CN101643723A - Extraction method of trypsin-chymotrypsin - Google Patents

Extraction method of trypsin-chymotrypsin Download PDF

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Publication number
CN101643723A
CN101643723A CN200910170682A CN200910170682A CN101643723A CN 101643723 A CN101643723 A CN 101643723A CN 200910170682 A CN200910170682 A CN 200910170682A CN 200910170682 A CN200910170682 A CN 200910170682A CN 101643723 A CN101643723 A CN 101643723A
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chymotrypsin
trypsin
supernatant liquor
pancreas
minutes
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马忠仁
印明善
冯玉萍
李明生
蔡翠萍
乔自林
冯若飞
阮小华
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马忠仁
印明善
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Abstract

The invention relates to the field of biological engineering, in particular to an extraction method of trypsin-chymotrypsin. In the invention, CaCl2 is adopted to activate chymotrypsinogen into the trypsin-chymotrypsin, and simultaneously (NH4)2SO4 is added to generate CaSO4 precipitation and adsorb the trypsin-chymotrypsin; the white freeze-dried powder can be obtained directly through the stepsof elution, salting out, ultrafiltration and freeze-drying, with no need of preparing by the following steps: firstly salting out multiple crystallization joint dialysis and an organic solvent methodof alcohol and acetone and the like is adopted to prepare the trypsin-chymotrypsin crude product, and then the complex steps of CM-cellulose column chromatography and affinity chromatography and the like are carried out for purifying preparation. The extraction method in the invention is convenient and feasible, and is applicable to industrial production, thus saving cost and shortening time whichis shortened to 3 to 4 days from the original required 7 to 8 days. 4.0-4.5 grams of freeze-dried trypsin-chymotrypsin powder can be obtained from per kilogram of pancreas, wherein, the specific activity of chymotrypsin is 355 U./mg protein (nitrogen) and the specific activity of trypsin is 1361 U./mg protein (nitrogen).

Description

A kind of extracting method of Trypsin-chymotrypsin
Technical field
The present invention relates to bioengineering field, more specifically to a kind of extracting method of Trypsin-chymotrypsin.
Background technology
Chymotrypsin and trypsinase all are typical serine proteases, and the character of the two is very similar, be difficult for separately, but different to the specificity of proteolysis.Trypsinase only acts on the C-terminal peptide bond that carboxyl constituted by basic aminoacids L-arginine or Methionin, and Chymotrypsin is the peptide bond be made up of aromatics or aliphatic amino acid (as phenylalanine, tyrosine) of hydrolysis optionally then.Therefore, they the two have the effect of synergetic hydrolysis protein peptide bond, than being used alone the effective of enzyme, the scope of application is wider.At present they have been done a large amount of research both at home and abroad, and, be applied to clinical as a kind of medicine of anti-inflammation detumescence.Other purposes are for example: leather industry is sloughed the hair on the animal skin, textile industry sericin removal and caseinhydrolysate system lactoalbumin hydrolysate etc.Be to adopt the organic solvent method of multiple crystallization of saltouing from the pancreas extraction and separation method generally in conjunction with dialysis and ethanol and acetone etc. with them.Earlier they are prepared into the raw product of Trypsin-chymotrypsin, and then by purifying such as CM-cellulose chromatography and affinity chromatographys, freeze-drying obtains the higher Trypsin-chymotrypsin of purity, but because its preparation technology's flow process complexity, the technical requirements height, the cost costliness is difficult for being extensive use of.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of extracting method of Trypsin-chymotrypsin, adopts CaCl 2Activate the former Trypsin-chymotrypsin that becomes of Trypsin-chymotrypsin, when becoming Trypsin-chymotrypsin, add (NH 4) 2SO 4, make it generate CaSO 4Precipitation absorption Trypsin-chymotrypsin to simplify its technological process of production, shortens the cycle of subsequent purification and reduces cost.
The present invention has following sequential steps:
A, be raw material, remove fat and reticular tissue, clean, rub with mincer again with clear water with fresh bovine pancreas or Pancreas Sus domestica or freezing Pancreas Bovis seu Bubali or freezing Pancreas Sus domestica.
