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A strain of Paenibacillus polymyxa swgc4112 and its cultivation method and application

The technology of a polymyx spore-like and culturing method is applied in the field of functional bacteria development, and can solve the problems of not being able to meet the requirements of industrial production, being difficult to purify, and being immature.

Active Publication Date: 2022-04-22
BIOLOGY INST OF HEBEI ACAD OF SCI
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  • Description
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  • Application Information

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Problems solved by technology

In 1955, Courtois, J.E. reported that 2,4-O-benzene-D-sorbitol was used as a raw material to synthesize R-3-aminobutyric acid by a two-step method of first oxidation and then hydrolysis. The shortcoming of this method is that the catalyst used Oxidants are extremely unstable, especially easy to decompose, and the reaction process is difficult to control
In 2003, it was reported in U.S. Patent 20030097029 that R-3-aminobutyric acid was hydrogenated and reduced under the action of a nail catalyst to generate R-3-aminobutyric acid. The reaction pressure was 5 MPa and the reaction time was 18 hours. The reaction pressure was high and the reaction time was long. And under this condition, R-3-aminobutyric acid will continue to be excessively reduced to generate xylitol, and the resulting monoxylose is not high in purity.
In 2017, Chinese patent 108276455A reported the use of 2,4-benzene-R-3-aminobutyric acid as a substrate to synthesize R-3-aminobutyric acid under the action of an acid catalyst, and the product yield was about 80%. , the product purity is 98%, there are many by-products, and the purification is difficult. At this stage, it is still limited to laboratory research and cannot meet the requirements of industrial production.
In 2017, Chinese patent 108165591A reported the research on the catalytic synthesis of R-3-aminobutyric acid by engineering bacteria using crotonic acid as a substrate, and the highest conversion rate was about 94%. It is still limited to laboratory research. If it is to be applied on a large scale in industry, it still needs the further development of genetic engineering to solve the problems of stability and reaction speed of biocatalysts.
[0005] Therefore provide a kind of synthetic method of R-3-aminobutyric acid, solve the synthesis technique pressure of R-3-aminobutyric acid in the prior art too high, the reaction time is long, the reaction process control is difficult, post-processing is loaded down with trivial details, not easy to industrialization Problem has become a problem to be solved urgently by those skilled in the art

Method used

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  • A strain of Paenibacillus polymyxa swgc4112 and its cultivation method and application
  • A strain of Paenibacillus polymyxa swgc4112 and its cultivation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Fermentation of Paenibacillus polymyxa SWGC4112

[0040] Seed medium: LB medium; fermentation medium: glucose 50g / L, yeast extract 50 g / L, MgSO 4 1.5g / L, (NH 4 ) 2 HPO 4 3g / L, KH 2 PO 4 3g / L, the solvent is water, and the pH value is adjusted to 7.0. The seed medium was sterilized at 121°C for 20 minutes, inoculated after cooling, and cultured in shake flasks with a liquid volume of 30%.

[0041] Specific cultivation steps: take the strains stored at -70°C and streak on the LB solid plate, pick a single colony and inoculate it in the LB seed liquid medium, cultivate it at 30°C and 250rpm for 12 hours, and inoculate the seeds according to 5% Inoculate a large amount in the fermentation medium, shake and culture at 30°C and 250rpm for 36 hours. After the cultivation, the fermentation broth is centrifuged and washed twice with saline, and the wet bacterial cells are collected. The wet weight of the bacterial cells reaches 20g / L.

[0042] (1) According to the resu...

Embodiment 2

[0049] Production of R-3-aminobutyric acid

[0050] In a 10L catalytic system, add 3kg crotonic acid, 15g magnesium sulfate, 600g ammonium sulfate, adjust the pH to 9.0 with ammonia water, stir, add 100g of Paenibacillus polymyxa SWGC4112 strain, and start the reaction. After 12 hours of reaction, the concentration of R-3-aminobutyric acid in the solution can reach 305.80 g / L, and the conversion rate can reach more than 85.1%. After the reaction, the reaction liquid was treated by membrane filtration, separated and purified, concentrated and crystallized, centrifuged and dried to obtain 2.95 kg of finished product. After testing, the chiral purity of the finished product R-3-aminobutyric acid is 100%, the product purity is 99.92%, and the product yield is 96.4%.

Embodiment 3

[0052] Production of R-3-aminobutyric acid

[0053] In a 10L catalytic system, add 3 kg of crotonic acid, 15 g of magnesium sulfate, and 300 g of ammonium chloride, then adjust the pH to 9.0 with ammonia water, stir, add 100 g of Paenibacillus polymyxa SWGC4112 strain, and start the reaction. After 10 hours of reaction, the concentration of R-3-aminobutyric acid in the solution can reach 35.90 g / L, and the conversion rate can reach 100%. After the reaction, the reaction solution was treated by membrane filtration, separated and purified, concentrated and crystallized, centrifuged and dried to obtain 3.46 kg of finished product. After testing, the chiral purity of the finished product R-3-aminobutyric acid is 100%, the product purity is 99.94%, and the product yield is 96.5%.

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Abstract

The invention provides a strain of Paenibacillus polymyxa SWGC4112 and its cultivation method and application, and relates to the technical field of functional bacteria development. The preservation number of Paenibacillus polymyxa (Paenibacillus polymyxa) SWGC4112 of the present invention is CGMCC No.13356, and the bacterial strain described in the present invention is a new one that can catalyze the synthesis of R-3-aminobutyric acid with crotonic acid as a substrate. The bacterial strain is capable of expressing and producing aspartase, and the high-purity preparation of R-3-aminobutyric acid can be realized through the bacterial strain, the conversion rate is 100%, and the optical purity reaches 100%. Using the bacterial strain of the present invention to carry out the asymmetric reduction process has mild reaction conditions, energy saving, environmental protection, and more importantly, economic benefit is significantly improved, and has good industrial application prospects.

Description

technical field [0001] The invention belongs to the technical field of functional bacteria development, and in particular relates to a Paenibacillus polymyxa SWGC4112 and its cultivation method and application. Background technique [0002] Xylose is a five-carbon sugar with two configurations, D- and L-. D-xylose mainly exists in plants and animals, R-3-aminobutyric acid does not exist in nature, and R-3-aminobutyric acid is widely used in medicine, food, chemical industry and other fields. As a pharmaceutical intermediate, R-3-aminobutyric acid plays an important role in anti-cancer, anti-virus, anti-inflammation, anti-diabetes, etc. In terms of food, R-3-aminobutyric acid can improve the microbial environment of the human body, improve It is an ideal sweetener for diabetics, and it can also be used as a raw material to synthesize xylitol, a healthy sweetener. [0003] Xylose is not a naturally occurring compound, only synthesized by chemical synthesis and biotransformat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P13/04C12R1/01
CPCC12N1/20C12P13/04
Inventor 贾振华张翔李冉宋聪宋水山孙劲冲
Owner BIOLOGY INST OF HEBEI ACAD OF SCI
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