CN101182495B - Joint production process for producing alkaline phosphatase and heparin sodium with pig small intestine as raw material - Google Patents
Joint production process for producing alkaline phosphatase and heparin sodium with pig small intestine as raw material Download PDFInfo
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Abstract
The invention discloses a co-production production process of alkaline phosphatase and heparin sodium, which uses porcine small intestinal as the raw material. The process comprises the following steps: (1) the porcine small intestinal is refined and grinded; (2) 0.05mol/LTris-HCl buffer is added for extraction; (3)butanol precipitation; (4)pressure filtration and centrifugal separation; (5) the supernatant which is isolated from step(4) is precipitated by acetone; then the alkaline phosphatase is obtained through resolution ion exchange chromatography separation, concentration, gel chromatography separation, concentration and lyophilization; (6) the filter residue isolated from step(4) is processed with extraction, enzymatic hydrolysis, filtration, ion exchange resin absorption, impurities removal process by washing and elution, so that the heparin is obtained. The crude heparin sodium is obtained through alcohol precipitation, dehydration and drying; and the refined heparin sodium is obtained through hydrogen peroxide treatment, filtration, alcohol precipitation of the filtrate and acetone dehydration and drying. The process suits for the industrial production. The process can extract both alkaline phosphatase and refined heparin sodium. The invention is beneficial for the realization of the comprehensive utilization of the raw material. The invention reduces the production cost and decreases the environmental pollution of tail materials.
Description
Technical field
The present invention relates to biomedicine field, particularly is the joint process of raw material production alkaline phosphatase and heparin sodium with chitterlings, can be widely used in biochemical reagents and biological medicine.
Background technology
(1) alkaline phosphatase (Alkline phosphatase, EC 3.1.3.1, be called for short ALP or AKP) extensively be present in human body, animal, plant and the microbe, participate in physiological processs such as the transfer of phosphate group and metabolism in vivo directly, to the absorption and the metabolism of calcium phosphorus in the body, keep calcium phosphorus ration suitable in the body and play an important role.Alkaline phosphatase is the lower phosphomonoesterase of a kind of Substratspezifitaet, energy hydrolysis mono phosphoric acid ester ester bond, but not hydrolysis phosphodiester bond.It can act on the multiclass substrate, as β-Phosphoric acid glycerol esters, Cori ester, and adenylic acid, uridine phosphoric acid and 5-lactofiavine phosphate etc.Mg
2+, Zn
2+It is the activator of enzyme; Zn
2+May be relevant with the catalytic activity of the structure of enzyme and enzyme.Other activator such as amino alcohol etc.; Inhibitor has inorganic phosphorus, thanomin etc.Its optimal pH about 10 promptly has higher activity under alkaline condition, alkaline phosphate ester is gained the name thus.
Alkaline phosphatase can be produced by tissues such as human liver, bone, intestines, kidney and placentas respectively and discharge outside courage through liver, but it is not single enzyme, but one group of isozyme.Some difference of ALP physico-chemical property of each organ, when pathology also polymer ALP may appear, and some variation ALPs relevant with tumour, but the amino acid structure of various alkaline phosphatases is similar, the antigenicity indifference, these enzyme electrophoresis rates with to the difference of different inhibitor response differences from contained glycosyl among the ALP.
Highly purified alkaline phosphatase is widely used in biological study and biological technical field, except that being used for enzymatic analysis (comprising enzyme reagent kit, enzyme linked immunological, hrp gene probe, Enzyme sensor or the like), also is used for the hydrolysis of biotechnology amplifying nucleic acid, as
32Remove 5 '-phosphoric acid of RNA or DNA before P mark 5 '-end with alkaline phosphatase, 5 '-phosphoric acid of removing carrier, dna fragmentation in the genetically engineered is to prevent recirculation etc.
