CN103555699A - Technology for improving valent of complex rennin - Google Patents

Technology for improving valent of complex rennin Download PDF

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Publication number
CN103555699A
CN103555699A CN201310552732.7A CN201310552732A CN103555699A CN 103555699 A CN103555699 A CN 103555699A CN 201310552732 A CN201310552732 A CN 201310552732A CN 103555699 A CN103555699 A CN 103555699A
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Prior art keywords
rennh
compound
solution
gained
tired
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CN201310552732.7A
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Inventor
陈猛
韩红蕊
华晓杰
肖艳红
刘东梅
王丽英
张丽丽
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Kuihua Pharmaceutical Group (tangshan) Biological Pharmaceutical Co Ltd
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Kuihua Pharmaceutical Group (tangshan) Biological Pharmaceutical Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6478—Aspartic endopeptidases (3.4.23)
    • C12N9/6483—Chymosin (3.4.23.4), i.e. rennin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/23—Aspartic endopeptidases (3.4.23)
    • C12Y304/23004—Chymosin (3.4.23.4), i.e. rennin

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention relates to a technology for improving the valent of complex rennin, belonging to the field of biopharmaceutical technology. The technology for improving the valent of complex rennin comprises the step of acidifying complex rennin solution through acid solution. In the technology, all steps and parameters are matched for combined action, so that the refining technology is short in reaction time, mild in reaction conditions, and free from polluting the environment with the use of a great amount of organic solvents, the in-vitro rennin vigor and protein vigor of the complex rennin are further improved, the medical effect of the complex rennin can be fully played on the original basis, and the clinical usage efficiency can be increased.

