CN1584025A - Method for extracting cydonic chimosin - Google Patents
Method for extracting cydonic chimosin Download PDFInfo
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- CN1584025A CN1584025A CN 200410046107 CN200410046107A CN1584025A CN 1584025 A CN1584025 A CN 1584025A CN 200410046107 CN200410046107 CN 200410046107 CN 200410046107 A CN200410046107 A CN 200410046107A CN 1584025 A CN1584025 A CN 1584025A
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- disken
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- ammonium sulfate
- supernatant liquor
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Abstract
A method for extracting chymopapain method relating enzyme preparation includes: 1) adding chitin into raw materials and keeping purified liquor after precipitating; 2) salting out ammonia sulfate with 30% saturating degree, removing precipitation and keeping supernatant fluid; 3) re-salting out ammonia sulfate with 50% saturating degree, removing precipitation and keeping supernatant fluid; 4) flocculating supernatant fluid by excess tartrate, removing precipitation and keeping supernatant fluid; 5) salting out purified liquor by ammonia sulfate with 70% saturating degree and collecting sludge; 6) dissolving sludge by pure water, concentrating solution by ultrafiltering membrane with trapping molecular weight 20000 and collecting concentrate; 7) separating concentrate by Weakly Acid cationic cellulose -92 and collecting eluent; 8) concentrating eluent by ultrafiltering membrane with trapping molecular weight 10000 and obtaining chymopapain concentrate. It achieves high activity and recovery.
Description
Technical field
The present invention relates to the extracting method of enzyme in the zymin field, particularly relate to a kind of extracting method of Disken.
Background technology
" papain " derives from the pulp of papaya (Carica Papaya) young fruit, is a kind of mixing sulfydryl enzyme that contains papoid (Papain), papoid III (Papaya Proteinase III), papoid IV (PapayaProteinase IV), Disken (Chymopapain) and pawpaw N,O-Diacetylmuramidase (Papaya Lysozyme).Various Seasonal results and " papain " preparation of producing through different process, the ratio drift of the various sulfydryl enzymes that contained is bigger, and enzymatic reaction kinetics is an on-fixed constant.
Use " papain " and carry out the existing nearly 60 years history of external blood group serology calibrating experiment, advance studies show that of phase, self-hydrolysis has taken place in the papoid III in the enzyme reagent solution under use and preservation condition, produced enzyme molecule segment, the instability that causes enzyme reagent solution vigor to tire with higher hydrolysis vigor; The red cell agglutination experiment shows that the pawpaw N,O-Diacetylmuramidase that antithesis nitrogenize albumin does not show hydrolytic activity has produced the hydrolysis modification to red corpuscle.Be subjected to the influence of above-mentioned zymetology quality factor, there is the probability that produces " reagent type---false positive " verification result in " papain " calibrating experiment.
Still do not have a kind of clear and definite enzymatic reaction kinetics feature that has at present, adapt to the protease preparation of stechiology, immunochemistry needs.
Summary of the invention
The method that the purpose of this invention is to provide a kind of effective extraction Disken.
The method of extraction Disken provided by the present invention comprises the steps:
1) add chitosan solution in papaya young fruit pulp raw material, post precipitation is removed throw out, keeps clear liquid;
2) adding ammonium sulfate in clear liquid is 30-40% to solution ammonium sulfate saturation ratio, removes precipitation, keeps supernatant liquor;
3) in supernatant liquor, add excess tartrate, behind the flocculation sediment, remove throw out, keep supernatant liquor;
4) adding ammonium sulfate in supernatant liquor is 65-70% to solution ammonium sulfate saturation ratio, the collecting precipitation thing;
5) with pure water dissolution precipitation thing,, collect concentrated solution to hold back the ultra-filtration membrane concentrated solution of 1-2 ten thousand molecular weight;
6) separate Disken with Weak-acid cation Mierocrystalline cellulose CM-C92 chromatography column, collect the 0.7mol/L sodium phosphate buffer elutriant of pH5.5;
7) concentrate the elutriant of Disken with the ultra-filtration membrane of holding back 1-2 ten thousand molecular weight, obtain the Disken concentrated solution.