(2) heparin is a class glycosaminoglycan, the mixture of the polysaccharide chain that the repetition disaccharide unit that is coupled together with 1 → 4 key by uronic acid and glucosamine is formed.Contain 10-30 disaccharide unit and do not wait, molecular weight 4000-20000, molecular-weight average 12000.The structure of heparin is complicated, it is generally acknowledged that it is to be combined into " tetrose " as structural unit by α-L-iduronic acid-2-sulfuric ester, the amino grape 6-of N-sulfo group-α-D-sulfuric ester, β-D-glucuronic acid and N-sulfo group-alpha-D aminoglucose-6-sulfuric ester with glycosidic link, aggregate into polysaccharide by " tetrose " again, thereby make the entire structure of heparin become complicated unusually, up to the present, the precision architecture of heparin is not clear.Heparin sodium belongs to macromolecular compound, by the sodium salt of the different sticking how smart phase blended sulfuric acid glycosaminoglycan of acidity of molecular weight, and white powder, water soluble is not soluble in the liquor-saturated and acetone and other organic solvent of second.
Since nineteen thirty-seven, heparin was used as antithrombotics clinically, heparin was a kind of main anticoagulant always.Find also that in recent years heparin has the inhibition smooth muscle cell proliferation, anti-inflammatory, biological function such as antitumor and antiviral.Because heparin sodium is by the crude substance that extracts in the organism, it or not the product of chemosynthesis, have no side effect, in clinical use, be subjected to doctor and patient's favor, be widely used in the various cerebrovascular diseases of control, stop the liquor-saturated former fibrin monomer that is transformed into of blood coagulation, prevent from hematoblasticly to gather and destroy, it is solid liquor-saturated to treat sudden thrombotic disease, arteriosclerosis and reduction courage, prevents that the metastasis of cancer cells from all having definite curative effect.Low molecular heparin (sodium) product of producing with highly purified heparin also has significant therapeutic action.
Heparin sodium is constantly popularized and expansion medical clinical application, and market demand is also in continuous increase.Statistics of export data presentation in 2006, China exported 113.60 tons of heparin sodiums in 2006 altogether, created the new peak over nearly 3 years.And prediction according to the insiders, China's 2007 outlet heparin sodium total amounts will reach the 130-150 ton.World market heparin sodium bulk drug total sales volume estimates that annual growth is approximately 10% at 2,000,000,000-2,500,000,000 dollars at present.From the eighties in last century,, developed the Low molecular heparin of treatment and pre-preventing thrombosis by chemically modified heparin sodium molecule.Low molecular heparin is the part degradation product of heparin, and it is with better function, and security is higher.The production of Low molecular heparin also needs the heparin of higher degree.Meanwhile, along with the aggravation of market competition, also more and more higher to the specification of quality of heparin sodium.Traditional in the past extraction production technology method must further be improved and be perfect, both can reduce the production cost of heparin, can make heparin be suitable for being processed into Low molecular heparin again.The present invention then can satisfy above-mentioned requirements.
Summary of the invention
Purpose of the present invention just at the situation of above-mentioned prior art and invent a kind of be the joint process of raw material joint production producing alkaline phosphatase and heparin sodium with chitterlings, this joint process is suitable for suitability for industrialized production, can extract alkaline phosphatase, also can extract refining heparin sodium, help realizing the comprehensive utilization of raw material, reduce production costs, reduce the pollution of tankage environment.
The objective of the invention is to be achieved through the following technical solutions: of the present invention is the joint process of raw material joint production producing alkaline phosphatase and heparin sodium with chitterlings, may further comprise the steps: 1. the chitterlings making beating is pulverized; 2. add the Tris-HCl damping fluid extraction that 0.05mol/L contains magnesium chloride, sodium-chlor and zinc chloride; 3. propyl carbinol precipitates; 4. press filtration or centrifugation; 5. the 4. middle separated liquid supernatant acetone precipitation of step promptly gets the alkaline phosphatase enzyme product by the separation of redissolution ion-exchange chromatography, vacuum concentration, gel chromatography separation, vacuum concentration, lyophilize more according to this; 6. step 4. in isolated filter residue obtain heparin, dehydrate to such an extent that crude heparin sodium, hydrogen peroxide processing, filtration, filtrate dehydrate with acetone with ethanol sedimentation, throw out and promptly gets refined heparin sodium through the alcohol precipitation again by lixiviate, enzymolysis, filtration, ion exchange resin absorption, washing decon, wash-out.
