Epoxide hydrolase and preparation method thereof

A technology of epoxide and hydrolase, which is applied in the field of genetic engineering, can solve problems such as temperature sensitivity, loss of activity, and enzyme instability, and achieve the effects of improving temperature stability, increasing service life, and high enzyme activity

Active Publication Date: 2011-01-12
HANGZHOU BIOKING BIOCHEM ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the enzyme is extremely unstable and is particularly sensitive to temperature. After being incubated at 35°C for 30 minutes, the enzyme activity is only 60% ...

Method used

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  • Epoxide hydrolase and preparation method thereof
  • Epoxide hydrolase and preparation method thereof
  • Epoxide hydrolase and preparation method thereof

Examples

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Effect test

Embodiment 1

[0041] Embodiment 1: Obtaining of Rhodococcus CGMCC1756 epoxide hydrolase gene

[0042] Rhodococcus CGMCC1756 was inoculated into the seed medium (1% glucose, 0.5% peptone, 0.5% beef extract, 1% sodium chloride, pH7.5), and cultured with shaking at 30°C for 24h. Genomic DNA of Rhodococcus CGMCC1756 was extracted with EZ-10 Column Genomic DNA Extraction Kit (purchased from Bio Basic Inc.) according to the instructions. According to the nucleotide sequence (accession number DQ471957) of Rhodococcus opacus ML-0004 (Rhodococcus opacusML-0004) epoxide hydrolase disclosed in the U.S. GenBank database, two primers (i.e. primer 1 and primer 2) were designed, primer 1 and The nucleotide sequences of primer 2 are respectively shown in SEQ ID NO: 1 and SEQ ID NO: 2. Using the extracted Rhodococcus CGMCC1756 genomic DNA as a template, using primers 1 and 2, a DNA fragment of about 800 bp was obtained by a commonly used PCR amplification technique. The PCR reaction system is: 40 μl of do...

Embodiment 2

[0043] Example 2: Construction of an expression system for epoxide hydrolase from Rhodococcus CGMCC1756

[0044] According to the sequencing result of Rhodococcus CGMCC1756 epoxide hydrolase gene in embodiment 1, redesign two primers (being primer 3 and primer 4), the nucleotide sequence of primer 3 and primer 4 is respectively as SEQID NO:5 and Shown in SEQ ID NO:6. Primer 3 contains Nco I restriction endonuclease site, 6 polyhistidine sequences and initiation codon. Primer 4 contains a Bam HI restriction endonuclease site and a stop codon. Using the Rhodococcus CGMCC1756 genomic DNA extracted in Example 1 as a template, using primers 3 and 4, a nucleotide fragment of about 800 bp was obtained by PCR amplification. The PCR reaction system is: 40 μl of double distilled water, 5 μl of 10×PCR buffer, 1 μl of 10 mmol / L dNTPs, 1 μl of 10 mmol / L primer 3, 1 μl of 10 mmol / L primer 4, 1 μl of Rhodococcus CGMCC1756 genomic DNA, 1 μl of Taq enzyme, A total of 50 μl of the reaction s...

Embodiment 3

[0046] Embodiment 3: Site-directed mutation of Rhodococcus CGMCC1756 epoxide hydrolase gene

[0047] The 163rd tyrosine (Tyr, corresponding codon is TAC) of the main amino acid sequence of Rhodococcus CGMCC1756 epoxide hydrolase is site-directed mutation to phenylalanine (Phe, corresponding codon is TTC or TTT). TTC and TTT are synonymous codons, and they all encode phenylalanine. The present invention uses the TTC codon as an example to design mutation primers (i.e., primer 5 and primer 6). The nucleotide sequences of primer 5 and primer 6 are as follows: Shown in SEQ ID NO: 7 and SEQ ID NO: 8. PCR amplification was performed using the recombinant vector pTrc99A-EH in Example 2 as a template, and primers 5 and 6 as mutation primers. The PCR reaction system is: 40 μl of double distilled water, 5 μl of 10×PCR buffer, 1 μl of 10 mmol / L dNTPs, 1 μl of 10 mmol / L primer 5, 1 μl of 10 mmol / L primer 6, 1 μl of recombinant vector pTrc99A-EH, 1 μl of Taq enzyme, A total of 50 μl of t...

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Abstract

The invention discloses an epoxide hydrolase and a preparation method thereof. The amino acid of the 163rd site of the main body amino acid sequence of the epoxide hydrolase is phenylalanine, the main body amino acid sequence of the epoxide hydrolase is SEQ ID No: 10, and the epoxide hydrolase is coded by DNA molecules (expressed as SEQ ID No: 9) of the main body amino acid sequence. The preparation method for the epoxide hydrolase comprises that: tyrosine of the 163rd site of the main body amino acid sequence of the wild epoxide hydrolase is substituted by using the phenylalanine, the main body amino acid sequence of the wild epoxide hydrolase is SEQ ID No: 4, and the wild epoxide hydrolase is coded by DNA molecules (expressed as SEQ ID No: 3) of the main body amino acid sequence. Compared with the wild epoxide hydrolase, the epoxide hydrolase has high temperature stability and maintains high enzyme activity, can be used for hydrolyzing cis-epoxysuccinic acid or L (+)-tartaric acid or salt thereof, and prolongs the service life of a biocatalyst in the industrialized production.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to an epoxide hydrolase obtained through site-directed mutation. Background technique [0002] Epoxide hydrolase (EH, EC 3.3.2.3) is a general term for a class of enzymes that catalyze the hydrolysis of epoxides or their salts into corresponding adjacent diols or their salts. The catalytic reaction does not require any coenzymes, prosthetic groups or metals Participation of ions. Epoxide hydrolases are widely distributed in mammals, plants and microorganisms. At present, most reports focus on epoxide hydrolases in mammals, mainly studying its role in cell detoxification and metabolism of important biologically active substances. In recent years, epoxide hydrolases derived from microorganisms have attracted much attention because of their good stereospecificity, fast enzymatic reaction, high optical purity and yield of products, and easy separation and purification of products. In 1...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N9/14C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10
Inventor 张建国鲍文娜潘海峰谢志鹏
Owner HANGZHOU BIOKING BIOCHEM ENG
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