CN103060296B - Method for extracting trypsin from animal pancreas - Google Patents

Abstract

The invention discloses a method for extracting trypsin from animal pancreas. According to the technical scheme of the invention, the zymogen in pancreas cells is extracted by using a sulfuric acid solution; subsequently according to the principle of isoelectric precipitation, the pH is adjusted, and the trypsinogen is deposited and precipitated in a classified salting-in and salting out mode by using ammonium sulfate. By utilizing the method, 6g trypsin can be extracted from 1kg of pancreas.

Description

From animal pancreas, extract tryptic method

Technical field

The present invention relates to biological technical field, relate in particular to and from animal pancreas, separate tryptic method.

Background technology

The one that trypsinase Trypsin (Parenzyme) is proteolytic enzyme, EC3.4.21.4.In vertebrates, work as digestive ferment.Be synthesized as the precursor trypsinogen of enzyme at pancreas.Secrete as the composition of pancreatic juice, be subject to enteropeptidase, or tryptic restriction being decomposed into activation trypsinase, is endopeptidase, and it can cut off the carboxyl side in Methionin in polypeptide chain and arginine residues.It not only plays digestive ferment, and can also limit the precursor of other enzymes such as decomposing chymotrypsinogen, proearboxypeptidase, phosphatide proenzyme, plays activation.Be the proteolytic enzyme that specificity is the strongest, in the amino acid that determines protein is arranged, it becomes indispensable instrument.

Trypsin-chymotrypsin and elastoser also have substantial connection aspect structure and catalyst mechanism, but its specificity is completely different.Trypsinase is a kind of proteolytic ferment extracting in ox, sheep or pig pancreas.China's drug standard regulation is pressed dry product and is calculated, and must not tire of every 1mg is less than 2500 units.By ox, sheep, Pancreas Sus domestica extract and a kind of endopeptidase, only rupture Methionin or arginic carboxyl participate in the peptide bond forming.White or beige crystalline powder.Water-soluble, be insoluble to ethanol, glycerine, chloroform and ether.Molecular weight 24000, pI10.5, optimum pH 7.8～8.5 left and right.The irreversible inactivation of pH > 9.0.Ca ²⁺enzymic activity is had to stabilization; Heavy metal ion, organo phosphorous compounds, DFP, natural trypsin inhibitor have strongly inhibited to its activity.Clinical in anti-inflammation detumescence, industrial for leather manufacture, raw silk processing, food-processing etc.

Trypsinase is proteolytic enzyme, the peptide chain selectively being formed by Methionin or arginic carboxyl in protein hydrolysate, can digestion dissolve sex change egg matter, without effect, therefore, can make the decomposition, thinning such as purulence, sputum, blood clot to unmodified protein, being easy to drainage gets rid of, the acceleration surface of a wound purifies, and promotes granulation tissue new life, also has in addition anti-inflammatory effect.Local edema, hemotoncus and the abscess etc. that produce for pyothorax, hemothorax, surgery inflammation, ulcer, traumatic damage, fistula etc. clinically.Spraying sucks, for respiratory tract disease.Also can be used for treating venomous snake bite.Also be usually used in the front processing to tissue of animal cell culture.

Thereby tryptic effect is to make intercellular proteolysis make cell dissociation.Different tissues or cell are different to the action-reaction of pancreatin.The activity of pancreatin cell dispersion is also relevant with its concentration, temperature and action time, is 8.0, temperature is while being 37 ℃ at pH, and the ability to function of pancreatin solution is the strongest.While using pancreatin, should get hold of concentration, temperature and time, in order to avoid digestion excessively causes cell injury.Because of Ca ²⁺, Mg ²⁺can reduce the activity of pancreatin with serum, protein, so should select while preparing pancreatin solution not containing Ca ²⁺, Mg ²⁺bSS, as D-Hanks liquid.Stop when digestion, availablely contain serum free culture system liquid or pancreatin inhibitor stops the effect of pancreatin to cell.

It is quite difficult extracting trypsinase by existing method.In July, 2006, A Min pharmaceutical Co. Ltd in Shanghai disclosed a kind of preparation method (200610023582.0) of Trypsin-chymotrypsin, it adopts low temperature acid to carry, gradient is saltoutd, the Product Process of frozen water dialysis, affinity chromatography, alcohol chromatography, obtains Trypsin-chymotrypsin.The loyal benevolence of in January, 2011 horse etc. has been applied for a kind of method (200910170682.X) of extracting Trypsin-chymotrypsin, and it has adopted CaCl ₂activate (the NH that adds saturation ratio when Trypsin-chymotrypsin is former becomes Trypsin-chymotrypsin ₄) ₂sO ₄, make it generate calcium sulfate precipitation absorption Trypsin-chymotrypsin, through wash-out, saltout, lyophilized powder that ultrafiltration and freeze-drying obtain white Trypsin-chymotrypsin.Product is the mixture of Trypsin-chymotrypsin, there is no simple extraction trypsinase.The present invention has reduced cost, and trypsinase can be extracted.