In the present invention, used Tris-HCl damping fluid contains 0.01mol/L magnesium chloride-0.01mol/L sodium-chlor-0.01mol/L zinc chloride.
Step of the present invention 5. in, it is that (3.5cm * 100cm), each application of sample amount is 30ml to DEAESepharose 4B post that described redissolution ion-exchange chromatography separates used exchange column; The used chromatography column of described gel chromatography separation is Sephadex G-150.
Step of the present invention 6. in, described enzymolysis process realizes that by adding fresh pig pancreas slurry or commodity trypsinase used ion exchange resin is the D-204 macroporous adsorbent resin.
The filter residue drying that step of the present invention twice in 6. filters gained is protein fodder.
In the present invention, the step detailed process 5. just separation and the purge process of alkaline phosphatase is: 1. 2. 3. 4. after the step, implementing following processing step through aforesaid again:
A, in filtrate, add the equal-volume cold acetone, behind the mixing under rotating speed 3000-6000r/min centrifugal 10-20min, abandoning supernatant;
B, add about 3-5 doubly measure (W/V) 0.05mol/L Tris-HCl damping fluid (pH7.5 includes 0.01mol/L magnesium chloride-0.01mol/L sodium-chlor-0.01mol/L zinc chloride) in precipitation, fully stirring makes its dissolving be gained alkaline phosphatase crude enzyme liquid;
C, crude enzyme liquid is added with 0.05mol/L Tris-HCl damping fluid (pH7.5, including 0.01mol/L magnesium chloride-0.01mol/L sodium-chlor-0.01mol/L zinc chloride) the DEAE Sepharose 4B post crossed of balance is (among the 3.5cm * 100cm), each application of sample amount is 30ml, employing contains 0.05mol/LTris2HCl damping fluid (pH 7.5) wash-out of NaCl, wherein contain NaCl concentration by 0 to 1mol/L straight line gradient elution, flow velocity is 0.5mL/min, part is collected, every pipe is collected 4mL, detect elution peak with 280nm, with disodium phenyl phosphate is the enzyme activity that substrate detects elution peak, and great-hearted elution peak is merged;
D, with the active eluant that merges with rotary film evaporator in vacuum concentration below 35 ℃, make wherein proteinic concentration reach about 1%;
E, the zymoprotein concentrated solution is added Sephadex G-150 gel filtration chromatography (2.5cm * 100cm), adopt constant pressure arrangement, pressure reduction is 0.01mol/L Tris 2HCl damping fluid (pH7.5 for the 20cm. elutriant, contain 0.2mol/L NaCl), flow velocity is 0.5mL/min, part is collected, the about 4mL of every pipe volume.Detecting elution peak with 280nm, is the enzyme activity that substrate detects elution peak with disodium phenyl phosphate, and great-hearted elution peak is merged;
F, with the active eluant that merges with rotary film evaporator in vacuum concentration below 35 ℃, when proteinic concentration wherein reach about 1% the time freezing after, adopt vacuum freeze-drying method to obtain the alkaline phosphatase pulvis.
In the present invention, the step detailed process 6. just extraction and the treating process of heparin is: 1. 2. 3. 4. after the step, implementing following processing step through aforesaid again:
A, filter residue is put into hydrolyzer, in 1: the 3-5 ratio adds 0.9% sodium chloride solution, transfer pH to 9.0 with sodium hydroxide after, be heated to 50-60 ℃, be incubated 2-3 hour;
B, enzymolysis: pH is adjusted into 8.0-8.5, temperature is adjusted into 37-40 ℃, add and be equivalent to the fresh pig pancreas oar that intestinal mucosa 0.8-1.0% has rubbed, also can add 0.25% commodity trypsinase, down insulation hydrolysis 3-4 hour of agitation condition, and keep by adding 10% sodium hydroxide solution that this pH's is stable.
