CN109097435A - A method of screening malaga carbohydrate oxidase strain from air - Google Patents
A method of screening malaga carbohydrate oxidase strain from air Download PDFInfo
- Publication number
- CN109097435A CN109097435A CN201811015932.8A CN201811015932A CN109097435A CN 109097435 A CN109097435 A CN 109097435A CN 201811015932 A CN201811015932 A CN 201811015932A CN 109097435 A CN109097435 A CN 109097435A
- Authority
- CN
- China
- Prior art keywords
- screening
- strain
- carbohydrate oxidase
- air
- medium
- Prior art date
- 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.)
- Pending
Links
Classifications
-
- 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
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Toxicology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The method that the present invention relates to a kind of to screen malaga carbohydrate oxidase strain from air.The present inventor has abandoned traditional water sample or soil sample bacterial screening method, target strain is screened directly from air, optimize the culture medium prescription and screening technology for primary dcreening operation, the sterilizing and sterile working for eliminating primary dcreening operation culture medium realize easy, quick, accurate, the efficient screening malaga carbohydrate oxidase strain from air.
Description
Technical field
The invention belongs to microbiological arts, are related to a kind of malaga carbohydrate oxidase bacterial strain, specifically a kind of from sky
The method of malaga carbohydrate oxidase strain is screened in gas.
Background technique
Glucose oxidase has extensive commercial application value, it is widely used in major neck such as food, medicine, feed
Domain.The production of glucose oxidase has the direct extraction method and microbe fermentation method in plant or animal tissue source, due to micro- life
Object breeding is fast, and is easy to cultivate, so the industrialized production of glucose oxidase all uses microbe fermentation method at present.Portugal at present
The production strain of grape carbohydrate oxidase is mainly mould and aspergillus niger, but enzymatic productivity is not high.Therefore, one plant of malaga is screened
The high bacterial strain of carbohydrate oxidase ability is current urgent problem to be solved.The primary dcreening operation of glucose oxidase producing strains mainly uses at present
Development process, principle are as follows: object bacteria secretes glucose oxidase to extracellular, glucose generation gluconic acid in catalysis plate, together
When consumption oxygen generate hydrogen peroxide, the iodide ion in hydrogen peroxide oxidation culture medium is at iodine, and starch meets iodine in culture medium
Aobvious blue, periphery of bacterial colonies blue circle is bigger, illustrates that the enzymatic productivity of bacterium is stronger.This method simple, intuitive, belongs to microorganism fungus kind
Commonly develop the color screening method in screening.The measurement of activity of glucose oxidase is frequently with titration, principle are as follows: grape is glycoxidative
Enzyme is a kind of aerobic dehydrogenase, can be catalyzed glucose and generate gluconic acid.With the glucose in excessive sodium hydroxide with generation
Acid, then with hydrochloric acid back titration, the yield of gluconic acid can be acquired, enzyme activity height is indicated according to malaga saccharic acid amount.
The screening of glucose oxidase producing strains at present be mostly from water and soil sample screening, the present inventor directly from
Target strain is screened in air, is optimized the culture medium prescription and screening technology for primary dcreening operation, is eliminated primary dcreening operation culture medium
Sterilizing and sterile working realize easy, quick, accurate, the efficient screening malaga carbohydrate oxidase strain from air.
Summary of the invention
The method that the object of the present invention is to provide a kind of directly to screen malaga carbohydrate oxidase strain from air, this method
Sample handling processes cumbersome in conventional bacterial classification screening process are not only eliminated, and screening and culturing medium is just sieved through without sterilizing
Journey is also not required to sterile working, easy to operate, low in cost, is conducive to the exploitation of glucose oxidase microorganism resource.
To realize that goal of the invention adopts the following technical scheme that, specific steps are as follows:
1) solid plate containing screening and culturing medium is directly exposed to air standing 5-10 days, generates blue to occur in plate
Enclose lawn.Screening and culturing medium composition are as follows: glucose 40-100g/L, calcium carbonate 3.5g/L, soluble starch 10g/L, potassium iodide
Naturally, solvent is tap water, culture medium is not necessarily to sterile working without sterilizing, operating process by 1.7g/L, agar 15-20g/L, pH.
