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Carbonyl reductase with self-assembled interface, and application of same in synthesis of (R)-3-hydroxy-3-phenylpropionic acid ethyl ester

A technology of ethyl phenylpropionate and carbonyl reductase, applied in the directions of oxidoreductase, microorganism-based methods, enzymes, etc., can solve the problems of containing precious metals, low substrate processing capacity, high price, etc., and reduce the enzyme activity. loss, improving catalytic efficiency, and improving the effect of conversion

Active Publication Date: 2018-12-14
ZHEJIANG UNIV OF TECH
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

[0006] (R)-3-Hydroxy-3-phenylpropionic acid ethyl ester can be obtained by chemical asymmetric reduction of 3-carbonyl-3-phenylpropionic acid ethyl ester, which requires the preparation of a chemical catalyst with asymmetric reduction ability Catalysts often contain precious metals and are expensive
It is also possible to asymmetrically split 3-hydroxyl-3-phenylpropionate ethyl ester by chemical method or enzymatic method to prepare (R)-3-hydroxyl-3-phenylpropionate ethyl ester. The theoretical maximum conversion rate of this method is only Can reach 50%
The asymmetric reduction of ethyl 3-carbonyl-3-phenylpropionate by microorganism whole cells can also obtain ethyl (R)-3-hydroxyl-3-phenylpropionate, and the highest conversion rate can reach 100% in theory, but The throughput of the substrate is low and the production efficiency is not high

Method used

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  • Carbonyl reductase with self-assembled interface, and application of same in synthesis of (R)-3-hydroxy-3-phenylpropionic acid ethyl ester
  • Carbonyl reductase with self-assembled interface, and application of same in synthesis of (R)-3-hydroxy-3-phenylpropionic acid ethyl ester
  • Carbonyl reductase with self-assembled interface, and application of same in synthesis of (R)-3-hydroxy-3-phenylpropionic acid ethyl ester

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Screening and identification of Candida tropicalis CGMCC No.15016

[0026] 1. Strain screening process:

[0027] (1) culture medium

[0028] Enriched plate medium (g / L): Glucose 20, (NH 4 ) 2 SO 4 1, KH 2 PO 4 0.5,K 2 HPO 4 1.5, NaCl1, MgSO 4 0.1, agar 20, the solvent is deionized water, and the pH value is natural.

[0029] Slant Medium (g / L): Glucose 20, (NH 4 ) 2 SO 4 1, KH 2 PO 4 0.5,K 2 HPO 4 1.5, NaCl 1, MgSO 4 0.1, agar 20, the solvent is deionized water, and the pH value is natural.

[0030] Seed medium (g / L): glucose 20, yeast powder 3, (NH 4 ) 2 SO 4 1, KH 2 PO 4 0.5,K 2 HPO 4 1.5, NaCl1, MgSO 4 0.1, the solvent is deionized water, and the pH value is natural. The composition of the fermentation medium is the same as that of the seed medium.

[0031] (2) Culture method

[0032] Incline culture: culture at 30°C for 3-5 days.

[0033] Seed cultivation: transfer the cultivated slant into a 100mL Erlenmeyer flask f...

Embodiment 2

[0057] Embodiment 2: the preparation of carbonyl reductase

[0058] 1. Wet bacteria

[0059] (1) Inoculate Candida tropicalis CGMCC No.15016 into the slant medium and culture at 30°C for 3-5 days to obtain slant bacteria; slant medium composition: glucose 20g / L, ammonium sulfate 1g / L, potassium dihydrogen phosphate 0.5g / L, dipotassium hydrogen phosphate 1.5g / L, sodium chloride 1g / L, magnesium sulfate 0.1g / L, agar 20g / L, the solvent is deionized water, and the pH value is natural.

[0060] (2) Inoculate the bacterium on the slope into a 100 mL Erlenmeyer flask filled with 25 mL of seed medium, and culture at 30° C. and 120 r / min for 24 hours to obtain seed liquid. Seed medium composition: glucose 46g / L, yeast juice 25g / L, potassium dihydrogen phosphate 4g / L, dipotassium hydrogen phosphate 4g / L, sodium chloride 0.1g / L, solvent is deionized water, pH value is natural.

[0061] (3) Put the seed solution cultivated for 24 hours into a 1000 mL Erlenmeyer flask filled with 250 mL ...

Embodiment 3

[0070] Example 3 Preparation and Application of Interfacial Self-Assembly Carbonyl Reductase

[0071] (1) Preparation of interfacial self-assembled carbonyl reductase

[0072] Add 5mg of carbonyl reductase enzyme solution (180U / mg) prepared in Example 2 into 6.25ml, pH 7.9, 0.05M Tris-HCl buffer solution to prepare 0.8g / L enzyme solution, then add 3.75g / L poly Styrene (Mw 10000Da) toluene solution 10ml was reacted for 1 hour in the dark at a shaker speed of 80r / min at 30°C. After the reaction is completed, centrifuge at 1200r / min for 10min, and the interfacial self-assembling enzyme is distributed on the two-phase interface of water and organic phase (that is, the upper layer is toluene, the middle is the interfacial self-assembling enzyme, and the lower layer is Tris-HCl buffer solution). The buffer and organic solvent were removed, and the middle layer was washed 3 times with toluene and pH 7.9, 0.05M Tris-HCl buffer solution successively to remove free carbonyl reductase a...

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Abstract

The invention discloses a carbonyl reductase with a self-assembled interface, and application of the same in synthesis of (R)-3-hydroxy-3-phenylpropionic acid ethyl ester. The carbonyl reductase withthe self-assembled interface is prepared by using the following steps: adding carbonyl reductase to a Tirs-HCl buffer, adding a toluene solution of polystyrene, shaking in a shaking bed for 1-4 hoursin the dark at 40-100 r / min and 20-40DEG C, and centrifuging to obtain a reaction system in which an organic phase is at the upper layer, the carbonyl reductase with the self-assembled interface is atthe middle layer, and an aqueous phase is at the lower layer; and washing the middle layer with toluene and Tirs-HCl buffer to obtain the carbonyl reductase with the self-assembled interface. The carbonyl reductase with the self-assembled interface, constructed in the invention, has the relatively good catalytic function at the interface between the organic phase and the aqueous phase, and can beused for converting 3-hydroxy-3-phenylpropionic acid ethyl ester to (R)-3-hydroxy-3-phenylpropionic acid ethyl ester, wherein the enantiomer excess value of (R)-3-hydroxy-3-phenylpropionicacidethylester is greater than 99%.

Description

(1) Technical field [0001] The invention relates to the preparation of a microbial-derived interface self-assembled carbonyl reductase and its use in the preparation of a non-central stimulant drug R-atomoxetine key chiral intermediate (R)-3-hydroxy-3-phenylpropane Application in ethyl acetate. (2) Background technology [0002] Interfacial self-assembly enzymes are polymer-enzyme complexes, which combine enzymes with macromolecular polymers to make the modified enzymes have an amphiphilic structure, which is similar to protein-based surfactants, containing both hydrophilic The protein head also contains a hydrophobic polymer tail. When it is placed in an oil-water system, the polymer part of the polymer-enzyme extends to the oil phase, while the enzyme part is embedded in the water phase, immobilizing the enzyme in the oil phase. - Catalyzes chemical reactions in the oil-water two-phase at the water interface. Combining carbonyl reductase and polymer at a suitable oil-wat...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12P7/62C12R1/74
CPCC12N9/0006C12P7/62C12Y101/01184
Inventor 欧志敏王莹泮佳莹杜理华
Owner ZHEJIANG UNIV OF TECH
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