Summary of the invention

The object of the invention is to extract trypsinase from the pancreas of animal, realize the separation of Trypsin-chymotrypsin.

Technical scheme of the present invention is: utilize sulphuric acid soln that the proenzyme containing in pancreatic cell is extracted, then according to the principle of isoelectric precipitation, regulate pH, ammonium sulfate classification salt is molten, saltout Precipitations such as trypsinogens, comprises the steps:

(1) abstraction process: fresh pancreas is rubbed into pancreas slurry, weigh.0.1～0.2mol/L the sulphuric acid soln that adds 2～3L by every kilogram of pancreas slurry, floods 20～28 hours, macerate is put to filter bag and filter, and after being filtered dry, discards filter residue, leaves and takes filtrate;

(2) the molten operation of salt: add ammonium sulfate in steeping fluid, reach 30～50% saturation ratios, leave standstill 10～16 hours, filter, leave and take filtrate;

(3) salting-out procedures: add ammonium sulfate in filtrate, reach 50～80% saturation ratios, leave standstill 10～16 hours, filter, leave and take filter cake;

(4) every gram of filter cake adds the water dissolution of 2～5ml, repeats above-mentioned (2), (3) operation;

(5) water dissolution of 1～3ml for every gram of filter cake, then adds the saturated ammonium sulphate solution of 0.1～1ml in every gram of filter cake, regulate pH to 4.0～6.0;

(6) Crystallization Procedure: solution leaves standstill 24～72 hours in 20～30 ℃ of temperature, has needle-like Chymotrypsin crystallization; Centrifugal, regulate pH to 2.0～5.0, in every 1000ml solution, add 300～400g solid ammonium sulfate, reach 50～80% saturation ratios, leave standstill 12～16 hours, suction filtration;

(7) drying process: taking precipitate, dry, obtain trypsinase crude product.

Embodiment

Below in conjunction with specific embodiment, the invention will be further described.

Embodiment 1

The described dilute acid soln that utilizes extracts the proenzyme containing in pancreatic cell, then according to the principle of isoelectric precipitation, regulate pH with precipitation remove a large amount of acid foreign proteins and non-albumen impurity, then with grade ammonium sulfate salting-out by Precipitations such as trypsinogens, comprise the steps:

1. abstraction process:

1.1 fresh pancreases, remove adipose connective tissue etc. with tweezers and scissors, put in pulverizer and rub, and 15Kg weighs;

1.2 rub thing puts in plastic tank, and with 0.125mol/L sulphuric acid soln, 30L floods 24 hours, the interim stirring per hour of dipping 1 time;

Macerate is put filter bag by 1.3 to be filtered, and filter residue floods 1 hour with 0.125mol/L sulphuric acid soln 15L again, after being filtered dry, discards filter residue;

2. molten, the salting-out procedures of salt:

2.1 merge twice steeping fluid, and volume is 44.9L, puts in plastic tank, in every 1000ml solution, adds solid ammonium sulfate 242g, reaches 40% saturation ratio, adds altogether 10.866Kg solid ammonium sulfate, leaves standstill 12 hours;

2.2 filter, and leave and take filtered liquid, and filtrate volume is 44L, and solid discards;

2.3 add solid ammonium sulfate 205g in every 1000ml clear liquid, reach 70% saturation ratio, share ammonium sulfate 9.020kg, leave standstill 12 hours;

2.4 filter, and leave and take filter cake, and the 269.2g that weighs, abandons filtrate;

2.5 use 807.6ml water dissolution, then by 2.1,2.2,2.3,2.4 step gradation reinforcing body ammonium sulfate nearly 40% and 70% saturation ratio precipitations;

2.670% saturation ratio throw out, the 178.5g that weighs, uses 267.8ml water dissolution, adds 89.3ml saturated ammonium sulphate solution, and with 5mol/L sodium hydroxide solution regulate pH to 5.00;

2.7 by 25 ℃ of thermostat containers of solution left standstill 48 hours, had the Chymotrypsin crude product crystallization of needle-like;

2.8 filter, 0.25mol/L sulphur acid for adjusting pH to 3.0 for mother liquor, and its volume is that 328.4ml adds ammonium sulfate 100.8g, leaves standstill 12 hours;

3. drying process:

Taking precipitate, puts vacuum drying oven inner drying, obtains trypsinase crude product, the 91.8g that weighs, and every kilogram of pancreas can obtain 6.12 grams of trypsinase.

