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Production and immobilization method of recombinant nitrilase and its application to the degradation of acetonitrile

A technology of nitrilase and immobilization method, which is applied in the field of environmental biology, can solve the problems of persistence and efficiency that cannot meet the needs of treatment applications, cannot remove biological toxicity, and high cost of use, so as to improve catalytic stability and prolong the service life , Green and efficient degradation effect

Active Publication Date: 2022-06-17
FOSHAN YUHUANG ECOLOGICAL ENVIRONMENT TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it has certain biological toxicity, especially in living organisms, it can be degraded by intracellular enzymes to produce inorganic cyanide ions, which brings greater toxicity, so the emission control of acetonitrile in polluted water is very strict, generally at 5.0 mg per liter the following
The chemical properties of acetonitrile are relatively active, but non-specific degradation or transformation usually cannot relieve the biological toxicity caused by this type of organic nitrile compounds
High-concentration acetonitrile pollution can be treated by adsorption, ozonation or adding high-concentration lye, but when the pollutant concentration is below gram per liter, this kind of physical and chemical treatment method not only has high cost, but also brings new pollution. Pollutants, such as cyanates produced by ozone treatment, are less toxic but not completely degraded to harmless substances
[0004] Many microorganisms, such as Pseudomonas and Rhodococcus, can degrade and utilize acetonitrile in water. Although these microorganisms have been studied for many years as an efficient biodegradation system for degrading low-concentration acetonitrile and other organic nitriles, they are limited by living microorganisms. Influenced by the survival rate of individuals in the sewage system, the persistence and efficiency of the biocatalytic method still cannot meet the needs of actual pollution treatment applications

Method used

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  • Production and immobilization method of recombinant nitrilase and its application to the degradation of acetonitrile
  • Production and immobilization method of recombinant nitrilase and its application to the degradation of acetonitrile
  • Production and immobilization method of recombinant nitrilase and its application to the degradation of acetonitrile

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Synthesis and cloning of recombinant nitrilase

[0035] The nucleotide sequence shown in SEQ ID NO.1 encodes the completed nitrilase fusion protein, and its amino terminus contains a cellulose binding domain (CBD) from Trichoderma cellulase, and its coding sequence is connected with the subsequent linker peptide (linker). ) is shown by SEQ ID NO.5, the carboxyl terminus of nitrilase is a homologous protein from archaea, and its coding sequence is shown by SEQ ID NO.2. The nitrilase fusion proteins also include secreted peptides from Bacillus subtilis. The nucleotide sequence encoding the amino acid of SEQ ID NO.5 and the polypeptide sequence of the nitrilase are prepared by chemical synthesis after codon optimization. The PCR product was amplified by molecular biology technology and ligated into Thermo Fisher pET101 Topo plasmid system, and then transformed into E. coli DH5α to obtain ampicillin-resistant positive clones; 6 E. coli monoclones were picked and ...

Embodiment 2

[0037] Example 2 Expression of fusion nitrilase and assay of its enzymatic activity

[0038] Pick 3 verified Bacillus subtilis transformants and inoculate them into LB liquid medium (containing 10 grams of NaCl, 10 grams of tryptone, and 5 grams of yeast extract per liter) respectively. When the cell density is about 1.5, add 10 g / L xylose was used as the inducer, and the culture was continued for 12 hours at 37°C, and 20 μL of the culture supernatant was taken to identify the performance of nitrilase. Another 20 μL of the culture supernatant was used for polyacrylamide gel electrophoresis. Protein electrophoresis results such as figure 2 As shown, the protein composition in the expression supernatant is single, and there is basically only one molecular weight corresponding to the expression product of the nitrilase fusion protein.

[0039] The specific process of enzyme activity determination is as follows: 10 μL of supernatant was placed on a water bath shaker at 30-70°C ...

Embodiment 3

[0040] Example 3 Immobilization of fusion nitrilase and its application to acetonitrile degradation

[0041] Pick a single clone of Bacillus subtilis transformant 168 / pMK-CbN20, culture in a shaker flask, inoculate it into a culture tube containing 50 ml LB, containing 5 μg / mL chloramphenicol, 37 °C, 200 r / min Shaker culture for about 16 hours, transfer 1-3% of the overnight culture bacterial solution to a 5L fermenter with 2.5L liquid LB medium, add 10 g / L xylose, 5 μg to the medium / mL chloramphenicol, maintain aerobic culture, pass 2-10 L / min of air, automatically control the speed at 100-700rpm, keep the dissolved oxygen value at 40% or below, continue to culture for 72 hours, maintain pH around 6.8, culture Add 10 g / L (NH 4 ) 2 PO 4 and 20 g / L glucose. After fermentation, the cells were removed by centrifugation, the recombinase in the supernatant was collected, the protein concentration was determined, and the enzyme was bio-fixed, such as image 3 shown. The immob...

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Abstract

The invention discloses a production and immobilization method of a recombinant nitrilase and its application to degradation of acetonitrile, and relates to the field of environmental biotechnology. The new industrial enzymes generated after integration can specifically bind to wood pulp cellulose to achieve enzyme immobilization and efficient and specific pollutant degradation. The invention discloses a recombinant expression system for expressing the polypeptide, including a recombinant vector, an expression host, a sequence of a cellulose binding domain and a connecting peptide segment, and a secreted peptide sequence for expressing the recombinant polypeptide. The nitrilase fusion protein recombinantly expressed by Bacillus subtilis was immobilized to wood pulp cellulose by one-step method to improve the stability of enzyme catalysis and realize the biological removal of acetonitrile in flowing sewage.

Description

technical field [0001] The invention belongs to the technical field of environmental biology, and in particular relates to the production and immobilization of a recombinant nitrilase and its application to the degradation of acetonitrile. Background technique [0002] Acetonitrile is a by-product of acrylonitrile production. Acrylonitrile is an important chemical raw material. It is a chemical monomer for synthesizing artificial wool, butyl rubber and synthetic resins. It can also produce chemicals such as acrylamide and acrylic acid. The raw material production of acrylonitrile is huge. Acetonitrile itself is also an industrial raw material, which can be used to produce organic nitrile pesticides, vitamin B1 and other compounds, and is also an extraction solvent for chemical products such as butadiene and aromatic hydrocarbons. In recent years, acetonitrile has also been widely used in metronidazole. Synthetic raw materials or solvents for pharmaceutical products such as ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/55C12N9/78C12N15/75C12N15/62C07K19/00C12N11/12C02F3/34C12R1/125C02F101/38
CPCC12N9/78C12N15/75C12N11/12C02F3/342C12Y305/05001C07K2319/02C07K2319/00C02F2101/38
Inventor 邱建贺翁雯周国彪
Owner FOSHAN YUHUANG ECOLOGICAL ENVIRONMENT TECH
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