Kluyvera intermedia ZJB-17004 and application thereof

A ZJB-17004, reaction technology, applied in the direction of bacteria, microorganisms, biochemical equipment and methods, etc., can solve the problems of high price of trimethylsilyl cyanide, waste of raw materials, low product yield and e.e. value

Active Publication Date: 2019-02-12
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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  • Kluyvera intermedia ZJB-17004 and application thereof
  • Kluyvera intermedia ZJB-17004 and application thereof
  • Kluyvera intermedia ZJB-17004 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Screening of Kluyvera intermedia ZJB-17004

[0030] 1. Primary screening

[0031] 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.

[0032] 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 of t...

Embodiment 2

[0047] Growth medium composition: sodium chloride 10g / L, peptone 10g / L, yeast powder 5g / L, solvent is deionized water. Slant culture medium, seed culture medium, fermentation medium composition are the same as embodiment 3. Embodiment 2: identification of bacterial strain ZJB-17004

[0048] 1. Morphological identification:

[0049] The bacterial 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 round or nearly round, soft texture, smooth surface, flat, neat edges, shiny and opaque 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.

[0050] 2. Physiological and biochemical identification:

[0051] Using the Biolog (GENⅢ) automatic microbial identification system, 94 kinds of phenotypic tests were carried out on strain ZJB-17...

Embodiment 3

[0061] Embodiment 3: the preparation of wet thalline

[0062] (1) Incline cultivation:

[0063] Inoculate the slant medium with Kluyverella intermedia ZJB-17004 and culture at 30°C for 48 hours to obtain slant bacteria;

[0064] The final concentration of the slant culture 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, the agar is 20g / L, the solvent is deionized water, and the pH value is 6.8.

[0065] (2) Seed cultivation

[0066] 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;

[0067] The final concentration of the seed medium consists 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 p...

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Abstract

The invention relates to a K. intermedia ZJB-17004 and application thereof. The enantio-selectivity value of the K. intermedia for catalyzing N-phenylacetyl-DL-amino acid to produce L-amino acid reaches 98%, and the enantio-selectivity value of the K. intermedia for catalyzing 2-N-phenylacetyl-4-[hydroxy(methyl)phosphoryl]-DL-butyric acid to prepare 2-amino-4-[hydroxy(methyl)phosphoryl]-L-butyricacid reaches 99%.

Description

(1) Technical field [0001] The present invention relates to a bacterial strain producing amidohydrolase - Kluyveraintermedia ZJB-17004, and its N-phenylacetyl catalyzing N-phenylacetyl amino acid to prepare chiral amino acid and catalyzing amino acid derivatives Application of substituents in the preparation of chiral amino acid derivatives. (2) Background technology [0002] 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 genetically modified crop i...

Claims

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Application Information

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Patent Type & Authority Applications(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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