CN103173421B - Preparation method of solid enzyme compound for testing content of fructose diphosphate sodium - Google Patents
Preparation method of solid enzyme compound for testing content of fructose diphosphate sodium Download PDFInfo
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- CN103173421B CN103173421B CN201310089764.8A CN201310089764A CN103173421B CN 103173421 B CN103173421 B CN 103173421B CN 201310089764 A CN201310089764 A CN 201310089764A CN 103173421 B CN103173421 B CN 103173421B
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Abstract
The invention discloses a preparation method of a solid enzyme compound for testing the content of fructose diphosphate sodium. A compound of aldolase (ALD), triosephosphate isomerase (TIM) and glycerol phosphatedehydrogenase (GDH) is extracted from rabbit meat serving as raw material through splitting and centrifuging methods, wherein the splitting method is an alkaline splitting method, and an alkaline solution with a concentration of 0.01-0.1M is added in every1mL/g of the raw material. By adopting the technical scheme, the final yield, the unit enzyme activity and the stability in volume production of the solid enzyme compound can be effectively increased.
Description
Technical field
the present invention relates to a kind of for measuring the preparation method of solid enzyme mixture of fructose diphosphate sodium content.
Background technology
fructose Diphosphate content assaying method mainly contains high performance liquid phase (HPLC) method, ultraviolet (UV) absorption process, gas-chromatography (GC) method, number of chemical colorimetry and enzymatic assays.Wherein enzymatic assays specificity by force, accurately rapid.But the zymohexase (ALD), triosephosphate isomerase (TIM) and the glycerolphos phate dehydrogenase (GDH) that relate to enzymatic assays are expensive, cause cost of determination very high.For head it off, present stage has patent 200910091934.X, from animal muscle, extract solid enzyme mixture substitute more than three kinds of enzymes, obtain certain achievement.But the method with enzyme in water extraction muscle of mentioning in this patent, must cause lysis incomplete, and extraction efficiency is low, finally causes underproduce.Filtered through gauze method is difficult at utmost filter the zyme extract in meat gruel, also can reduce ultimate capacity; And meat gruel utmost point thickness, manual compression gauze efficiency is difficult to stdn, affects subsequent extracted operation.In ammonium sulfate precipitation method, do not provide concrete data, be unfavorable for the stability of batch production; And deposition condition can not effectively improve unit enzyme lives, and ammonium sulfate saturation ratio reaches 70% above postprecipitation thing and mostly is foreign protein.Therefore original technical scheme production efficiency is low and cost is high.
Summary of the invention
technical problem to be solved by this invention is to provide a kind of improved for measuring the preparation method of solid enzyme mixture of fructose diphosphate sodium content.
for solving above technical problem, the present invention adopts following technical scheme:
a kind of for measuring the preparation method of solid enzyme mixture of fructose diphosphate sodium content, taking rabbit meat as raw material, from described raw material, extract zymohexase (ALD), triosephosphate isomerase (TIM) and glycerolphos phate dehydrogenase (GDH) mixture by cracking, centrifugal method, the described alkaline lysis that is cracked into, adds the basic solution of 0.01-0.1M in the raw material described in every 1mL/g.
preferably, described for measuring the preparation method of solid enzyme mixture of fructose diphosphate sodium content, comprise the following steps of carrying out successively:
step 1, described raw material is broken into muddy flesh;
step 2, described muddy flesh is placed in to container, in the raw material described in every 1mL/g, adds the described basic solution of 0.01-0.1M, stir 10-20 minute;
step 3, frozen centrifugation 10-20 minute, be transferred to supernatant liquor in new container;
step 4, the throw out after centrifugal is carried out repeatedly to alkaline lysis, frozen centrifugation with the described basic solution of 0.01-0.1M again, repeatedly centrifugal supernatant liquor is transferred in described new container;
step 5, in described new container, slowly add the saturated ammonium sulphate solution of mass content at 90-97%, the saturation ratio that limit edged is stirred to final ammoniumsulphate soln is 30-40%, regulates pH to 5.0 ± 0.5, places 20-40 minute;
step 6, by the solution frozen centrifugation 10-20 minute of step 5 gained, getting supernatant liquor, to add mass content be 50-60% in the saturated ammonium sulphate solution of 90-97% to the saturation ratio of final ammoniumsulphate soln again, regulates pH to 3.5 ± 0.5, places 20-40 minute;
step 7, by the solution frozen centrifugation 10-20 minute of step 6 gained, by throw out, in-20 DEG C of pre-freezes, then freeze-drying obtains described solid enzyme mixture.
