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A strain producing nitrile hydratase and its method for producing p-hydroxyphenylacetamide

A technology of nitrile hydratase and bacterial strains, which is applied in the field of preparing p-hydroxyphenylacetamide and producing nitrile hydratase strains, can solve the problems of complex steps, pollution, low enzyme activity, etc., achieve low raw material cost, simple medium composition, The effect of high enzyme activity

Active Publication Date: 2020-04-21
SHANDONG YANGCHENG BIOLOGY TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

p-Hydroxyphenylacetamide is a key intermediate for the synthesis of the β-adrenergic receptor blocker atenolol, and has good market application prospects. At present, it is mainly synthesized by chemical methods at home and abroad, and there are problems such as complicated steps and serious pollution. Bioenzyme is a good alternative
Cai Qian et al. used p-hydroxyphenylacetonitrile as a substrate to screen a strain of Rhodococcus rubrum, which has the ability to transform and produce p-hydroxyphenylacetamide, but the enzyme activity is not high, and there is serious substrate inhibition.

Method used

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  • A strain producing nitrile hydratase and its method for producing p-hydroxyphenylacetamide
  • A strain producing nitrile hydratase and its method for producing p-hydroxyphenylacetamide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Classification of Bacillus arborii strains

[0022] 1) Characteristics of strains

[0023] On the preservation medium, the colonies are milky white or yellowish, nearly round, with smooth, moist and raised surfaces, and irregular edges; observed under a microscope, the bacteria are rod-shaped, have spores, can move, and are Gram-positive; physiological and biochemical indicators As shown in Table 1.

[0024] Table 1 Physiological and biochemical characteristics of strain Jn-102

[0025]

[0026] 2) Strain identification

[0027] The length of the 16S rRNA gene sequence obtained by DNA extraction, PCR amplification and sequencing is 1382bp, and the BLAST comparison on GenBank shows that the base similarity between the strain and Bacillus aryabhattai (EF114313) reaches 99%, combined with physiological and biochemical indicators, The Jn-102 strain was identified as Bacillus aryabhattai. The 16S rRNA sequence information is as follows:

[0028]gcttctatgac...

Embodiment 2

[0037] Embodiment 2 bacterial strain is cultivated and p-hydroxyphenylacetamide is synthesized

[0038] 1) Strain activation: pick the strains and inoculate them into solid preservation medium: yeast extract powder 5g / L, peptone 10g / L, NaCl 5g / L, agar 20g / L, pH 7.0, sterilize at 121℃ for 20min ; Streak inoculation in a constant temperature incubator at 30°C for 20 hours;

[0039] 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, sterilized by high-pressure steam at 121°C for 20 minutes; sealed with eight layers of gauze, and cultured at 30°C for 18 hours on a shaker at 200 r / min to obtain liquid seeds;

[0040] 3) liquid fermentation: according to 5% inoculation amount, the components are glucose 10g / L, p-hydroxybenzonitrile 5g / L, peptone 10g / L, yeast extract powder 5g / L, MgSO 4 0.3g / ...

Embodiment 3

[0042] Embodiment 3 bacterial strain is cultivated and p-hydroxyphenylacetamide is synthesized

[0043] 1) Strain activation: pick the strains and inoculate them into solid preservation medium: yeast extract powder 5g / L, peptone 10g / L, NaCl 5g / L, agar 20g / L, pH 7.0, cultivate in a constant temperature incubator at 30°C 20h;

[0044] 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, sterilized by high-pressure steam at 121°C for 20 minutes; sealed with eight layers of gauze, and cultured at 30°C for 18 hours on a shaker at 200 r / min to obtain liquid seeds;

[0045] 3) Liquid fermentation: by volume ratio of 5% inoculum, the composition is glucose 10g / L, p-hydroxyphenylacetonitrile 5g / L, peptone 5g / L, yeast extract powder 5g / L, MgSO 4 0.1g / L, ammonium sulfate 2g / L, KH 2 PO 4 0.15g / L,...

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Abstract

The invention relates to a strain for producing nitrile hydratase and a method for producing p-hydroxyphenylacetamide by using the strain and belongs to the technical field of biology. The classification name of the strain is bacillus aryabhattai Jn-102; the strain is preserved in China General Microbiological Culture Collection Center (CGMCC) on 26th, September, 2017; the preservation number is CGMCC No. 14668. The bacillus aryabhattai Jn-102 for producing the nitrile hydratase, provided by the invention, is high in genetic stability and low in culture cost; the produced nitrile hydratase ishigh in enzyme activity and wide in applicable substrate spectrum; the strain can be directly used for synthesizing the p-hydroxyphenylacetamide by directly converting p-hydroxybenzylcyanide; membranepermeating agents such as surfactants and organic solvents do not need to be added during catalytic synthesis, so that the introduction of exogenous impurities is avoided; the operations of extraction and separation are facilitated; the industrial application of the strain has advantages.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a bacterial strain producing nitrile hydratase and a method for catalyzing the preparation of p-hydroxyphenylacetamide by using the nitrile hydratase produced by the bacterial strain. Background technique [0002] Nitrile is an important class of compounds, which widely exist in plants in nature, and its hydrolysis reaction is widely used in the synthesis of amino acids, amides, carboxylic acids and their derivatives, and occupies an extremely important position in organic synthesis. Nitrile hydrolysis methods mainly include chemical hydrolysis and biotransformation. The chemical hydrolysis of nitrile requires strong acid or strong alkali, high temperature, high pressure and other reaction conditions, and there are many by-products, low yield and serious environmental pollution, which greatly limits its industrial application. In contrast, the biotransformation method has the advant...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P13/02C12R1/07
Inventor 王东阳冯志彬张娟刘树立
Owner SHANDONG YANGCHENG BIOLOGY TECH CO LTD
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