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Nitrilase, encoding genes, carrier and application

A nitrilase and coding gene technology, which is applied in the application field of preparing -3-cyano-5-methylhexanoic acid, can solve the problems of difficult separation and purification, environmental pollution and the like, and achieves high atom economy and environmental friendliness. , mild conditions

Inactive Publication Date: 2015-10-07
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional chemical hydrolysis of cyano group usually requires conditions such as strong acid, strong alkali and high temperature reflux, and is accompanied by the formation of a large number of inorganic salts, which not only brings difficulties to separation and purification, but also causes environmental pollution

Method used

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  • Nitrilase, encoding genes, carrier and application
  • Nitrilase, encoding genes, carrier and application
  • Nitrilase, encoding genes, carrier and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1: the acquisition of nitrilase

[0053] A nitrilase gene sequence was screened using protein PDB and NCBI database data to obtain a nitrilase gene (NCBI Reference Sequence: NP_001288810.1). The nitrilase is derived from Brassica rapa, a Brassicaceae plant. According to the amino acid sequence of the nitrilase, codon optimization is carried out according to the codon preferred by Escherichia coli, and according to the expression vector pET28b(+) Features The enzyme cutting sites Xho I and Xba I were designed, and the nitrilase gene BrNit (shown in SEQ ID NO: 2) was synthesized by the method of total synthesis through the conventional operation of genetic engineering. The amino acid sequence of the encoded enzyme is shown in SEQ ID NO: 2. ID NO: 1.

Embodiment 2

[0054] Example 2: Construction of recombinant expression vector pET28b(+)-BrNit and construction of recombinant engineering bacteria

[0055] Use Xho I and Xba I restriction endonucleases to carry out double digestion and recycling of the BrNit gene fragment, and use T4 DNA ligase to combine the fragment with the commercial vector pET28b(+) treated with the same restriction endonuclease. Ligation was carried out at 16°C for 16 hours, thereby constructing the intracellular recombinant expression vector pET28b(+)-BrNit. Transform the constructed intracellular expression vector pET28b(+)-BrNit into E.coli BL21(DE3) (Invitrogen) recipient bacteria, spread on LB agar plates containing kanamycin (final concentration: 50 μg / mL) On the plate, cultivate overnight at 37°C, and on the next day, clones were randomly selected from the colonies grown on the plate, and the plasmids were extracted for agarose gel electrophoresis identification, and the recombinant genetically engineered bacte...

Embodiment 3

[0056] Embodiment 3: the preparation that contains nitrilase BrNit bacterial cell

[0057] The genetically engineered bacteria E.coli BL21(DE3) / pET28b(+)-BrNit constructed in Example 2 was inoculated into LB liquid medium containing 50 μg / mL kanamycin, cultivated at 37°C for 12h, and then inoculated with 2% The amount (v / v) was inoculated into fresh LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C to the cell concentration OD 600 About 0.6, then add IPTG with a final concentration of 0.2mM to the LB liquid medium, and after inducing culture at 28°C for 10h, centrifuge the culture solution at 4°C and 12000rpm for 5min, discard the supernatant, and collect the recombinant nitrilase-containing The wet cells of Escherichia coli BL21(DE3) / pET28b-BrNit containing intracellular expression of recombinant nitrilase. Then SDS-PAGE electrophoresis was carried out to verify the size and expression of the target protein (see figure 2 ).

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Abstract

The invention discloses nitrilase from brassica rapa, encoding genes and an application of the encoding genes to the preparation of pregabalin chiral intermediate (S)-3-cyan-5-methylhexanoic acid through biocatalysis, and an amino acid sequence of the nitrilase is shown as SEQ ID NO:1. The invention provides novel nitrilase for preparing the (S)-3-cyan-5-methylhexanoic acid with high region and high stereoselectivity through hydrolysis, the concentration of hydrolysis substrates of the nitrilase can reach more than 1 M, and an ee (enantiomeric excess) value is kept more than 99 percent. The pregabalin important chiral intermediate (S)-3-cyan-5-methylhexanoic acid can be prepared through the nitrilase; a preparation method of the (S)-3-cyan-5-methylhexanoic acid has the advantages of high atom economy, mild conditions, environmental friendliness and the like and has high industrial application potential.

Description

(1) Technical field [0001] The invention relates to a nitrilase, in particular to a nitrilase for the biocatalytic preparation of (S)-3-cyano-5-methylhexanoic acid, a key chiral intermediate of pregabalin, and the preparation of (S)- Application of 3-cyano-5-methylhexanoic acid. (2) Background technology [0002] Nitrilase (Nitrilase, EC 3.5.5.1) is an important conversion enzyme of nitrile compounds, which catalyzes the hydrolysis of nitrile compounds into corresponding carboxylic acids. The traditional chemical hydrolysis of cyano groups usually requires conditions such as strong acid, strong alkali and high temperature reflux, and is accompanied by the formation of a large number of inorganic salts, which not only brings difficulties to separation and purification, but also causes environmental pollution. Nitrilase biocatalysis is not only efficient, mild reaction conditions, and environmentally friendly, but also has unique advantages such as strict stereo and regiosele...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/78C12N15/55C12P13/00
CPCC12N9/78C12P13/002C12Y305/05001
Inventor 郑裕国郑仁朝张琴柳志强徐明波
Owner ZHEJIANG UNIV OF TECH
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