In order to make isolating better effects if, in described step 2) supernatant liquor in, add before the excess tartrate, add ammonium sulfate earlier to the 40-50% saturation ratio, remove precipitation, keep supernatant liquor.The pH of described excess tartrate solution should be adjusted to＜and 2.5.
After ultrafiltration and concentration finishes, in the Disken concentrated solution, add Propiram and halfcystine as protective material, through lyophilize, the dry thing of preparation Disken.
The chitosan solution final concentration of the described adding of step 1) is 0.05-0.1%.
The present invention has the following advantages owing to adopted above technical scheme:
1, utilizes chitosan to remove impurity such as pectin, tannin, can significantly improve the solution transparence, remove the heavy metal in the solution, reduce solution colourity.
2, utilize tartrate as flocculation agent, significantly improve flocculation-sedimentary separating effect foreign protein.
3, utilize ammonium sulfate 30-40%, the 40-50% saturation ratio technology of saltouing for twice, can significantly reduce proteinic co-precipitation effect, improve the Disken yield.
4, abolish technologies such as traditional alkali neutralization, dialysis, utilize the polysulfone hollow fibre ultra-fine filter of holding back the 1-2W molecule, enzyme solution is directly carried out depickling, desalination, removal small molecular weight impurity, concentrates, can reduce the technology cost effectively, the protective enzyme activity.
5, adopt Weak-acid cation Mierocrystalline cellulose CM-C92 as chromatographic material, have characteristics such as loading capacity is big, wash-out is convenient, can significantly improve technological effect.
6, utilize Propiram (a kind of linear pattern macromolecular substance) as the auxiliary material of zymin, can improve the stability of zymin at processing, storage process.
The present invention adopts combination technological method, improves the stable processing technique of product, is suitable for the large-scale production of Disken reagent raw material, and the Disken of preparation meets the needed qualitative characteristics of external blood group serum calibrating experiment.
Embodiment
Embodiment 1, enzyme activity tire analytical procedure and condition
One, reagent
1) enzyme activation-stablizer
Prescription
L-halfcystine 0.025mol
EDTA-2K 0.03mol
N.F,USP MANNITOL 0.3mol
Propiram (10W) 0.05mmol
Potassium primary phosphate 0.064mol
Sodium phosphate dibasic 0.003mol
Compound method
Above-mentioned solute is dissolved in an amount of pure water, is diluted to the 1000ml constant volume with pure water.
2) sample solution
Take by weighing Disken 30mg,, be diluted to the 100ml constant volume with the dissolving of enzyme activation-stablizer.
3) substrate solution
Take by weighing Sodium phosphate dibasic 0.93mol, SODIUM PHOSPHATE, MONOBASIC 0.06mol uses dissolved in distilled water, is diluted to the 1000ml constant volume.
Take by weighing azo albumin 2.0g, be dissolved in the above-mentioned 100ml phosphate solution.
Two, enzyme activity titration method
1, with sample solution, substrate solution is preheated to 37 ℃ ± 0.5 ℃.
2, absorb substrate solution 1.0ml with sample injector, in the sample solution 100 μ l injecting tubes, put 37 ℃ ± 0.5 ℃ water bath reaction 10 minutes.
3, add 5% trichoroacetic acid(TCA) solution 1ml, mix.
4,1000 * g is centrifugal 5 minutes, draws supernatant liquor 1ml.
5, add 0.5mol/L sodium hydroxide solution 1ml, mix.
6, working sample 430nm OD value.
7, enzyme activity definition:
What under these conditions, hydrolysate produced 0.002 light absorption value at 430nm changes into an enzyme activity unit (PU).
8, the vigor marker method of tiring:
The vigor relative enzyme activity that every milligram of enzyme sample of marker method I:PU/min/mg=had in 1/10 minute of tiring is tired.
The vigor relative enzyme activity that every milligram of enzyme sample of marker method II:PU/mg=had in 10 minutes of tiring is tired.
The extraction of embodiment 2, Disken
1, the papaya young fruit of getting 1000g is starched freezing thing, melts with microwave heating, adds 1000ml 0.02mol/L sodium phosphate buffer (pH5.5), homogenate in homogenizer.
