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Nitrilase XiNit2 and its coding gene and application thereof

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

Active Publication Date: 2019-09-06
CHINA UNIV OF PETROLEUM (EAST CHINA)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 thereof
  • Nitrilase XiNit2 and its coding gene and application thereof
  • Nitrilase XiNit2 and its coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 3

[0043] Example 3 Isolation, identification and preservation of Fang Fang Fang DLY26

[0044] 1. Separation

[0045] Take about 1 ml of activated sludge sample (taken from the petrochemical refining wastewater treatment system), and dilute to 10 times, 20 times, 50 times, 100 times, and 1000 times volume with sterile distilled water. Spread 100 μl of the diluted sample on the surface of the solid culture medium by the spreading plate method, and incubate at 30°C. Observe the growth of colonies on the surface every day. The colonies with different colors and morphologies are picked, purified and cultured by the three-zone streaking method, until a single colony with the same morphology, size and other characteristics can be observed on the surface of the medium.

[0046] 2. Identification

[0047] The purified strain was inoculated on a TSA plate, cultured at 30°C for 24h, and then the morphology, size and other characteristics of the cells were observed using a transmission electron m...

Embodiment 2

[0064] Example 2. Degradation of acrylonitrile by Fang Fang Fang DLY26

[0065] Na 2 HPO 4 / NaH 2 PO 4 Buffer formulation: 14.2g disodium hydrogen phosphate, 12.2g sodium dihydrogen phosphate, dissolved in 800mLddH 2 O, use ddH 2 Set the volume of O to 1L and adjust the pH to 7.0.

[0066] Recipe of activation medium: Tryptone Soy Broth (TSB) 30g, use ddH 2 Dissolve O and dilute to 1L, adjust the pH to 7.0.

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

[0068] 1. Preparation of seed liquid

[0069] Inoculating Fang Fang Fang DLY26 into the activation medium, and shaking culture at 30° C. and 150 rpm to obtain seed liquid. OD of seed liquid 600nm Value = 0.6-0.8.

[0070] 2. Make a standard curve of dry cell weight

...

Embodiment 4

[0110] Example 4 Enzymatic properties of nitrilase (XiNit2 protein)

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

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

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

[0114] The preparation of sodium nitrosoferricyanide 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 nitrosoferricyanide solution.

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

[0116] Preparation of ammonia standard solution: the raw materials are ammonium chloride and water to prepare different ammonia (NH 3 ) Ammonia standard solution of concentra...

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Abstract

The present invention discloses a nitrilase XiNit2 and its encoding gene and an application thereof. The protein provided by the invention is derived from Xinfangfangia sp., is the nitrilase, designated as XiNit2 protein, and is the protein comprising an amino acid sequence shown in SEQ ID NO: 1 in a sequence table; and the protein comprising the amino acid sequence shown in SEQ ID NO: 3 in the sequence table. The invention also provides the application of the XiNit2 protein as the nitrilase. The present invention also provides a preparation for degrading a nitrile compound, and the active ingredient of which is the XiNit2 protein. The nitrilase XiNit2 has great application prospects for related chemical fields and environmental pollution control fields.

Description

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

Claims

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Application Information

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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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