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Nitrilase as well as preparation method and application thereof

A technology of nitrilase and hydroxybutyronitrile, which is applied in the field of enzymology, can solve the problems of expensive metal catalysts, harsh reaction conditions, and high equipment requirements, and achieve high industrial production potential, mild reaction conditions, and less hydrolysis by-products.

Active Publication Date: 2011-04-27
SHANGHAI PESTICIDE RES INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] However, in the existing technology, the preparation of 3-hydroxybutyric acid is mainly through chemical methods, such as hydrolysis of β-butyrolactone, Reformatsky reaction, reduction method, hydration of crotonic acid, preparation of naphthalene lithium catalyst, hydrolysis or oxidation, etc. preparation, but the above-mentioned chemical method has the following disadvantages: the synthesis process is complicated, the reaction conditions are relatively harsh, the requirements for equipment are high, the metal catalyst is expensive, easy to cause pollution, and is unfavorable to environmental protection, etc.
[0010] The biological method catalyzes nitrile hydrolysis with few by-products and mild reaction conditions, but none of the nitrilases found so far can hydrolyze 3-hydroxybutyronitrile to produce 3-hydroxybutyrate

Method used

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  • Nitrilase as well as preparation method and application thereof
  • Nitrilase as well as preparation method and application thereof
  • Nitrilase as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 , Fermentation strain culture and fermentation liquid detection

[0059] 1.1. Fermentation strain culture

[0060] Pick Arthrobacter nitroguajacolicus CGMCC No.2405, inoculate it into the slant medium, and cultivate it at 28°C for 4 days; then, pick the cultured strain from the slant, transfer it to the fermentation medium, and cultivate it at 220rpm and 28°C for 3-4 days , to obtain the fermentation broth.

[0061] 1.2. Stability detection of enzyme solution

[0062] Take 25ml of the fermentation broth obtained in step 1.1, concentrate it 5 times, use 10mM Tris-HCl buffer to suspend the cells, and perform cell disruption. The cell disruption conditions are as follows:

[0063] Ultrasound for 2 seconds, intermittent for 5 seconds, power 700-800W, time 0.5 hours.

[0064] The broken cell solution was centrifuged at 15,000 rpm for 30-40 minutes, and the upper layer of crude enzyme solution was collected.

[0065] After the collected centrifuged supernatant...

Embodiment 2

[0073] Example 2 ,protein purification

[0074] The crude enzyme solution in the upper layer obtained in Example 1 was subjected to fractional precipitation with saturated ammonium sulfate, and the precipitate at 0-35% saturation was collected.

[0075] Then, dissolve the crude enzyme precipitate with 0.5M pH7.2 potassium phosphate buffer solution, purify the obtained crude enzyme solution through a phenyl hydrophobic column, collect the eluate, and detect the enzyme activity and protein content of the eluate at the same time, and then The collected fraction with higher specific enzyme activity was purified using a butyl hydrophobic column, and the specific purification conditions were as follows:

[0076] 1. Phenyl hydrophobic column (Phenyl-650C)

[0077] Column bed: height 12cm×diameter 1cm, volume 9.4ml;

[0078] Flow rate: 0.1ml / min adsorption, 1ml / min washing column (0.5M potassium phosphate buffer, pH7.2), 0.1ml / min elution (deionized water);

[0079] Column volume...

Embodiment 3

[0097] Example 3 , protein detection

[0098] 3.1. N-terminal amino acid sequence determination

[0099] The N-terminal sequencing of the enzyme obtained by purifying the butyl hydrophobic column in Example 2 showed that the sequence of twenty amino acids at the N-terminal of the enzyme was NH 2 -THPQ FKAAVVQAAPVFLNLD .

[0100] Then, the determined sequence was compared with Blastp, and the result of the comparison showed that the amino acid sequence of the enzyme obtained in Example 2 had a high homology with the nitrilase, therefore, it might be the target protein-nitrilase.

[0101] 3.2. Determination of full-length DNA sequence

[0102] The genome of the strain obtained in Example 1.1 was extracted with reference to the experimental method in "Refined Molecular Biology Experiment Guide (Fourth Edition)" (Science Press).

[0103] The strain genome and vector pUC18 plasmid (purchased from Shanghai Sangong) were completely digested with BamHI, the digested genome and ...

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Abstract

The invention discloses a nitrilase as well as a preparation method and application thereof. The nitrilase provided in the invention has the amino acid sequence shown as SEQ ID NO: 20. The nitrilase can be obtained through fermenting Arthrobacternitroguajacolicus bacteria and can be used for hydrolyzing 3-hydroxybutyronitrile to produce 3-hydroxybutyric acid. The reaction has less hydrolysis by-products, mild condition and high space time yield, in addition, the 3-hydroxybutyronitrile has favorable water solubility, and the reaction can be carried out in a pure water phase and facilitate the enzyme stability, therefore, the nitrilase has very high industrialized production potential.

Description

technical field [0001] The invention belongs to the field of enzymology, and in particular relates to a microbial-sourced nitrilase, a preparation method and application thereof. Background technique [0002] Nitrilases are biocatalysts with broad substrate adaptability that can catalyze the hydrolysis of nitriles to the corresponding hydroxyacids. Due to the widespread existence of natural nitriles, many microorganisms have more or less the ability to degrade nitriles. At present, nitrilases from a variety of microbial sources have been reported, and their reaction substrates are diverse, as shown in Table 1 below: [0003] Table 1. Strains used to prepare nitrilase and their corresponding hydrolyzed substrates [0004] [0005] 3-hydroxybutyric acid is commonly known as β-hydroxybutyric acid, the English name is 3-hydroxybutyric acid or DL-β-hydroxybutyric acid, and its molecular formula is C 4 h 8 o 3 , molecular weight 104.10, CAS number 625-71-8, structural form...

Claims

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Application Information

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IPC IPC(8): C12N9/78C12P7/42C12R1/06
Inventor 薛建萍唐璐敏朱健郝婷婷
Owner SHANGHAI PESTICIDE RES INST
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