical field of bioengineering. Background technique [0002] γ-polyglutamic acid is an anionic polyamino acid produced by microbial fermentation in nature. It is a polymer formed by D-type and L-type glutamic acid through α-amino and γ-carboxyl in the form of amide bonds. γ-polyglutamic acid and its derivatives are environmentally friendly biopolymers with good water solubility, can be completely biodegraded, and are non-toxic to humans and the environment. Therefore, they are used in agriculture, food, medicine, environmental protection, fiber light Chemical industry and other fields have broad application prospects. [0003] At present, the biosynthesis of γ-polyglutamic acid is mainly carried out by Bacillus licheniformis ( Bacillus licheniformis ) and Bacillus sub...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/75C12P13/02C12R1/125
Inventor 肖磊黄静李娟周杰汪正华朱蓓霖赵芸
Owner EAST CHINA NORMAL UNIV
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