Process for the preparation of immobilized recombinant penicillin acylase catalyst from Achromobacter sp. CCM 4824 expressed in E. coli BL 21 CCM 7394 and its use for the synthesis of beta-lactam antibiotics

A BL21CCM7394, penicillin acylase biological technology, applied in the field of penicillin acylase, can solve the problems of increasing time and cost, hindering the use, etc.

Active Publication Date: 2010-08-11
FERMENTA BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Usually covalently immobilized, the target of the enzyme must be fully purified, which increases time and cost
In addition, specific characteristics of carrier materials need to be investigated to predict suitability for a particular protein, as these carriers tend to alter the protein's secondary, tertiary, or quaternary structure, hindering its subsequent specific use
[0011] This also leads to the possibility of detecting other non-covalently anchored forms

Method used

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  • Process for the preparation of immobilized recombinant penicillin acylase catalyst from Achromobacter sp. CCM 4824 expressed in E. coli BL 21 CCM 7394 and its use for the synthesis of beta-lactam antibiotics
  • Process for the preparation of immobilized recombinant penicillin acylase catalyst from Achromobacter sp. CCM 4824 expressed in E. coli BL 21 CCM 7394 and its use for the synthesis of beta-lactam antibiotics
  • Process for the preparation of immobilized recombinant penicillin acylase catalyst from Achromobacter sp. CCM 4824 expressed in E. coli BL 21 CCM 7394 and its use for the synthesis of beta-lactam antibiotics

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0122] Disintegration of cells under high pressure homogenization

[0123] Escherichia coli BL21CCM7394 was obtained from the culture medium by centrifugation as a cytoplasm (volume centrifuge) or as a cell suspension (alpha centrifuge) with the activity of the penicillin acylase obtained from Achromobacter sp. CCM4824. The microorganisms obtained were suspended in 0.02M sodium phosphate to give 5% (cdw / v), the pH of the suspension was adjusted to 7 and kept constant with 20% w / v sodium hydroxide. The cell suspension (2350ml, 4.9% cdw / v, AHPenGof 86.5U / ml) cooled to 8-13°C started to rupture. (uniform, pressure 40-50MPA, three consecutive cycles). After each cycle, the temperature of the homogeneous cell solution was lowered to 8-13 °C and the pH was adjusted to 7.0. Cellular debris and a small fraction of protein debris were removed by thermal coagulation, which involved adjusting the pH to 5.0 with 50% acetic acid, adding Sedipur CL 930 flocculant (2-4 g / L) and maintaining...

Embodiment 2

[0125] A. Extraction of Enzymes by Mild Chemical Extraction

[0126] In another specific embodiment of the invention, the cell suspension (1050ml, 5% cdw / v, AHpenG 10.2U / ml) was diluted with 0.1M sodium phosphate buffer (pH7.5) at 20°C. Gentle chemical lysis is performed in a three-necked glass reactor with pH electrodes, temperature probes and variable stirring devices. After adjusting the pH to 7.0 with dilute ammonia solution, the temperature reached 28°C. To dilute the biomass, 1% solvents such as toluene, ethyl acetate, chloroform, and butyl acetate were added to the initial cell lysis reaction, and the pH of the reaction was monitored at constant temperature. Samples were taken periodically to assess the extent of cell lysis. The reaction was continued for 2 hours at pH 7.0 and 28°C. The degree of lysis due to solubility was determined by analyzing the sample surface for AHpenG and compared to the initial activity of the cell suspension (% cell lysis, Table 1).

[01...

Embodiment 3

[0140] Preparation of concentrated enzymes

[0141] Prepare 350 gm of cell slurry as in Example 1 and dilute with distilled water to a final concentration of 5% (cdw / v) at 20-25°C. The pH of the cell suspension was adjusted to 7.0 with 2% (v / v) glacial chloroform, and 0.2% (w / v) sodium lauryl sulfate was added to the initial lysis. Samples were drawn periodically during the total time period of 5 hours. The lysed material was stored at 5°C for 12 hours prior to the protein isolation process from the lysed cells. The temperature of the suspension was raised to 28-30°C and diluted 2-fold with distilled water. The pH was adjusted to 5.0 with 2M acetic acid. Acidic protein coagulum was separated from the suspension by adding 1% polyethyleneimine for one hour at 40°C. The flocs were separated from the solution in a centrifuge at 7000 rpm. The pH of the supernatant was adjusted to 7.5 with sodium tetrahydroxide solution, and the resulting solution was called soluble enzyme (SE)...

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Abstract

The present invention discloses isolation of Penicillin Acylase (PA) from Achromobacter sp CCM 4824 expressed in recombinant strain E. coli BL21 CCM 7394 bearing the recombinant plasmid pKXIP1 and processing of PA into biocatalyst useful for the industrial synthesis of antibiotics. More particularly the invention discloses a synthesis of semi-synthetic [beta]-lactam antibiotics in the reaction mixture consisting of activated acyl-donor (D-p-hydroxyphenylglycine methyl ester or amide for Amoxicillin and Cefadroxil; D-phenylglycine methyl ester or amide for Ampicillin and Cephalexin) and nucleophile (6-APA or 7-ADCA) catalyzed by PA obtained from recombinant E. coli BL21 CCM 7394 as the biocatalyst.

Description

【Technical field】 [0001] The invention relates to the penicillin acylase prepared from the achromobacter CCM4824 in recombinant Escherichia coli BL21CCM7394, and relates to the biocatalysis of the preparation of the recombinant plasmid pKXIP1 and the penicillin acylase in the industrial synthesis of antibiotics. In particular, it relates to semi-synthetic β-lactam antibiotics synthesized from reaction mixtures obtained from cefadroxil (preparation of hydroxyphenylmethyl esters or amino compounds of amoxicillin and cefadroxil) catalyzed by penicillin acylase ; Preparation of ampicillin and cephalexin (phenylglycine or amino compound) and nucleophile (6-APA or 7-ADCA) composition, penicillin acylase is derived from recombinant Escherichia coli BL21CCM7394 as a biocatalyst. 【Background technique】 [0002] Penicillin G acylase has been used commercially for decades to hydrolyze the penicillin G acyl group and its six different components, the acetoepoxycephalosporin G, for the p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P35/04C12P37/04
CPCC12P35/04C12P37/04
Inventor 阿奴帕马·达特拉威瑟瑞阿尼·威廉姆斯·瑞杰斯克帕维尔·尅斯里克斯坦尼斯拉伍·贝克阿夏尔·特瑞特·克瑞斯纳克德赞博瑞·苏加特·优格西克犹马·尼昆杰
Owner FERMENTA BIOTECH
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