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Nitrilase and gene and application thereof

A technology of nitrilase and hydrolysis reaction, applied in the field of bioengineering, can solve the problems of low yield and poor tolerance, and achieve the effects of high yield, high ee value, and good substrate tolerance

Inactive Publication Date: 2012-07-04
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

And at present, known nitrilase is generally poor for the substrate tolerance of o-chloromandelonitrile, and majority is below 50mmol / L (J Am Chem Soc, 2002,124:9024-9025; J Ind Microbiol Biotechnol, 2010 , 37:741-750; Enzyme Microb Technol, 2010, 47:140-146)
Although it has been reported in the literature that nitrilase can hydrolyze 200mM o-chloromandelonitrile in water-toluene two-phase system, its productive rate and ee are low, product concentration 157mmol / L, ee=90.4% (J Biotechnol, 2011,152 :24-29)

Method used

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  • Nitrilase and gene and application thereof
  • Nitrilase and gene and application thereof
  • Nitrilase and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1 process such as figure 2 shown.

[0064] Cloning of embodiment 1 nitrilase gene

[0065] According to the gene sequence Genbank Accession NZ_AAUW01000019.1 of Labrenzia aggregate included in Genbank, PCR primers were designed as follows:

[0066] Primers (amplification of LaN gene):

[0067] Upstream primer: 5'-CCG GAATTC ATGAAAGCTATCAAGGTTGCCG-3',

[0068] Downstream primer: 5'-CCG CTCGAG CTACTCCTCGACCTCAAAAAGGC-3'.

[0069] Wherein, the underlined part of the upstream primer is the EcoRI restriction site, the underlined part of the downstream primer is the XhoI restriction site, and CCG is the protection base.

[0070] Genomic DNA of Labrenzia aggregate DSM 13394 (purchased from DSMZ) was used as a template, and primers were added for polymerase chain reaction (PCR). The PCR system is: 15 μl of 2×Taq PCR MasterMix, 1 μl (0.3 μmol / L) of each upstream primer and downstream primer, 1 μl (0.1 μg) of DNA template and ddH 2 O 12 μl. PCR amplification...

Embodiment 2

[0071] Embodiment 2 Preparation of recombinant expression plasmid and recombinant expression transformant

[0072] The PCR product obtained in Example 1 was double-digested with restriction endonucleases EcoRI and XhoI at 37°C for 6 h, purified by agarose gel electrophoresis, and the target fragment was recovered using an agarose gel DNA recovery kit, which contained the correct Insert snippet. The target fragment was mixed with the plasmid pET28a digested by EcoR1 and XhoI, and ligated overnight at 4°C under the action of T4 DNA ligase to obtain the recombinant expression plasmid pET-LaN.

[0073] The above recombinant expression plasmids were transformed into E. coli DH5α competent cells. On the resistance plate containing kanamycin (medium composition LB medium, peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L and agar 2%, antibiotic content 50mg / L) to positive recombination Screening was carried out, single clones were picked, and recombinant strains were cultiva...

Embodiment 3

[0074] Expression of embodiment 3 recombinant nitrilase

[0075] The recombinant Escherichia coli obtained in Example 2 was inoculated into LB medium containing kanamycin (50 mg / L), and cultured with shaking at 37° C. overnight. According to the inoculum amount of 1% (v / v), it is inserted into a 250ml Erlenmeyer flask equipped with 50ml LB medium, and cultured on a shaker at 37°C and 180rpm. When the OD of the culture medium 600 When it reaches 0.6, add IPTG with a final concentration of 0.5mmol / L for induction. After induction at 16°C for 24 hours, the culture medium was centrifuged, the cells were collected, and washed twice with normal saline to obtain 0.1-0.5 g of wet cells with a total viability of 5-40 U.

[0076] Preparation of recombinant nitrilase: suspend the resulting resting cells in phosphate buffer (100mmol / L, pH8.0), and ultrasonically break in an ice-water bath (power 400W, work 6s, gap 4s, ultrasonic 99 times) . Centrifuge at 8800 rpm for 20 minutes to col...

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Abstract

The invention discloses a new nitrilase and gene thereof, a recombinant expression vector and a recombinant expression transformant containing the gene, a method for preparing the recombinant nitrilase or microbial cell containing the recombinant nitrilase by use of the recombinant expression transformant, and application of the microbial cell in dehydrating ortho-chlorine mandelonitrile or other analogues and producing chiral ortho-chlorine mandelonitrile or other analogues. The recombinant nitrilase disclosed by the invention comes from Labrenzia aggregate and can be used as a catalyst for dehydrating and splitting ortho-chlorine mandelonitrile or other analogues; and the recombinant nitrilase has the advantages of high catalysis efficiency, strong enantioselectivity, mild reaction conditions, environmental friendliness and the like.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and particularly relates to a new nitrilase and its gene, a recombinant expression vector and a recombinant expression transformant containing the gene, a method for preparing a recombinant enzyme using a recombinant expression transformant, and the recombinant nitrilase The application of the enzyme in hydrolyzing o-chloromandelonitrile or its structural analogues to prepare (R)-o-chloromandelic acid or its structural analogues. Background technique [0002] (R)-o-chloromandelic acid is an important chiral intermediate of the anticoagulant drug clopidogrel. And its analogs are an important class of chiral synthetic building blocks, which are widely used in the synthesis of various drugs. Such as cephalosporin semi-synthetic antibiotics, important intermediates of anti-tumor and anti-aging drugs; it can also be used as a chiral resolution reagent for the resolution of other chiral drugs o...

Claims

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Application Information

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IPC IPC(8): C12N9/78C12N15/55C12N15/63C12N1/21C12N15/70C12P7/42C12R1/19C12R1/01
Inventor 许建和张陈胜潘江李春秀张志钧
Owner EAST CHINA UNIV OF SCI & TECH
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