In b, the pancreas immigration stainless steel cask, add the tap water W/V of 3 times of volumes, H with 15% with rubbing 2SO 4Solution V/V, it is adjusted to pH value is 3.0, at room temperature constantly stirs 24 hours, uses 4000 rev/mins centrifugal 30 minutes of speed then, obtains supernatant liquor I and pancreas residue deposit I.
C, pour in the supernatant liquor bucket supernatant liquor I that obtains standby, being 3.0 tap water with pancreas residue deposit I pH value carried out agitator treating 20 minutes with the amount W/V of 1 times of volume, use 4000 rev/mins centrifugal 30 minutes of speed then, obtain supernatant liquor II and pancreas residue deposit II.
D, supernatant liquor II is poured in the supernatant liquor bucket, being 3.0 tap water with pancreas residue deposit II pH value carried out agitator treating 20 minutes with the amount W/V of 1 times of volume, use 4000 rev/mins centrifugal 30 minutes of speed then, obtain supernatant liquor III and pancreas residue deposit III, at last supernatant liquor III is poured in the supernatant liquor bucket, discard pancreas residue deposit III.
E, in the supernatant liquor bucket, add 25%～30% (NH 4) 2SO 4W/V stirs, left standstill 2 hours, and with 4000 rev/mins speed centrifugal 30 minutes, discard the foreign protein precipitation, obtained clear supernatant liquor.
F, in the clear supernatant liquor that obtains, add 70%～75% (NH 4) 2SO 4W/V, with 4000 rev/mins speed centrifugal 30 minutes, abandoning supernatant after the sedimentation obtained the former precipitation of Trypsin-chymotrypsin.
G, be behind 3.0 the deionized water dissolving at the former precipitation pH value of Trypsin-chymotrypsin that obtains, adding 4%～6%CaCl 2W/V stirs, and under 4 ℃～5 ℃, leaves standstill 10 hours, makes Ca ++The former Trypsin-chymotrypsin that becomes of ion-activated Trypsin-chymotrypsin; And then add 10%～15% (NH 4) 2SO 4W/V stirs, and heats to 37 ℃, makes Ca in the supernatant liquor ++Ion generates CaSO 4Precipitation (
Figure G200910170682XD00031
Figure G200910170682XD00032
), the Trypsin-chymotrypsin in the adsorbent solution, leave standstill 10 hours after, with 4000 rev/mins speed centrifugal 30 minutes, after the sedimentation, abandoning supernatant was collected CaSO 4Precipitation.
H, with the CaSO of deionized water to post precipitation 4Wash-out is three times repeatedly, merges together, adds 70%～75% (NH then 4) 2SO 4W/V stirs, and leaves standstill under 4 ℃ 10 hours, uses 4000 rev/mins centrifugal 30 minutes of speed again, abandoning supernatant, the Trypsin-chymotrypsin precipitation.With ultrafiltration behind the deionized water dissolving, lyophilize has obtained white Trypsin-chymotrypsin lyophilized powder at last.
Advantage of the present invention is: extraction and separation method is simple and feasible, is suitable for suitability for industrialized production, is using CaCl 2Activate when Trypsin-chymotrypsin is former to become Trypsin-chymotrypsin, add (NH 4) 2SO 4Make it generate CaSO 4Precipitation absorption Trypsin-chymotrypsin, separate with elastoser and pancreatin etc., do not need to adopt earlier the multiple crystallization of saltouing to be prepared into the raw product of Trypsin-chymotrypsin in conjunction with dialysis and ethanol and acetone and other organic solvent method, again by complex steps such as CM-cellulose chromatography and affinity chromatographys, and directly pass through wash-out, saltout, ultrafiltration and step of freeze drying get final product.Further separate Trypsin-chymotrypsin if desired, then can adopt the CM-Mierocrystalline cellulose that they are separated into Chymotrypsin and trypsinase, so not only provided cost savings but also shortened the time, by needing 7～8 day time shorten to 3～4 days approximately originally.The per kilogram pancreas can obtain freeze-drying Trypsin-chymotrypsin powder 4.0～4.5 grams, and wherein the ratio vigor of Chymotrypsin is 355U./mg albumen (nitrogen), and tryptic is 1361U./mg albumen (nitrogen) than vigor.