preferably, rotating speed when centrifugal in described step 3 is 5500-7000rpm.
preferably, rotating speed when centrifugal in described step 4 is 5500-7000rpm.
preferably, rotating speed when centrifugal in described step 6 is 8000-10000rpm.
preferably, rotating speed when centrifugal in described step 7 is 8000-10000rpm.
preferably, described basic solution is KOH solution or NaOH solution.
preferably, the preparation process of described solid enzyme mixture is carried out at 0-4 DEG C.
preferably, the muscle of back that described raw material is rabbit or huckle muscle or both mixtures.
beneficial effect of the present invention is:
by adopting the technical program, can effectively improve the stability of ultimate capacity, unit enzyme activity and the batch production of solid enzyme mixture.
Brief description of the drawings
accompanying drawing 1 is the impact of different saturation gradient ammoniumsulphate soln on unit enzyme activity in precipitation;
accompanying drawing 2 is the impacts of different pH values on unit enzyme activity in precipitation;
accompanying drawing 3 is embodiment 1 and the output of comparative example 1, the comparison of unit enzyme activity.
Embodiment
the present invention is further elaborated below.
embodiment 1
get house and exempt from one, fixing, after sudden death, peeling extracts back and huckle muscle, is placed on trash ice, shreds with scissors simultaneously.Meat mincing are broken into muddy flesh by the mincer that is used in-20 DEG C of refrigerator precoolings, weighs.Be placed in beaker, add the KOH solution of 0.05M according to the amount of 1mL/g meat mincing, uniform stirring after 15 minutes in centrifugal 10 minutes of 4 DEG C of refrigerated centrifuge 7000rpm.Supernatant is transferred in new beaker, meat gruel precipitation again with the 0.05M KOH same method extracting of half amount and centrifugal after, merge two times centrifugal supernatant liquor, above operation is all carried out on trash ice or under 4 DEG C of environment.Slowly add saturated ammonium sulphate solution (706g/L, 4 DEG C), limit edged is stirred to final 40% saturation ratio, regulates pH to 5.0, places 30 minutes on trash ice.4 DEG C afterwards, 8000rpm frozen centrifugation 15 minutes, gets supernatant liquor and adds extremely final 60% saturation ratio of saturated ammonium sulphate solution again, regulates pH to 3.5, places 30 minutes on trash ice.Finally, 4 DEG C, 10000rpm frozen centrifugation 20 minutes, obtains and is deposited in-20 DEG C of pre-freezes, the freeze drier freeze-drying of spending the night.Packing after lyophilized powder porphyrize, measures its unit enzyme according to official method and lives, and is greater than 40 μ g as qualified product taking the amount of every 1g solid enzyme mixture per minute catalysis Fructose Diphosphate, and-20 DEG C of preservations, for the mensuration of fructose diphosphate sodium content.