Description

A kind ofly improve the technique that Compound rennh is tired
Technical field
The present invention relates to a kind of technique that biological medicament is tired that improves, relate in particular to a kind of technique that Compound rennh is tired that improves, belong to biological pharmacy technical field.
Background technology
Disease of stomach is the organic or functional disease that betides stomach.Common are clinically chronic gastritis, peptide ulceration, gastroptosis and cancer of the stomach etc.The cause of disease is very complicated, comprises physicochemical irritation, infection, toxin, heredity, mental element, dysplasia, operation impact etc.Cardinal symptom has epigastric pain, the heart that burns, feel sick, vomiting, belch, sour regurgitation, poor appetite, spitting blood, have blood in stool, lump etc.Disease of stomach is having a strong impact on the healthy of people.
Biochemical drug is because it is from organism, composition belongs to biomacromolecule more, easily absorbed by the body, and participate in the metabolism of human body directly, can regulate, supplement, recover and maintain the normal function of human body, therefore have good effect, side effect is little, targeting is strong, the advantages such as instant effect, are more and more paid attention to by people.
Compound rennh is a kind of biochemical drug, and it take lamb sheep fourth stomach as main extract.The external curdled milk vigor of every 1g Compound rennh must not be less than 800 curdled milk units; Containing proteinase activity, must not be less than 750 units.Yet, in existing document, there is no report and further improve the technology that Compound rennh is tired, for giving full play to the medical functions of Compound rennh, research further improves its technology of tiring and is very important.
Summary of the invention
Technical problem to be solved by this invention is that the defect that overcomes prior art provides a kind of technique that Compound rennh is tired that improves, and this technique has that the time is short, reaction conditions is gentle, free from environmental pollution, the further advantages of higher of the external curdled milk vigor of gained Compound rennh.
Technical problem of the present invention is realized by following technical scheme.
Improve the technique that Compound rennh is tired, it comprises the step with acid solution acidifying Compound rennh solution.
The technique that above-mentioned raising Compound rennh is tired, described acid solution is 5%--10% hydrochloric acid or glacial acetic acid solution.
The technique that above-mentioned raising Compound rennh is tired, described acidifying is pH value to 3.5-4.5.
The technique that above-mentioned raising Compound rennh is tired, it comprises the steps:
(1) former material is prepared: get Compound rennh meal 15-20g, add 8-10 times of distilled water, and after stirring, solution for standby;
(2) acidifying: get step (1) gained solution and regulate pH value to 3.5-4.5 with 5%--10% hydrochloric acid or glacial acetic acid solution, and constantly stir 2 hours in 30 ℃, standby;
(3) collecting precipitation: step (2) gained souring soln is crossed to 60 mesh sieves, in filtrate, add filtrate weight 3-4 acetone or alcohol doubly, after stirring, standing 10 minutes, put suction filtration in Büchner funnel, and with original material amount 1-2 acetone or alcohol washing doubly 3-4 time, put in vacuum drying oven 40 ℃ and be dried, obtain Compound rennh.
The technique that above-mentioned raising Compound rennh is tired, it comprises the steps:
(1) former material is prepared: get Compound rennh 17g, add 9 times of distilled water, and after stirring, solution for standby;
(2) acidifying: get step (1) gained solution and regulate pH value to 4 with 8% hydrochloric acid or glacial acetic acid solution, and constantly stir 2 hours in 30 ℃, standby;
(3) collecting precipitation: step (2) gained souring soln is crossed to 60 mesh sieves, to the acetone or alcohol that adds 3.5 times of filtrate weight in filtrate, after stirring, standing 10 minutes, put suction filtration in Büchner funnel, and with 1.5 times of acetone or alcohols of original material amount washing 3 times, put in vacuum drying oven 40 ℃ dry, obtain Compound rennh.
A kind of technique that Compound rennh is tired that improves of the present invention, between each step, each parameter, cooperatively interact, acting in conjunction, make this technique have that not only the reaction times is short, reaction conditions is gentle, do not use a large amount of organic solvents free from environmental pollution, the external curdled milk vigor of gained Compound rennh is up to 1070 curdled milk units, protein vigor, up to 1000 units, can be given full play to the medical functions of Compound rennh on the original basis, and its clinical service efficiency is provided.
Embodiment
Below in conjunction with embodiment, the present invention is described in further details.
The preparation of embodiment 1 Compound rennh meal
Step is as follows:
(1) former material preparation: get lamb sheep fourth stomach 300g, put the broken end that is twisted into 180mm diameter in mincer, standby;
(2) thorn activating signal activation: the broken end of step (1) gained is put into beaker and added purified water 350ml, then put in water-bath and heat, be warmed up to 41 ℃, after stirring, hydrochloric acid soln with 8% regulates pH value to 4, constantly stirs and activates 3.5 hours, lets cool, 80 order filter-cloth filterings, collect filtrate, standby;
(3) collecting precipitation: the acetone to adding 3.5 times of filtrate weight in step (2) gained filtrate, stir 20 minutes, standing 20 minutes, the supernatant liquor collecting precipitation thing that inclines, standby;
(4) target product: step (3) gained throw out is put in beaker and is added 750ml acetone, stir 10 minutes, the acetone that inclines, by this method repetitive scrubbing 5 times, then washes is put in vacuum drying oven 40 ℃ dry, obtain 77 grams of Compound rennhs, yield 25.6%.
The preparation of embodiment 2 Compound rennh meal
Step is as follows:
(1) former material preparation: get lamb sheep fourth stomach 300g, put the broken end that is twisted into 180mm diameter in mincer, standby;
(2) thorn activating signal activation: the broken end of step (1) gained is put into beaker and added purified water 350ml, then put in water-bath and heat, be warmed up to 41 ℃, after stirring, glacial acetic acid solution with 8% regulates pH value to 4, constantly stirs and activates 3.5 hours, lets cool, 80 order filter-cloth filterings, collect filtrate, standby;
(3) collecting precipitation: the ethanol to adding 3.5 times of filtrate weight in step (2) gained filtrate, stir 20 minutes, standing 20 minutes, the supernatant liquor collecting precipitation thing that inclines, standby;
(4) target product: step (3) gained throw out is put in beaker and is added 750ml ethanol, stir 10 minutes, the ethanol that inclines, by this method repetitive scrubbing 5 times, then washes is put in vacuum drying oven 40 ℃ dry, obtain 78 grams of Compound rennhs, yield 26.3%.
The raising technique that embodiment 3 embodiment 1 gained Compound rennh meal are tired
Step is as follows:
(1) former material is prepared: get Compound rennh meal 15g, add 8 times of distilled water, and after stirring, solution for standby;
(2) acidifying: get step (1) gained solution and regulate pH value to 3.5 with 5% hydrochloric acid soln, and constantly stir 2 hours in 30 ℃, standby;