Embodiment
Embodiment 1
The present invention has following sequential steps:
A, to get 1 kilogram of fresh bovine pancreas be raw material, removes fat and reticular tissue, cleans with clear water, rubs with mincer again.
In b, the Pancreas Bovis seu Bubali immigration stainless steel cask, add 3000 milliliters tap water W/V, H with 15% with rubbing 2SO 4Solution V/V, it is adjusted to pH value is 3.0, at room temperature constantly stirs 24 hours, uses 4000 rev/mins centrifugal 30 minutes of speed then, obtains supernatant liquor I and pancreas residue deposit I.
C, 3000 milliliters of supernatant liquor I that will obtain pour in the supernatant liquor bucket standby, with pancreas residue deposit I pH value is that 1000 milliliters of W/V of tap water of 3.0 carried out agitator treating 20 minutes, use 4000 rev/mins centrifugal 30 minutes of speed then, obtain supernatant liquor II and pancreas residue deposit II.
D, supernatant liquor II is poured in the supernatant liquor bucket, with pancreas residue deposit II pH value is that 1000 milliliters of W/V of tap water of 3.0 carried out agitator treating 20 minutes, use 4000 rev/mins centrifugal 30 minutes of speed then, obtain supernatant liquor III and pancreas residue deposit III, at last supernatant liquor III is poured in the supernatant liquor bucket, discard pancreas residue deposit III, make his usefulness in addition, in order to avoid contaminate environment.
E, in the supernatant liquor bucket, add 25% (NH 4) 2SO 4W/V stirs, left standstill 2 hours, and with 4000 rev/mins speed centrifugal 30 minutes, discard the foreign protein precipitation, obtained clear supernatant liquor.
F, in the clear supernatant liquor that obtains, add 75% (NH 4) 2SO 4W/V, with 4000 rev/mins speed centrifugal 30 minutes, abandoning supernatant after the sedimentation obtained the former precipitation of Trypsin-chymotrypsin.
G, be after 200 milliliters of dissolvings of deionized water of 3.0 with the former precipitation pH value of Trypsin-chymotrypsin that obtains, adding 6%CaCl 2W/V stirs, and under 4 ℃～5 ℃, leaves standstill 10 hours, makes Ca ++The former Trypsin-chymotrypsin that becomes of ion-activated Trypsin-chymotrypsin; And then add 10% (NH 4) 2SO 4W/V stirs, and heats to 37 ℃, makes Ca in the supernatant liquor ++Ion generates CaSO 4Precipitation (
Figure G200910170682XD00041
Figure G200910170682XD00042
), the Trypsin-chymotrypsin in the adsorbent solution, leave standstill 10 hours after, with 4000 rev/mins speed centrifugal 30 minutes, after the sedimentation, abandoning supernatant was collected CaSO 4Precipitation.
H, with 400 milliliters of deionized waters to CaSO 4Precipitate repeatedly wash-out three times, merge together, add 75% (NH then 4) 2SO 4W/V stirs, and leaves standstill under 4 ℃ 10 hours, uses 4000 rev/mins centrifugal 30 minutes of speed again, abandoning supernatant, the Trypsin-chymotrypsin precipitation; With ultrafiltration behind the deionized water dissolving, lyophilize has obtained white Trypsin-chymotrypsin lyophilized powder at last.
Embodiment 2
The present invention has following sequential steps:
A, to get 2 kilograms of freezing Pancreas Bovis seu Bubalis be raw material, removes fat and reticular tissue, cleans with clear water, rubs with mincer again.
