Boron-resistant lysine bacillus zjb-17007 and its application

Abstract

The present invention relates to a strain of boron-resistant lysine bacillus (Lysinibacillus boronitolerans) ZJB-17007 and its application. The enantiomeric selectivity value for catalyzing N-phenylacetyl-DL-amino acid to produce L-amino acid reaches 99%, and it is effective for catalyzing 2 Enantioselectivity values ​​for the preparation of 2-amino-4-[hydroxy(methyl)phosphoryl]-L-butyric acid from ‑N‑phenylacetyl‑4‑[hydroxy(methyl)phosphoryl]‑DL‑butyric acid up to 99%.

Description

[0001] (1) Technical field

[0002] The present invention relates to a bacterial strain producing amidohydrolase——Lysinibacillus boronitolerans ZJB-17007, and its N-benzene catalyzed preparation of chiral amino acid and catalyzed amino acid derivatives from N-phenylacetyl amino acid. Application of acetyl substituents in the preparation of chiral amino acid derivatives.

[0003] (2) Background technology

[0004] The chemical name of L-glufosinate-ammonium is: 4-[hydroxyl (methyl)phosphono]-L-homoalanine, which is a structural analog of L-glutamic acid and can inhibit glutamine synthetase (GS ) activity, the accumulation of ammonia in plants, the accumulation of high-concentration ammonia gas blocks the photorespiration of plants, the destruction of chloroplast structure and the vesicleization of matrix, and the synthesis of amino acids is blocked, resulting in cell membrane damage and cell death, thus killing weeds . It has a broad spectrum and is the second most herbicide-t...

Examples

Embodiment 1

[0033] Example 1: Screening of Boron-resistant Lysine Bacillus (Lysinibacillus boronitolerans) ZJB-17007

[0034] 1. Primary screening

[0035] The present invention takes soil samples from all over the country, and takes 80 parts of soil samples altogether. The specific method of screening: Weigh 1g of soil sample and place it in 10mL of 0.85% physiological saline, shake it and let it stand still, take the supernatant into the enrichment medium, and cultivate it at 30°C and 150r / min for 2-3 days with shaking . Take 1mL of the enrichment solution and add it to 50mL of fresh enrichment medium, and repeat this process 3 times before separation and purification.

[0036] Bromothymol blue filter paper was selected as the indicator filter paper to detect the colony capable of degrading the substrate. The principle is: after the colony containing the target enzyme degrades the substrate, acidic by-products (phenylacetic acid, acetic acid, formic acid, benzoic acid) will be produc...

Embodiment 2

[0052] Embodiment 2: identification of bacterial strain ZJB-17007

[0053] 1. Morphological identification:

[0054] The bacterial strain ZJB-17007 screened in Example 1 of the present invention forms a round or nearly round, soft texture, smooth surface, flat, neat edges, and shiny light yellow colony after being cultivated on a solid medium for 24 hours at 37°C , 2-4mm in diameter. Observation by Gram staining: purple long rod with spores. Composition of solid medium: sodium chloride 10g / L, peptone 10g / L, yeast powder 5g / L, agar 20g / L, solvent is deionized water.

[0055] 2. Physiological and biochemical identification:

[0056] Using the Biolog (GENⅢ) automatic microbial identification system, 94 phenotypic tests were performed on strain ZJB-17007, including 71 carbon source utilization tests and 23 chemical sensitivity tests: the strains were inoculated on a specific plate medium at 33 °C Cultivate at constant temperature for 2 days, wash the bacteria on the plate with...

Embodiment 3

[0066] Embodiment 3: the preparation of wet thalline

[0067] (1) Incline cultivation:

[0068] Inoculate boron-resistant Bacillus lysine ZJB-17007 into the slant medium and culture at 30°C for 48 hours to obtain slant bacteria;

[0069] The final concentration of the slant culture medium is: casein peptone 17g / L, Na 2 HPO 4 3.0g / L, K 2 HPO 4 1.5g / L, soybean peptone 3g / L, glucose 2.5g / L, NaCl 5g / L, agar 20g / L, solvent is deionized water, pH value is 7.0.

[0070] (2) Seed cultivation:

[0071] Pick an inoculation loop of bacteria from the slant and inoculate it into the seed medium, and cultivate it at 30°C for 24 hours to obtain the seed liquid;

[0072] The final concentration of the seed medium consists of: casein peptone 17g / L, Na 2 HPO 4 3.0g / L, K 2 HPO 4 1.5g / L, soybean peptone 3g / L, glucose 2.5g / L, NaCl 5g / L, solvent is deionized water, pH value is 7.0.