Embodiment 2

1. abstraction process:

1.1 fresh pancreases, remove adipose connective tissue etc. with tweezers and scissors, put in pulverizer and rub, and 10Kg weighs;

1.2 rub thing puts in plastic tank, and with 0.150mol/L sulphuric acid soln, 25L floods 24 hours, the interim stirring per hour of dipping 1 time;

Macerate is put filter bag by 1.3 to be filtered, and filter residue floods 1 hour with 0.150mol/L sulphuric acid soln 12.5L again, after being filtered dry, discards filter residue;

2. molten, the salting-out procedures of salt:

2.1 merge twice steeping fluid, and volume is 37.4L, puts in plastic tank, in every 1000ml solution, adds solid ammonium sulfate 225g, reaches 35% saturation ratio, and power 8.415Kg solid ammonium sulfate altogether, leaves standstill 12 hours;

2.2 filter, and leave and take filtered liquid, and filtrate volume is 36.8L, and solid discards;

2.3 add solid ammonium sulfate 186g in every 1000ml clear liquid, reach 65% saturation ratio, share ammonium sulfate 6.845kg, leave standstill 12 hours;

2.4 filter, and leave and take filter cake, and the 180.2g that weighs, abandons filtrate;

2.5 use 720.8ml water dissolution, then by 2.1,2.2,2.3,2.4 step gradation reinforcing body ammonium sulfate nearly 35% and 65% saturation ratio precipitations;

2.6 get 65% saturation ratio throw out, and the 90.5g that weighs, uses 181ml water dissolution, add 54.3ml saturated ammonium sulphate solution, and with 5mol/L sodium hydroxide solution regulate pH to 5.10;

2.7 by 25 ℃ of thermostat containers of solution left standstill 48 hours, had the Chymotrypsin crude product crystallization of needle-like;

2.8 filter, 0.25mol/L sulphur acid for adjusting pH to 2.9 for mother liquor, and its volume is that 234.7ml adds ammonium sulfate 82.1g, leaves standstill 15 hours;

3. drying process:

Taking precipitate, puts vacuum drying oven inner drying, obtains trypsinase crude product, the 60.9g that weighs, and every kilogram of pancreas can obtain 6.09 grams of trypsinase

The above, be only preferred embodiment of the present invention, is not the restriction of the present invention being made to other form, and any those skilled in the art may utilize the technology contents of above-mentioned announcement to be changed or be modified as the equivalent embodiment of equivalent variations.But every technical solution of the present invention content that do not depart from, any simple modification, equivalent variations and the remodeling above embodiment done according to technical spirit of the present invention, still belong to the protection domain of technical solution of the present invention.

Claims (1)

1. from animal pancreas, extract a tryptic method, it is characterized in that, the method comprises the steps:

1. abstraction process:

1.1 fresh pancreases, remove adipose connective tissue with tweezers and scissors, put in pulverizer and rub, and 15Kg weighs;

1.2 rub thing puts in plastic tank, and with 0.125mol/L sulphuric acid soln, 30L floods 24 hours, the interim stirring per hour of dipping 1 time;

Macerate is put filter bag by 1.3 to be filtered, and filter residue floods 1 hour with 0.125mol/L sulphuric acid soln 15L again, after being filtered dry, discards filter residue;

2. molten, the salting-out procedures of salt:

2.1 merge twice steeping fluid, and volume is 44.9L, puts in plastic tank, in every 1000ml solution, adds solid ammonium sulfate 242g, reaches 40% saturation ratio, adds altogether 10.866Kg solid ammonium sulfate, leaves standstill 12 hours;

2.2 filter, and leave and take filtered liquid, and filtrate volume is 44L, and solid discards;

2.3 add solid ammonium sulfate 205g in every 1000ml clear liquid, reach 70% saturation ratio, share ammonium sulfate 9.020kg, leave standstill 12 hours;

2.4 filter, and leave and take filter cake, and the 269.2g that weighs, abandons filtrate;

2.5 use 807.6ml water dissolution, then by 2.1,2.2,2.3,2.4 step gradation reinforcing body ammonium sulfate nearly 40% and 70% saturation ratio precipitations;

2.670% saturation ratio throw out, the 178.5g that weighs, uses 267.8ml water dissolution, adds 89.3ml saturated ammonium sulphate solution, and with 5mol/L sodium hydroxide solution regulate pH to 5.00;

2.7 by 25 ℃ of thermostat containers of solution left standstill 48 hours, had the Chymotrypsin crude product crystallization of needle-like;

2.8 filter, 0.25mol/L sulphur acid for adjusting pH to 3.0 for mother liquor, and its volume is that 328.4ml adds ammonium sulfate 100.8g, leaves standstill 12 hours;

3. drying process:

Taking precipitate, puts vacuum drying oven inner drying, obtains trypsinase crude product, the 91.8g that weighs, and every kilogram of pancreas can obtain 6.12 grams of trypsinase.