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Nitrilase xinit1 and its coding gene and application

A technology of nitrilase and gene, applied in nitrilase XiNit1 and its coding gene and application fields, to achieve the effects of high tolerance, wide reaction pH and high enzyme activity

Inactive Publication Date: 2020-09-15
CHINA UNIV OF PETROLEUM (EAST CHINA)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Generally speaking, the existing reported nitrilases can only specifically degrade a class of nitrile compounds

Method used

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  • Nitrilase xinit1 and its coding gene and application
  • Nitrilase xinit1 and its coding gene and application
  • Nitrilase xinit1 and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1, Isolation, Identification and Preservation of Xingfangfangbacterium DLY26

[0046] 1. Separation

[0047] 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.

[0048] 2. Identification

[0049] 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

[0066] Embodiment 2, the degradation action of Xinfangfang bacteria DLY26 to acrylonitrile

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 1. Preparation of seed solution

[0071] Inoculate Xingfangfang bacteria DLY26 into the activation medium, shake culture at 30°C and 150rpm, and obtain se...

Embodiment 3

[0091] Embodiment 3, the preparation of nitrilase (XiNit1 protein)

[0092]After a large number of sequence analysis, alignment and functional verification, a new protein was found from DLY26 of Xingfangfang bacteria, which was named as XiNit1 protein, as shown in sequence 1 of the sequence table. The gene encoding XiNit1 protein in Xingfangxiang bacteria DLY26 is named as xinit1 gene, and its coding frame is shown in sequence 2 of the sequence list.

[0093] 1. Construction of recombinant plasmids

[0094] 1. Using the genomic DNA of Xingfangfang bacteria DLY26 as a template, PCR amplification is carried out with primer pairs composed of DN1-F and DN1-R, and the PCR amplification products are recovered.

[0095] DN1-F: 5'- CACC ATGCTGGCGGAAGG-3';

[0096] DN1-R: 5'-CGGCCAGCTCGTCATGAC-3'.

[0097] 2. Take the PCR amplification product obtained in step 1 and connect it to the pDE1 vector to obtain the recombinant plasmid pDE1-xinit1.

[0098] The pDE1 vector (pDE1Vector) ...

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Abstract

The invention discloses nitrilase XiNit1 and a coding gene and application thereof. Protein provided by the invention comes from Xinfangfangiasp, is nitrilase, is named as XiNit1 protein, is protein composed of an amino acid sequence shown as a sequence 1 in a sequence table and protein composed of an amino acid sequence shown as a sequence 3 in the sequence table. The invention further disclosesapplication of the XiNit1 protein as nitrilase and a preparation for degrading nitrile compounds. The XiNit1 protein serves as an active ingredient of the preparation. The nitrilase has great application prospect in the field of related chemical industry and the field of environment pollution control.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to nitrilase XiNit1 and its coding gene and application. Background technique [0002] Nitrile compound is an important organic chemical raw material, but also a toxic substance with pungent smell. There are many different types of nitrile compounds in the sewage of synthetic rubber and petroleum refining process, and acrylonitrile is one of the important toxic components. Compared with the by-products of physical and chemical methods in the process of treating nitrile compounds, people prefer to use environmentally friendly biological methods to treat nitrile compounds in sewage. Compared with physical and chemical methods, biological methods have the characteristics of mild reaction conditions, stable operation and low cost. Since the 19th century, the use of biological methods to treat organic matter in wastewater has become the main method in all countries in the world...

Claims

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Application Information

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IPC IPC(8): C12N9/78C12N15/55
CPCC12N9/78C12Y305/05001
Inventor 郗丽君刘建国刘德健张静静王佳宇苏石晶窦珂王明谭雯斐
Owner CHINA UNIV OF PETROLEUM (EAST CHINA)
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