CN101831417A

CN101831417A - Preparation method of L-asparaginase

Info

Publication number: CN101831417A
Application number: CN 201010176205
Authority: CN
Inventors: 明飞平; 黄乐天; 梁淑娃; 夏枫耿; 蓝碧峰; 谭颖嫦; 谢鹏
Original assignee: GUANGZHOU CITY INSTITUTE OF MICROBIOLOGY
Current assignee: GUANGZHOU CITY INSTITUTE OF MICROBIOLOGY
Priority date: 2010-05-11
Filing date: 2010-05-11
Publication date: 2010-09-15

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

A kind of preparation method of L-Asparaginase

Technical field

The present invention relates to the preparation field of L-Asparaginase, be specifically related to a kind of preparation method of simple, fast and efficient L-Asparaginase.

Background technology

L-Asparaginase (L-asparaginase, L-ASP), have another name called altheine enzyme or L-Asnase, commodity are called L-asparaginase, be mainly used in clinically at present the treatment children acute lymphoblastic leukaemia (childhood acute lymphoblastic leukemia, ALL).It is 60% that L-ASP uses the efficient of ALL separately, and during with vinealeucoblastine(VLB) (vincristine) and the medication combined uses such as (corticosteroids) of corticosteroid medicine, efficient up to 95% to ALL is the leukemic important drugs of clinical treatment.

L-ASP antineoplastic action mechanism is: reduce the concentration of interior altheine of human body and L-days glutamine, this two seed amino acid is the important component part of purine biosynthesis ring and pyrimidine ring.Tumour cell lacks asparagine synthetase, can not synthesize altheine, and cancer cells synthesizing ribonucleotide and proteinic ability will significantly reduce, and suppresses the tumor cell proliferation effect thereby reach.

The method of producing the L-Asparaginase all adopts microbial fermentation technology both at home and abroad, promptly obtains by the microorganism culturing technology to contain the proteic cell of L-Asparaginase in a large number, obtains highly purified L-amino-succinamic acid zymoprotein by extracting purifying then.The separation purifying technique of open report generally is: cell wall breaking---albumen precipitation---dialysis---affinity chromatography.The method of cell wall breaking mainly contains acetone broken wall method, osmotic shock method, multigelation method.The method of albumen precipitation mainly contains ammonium sulfate precipitation method and ethanol precipitation method.The above-mentioned several broken walls and the precipitator method or technology is loaded down with trivial details, inefficiency, or consumes energy, not environmental protection are difficult to realize the production of industrially scalable.

Summary of the invention

The objective of the invention is to the how not environmental protection of, inefficiency loaded down with trivial details, consumes energy, be difficult to realize the defective of suitability for industrialized production according to the technology that exists among the existing L-Asparaginase preparation method, provide a kind of simple, the efficient height, recovery rate can reach more than 60%, and the purity of the L-Asparaginase that makes reaches the preparation method of the L-Asparaginase more than 99%.

The object of the invention is achieved by the following technical programs:

A kind of preparation method of L-Asparaginase comprises the steps:

(1) gets the Erwinia thalline, add the SODIUM PHOSPHATE, MONOBASIC extract, regulate pH value to 4.8～5.2, through stirring, the centrifugal supernatant liquor of collecting;

(2), carry out adsorption chromatography by the cation-exchange chromatography post with supernatant liquid filtering;

(3) use buffer solution elution;

(4) elutriant is filtered, carry out adsorption chromatography by affinity column;

(5) use buffer solution elution, obtain the L-Asparaginase.

As a kind of preferred version, among the above-mentioned preparation method, in the step (1), described SODIUM PHOSPHATE, MONOBASIC extract is that lysozyme content is the phosphate sodium dihydrogen buffer solution of the 50mmol/L of 0.2mg/ml, and the pH value of adjusting most preferably is 5.0.

As a kind of preferred version, among the above-mentioned preparation method, in the step (1), the time of described stirring is 20～30min, and most preferred churning time is 25min.

As a kind of preferred version, among the above-mentioned preparation method, in the step (2), described filtration is through 1.2 μ m membrane filtrations, adsorbs in the cation-exchange chromatography post of velocity flow through being balanced of the supernatant liquor after the filtration with 50ml/min.

As a kind of preferred version, among the above-mentioned preparation method, in the step (2), described cation-exchange chromatography post is a CM-Mierocrystalline cellulose cation-exchange chromatography post.

As a kind of preferred version, among the above-mentioned preparation method, as follows with the buffer solution elution method described in the step (3): with pH is 7.0 50mmol/L phosphate sodium dihydrogen buffer solution washing cation-exchange chromatography post gel, to elutriant OD ₂₈₀＜0.1 o'clock, changing elutriant was that pH is 8.8 100mmol/L phosphate sodium dihydrogen buffer solution wash-out, collected OD ₂₈₀＞0.1 elutriant.

As a kind of preferred version, among the above-mentioned preparation method, in the step (4), described filtration is through 0.2 μ m membrane filtration, filters the back and adsorbs in the good affinity column of balance with the velocity flow of 50ml/min.

As a kind of preferred version, among the above-mentioned preparation method, in the step (4), described affinity column is the DEAE-Sephadex affinity column.

