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Enzyme preparation for removing o-nitrobenzoic acid (2-NBA) and application

A technology of o-nitrobenzoic acid and enzyme preparation, which is applied in the fields of enzyme genetic engineering and enzyme engineering, can solve problems such as the reduction of decomposition rate of organic matter, and achieve the effects of excellent effect, mild reaction conditions and high enzyme activity

Inactive Publication Date: 2014-01-08
ZHEJIANG FORESTRY UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the acidification of water body will also lead to changes in the composition and structure of aquatic organisms, the increase of acid-resistant algae and fungi, the reduction of rooted plants, bacteria and vertebrates, and the reduction of the decomposition rate of organic matter

Method used

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  • Enzyme preparation for removing o-nitrobenzoic acid (2-NBA) and application
  • Enzyme preparation for removing o-nitrobenzoic acid (2-NBA) and application
  • Enzyme preparation for removing o-nitrobenzoic acid (2-NBA) and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The extraction of embodiment 1Pseudomonas sp.ONBA-17 total DNA (Genomic DNA)

[0024] 1.1 Expanded cultivation of Pseudomonas sp.ONBA-17

[0025] Under a sterile operating environment, small pieces of Pseudomonas sp.ONBA-17 stored at -70°C were picked with an inoculation needle, placed and spread on Luria-Bertani (LB) solid medium. 28 ℃, static culture 3d. A single colony was selected, and after two transfers, it was observed that the colonies on the plate had the same shape (no bacteria). The "plate" and Luria-Bertani (LB) solid / liquid medium described here are all common terms, medium, techniques and methods in the field of microbial research / production. For details, see "Molecular Cloning Experiment Guide" (J. Sam Brook, et al. Molecular Cloning Experiment Guide (Third Edition) [M]. Huang Peitang, et al. Translated. Beijing: Science Press, 2008). Unless otherwise specified, the techniques, methods, culture media, reagents and medicines used in microbial research a...

Embodiment 22

[0029] The acquisition of embodiment 22-NBA degrading enzyme coding gene nbaB

[0030] After a large number of analysis experiments, a pair of effective amplification primers (denoted as SEQ ID No.3 and SEQ ID No.4 respectively) were screened out from 24 pairs of self-designed primers.

[0031] nbaB-f,5'-ACGAC CATATG AGTTACCAAAACTTAG-3' (the underline is the NdeI restriction site, and the gene sequence is SEQ ID No.3);

[0032] nbaB-r, 5'-CATCA GAATTC GGAAGACCAGGAGC-3' (the underline is the EcoRI restriction site, and the gene sequence is SEQ ID No.4).

[0033] 25 μL amplification system: 2.5 μL of 10×Taq DNA polymerase reaction buffer; 2 μL of dNTP (25 mM); 0.5 μL of each primer (25 pmol / μL); Mg 2+ Buffer (25mM) 2.5μL; total DNA obtained in Example 1 0.5μL (about 100ng); Taq enzyme (5U / μL) 0.3μL; ddH 2 016.2 μL. Reaction parameters: pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 sec, annealing at 52°C for 30 sec, extension at 72°C for 1 min, amplificat...

Embodiment 3

[0037] Construction, expression and purification of embodiment 3 expression vector

[0038] The amplified product of the nbaB gene in Example 2 was digested with Nde I and EcoRI, and then the fragment was inserted into the pET21b(+) vector digested with the same restriction endonuclease. In the recombinant plasmid, the nbaB gene is driven by the T7 promoter, the C-terminus of the expression product has 6 histidine tags, and ampicillin is the selection marker. The joined fragments were verified by Shanghai Sangon sequencing to ensure that no mutations were introduced during the PCR process.

[0039] The recombinant plasmid was transferred into Escherichia coli BL21 (DE3), and then the strain containing the recombinant plasmid (numbered Y-6) was selected, and the foreign gene introduction was verified by Shanghai Sangon sequencing. Y-6 was cultured with LB based on 37°C culture, after A 600nm When the value was 0.6, IPTG was added to a final concentration of 1 mM, followed by ...

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Abstract

The invention belongs to the field of enzyme gene engineering and enzyme engineering and relates to an enzyme preparation for removing o-nitrobenzoic acid (2-NBA) and an application. The enzyme preparation is an aqueous solution or a buffer solution and comprises a 2-NBA degrading enzyme and MgCl2, wherein the amino acid sequence of the 2-NBA degrading enzyme is shown in SEQ ID No.1; the final concentration of MgCl2 is 0.1-0.3mM; the pH value of a reaction system is 6.0-8.0; when the enzyme preparation is the buffer solution, the buffer solution is generally a sodium phosphate buffer solution or a potassium phosphate buffer solution with pH value of 7.2-7.7 and concentration of 50-100mM.

Description

technical field [0001] The invention belongs to the fields of enzyme genetic engineering and enzyme engineering, and relates to an enzyme preparation for removing o-nitrobenzoic acid and its application. Background technique [0002] With the rapid development of my country's chemical industry, the amount of three wastes (waste water, waste gas, waste residue) produced by organic synthesis is increasing year by year. There are a large number of substances that are toxic to organisms in the above-mentioned three wastes, which have caused extremely serious pollution and harm to the environment, and threatened the safety and health of humans and other organisms. Among them, the pollution problems related to nitroaromatic compounds are the most prominent. [0003] Nitroaromatic compounds widely exist in nature and are mainly used in the production of dyes, insecticides, explosives, pesticides, medicines and other chemical products. With the rapid development of industry and ag...

Claims

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Application Information

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IPC IPC(8): C12N9/04B09C1/10
CPCB09C1/10C12N9/0069C12N9/0071
Inventor 虞方伯管莉菠骆林平单胜道
Owner ZHEJIANG FORESTRY UNIVERSITY
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