2, add 1% chitosan solution 100ml, homogenate is stirred, and room temperature leaves standstill 2 hours after-filtration, removes precipitation, keeps filtrate.
3, add ammonium sulfate to 30% saturation ratio in filtrate, room temperature left standstill 60 minutes, centrifugal 20 minutes of 2-8 ℃, 10000 * g, removed precipitation; Add ammonium sulfate to 50% saturation ratio in centrifuged supernatant, room temperature left standstill 60 minutes, centrifugal 20 minutes of 2-8 ℃, 10000 * g, removed throw out, kept supernatant liquor.
4, add excess tartrate solution to supernatant liquor, regulate pH to＜2.5, room temperature left standstill 60 minutes, after flocculation sediment occurring, centrifugal 20 minutes of 10000 * g discards throw out, in supernatant liquor, add ammonium sulfate to 70% saturation ratio, room temperature left standstill 60 minutes, centrifugal 20 minutes of 10000 * g, collecting precipitation thing.
5, throw out being dissolved with the 500ml pure water, is 20,000 polysulfone hollow fibre ultra-filtration membrane ultrafiltration module then with molecular weight cut-off, and gradation adds 2-8 ℃ of pure water in ultra-filtration process, and loop ultrafiltration 1h under the condition of 0.1 MPa obtains ultrafiltration and concentration liquid.
6, the 0.2mol/L sodium phosphate buffer (pH5.5) that in above-mentioned solution, adds equivalent, mixing.With this sodium phosphate buffer pre-balance granule type Weak-acid cation CM-C92 chromatography column, then with the speed of 5ml/min, with the solution upper prop, with 0.7mol/L sodium phosphate buffer (pH5.5) is elutriant, collecting this elutriant, is 10,000 polysulfone hollow fibre ultra-filtration membrane concentrate eluant with molecular weight cut-off.
7, add Propiram (10W) 0.1mmol, L-halfcystine 0.025mol to concentrated solution, lyophilize obtains the dry thing 30g of Disken.
8, measure the product enzyme activity with measuring method among the embodiment 1.
Tire and be 1500PU/mg with tire enzyme activity that marker method I draws of vigor;
Tire and be 15000PU/mg with tire enzyme activity that marker method II draws of vigor.
The extraction of embodiment 3, Disken
Get the dry thing 600g of pulp of papaya young fruit, be dissolved in 0.3mol/L phosphate buffered saline buffer (pH5.5) 2L, add 3% chitosan aqueous solution 100ml, room temperature leaves standstill 2 hours after-filtration, removes precipitation, keeps filtrate.
Add ammonium sulfate to 40% saturation ratio in filtrate, room temperature left standstill 60 minutes, centrifugal 20 minutes of 2-8 ℃, 10000 * g, removed precipitation; Add ammonium sulfate to 50% saturation ratio in centrifuged supernatant, room temperature left standstill 60 minutes, centrifugal 20 minutes of 2-8 ℃, 10000 * g, removed throw out, kept supernatant liquor.
Add excess tartrate solution to supernatant liquor, regulate pH to＜2.5, room temperature left standstill 60 minutes, flocculation sediment appears and after, centrifugal 20 minutes of 10000 * g discards throw out; Add ammonium sulfate to 65% saturation ratio in supernatant liquor, room temperature left standstill 60 minutes, centrifugal 20 minutes of 10000 * g, collecting precipitation thing.
Throw out is dissolved with the 500ml pure water, is 20,000 polysulfone hollow fibre ultra-filtration membrane ultrafiltration module then with molecular weight cut-off, and gradation adds 2-8 ℃ of pure water in ultra-filtration process, and loop ultrafiltration 1h under the condition of 0.1 MPa obtains ultrafiltration and concentration liquid.
The 0.2mol/L sodium phosphate buffer (pH5.5) that in above-mentioned ultrafiltration and concentration liquid, adds equivalent, mixing.With this sodium phosphate buffer pre-balance granule type Weak-acid cation CM-C92 chromatography column, with the speed of 5ml/min, with the solution upper prop, (pH5.5) is elutriant with the 0.7mol/L sodium phosphate buffer, collects this elutriant then.
