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A high-yielding aspartate decarboxylase strain and its method for producing alanine

A technology of aspartic acid and decarboxylase, applied in the biological field, can solve problems such as low enzyme activity, and achieve the effects of high enzyme activity, simple medium composition and good genetic stability

Active Publication Date: 2018-06-19
山东中酶生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above reports are limited by the low enzyme activity, and there is still a long way to go before industrial application

Method used

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  • A high-yielding aspartate decarboxylase strain and its method for producing alanine
  • A high-yielding aspartate decarboxylase strain and its method for producing alanine
  • A high-yielding aspartate decarboxylase strain and its method for producing alanine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Classification of Bacillus tequila strains

[0019] 1) Characteristics of strains

[0020] On the preservation medium, the colonies are milky white, nearly round, with dry surface, opaque, wrinkled, dull, with irregular lobed edges, easy to pick; through microscope observation, the bacteria are rod-shaped, single and not in chains, Gram-positive, after 48 hours of culture, it is observed that the spores are attached to the side of the bacterium or exist freely, without swelling, oval or columnar.

[0021] 2) Strain identification

[0022] The length of the 16S rDNA gene sequence obtained by DNA extraction, PCR amplification and sequencing is 1459bp, and the BLAST comparison on GenBank shows that the base similarity between the strain and Bacillus tequilensis (JX412373) reaches 99%. The sequence information is as follows:

[0023]tggctttgcgcttgctatacatgcaagtcgagcggacagatgggagcttgctccctgatgttagcggcggacgggtgagtaacacgtgggtaacctgcctgtaagactgggataactccgggaaaccgg...

Embodiment 2

[0027] 1) Strain activation: pick strains to solid preservation medium: yeast extract powder 5g / L, peptone 10g / L, NaCl 5g / L, agar 20g / L, pH 7.0, streak inoculation in a constant temperature incubator at 30°C 20h;

[0028] 2) Preparation of liquid seeds: pick 1 ring of activated strains and inoculate them in a 500ml Erlenmeyer flask containing 50ml of seed medium. The components of liquid seed medium: yeast extract powder 5g / L, peptone 10g / L, NaCl 5g / L, pH 7.0, sealed with eight layers of gauze, cultured at 30°C, 200r / min shaker for 18h, to obtain liquid seeds;

[0029] 3) liquid fermentation: according to 5% inoculum size, the components are glucose 20g / L, peptone 20g / L, aspartic acid 2g / L, MgSO 4 0.2g / L, KH 2 PO 4 0.5g / L, ammonium sulfate 3g / L fermentation medium was inserted into liquid seeds, the rotation speed was 200-700r / min, the dissolved oxygen was controlled at 20-30%, pH 7.0, temperature 30°C, aerated for 24 hours, and L-days were obtained. Fermentation broth of ...

Embodiment 3

[0034] 1) Strain activation: pick strains to solid preservation medium: yeast extract powder 5g / L, peptone 10g / L, NaCl 5g / L, agar 20g / L, pH 7.0, streak inoculation in a constant temperature incubator at 30°C 20h;

[0035] 2) Preparation of liquid seeds: pick 1 ring of activated strains and inoculate them in a 500ml Erlenmeyer flask containing 50ml of seed medium. The components of liquid seed medium: yeast extract powder 5g / L, peptone 10g / L, NaCl 5g / L, pH7.0. Eight layers of gauze were sealed, and cultured at 30°C and shaken at 200r / min for 18 hours to obtain liquid seeds;

[0036] 3) Liquid fermentation: according to 7% inoculum size, the components are glucose 20g / L, peptone 20g / L, aspartic acid 2g / L, MgSO 4 0.2g / L, KH 2 PO 4 0.5g / L, ammonium sulfate 3g / L fermentation medium was inserted into liquid seeds, the rotation speed was 200-700r / min, dissolved oxygen was controlled at 25-30%, pH 7.0, temperature 30°C, aerated for 28 hours, and L-days were obtained. Fermentation...

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Abstract

The present invention relates to a strain with high production of aspartate decarboxylase and a method for producing alanine, which belongs to the field of biotechnology. The strain is classified and named as Bacillus tequila PanD37, which was published on February 2, 2015. Preserved in China General Microorganism Culture Collection Management Center, preservation number: CGMCC No.10506. The Bacillus tequila producing L-aspartic acid α-decarboxylase provided by the present invention is reported for the first time. The bacterial strain PanD37 has good genetic stability and low cultivation cost, and the produced L-aspartic acid α-decarboxylase enzyme With high activity, the fermentation broth of this strain can be used to directly convert β-alanine. No need to add surfactants, organic solvents and other membrane-permeable agents during the catalytic synthesis, avoiding the introduction of exogenous impurities, which is conducive to extraction and separation operations, and has advantages in industrial applications .

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a bacterial strain producing L-aspartic acid α-decarboxylase, and using the fermentation liquid of L-aspartic acid α-decarboxylase produced by the bacterial strain to catalyze the preparation of β-alanine Methods. Background technique [0002] β-alanine is the only β-type non-protein amino acid that exists in nature. It is used in the synthesis of pharmaceutical products such as carnosine, calcium pantothenate, balsalazide and sodium pamidronate, and has a wide range of uses. At present, β-alanine mainly adopts chemical synthesis method, which has disadvantages such as high cost, unfriendly conditions and production environment. The development of green and safe biological production process will have very obvious economic and social benefits. L-aspartate α-decarboxylase (L-aspartate α-decarboxylase, EC4.1.1.11, PanD) is a key enzyme in the biosynthetic pathway of pantothenic acid, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N9/88C12P13/06C12R1/07
CPCC12N9/88C12P13/06C12Y401/01011C12N1/205C12R2001/07
Inventor 冯志彬陈国忠张娟
Owner 山东中酶生物科技有限公司
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