CN108165605A - Culture medium and method a kind of while that screen protease and cellulase - Google Patents
Culture medium and method a kind of while that screen protease and cellulase Download PDFInfo
- Publication number
- CN108165605A CN108165605A CN201810032356.1A CN201810032356A CN108165605A CN 108165605 A CN108165605 A CN 108165605A CN 201810032356 A CN201810032356 A CN 201810032356A CN 108165605 A CN108165605 A CN 108165605A
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- CN
- China
- Prior art keywords
- culture medium
- result
- cellulase
- bacterial strain
- tablet
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- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
Abstract
The invention discloses a kind of while screen the culture medium and method of protease and cellulase.Culture medium of the present invention includes following components：1 ~ 3g sodium nitrate, 0.8 ~ 1.2g dipotassium hydrogen phosphates, 0.4 ~ 0.6g magnesium sulfate, 0.4 ~ 0.6g potassium chloride, 1 ~ 3g carboxymethyl celluloses or microcrystalline cellulose, 8 ~ 12g gelatin, 1 ~ 3g yeast extracts, 16 ~ 18g agar or agarose, add water to be settled to 1000ml.The method of the present invention includes the following steps：（1）Ago-Gel tablet is made in culture medium, select two tablets and is inoculated with identical bacterial strain or its zymotic fluid culture for 24 hours；One tablet is first carried out to observe and record after iodine is smoked as a result, iodine smoke after color fade carry out again coomassie brilliant blue staining decoloration observe and record result；Result is observed and recorded after directly carrying out coomassie brilliant blue staining decoloration to another tablet；Record result judges the bacterial strain of had proteinase activity, cellulase activity according to the observation.The present invention it is environmental-friendly, can detect simultaneously with screen fibre element enzyme and proteinase activity bacterial strain, it is easy to operate efficiently.
Description
Technical field
The invention belongs to biotechnology more particularly to culture medium that is a kind of while screening protease and cellulase and
Method.
Background technology
It is all using single tablet to screen protease or cellulase activity now, and preparation can screen different activities
The tablet of enzyme or screening protease or screen fibre element enzyme etc..In addition, the more of Screening Protease is defatted milk
Powder directly can be seen that transparent circle, but if combining with other methods, be easy to cause interference.And screen fibre
Plain enzymatic activity, logical the most commonly used is substrate carboxymethyl cellulose or microcrystalline celluloses, then pass through what Congo red or iodine solution was disseminated
Method obtains as a result, iodine solution dip dyeing is sensitiveer than Congo red, but needs to consume a large amount of iodine solution and have certain influence on environment.
Invention content
The purpose of the present invention is to provide a kind of while screen the culture medium and method of protease and cellulase, it is intended to solve
Certainly protease and cellulase activity need the problem of separately screening and each screening technique equal Shortcomings.
The invention is realized in this way culture medium that is a kind of while screening protease and cellulase, the culture medium include
Following components：1 ~ 3g sodium nitrate, 0.8 ~ 1.2g dipotassium hydrogen phosphates, 0.4 ~ 0.6g magnesium sulfate, 0.4 ~ 0.6g potassium chloride, 1 ~ 3g carboxylics
Methylcellulose or microcrystalline cellulose, 8 ~ 12g gelatin, 1 ~ 3g yeast extracts, 16 ~ 18g agar or agarose, add water to be settled to
1000ml。
Preferably, which includes following components：2g sodium nitrate, 1g dipotassium hydrogen phosphates, 0.5g magnesium sulfate, 0.5g chlorine
Change potassium, 2g carboxymethyl celluloses or 2g microcrystalline celluloses, 10g gelatin, 2g yeast extracts, 17g agar or agarose, add water constant volume
To 1000ml.
Invention further provides method that is a kind of while screening protease and cellulase, this method includes following step
Suddenly：
（1）Ago-Gel tablet is made in above-mentioned culture medium, select two tablets and is inoculated with identical bacterial strain or its zymotic fluid
Culture is for 24 hours；
（2）It first carries out iodine to a tablet to smoke 5 ~ 10 minutes, then as a result, photographing to record, iodine is smoked after color fade and carried out again for observation
Coomassie brilliant blue staining decoloration observation is as a result, photograph to record result；
（3）Another tablet is directly carried out to observe result after coomassie brilliant blue staining decoloration, is photographed to record；
（4）According to step（2）、（3）The result that observes and records of gained judges the bacterial strain with proteinase activity, with cellulose
The bacterial strain of enzymatic activity or the bacterial strain simultaneously with proteinase activity and cellulase activity.