2) aseptically, picking generates the lawn of blue circle, in the solid plate containing scribing line isolation medium
Scribing line culture generates the single colonie of blue circle to occur in plate.Separation screening culture medium of crossing forms are as follows: glucose 80g/L,
Peptone 3g/L, ammonium sulfate 0.4g/L, potassium dihydrogen phosphate 0.19g/L, magnesium sulfate 0.16g/L, calcium carbonate 3.5g/L, soluble shallow lake
Powder 10g/L, potassium iodide 1.7g/L, agar powder 15-20g/L, solvent are deionized water, pH 5-6,121 DEG C of sterilizing 25min, training
Supporting temperature is 25-28 DEG C, and incubation time is 48-72 hours.
3) aseptically, picking generates the single colonie of blue circle, and it is oblique to be inoculated in potato agar medium (PDA) solid
Face activates 2-3 generation under the conditions of 25-28 DEG C, and strain after picking activation, is inoculated in potato fluid nutrient medium (PDY) under aseptic condition,
25-28 DEG C, 150-200rpm shaking flask culture 24-48h, obtained seed liquor is inoculated in liquid fermentation medium, inoculum concentration 5-
10%, 150-200rpm, cultivation temperature are 25-28 DEG C, and harvest fermentation liquid is stand-by after 48-72h.Liquid fermentation medium composition are as follows:
Glucose 80g/L, peptone 3g/L, ammonium sulfate 0.4g/L, potassium dihydrogen phosphate 0.19g/L, magnesium sulfate 0.16g/L, calcium carbonate
3.5g/L, solvent are deionized water, pH 5-6,121 DEG C of sterilizing 25min.
4) it draws 2mL fermentation liquid with liquid-transfering gun to be placed in 2ml eppendorf centrifuge tube, 5000-8000rpm is centrifuged 5-
10min obtains supernatant after removing thallus, then using glucose oxidase enzyme activity in titration measuring supernatant.
Specific embodiment:
The present invention is more specifically described in detail combined with specific embodiments below, but embodiments of the present invention are not limited to
This can refer to routine techniques progress for not specifically specified technological parameter.
1 plate primary dcreening operation of embodiment
Screening and culturing medium heating inverted plate (9cm diameter) immediately after agar dissolution, in order to avoid culture medium is dry and cracked, every piece of plate
Middle culture medium pours into about 30mL culture medium.Solid plate containing screening and culturing medium is directly exposed to air standing 5-10 days,
To occur producing blue circle lawn in plate.
Screening and culturing medium composition are as follows: glucose 80g/L, calcium carbonate 3.5g/L, soluble starch 10g/L, potassium iodide 1.7g/
L, naturally, solvent is tap water, culture medium is not necessarily to sterile working without sterilizing, operating process by agar 15-20g/L, pH.
2 shaking flask secondary screening of embodiment
Aseptically, picking generates the lawn of blue circle, training of crossing in the solid plate containing scribing line isolation medium
It supports, generates the single colonie of blue circle, scribing line separation screening culture medium composition are as follows: glucose 80g/L, peptone to occur in plate
3g/L, ammonium sulfate 0.4g/L, potassium dihydrogen phosphate 0.19g/L, magnesium sulfate 0.16g/L, calcium carbonate 3.5g/L, soluble starch 10g/
L, potassium iodide 1.7g/L, agar powder 20g/L, solvent are deionized water, 5,121 DEG C of sterilizing 25min of pH, cultivation temperature 28
DEG C, incubation time is 72 hours.
Aseptically, picking generates the single colonie of blue circle, and it is oblique to be inoculated in potato agar medium (PDA) solid
Face activated for 3 generations under the conditions of 28 DEG C, and strain after picking activation, is inoculated in potato fluid nutrient medium (PDY), 28 DEG C under aseptic condition,
For 24 hours, obtained seed liquor is inoculated in liquid fermentation medium, inoculum concentration 10%, 150rpm, cultivation temperature to the culture of 150rpm shaking flask
It is 28 DEG C, it is stand-by that fermentation liquid is harvested after 72h.Liquid fermentation medium composition are as follows: glucose 80g/L, peptone 3g/L, ammonium sulfate
0.4g/L, potassium dihydrogen phosphate 0.19g/L, magnesium sulfate 0.16g/L, calcium carbonate 3.5g/L, solvent are deionized water, pH 5,121
DEG C sterilizing 25min.
It draws 2mL fermentation liquid with liquid-transfering gun to be placed in 2ml eppendorf centrifuge tube, 8000rpm is centrifuged 5min, removes bacterium
Supernatant is obtained after body, then using glucose oxidase enzyme activity in titration measuring supernatant.
Purpose bacterium is obtained using plate primary dcreening operation, enzyme activity has been measured after shaking flask secondary screening, and primary dcreening operation purpose bacterium blue circle becomes
Color range size and measurement enzyme activity size are positively correlated, this illustrates that prescreening method is reliable in this invention.