As a kind of preferred version, among the above-mentioned preparation method, as follows with the buffer solution elution method described in the step (5): with pH is 7.0 50mmol/L phosphate sodium dihydrogen buffer solution washing affinity column gel, to elutriant OD ₂₈₀＜0.1 o'clock, changing elutriant was that pH is 8.8 100mmol/L phosphate sodium dihydrogen buffer solution, and wash-out is collected OD ₂₈₀＞0.1 elutriant.

The L-Asparaginase for preparing by the inventive method detects 〉=99% through HPLC, reaches " the requirement of Chinese pharmacopoeia (2005 editions).

Compared with prior art, the present invention has following beneficial effect:

The preparation method of L-Asparaginase of the present invention is simple, step is simple, do not relate to hazardous agents such as inflammable and explosive, not only economy but also environmental protection, the purity of the L-Asparaginase that makes reaches more than 99%, reach that " requirement of Chinese pharmacopoeia (2005 editions) is applicable to large-scale L-Asparaginase suitability for industrialized production.

Embodiment

Further explain the present invention below in conjunction with embodiment, but embodiment does not do any type of qualification to the present invention.

1. get the thalline 3kg of the centrifugal collection of having fermented, add the SODIUM PHOSPHATE, MONOBASIC extract of its weight 30L, adjust pH is 5.0, mechanical stirring 25min, the centrifugal supernatant liquor of collecting;

2. the supernatant liquor that step 1 is collected is behind 1.2 μ M membrane filtrations, to adsorb in the CM-Mierocrystalline cellulose cation-exchange chromatography post of 50ml/min velocity flow through being balanced;

3. be 7.0 50mmol/L phosphate sodium dihydrogen buffer solution with 10L pH, washing step 2 chromatography column gels are to elutriant OD ₂₈₀＜0.1 o'clock, change elutriant and be pH and be the phosphate sodium dihydrogen buffer solution wash-out of 8.8 100mmol/L, collect OD ₂₈₀＞0.1 elutriant;

4. the elutriant that step 3 is collected filters the back and adsorbs in the good DEAE-Sephadex affinity column of balance with the 50ml/min velocity flow through 0.2 μ m membrane filtration;

5. be the phosphate sodium dihydrogen buffer solution of 7.0 50mmol/L with 10L pH, washing step 4 affinity column gels are to elutriant OD ₂₈₀＜0.1 o'clock, change elutriant and be pH and be the phosphate sodium dihydrogen buffer solution of 8.8 100mmol/L, wash-out is collected OD ₂₈₀＞0.1 elutriant promptly gets highly purified L-Asparaginase.

The ratio work of measuring the L-Asparaginase is 448 IU/ml, and the separation and purification yield is 61%, and purity is 99.5%.

Claims

1. the preparation method of a L-Asparaginase is characterized in that comprising the steps:

(3) use buffer solution elution;

(5) use buffer solution elution, obtain the L-Asparaginase.

2. the preparation method of L-Asparaginase according to claim 1 is characterized in that in the step (1), and described SODIUM PHOSPHATE, MONOBASIC extract is that lysozyme content is the phosphate sodium dihydrogen buffer solution of the 50mmol/L of 0.2mg/ml.

3. the preparation method of L-Asparaginase according to claim 1 is characterized in that in the step (1), the time of described stirring is 20～30min.

4. the preparation method of L-Asparaginase according to claim 1, it is characterized in that in the step (2), described filtration is through 1.2 μ m membrane filtrations, adsorbs in the cation-exchange chromatography post of velocity flow through being balanced of the supernatant liquor after the filtration with 50ml/min.

5. according to the preparation method of claim 1 or 4 described L-Asparaginases, it is characterized in that in the step (2) that described cation-exchange chromatography post is a CM-Mierocrystalline cellulose cation-exchange chromatography post.

6. the preparation method of L-Asparaginase according to claim 1, it is characterized in that described in the step (3) as follows with the buffer solution elution method: with pH is 7.0 50mmol/L phosphate sodium dihydrogen buffer solution washing cation-exchange chromatography post gel, to elutriant OD ₂₈₀＜0.1 o'clock, changing elutriant was that pH is 8.8 100mmol/L phosphate sodium dihydrogen buffer solution wash-out, collected OD ₂₈₀＞0.1 elutriant.

7. the preparation method of L-Asparaginase according to claim 1 is characterized in that in the step (4), and described filtration is through 0.2 μ m membrane filtration, filters the back and adsorbs in the good affinity column of balance with the velocity flow of 50ml/min.

8. according to the preparation method of claim 1 or 7 described L-Asparaginases, it is characterized in that in the step (4), described affinity column is the DEAE-Sephadex affinity column.

9. the preparation method of L-Asparaginase according to claim 1 is characterized in that described in the step (5) as follows with the buffer solution elution method: with pH is 7.0 50mmol/L phosphate sodium dihydrogen buffer solution washing affinity column gel, to elutriant OD ₂₈₀＜0.1 o'clock, changing elutriant was that pH is 8.8 100mmol/L phosphate sodium dihydrogen buffer solution, and wash-out is collected OD ₂₈₀＞0.1 elutriant.