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Nitrilase xinit2 and its coding gene and application

A nitrilase and gene technology, applied in nitrilase XiNit2 and its coding gene and application field, can solve the problems of low substrate degradation efficiency, narrow pH range, poor thermal stability, etc., and achieve wide reaction temperature and high enzyme activity , high tolerance effect

Active Publication Date: 2021-01-01
CHINA UNIV OF PETROLEUM (EAST CHINA)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, according to current research reports, nitrilases generally have problems such as poor thermal stability, narrow substrate spectrum and weak tolerance, and only nitrilases derived from thermophilic bacteria Pyrococcus sp. have higher temperature Stability (60-90°C), but the pH range is extremely narrow (6-8), and the degradation efficiency of the substrate is very low

Method used

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  • Nitrilase xinit2 and its coding gene and application
  • Nitrilase xinit2 and its coding gene and application
  • Nitrilase xinit2 and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 3

[0043] Example 3, Isolation, Identification and Preservation of Xingfangfangbacterium DLY26

[0044] 1. Separation

[0045] Take about 1ml of activated sludge sample (taken from the petrochemical refinery sewage treatment system), and dilute to 10 times volume, 20 times volume, 50 times volume, 100 times volume and 1000 times volume in turn with sterile distilled water. Spread 100 μl of the diluted sample on the surface of the solid medium by the coating plate method, incubate at 30°C, and observe the growth of the colonies on the surface every day. Pick colonies with different colors and shapes, and purify and culture them by the three-section line method until a single colony with the same shape and size can be observed on the surface of the culture medium.

[0046] 2. Identification

[0047] The purified bacterial strains were inoculated on TSA plates, cultivated at 30°C for 24h, and then observed the morphology, size and other characteristics of the cells using a transmi...

Embodiment 2

[0064] Embodiment 2, the degradation action of Xinfangfang bacteria DLY26 to acrylonitrile

[0065] Na 2 HPO 4 / NaH 2 PO 4 The formula of the buffer solution: disodium hydrogen phosphate 14.2g, sodium dihydrogen phosphate 12.2g, dissolved in 800mLddH 2 O, with ddH 2 O was adjusted to 1L, and the pH was adjusted to 7.0.

[0066] The formula of the activation medium: tryptone soybean broth medium (TSB) 30g, with ddH 2 O was dissolved and the volume was adjusted to 1L, and the pH was adjusted to 7.0.

[0067] Basal medium: 2.0g disodium hydrogen phosphate, 1.0g potassium dihydrogen phosphate, 0.5g yeast powder, 0.2g magnesium sulfate, 0.03g ferrous sulfate, dissolved in 800mL Na 2 HPO 4 / NaH 2 PO 4 buffer, then add 3.25mL of glycerol and mix well, then use Na 2 HPO 4 / NaH 2 PO 4 Make up to 1L of buffer.

[0068] 1. Preparation of seed solution

[0069] Inoculate Xingfangfang bacteria DLY26 into the activation medium, shake culture at 30°C and 150rpm, and obtain se...

Embodiment 4

[0110] Embodiment 4, the enzymatic property of nitrilase (XiNit2 protein)

[0111] Ammonia-free water: After boiling deionized water, keep it boiling for 15 minutes.

[0112] The water in this embodiment all refers to ammonia-free water.

[0113] Preparation of sodium phenoxide solution: Dissolve 25g of phenol in 800mL of water, then add 78mL of 4M aqueous sodium hydroxide solution, and then dilute to 1L with water.

[0114] Preparation of sodium nitroferricyanide solution: 1 g of sodium nitroferricyanide was dissolved in 100 mL of water to obtain a mother liquor, which was diluted with water to 100 times the volume before use to obtain a sodium nitroferricyanide solution.

[0115] Preparation of sodium hypochlorite solution: 19.4 mL of commercially available 10% sodium hypochlorite stock solution, dilute to 1 L with water.

[0116] Preparation of ammonia standard solution: the raw materials are ammonium chloride and water, and different ammonia (NH 3 ) concentration of amm...

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Abstract

The invention discloses nitrilase XiNit2 and its coding gene and application. The protein provided by the present invention is derived from Xinfangfangia sp., and is a nitrilase named as XiNit2 protein, which is a protein composed of the amino acid sequence shown in sequence 1 in the sequence listing; it is composed of sequence 3 in the sequence listing Proteins composed of the amino acid sequences shown. The invention also protects the application of XiNit2 protein as nitrilase. The invention also protects a preparation for degrading nitrile compounds, the active ingredient of which is XiNit2 protein. The present invention has great application prospects for related chemical industry fields, environmental pollution control fields and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to nitrilase XiNit2 and its coding gene and application. Background technique [0002] In the field of chemistry, nitrile compounds are not only widely used as fine chemical raw materials, but also intermediates of many chemical products. For example, nitrile compounds are used in the production of amines, amino compounds, carboxylic acids and heterocyclic compounds, but nitrile compounds are also It is also a highly toxic pollutant, which poses a serious threat to the health of animals, plants and humans themselves. The chemical treatment of nitrile compounds needs to be carried out under the conditions of strong acid (strong base), high pressure and high temperature, which will be accompanied by the formation of a large amount of salt, which will make the separation difficult in the later stage, and the residue will continue to cause harm. Biological degradation of nitril...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/78C12N15/55C12N1/20A62D3/02A62D101/26A62D101/28C12R1/01
CPCA62D3/02A62D2101/26A62D2101/28C12N9/78C12Y305/05001C12N1/205C12R2001/01
Inventor 刘建国刘德健苏石晶贾庆珠谭雯斐陈京生郗丽君窦珂王明
Owner CHINA UNIV OF PETROLEUM (EAST CHINA)
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