CN104862374A - Method for detecting collagen enzymolysis activity of trepang cathepsin - Google Patents
Method for detecting collagen enzymolysis activity of trepang cathepsin Download PDFInfo
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- CN104862374A CN104862374A CN201510241949.5A CN201510241949A CN104862374A CN 104862374 A CN104862374 A CN 104862374A CN 201510241949 A CN201510241949 A CN 201510241949A CN 104862374 A CN104862374 A CN 104862374A
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- enzymolysis
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- sea cucumber
- kethepsin
- cathepsin
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Abstract
The invention discloses a method for detecting collagen enzymolysis activity of trepang cathepsin, wherein the method comprises: adopting the trepang cathepsin to carry out enzymolysis effectiveness evaluation to the sunken diameter generated by enzymolysis on the surface of collagen gel. An enzymolysis diameter or area is adopted to carry out evaluation to enzymatic activity, and a semiquantitative result can be obtained. The method for detecting the collagen enzymolysis activity of the trepang cathepsin can meet a large proportion of enzymatic activity evaluation requirement, and is relatively accurate. Therefore, the method for detecting the collagen enzymolysis activity of the trepang cathepsin has the advantages of being suitable for trepang protease, able to carry out semiquantitative evaluation to the collagen enzymolysis activity of the trepang cathepsin, and convenient for obtaining raw materials.
Description
Technical field
The present invention relates to a kind of enzymolysis activity detection method, particularly relate to the collagen enzymolysis activity test method of sea cucumber kethepsin.
Background technology
Collagen protein is the structural protein of body tissue, and its many physiological function is by systematic study and extensively accreditation.Current domestic collagen protein industry development is rapid, and along with the fast development of collagen protein industry, the demand of the crucial production factors-proteolytic enzyme in collagen protein enzymolysis technique also constantly increases.Collagenase comparatively conventional at present can be divided into plant protease (as bromeline, papoid etc.), animal protease (as trypsinase, stomach en-etc.), microbial protease etc. from source.The shortcoming that current above-mentioned enzyme classes ubiquity collagen protein enzymolysis efficiency is not high, therefore, researches and develops new and effective Collagenase and has important using value.And as the ring in this R&D work, the collagen enzymolysis active function evaluation analysis technology of Collagenase is extremely important, at present in the urgent need to having a set of simple possible and application oriented method and technology.The present invention is specifically designed to artificial sea cucumber kethepsin collagen protein enzymolysis activity rating of expressing, and has simple and easy, feature accurately and reliably, has very high using value.
Summary of the invention
The object of the invention is to provide a kind of for prior art and be applicable to sea cucumber proteolytic enzyme, the collagen enzymolysis activity of semidefinite quantitatively evaluating sea cucumber kethepsin, raw material can obtain the collagen enzymolysis activity test method of sea cucumber kethepsin easily.
The present invention solves the problems of the technologies described above adopted technical scheme: the collagen enzymolysis activity test method of sea cucumber kethepsin, comprise, wherein: adopt sea cucumber kethepsin to carry out hydrolysis result evaluation to the recess diameter that collagen gel surface enzymolysis produces.Adopt enzymolysis diameter or area to evaluate to enzymic activity, semiquantitative result can be obtained.Most enzymic activity evaluation requirements can be met, and comparatively accurate.
