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Epoxide hydrolase

An epoxide, hydrolase technology, applied in hydrolase, microorganism, microorganism-based methods, etc., can solve the problem of not describing the amino acid sequence, etc.

Inactive Publication Date: 2001-01-10
普瑞图斯股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But the article did not describe the amino acid sequence of this enzyme and the possible nucleotide sequence encoding this enzyme

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1: Partial purification of epoxide hydrolase

[0070] strain

[0071] Rhodococcus rhodochrous LMGP-18079 was routinely maintained on agar slants containing 0.4% glucose, 0.4% yeast extract, 1% malt extract and 2% agar.

[0072] Determination of cis-epoxysuccinate hydrolase activity

[0073] Carry out the enzymatic reaction at 37°C in a system with a final volume of 9.3ml, which includes the following components: 0.7ml 0.1N Tris. HCl buffer (pH 8.0), 2.6ml 1.14% Triton-X100 solution, 5.0ml 30% sodium cis-epoxysuccinate solution and 1.0ml cell suspension. H 3 PO 4 The reaction was terminated by diluting the mixture 100-500 times with water acidified to pH 2.2.

[0074] L-tartaric acid formed during the reaction was determined by HPLC (flow rate between 400-600 μl / min, sample volume 20 μl) on a Vydac C18 column (Cat. No. 201HS3410) at room temperature. The solvent is the same as that used to dilute the sample. cis-epoxysuccinic acid and tartaric acid were de...

Embodiment 2

[0094] Embodiment 2: Determination of amino acid sequence of cis-epoxysuccinate hydrolase

[0095] After electrophoresis by SDS-PAGE and electroblotting on PVDF Immobilon-P membrane (Millipore), the amino-terminus of the protein was sequenced according to the general procedure. Sequencing was performed using a 477A protein automatic sequencer (Applied Biosystem) coupled to an HPLC 120A analyzer.

[0096] To determine the sequence of internal fragments, the protein was first digested with trypsin on the membrane. The resulting peptides were separated by reverse phase chromatography on HPLC, followed by amino-terminal sequencing as described above.

[0097] resulting in the following sequence:

[0098] Amino terminus: MQLNNANDNTQF SEQ ID NO 1

[0099] First internal peptide: SWPDVPSGLEQLR SEQ ID NO 2

[0100] Second internal peptide: RPLEYGPTGR SEQ ID NO 3

Embodiment 3

[0101] Example 3: Identification of cis-epoxysuccinate hydrolase gene

[0102] 1. Design oligonucleotide probes

[0103] According to the amino-terminal sequence of the protein (SEQ ID NO 1), a digoxigenine-labeled oligonucleotide was synthesized. The oligonucleotide sequence is as follows:

[0104] 5′AAYAAYGCNAAYGAYAAYAC 3′ SEQ ID NO 4

[0105] Y stands for C or T, and N stands for any one of the four bases.

[0106] 2. Hybridization of oligonucleotides with total DNA of Rhodococcus rhodochrous

[0107] 2.1 Isolation of genomic DNA

[0108] Rhodococcus rhodochrous LMGP-18079 strain was cultured overnight at 37°C in 200 ml LBroth. After centrifugation, the cells were washed at 80°C with TE buffer (Tris-HCl 10 mM, EDTA 1 mM, pH 8.0) for 30 minutes, centrifuged again, and resuspended in 15 ml of TSE buffer (Tris-HCl) containing 120 mg lysozyme. 50mM, sucrose 200mM, EDTA 1mM, pH8.0), and incubated at 37°C for 2 hours, then added 1.5ml of 250mM EDTA (pH8.0). The mixture w...

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Abstract

The present invention is related to an isolated and purified nucleotide sequence from microbial origin, encoding an epoxide hydrolase, the vector comprising said nucleotide sequence, the recombinant host cell transformed by said nucleotide sequence and the epoxide hydrolase amino acid sequence encoded by said nucleotide sequence and / or expressed by said recombinant host cell.

Description

field of invention [0001] The present invention relates to the nucleotide and amino acid sequences of epoxide hydrolases and their use in the enantiomeric hydrolysis of epoxides. Background of the invention [0002] epoxy [0003] Epoxides are used as chiral building blocks in the organic synthesis of refined compounds, especially enantiomerically pure compounds. They are reactive molecules because their rings can be easily opened to generate a variety of products. For this reason, they are precursors of important drugs and specialty chemicals. Some chemistries exist to prepare these molecules from optically active precursors, but there is still a lack of efficient asymmetric synthesis (involving asymmetry) and Methods of resolution (Journal of the American Chemical Society 102, P. 5974 (1980)). The use of biological reactions for epoxide synthesis has been studied (see, for example, the review by de Bont J.A.M., Tetrahedron: Asymmetry 4, p. 1331 (1993)). [0004] Other...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N1/19C12N1/21C12N9/14C12P7/46C12R1/01C12R1/865
CPCC12N9/14
Inventor T·道威里P·德斯里
Owner 普瑞图斯股份有限公司
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