(3) collecting precipitation: step (2) gained souring soln is crossed to 60 mesh sieves, to the acetone that adds 3 times of filtrate weight in filtrate, after stirring, standing 10 minutes, put suction filtration in Büchner funnel, and with the washing with acetone of 1 times of original material amount 3 times, put in vacuum drying oven 40 ℃ dry, obtain 13.5 grams of Compound rennhs.
The raising technique that embodiment 4 embodiment 2 Compound rennh meal are tired
Step is as follows:
(1) former material is prepared: get Compound rennh meal 20g, add 10 times of distilled water, and after stirring, solution for standby;
(2) acidifying: get step (1) gained solution and regulate pH value to 4.5 with 10% glacial acetic acid solution, and constantly stir 2 hours in 30 ℃, standby;
(3) washing: step (2) gained souring soln is crossed to 60 mesh sieves, to the acetone that adds 4 times of filtrate weight in filtrate, after stirring, standing 10 minutes, put suction filtration in Büchner funnel, and with the washing with acetone of 2 times of original material amounts 4 times, put in vacuum drying oven 40 ℃ dry, obtain 18.4 grams of Compound rennhs.
The raising technique that embodiment 5 embodiment 1 gained Compound rennh meal are tired
Step is as follows:
(1) former material is prepared: get Compound rennh meal 17g, add 9 times of distilled water, and after stirring, solution for standby;
(2) acidifying: get step (1) gained solution and regulate pH value to 4 with 8% hydrochloric acid soln, and constantly stir 2 hours in 30 ℃, standby;
(3) washing: step (2) gained souring soln is crossed to 60 mesh sieves, to the ethanol that adds 3.5 times of filtrate weight in filtrate, after stirring, standing 10 minutes, put suction filtration in Büchner funnel, and by the washing with alcohol of 1.5 times of original material amounts 3 times, put in vacuum drying oven 40 ℃ dry, obtain 16.3 grams of Compound rennhs.
The raising technique that embodiment 6 embodiment 2 gained Compound rennh meal are tired
Step is as follows:
(1) former material is prepared: get Compound rennh meal 17g, add 9 times of distilled water, and after stirring, solution for standby;
(2) acidifying: get step (1) gained solution and regulate pH value to 4 with 8% glacial acetic acid solution, and constantly stir 2 hours in 30 ℃, standby;
(3) washing: step (2) gained souring soln is crossed to 60 mesh sieves, to the ethanol that adds 3.5 times of filtrate weight in filtrate, after stirring, standing 10 minutes, put suction filtration in Büchner funnel, and by the washing with alcohol of 1.5 times of original material amounts 3 times, put in vacuum drying oven 40 ℃ dry, obtain 16.2 grams of Compound rennhs.
The raising technique that the commercially available Compound rennh of embodiment 7 is tired
Step is as follows:
(1) former material is prepared: get Compound rennh 17g, add 9 times of distilled water, and after stirring, solution for standby;
(2) acidifying: get step (1) gained solution and regulate pH value to 4 with 8% glacial acetic acid solution, and constantly stir 2 hours in 30 ℃, standby;
(3) washing: step (2) gained souring soln is crossed to 60 mesh sieves, to the acetone that adds 3.5 times of filtrate weight in filtrate, after stirring, standing 10 minutes, put suction filtration in Büchner funnel, and with the washing with acetone of 1.5 times of original material amounts 3 times, put in vacuum drying oven 40 ℃ dry, obtain 15.9 grams of Compound rennhs.
Embodiment 8 gets embodiment 1 to 7 gained and commercially available Compound rennh carries out titration
[rennin]
1. whole milk powder 12g is got in the preparation of substrate solution, adds calcium chloride solution and (gets sodium-acetate 2.05g, calcium chloride 1.11g, add water and make to be dissolved into 1000ml, regulate pH value to 6.3 ± 0.1) appropriate, grind evenly, put in 100ml measuring bottle, add calcium chloride solution to scale, shake up.This liquid should face with newly joining.
2. rennet standard product are got in the preparation of standard solution, accurately weighed, add phosphate buffered saline buffer (PH6.3) and (get SODIUM PHOSPHATE, MONOBASIC 7.8g, Sodium phosphate dibasic 2.7g, add water and make to be dissolved into 1000ml, regulate pH value to 6.3 ± 0.05) dissolve and be diluted in every 1ml the solution containing 0.4～0.6 unit of activity.This liquid should remain on below 20 ℃, and uses in latter 2 hours of preparation.
3. Preparation Example 1 to 7 gained and the commercially available Compound rennh of need testing solution proceed as follows respectively; get the about 0.1g of this product; accurately weighed; put in mortar; add phosphate buffered saline buffer (PH6.3) a small amount of, grind evenly, put in 100ml measuring bottle; add phosphate buffered saline buffer (PH6.3) to scale, shake up.This liquid should remain on below 20 ℃, faces with newly joining.
4. assay method precision measures substrate solution 10ml, put in test tube, in 30 ℃ ± 0.5 ℃ water bath with thermostatic control, be incubated 10 minutes, precision adds standard solution 1ml, shake up and timing immediately, with glass stick, constantly dip solution and flow down along tube wall, make into even thin layer, when visible particle appears in thin layer, show curdled milk, accurate recording after standard solution adds to the time that occurs curdled milk phenomenon.Replication three times, the relative standard deviation of three measured values must not be greater than 5%.Get the mean value of three times as curdled milk time of standard solution.Take embodiment 1 to 7 gained and the prepared need testing solution 1ml of commercially available Compound rennh, operation, is calculated as follows in accordance with the law.
Figure BDA0000411472710000051
The vigor of S-------standard substance in formula, Ru/mg;
The average curdled milk time of Ts-------standard solution, second;
The average curdled milk time of T--------need testing solution, second;
In the every 1ml of Ws--------standard solution, contain the amount of rennet standard product, g;
W-------trial-product sampling amount, g;
N--------trial-product extension rate.
Note: curdled milk time circulation ratio in the time of 180～240 seconds is best.Otherwise test liquid concentration should be adjusted.
[proteolytic enzyme]
1. the preparation precision of reference product solution takes that through 105 ℃, to be dried to constant weight tyrosine appropriate, adds hydrochloric acid soln (get 1mol/L hydrochloric acid soln 65ml, add water to 1000ml) and makes in every 1ml the solution containing 0.4mg.
2. Preparation Example 1 to 7 gained of need testing solution and commercially available Compound rennh proceed as follows respectively and get the about 0.2g of this product, accurately weighed, with above-mentioned hydrochloric acid soln, grind and make to dissolve, and put in 100ml measuring bottle, add above-mentioned hydrochloric acid soln to scale, shake up.
3. assay method is measured according to the method under stomach egg enzyme activity determination item (Chinese Pharmacopoeia 2010 version two 530 pages).
[result] is in Table 1.
Tiring of table 1 embodiment 1 to 7 gained and commercially available Compound rennh
? Commercially available 1 2 3 4 5 6 7
External curdled milk vigor (curdled milk unit) 800 950 940 980 970 1050 1070 1030
Proteinase activity (unit) 750 900 900 930 940 990 1000 990
Above-described embodiment is only explanation technical conceive of the present invention and advantage; the present invention also can have other variation; as well known to the skilled person; above-described embodiment only plays the exemplary role in foregoing invention protection domain; for those of ordinary skills; in the protection domain limiting in the present invention, also have a lot of conventional distortion and other embodiment, these distortion and embodiment are by within the protection domain awaiting the reply in the present invention.

