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Nitrilase mutant and application thereof in preparation of nicotinic acid

A nitrilase and mutant technology, applied in the fields of molecular biology and biology, can solve the problems of complex reaction process, serious environmental pollution and high energy consumption, and achieve the effect of high enzymatic reaction rate

Active Publication Date: 2016-11-23
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages are that the reaction needs to be carried out at high temperature, the energy consumption is high, the reaction process is complicated, and the environmental pollution is serious.

Method used

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  • Nitrilase mutant and application thereof in preparation of nicotinic acid
  • Nitrilase mutant and application thereof in preparation of nicotinic acid
  • Nitrilase mutant and application thereof in preparation of nicotinic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1. Construction of Prototype Nitrilase Recombinant Bacteria E.coli BL21pET28a-nitA

[0018] Using the synthetic gene SEQ ID NO.1 (NCBI GenBank No.DQ444267, synthesized by GenScript) as a template, according to the gene sequence of SEQ ID NO.1 and the polyclonal enzyme of Escherichia coli expression vector pET-28a (+) Cut site design primers.

[0019] Upstream primer: P1: 5'-GGTGCCGCGCGGCAGCCATATGGTTTCGTATAACAGCA-3'.

[0020] Downstream primer: P2: 5'-GCGGCCGCAAGCTTGTCGACCTTTGCTGGGACCGGTTCT-3'.

[0021] The PCR reaction conditions are:

[0022]

[0023] The reaction conditions of PCR were: cover temperature at 99°C; pre-denaturation at 94°C for 10 min; denaturation at 94°C for 45 s, annealing at 50°C for 45 s, and extension at 72°C for 2 min, a total of 32 cycles. The PCR product was verified using 0.8% agarose gel, and after the verification was correct, the fragment was cut using the Biomiga DNA gel recovery kit to recover the fragment.

[0024] Escheric...

Embodiment 2

[0028] Embodiment two, the construction of mutant bacteria

[0029] Using the synthetic gene SEQ ID NO.1 in Example 1 as a template, primers were designed according to the gene sequence of SEQ ID NO.1 and the nucleic acid sequence of the mutation site.

[0030]

[0031] The recombinant plasmid pET-28a(+)-Nit containing the target gene fragment was used as a template, and the template was amplified according to the method of overlap extension PCR, and the principle of the method was the same as in Example 1. The whole amplification process is carried out in two steps. First, primers P1 and F168V-F were added respectively, and the template was amplified with P2 and F168V-R to obtain two DNA sequences. Then add the two sequences into a PCR system, add primers P1 and P2, and continue PCR amplification to obtain the full-length F168V mutant DNA sequence. On the basis of the mutant at position 168, the amino acid at position 192 was mutated according to the above method to obta...

Embodiment 3

[0033] Embodiment three, the expression of nitrilase gene

[0034] Pick a single colony and insert it into a 5ml liquid LB medium test tube, and the medium contains 0.1g / L kanamycin. Culture overnight at 37°C, 200 rpm, with shaking. The next day, the liquid seeds in the test tube were transferred to fresh LB liquid medium containing 0.1g / L kanamycin with an inoculum size of 3% by volume, and cultivated at 37°C until the OD of the bacteria 600 When it is about 0.4-0.8, add the inducer IPTG (final concentration is 0.1mM) to the above LB liquid medium, induce for 20h at 25°C, then centrifuge the fermentation broth at 4°C and 8000rpm for 10min, and collect the wet cells. The bacterial cells were washed twice with PBS buffer solution of pH=7.0, and the bacterial cells were collected.

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Abstract

The invention discloses a nitrilase mutant and application thereof in preparation of nicotinic acid. The mutant can be used for mutating phenylalanine at position 168 of an amino acid sequence shown in SEQ ID NO: 2 into valine and serine at position 192 into phenylalanine. A wet cell obtained by induced expression of a recombinant bacterial strain containing an itrilase mutant encoding gene is used as an enzyme source, 3-cyanopyridine is used as a reaction substrate, and a PBS buffer with pH of 7.0 is used as a reaction medium to form a reaction system, a reaction is carried out in a 40 DEG C water bath, and the nicotinic acid is obtained after the reaction is finished.

Description

technical field [0001] The invention belongs to the field of molecular biology and biotechnology, and mainly relates to a nitrilase mutant and its application in nicotinic acid synthesis. Background technique [0002] Nicotinic acid, also known as nicotinic acid, is chemically named pyridine 3-carboxylic acid. It has a simple chemical structure and stable physical and chemical properties. Together with nicotinamide, it is called vitamin B3 or vitamin PP. It has important applications in food, medicine and industry. In the field of medicine, niacin can be directly used as an effective drug to prevent and treat skin diseases such as pellagra and similar vitamin deficiency, and can also be used to treat diseases such as atherosclerosis and peripheral nerve vasospasm; niacin can be used as a pharmaceutical intermediate to synthesize phthalate Amines and acyls. In the food field, niacin is often used as an additive in dairy products, cakes, corn flour, etc. Niacin can also be u...

Claims

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Application Information

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IPC IPC(8): C12N9/78C12N15/55C12N15/70C12N1/21C12P17/12C12R1/19
CPCC12N9/78C12P17/12C12Y305/05001
Inventor 姜岷李禾董维亮马江锋章文明信丰学
Owner NANJING UNIV OF TECH
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