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Lysostaphin industrial purifying process

A lysostaphin enzyme and process technology, which is applied in the field of enzyme purification process, can solve the problems of unsatisfactory yield and purity, weak rigidity of chromatography medium, and the flow rate of chromatography process cannot be too fast, etc.

Active Publication Date: 2006-03-08
昆山博青生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 1987, Fudan University successfully expressed it in Bacillus sphaericus, and established a separation and purification method for the enzyme, which included anion chromatography using DEAE cellulose and cation chromatography using CM cellulose. Due to the weak rigidity of the chromatographic medium, the protein Due to the low load capacity, the flow rate of the chromatography process cannot be too fast, and only small-volume samples can be processed. It takes 4 days to process 10 liters of samples, and the yield and purity are not ideal, so it cannot meet the needs of industrial production.

Method used

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  • Lysostaphin industrial purifying process

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Ion exchange chromatography:

[0028] A chromatographic column containing 10 ml of strong cationic agarose gel (manufactured by Amersia Biotechnology Co., Ltd., trade name SP Sepharose BigBead (please provide specific standardized chemical name)) medium with a particle size of 100-300 μm, layer The diameter of the analysis column is 2.5 cm.

[0029] Sample: 200 ml culture supernatant of Bacillus sphaericus containing lysostaphin gene; containing 9750 units of lysostaphin.

[0030] Chromatography equipment: AKTA explorer 100 produced by Amersya Biotechnology Co., Ltd.;

[0031] Equilibrium and washing buffer: 15mmol / L phosphate buffer at pH6.5;

[0032] Elution buffer: 15mmol / L pH6.5 phosphate buffer containing 1mol / L NaCl.

[0033] Equilibration: Use 15mmol / L phosphate buffer solution with a pH of 6.5 to equilibrate the chromatographic column containing 10ml volume of SPSepharose BigBead medium, equilibrate 8 column volumes at a flow rate of 120cm per hour, and ensur...

Embodiment 2

[0042] Hydrophobic chromatography

[0043] A chromatographic column with 1 ml of agarose gel (produced by Amersia Biotechnology Co., Ltd., trade name Phenyl Sepharose 6 fast flow) medium with a particle size of 20-165 μm and a phenyl group, the diameter of the chromatographic column is 0.6 cm.

[0044] Sample: Treat 10 ml of eluate from ion exchange chromatography; contains 8550 units of lysostaphin.

[0045] Chromatography equipment: AKTA explorer 100 produced by Amersya Biotechnology Co., Ltd.;

[0046] Equilibrium buffer: a phosphate buffer containing 1.5mol / L sodium chloride and 50mmol / L glycerol with a pH of 6.5;

[0047] Washing buffer: phosphate buffer containing 1.8mol / L sodium chloride and 50mmol / L glycerol with a pH of 6.5;

[0048] Elution buffer: phosphate buffer containing 50mmol / L glycerol and pH 6.5;

[0049] Equilibration: equilibrate the chromatography column with 1ml volume of Phenyl Sepharose 6 fast flow medium with equilibration buffer, equilibrate 5 co...

Embodiment 3

[0058] Ion exchange chromatography:

[0059] A chromatographic column with 3000ml of SP Sepharose BigBead medium is installed, and the diameter of the chromatographic column is 15 cm.

[0060] Sample: 45 liters of culture supernatant of Bacillus sphaericus containing lysostaphin gene; containing 4.95 million units of lysostaphin.

[0061] Chromatography equipment: 6mm Manual Process SystemTC from Amersya Biotechnology Co., Ltd.;

[0062] Equilibrium and washing buffer: 15mmol / L phosphate buffer at pH6.5;

[0063] Elution buffer: 15mmol / L pH6.5 phosphate buffer containing 1mol / L NaCl.

[0064] Balance: Use 15mmol / L pH 6.5 phosphate buffer to balance the chromatographic column equipped with 3000 ml volume of SP Sepharose BigBead medium, balance 6 column volumes at a flow rate of 120 cm per hour, and ensure that the chromatographic column is complete by ultraviolet detection. Equilibrate to baseline.

[0065] Sample pretreatment: Dilute 45 liters of sample (culture supernatan...

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Abstract

This invention discloses a purification technology for lysostaphin industry, which cultures supernatant purified lysostaphins from sphere bacilli containing the lysostaphin gene. Said method includes ionic exchange and drain chromatograph and needs two days only to process 45L sample, the total recovery rate is 50%, the specific activity is greater than 1000V / mg proteins and the purity is over 90% displayed on the non-recovery SDS-PAGE electrophoresis result.

Description

technical field [0001] The invention relates to an enzyme purification process, in particular to an industrial purification process of lysostaphin. Background technique [0002] Lysostaphin (Lysostaphin) is an enzyme secreted by Staphylococus Simulans that can lyse Staphylococcus, and its action site is the glycine pentapeptide bridge in the cell wall of Staphylococcus. In 1987, Fudan University successfully expressed it in Bacillus sphaericus, and established a separation and purification method for the enzyme, which included anion chromatography using DEAE cellulose and cation chromatography using CM cellulose. Due to the weak rigidity of the chromatographic medium, the protein Due to the low load capacity, the flow rate of the chromatography process cannot be too fast, and only small-volume samples can be processed. It takes 4 days to process 10 liters of samples, and the yield and purity are not ideal, so it cannot meet the needs of industrial production. Contents of t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/36
Inventor 黄青山陆海荣
Owner 昆山博青生物科技有限公司
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