[0073] (3) Fermentation culture:

[0074] Inoculate the seed liquid into the fermentation medium wit...

[0001] (1) Technical field

[0002] The present invention relates to a bacterial strain producing amidohydrolase——Lysinibacillus boronitolerans ZJB-17007, and its N-benzene catalyzed preparation of chiral amino acid and catalyzed amino acid derivatives from N-phenylacetyl amino acid. Application of acetyl substituents in the preparation of chiral amino acid derivatives.

[0003] (2) Background technology

[0004] The chemical name of L-glufosinate-ammonium is: 4-[hydroxyl (methyl)phosphono]-L-homoalanine, which is a structural analog of L-glutamic acid and can inhibit glutamine synthetase (GS ) activity, the accumulation of ammonia in plants, the accumulation of high-concentration ammonia gas blocks the photorespiration of plants, the destruction of chloroplast structure and the vesicleization of matrix, and the synthesis of amino acids is blocked, resulting in cell membrane damage and cell death, thus killing weeds . It has a broad spectrum and is the second most herbicide-t...

Examples

Embodiment 1

[0033] Example 1: Screening of Boron-resistant Lysine Bacillus (Lysinibacillus boronitolerans) ZJB-17007

[0034] 1. Primary screening

[0035] The present invention takes soil samples from all over the country, and takes 80 parts of soil samples altogether. The specific method of screening: Weigh 1g of soil sample and place it in 10mL of 0.85% physiological saline, shake it and let it stand still, take the supernatant into the enrichment medium, and cultivate it at 30°C and 150r / min for 2-3 days with shaking . Take 1mL of the enrichment solution and add it to 50mL of fresh enrichment medium, and repeat this process 3 times before separation and purification.

[0036] Bromothymol blue filter paper was selected as the indicator filter paper to detect the colony capable of degrading the substrate. The principle is: after the colony containing the target enzyme degrades the substrate, acidic by-products (phenylacetic acid, acetic acid, formic acid, benzoic acid) will be produc...

Embodiment 2

[0052] Embodiment 2: identification of bacterial strain ZJB-17007

[0053] 1. Morphological identification:

[0054] The bacterial strain ZJB-17007 screened in Example 1 of the present invention forms a round or nearly round, soft texture, smooth surface, flat, neat edges, and shiny light yellow colony after being cultivated on a solid medium for 24 hours at 37°C , 2-4mm in diameter. Observation by Gram staining: purple long rod with spores. Composition of solid medium: sodium chloride 10g / L, peptone 10g / L, yeast powder 5g / L, agar 20g / L, solvent is deionized water.

[0055] 2. Physiological and biochemical identification:

[0056] Using the Biolog (GENⅢ) automatic microbial identification system, 94 phenotypic tests were performed on strain ZJB-17007, including 71 carbon source utilization tests and 23 chemical sensitivity tests: the strains were inoculated on a specific plate medium at 33 °C Cultivate at constant temperature for 2 days, wash the bacteria on the plate with...

Embodiment 3

[0066] Embodiment 3: the preparation of wet thalline

[0067] (1) Incline cultivation:

[0068] Inoculate boron-resistant Bacillus lysine ZJB-17007 into the slant medium and culture at 30°C for 48 hours to obtain slant bacteria;

[0069] The final concentration of the slant culture medium is: casein peptone 17g / L, Na 2 HPO 4 3.0g / L, K 2 HPO 4 1.5g / L, soybean peptone 3g / L, glucose 2.5g / L, NaCl 5g / L, agar 20g / L, solvent is deionized water, pH value is 7.0.

[0070] (2) Seed cultivation:

[0071] Pick an inoculation loop of bacteria from the slant and inoculate it into the seed medium, and cultivate it at 30°C for 24 hours to obtain the seed liquid;

[0072] The final concentration of the seed medium consists of: casein peptone 17g / L, Na 2 HPO 4 3.0g / L, K 2 HPO 4 1.5g / L, soybean peptone 3g / L, glucose 2.5g / L, NaCl 5g / L, solvent is deionized water, pH value is 7.0.

[0073] (3) Fermentation culture:

[0074] Inoculate the seed liquid into the fermentation medium wit...

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