For optimizing technique scheme, the measure taked also comprises: detection method has following steps:
1) collagen gel makes: to utilize in daily life the common collagen food raw material that is rich in as making material, and through boiling, degrease, filtration impurity elimination, pH value adjustment, solid substance measure, the process of a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices congealing step, makes collagen gel;
2) protein enzyme solution preparation: protease standards is carried out dissolving with the concentration of 10mg/mL, 1mg/mL, 0.1mg/mL, 0.01mg/mL and 0.001mg/mL and prepares enzyme liquid, the sea cucumber kethepsin of test adopts one of concentration of protease standards configuration to prepare;
3) enzymolysis curve plotting: take a morsel the standard protein enzyme and sea cucumber tissue protein enzyme solution prepared, drips in collagen gel surface; Constant temperature enzymolysis after covered and enclosed; Rapidly the flat board after enzymolysis is put into low temperature environment, after cooling, Quick Measurement enzymolysis forms the surface diameter of circular depressed, statistical study diameter data, makes enzymolysis curve;
4) sea cucumber cathepsin active is analyzed: the enzymolysis curve of the recess diameter comparing standard protein enzyme enzymolysis data creating formed with sea cucumber kethepsin enzymolysis, search the standard protein enzyme concn feature that sea cucumber kethepsin is corresponding, convert and check in sea cucumber cathepsin active relative value.In step 1), be rich in collagen food raw material and be selected from fish-skin or shank or pigskin; The described process temperature that boils is 85-125 DEG C, and the time of boiling is 5-30min, by centrifugal and freezing removal fat.In step 1), the soda acids such as HCl and NaOH can be utilized to carry out solution ph adjustment, according to different collagen protein raw material characteristic, adjustment solid concentration is 10-50%, utilizes conventional dish, culture dish etc. as a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices vessel.Step 2) in, the proteolytic enzyme used generally selects the active stronger prozyme of collagen protein enzymolysis as standard substance, the enzyme liquid prepared need adjust pH to enzymic activity top condition, and the pH of sea cucumber kethepsin sample configuration is generally 3.5-8.5, and preparation final concentration is 1mg/mL.In step 3), protein enzyme solution generally gets 5-500 microlitre, uses closed protein enzyme liquid and the collagen gels such as culture dish lid, preservative film, is carefully transferred in the equipment such as water-bath, constant incubator, keeps 20-60 DEG C of condition enzymolysis 10-60min.In described step, flat board should be put into rapidly sub-zero temperature environment after enzymolysis terminates, to suppress enzymolysis; Measure the surface diameter that enzymolysis forms circular depressed afterwards, statistical study diameter data, make enzymolysis curve.
Sea cucumber kethepsin is adopted to carry out hydrolysis result evaluation to the recess diameter that collagen gel surface enzymolysis produces owing to present invention employs.Adopt enzymolysis diameter or area to evaluate to enzymic activity, semiquantitative result can be obtained.Most enzymic activity evaluation requirements can be met, and comparatively accurate.Thus the present invention has and is applicable to sea cucumber proteolytic enzyme, the collagen enzymolysis activity of semidefinite quantitatively evaluating sea cucumber kethepsin, raw material can obtains advantage easily.
Accompanying drawing explanation
View when Fig. 1 is embodiment of the present invention dropping enzyme liquid;
Fig. 2 is the circular enzymolysis depression formed after embodiment of the present invention enzymolysis 13min.
Embodiment
Below in conjunction with attached embodiment, the present invention is described in further detail.
Embodiment: referring to figs. 1 through Fig. 2, the collagen enzymolysis activity test method of sea cucumber kethepsin, comprises, wherein: adopt sea cucumber kethepsin to carry out hydrolysis result evaluation to the recess diameter that collagen gel surface enzymolysis produces.Adopt enzymolysis diameter or area to evaluate to enzymic activity, semiquantitative result can be obtained.Most enzymic activity evaluation requirements can be met, and comparatively accurate.Detection method has following steps:
1) collagen gel makes: to utilize in daily life the common collagen food raw material that is rich in as making material, and through boiling, degrease, filtration impurity elimination, pH value adjustment, solid substance measure, the process of a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices congealing step, makes collagen gel;
2) protein enzyme solution preparation: protease standards is carried out dissolving with the concentration of 10mg/mL, 1mg/mL, 0.1mg/mL, 0.01mg/mL and 0.001mg/mL and prepares enzyme liquid, the sea cucumber kethepsin of test adopts one of concentration of protease standards configuration to prepare;
3) enzymolysis curve plotting: take a morsel the standard protein enzyme and sea cucumber tissue protein enzyme solution prepared, drips in collagen gel surface; Constant temperature enzymolysis after covered and enclosed; Rapidly the flat board after enzymolysis is put into low temperature environment, after cooling, Quick Measurement enzymolysis forms the surface diameter of circular depressed, statistical study diameter data, makes enzymolysis curve;
4) sea cucumber cathepsin active is analyzed: the enzymolysis curve of the recess diameter comparing standard protein enzyme enzymolysis data creating formed with sea cucumber kethepsin enzymolysis, search the standard protein enzyme concn feature that sea cucumber kethepsin is corresponding, convert and check in sea cucumber cathepsin active relative value.In step 1), be rich in collagen food raw material and be selected from fish-skin or shank or pigskin; The described process temperature that boils is 85-125 DEG C, and the time of boiling is 5-30min, by centrifugal and freezing removal fat.In step 1), the soda acids such as HCl and NaOH can be utilized to carry out solution ph adjustment, according to different collagen protein raw material characteristic, adjustment solid concentration is 10-50%, utilizes conventional dish, culture dish etc. as a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices vessel.Step 2) in, the proteolytic enzyme used generally selects the active stronger prozyme of collagen protein enzymolysis as standard substance, the enzyme liquid prepared need adjust pH to enzymic activity top condition, and the pH of sea cucumber kethepsin sample configuration is generally 3.5-8.5, and preparation final concentration is 1mg/mL.In step 3), protein enzyme solution generally gets 5-500 microlitre, uses closed protein enzyme liquid and the collagen gels such as culture dish lid, preservative film, is carefully transferred in the equipment such as water-bath, constant incubator, keeps 20-60 DEG C of condition enzymolysis 10-60min.In described step 3, flat board should be put into rapidly sub-zero temperature environment after enzymolysis terminates, to suppress enzymolysis; Measure the surface diameter that enzymolysis forms circular depressed afterwards, statistical study diameter data, make enzymolysis curve.
