Method for purifying chymotrypsin through affinity chromatography and stepwise elution

A technology of chymotrypsin and segmented elution, applied in biochemical equipment and methods, enzymes, peptidases, etc., can solve problems such as low specificity, and achieve the effect of saving costs and improving economic benefits

Active Publication Date: 2014-03-26
QINGDAO KANGYUAN PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Chymotrypsin, also known as chymotrypsin, has a molecular weight of 42,000 Daltons. It is used for the enzymatic hydrolysis of peptide chains. It

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] 1) Multiple crystallization

[0045] 1.1) Crystal I

[0046] Put 30kg of chymotrypsinogen into the reaction tank, add 210L of purified water and stir to dissolve, add 500mL of 2.5mol / L sulfuric acid and stir for 8 hours, then adjust the pH value to 2.5 with 2.5mol / L sulfuric acid; add 60L of saturated ammonium sulfate solution, stir For 15 minutes, adjust the pH value to 4.5 with 5mol / L NaOH, place it at 25°C, and filter after 48 hours at a constant temperature to collect 23.6kg of filter cake;

[0047] 1.2) Crystal II

[0048] Put the obtained filter cake into the reaction tank, add 70.8L purified water and stir to dissolve the filter cake, adjust the pH value to 3.0 with 2.5mol / L sulfuric acid; add 23.6L saturated ammonium sulfate solution, stir for 15 minutes, adjust with 5mol / L NaOH When the pH value reaches 5.0, put it under the condition of 25°C, keep the temperature for 24 hours, and then filter, and collect 20.5kg of filter cake;

[0049] 1.3) Crystal III

...

Embodiment 2

[0077] 1) Multiple crystallization

[0078] 1.1) Crystal I

[0079] Put 28kg of chymotrypsinogen into the reaction tank, add 196L of purified water and stir to dissolve, add 2.5mol / L sulfuric acid 500mL and stir for 8 hours, then adjust the pH value to 2.5 with 2.5mol / L sulfuric acid; add 56L of saturated ammonium sulfate solution, stir After 15 minutes, adjust the pH value to 4.5 with 5mol / L NaOH, place it at 25°C, and filter after 48 hours at a constant temperature to collect 23.2kg of filter cake;

[0080] 1.2) Crystal II

[0081] Put the obtained filter cake into the reaction tank, add 69.6L purified water and stir to dissolve the filter cake, adjust the pH value to 3.0 with 2.5mol / L sulfuric acid; add 23.2L saturated ammonium sulfate solution, stir for 15 minutes, adjust the pH with 5mol / L NaOH value to 5.0, put it under the condition of 25 ℃, filter after constant temperature for 24 hours, and collect 20.2kg of filter cake;

[0082] 1.3) Crystal III

[0083] Put the ...

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Abstract

The invention discloses a method for purifying chymotrypsin through affinity chromatography and stepwise elution. The method comprises the process steps as follows: (1) multiple crystallization; (2) activation; (3) salting out; (4) ultra-filtration; (5) pyrogen treatment; (6) dialysis; (7) affinity chromatography; and (8) freeze-drying and preparation of the chymotrypsin. According to the method, the production technology of the chymotrypsin is continuously improved, technological parameters are studied repeatedly through experiments and optimized continuously in particular, a more scientific large-scale production technology is established finally, the titer of the chymotrypsin purified through affinity chromatography is up to 1500-1800 iu/mg, and all indicators conform to regulations of Chinese Pharmacopoeia.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for purifying chymotrypsin by using multiple crystallization, activation, salting out, dialysis, affinity chromatography and the like. Background technique [0002] Chymotrypsin, also known as chymotrypsin, has a molecular weight of 42,000 Daltons. It is used for the enzymatic hydrolysis of peptide chains. It specifically hydrolyzes peptide bonds. It belongs to endopeptidases and has a high hydrolysis yield, but its specificity is lower than that of trypsin. . Its solid state is relatively stable, showing white rod-like crystals; while its aqueous solution is extremely unstable, when the pH is 5-8, its activity is the strongest, and it is also most likely to lose its activity. [0003] Chymotrypsin is a proteolytic enzyme secreted by the pancreas, which can quickly decompose denatured proteins. Its function and use are similar to trypsin, but it has stronger decomposition a...

Claims

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Application Information

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IPC IPC(8): C12N9/76
CPCC12N9/6427C12Y304/21001
Inventor 刘乃山林晓磊葛翠凤刘翠珍
Owner QINGDAO KANGYUAN PHARMA
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