C, filtration: after enzymolysis finished, adjusting pH with 10% dilute hydrochloric acid was 5.5-6.0,, be warming up to 90-95 ℃ then, agitation condition adds sodium-chlor to 5% down, and insulation 30min, and filtered while hot is removed insoluble impurities.Filter residue washs once with 5% sodium chloride solution filtrate again, and merging filtrate is the heparin crude extract.Filter residue is used for animal protein feed.
D, ion exchange resin absorption: the heparin crude extract is cooled to about 10 ℃, remove the surface solidification layer that may occur after leaving standstill, be heated to 40-45 ℃ then, after adjustment pH value is 8.5-9.0, slowly agitation condition adds in the D-204 macroporous adsorbent resin down, the consumption of resin is about the 2.5-3.0% of feed liquid, and adsorption time is 3-5 hour.Then, filter liquid, collect resin (heparin absorption is arranged) with the nylon cloth bag.
E, washing: water washes the resin after the absorption repeatedly, till washing fluid becomes clearly.
F, wash-out: remove partial impurities with the sodium chloride solution washing of 3-4% earlier; Remove dermatan sulfate with the washing of 12-15% sodium chloride solution again; Last resin is dipped in the sodium chloride solution of 25-30%, and after stirring 5-8 hour gently, filters, and relaunders resin one time with this sodium chloride solution, merges and collects filtrate.
G, alcohol precipitation: under poly-the stirring, in filtrate, add ethanol, make its final concentration reach 40-45%, leave standstill siphon upper strata alcoholic solution after 3-5 hour (the recyclable utilization of ethanol), get the precipitation part, after about 2 times of 1% sodium chloride solution redissolution, with ethanol secondary sedimentation 5-8 hour
H, dehydrate: throw out is with 95% or the dehydrated alcohol dehydration secondary of 2 times of amounts, and with the acetone dehydration once, filter is done again, and is slowly dry in the vacuum drying oven below 50 ℃.At this moment, tiring of heparin sodium can be more than 90USP u/mg.
I, crude heparin sodium is mixed with 10% solution with 1.5% sodium-chlor, adjust pH 1-2 with hydrochloric acid, filter rapidly, supernatant liquor is adjusted to pH 10-12 with sodium hydroxide solution again, add 30% hydrogen peroxide according to 3.5% amount, place more than 40 hours down, keep pH10-12 therebetween in 25 ℃, filter and collect filtrate then
After j, filtrate adjust pH6-7 with dilute hydrochloric acid, add the dehydrated alcohol of equivalent, leave standstill more than 12 hours after stirring evenly gently, get the precipitation part, and, be refined heparin sodium with the acetone after drying that dewaters.
Innovative point of the present invention is as follows:
(1) technology of the present invention can make the starting material chitterlings be utilized effectively, and can extract alkaline phosphatase, also can extract refining heparin sodium, helps realizing the comprehensive utilization of raw material, reduces production costs, and reduces the pollution of tankage to environment.
(2) there are many pieces of data to introduce the separation purification method and the character thereof of alkaline phosphatase, various animals and plants of research material order and microorganism.But these researchs all are to carry out under laboratory level, and the starting material treatment capacity is few, is not suitable for suitability for industrialized production.The present invention is according to the practical situation of industrial-scale production, the advantage of comprehensive the whole bag of tricks on the basis of forefathers' research, operational path and the technical parameter different have been adopted with present bibliographical information, can be under fairly large condition the separation and purification alkaline phosphatase, extract on this basis and refining heparin sodium simultaneously.This research has with the several remarkable difference of bibliographical information:
1. the document person adopts ammonium sulfate method fractionation precipitation alkaline phosphatase mostly, and the present invention adopts acetone precipitation;
2. in the document with the Tris-HCl damping fluid of sodium-chlor as zyme extract, the present invention is then containing the Tris-HCl buffer extraction enzyme of finite concentration magnesium chloride, sodium-chlor and zinc chloride, its advantage be can more effective protective enzyme activity.