1) collagen gel makes
Get freezing shank 300g as making material, add water 1L, 100 DEG C boil 30min after, room temperature cooling precipitation, take off a layer solution 30mL and be placed in the centrifugal 10min of 50mL centrifuge tube 7000rpm normal temperature, use syringe to draw middle level solution 10mL and be placed in 1 clean beaker container, mixing rear detection solid content is 24.7%, be divided into two parts again, use NaOH and HCl solution adjusted to ph to 8.5 and 6.0 respectively, get 3mL solution to fall in the plastic culture dish of diameter 4cm, room temperature cooling condensation, completes collagen gel and makes.
2) protein enzyme solution preparation
Utilize trypsin traditional Chinese medicines, CAS:9002-07-7) as protease standards, carry out dissolving with 10mg/mL, 1mg/mL, 0.1mg/mL, 0.01mg/mL and 0.001mg/mL isoconcentration and prepare enzyme liquid (pH is for 8.5), the sea cucumber kethepsin of experiment test carries out preparing (pH is for 6.0) with 1mg/mL.
3) enzymolysis curve plotting
Get standard protein enzyme and sea cucumber tissue protein enzyme solution that 50 microlitres have prepared, drip in collagen gel surface; Be positioned over after covered and enclosed in 37 DEG C of thermostat water baths and carry out constant temperature enzymolysis; Rapidly the flat board after enzymolysis is put into-20 DEG C of refrigerator-freezers after 15min, be put in ice chest after cooling 1min, use vernier caliper measurement enzymolysis to form the surface diameter of circular depressed, utilize the software analysis diameter data such as Spass and Sigmaplot, make enzymolysis curve.After enzymolysis procollagen state and enzymolysis, collagen protein state asks for an interview Fig. 1.
4) sea cucumber cathepsin active is analyzed
The enzymolysis curve of the recess diameter comparing standard protein enzyme enzymolysis data creating formed with sea cucumber kethepsin enzymolysis, searches the standard protein enzyme concn feature that 1mg/mL sea cucumber kethepsin is corresponding, converts and checks in sea cucumber cathepsin active relative value.Record the collagen protein enzymolysis activity of sea cucumber kethepsin for traditional Chinese medicines trypsin CAS:9002-07-7) 12.4%.
Although describe the present invention in conjunction with preferred embodiment; so itself and be not used to limit the present invention; any those skilled in the art; without departing from the spirit and scope of the present invention; can implement various change, the displacement of coordinator and amendment here to the theme listed, therefore protection scope of the present invention be as the criterion when the scope limited depending on proposed claim.
Claims (7)
1. the collagen enzymolysis activity test method of sea cucumber kethepsin, is characterized in that: adopt sea cucumber kethepsin to carry out enzymolysis activity evaluation to the recess diameter that collagen gel surface enzymolysis produces.
