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Preparation method of L-asparaginase

An asparaginase and chromatography column technology, which is applied in the field of preparation of L-asparaginase, can solve the problems of difficulty in industrialized production, low efficiency, cumbersome process and the like, and achieves simple and easy steps and simple and easy preparation methods. row effect

Inactive Publication Date: 2010-09-15
广州市微生物研究所
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Problems solved by technology

[0005] The purpose of the present invention is to provide a simple, efficient, The yield can reach more than 60%, and the preparation method of L-asparaginase whose purity reaches more than 99%

Method used

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Examples

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Embodiment Construction

[0024] The present invention is further explained below in conjunction with the examples, but the examples do not limit the present invention in any form.

[0025] 1. Take 3 kg of bacteria collected by centrifugation after fermentation, add 30 L of sodium dihydrogen phosphate extract, adjust the pH to 5.0, stir mechanically for 25 minutes, and collect the supernatant by centrifugation;

[0026] 2. After filtering the supernatant collected in step 1 through a 1.2 μM filter membrane, flow through the balanced CM-cellulose cation exchange chromatography column at a speed of 50ml / min for adsorption;

[0027] 3. Use 10L of 50mmol / L sodium dihydrogen phosphate buffer solution with a pH of 7.0 to wash the chromatography column gel in step 2 to the eluent OD 280 When 280 >0.1 eluent;

[0028] 4. Filter the eluate collected in step 3 through a 0.2 μm membrane filter, and then flow through a well-balanced DEAE-Sephadex affinity chromatography column at a rate of 50 ml / min for adsorptio...

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Abstract

The invention discloses a preparation method of L-asparaginase. The preparation method comprises the following steps of: carrying out wall breaking on Erwinia thalli by using an extracting solution containing lysozyme; and obtaining high-purity L-asparaginase through ion-exchange chromatography and affinity chromatography. The preparation method of the L-asparaginase is simple and easy to apply and has high efficiency, the yield can reach higher than 60 percent, and the purity of the prepared L-asparaginase can is higher than 99 percent and meets the requirement of the Chinese Pharmacopoeia (2005 Version). The preparation method has no pollution to the environment, is economical and environmenta friendly and can be applied to industrial production.

Description

technical field [0001] The invention relates to the field of preparation of L-asparaginase, in particular to a simple, fast and efficient preparation method of L-asparaginase. Background technique [0002] L-asparaginase (L-asparaginase, L-ASP), also known as L-asparaginase or L-asparaginase, trade name L-asparaginase, is currently mainly used in clinical treatment of children Acute lymphoblastic leukemia (childhood acute lymphoblastic leukemia, ALL). L-ASP alone has an effective rate of 60% for ALL, and when used in combination with drugs such as vincristine and corticosteroids, the effective rate for ALL is as high as 95%. It is an important drug for clinical treatment of leukemia . [0003] The anti-tumor mechanism of L-ASP is to reduce the concentration of L-asparagine and L-asynamide in the human body. These two amino acids are important components for the synthesis of purine and pyrimidine rings. Tumor cells lack asparagine synthetase and cannot synthesize L-asparag...

Claims

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Application Information

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IPC IPC(8): C12N9/82C12R1/18
Inventor 明飞平黄乐天梁淑娃夏枫耿蓝碧峰谭颖嫦谢鹏
Owner 广州市微生物研究所
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