3. the present invention adopts DEAE Sepharose 4B ion-exchange chromatography and Sephadex G-150 gel filtration chromatography column combination separation and purification alkaline phosphatase, and all chromatographic columns and eluent all are suitable for this zymin of mass production.
4. the present invention adopts the cryodesiccated method of first concentrate under reduced pressure at low temperature final vacuum to prepare zymin, and the advantage of this method is: the activity that both can guarantee enzyme is not suffered a loss, and can enhance productivity again, thereby reach the extraction effect of getting twice the result with half the effort.
(3) heparin sodium production at present adopts salt to separate technology mostly.The present invention is a raw material with the filter residue that extracts behind the alkaline phosphatase then, employing enzymolysis process and the enzymolysis parameter optimized through salt is molten after, and hydrolysis effect is better, and the yield of heparin is higher.The present invention adopts branch's elution technique in elution process, can will have the composition of different bonding forces progressively to wash off with ion exchange resin, thereby obtains refining heparin sodium.
Description of drawings
Accompanying drawing is a process flow sheet of the present invention.
Embodiment
The present invention is described further below in conjunction with the concrete preparation method and the process of each target compound, but does not limit protection scope of the present invention.
1, the separation of alkaline phosphatase and purifying
(1) chitterlings is cleaned up, get its mucous membrane after shredding, the 0.05mol/L Tris-HCl damping fluid (pH7.5 that adds 4-10 ℃ of precooling by solid-to-liquid ratio at 1: 3, include 0.01mol/L magnesium chloride-0.01mol/L sodium-chlor-0.01mol/L zinc chloride), fully homogenate is placed more than the 10h slurries in 4-10 ℃ of low temperature thereafter in hollander.
(2) propyl carbinol that adds precooling in the homogenate makes the concentration of propyl carbinol reach 20%-25% (V/V), and after abundant stirring, insulated and stirred 20-30min in not being higher than 37 ℃ of water-baths at room temperature places 8-10h then again.
(3) with behind plate-and-frame filter press press filtration or the whizzer centrifugal (3000-5000r/m), collect filtrate, precipitation is used to extract heparin.
(4) under agitation condition, in filtrate, add the equal-volume cold acetone, behind the mixing under rotating speed 3000-6000r/min centrifugal 10-20min, abandoning supernatant.
(5) add about 3-5 in precipitation and doubly measure (W/V) 0.05mol/L Tris-HCl damping fluid (pH7.5 includes 0.01mol/L magnesium chloride-0.01mol/L sodium-chlor-0.01mol/L zinc chloride), fully stirring makes its dissolving be gained alkaline phosphatase crude enzyme liquid.
(6) crude enzyme liquid is added with 0.05mol/L Tris-HCl damping fluid (pH7.5, including 0.01mol/L magnesium chloride-0.01mol/L sodium-chlor-0.01mol/L zinc chloride) the DEAESepharose 4B post crossed of balance is (among the 3.5cm * 100cm), each application of sample amount is 30ml, employing contains 0.05mol/L Tris2HCl damping fluid (pH 7.5) wash-out of NaCl, wherein contain NaCl concentration by 0 to 1mol/L straight line gradient elution, flow velocity is 0.5mL/min, part is collected, every pipe is collected 4mL, detect elution peak with 280nm, with disodium phenyl phosphate is the enzyme activity that substrate detects elution peak, and great-hearted elution peak is merged.