The present invention overcomes the deficiencies of the prior art and provide culture medium that is a kind of while screening protease and cellulase and side
Method by the way of protease substrate glutin and cellulase substrate carboxymethyl cellulose is added simultaneously, prepares culture medium,
The method that rear coomassie brilliant blue staining is smoked with reference to first iodine, foundation detection simultaneously and screen fibre element enzyme and proteinase activity bacterial strain
Quick method.The more of Screening Protease is skimmed milk power, directly it can be seen that transparent circle, but and if other methods
If combining, interference is be easy to cause, the present invention has selected glutin, using coomassie brilliant blue staining, is through overtesting
Feasible；And screen fibre element enzymatic activity, logical the most commonly used is substrate carboxymethyl cellulose or microcrystalline celluloses, then Congo red
Or the method for iodine solution dip dyeing, iodine solution dip dyeing is sensitiveer than Congo red, but needs to consume a large amount of iodine solution and have a fixing to environment
It ringing, iodine solution dip dyeing is changed to iodine solution and exerts a gradual, corrupting influence on similary sensitive quick, and iodine solution consumption is reduced, and environmental pollution is few, so
It in the present invention, integrates and is combined with glutin and carboxymethyl cellulose, then first iodine smokes the dyeing side of rear Coomassie brilliant blue
Method, the two, which combines, can screen protease and cellulase, it is contemplated that iodine, which is smoked, to cause coomassie brilliant blue staining
As a result, experiment has been done, the two does not interfere with, so method is feasible, this also adds the screening of other substrates again to be follow-up
Other activity lay the foundation.
Compared with the prior art the shortcomings that and deficiency, the invention has the advantages that：The present invention is environmental-friendly, can be same
When detection and screen fibre element enzyme and proteinase activity bacterial strain, it is easy to operate efficiently.
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Embodiment 1
Culture medium prescription is as follows：2g sodium nitrate 1g dipotassium hydrogen phosphate 0.5g magnesium sulfate 0.5g potassium chloride 2g carboxymethyl celluloses
Element or 2g microcrystalline cellulose 10g gelatin 2g yeast extract 17g agar or agarose, add water to be settled to 1000ml.
Embodiment 2
Culture medium prescription is as follows：1g sodium nitrate, 0.8g dipotassium hydrogen phosphates, 0.4g magnesium sulfate 0.6g potassium chloride, 3g carboxymethyls are fine
Dimension element or 3g microcrystalline celluloses, 8g gelatin, 1g yeast extracts, 18g agar or agarose, add water to be settled to 1000ml.
Embodiment 3
Culture medium prescription is as follows：3g sodium nitrate, 1.2g dipotassium hydrogen phosphates, 0.6g magnesium sulfate, 0.6g potassium chloride, 1g carboxymethyls are fine
Dimension element or 1g microcrystalline celluloses, 12g gelatin, 1g yeast extracts, 16g agar or agarose, add water to be settled to 1000ml.
Embodiment 4
（1）Ago-Gel tablet is made in 1 culture medium of embodiment, select two tablets and is inoculated with identical bacterial strain or its fermentation
Liquid culture is for 24 hours；
（2）Iodine is first carried out to a tablet to smoke 5 ~ 10 minutes（The tablet is tipped upside down on into iodine on the plate lid equipped with Gram's iodine solution
5 ~ 10 minutes smoked, the screening of cellulase is typically to use with carboxymethyl cellulose（CMC）Or microcrystalline cellulose（Avicel）For
Ago-Gel tablet made of substrate combines Congo red or iodine staining method）, for verifying that whether iodine is smoked to later
Coomassie brilliant blue staining impacts, and then observation is as a result, photograph to record；Iodine is smoked carries out Coomassie brilliant blue dye again after color fade
Color decoloration observation is as a result, photograph to record result.
