Kluyver intermedia zjb-17004 and its application

A ZJB-17004, wet cell technology, applied in bacteria, microorganisms, microorganisms, etc., can solve the problems of unconverted D-glufosinate, high price of trimethylsilicon, low product yield and e.e. value, etc.

Active Publication Date: 2020-06-23
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

3) Asymmetric synthesis method, asymmetric catalytic hydrogenation - large amount of catalyst and expensive trimethylsilyl cyanide; asymmetric Strecker reaction - use of highly toxic cyanide; asymmetric Michael addition - large amount of catalyst, And the product yield and e.e. value are low
4) The racemate resolution method, the highest yield of this method is 86%, the highest e.e. value is 99%, but after the resolution, D-glufosinate-ammonium is not converted, wasting raw materials

Method used

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  • Kluyver intermedia zjb-17004 and its application
  • Kluyver intermedia zjb-17004 and its application
  • Kluyver intermedia zjb-17004 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Screening of Kluyvera intermedia ZJB-17004

[0035] 1. Primary screening

[0036] The invention takes soil samples from all parts of the country, a total of 80 soil samples are taken. The specific method of screening: weigh 1g of soil sample and place it in 10mL 0.85% normal saline, shake it and let it stand, take the supernatant into the enriched medium, culture at 30℃, 150r / min for 2-3 days . Take 1 mL of the enrichment solution and add it to 50 mL of fresh enrichment medium, repeat the procedure three times, and then perform separation and purification.

[0037] Bromothymol blue filter paper was selected as the indicator filter paper to detect the colonies that can degrade the substrate. The principle is: after the colony containing the target enzyme degrades the substrate, it will produce acidic by-products (phenylacetic acid, acetic acid, formic acid, benzoic acid), thereby changing the local pH of the indicator filter paper. The indicator bromothymol blue ...

Embodiment 2

[0052] Growth medium composition: sodium chloride 10g / L, peptone 10g / L, yeast powder 5g / L, and the solvent is deionized water. The composition of the slant medium, seed medium, and fermentation medium is the same as in Example 3. Example 2: Identification of strain ZJB-17004

[0053] 1. Morphological identification:

[0054] The strain ZJB-17004 screened in Example 1 of the present invention was cultured on a solid medium at 37°C for 24 hours to form a round or nearly round, soft texture, smooth surface, flat, neat edges, shiny and opaque milky white colonies , 2-4mm in diameter. Gram staining observation: pink short rod shape, no spores. Solid medium composition: sodium chloride 10g / L, peptone 10g / L, yeast powder 5g / L, agar 20g / L, and the solvent is deionized water.

[0055] 2. Physiological and biochemical identification:

[0056] Using the Biolog (GENⅢ) automatic microbial identification system, 94 phenotypic tests were conducted on the strain ZJB-17004, including 71 carbon sou...

Embodiment 3

[0066] Example 3: Preparation of wet bacteria

[0067] (1) Inclined surface cultivation:

[0068] Inoculate the intermediate Kluyver bacterium ZJB-17004 into the slant medium, and culture at 30°C for 48 hours to obtain the slant bacteria;

[0069] The final concentration of the slant medium is: lactose 8g / L, peptone 6g / L, Na 2 HPO 4 3.2g / L, K 2 HPO 4 1.5g / L, Na 2 EDTA0.06g / L, MnSO 4 0.002g / L, MgSO 4 0.001g / L, ZnSO 4 0.002g / L, FeSO 4 0.01g / L, 20g / L agar, deionized water as solvent, and pH 6.8.

[0070] (2) Seed cultivation

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

[0072] The final concentration of the seed culture medium is composed of: lactose 8g / L, peptone 6g / L, Na 2 HPO 4 3.2g / L, K 2 HPO 4 1.5g / L, Na 2 EDTA 0.06g / L, MnSO 4 0.002g / L, MgSO 4 0.001g / L, ZnSO 4 0.002g / L, FeSO 4 0.01g / L, the solvent is deionized water, and the pH is 6.8.

[0073] (3) Fermentation culture

[0074...

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Abstract

The present invention relates to a Kluyvera intermedia ZJB-17004 and its application, its enantiomeric selectivity value for catalyzing N-phenylacetyl-DL-amino acid to produce L-amino acid reaches 98%, and its enantiomeric selectivity 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 strain producing amide hydrolase-Kluyvera intermedia ZJB-17004, and N-phenylacetyl catalyzing the preparation of chiral amino acids and catalyzing amino acid derivatives from N-phenylacetyl amino acids The application of base substituents in the preparation of chiral amino acid derivatives. [0003] (2) Background technology [0004] The chemical name of L-glufosinate-ammonium is: 4-[hydroxy(methyl)phosphono]-L-homoalanine, which is a structural analogue of L-glutamic acid and can inhibit glutamine synthetase (GS ) Activity, the accumulation of ammonia in plants, the accumulation of high concentrations of ammonia gas blocks the photorespiration of plants, the destruction of the chloroplast structure and the vesicleization of the matrix, while the amino acid synthesis is blocked, resulting in cell membrane damage and cell death, thereby killing weeds . It has a broad spectrum and is the world's second largest gen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P13/04C12P41/00C12P13/06C12P13/08C12P13/12C12P13/14C12P13/20C12P13/22C12R1/01
CPCC12P13/04C12P13/06C12P13/08C12P13/12C12P13/14C12P13/20C12P13/222C12P13/225C12P41/001C12N1/205C12R2001/01
Inventor 柳志强郑裕国康雪梅张晓健金利群
Owner ZHEJIANG UNIV OF TECH
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