(7) use rotary film evaporator in vacuum concentration below 35 ℃ the active eluant that merges, make wherein proteinic concentration reach about 1%.
(8) the zymoprotein concentrated solution is added Sephadex G-150 gel filtration chromatography (2.5cm * 100cm), adopt constant pressure arrangement, pressure reduction is 0.01mol/L Tris 2HCl damping fluid (pH7.5 for the 20cm. elutriant, contain 0.2mol/L NaCl), flow velocity is 0.5mL/min, part is collected, the about 4mL of every pipe volume.Detecting elution peak with 280nm, is the enzyme activity that substrate detects elution peak with disodium phenyl phosphate, and great-hearted elution peak is merged.
(9) with the active eluant that merges with rotary film evaporator in vacuum concentration below 35 ℃, when proteinic concentration wherein reach about 1% the time freezing after, adopt vacuum freeze-drying method to obtain the alkaline phosphatase pulvis.
2, the extraction of heparin and refining
(1) lixiviate with intestinal mucosa extract alkaline phosphatase after filter residue put into hydrolyzer, in 1: the 3-5 ratio adds 0.9% sodium chloride solution, transfer pH to 9.0 with sodium hydroxide after, be heated to 50-60 ℃, be incubated 2-3 hour,
(2) enzymolysis: pH is adjusted into 8.0-8.5, temperature is adjusted into 37-40 ℃, add and be equivalent to the fresh pig pancreas oar that intestinal mucosa 0.8-1.0% has rubbed, also can add 0.25% commodity trypsinase, down insulation hydrolysis 3-4 hour of agitation condition, and keep by adding 10% sodium hydroxide solution that this pH's is stable.
(3) filter: after enzymolysis finished, adjusting pH with 10% dilute hydrochloric acid was 5.5-6.0,, be warming up to 90-95 ℃ then, agitation condition adds sodium-chlor to 5% down, and insulation 30min, and filtered while hot is removed insoluble impurities.Filter residue washs once with 5% sodium chloride solution filtrate again, and merging filtrate is the heparin crude extract.Filter residue is used for animal protein feed.
(4) ion exchange resin absorption: the heparin crude extract is cooled to about 10 ℃, remove the surface solidification layer that may occur after leaving standstill, be heated to 40-45 ℃ then, after adjustment pH value is 8.5-9.0, slowly agitation condition adds in the D-204 macroporous adsorbent resin down, the consumption of resin is about the 2.5-3.0% of feed liquid, and adsorption time is 3-5 hour.Then, filter liquid, collect resin (heparin absorption is arranged) with the nylon cloth bag.
(5) washing: water washes the resin after the absorption repeatedly, till washing fluid becomes clearly.
(6) wash-out: remove partial impurities with the sodium chloride solution washing of 3-4% earlier; Remove dermatan sulfate with the washing of 12-15% sodium chloride solution again; Last resin is dipped in the sodium chloride solution of 25-30%, and after stirring 5-8 hour gently, filters, and relaunders resin one time with this sodium chloride solution, merges and collects filtrate.
(7) alcohol precipitation: under poly-the stirring, in filtrate, add ethanol, make its final concentration reach 40-45%, leave standstill siphon upper strata alcoholic solution after 3-5 hour (the recyclable utilization of ethanol), get the precipitation part, after about 2 times of 1% sodium chloride solution redissolution, with ethanol secondary sedimentation 5-8 hour
(8) dehydrate: throw out with the acetone dehydration once, is filtered and done again with 95% or the dehydrated alcohol dehydration secondary of 2 times of amounts, and is slowly dry in the vacuum drying oven below 50 ℃.At this moment, tiring of heparin sodium can be more than 90USP u/mg.
