CN1056884C - Method of identifying, screening, quantitating and locating protease by use of protein gel coating soft film - Google Patents
Method of identifying, screening, quantitating and locating protease by use of protein gel coating soft film Download PDFInfo
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- CN1056884C CN1056884C CN92101774A CN92101774A CN1056884C CN 1056884 C CN1056884 C CN 1056884C CN 92101774 A CN92101774 A CN 92101774A CN 92101774 A CN92101774 A CN 92101774A CN 1056884 C CN1056884 C CN 1056884C
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Abstract
The present invention relates to a method for identifying screening and quantitating proteinase by using a film with a protein gel coating layer as a substrate, particularly to a method for positioning proteinase of animals and plants. In the method, a transparent film accompanied by protein gel, such as gelatin, etc., a black silver particle layer, etc. is used as a substrate; a fresh tissue to be tested is taken out and is pressed into juice or is sliced; the juice is dropped or the slices are appressed on a medicine membrane surface of the film; after a period of time, the film is washed with clear water, and the coating layer of the film is hydrolyzed by proteinase to form eroded plaque. The enzyme containing state and the enzyme content of the tested tissue can be judged or positioned according to whether the plaque appears or not and the transparence magnitude of the plaque. The present invention has the characteristics of simple method, low cost, suitability for field operation, etc.
Description
The present invention relates to a kind of with gelatin and so on protein gel coating film do the evaluation, screening of substrate, quantitatively, the method for locating protease.
Biology, especially microorganism and plant protease often have aboundresources, cheap characteristics, thereby easily developed in a large number.As the Chymotrypsin of selling on the market, papoid, bromeline, ficins etc. are exactly exemplary.
Nature biotechnology is of a great variety.Different biological interior contained proteolytic enzyme usually can be had different application because of having different point of contacts with different character such as optimum reaction conditions.And screening these different propertiess of exploitation even the special new albumen enzyme of character just have been significant.Sought the new enzyme that value of exploiting and utilizing is arranged, mainly be to adopt the casein method in the past, and this class artificial substrates of DHT casein method is measured protease activity.When adopting the casein method, need expensive equipment, and adopt the cost of synthetic substrate also very high.Need 10 yuans as the every gram of DHT casein, little peptide such as BAEE, BTEE even every gram reach up to a hundred dollars.Singly do once and to measure, reagent cost just needs up to tens of and even hundreds of yuans.Simultaneously, because biological majority all contains pigment, such as chlorophyll, anthocyanidin, carotene, protoheme etc., their existence has influenced conventional casein method consumingly and has surveyed periodic ultraviolet colorimetric.Obviously, proteolytic enzyme screens this large-scale test job, owing to need complicated plant and instrument, and expensive reagent cost and be difficult to carry out in the open air.
It is simple to the objective of the invention is to seek a kind of method, with low cost, the screening of easy handling, quantitatively and the novel method of locating protease.Whether this method can the transparency of protease hydrolysis spot and hydrolysis spot and size shape occur and identify directly, intuitively whether this analyte contains enzyme by film substrate and determined microorganism homogenate or animal vegetable tissue reaction back film, contain the enzyme position and contain what of enzyme amount, and be suitable for field operation.
The present invention adopts with the quasi-protein glue of gelatin and the residuite film that silver particles or carbon black and so on coloring matter layer is done the medicine film to do substrate; Also can use the X-ray film, take a picture or cinestrip is made exposure, development, photographic fixing and handled and make the complete complete black film of sheet base; Or directly select the complete complete black film of sheet base.The specification size depends on the needs.Before the test, film is put into clear water earlier and was soaked 5-60 minute, treats to pick up after it soaks into standby.During test, because the counterdie of film is a gelatin coating, this is the dried glue of one deck protein,, can be come off by protease hydrolysis during in certain temperature in its moistening back.Insoluble silver of this related its surperficial treated one-tenth black of process meeting that comes off or carbon black and so on material coating is sloughed and is made film expose transparent plaque.
The present invention adopts the tissue of microorganism homogenate to be identified or animals and plants fresh and healthy cleaning and presses down pressed juice, maybe will organize with scraper and cut into slices.During test, the biological homogenate in emblem or expressed organized more than juice 10 microlitres directly from point of fixity drip on the medicine face that the film that keeps flat scribbles gelatin layer, the juice amount is as the criterion outward with slice not.In order to reduce volatilization, can the film left-hand thread be covered with culture dish after dripping.Use flushing with clean water in reaction under the 10-40 ℃ of room temperature after 20 minutes to 24 hours, that can observe enzymic hydrolysis gelatin counterdie on the film transparently loses the spot situation.Direct slicing is measured in this way, then the cross section with the section of animals and plants organ-tissue clings on the medicine face of film, with clip fastening after, carefully wipe and organize the liquid pearl that to leach with the film junction, pack in the plastics bag, reaction was taken out from bag after 1-10 hour under 10-40 ℃ of room temperature, removed test organization, use the flushing with clean water film, observe hydrolysis and lose the situation that spot occurs.