In b, the Pancreas Bovis seu Bubali immigration stainless steel cask, add 6000 milliliters tap water W/V, H with 15% with rubbing 2SO 4Solution V/V, it is adjusted to pH value is 3.0, at room temperature constantly stirs 24 hours, uses 4000 rev/mins centrifugal 30 minutes of speed then, obtains supernatant liquor I and Pancreas Bovis seu Bubali residue deposit I.
C, 6000 milliliters of supernatant liquor I that will obtain pour in the supernatant liquor bucket standby, with Pancreas Bovis seu Bubali residue deposit I pH value is that 2000 milliliters of W/V of tap water of 3.0 carried out agitator treating 20 minutes, use 4000 rev/mins centrifugal 30 minutes of speed then, obtain supernatant liquor II and Pancreas Bovis seu Bubali residue deposit II.
D, supernatant liquor II is poured in the supernatant liquor bucket, with Pancreas Bovis seu Bubali residue deposit II pH value is that 2000 milliliters of W/V of tap water of 3.0 carried out agitator treating 20 minutes, use 4000 rev/mins centrifugal 30 minutes of speed then, obtain supernatant liquor III and Pancreas Bovis seu Bubali residue deposit III; Then supernatant liquor III is poured in the supernatant liquor bucket, discard Pancreas Bovis seu Bubali residue deposit III, make his usefulness in addition, in order to avoid contaminate environment.
E, in the supernatant liquor bucket, add 30% (NH 4) 2SO 4W/V stirs, left standstill 2 hours, and with 4000 rev/mins speed centrifugal 30 minutes, discard the foreign protein precipitation, obtained clear supernatant liquor.
F, in the clear supernatant liquor that obtains, add 70% (NH 4) 2SO 4W/V stirs, and leaves standstill 2 hours, and with 4000 rev/mins speed centrifugal 30 minutes, abandoning supernatant after the sedimentation obtained the former precipitation of Trypsin-chymotrypsin.
G, be after 400 milliliters of dissolvings of deionized water of 3.0 with the former precipitation pH value of Trypsin-chymotrypsin that obtains, adding 4%CaCl 2W/V stirs, and under 4 ℃～5 ℃, leaves standstill 10 hours, makes Ca ++The former Trypsin-chymotrypsin that becomes of ion-activated Trypsin-chymotrypsin; And then add 15% (NH 4) 2SO 4W/V stirs, and heats to 37 ℃, makes Ca in the supernatant liquor ++Ion generates CaSO 4Precipitation (
Figure G200910170682XD00061
Figure G200910170682XD00062
), the Trypsin-chymotrypsin in the adsorbent solution, leave standstill 10 hours after, with 4000 rev/mins speed centrifugal 30 minutes, after the sedimentation, abandoning supernatant was collected CaSO 4Precipitation.
H, with 600 milliliters of deionized waters to CaSO 4Precipitate repeatedly wash-out three times, merge together, add 70% (NH then 4) 2SO 4W/V stirs, and leaves standstill under 4 ℃ 10 hours, uses 4000 rev/mins centrifugal 30 minutes of speed again, abandoning supernatant, the Trypsin-chymotrypsin precipitation.With ultrafiltration behind the deionized water dissolving, lyophilize, obtained white Trypsin-chymotrypsin lyophilized powder at last.
Embodiment 3
The present invention has following sequential steps:
A, to get 5 kilograms of fresh bovine pancreases be raw material, removes fat and reticular tissue, cleans with clear water, rubs with mincer again.
In b, the Pancreas Bovis seu Bubali immigration stainless steel cask, add 15,000 milliliters tap water W/V, H with 15% with rubbing 2SO 4Solution V/V, it is adjusted to pH value is 3.0, at room temperature constantly stirs 24 hours, uses 4000 rev/mins centrifugal 30 minutes of speed then, obtains supernatant liquor I and pancreas residue deposit I.
C, will obtain 15,000 milliliter of supernatant liquor I pours in the supernatant liquor bucket standby, with pancreas residue deposit I pH value is that 3000 milliliters of W/V of tap water of 3.0 carried out agitator treating 20 minutes, uses 4000 rev/mins centrifugal 30 minutes of speed then, obtains supernatant liquor II and pancreas residue deposit II.