(9) crude heparin sodium is mixed with 10% solution with 1.5% sodium-chlor, adjust pH 1-2 with hydrochloric acid, filter rapidly, supernatant liquor is adjusted to pH 10-12 with sodium hydroxide solution again, add 30% hydrogen peroxide according to 3.5% amount, place more than 40 hours down, keep pH10-12 therebetween in 25 ℃, filter and collect filtrate then
(10) after filtrate is adjusted pH6-7 with dilute hydrochloric acid, add the dehydrated alcohol of equivalent, leave standstill more than 12 hours after stirring evenly gently, get the precipitation part, and, be refined heparin sodium with the acetone after drying that dewaters.
3, the production of protein feed
Step (3) and step (9) gained throw out in the extraction of above-mentioned heparin and the treating process are promptly got protein fodder through super-dry.
Claims (2)
1. one kind is the technology of raw material joint production producing alkaline phosphatase and heparin sodium with the pig intestinal mucosa, and it is characterized in that: it may further comprise the steps:
1. the pig intestinal mucosa making beating is pulverized;
2. add the Tris-HCl damping fluid extraction that 0.05mol/L contains magnesium chloride, sodium-chlor and zinc chloride, this Tris-HCl damping fluid contains 0.01mol/L magnesium chloride, 0.01mol/L sodium-chlor, 0.01mol/L zinc chloride;
3. propyl carbinol precipitates;
4. press filtration or centrifugation;
5. step 4. in the separated liquid supernatant acetone precipitation, again according to this separation of DEAE-Sepharose 4B post, the vacuum concentration by 3.5cm * 100cm, promptly get the alkaline phosphatase enzyme product through the separation of Sephadex G-150 gel filtration chromatography, vacuum concentration, lyophilize, its detailed process is:
A, in filtrate, add the equal-volume cold acetone, behind the mixing under rotating speed 3000-6000r/min centrifugal 10-20min, abandoning supernatant;
B, add a small amount of 0.05mol/L Tris-HCl damping fluid in precipitation, this pH of buffer 7.5 includes 0.01mol/L magnesium chloride, 0.01mol/L sodium-chlor, 0.01mol/L zinc chloride, fully stirs to make its dissolving be gained alkaline phosphatase crude enzyme liquid;
C, crude enzyme liquid is added in the described DEAE-Sepharose 4B post of crossing with 0.05mol/L Tris-HCl damping fluid balance, described pH of buffer 7.5, include the 0.01mol/L magnesium chloride, 0.01mol/L sodium-chlor, 0.01mol/L zinc chloride, each application of sample amount is 30ml, employing contains the 0.05mol/L Tris-HCl buffer solution elution of the pH 7.5 of NaCl, wherein NaCl concentration by 0 to 1mol/L, linear gradient elution, flow velocity is 0.5mL/min, part is collected, and every pipe is collected 4mL, detects elution peak with 280nm, with disodium phenyl phosphate is the enzyme activity that substrate detects elution peak, and great-hearted elution peak is merged;
D, with the active eluant that merges with rotary film evaporator in vacuum concentration below 35 ℃, make wherein proteinic concentration reach about 1%;
E, the zymoprotein concentrated solution added the Sephadex G-150 gel filtration chromatography of 2.5cm * 100cm, adopt constant pressure arrangement, pressure reduction is 20cm, elutriant is pH 7.5, contains the 0.01mol/L Tris-HCl damping fluid of 0.2mol/L NaCl that flow velocity is 0.5mL/min, the part collection, the about 4mL of every pipe volume, detecting elution peak with 280nm, is the enzyme activity that substrate detects elution peak with disodium phenyl phosphate, and great-hearted elution peak is merged;
F, with the active eluant that merges with rotary film evaporator in vacuum concentration below 35 ℃, when proteinic concentration wherein reach about 1% the time freezing after, adopt vacuum freeze-drying method to obtain the alkaline phosphatase pulvis;