After adopting the inventive method test, whether occur to judge according to the film plaque whether this microorganism homogenate or animal vegetable tissue contain enzyme.When the biology of being tested did not contain enzyme, plaque did not appear in film; When containing enzyme, losing the transparency size of spot on the film and containing the enzyme amount has proportional relationship.Contain the low biology of enzyme amount, the lower even slight point-like plaque of transparency appears in test back film, the biology that the enzyme amount is high, and the bright plaque of full impregnated appears in test back film.
Table 1 is a papoid, and subtilisin, the different activities of Chymotrypsin drip the back and lost plaque transmittance relation with film.Table 1:
Connect table 1
| Papoid | 0 | 2 | 3 | 4.5 | 15 | 97 | 100 |
| Subtilisin | 0 | 0 | 2 | 4 | 10 | 93 | 98 |
| Chymotrypsin | 0 | 4 | 8 | 15 | 30 | 98 | 100 |
Fig. 1,2,3 is respectively a papoid, subtilisin, Chymotrypsin is used PH7.0 respectively, 0.05mol/L phosphoric acid buffer is made into every milliliter 0,50,100,150,200,300,400 unit enzyme liquid, respectively get 100 microlitres and drip to from point of fixity on the film medicine face that keeps flat in 30 ℃ of reactions after 30 minutes, plaque standard relationship figure is lost in the hydrolysis of proteolytic enzyme amount and film.
As long as the erosion plaque that new specimen adopt occurs after the corresponding film test and Fig. 1,2,3 standard film plaque figure and the table values of table 1 compare, and can see intuitively the test sample product enzymatic activity that makes new advances.
If the biology of test contains the enzyme amount when too high, also can do suitably act on film after the dilution by its juice, through knowing relative activity with the standard items comparison. Every batch of film can compare repeatedly as long as make a standard chart. If the same batch of abundant film sheet that gives up is arranged, tens thousand ofly do the field measuring screening inferiorly and all be not a problem.
Because the method that the present invention adopts has suitable sensitivity, if contain enzyme animal vegetable tissue section and be close on the film medicine face after a period of time fresh, take off sample, the corresponding position that plant tissue on film contains the enzyme place can occur losing intuitively spot, still can contain the enzyme position to plant tissue and do Position Research.
The present invention is because directly with after microorganism homogenate or animal vegetable tissue juice and section and scribbling the transparent film reaction of medicine film, transparency size whether occurs reaching and judge whether this tester contains enzyme, contain the enzyme amount and animal vegetable tissue contains the enzyme position according to losing spot on the film, therefore it is simple to have method, accurately, with low cost, be suitable for the characteristics such as open-air large-scale test job.
Embodiment 1:
Make exposure, development, photographic fixing with the X-ray film and handle and make the complete complete black film of sheet base, put into clear water and soak after 60 minutes and pick up, be cut into 4 * 2cm small pieces.Palmae gomuti palm fruit is extruded juice with squeezing machine, and taking juice 200 microlitres scribble on the one side of gelatin medicine film from the X-ray film that point of fixity drips to through above processing, cover with the culture dish left-hand thread immediately.After 30 minutes, take out X-ray film flushing with clean water in reaction under 30 ℃ of room temperatures, droplet have the X-ray film position of juice promptly reveal by the full impregnated behind the enzymic hydrolysis gelatin is bright and lose spot.
Embodiment 2:
Make exposure, development, photographic fixing with cinestrip and handle and make the complete full black matrix sheet of sheet base, put into clear water and soak after 30 minutes and pick up, be cut into long 4cm small pieces.With four ribs of Vitaceae from powder rattan chylocaulous, palmaceous gomuti palm pulp, queen certain herbaceous plants with big flowers pulp, the careless pineapple pulp of pineapple section, the papaya fruit of Caricaceae, Indonesia's fig fruit of Moraceae, the tender skill of milk fruit green pulp, the goat's horn stem of Agavaceae, golden-rimmed dragon tree leaf green palisade tissue, the green palisade tissue of liliaceous Yucca gloriosa leaf.Above-mentioned plant tissue is cut into the small pieces that thickness is about 1cm with slicing knife respectively, is close on the one side that treated sensitive film scribbles gelatin medicine film with being about to tangent plane, is fixed with wooden clamp.Put into plastics bag after carefully wiping the liquid pearl that to leach in plant tissue and film junction.Remove plant tissue in reaction under 30 ℃ of room temperatures after 3 hours, use the flushing with clean water film, can see the erosion plaque situation that occurs behind the enzymic hydrolysis gelatin on the film intuitively.Various test plants lose shape of spot and corresponding plant tissue the results are shown in Table 2 with casein method comparative determination protease activity.