（3）Another tablet is directly carried out to observe result after coomassie brilliant blue staining decoloration, is photographed to record；
（4）According to step（2）、（3）The result that observes and records of gained judges the bacterial strain with proteinase activity, with cellulose
The bacterial strain of enzymatic activity or the bacterial strain simultaneously with proteinase activity and cellulase activity；Wherein, defatted milk is replaced with gelatin
Culture medium of the powder with screening protease after inoculating strain or its zymotic fluid, cannot visually observe dissolving circle, and coomassie can be used bright
Blue solution is to tablet dyeing-decolorzing, and having does not have albumen around dissolving circle, blue is taken off after dyeing-decolorzing, does not dissolve the ground of circle
Fang Ze has a large amount of albumen that will dye blue.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
All any modification, equivalent and improvement made within refreshing and principle etc., should all be included in the protection scope of the present invention.
Claims (3)
1. culture medium that is a kind of while screening protease and cellulase, which is characterized in that the culture medium includes following components：1~
3g sodium nitrate, 0.8 ~ 1.2g dipotassium hydrogen phosphates, 0.4 ~ 0.6g magnesium sulfate, 0.4 ~ 0.6g potassium chloride, 1 ~ 3g carboxymethyl celluloses
Or microcrystalline cellulose, 8 ~ 12g gelatin, 1 ~ 3g yeast extracts, 16 ~ 18g agar or agarose, water is added to be settled to 1000ml.
2. the culture medium as described in claim 1 for screening protease and cellulase simultaneously, which is characterized in that the culture medium packet
Include following components：2g sodium nitrate, 1g dipotassium hydrogen phosphates, 0.5g magnesium sulfate, 0.5g potassium chloride, 2g carboxymethyl celluloses or crystallite
Cellulose, 10g gelatin, 2g yeast extracts, 17g agar or agarose, add water to be settled to 1000ml.
3. method that is a kind of while screening protease and cellulase, which is characterized in that this method includes the following steps：
（1）Ago-Gel tablet is made in culture medium described in 1 or 1 claim 2 of claim, select two tablets and is connect
The identical bacterial strain of kind or its zymotic fluid culture are for 24 hours；
（2）It first carries out iodine to a tablet to smoke 5 ~ 10 minutes, then as a result, photographing to record, iodine is smoked after color fade and carried out again for observation
Coomassie brilliant blue staining decoloration observation is as a result, photograph to record result；
（3）Another tablet is directly carried out to observe result after coomassie brilliant blue staining decoloration, is photographed to record；
（4）According to step（2）、（3）The result that observes and records of gained judges the bacterial strain with proteinase activity, with cellulose
The bacterial strain of enzymatic activity or the bacterial strain simultaneously with proteinase activity and cellulase activity.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810032356.1A CN108165605A (en) | 2018-01-12 | 2018-01-12 | Culture medium and method a kind of while that screen protease and cellulase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810032356.1A CN108165605A (en) | 2018-01-12 | 2018-01-12 | Culture medium and method a kind of while that screen protease and cellulase |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108165605A true CN108165605A (en) | 2018-06-15 |
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ID=62514759
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810032356.1A Pending CN108165605A (en) | 2018-01-12 | 2018-01-12 | Culture medium and method a kind of while that screen protease and cellulase |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110734954A (en) * | 2019-11-21 | 2020-01-31 | 河北省微生物研究所 | Rapid identification culture medium and application thereof in cellulase qualitative detection |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1387578A (en) * | 1999-09-03 | 2002-12-25 | 三共株式会社 | Novel compound F-15078 |
| EP3209132A1 (en) * | 2014-10-23 | 2017-08-30 | Futureco Bioscience, S.A. | Bacteria with nematicidal activity and the ability to promote plant growth |
-
2018
- 2018-01-12 CN CN201810032356.1A patent/CN108165605A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1387578A (en) * | 1999-09-03 | 2002-12-25 | 三共株式会社 | Novel compound F-15078 |
| EP3209132A1 (en) * | 2014-10-23 | 2017-08-30 | Futureco Bioscience, S.A. | Bacteria with nematicidal activity and the ability to promote plant growth |
Non-Patent Citations (3)
| Title |
|---|
| 刘丽莉等: "胶原蛋白酶产生菌的筛选及酶的分离纯化", 《生物工程学报》 * |
| 陈晨等: "一种筛选胞外纤维素酶产生菌的快捷、灵敏、有效的方法", 《微生物学通报》 * |
| 韩锐: "《抗癌药物研究与实验技术》", 30 April 1997, 北京医科大学 中国协和医科大学联合出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110734954A (en) * | 2019-11-21 | 2020-01-31 | 河北省微生物研究所 | Rapid identification culture medium and application thereof in cellulase qualitative detection |
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Application publication date: 20180615 |