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Method for performing crosslinking immobilized modification on lipase Novozyme 435 by using glutaraldehyde

A technology of glutaraldehyde cross-linking and lipase, which is applied in the direction of fixing on/in the organic carrier, can solve the problems of reduced enzyme recovery, enzyme molecule shedding, and impact on catalyst cost, and achieves the effect of improving stability

Active Publication Date: 2014-06-11
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since Novozyme 435 is an immobilized enzyme obtained by adsorption of Candida antarctica lipase B (lipase CAL-B) through a macroporous adsorption resin, enzyme molecules will fall off during the reaction process, resulting in a decrease in the amount of enzyme recovered , affecting the reuse effect of the enzyme, thereby affecting the catalyst cost of the bioseparation process

Method used

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  • Method for performing crosslinking immobilized modification on lipase Novozyme 435 by using glutaraldehyde
  • Method for performing crosslinking immobilized modification on lipase Novozyme 435 by using glutaraldehyde
  • Method for performing crosslinking immobilized modification on lipase Novozyme 435 by using glutaraldehyde

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1: Cross-linking modification of lipase Novozyme 435 by glutaraldehyde

[0026] Take three parts of 160mg lipase Novozyme 435 (Novo Nordisk, Bagsvard, Denmark) in parallel and place them in 50ml ground-mouth Erlenmeyer flasks. The joint reaction was carried out for 45 minutes. After the reaction was completed, it was filtered, washed and dried naturally.

Embodiment 2

[0027] Example 2: Determination of enzyme activity of "modified lipase Novozyme 435" after glutaraldehyde crosslinking

[0028] The cross-linked modified lipase obtained after drying in Example 1 was used for the conversion reaction, which was added to 10 mL of 1mol / L pH 7.2 potassium phosphate buffer, and then 5 g / L of the substrate 2,2- Ethyl dimethylcyclopropanecarboxylate undergoes asymmetric hydrolysis. The reaction conditions are as follows: the rotating speed of the shaker is 200 rpm, the conversion temperature is 30° C., and the conversion time is 2 h. After the reaction, the reaction solution was extracted with an equal volume of ethyl acetate, and the extract was analyzed by gas chromatography to measure the enzyme activity after cross-linking modification.

Embodiment 3

[0029] Example 3: Determination of yield and ee value of asymmetric hydrolysis reaction of ethyl 2,2-dimethylcyclopropanecarboxylate catalyzed by cross-linking modified lipase Novozyme 435

[0030] The cross-linked modified lipase obtained after drying in Example 1 was used for the conversion reaction, which was added to 10 mL of 1mol / L pH 7.2 potassium phosphate buffer, and then 10 g / L of the substrate 2,2- Ethyl dimethylcyclopropanecarboxylate undergoes asymmetric hydrolysis. The reaction conditions are as follows: the rotation speed of the shaker is 200 rpm, the conversion temperature is 30° C., and the conversion time is 64 h. After the reaction, each reaction solution was extracted with 2 times the volume of ethyl acetate, analyzed by gas chromatography, and the product yield and ee value were determined.

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Abstract

The invention provides a method for performing crosslinking immobilized modification on lipase Novozyme 435 by using glutaraldehyde. The method comprises the following steps: adding lipase Novozyme 435 with the dose of 10 to 20 g / L into a glutaraldehyde aqueous solution at the mass concentration of 0.5 to 10 percent; performing crosslinking reaction at the temperature of between 20 and 50 DEG C and at the rotating speed of 40 to 240 rpm for 15 to 120 minutes; after the reaction is finished, washing the unreacted glutaraldehyde completely by using distilled water; and drying to obtain the lipase Novozyme 435 subjected to crosslinking modification by the glutaraldehyde. The lipase Novozyme 435 is subjected to chemical crosslinking by the glutaraldehyde, so the stability of the lipase Novozyme 435 subjected to secondary modification is improved; according to investigation and the catalytic property of the lipase Novozyme 435 repeatedly used for catalyzing asymmetrical hydrolysis of 2,2-dimethyl cyclopropane ethyl formate for five times, after the lipase Novozyme 435 subjected to secondary modification is repeatedly used for five times, the yield is increased by 49.3 percent compared with that of the lipase Novozyme 435 not subjected to crosslinking modification.

Description

(1) Technical field [0001] The invention relates to a method for modifying lipase Novozyme 435 by cross-linking and immobilizing glutaraldehyde, and using the modified lipase Novozyme 435 to catalyze the asymmetric hydrolysis synthesis of racemic 2,2-dimethylcyclopropane ethyl formate The key chiral intermediate of cilastatin (S)-(+)-2,2-dimethylcyclopropanecarboxylic acid. (2) Background technology [0002] S-(+)-2,2-Dimethylcyclopropanecarboxylic acid is an important chiral intermediate in the synthesis of cilastatin. Cilsatatin (Cilsatatin) chemical name: (+)-(Z)-7-[(2R)-(2-amino-2-carboxyethyl)sulfur]-2-[(1S)-(2,2 -Dimethylcyclopropanecarboxamido)]-2-heptenoic acid, the first renal dehydrodipeptidase inhibitor to be used clinically. Compound agent Taineng made of cilastatin and carbapenem antibiotic imipenem (Imipenem) (Tienam) not only has strong broad-spectrum antibacterial activity, but also has β-lactamase inhibitory effect, and has strong antibacterial effect on...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/08
Inventor 王普何军邀黄金梁法勇
Owner ZHEJIANG UNIV OF TECH