experimental result is shown in accompanying drawing 1 and accompanying drawing 2, and accompanying drawing 1 is family's rabbit muscle centrifugal gained supernatant after the KOH of 0.05M cracking, successively it is precipitated with the ammoniumsulphate soln of different saturation, surveys the unit enzyme activity in mixture after precipitating freeze-drying.In figure, the ammoniumsulphate soln of 40-50% and 50-60% saturation ratio gradient is conducive to the raising of unit enzyme activity in precipitation;
accompanying drawing 2 is family's rabbit muscle centrifugal gained supernatant after the KOH of 0.05M cracking, under the ammoniumsulphate soln condition of 40% saturation ratio, regulates different pH values to precipitate it, after precipitation freeze-drying, surveys the unit enzyme activity in mixture.In figure, under pH4 condition, in precipitation, unit enzyme activity significantly improves.
comparative example 1
get house and exempt from one, fixing, after sudden death, peeling extracts back and huckle muscle, under low temperature, be twisted into muddy flesh (500 milliliters) with mincer, be contained in 1000 ml beakers, add equal-volume water to stir after 15 minutes and carry out coarse filtration with medical 3-4 layer gauze extruding, filter residue is used half water gaging secondary extracting again, merge filtrate twice, add standard reagent (content is more than 98%) solid ammonium sulfate to saturated (solid ammonium sulfate does not dissolve), limit edged is mixed, after adding, putting refrigerator cold-storage (5 DEG C of left and right) deposits 30 minutes, liquid storage frozen centrifugation 30 minutes, rotating speed is 4000 revs/min (when rotating speed is excessive, macromole is by separated), get centrifuged supernatant again fine filtering " by asbestos filter plate Büchner funnel " remove the precipitation suspended substance remaining.In clear liquid, add again the saturation ratio of standard reagent (content is more than 98%) solid ammonium sulfate to 80%.Then by this liquid with 7000 revs/min of (this rotating speed can separate effective macromole) frozen centrifugations 30 minutes, be precipitated as pink mashed prod and be divided in culture dish, lyophilize obtains pink solid.Packing after porphyrize, measures its unit enzyme according to official method and lives, and is greater than 40 μ g as qualified product taking the amount of every 1g solid enzyme mixture per minute catalysis Fructose Diphosphate, and-20 DEG C of preservations, for the mensuration of fructose diphosphate sodium content.
embodiment 2
the vigour-testing method of special solid prozyme:
state's pharmacopeia is sent out in (2009) No. 12 files (Fructose Diphosphate and preparation quality standard exposure draft thereof): the solid complex enzyme reagent in Fructose Diphosphate (FDP) detection method.Experimental result is shown in accompanying drawing 3, adopts respectively two kinds of methods to extract the solid enzyme mixture in rabbit motion muscle, after freeze-drying, it is weighed and analytical unit enzyme activity.After in figure, solid enzyme mixture adopts embodiment 1 preparation, output significantly improves approximately 2.0 times, and unit enzyme activity also significantly improves approximately 1.9 times, * P<0.001.
above-described embodiment is only explanation technical conceive of the present invention and feature, and its object is to allow person skilled in the art can understand content of the present invention and implement according to this, can not limit the scope of the invention with this.All equivalences that spirit is done according to the present invention change or modify, within all should being encompassed in protection scope of the present invention.
Claims (8)
1. for measuring the preparation method of solid enzyme mixture of fructose diphosphate sodium content, taking rabbit meat as raw material, from described raw material, extract zymohexase (ALD), triosephosphate isomerase (TIM) and glycerolphos phate dehydrogenase (GDH) mixture by cracking, centrifugal method, it is characterized in that: the described alkaline lysis that is cracked into, adds the basic solution of 1ml 0.01-0.1M concentration range in the raw material described in every 1g; Comprise the following steps of carrying out successively:
Step 1, described raw material is broken into muddy flesh;
Step 2, described muddy flesh is placed in to container, in the raw material described in every 1mL/g, adds the described basic solution of 0.01-0.1M concentration range, stir 10-20 minute;
Step 3, frozen centrifugation 10-20 minute, be transferred to supernatant liquor in new container;
Step 4, the throw out after centrifugal is carried out repeatedly to alkaline lysis, frozen centrifugation with the described basic solution of 0.01-0.1M concentration range again, repeatedly centrifugal supernatant liquor is transferred in described new container;
Step 5, in described new container, slowly add the saturated ammonium sulphate solution of mass content at 90-97%, the saturation ratio that limit edged is stirred to final ammoniumsulphate soln is 30-40%, regulates pH to 5.0 ± 0.5, places 20-40 minute;
Step 6, by the solution frozen centrifugation 10-20 minute of step 5 gained, getting supernatant liquor, to add mass content be 50-60% in the saturated ammonium sulphate solution of 90-97% to the saturation ratio of final ammoniumsulphate soln again, regulates pH to 3.5 ± 0.5, places 20-40 minute;
Step 7, by the solution frozen centrifugation 10-20 minute of step 6 gained, by throw out, in-20 DEG C of pre-freezes, then freeze-drying obtains described solid enzyme mixture.