6. step 4. in isolated filter residue by lixiviate, enzymolysis, filtration, obtain heparin, dehydrate to such an extent that crude heparin sodium, hydrogen peroxide processing, filtration, filtrate dehydrate with acetone with ethanol sedimentation, throw out and promptly gets refined heparin sodium through the alcohol precipitation again through D-204 absorption with macroporous adsorbent resin, washing decon, wash-out, its detailed process is:
A, filter residue are put into hydrolyzer, and in 1: the 3-5 ratio adds 0.9% sodium chloride solution, behind sodium hydroxide accent pH to 9.0, are heated to 50-60 ℃, are incubated 2-3 hour;
B, enzymolysis: pH is adjusted into 8.0-8.5, temperature is adjusted into 37-40 ℃, add and be equivalent to the fresh pig pancreas oar that intestinal mucosa 0.8-1.0% has rubbed, or add 0.25% commodity trypsinase, down insulation hydrolysis 3-4 hour of agitation condition, and keep by adding 10% sodium hydroxide solution that this pH's is stable;
C, filtration: after enzymolysis finishes, adjusting pH with 10% dilute hydrochloric acid is 5.5-6.0, be warming up to 90-95 ℃ then, agitation condition adds sodium-chlor to 5% down, and insulation 30min, filtered while hot is removed insoluble impurities, and filter residue washs once with 5% sodium chloride solution again, merging filtrate is the heparin crude extract, and filter residue is used for animal protein feed;
D, ion exchange resin absorption: the heparin crude extract is cooled to about 10 ℃, remove the surface solidification layer that may occur after leaving standstill, be heated to 40-45 ℃ then, after adjustment pH value was 8.5-9.0, slowly agitation condition added in the D-204 macroporous adsorbent resin down, and the consumption of resin is about the 2.5-3.0% of feed liquid, adsorption time is 3-5 hour, then, filter liquid, collect the resin that has adsorbed heparin with the nylon cloth bag;
E, washing: water washes the resin after the absorption repeatedly, till washing fluid becomes clearly;
F, wash-out: remove partial impurities with the sodium chloride solution washing of 3-4% earlier; Remove dermatan sulfate with the washing of 12-15% sodium chloride solution again; Last resin is dipped in the sodium chloride solution of 25-30%, and after stirring 5-8 hour gently, filters, and relaunders resin one time with this sodium chloride solution, merges and collects filtrate;
G, alcohol precipitation: under poly-the stirring, in filtrate, add ethanol, make its final concentration reach 40-45%, leave standstill siphon upper strata alcoholic solution after 3-5 hour, get the precipitation part, after redissolving with about 2 times of 1% sodium chloride solution, with ethanol secondary sedimentation 5-8 hour;
H, dehydrate: throw out is with 95% or the dehydrated alcohol dehydration secondary of 2 times of amounts, and with the acetone dehydration once, filter is done again, and slowly dry in the vacuum drying oven below 50 ℃, at this moment, tiring of heparin sodium can be more than 90USP u/mg;
I, crude heparin sodium is mixed with 10% solution with 1.5% sodium-chlor, adjust pH 1-2 with hydrochloric acid, filter rapidly, supernatant liquor is adjusted to pH 10-12 with sodium hydroxide solution again, add 30% hydrogen peroxide according to 3.5% amount, place more than 40 hours down in 25 ℃, keep pH10-12 therebetween, filter and collect filtrate then;
After j, filtrate adjust pH6-7 with dilute hydrochloric acid, add the dehydrated alcohol of equivalent, leave standstill more than 12 hours after stirring evenly gently, get the precipitation part, and, be refined heparin sodium with the acetone after drying that dewaters.
2. the joint process of production alkaline phosphatase according to claim 1 and heparin sodium is characterized in that: the filter residue drying that step twice in 6. filters gained is protein fodder.
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| CN101773522B (en) * | 2010-03-03 | 2011-12-21 | 中国农业科学院饲料研究所 | Animal intestinal mucosa extract and preparation method as well as application thereof |
| CN102643792B (en) * | 2011-02-17 | 2015-04-22 | 深圳华大基因科技服务有限公司 | RNA fragmentation reagent and application thereof |
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