Table 2
Connect table 2
| Botanical name | Sensitive film is measured the plaque shape | Casein method comparative determination enzymic activity n/g |
| Four rib Cissus repens | The bright erosion plaque of full impregnated | 1000-3000 |
| Gomuti palm | The bright erosion plaque of full impregnated | 60000 |
| The queen certain herbaceous plants with big flowers | The low erosion plaque of transparency | 500 |
| The grass pineapple | The bright erosion plaque of full impregnated | 2000-5000 |
| Botanical name | Sensitive film is measured the plaque shape | Ester gp method comparative determination enzymic activity n/g |
| Papaya | The bright erosion plaque of full impregnated | 150,000-250,000 |
| Indonesia's Fructus Fici | Plaque does not appear losing | 0 |
| The milk fruit | The bright erosion plaque of full impregnated | 1000-2000 |
| Goat's horn | The transparent erosion plaque of point-like | 100-500 |
| Golden-rimmed dragon tree | The bright erosion plaque of full impregnated | 1000-2000 |
| Yucca gloriosa L. | The bright erosion plaque of full impregnated | 3000-10000 |
Embodiment 3:
Successively coat gelatin layer and carbon black powder with transparent film, make the gelatin coating film, be cut into long 4cm small pieces.Use sisal hemp leaf, be close on the gelfilm medicine face after the cane section of margosa, fix with clip, make it not loosening, be not shifted, and carefully wipe the liquid pearl that plant tissue and film junction may be leached, reacted 5 hours, remove test organization, can see very intuitively on film: the green palisade tissue of sisal leaves table, the correspondence position of the bark endodermis of margosa all have the transparent plaque of being lost＜see Fig. 4,5 〉.
Claims (6)
1. method with the quantitative locating protease of protein gel coating film evaluation and screening, it is characterized in that with having gelatin and silver particles or carbon black coloring matter coating on the transparent film, or X-ray film, or take a picture, cinestrip exposes, develop, photographic fixing is handled the film of making the sheet base and is done substrate, with biological homogenate to be measured, animal vegetable tissue juice or section directly drip to or cling on the medicine face that film scribbles, after the reaction, use the flushing with clean water film, be hydrolyzed the size whether the plaque transparency appears reaching in back erosion plaque according to film and judge whether tester contains enzyme and how much contain the enzyme amount.
2. by the described method of claim 1, film specification size is unrestricted when it is characterized in that testing.
3. by the described method of claim 1, used tissue or juice amount are unrestricted when it is characterized in that testing, and are as the criterion outward with slice not.
4. by the described method of claim 1, it is characterized in that test is to react 1-24 hour under 10-40 ℃ of room temperature.
5. by the described method of claim 1, it is characterized in that testing the transparency that loses spot on the film of back and size shape contains the proportional example of enzyme amount with the biology of being tested and concerns, contain the enzyme amount when low, lower even the slight point-like plaque of transparency appears in film, contain the enzyme amount when high, occur that full impregnated is bright to lose spot.
6. by the described method of claim 1, it is characterized in that enzyme liquid with known activity is made into one group of step concentration and acts on film respectively, obtain one group of film enzyme and lose the standard map that, lose spot relatively, measure the latter's activity with film behind the sample testing of itself and unknown enzyme amount.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN92101774A CN1056884C (en) | 1992-03-14 | 1992-03-14 | Method of identifying, screening, quantitating and locating protease by use of protein gel coating soft film |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN92101774A CN1056884C (en) | 1992-03-14 | 1992-03-14 | Method of identifying, screening, quantitating and locating protease by use of protein gel coating soft film |
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| CN1065095A CN1065095A (en) | 1992-10-07 |
| CN1056884C true CN1056884C (en) | 2000-09-27 |
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| CN92101774A Expired - Fee Related CN1056884C (en) | 1992-03-14 | 1992-03-14 | Method of identifying, screening, quantitating and locating protease by use of protein gel coating soft film |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0454046A2 (en) * | 1990-04-26 | 1991-10-30 | Difco Laboratories Incorporated | Diagnostic test slide |
| US5073487A (en) * | 1989-01-30 | 1991-12-17 | Athens Research And Technology, Inc. | Rapid assay for functional human α1 -proteinase inhibitor |
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1992
- 1992-03-14 CN CN92101774A patent/CN1056884C/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5073487A (en) * | 1989-01-30 | 1991-12-17 | Athens Research And Technology, Inc. | Rapid assay for functional human α1 -proteinase inhibitor |
| EP0454046A2 (en) * | 1990-04-26 | 1991-10-30 | Difco Laboratories Incorporated | Diagnostic test slide |
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| CN1065095A (en) | 1992-10-07 |
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