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Recombinant cyanohydrin lyase and its application for preparing optically pure chiral cyanohydrins

A technology of cyanohydrin lyase and cyanohydration reaction, which is applied in the field of catalyzing the asymmetric cyanohydration of aromatic aldehydes to prepare optically pure chiral α-cyanohydrins, which can solve the problems of differences in catalytic properties and achieve stereoselectivity Strong performance, good catalytic effect and high catalytic efficiency

Active Publication Date: 2019-08-06
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The molecular characteristics and catalytic performance of the cyanohydrin lyase contained in Badam have not been reported so far.
Moreover, similar to bitter almonds, badalwood cyanohydrin lyase exists in the form of isozymes, and different isozymes may have great differences in catalytic properties

Method used

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  • Recombinant cyanohydrin lyase and its application for preparing optically pure chiral cyanohydrins
  • Recombinant cyanohydrin lyase and its application for preparing optically pure chiral cyanohydrins
  • Recombinant cyanohydrin lyase and its application for preparing optically pure chiral cyanohydrins

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Experimental program
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Effect test

Embodiment 1

[0045] The cloning of embodiment 1 badam cyanohydrin lyase gene

[0046] Soak fresh Badam seed cores in hot water at 80°C for 0.5 hours, take them out and cool them in ice water, repeat 3 times, take them out and bury them in the sand, the thickness of the sand on the surface is about 0.5cm, water intermittently to keep the sand After the seeds germinated, the genomic DNA of the young leaves was extracted by the phenol-chloroform method. Since the gene sequence of Badamu cyanohydrin lyase has not been reported in the literature, according to the known gene sequence of Rosaceae cyanohydrin lyase (Genbank, Accession AF412329.1, AF040079.1, U51562.1, AY321296.1, GU126428. 1. U78814.1) analyzed the conserved base segment that may exist in the entire Rosaceae family HNL, based on which degenerate primers were designed, using the Badamu genome as a template, and a method similar to that reported in the literature (JBiosci.Bioeng., 2011, 112, 321-325) walked on its chromosome to obt...

Embodiment 2

[0055] Example 2 Preparation of recombinant plasmid pPICZαA-PcHNL

[0056] The construction process of the recombinant plasmid pPICZαA-PcHNL is as follows: figure 2 shown.

[0057] The complete gene of Badam cyanohydrin lyase obtained by cloning in Example 1 contains introns. By comparing with the conserved introns characteristic of Rosaceae cyanohydrin lyases reported in Genbank, Badam cyanohydrin lyase was identified. Three introns contained in cyanohydrin lyase. Subsequently, using the plasmid pMD-19T-HNL prepared in Example 1 as a template, the three introns included in the original gene sequence were deleted by using primer ligation overlap extension PCR technology. The specific operation steps are as follows:

[0058] Step 1, use PrimeStar HS premix for PCR amplification, and use three sets of primers to amplify the three exons of cyanohydrin lyase PcHNL respectively. PCR system: 2×PrimeStar HS premix 15μl, upstream and downstream primers 1.5μl each, pMD-19T-HNL tem...

Embodiment 3

[0066] Example 3 Preparation of recombinant Pichia pastoris P.pastoris / pPICZαA-PcHNL

[0067] The recombinant plasmid pPICZαA-PcHNL obtained in Example 2 was double-digested with restriction endonuclease Sac I at 37°C for 4 hours, and after electrophoresis verified that the recombinant plasmid was completely linearized, the target DNA fragment was recovered using a PCR purification and recovery kit (concentration> 100ng / μl).

[0068] Mix 80 μl competent cells of Pichia pastoris X33 and 1 μg linearized plasmid DNA sample, transfer to a pre-cooled electroporation cup (electrode distance 0.2 cm), ice bath for 5 min; then pulse electric shock once under the condition of 2kV, 5ms ; After that, quickly add 0.5ml ice-cold sorbitol solution (1M) to the electrocup, and transfer the bacterial solution in the electrocup to 0.5ml YPD liquid medium (peptone: 20g / L, yeast extract: 10g / L, glucose: 20g / L, pH 6.0) in a 1.5ml Eppendorf tube, incubate at 30°C, 200rpm for 2-3h; use a pipette gun...

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Abstract

The invention discloses hydroxynitrile lyase from Prunus communis L.var.dulcis Borkh, a gene of the hydroxynitrile lyase, a recombinant expression vector and a recombinant expression transformant which contain the gene, a recombinase of the hydroxynitrile lyase, a preparation method of the recombinant hydroxynitrile lyase and an application of the recombinant hydroxynitrile lyase as a catalyst in catalysis of asymmetric hydroxyl cayaniding of latent chiral aromatic aldehyde compounds for preparation of chiral cyanohydrin as a medical precursor. The hydroxynitrile lyase has the advantages of high catalytic efficiency and good stereoselectivity; compared with a wild Prunus communis kernel powder catalyst prepared by degreasing, the hydroxynitrile lyase catalyst has a better catalytic effect and broader substrate adaptability. The enzymatic reaction can be performed at the room temperature, so that the defect of a conventional cyanidation reaction is required to be performed at the low temperature of is overcome, the operation is simple, and good industrial application prospect is realized.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a cyanohydrin lyase and its gene, a recombinant expression vector and a recombinant expression transformant containing the gene, the preparation of the recombinant cyanohydrin lyase, and the use of the recombinant cyanohydrin lyase as Application of catalysts in catalyzing the asymmetric hydroxycyanation of aromatic aldehydes to prepare optically pure chiral α-cyanohydrins. Background technique [0002] The α-carbon of optically pure α-cyanohydrin is a chiral carbon atom, which connects a hydroxyl group and a nitrile group, and these groups can be converted into other various groups while maintaining chirality, plus the cyanohydrin molecule Transformation involving other functional groups in the compound can generate a variety of chiral compound molecules, many of which are biologically active. Therefore, optically pure α-cyanohydrins occupy an important positi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12N15/60C12N1/19C12P13/00C12R1/84
CPCC12N9/88C12P13/004C12Y401/02047
Inventor 郁惠蕾郑宇璁许建和潘江
Owner EAST CHINA UNIV OF SCI & TECH
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