Enterobacter aerogenes zjb-17003 and its application

Abstract

The present invention relates to a strain of Enterobacter aerogenesa (Enterobacter aerogenesa) ZJB-17003 and its application, which catalyzes N-phenylacetyl-DL-amino acid to prepare L-amino acid with an enantiomer selectivity value of 99%, and catalyzes 2-N-amino acid The enantioselectivity value of 2-amino-4-[hydroxy (methyl) phosphoryl] butyric acid from phenylacetyl-4-[hydroxyl (methyl) phosphoryl] butanoic acid reaches 99%.

Description

[0001] (1) Technical field

[0002] The present invention relates to a bacterial strain producing amidohydrolase—Enterobacteraerogenesa (Enterobacteraerogenesa) ZJB-17003, and its use in catalyzing N-phenylacetyl amino acid to prepare chiral amino acid and catalyzing N-phenylacetyl-DL-amino acid derivatives Application of N-phenylacetyl-L-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-tolerant ...

Examples

Embodiment 1

[0034] Example 1: Screening of Enterobacter aerogenesa ZJB-17003

[0035] 1. Primary screening

[0036] 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.

[0037] 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 produced, thereby changing the local pH value ...

Embodiment 2

[0053] Embodiment 2: identification of bacterial strain ZJB-17003

[0054] 1. Morphological identification:

[0055] The bacterial strain ZJB-17003 screened in Example 1 of the present invention was inoculated on a solid medium and cultured at 37°C for 24 hours to form round or nearly round, soft texture, smooth surface, flat, neat edges, and shiny milky white colonies , 2-4mm in diameter. Observation by Gram staining: short pink rods without spores. Composition of solid medium: sodium chloride 10g / L, peptone 10g / L, yeast powder 5g / L, agar 20g / L, solvent is deionized water.

[0056] 2. Physiological and biochemical identification:

[0057] Using the Biolog (GENⅢ) automatic microbial identification system, 94 kinds of phenotypic tests were carried out on the strain ZJB-17003, including 71 kinds of carbon source utilization detection and 23 kinds of chemical sensitivity detection: the strain ZJB-17003 was inoculated on the BUG plate medium ( BIOLOG UNIVERSAL GROWTH AGAR), cu...

Embodiment 3

[0067] Embodiment 3: the preparation of wet thalline

[0068] (1) Incline cultivation:

[0069] Enterobacter aerogenes (Enterobacter aerogenesa) ZJB-17003 was inoculated into the slant culture medium and cultured at 30°C for 48 hours to obtain slant cells;

[0070] The final concentration of the slant culture medium is: mannitol 10g / L, sodium glutamate 7g / L, yeast extract 3g / L, K 2 HPO 4 0.75g / L, KH 2 PO 4 0.75g / L, MgSO 4 0.5g / L, caprolactam 1g / L, agar 20.0g / L, solvent is deionized water, pH 7.0~7.5.

[0071] (2) Seed cultivation

[0072] 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;

[0073] The final concentration of the seed medium consists of: mannitol 10g / L, sodium glutamate 7g / L, yeast extract 3g / L, K 2 HPO 4 0.75g / L, KH 2 PO 4 0.75g / L, MgSO 4 0.5g / L, caprolactam 1g / L, solvent is deionized water, pH 7.0~7.5.

[0074] (3) Fermentation culture

[...

[0001] (1) Technical field

[0002] The present invention relates to a bacterial strain producing amidohydrolase—Enterobacteraerogenesa (Enterobacteraerogenesa) ZJB-17003, and its use in catalyzing N-phenylacetyl amino acid to prepare chiral amino acid and catalyzing N-phenylacetyl-DL-amino acid derivatives Application of N-phenylacetyl-L-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-tolerant ...

Examples

Embodiment 1

[0034] Example 1: Screening of Enterobacter aerogenesa ZJB-17003

[0035] 1. Primary screening

[0036] 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.

[0037] 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 produced, thereby changing the local pH value ...

Embodiment 2

[0053] Embodiment 2: identification of bacterial strain ZJB-17003

[0054] 1. Morphological identification:

[0055] The bacterial strain ZJB-17003 screened in Example 1 of the present invention was inoculated on a solid medium and cultured at 37°C for 24 hours to form round or nearly round, soft texture, smooth surface, flat, neat edges, and shiny milky white colonies , 2-4mm in diameter. Observation by Gram staining: short pink rods without spores. Composition of solid medium: sodium chloride 10g / L, peptone 10g / L, yeast powder 5g / L, agar 20g / L, solvent is deionized water.

[0056] 2. Physiological and biochemical identification:

[0057] Using the Biolog (GENⅢ) automatic microbial identification system, 94 kinds of phenotypic tests were carried out on the strain ZJB-17003, including 71 kinds of carbon source utilization detection and 23 kinds of chemical sensitivity detection: the strain ZJB-17003 was inoculated on the BUG plate medium ( BIOLOG UNIVERSAL GROWTH AGAR), cu...

Embodiment 3

[0067] Embodiment 3: the preparation of wet thalline

[0068] (1) Incline cultivation:

[0069] Enterobacter aerogenes (Enterobacter aerogenesa) ZJB-17003 was inoculated into the slant culture medium and cultured at 30°C for 48 hours to obtain slant cells;

[0070] The final concentration of the slant culture medium is: mannitol 10g / L, sodium glutamate 7g / L, yeast extract 3g / L, K 2 HPO 4 0.75g / L, KH 2 PO 4 0.75g / L, MgSO 4 0.5g / L, caprolactam 1g / L, agar 20.0g / L, solvent is deionized water, pH 7.0~7.5.

[0071] (2) Seed cultivation

[0072] 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;

[0073] The final concentration of the seed medium consists of: mannitol 10g / L, sodium glutamate 7g / L, yeast extract 3g / L, K 2 HPO 4 0.75g / L, KH 2 PO 4 0.75g / L, MgSO 4 0.5g / L, caprolactam 1g / L, solvent is deionized water, pH 7.0~7.5.

[0074] (3) Fermentation culture

[...

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