Method for preparing (R)-phenylglycol (PED) from racemic styrene oxide (rac-SO) under catalytic actions of double enzymes

A technology of racemic styrene oxide and styrene glycol, which is applied in the field of biocatalysis, can solve the problems of low substrate solubility, catalytic substrate concentration, catalytic efficiency, low enantiomeric purity of product yield, and enzyme-to-substrate Specific effects and other issues, to achieve the effect of increasing concentration, large industrial application potential and economic value

Active Publication Date: 2016-12-21
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, although there are some methods related to the hydrolysis of styrene oxide substrates using double enzymes, there are generally defects such as catalytic substrate concentration, catalytic eff...

Method used

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  • Method for preparing (R)-phenylglycol (PED) from racemic styrene oxide (rac-SO) under catalytic actions of double enzymes
  • Method for preparing (R)-phenylglycol (PED) from racemic styrene oxide (rac-SO) under catalytic actions of double enzymes

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Experimental program
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Effect test

Embodiment 1

[0020] See GenBank: KR013755.1 for the nucleic acid sequence of the gene encoding VrEH3. The construction steps of E.coli / VrEH3 engineering bacteria expressing VrEH3 are as follows: synthesize the target gene, use pET-28a as the expression vector, and use E.coli BL21(DE3) as the expression host to construct the recombinant strain E.coli / VrEH3. Pick a single colony of E.coli / VrEH3 and culture it in 2mL LB medium at 37°C for 12h, then transfer it to fresh 100mL LB medium with an inoculum size of 2% (v / v), and culture it at 37°C After 2h, add the inducer IPTG to a final concentration of 0.05mM, incubate at 35°C for 10h, induce the high expression of VrEH3, collect the bacteria by centrifugation at 8000rpm, add 100mM, pH 7.0 phosphate buffer according to the ratio of 1g wet bacteria to 10mL buffer The suspension was prepared into a bacterial suspension with a bacterial concentration of 100 mg / mL and stored for later use.

[0021] For the amino acid sequence of AuEH2 and the const...

Embodiment 2

[0023] Determination of the specific enzyme activity of VrEH3 whole cells: Add 800 μL of VrEH3 bacterial suspension and 150 μL of potassium phosphate buffer (100 mM, pH=7.0) to a 1.5 mL EP tube, preheat at 25°C for 5 minutes; add 50 μL of 200 mM rac-SO After reacting for 15 minutes, 100 μL was extracted in 1 mL ethyl acetate containing (1 mg / mL n-hexanol as internal standard), dried over anhydrous magnesium sulfate, and passed through a 0.22 μm organic membrane.

[0024] AuH2 A250I Determination method of specific enzyme activity: add 33μLAuEH2 to 1.5mL EP tube A250I Bacteria suspension and 917 μL potassium phosphate buffer solution (100 mM, pH=7.0), preheated at 35°C for 2 min; add 50 μL 200 mM rac-SO, react for 15 min, take 100 μL in 1 mL ethyl acetate containing (1 mg / mL n-hexanol is internal standard) for extraction, dried over anhydrous magnesium sulfate, and passed through a 0.22 μm organic membrane.

[0025] The above-mentioned samples after organic membrane filtratio...

Embodiment 3

[0026]Embodiment 3 dual enzymes add sequence to conversion rate and product ee p Impact

[0027] The two enzymes were added to the catalytic system in sequence: in a 1mL reaction system, 400μl of VrEH3 whole-cell bacterial suspension was added to 517μl of 100mmol / L, pH 7.0 phosphate buffer, preheated at 25°C for 5min, and then added 50μl of rac-SO (final concentration was 10mmol / L L) Start the reaction, take regular samples for GC detection until the hydrolysis of (S)-SO is complete, then add 33 μl AuEH2 A250I Whole-cell bacterial suspensions were used to catalyze the remaining conformational substrate (R)-SO.

[0028] Two enzymes are added to the catalytic system at the same time: in a 1mL reaction system, 400μl VrEH3 and 33μl AuEH2 A250I The whole cell suspension was added to 517 μl of 100 mmol / L, pH 7.0 phosphate buffer, preheated at 25° C. for 5 min, and 50 μl of rac-SO (final concentration 10 mmol / L) was added to start the reaction. During the reaction process, samples...

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Abstract

The invention discloses a method for preparing (R)-phenylglycol (PED) from racemic styrene oxide (rac-SO) under catalytic actions of double enzymes, belonging to the technical field of biological catalysis. By using rac-SO as the substrate, double epoxide hydrolases (epoxide hydrolase mutant AuEH2A250I and epoxide hydrolase VrEH3) are utilized to perform enantioconvergent hydrolysis on the rac-SO, thereby preparing the high-enantiopurity product (R)-PED. The VrEH3 derived from mung beans and AuEH2A250I derived from Aspergillus usamii are combined to perform enantioconvergent hydrolysis on the rac-SO for the first time in the invention. The ee value for preparing (R)-PED by using catalyzing 10mM rac-SO is 96.0%. The method has ideal behaviors applicable to industrial application, lays theoretical foundation for industrialized production of enzymes, and has higher industrialized application potential and economic value.

Description

technical field [0001] The invention relates to a method for preparing (R)-phenylethylene glycol from racemic styrene oxide by double-enzyme catalysis, and belongs to the technical field of biocatalysis. Background technique [0002] Chiral epoxides and vicinal diols can react with a variety of nucleophiles, electrophiles, acids, and bases, and are a class of high-value-added multifunctional synthons or building blocks that can be used in pharmaceuticals and fine chemicals Synthesis of , pesticides and functional materials, such as leukotrienes, insect pheromones, steroids, β-adrenergic blocking agents, neuroprotective agents and HIV protease inhibitors, etc. [0003] Epoxide hydrolases (Epoxide hydrolases, EHs, EC 3.3.2.-) can specifically catalyze the hydrolytic kinetic resolution or enantionormalized hydrolysis of racemic epoxides to obtain enantiomerically pure epoxides or the corresponding vicinal diols. EHs is a potential biocatalyst due to its wide source, high enan...

Claims

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Application Information

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IPC IPC(8): C12P7/22C12R1/19
CPCC12P7/22
Inventor 邬敏辰王瑞胡蝶李闯宗讯成李剑芳
Owner JIANGNAN UNIV
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