D, supernatant liquor II is poured in the supernatant liquor bucket, with pancreas residue deposit II pH value is that 3000 milliliters of W/V of tap water of 3.0 carried out agitator treating 20 minutes, use 4000 rev/mins centrifugal 30 minutes of speed then, obtain supernatant liquor III and pancreas residue deposit III, at last supernatant liquor III is poured in the supernatant liquor bucket, discard pancreas residue precipitation III, make his usefulness in addition, in order to avoid contaminate environment.
In e, the supernatant liquor in the supernatant liquor bucket, add 25% (NH 4) 2SO 4W/V stirs, left standstill 2 hours, and with 4000 rev/mins speed centrifugal 30 minutes, discard the foreign protein precipitation, obtained clear supernatant liquor.
F, in the clear supernatant liquor that obtains, add 75% (NH 4) 2SO 4W/V, with 4000 rev/mins speed centrifugal 30 minutes, abandoning supernatant after the sedimentation obtained the former precipitation of Trypsin-chymotrypsin.
G, be after 1000 milliliters of dissolvings of deionized water of 3.0 with the former precipitation pH value of Trypsin-chymotrypsin that obtains, adding 6%CaCl 2W/V stirs, and under 4 ℃～5 ℃, leaves standstill 10 hours, makes Ca ++The former Trypsin-chymotrypsin that becomes of ion-activated Trypsin-chymotrypsin; And then add 10% (NH 4) 2SO 4W/V stirs, and heats to 37 ℃, makes Ca in the supernatant liquor ++Ion generates CaSO 4Precipitation (
Figure G200910170682XD00071
Figure G200910170682XD00072
), the Trypsin-chymotrypsin in the adsorbent solution, leave standstill 10 hours after, with 4000 rev/mins speed centrifugal 30 minutes, after the sedimentation, abandoning supernatant was collected CaSO 4Precipitation.
H, with 1000 milliliters of deionized waters to CaSO 4Precipitate repeatedly wash-out three times, merge together, add 75% (NH then 4) 2SO 4W/V stirs, and leaves standstill under 4 ℃ 10 hours, uses 4000 rev/mins centrifugal 30 minutes of speed again, abandoning supernatant, the trypsinase precipitation.With ultrafiltration behind the deionized water dissolving, lyophilize, obtained white Trypsin-chymotrypsin lyophilized powder at last.
Embodiment 4
Getting 1 kilogram of fresh pig pancreas is raw material, and the method for this embodiment is identical with embodiment 1, and different is that raw material is a Pancreas Sus domestica, has also obtained white Trypsin-chymotrypsin lyophilized powder at last.
In sum: the Trypsin-chymotrypsin that last lyophilize obtains is a kind of lyophilized powder of white, and is water-soluble, aqueous solution clear, but inactivation very easily at room temperature, and lyophilized powder is stored in about 4 ℃ more stable.

