Immobilized alpha-amino-acid ester hydrolase, preparation and application thereof

A technology of amino acid esters and hydrolytic enzymes, applied in hydrolytic enzymes, immobilized on/in organic carriers, biochemical equipment and methods, etc., can solve problems of desorption, poor thermal stability of immobilized enzymes, and heterogeneous biocatalysts Low activity and other problems, to achieve the effect of increasing activity yield and simplifying the preparation process

Inactive Publication Date: 2013-12-25
SICHUAN INDAL INST OF ANTIBIOTICS CHINA NAT PHARMA GROUP CORP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0025] 3. Low activity of heterogeneous biocatalysts as end products
[0026] 4. Since the enzyme is immobilized by adsorption, the operational stability of the biocatalyst is low, and it cannot be guaranteed that the combination of the enzyme and the carrier is multi-site, firm and irreversible, making the thermal stability of the immobilized enzyme poor. Desorption of enzymes and other proteins in the medium is susceptible to substrate (especially for high concentration substrate) solutions

Method used

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  • Immobilized alpha-amino-acid ester hydrolase, preparation and application thereof
  • Immobilized alpha-amino-acid ester hydrolase, preparation and application thereof
  • Immobilized alpha-amino-acid ester hydrolase, preparation and application thereof

Examples

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preparation example Construction

[0056] The preparation process of the heterogeneous biocatalyst is illustrated by the following examples and the usage of the enzyme product. In the following examples, the activity of the enzyme preparations is calculated relative to cephalexin synthesis. The initial velocity of target product formation was measured under the following conditions: pH6.0-6.2, 40±1°C, 7-ADCA concentration 0.040±0.002mole / l, Methyl Ester Phenyl Glycine (MEPhGl) 0.080±0.004mole / 1.

[0057] The amount of dry matter contained in the preparation is determined after drying the preparation to constant weight at 105±1°C.

Embodiment 1

[0058] Embodiment 1. prepare biocatalyst method with Xanthomonas rhodophylla bacterial strain VKPM B-9915

[0059] The same bacterial strain used in the present invention has been submitted to the "China Microbiological Culture Collection Management Committee General Microbiology Center (CGMCC)" and "All-Russia Industrial Microbiology Preservation Department (VKPM)" for preservation, and the bacterial strains given by CGMCC and VKPM have been obtained. No. CGMCC №.2339 and VKPM B-9915, the proposed taxonomic designations are Xanthomonas rubrilineans and Xanthomonas rubrilineans K40, respectively.

[0060] Get 273g (wet weight) Xanthomonas rhodoprint strain VKPMB-9915 cell biomass as the initiator of heterogeneous biocatalyst, its content is 18.5% (dry weight), synthase activity 83.3U / g (wet weight ), 450U / g (dry weight).

[0061] Put the cell biomass into a plastic container, freeze at -28°C for 3 hours, take out the frozen plastic container, let the cell biomass thaw at room...

Embodiment 2

[0067] Embodiment 2. the method for preparing biocatalyst with Xanthomonas rhodoprint bacterial strain CGMCC No. 2339

[0068] Get 91.2g (wet weight) Xanthomonas rhodoprint strain CGMCC No. 2339 cell biomass as the starting material of heterogeneous biocatalyst, its dry weight content 19.5%, synthase activity 70.2U / g (wet weight) , 360U / g (dry weight).

[0069] The cell biomass was treated according to the procedure in Example 1 and kept at -30°C for 5 hours.

[0070] The process of treating the cells with butyl acetate and extracting the enzyme into the aqueous phase is carried out according to the method of Example 1, the conditions are: the suspended cell biomass concentration is 50 mg (dry weight) / ml, the butyl acetate concentration is 4.0% by volume), 28 ° C , pH gradient, continuous stirring. The amount of reagents used: buffer 50ml, mercaptoethanol 50ml, butyl acetate 14ml, mixed to obtain 355ml of suspension. The enzyme extraction process takes 6 hours and 10 minute...

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Abstract

The invention discloses a heterogeneous biocatalyst taking alpha-amino-acid ester hydrolase as basis, a preparation method thereof and application in synthesizing amino beta-lactams. The activity of catalyst synthetase is 2,200 U / g by dry weight, and the content of protein is 0.95 g / g by dry weight; and xanthomonas rubrilineans cell biomass is processed in an organic solvent under a pH gradient through low temperature effect to extract enzyme, and enzyme aggregates are deposited and cross-linked to obtain the hydrolase. The alpha-amino-acid ester hydrolase as a catalyst can be synthesized into amino penicillin and amino cephalosporin drugs through acylating a beta-lactam compound by D-phenylglycine methyl ester derivatives in water or in a mixed medium of water and an organic solvent.

Description

field of invention [0001] The invention relates to a preparation method of a heterogeneous biocatalyst for enzymatically synthesizing aminocephalosporins and aminopenicillins. technical background [0002] α-amino acid ester hydrolases (AEHs) are a class of enzymes that can catalyze the hydrolysis of α-amino acid esters and the amide bonds of various cephalosporins and penicillins, and can also undergo α-amino acid derivatives ( ester or amide) for N-acylation of 7-aminocephalosporins and 6-aminopenicillanes. The similar substrate characteristics make this kind of enzymes can be used as biocatalysts (biocatalysts) for the synthesis of cephalosporins and penicillins (namely, aminocephalosporins and aminopenicillins) containing amino groups in the side chain α-position. [0003] The study on the structure of α-amino acid ester hydrolase shows that this type of enzyme belongs to α / β-fold hydrolase, which contains Ser-His-Asp three-way catalysis in the active site. combination...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/16C12N11/02C12P35/00C12P37/00C12R1/64
CPCY02P20/50
Inventor 熊辉斯克利亚仁科·安娜蒋用库偌什金娜·瓦连金娜王明蓉萨塔偌娃·热尼胡远辉克列斯季安洛瓦·伊琳娜
Owner SICHUAN INDAL INST OF ANTIBIOTICS CHINA NAT PHARMA GROUP CORP
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