It with molecular weight cut-off 10,000 huge sulfone hollow fiber ultrafiltration membrane concentrate eluant.Add Propiram 0.1mmol, L-halfcystine 0.025mol to concentrated solution, lyophilize obtains the dry thing 20g of Disken.
Measuring the product vigor with measuring method among the embodiment 1 tires.
With the embodiment 1 vigor relative enzyme activity that marker method I draws of tiring is 1300PU/mg;
With the embodiment 1 vigor relative enzyme activity that marker method II draws of tiring is 13000PU/mg.
The application of embodiment 4, Disken
With the dry thing of the Disken of above-mentioned manufacture method, as raw material, be used to produce the Disken reagent of tailored version, be used for external blood group serology calibrating experiment.Its technological process is as follows.
One, Disken preparation
1, prescription
Propiram 0.05mmol
N.F,USP MANNITOL 0.3mol
Trehalose 0.02mol
Sodium ascorbate 0.02mol
L-halfcystine 0.05mol
Disken 200000PU
2, preparation technology and process
Quantitatively take by weighing above-mentioned solute, be dissolved in the pure water, be diluted to the 1000ml constant volume,, divide by every bottle of 10ml then to be filled to clean cillin bottle with 0.2 μ m filtering with microporous membrane degerming.Be cooled to-40 ℃ rapidly with freeze drier, kept 4 hours; Start drying program then,, frozen product intensification was kept 3 hours for 33 ℃, stop decompression, finished product is packed, obtain finished product Disken preparation with the 5 ℃ of speed drying under reduced pressure that per hour heat up.
Two, Disken solvent
Potassium primary phosphate 0.064mol
Sodium phosphate dibasic 0.003mol
Dissolve above-mentioned solute with pure water, be diluted to the 1000ml constant volume,, divide by every bottle of 10ml then to be filled to polyethylene bottle sealing with 0.2 μ m filtering with microporous membrane degerming.
With Disken dissolution with solvents Disken preparation, form the Disken reagent solution before using, detect the vigor of Disken reagent solution with 2% even egg albumin-phosphate solution and tire.
According to the following vigor identification method I that tires, the vigor of every bottle of Disken preparation is tired and is the 2000PU/ bottle; By the judgement criteria of external blood group serology experiment calibrating sensitivity, the vigor that per 50 μ l Disken reagent solutions should possess is tired and is 10-16PU.
According to the following vigor identification method II that tires, the vigor of every bottle of Disken preparation is tired and is the 20000PU/ bottle; By the judgement criteria of external blood group serology experiment calibrating sensitivity, the vigor that per 50 μ l Disken reagent solutions should possess is tired and is 100-160PU.
The vigor vigor that the per 50 μ l reagent solutions of identification method I:PU=were had in 1/10 minute of tiring is tired.
The vigor vigor that the per 50 μ l reagent solutions of identification method II:PU=were had in 10 minutes of tiring is tired.
The step of utilizing mentioned reagent to carry out blood group antibody specificity identification experiment is:
1, get test tube, mark I, II, III, IV, V, VI, VII, VIII, IX, X, XI put test-tube stand successively and arrange.
2, add 3% blood group spectrum cell suspension, the 50 μ l that are consistent with its mark to above-mentioned test tube, Disken reagent solution 50 μ l mix.
3, test tube being moved into 37 ± 0.5 ℃ of water-baths hatched 5 minutes.
4, respectively add enzyme inhibitors-64 reagent solution 50 μ l to whole test tubes; Respectively add serum specimen 50 μ l to whole test tubes, or red corpuscle diffuses liquid 100 μ l, mix.
5, test tube being moved into 37 ± 0.5 ℃ of water-baths hatched 7 minutes.
6, test tube is moved into whizzer, centrifugal 1 minute of 120 * g.
7, shake test tube gently, Red Blood Cells Suspension, visual inspection experimental identification result.According to whether being examined aggegation that serum produces at different blood groups spectrum cells, judge the blood group antibody specificity of being examined serum.
