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Epoxide hydrolase mutant as well as gene and application of epoxide hydrolase mutant

An epoxide hydrolase and epoxide technology, applied in the field of bioengineering, can solve the problems of low catalytic activity and selectivity of naphthalene-based substrates, and achieve the effects of high enantioselectivity and high catalytic activity

Inactive Publication Date: 2013-04-03
EAST CHINA UNIV OF SCI & TECH +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Therefore, the technical problem to be solved by the present invention is to provide a kind of epoxy hydrolase mutant protein and gene thereof, aiming at the problem that the catalytic activity and selectivity of the currently found epoxy hydrolase to naphthalene base substrates are low. The recombinant expression vector and recombinant expression transformant containing the gene, the method for preparing the epoxyhydrolase mutant protein, the cell containing the epoxyhydrolase mutant protein, and the epoxyhydrolase mutant protein or expressing the epoxyhydrolase mutant protein Application of recombinant transformants of epoxyhydrolase mutant proteins as catalysts in catalyzing the hydrolysis and resolution of racemic epoxides to prepare optically pure chiral compounds

Method used

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  • Epoxide hydrolase mutant as well as gene and application of epoxide hydrolase mutant
  • Epoxide hydrolase mutant as well as gene and application of epoxide hydrolase mutant
  • Epoxide hydrolase mutant as well as gene and application of epoxide hydrolase mutant

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Embodiment 1

[0045] The preparation of embodiment 1 epoxyhydrolase mutant

[0046] Site-directed mutagenesis using II Site-Directed Mutagenesis Kit (Stratagene, Catalog #200522) described the protocol to operate. First design mutation primers containing mutation points, as shown in Table 1, including the 98th, 101st, 123rd, 128th, 132nd, 144th, 145th in the sequence shown in SEQ ID NO: 1 in the sequence table , 168, 169, 203, 206, 208, 219, 220, 221, 242 and 268 primers for introducing mutations, and saturation mutations at 128 and 145 (the 128th The phenylalanine is mutated to any of the 20 amino acids and the 145th methionine is mutated to any of the 20 amino acids and) two pairs of degenerate primers, the sequences of the above primers are shown in the sequence table Shown in SEQ ID NO:2~SEQ ID NO:43:

[0047] Table 1 Mutation primers of epoxyhydrolase

[0048]

[0049]

[0050] PCR reaction system (50μl): template 0.5~20ng, 5μl 10×KOD plus buffer, 5μl dNTP (each 2.0mM), 2μl ...

Embodiment 2

[0054] Example 2 Expression and Purification of Bacillus megaterium Epoxyhydrolase Mutant

[0055] 1. Expression of Epoxyhydrolase Mutants

[0056] The expression strain of the mutant obtained in Example 1 was inoculated in 1 L of LB liquid medium containing 50 μg / ml kanamycin, cultivated at 37° C. at 200 rpm until the OD600 reached 0.6 to 0.8, cooled to 16° C. and added with a final concentration of 0.4mM IPTG, continue to culture for 20h to induce expression. Collect bacteria by centrifugation at 6000×g for 10 minutes, resuspend in 50mM pH8.0 sodium phosphate buffer (containing 500mM NaCl and 5mM β-mercaptoethanol), homogenate under high pressure, and centrifuge at 30,000×g for 45 minutes, then take the supernatant to obtain Crude enzyme solution of epoxyhydrolase mutant protein. The obtained crude enzyme solution is freeze-dried to obtain the crude enzyme powder of the epoxyhydrolase mutant.

[0057] 2. Purification of Epoxyhydrolase Mutants

[0058] Use the Ni affinity...

Embodiment 3

[0065] The activity measurement of embodiment 3 epoxyhydrolase mutants

[0066] We use high performance liquid chromatography (HPLC) to separate the substrate and product in the reaction mixture, measure the decrease of substrate concentration and the increase of product concentration in the reaction system before and after the reaction, and determine the catalytic activity of epoxyhydrolase. One unit of enzyme activity is defined as the amount of enzyme required to hydrolyze 1 μmol of substrate per minute under the above reaction conditions. Specific activity is defined as the number of units of activity per milligram of protein.

[0067] The detection method of epoxyhydrolase activity is as follows: the total reaction system is 0.5ml, which contains 2mM substrate, co-solvent is 10% DMSO, 100mM phosphate buffer (pH7.0). In a 2ml EP tube, react at 30°C and 1000rpm. After preheating for 5 minutes, add enzyme solution that was also preheated at 30°C for 5 minutes. Take 100μl of...

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Abstract

The invention discloses an epoxide hydrolase mutant as well as a gene and an application of the epoxide hydrolase mutant. The epoxide hydrolase mutant is protein which is obtained by substituting one amino acid at one site or multiple sites of the 123th, 128th, 144th 145th, 168th, 219th and 221st amino acid of the protein with an amino acid sequence as shown in SEQ ID No: 1 in a sequence table and deriving from the protein with the amino acid sequence as shown in SEQ ID No: 1 and has epoxide hydrolase activity. Compared with wild type epoxide hydrolase, the epoxide hydrolase mutant provided by the invention can greatly improve hydrolytic activity and enantioselectivity of the racemization epoxide, and is particularly suitable for splitting and catalyzing the racemization naphthyl glycidyl ether to prepare (S)- naphthyl glycidyl ether and further synthesize racemic medicament (S)-propranolol. The hydrolase mutant has good industrial application prospect.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to an epoxy hydrolase mutant and its gene and application. Background technique [0002] Biocatalysis is a research field that intersects many disciplines such as organic chemistry, biochemistry, microbiology and process engineering. Biocatalyst has high-efficiency and specific catalytic activity, and its biggest advantage lies in unparalleled chemoselectivity and stereoselectivity. Under the action of enzymes, the optical purity of the product can be as high as 99% ee, which is particularly suitable for the synthesis of pharmaceutical intermediates. important. The reaction conditions of biocatalysis are very mild, suitable for the reaction at normal temperature and pressure, neutral pH and water phase, which is in line with the goals of industrial development such as "sustainable development", "green chemistry" and "environmentally friendly manufacturing". Bioc...

Claims

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Application Information

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IPC IPC(8): C12N9/14C12N15/55C12N15/63C12N1/21C12P41/00C12P17/16C12P17/02C12P17/10C12P7/22C12P13/00C12P17/12C12R1/11
Inventor 孔旭东许建和周佳海潘江
Owner EAST CHINA UNIV OF SCI & TECH
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