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High-yield strain for producing L-alanine by fermentation and preparation method thereof

A technology of high-yield strains and alanine, applied in the field of microorganisms, can solve the problems of stable genetic engineering strains and preservation, difficulty in grasping the dose of radiation absorbed by microorganisms, and poor ultraviolet effect, etc., to achieve low prices and reduce production costs , the effect of stable properties

Inactive Publication Date: 2014-02-26
淮北新旗氨基酸有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The enzymatic conversion process is characterized by high enzyme activity, low equipment investment, and simple extraction process, but its main raw material L-aspartic acid is expensive, resulting in high production costs
Pascal Hols et al reported the use of genetic engineering to transform lactic acid bacteria to ferment and produce L-alanine with glucose, sucrose or lactose as raw materials (US 6 627 420 B1), but the process of constructing strains by genetic engineering is complicated and requires high technology , and the obtained genetically engineered strains are difficult to stably inherit and preserve
Gao Li et al. reported the use of ultraviolet and laser combined mutagenesis to screen high-yield strains of L-alanine (Journal of Anhui Institute of Engineering Science and Technology, 2008, 23 (1): 19-24), but due to the ultraviolet light on various The mutagenic effect of microorganisms is different, and it is difficult to grasp the dose of radiation absorbed by microorganisms, and photorepair is prone to occur after mutation, so the effect of ultraviolet mutagenesis is not good, the positive mutation rate is low, and the mortality rate is high

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Mutagenesis of Lactobacillus delbrueckii subsp. bulgaricus mutants with L-alanine.

[0023] 1. Medium components:

[0024] 1.1 The composition of the slant medium: peptone 1%, yeast extract 0.5%, sodium acetate 0.5%, Tween 80 0.1%, magnesium sulfate heptahydrate 0.02%, calcium carbonate 2%, beef extract 1%, glucose 1%, lemon Acid diamine 0.2%, dipotassium hydrogen phosphate 0.2%, manganese sulfate heptahydrate 0.005%, agar 2%, sodium hydroxide to adjust the pH to 7.0, 121 ℃ steam sterilization for 20min.

[0025] 1.2 Composition of fermentation medium: glucose 6%, potassium dihydrogen phosphate 0.2%, dipotassium hydrogen phosphate 1.2%, anhydrous magnesium sulfate 0.02%, ammonia water to adjust pH to 7.0, steam sterilization at 115°C for 20 minutes.

[0026] During the preparation of the fermentation medium, no alanine-containing organic matter was incorporated, in order to avoid interference with the next step of screening L-alanine-producing bacteria.

[0027] 2. O...

Embodiment 2

[0035] Mutagenesis of an L-alanine-producing Lactobacillus delbrueckii subsp. bulgaricus mutant.

[0036] 1, medium component: the composition of slant medium and fermentation medium is the same as embodiment 1.

[0037] 2. Operation steps: according to the method in Example 1, the starting bacterial strain was made into a bacterial film, and after microscopic inspection without cell overlap, ion implantation was performed under vacuum conditions. The implanted ions were N ions, the ion energy was 20keV, and the implantation dose was 6.0×10 15 ions / cm 2 , the injection time is 5s, and the interval time is 30s.

[0038] The bacterial film after the ion implantation treatment was eluted with 1 mL of sterile saline, and then 0.1 mL of the eluate was applied to the slant medium, and placed in a 37 ° C incubator for 72 h.

[0039] The grown single colonies were numbered, and each single colony was picked into the corresponding numbered fermentation medium with an inoculation loo...

Embodiment 3

[0043] Mutagenesis of an L-alanine-producing Lactobacillus delbrueckii subsp. bulgaricus mutant.

[0044] 1, medium component: the composition of slant medium and fermentation medium is the same as embodiment 1.

[0045] 2. Operation steps: according to the method of Example 1, the starting bacterial strain was made into a bacterial film, and after microscopic inspection without cell overlap, ion implantation treatment was performed under vacuum conditions. The implanted ions were N ions, the ion energy was 20keV, and the implantation dose was 5.0×10 16 ions / cm 2 , the injection time is 5s, and the interval time is 40s.

[0046] The bacterial pellicle after ion implantation treatment was eluted with 0.5 mL of sterile saline, and then 0.1 mL of the eluate was spread on the slant medium, and placed in a 37 ° C incubator for 96 h.

[0047] The grown single colonies were numbered, and each single colony was picked into the corresponding numbered fermentation medium with an inoc...

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Abstract

The invention relates to a high-yield strain Lds.0108 for producing L-alanine by fermentation. The high-yield strain is deposited in the China Center for Type Culture Collection with an accession number of CCTCC M2013361. The strain is a mutant strain by using Lactobacillus delbrueckii subsp. Bulgaricus (GIMI.155) as an initial strain, mutagenizing cells by utilization of a low energy ion implantation method, and screening by a reversible inhibition assay method. The growth temperature range of the strain is 35-40 DEG C. The optimum growth temperature is 37 DEG C and the optimum pH value is 7.0. A slope culture medium used is an MRS culture medium. Glucose and other carbohydrates are used as substrates for the strain. The L-alanine is produced by fermentation under anaerobic conditions. Impurities are removed by ultrafiltration membrane filtration and nanofiltration membrane filtration in a purifying process, thus guaranteeing the product quality. The strain and the preparation method are suitable for large-scale industrial production.

Description

technical field [0001] The invention relates to a high-yield bacterial strain for fermenting and producing L-alanine and a preparation method thereof, belonging to the technical field of microorganisms. Background technique [0002] L-alanine is one of the amino acids that make up protein. Although it is a non-essential amino acid for the human body, it is the amino acid with the highest content in human blood. It has a wide range of uses in the fields of medicine, polymer materials, and food. In the field of medicine: as a pharmaceutical intermediate, L-alanine is the main raw material for the production of vitamin B6 and L-aminopropanol; it is closely related to sugar metabolism and is the main amino donor in the transamination reaction, which has important physiological functions. Function, often added to the infusion in clinical practice; In addition, L-alanine is also a good diuretic. In the field of polymer materials: In recent years, it has been found that adding L-a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N13/00C12N15/01C12P13/06C12R1/225
Inventor 黄建坡苗位云孟凡会刘焕书朱传厚韩秀丽郭志远徐龙
Owner 淮北新旗氨基酸有限公司
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