Through adopt room temperature saline experiment, 37 saline experiments, blood group antibody specificity identification experiment method such as antihuman globulin experiment and experimental technique of the present invention be as parallel test indirectly, to the people source type Rh-that originates from Immucor anti--D, anti--C, anti--E, anti--c, anti--e, Kell-is anti--K, anti--k, kiad-resist-JK
a, anti--JK
b, Lewis-is anti--Le
a, anti--Le
b, anti--Le
A+b, P-is anti--P
1Deng blood group antibody serum, and clinical sample serum carries out the antibodies specific evaluation.Experimental result shows that this experimental technique is 100% to the experimental identification accuracy rate of above-mentioned blood group antibody.
Claims (7)
1, a kind of method of extracting pawpaw albumen curd enzyme comprises the steps:
1) add chitosan solution in papaya young fruit pulp raw material, post precipitation is removed throw out, keeps clear liquid;
2) adding ammonium sulfate in clear liquid is 30-40% to solution ammonium sulfate saturation ratio, removes precipitation, keeps supernatant liquor;
3) in supernatant liquor, add excess tartrate, behind the flocculation sediment, remove throw out, keep supernatant liquor;
4) adding ammonium sulfate in supernatant liquor is 65-70% to solution ammonium sulfate saturation ratio, the collecting precipitation thing;
5) with pure water dissolution precipitation thing, be 0.9-1.1 ten thousand daltonian ultra-filtration membrane concentrated solutions, collect concentrated solution with the molecular weight cut-off;
6) separate Disken with cationic cellulose CM-C92 chromatography column, collect the 0.7mol/L sodium phosphate buffer elutriant of pH5.5;
7) be the elutriant of the concentrated Disken of ultra-filtration membrane of 1-2 ten thousand with the molecular weight cut-off, obtain the Disken concentrated solution.
2, the method for extraction pawpaw albumen curd enzyme according to claim 1, it is characterized in that: in described step 2) supernatant liquor in, add before the excess tartrate, adding ammonium sulfate earlier is 40-50% to solution ammonium sulfate saturation ratio, remove precipitation, keep supernatant liquor.
3, the method for extraction pawpaw albumen curd enzyme according to claim 1 and 2, it is characterized in that: the pH regulator of described excess tartrate solution is to＜2.5.
4, according to claim 1 or the 2 or 3 described methods of extracting pawpaw albumen curd enzymes; it is characterized in that: also need in the Disken concentrated solution, to add Propiram and halfcystine as protective material, obtain the dry thing of Disken through lyophilize.
5, the method for extraction pawpaw albumen curd enzyme according to claim 4, it is characterized in that: the Propiram concentration of described adding is 0.05-0.1mmol; The concentration of the halfcystine of described adding is 0.025-0.05mol.
6, according to the method for claim 1 or 2 or 3 described extraction pawpaw albumen curd enzymes, it is characterized in that: the chitosan solution final concentration of the described adding of step 1) is 0.05-0.1%.
7, according to the method for claim 1 or 2 or 3 described extraction pawpaw albumen curd enzymes, it is characterized in that: it is 1/10 minute 10-16PU/50 μ l that the vigor of described Disken is tired, or 10 minutes 100-160PU/50 μ l.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410046107 CN1264976C (en) | 2004-05-31 | 2004-05-31 | Method for extracting cydonic chimosin |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410046107 CN1264976C (en) | 2004-05-31 | 2004-05-31 | Method for extracting cydonic chimosin |
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| CN1584025A true CN1584025A (en) | 2005-02-23 |
| CN1264976C CN1264976C (en) | 2006-07-19 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321601A (en) * | 2011-08-26 | 2012-01-18 | 甘肃农业大学 | The preparation method of bacillus amyloliquefaciens rennin lyophilized powder |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2556985T3 (en) | 2011-01-11 | 2016-01-21 | Capsugel Belgium Nv | New hard capsules comprising pululane |
| CA3059527A1 (en) | 2017-04-14 | 2018-10-18 | Capsugel Belgium Nv | Pullulan capsules |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321601A (en) * | 2011-08-26 | 2012-01-18 | 甘肃农业大学 | The preparation method of bacillus amyloliquefaciens rennin lyophilized powder |
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| CN1264976C (en) | 2006-07-19 |
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Granted publication date: 20060719 Termination date: 20100531 |