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Method for catalyzing and splitting 2-aryl ethylformic acid antimer by using Serratieae fat enzyme

A technology of Serratia and arylpropionic acid, which is applied in the field of separation of 2-arylpropionic acid enantiomers, can solve the problems of increased catalyst cost, complicated treatment process, and failure to meet the high purity requirements of the pharmaceutical industry. To achieve the effect of reducing labor intensity and environmental pollution and reducing costs

Inactive Publication Date: 2007-10-31
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the process of this method is very complicated, the cost of the catalyst is greatly increased, which brings certain difficulties to industrial application.
[0005] Chinese patent ZL98121942.X directly uses the Lipase OF crude enzyme produced by Japan Mingtang Company as the catalyst of the enzymatic hydrolysis reaction for the resolution of racemic ketoprofen, although the reaction is carried out under high acidity conditions, it can be further Improve the optical purity of product (S)-ketoprofen, but the high acidity makes the stability of the enzyme decrease, and the use of commercial enzymes also increases the cost of industrial production
But enantioselectivity is not ideal, the content of (S)-configuration product is only slightly greater than 90%, can't reach the high purity requirement of pharmaceutical industry

Method used

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  • Method for catalyzing and splitting 2-aryl ethylformic acid antimer by using Serratieae fat enzyme
  • Method for catalyzing and splitting 2-aryl ethylformic acid antimer by using Serratieae fat enzyme
  • Method for catalyzing and splitting 2-aryl ethylformic acid antimer by using Serratieae fat enzyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Cultivate wild bacteria:

[0047] The wild Serratia S. marcescens ECU1010 (strain collection number: CGMCC No. 1219) was cultured in the fermentation medium, the culture time was 24 hours, the temperature was 30 ° C, and the shaking speed was 160 rpm, and then the supernatant was collected by centrifugation. 2000ml of enzyme solution, concentrated to 300ml by ultrafiltration, added 58.2 grams of ammonium sulfate until the saturation of ammonium sulfate was 35% to precipitate protein;

[0048] Using DEAE-Toyopearl 650M ion chromatography, then using Sephadex G150 gel chromatography, and then using Pheneyl-Toyopearl 650M hydrophobic chromatography to purify to obtain SDS electrophoresis pure protein, and HPLC detection shows that its purity is almost 100%, see figure 1.

[0049] The biochemical experiments of this enzyme show that the molecular weight of its subunit is 65kDa, as shown in Figure 2 lipase SDS-PAGE electrophoresis map; the isoelectric point is 4.2, as shown...

Embodiment 2

[0051] Extraction of S. marcescens ECU1010 lipase gene and acquisition of recombinant strain BL21(DE3) / pBCLipE1:

[0052] (1) Acquisition of S. marcescens ECU1010 lipase gene

[0053] The wild bacterium S. marcescens ECU1010 (strain collection number: CGMCC No. 1219) was cultivated, and the cultivation method was as follows: the solid plate was placed in an incubator at 30° C. for inverted cultivation;

[0054] The components and contents of said culture medium are:

[0055] Liquid medium (LM): (g / L) tryptone 10g, beef extract 5g, NaCl 5g, pH 7.0

[0056] Solid medium (SM): LM+1.5% (w / v) agar powder

[0057] All reagents used were domestic analytical grade.

[0058] Total bacterial DNA was extracted using standard methods (see Nucleic Acids Res. 1993, 21:2260-2263). Then, using the total DNA as a template, the amplification primers P1 and P2 of the lipase gene were designed according to the literature J Bacterial.

[0059] P1: 5' ccg cat acc aat aac gtt tca tca 3' (SEQ ID...

Embodiment 3～8

[0118] Using the lipase produced by the S.marcescens ECU1010 of Example 1 and the recombinant bacterium BL21(DE3) / pBCLipE1 of Example 2 as a biocatalyst, split the racemic ketoprofen esters of different carbon chain lengths to obtain optical Pure (S)-ketoprofen.

[0119] Reaction condition is: the enzyme liquid 1ml that embodiment 1 and 2 obtains, wherein contains the enzyme amount of 6U, adds 2.8mg racemic ketoprofen axetil, makes its final concentration be 10mM, then adds 50ul of DMSO (dimethyl methylene Sulfone) (final concentration is 5% (v / v)) to aid in dissolution, and reacted for 24 hours at 30° C. under the condition of 1000 rpm.

[0120] The obtained sample was acidified with 6mol / L HCl to pH = 1, then extracted with an equal volume of ethyl acetate, and then extracted with a chiral chromatographic column Chiracel OJ-H column (Daicel Japan, Φ0.45×25cm), and separated by n-hexane-iso Propanol-acetic acid (volume ratio is 90: 10: 0.2) is mobile phase (1ml / min), on high...

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Abstract

The invention discloses a method to resolve 2-aryl radical propanoic acid antimer with Serratieae lipase, which comprises the following steps: touching Serratieae lipase with racemic 2-aryl propionic ester; proceeding enzymatic reaction with temperature at 20-60 deg. c and pH value at 5-11; collecting (S)-2-aryl propionic acid and un-reacting (R)-2-aryl propionic acid; using the (R)-2-aryl propionic acid to enzymatic resolution repeatedly. This invention can leases labor strength and environmental contamination, which can be used to antiphlogistic anodyne firm.

Description

technical field [0001] The invention relates to a method for separating 2-arylpropionic acid enantiomers, in particular to a method for catalyzing and separating 2-arylpropionic acid enantiomers by using Serratia lipase. Background technique [0002] 2-Arylpropionic acid can be used in the preparation of important non-steroidal anti-inflammatory drugs such as ketoprofen and naproxen. These compounds are all chiral compounds, and the pharmacological activities of their different enantiomers are different. Dex-ketoprofen, or (S)-ketoprofen, is widely used in the pharmaceutical industry; and lev-ketoprofen, or (R)-ketoprofen Fen can be added to toothpaste for the prevention and treatment of osteoporosis. The (S)-(+)-enantiomer of naproxen is 28 times more active than the (R)-(-)-enantiomer. In order to improve drug efficacy, reduce drug side effects, expand drug safety range, reduce complications and correctly evaluate drugs, it is necessary to prepare optically pure single ...

Claims

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Application Information

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IPC IPC(8): C12P7/62C12P7/40C12P41/00C12R1/425
Inventor 许建和赵丽丽龙章德潘江
Owner EAST CHINA UNIV OF SCI & TECH
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