2. according to claim 1 for measuring the preparation method of solid enzyme mixture of fructose diphosphate sodium content, it is characterized in that: rotating speed when centrifugal in described step 3 is 5500-7000rpm.
3. according to claim 1 for measuring the preparation method of solid enzyme mixture of fructose diphosphate sodium content, it is characterized in that: rotating speed when centrifugal in described step 4 is 5500-7000rpm.
4. according to claim 1 for measuring the preparation method of solid enzyme mixture of fructose diphosphate sodium content, it is characterized in that: rotating speed when centrifugal in described step 6 is 8000-10000rpm.
5. according to claim 1 for measuring the preparation method of solid enzyme mixture of fructose diphosphate sodium content, it is characterized in that: rotating speed when centrifugal in described step 7 is 8000-10000rpm.
6. according to claim 1 for measuring the preparation method of solid enzyme mixture of fructose diphosphate sodium content, it is characterized in that: described basic solution is KOH solution or NaOH solution.
7. according to claim 1 for measuring the preparation method of solid enzyme mixture of fructose diphosphate sodium content, it is characterized in that: the preparation process of described solid enzyme mixture is carried out at 0-4 DEG C.
8. according to claim 1 for measuring the preparation method of solid enzyme mixture of fructose diphosphate sodium content, it is characterized in that: the muscle of back that described raw material is rabbit or huckle muscle or both mixtures.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5162221A (en) * | 1989-12-07 | 1992-11-10 | Forschungszentrum Juelich Gmbh | Fructose-1,6-bisphosphate aldolase, a process for the preparation thereof and its use |
| CN101691566B (en) * | 2009-09-02 | 2010-09-01 | 赵军 | Special solid complex enzyme (ALD, TIM and GDH) reagent for determining content of fructose diphosphate sodium (FDP) |
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2013
- 2013-03-20 CN CN201310089764.8A patent/CN103173421B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5162221A (en) * | 1989-12-07 | 1992-11-10 | Forschungszentrum Juelich Gmbh | Fructose-1,6-bisphosphate aldolase, a process for the preparation thereof and its use |
| CN101691566B (en) * | 2009-09-02 | 2010-09-01 | 赵军 | Special solid complex enzyme (ALD, TIM and GDH) reagent for determining content of fructose diphosphate sodium (FDP) |
Non-Patent Citations (4)
| Title |
|---|
| CRYSTALLINE ALDOLASE;John Fuller Taylor, 等;《The Journal of Biological Chemistry》;19480401;第173卷;第594页最后一段，第594页第1段和表I * |
| John Fuller Taylor, 等.CRYSTALLINE ALDOLASE.《The Journal of Biological Chemistry》.1948,第173卷591-604. |
| 关于果糖二磷酸钠及其制剂质量标准有关事宜的通知;无;《国药典化发[2009]12号》;20090109;第4页第1行－第5页第14行 * |
| 无.关于果糖二磷酸钠及其制剂质量标准有关事宜的通知.《国药典化发[2009]12号》.2009, |
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