2. the collagen enzymolysis activity test method of sea cucumber kethepsin according to claim 1, is characterized in that: described detection method has following steps:
1) collagen gel makes: to utilize in daily life the common collagen food raw material that is rich in as making material, and through boiling, degrease, filtration impurity elimination, pH value adjustment, solid substance measure, the process of a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices congealing step, makes collagen gel;
2) protein enzyme solution preparation: protease standards is carried out dissolving with the concentration of 10mg/mL, 1mg/mL, 0.1mg/mL, 0.01mg/mL and 0.001mg/mL and prepares enzyme liquid, one of concentration of the protease standards configuration described in the employing of sea cucumber kethepsin of test is prepared;
3) enzymolysis curve plotting: take a morsel the standard protein enzyme and sea cucumber tissue protein enzyme solution prepared, drips in collagen gel surface; Constant temperature enzymolysis after covered and enclosed; Rapidly the flat board after enzymolysis is put into low temperature environment, after cooling, Quick Measurement enzymolysis forms the surface diameter of circular depressed, statistical study diameter data, makes enzymolysis curve;
4) sea cucumber cathepsin active is analyzed: the enzymolysis curve of the recess diameter comparing standard protein enzyme enzymolysis data creating formed with sea cucumber kethepsin enzymolysis, search the standard protein enzyme concn feature that sea cucumber kethepsin is corresponding, convert and check in sea cucumber cathepsin active relative value.
3. the collagen enzymolysis activity test method of sea cucumber kethepsin according to claim 2, is characterized in that: in described step 1), is rich in collagen food raw material and is selected from fish-skin or shank or pigskin; The described process temperature that boils is 85-125 DEG C, and the time of boiling is 5-30min, by centrifugal and freezing removal fat.
4. the collagen enzymolysis activity test method of sea cucumber kethepsin according to claim 2, it is characterized in that: in described step 1), the soda acids such as HCl and NaOH can be utilized to carry out solution ph adjustment, according to different collagen protein raw material characteristic, adjustment solid concentration is 10-50%, utilizes conventional dish, culture dish etc. as a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices vessel.
5. the collagen enzymolysis activity test method of sea cucumber kethepsin according to claim 2, it is characterized in that: described step 2) in, the proteolytic enzyme used generally selects the active stronger prozyme of collagen protein enzymolysis as standard substance, the enzyme liquid prepared need adjust pH to enzymic activity top condition, the pH of sea cucumber kethepsin sample configuration is generally 3.5-8.5, and preparation final concentration is 1mg/mL.
6. the collagen enzymolysis activity test method of sea cucumber kethepsin according to claim 2, it is characterized in that: in described step 3), protein enzyme solution generally gets 5-500 microlitre, use closed protein enzyme liquid and the collagen gels such as culture dish lid, preservative film, carefully be transferred in the equipment such as water-bath, constant incubator, keep 20-60 DEG C of condition enzymolysis 10-60min.
7. the collagen enzymolysis activity test method of sea cucumber kethepsin according to claim 2, is characterized in that: in described step (3), flat board should be put into rapidly sub-zero temperature environment, to suppress enzymolysis after enzymolysis terminates; Measure the surface diameter that enzymolysis forms circular depressed afterwards, statistical study diameter data, make enzymolysis curve.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1763219A (en) * | 2005-10-11 | 2006-04-26 | 广西医科大学 | Fibrinogen plate assay for detection of thrombin, thrombin-like enzyme and thrombin inhibitor |
| CN101871008A (en) * | 2010-05-31 | 2010-10-27 | 浙江工商大学 | Fungal acid nuclease detection reagent, preparation method thereof, and method for detecting fungal acid nuclease |
| CN102911999A (en) * | 2012-08-10 | 2013-02-06 | 浙江工业大学 | Method for quantitatively determining activity of L-amino acid oxidase by using Prussian blue plate |
| CN103361397A (en) * | 2013-06-21 | 2013-10-23 | 浙江工业大学 | Quantitative detection method of activity of L-amino acid oxidase |
| CN104846060A (en) * | 2015-05-11 | 2015-08-19 | 浙江海洋学院 | Collagen enzymolysis activity detection method of sea cucumber cathepsin |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1763219A (en) * | 2005-10-11 | 2006-04-26 | 广西医科大学 | Fibrinogen plate assay for detection of thrombin, thrombin-like enzyme and thrombin inhibitor |
| CN101871008A (en) * | 2010-05-31 | 2010-10-27 | 浙江工商大学 | Fungal acid nuclease detection reagent, preparation method thereof, and method for detecting fungal acid nuclease |
| CN102911999A (en) * | 2012-08-10 | 2013-02-06 | 浙江工业大学 | Method for quantitatively determining activity of L-amino acid oxidase by using Prussian blue plate |
| CN103361397A (en) * | 2013-06-21 | 2013-10-23 | 浙江工业大学 | Quantitative detection method of activity of L-amino acid oxidase |
| CN104846060A (en) * | 2015-05-11 | 2015-08-19 | 浙江海洋学院 | Collagen enzymolysis activity detection method of sea cucumber cathepsin |
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