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Penicillin acylated enzyme crystal and fixed penicillin acylated enzyme

A penicillin acylase, crystal technology, applied in the direction of immobilization on/in organic carriers, hydrolase, etc., can solve the problems of poor stability and insufficient stability, achieve enhanced mechanical strength, improve reaction speed and reaction specificity. One-size-fits-all, easy-to-use effects

Inactive Publication Date: 2009-06-17
SHANGHAI INST OF PHARMA IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although penicillin acylase immobilized by traditional methods has been widely used, it still has insufficient stability, especially in extreme pH, high temperature, and poor stability in organic solvents, thus limiting its application.

Method used

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  • Penicillin acylated enzyme crystal and fixed penicillin acylated enzyme
  • Penicillin acylated enzyme crystal and fixed penicillin acylated enzyme
  • Penicillin acylated enzyme crystal and fixed penicillin acylated enzyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Preparation of penicillin acylase crystals

[0028] (1) Preparation of Penicillin Acylase Purification Solution

[0029] At room temperature, the solution-state penicillin acylase (purchased from Zhejiang Haider Company) was first purified, and the penicillin acylase could be purified to More than 25u / mg, the penicillin acylase of this purity can be relatively easy to use the crystallization method to obtain crystals. After purification, store in the protein stock solution in the refrigerator at 4°C, centrifuge at 12000rpm for 10min at 4°C to remove denatured protein and impurity precipitates, filter the supernatant with a 0.22μm membrane, and determine the protein concentration by Lowry method: 105mg / ml;

[0030] (2) Preparation of penicillin acylase crystals

[0031] Take 1.14ml of protein stock solution, add 7.06ml of pH6.0, 0.1mol / L phosphate buffered saline (PBS), slowly add 1.8ml of 50(w / v)% PEG-8000 stock solution under stirring at 100rpm, and use 6mo...

Embodiment 2

[0032] Example 2 Preparation of penicillin acylase crystals

[0033] According to the method and steps of Example 1, the precipitant PEG-8000 is replaced with PEG-4000, so that the protein concentration of the crystallization mother liquor is 8 mg / ml, the PEG concentration is 13%, the crystallization period is 3 days, and other conditions are the same, and about 50 μm is obtained. Penicillin acylase crystals.

Embodiment 3

[0034] Example 3 Preparation of penicillin acylase crystals

[0035] According to the method and steps of Example 1, the protein concentration of the crystallization mother solution was 10 mg / ml, the concentration of PEG-8000 was 12%, and other conditions were the same, and penicillin acylase crystals of about 100 μm were obtained.

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Abstract

The invention published a kind of penicillin acylatease crystals and the immobilized penicillin acylatease.You can get them by the following method: first put the purified penicillin acylatease solution in buffer solution in order to make them crystal.The purified penicillin acylatease solution contains 8~12mg / ml protein. The rate of purified protein is over 25u / mg. It contains precipitation reagent called polyethylene glycol in buffer solution and its pH is 6.0~7.0. The polyethylene glycol occupied 9~13% probability of the buffer solution, weight in volume.Then put the above-mentioned penicillin acylatease crystals into buffer system which contains double fuction cross-linking agent.The immobilized penicillin acylatease was got after the cross linking reaction.There are many characters of the immobilized penicillin acylatease :It is insensitive to temperature ,acid and alkali, organic solvent and proteinase.It also has strong mechanical robustness ,high enzyme content and good purity. The immobilized penicillin acylatease can be used very convenient and the shape of the granule is regular.

Description

technical field [0001] The invention relates to a penicillin acylase crystal and immobilized penicillin acylase. Background technique [0002] The production of semi-synthetic β-lactam antibiotics mainly includes chemical and enzymatic methods. The production of domestic semi-synthetic β-lactam antibiotics mainly adopts chemical methods, through 6-APA (6-aminopenicillin alkanoic acid), 7-ADCA (7-aminodeacetoxycephalosporanic acid), 7-ACA (7 -Aminocephalosporanic acid) and other acyl acceptors are synthesized by chemical acylation of acyl donors. The chemical synthesis steps are cumbersome and need to be carried out in highly toxic organic solvents such as dichloromethane. Compared with chemical methods, enzymatic synthesis of β-lactam antibiotics has many advantages: 1. The acyl donor does not need to be activated by the carboxyl group with a very toxic reagent, or only needs milder conditions, such as in the form of acid or ester ; 2, the specificity of the enzyme-cataly...

Claims

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Application Information

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IPC IPC(8): C12N9/78C12N11/02
Inventor 严惠敏蒋建华张蓓蕾
Owner SHANGHAI INST OF PHARMA IND