Claims (1)

1, a kind of extracting method of Trypsin-chymotrypsin, this method has following sequential steps:
A, be raw material, remove fat and reticular tissue, clean, rub with mincer again with clear water with fresh bovine pancreas or Pancreas Sus domestica or freezing Pancreas Bovis seu Bubali or freezing Pancreas Sus domestica;
In b, the pancreas immigration stainless steel cask, add the tap water W/V of 3 times of volumes, H with 15% with rubbing 2SO 4Solution V/V, it is adjusted to pH value is 3.0, at room temperature constantly stirs 24 hours, uses 4000 rev/mins centrifugal 30 minutes of speed then, obtains supernatant liquor I and pancreas residue deposit I;
C, pour in the supernatant liquor bucket supernatant liquor I that obtains standby, being 3.0 tap water with pancreas residue deposit I pH value carried out agitator treating 20 minutes with the amount W/V of 1 times of volume, use 4000 rev/mins centrifugal 30 minutes of speed then, obtain supernatant liquor II and pancreas residue deposit II;
D, supernatant liquor II is poured in the supernatant liquor bucket; Being 3.0 tap water with pancreas residue deposit II pH value carried out agitator treating 20 minutes with the amount W/V of 1 times of volume, used 4000 rev/mins centrifugal 30 minutes of speed then, obtained supernatant liquor III and pancreas residue deposit III; At last supernatant liquor III is poured in the supernatant liquor bucket, discard pancreas residue deposit III;
E, in the supernatant liquor bucket, add 25%～30% (NH 4) 2SO 4W/V stirs, and leaves standstill 2 hours, uses 4000 rev/mins centrifugal 30 minutes of speed then, discards the foreign protein precipitation, has obtained clear supernatant liquor;
F, in the clear supernatant liquor that obtains, add 70%～75% (NH 4) 2SO 4W/V, with 4000 rev/mins speed centrifugal 30 minutes, abandoning supernatant after the sedimentation obtained the former precipitation of Trypsin-chymotrypsin;
G, be behind 3.0 the deionized water dissolving with the former precipitation pH value of Trypsin-chymotrypsin that obtains, adding 4%～6%CaCl 2W/V stirs, and under 4 ℃～5 ℃, leaves standstill 10 hours, makes Ca ++The former Trypsin-chymotrypsin that becomes of ion-activated Trypsin-chymotrypsin; And then add 10%～15% (NH 4) 2SO 4W/V stirs, and heats to 37 ℃, makes Ca in the supernatant liquor ++Ion generates CaSO 4Precipitation
Figure A2009101706820003C1
Figure A2009101706820003C2
Trypsin-chymotrypsin in the adsorbent solution, leave standstill 10 hours after, with 4000 rev/mins speed centrifugal 30 minutes, after the sedimentation, abandoning supernatant was collected CaSO 4Precipitation;
H, with deionized water to CaSO 4Precipitate repeatedly wash-out three times, merge together, add 70%～75% (NH then 4) 2SO 4W/V stirs, and leaves standstill under 4 ℃ 10 hours, uses 4000 rev/mins centrifugal 30 minutes of speed again, abandoning supernatant, the Trypsin-chymotrypsin precipitation; With ultrafiltration behind the deionized water dissolving, lyophilize has obtained white Trypsin-chymotrypsin lyophilized powder at last.
CN200910170682A 2009-08-27 2009-08-27 Extraction method of trypsin-chymotrypsin Pending CN101643723A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102174495A (en) * 2011-01-20 2011-09-07 马忠仁 Method for extracting chymocotrypsin
CN103060296A (en) * 2012-12-30 2013-04-24 青岛九龙生物医药有限公司 Method for extracting trypsin from animal pancreas
CN108118046A (en) * 2017-12-19 2018-06-05 浙江丰安生物制药有限公司 A kind of trypsase and chymotrypsin combined extraction method and its application
CN109085298A (en) * 2018-08-23 2018-12-25 中国农业科学院北京畜牧兽医研究所 A kind of highly-water-soluble chitterlings digestive ferment pulvis and the preparation method and application thereof

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102174495A (en) * 2011-01-20 2011-09-07 马忠仁 Method for extracting chymocotrypsin
CN103060296A (en) * 2012-12-30 2013-04-24 青岛九龙生物医药有限公司 Method for extracting trypsin from animal pancreas
CN103060296B (en) * 2012-12-30 2014-05-21 青岛九龙生物医药有限公司 Method for extracting trypsin from animal pancreas
CN108118046A (en) * 2017-12-19 2018-06-05 浙江丰安生物制药有限公司 A kind of trypsase and chymotrypsin combined extraction method and its application
CN108118046B (en) * 2017-12-19 2020-06-05 浙江丰安生物制药有限公司 Trypsin and chymotrypsin combined extraction method and application thereof
CN109085298A (en) * 2018-08-23 2018-12-25 中国农业科学院北京畜牧兽医研究所 A kind of highly-water-soluble chitterlings digestive ferment pulvis and the preparation method and application thereof

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