Claims (5)

1. improve the technique that Compound rennh is tired, it comprises the step with acid solution acidifying Compound rennh solution.
2. the technique that raising Compound rennh according to claim 1 is tired, is characterized in that, described acid solution is 5%--10% hydrochloric acid or glacial acetic acid solution.
3. the technique that raising Compound rennh according to claim 2 is tired, is characterized in that, described acidifying is pH value to 3.5-4.5.
4. the technique that raising Compound rennh according to claim 3 is tired, is characterized in that, it comprises the steps:
(1) former material is prepared: get Compound rennh meal 15-20g, add 8-10 times of distilled water, and after stirring, solution for standby;
(2) acidifying: get step (1) gained solution and regulate pH value to 3.5-4.5 with 5%--10% hydrochloric acid or glacial acetic acid solution, and constantly stir 2 hours in 30 ℃, standby;
(3) washing: step (2) gained souring soln is crossed to 60 mesh sieves, in filtrate, add filtrate weight 3-4 acetone or alcohol doubly, after stirring, standing 10 minutes, put suction filtration in Büchner funnel, and with original material amount 1-2 acetone or alcohol washing doubly 3-4 time, put in vacuum drying oven 40 ℃ and be dried, obtain Compound rennh.
5. the technique that raising Compound rennh according to claim 4 is tired, is characterized in that, it comprises the steps:
(1) former material is prepared: get Compound rennh 17g, add 9 times of distilled water, and after stirring, solution for standby;
(2) acidifying: get step (1) gained solution and regulate pH value to 4 with 8% hydrochloric acid or glacial acetic acid solution, and constantly stir 2 hours in 30 ℃, standby;
(3) washing: step (2) gained souring soln is crossed to 60 mesh sieves, to the acetone or alcohol that adds 3.5 times of filtrate weight in filtrate, after stirring, standing 10 minutes, put suction filtration in Büchner funnel, and with the acetone or alcohol washing of 1.5 times of original material amounts 3 times, put in vacuum drying oven 40 ℃ dry, obtain Compound rennh.
CN201310552732.7A 2013-11-11 2013-11-11 Technology for improving valent of complex rennin Pending CN103555699A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1924012A (en) * 2006-09-01 2007-03-07 上海阿敏生物技术有限公司 Preparation method for rennin

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1924012A (en) * 2006-09-01 2007-03-07 上海阿敏生物技术有限公司 Preparation method for rennin

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
KLEINER I. S.ET AL: "Studies on renin II,The isolating of prorennin", 《CHEM》 *
张富新: "羔羊凝乳酶提取分离及特性的研究", 《中国博士学位论文全文数据库》 *
张富新等: "羔羊皱胃酶提取技术的研究", 《工艺技术》 *
赵胜娟等: "羔羊皱胃酶提取工艺研究", 《河南农业科学》 *

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Application publication date: 20140205