Enzyme preparation for removing o-nitrobenzaldehyde (ONBA) and application

The technology of o-nitrobenzaldehyde and enzyme preparation is applied in the fields of enzyme genetic engineering and enzyme engineering, which can solve the problems of high cost, easy corrosion of acid and alkali, and achieve the effects of low cost, good effect and high enzyme activity.

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

AI Technical Summary

Problems solved by technology

Although physical and chemical methods can effectively remove such substances, they are costly and require special instruments and equipment, and acids and alkalis are easy to corrode and have certain risks.

Method used

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  • Enzyme preparation for removing o-nitrobenzaldehyde (ONBA) and application
  • Enzyme preparation for removing o-nitrobenzaldehyde (ONBA) and application
  • Enzyme preparation for removing o-nitrobenzaldehyde (ONBA) and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] The extraction of embodiment 1 Pseudomonas sp.ONBA-17 total DNA (Genomic DNA)

[0023] 1.1 Expanded cultivation of Pseudomonas sp.ONBA-17

[0024] 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 ...

Embodiment 2

[0028] The acquisition of embodiment 2 ONBA degrading enzyme coding gene nbaA

[0029] 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) was screened out from 40 pairs of self-designed primers.

[0030] nbaA-f, 5'-ACGAC CATATG AAGGTACAACTGGTGAT-3' (the underline is the NdeI restriction site, and the gene sequence is SEQ ID No.3);

[0031] nbaA-r, 5'-CATCA AAGCTT GAAGGGATAAGGCTGGCC-3' (the underline is the HindIIII restriction site, and the gene sequence is SEQ ID No.4).

[0032] 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 O16.2 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, am...

Embodiment 3

[0035] Construction, expression and purification of embodiment 3 expression vector

[0036] The amplified product of the nbaA gene in Example 2 above was digested with NdeI and HindIII, and then the fragment was inserted into the pET21b(+) vector digested with the same endonuclease above. In the recombinant plasmid, the nbaA 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.

[0037] The recombinant plasmid was transferred into Escherichia coli BL21 (DE3), and then the strain containing the recombinant plasmid (numbered K-7) was selected, and the introduction of the foreign gene was verified by Shanghai Sangon sequencing, which confirmed the success of the foreign gene introduction.

[0038] K-7 was cultured with LB based on 37°C culture, after A 600nm When t...

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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-nitrobenzaldehyde (ONBA) and an application. The enzyme preparation is characterized in that the enzyme preparation is an aqueous solution or a buffer solution and comprises an ONBA degrading enzyme and MgCl2; the amino acid sequence of the ONBA 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 6.5-8.0; 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; the pure enzyme content of the ONBA degrading enzyme in the enzyme preparation is 0.03-0.1mg/mL. The enzyme preparation has the beneficial effects that by preferably adding metallic compounds, the enzyme preparation has high enzyme activity and excellent ONBA degrading effects; the adopted microbial degrading enzyme is prepared by means of gene engineering, is low in cost and is beneficial to large-scale popularization; by adopting an enzymic method to degrade ONBA, the enzyme preparation not only has good effects but also is simple and safe to operate, is mild in required reaction conditions and does not cause secondary pollution.

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-nitrobenzaldehyde and its application. Background technique [0002] With the acceleration of my country's industrialization process, the discharge of organic industrial synthetic wastewater is increasing. Organic industrial synthetic wastewater not only contains a large amount of organic matter, but also has a large amount of substances that are toxic to organisms, which has caused serious pollution to the environment and threatened human life and health. According to the acute toxicity classification standard, o-nitrobenzaldehyde (ONBA) is a moderately toxic compound. ONBA is an important fine organic chemical intermediate, widely used in medicine, dyes and organic synthesis, such as for the synthesis of anti-angina pectoris drug nitropyridine (nifedin), o-nitrostyrene and o-nitrocinnamic acid series products ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04B09C1/10
CPCB09C1/10C12N9/0008C12Y102/01065
Inventor 虞方伯单胜道管莉菠叶正钱骆林平
Owner ZHEJIANG FORESTRY UNIVERSITY
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