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Ester hydrolase with R-isomer stereoselectivity, gene and recombination enzyme thereof

A stereoselective, ester hydrolase technology, applied in the field of bioengineering, can solve problems such as high cost, complexity, and difficulty in adopting

Active Publication Date: 2007-10-24
SHANGHAI INST OF PHARMA IND CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To solve this problem, the occurrence of side reactions can be reduced by means of engineering regulation (PerBerglund, Biomolecular Engineering, 18, 2001: 13-22), and a single enzyme preparation can be obtained by separation and purification to catalyze the reaction, but these processes are often Complicated and costly, not easily adopted in production

Method used

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  • Ester hydrolase with R-isomer stereoselectivity, gene and recombination enzyme thereof
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  • Ester hydrolase with R-isomer stereoselectivity, gene and recombination enzyme thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Cloning of the BSEST gene from Bacillus

[0033] According to the DNA sequence of Bacillus sp H-257 lipase (J Biochem, 2001, 129:397) and the DNA sequence of Bacillus cereus C71 lipase (Genbank: AY896293), degenerate primers 1 and 2 were designed. A NdeI site was added to primer 1, and a HindIII site was added to primer 2.

[0034]Primer 1 sequence: GGAATTC CATATG AGCGAMCABTACHGGTGCTCGKGC

[0035] Nd

[0036] Primer 2 sequence: CCC AAGCTT TCCNGCNTGCTTBKCGNAAAATKCGAGA

[0037] Hind III

[0038] steps

[0039] 3

[0040] Use 0.7% agarose gel to separate the PCR product, reclaim the DNA fragment (as shown in Figure 1) of 750bp from the gel, be called for short the BSEST gene, connect with the pMD18-T carrier, the gained plasmid is called pBSEST-T, and the connected product transforms Competent Escherichia coli DH5a. Plasmid pBSEST-T was analyzed by HindIII and NdeI double enzyme digestion, which prove...

Embodiment 2

[0041] Embodiment 2: Construction of expression vector and transformant

[0042] After the pBSEST-T plasmid and pET22b(+) were digested with NdeI and HindIII respectively, the digested products were separated with 0.7% agarose gel, and the 750kbp and 4.8kbp DNA fragments were recovered from the gel, and connected with T4 DNA ligase , the obtained plasmid is called pBSEST-PET plasmid, and the ligation product is transformed into competent Escherichia coli E.coli BL21 (DE3). The positive clones are screened by resistance medium and identified by HindIII and NdeI double digestion analysis, which proves that they contain the correct The insert fragment (as shown in FIG. 3 ), thereby obtaining a transformant-a genetically engineered strain E.coliBL21(DE3) / pBSEST-PET CGMCC No.1648 expressing the esterase of the present invention.

[0043] The process of Embodiments 1-2 is shown in Figure 6.

Embodiment 3

[0044] Example 3: Expression of Esterhydrolase

[0045] The genetically engineered strain E.coli BL21(DE3) / pBSEST-PET CGMCC No.1648 was inoculated in 5ml of LB medium containing 50ug / ml ampicillin, and cultured overnight at 37°C with shaking. Take 50ul culture solution and connect to 5ml fresh LB medium containing 50ug / ml ampicillin, culture to OD 600 =0.6 or so, add IPTG until its concentration is 1mmol / L for induction, and continue to cultivate for 4h. The bacteria were collected by centrifugation, and the enzyme activity was detected with 1 mmol / L flurbiprofen ethyl ester as the substrate, and the enzyme activity was 20 U / g dry bacteria.

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Abstract

The invention discloses a Bacillus sp. Code with R-isomer stereoselectivity of esterase gene BSEST, which provides BSEST sequence and amino acid sequence, the expression carrier Pbsest-pet with BSEST and the gene engineering bacterial with the carrier(E. coli BL21(DE3) / pBSEST-PET CGMCC No. 1648), and also provides the recombinant method of the esterase. The recombinant esterase can apply to the synthesis of stereoselectivity catalytic chirality compounds(R shape).

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to an ester hydrolase with R-isomer stereoselectivity and its gene and recombinant enzyme, including an expression vector of the ester hydrolase gene, a method for preparing its recombinant enzyme and its genetic engineering Bacterial strain, and the application of the ester hydrolase in the synthesis of stereoselectively catalyzed chiral compounds (R type). Background technique [0002] Ester hydrolases include lipase and esterase, which are a class of biocatalysts with important uses in chiral synthesis. These enzymes can recognize a wide range of substrates. At present, more than 40% of biological object synthesis reactions can be catalyzed by ester hydrolases. These reactions have the characteristics of mild conditions, high regioselectivity and high stereoselectivity. It has been widely used in enzymatic kinetic resolution of racemic esters, amines and conversion of prechiral alcoh...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N9/16C12N15/63C12N1/21C12P41/00
Inventor 陈少欣白骅史炳照林剑秋钱莉莉
Owner SHANGHAI INST OF PHARMA IND CO LTD
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