Claims (5)
1. a kind of method for screening malaga carbohydrate oxidase strain from air, it is characterised in that include the following steps:
1) solid plate containing screening and culturing medium is directly exposed to air standing 5-10 days, generates blue to occur in plate
Enclose lawn；
2) aseptically, picking generates the lawn of blue circle, crosses in the solid plate containing scribing line isolation medium
Culture generates the single colonie of blue circle to occur in plate；
3) aseptically, picking generates the single colonie of blue circle, after strain is activated, prepares seed liquor, seed liquor inoculation
In liquid fermentation medium, shake flask fermentation is carried out；
4) fermentation liquid is harvested, centrifugation surveys enzyme activity using titration, completes entire screening process.
2. screening the method for malaga carbohydrate oxidase strain in air according to claim 1, it is characterised in that step 1)
Be not required to sterile working, and screening and culturing medium forms are as follows: glucose 40-100g/L, calcium carbonate 3.5g/L, soluble starch 10g/L,
Potassium iodide 1.7g/L, agar 15-20g/L, pH are naturally, solvent is tap water, and culture medium is without sterilizing.
3. screening the method for malaga carbohydrate oxidase strain in air according to claim 1, it is characterised in that step 2
Middle scribing line isolation medium composition are as follows: glucose 80g/L, peptone 3g/L, ammonium sulfate 0.4g/L, potassium dihydrogen phosphate 0.19g/L,
Magnesium sulfate 0.16g/L, calcium carbonate 3.5g/L, soluble starch 10g/L, potassium iodide 1.7g/L, agar powder 15-20g/L, solvent are
Deionized water, pH5-6,121 DEG C of sterilizing 25min, cultivation temperature are 25-28 DEG C, and incubation time is 48-72 hours.
4. screening the method for malaga carbohydrate oxidase strain in air according to claim 1, it is characterised in that step 3)
Middle liquid fermentation medium composition are as follows: glucose 80g/L, peptone 3g/L, ammonium sulfate 0.4g/L, potassium dihydrogen phosphate 0.19g/L,
Magnesium sulfate 0.16g/L, calcium carbonate 3.5g/L, solvent are deionized water, pH 5-6,121 DEG C of sterilizings 25min, inoculum concentration 5%-
10%, cultivation temperature is 28 DEG C, shaking flask revolving speed 150-200rpm, and incubation time is 48-72 hours.
5. screening the method for malaga carbohydrate oxidase strain in air according to claim 1, it is characterised in that step 4)
Middle centrifuge tube is 2ml eppendorf centrifuge tube, revolution 5000-8000rpm, time 5-10min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811015932.8A CN109097435A (en) | 2018-09-01 | 2018-09-01 | A method of screening malaga carbohydrate oxidase strain from air |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811015932.8A CN109097435A (en) | 2018-09-01 | 2018-09-01 | A method of screening malaga carbohydrate oxidase strain from air |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109097435A true CN109097435A (en) | 2018-12-28 |
Family
ID=64864758
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811015932.8A Pending CN109097435A (en) | 2018-09-01 | 2018-09-01 | A method of screening malaga carbohydrate oxidase strain from air |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109097435A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110066849A (en) * | 2019-04-04 | 2019-07-30 | 大连大学 | A kind of screening technique producing cholesterol oxidase bacterial strain |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0125698A2 (en) * | 1983-05-16 | 1984-11-21 | NABISCO BRANDS, Inc. | A process for screening microorganisms producing glucose-2-oxidase |
| CN102382773A (en) * | 2011-10-26 | 2012-03-21 | 江南大学 | Aspergillus niger strain capable of producing glucose oxidase and application thereof |
| CN102925362A (en) * | 2012-09-17 | 2013-02-13 | 中国农业科学院农业环境与可持续发展研究所 | Screening method of fusarium oxysporum and application thereof |
| CN103409340A (en) * | 2012-03-07 | 2013-11-27 | 昆明金泽实业有限公司 | Microbial strain and application of bactericide thereof in biological treatment of phenolic wastewater |
| CN106434393A (en) * | 2016-09-09 | 2017-02-22 | 南京百斯杰生物工程有限公司 | Recombinant Aspergillus niger expression strain |
-
2018
- 2018-09-01 CN CN201811015932.8A patent/CN109097435A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0125698A2 (en) * | 1983-05-16 | 1984-11-21 | NABISCO BRANDS, Inc. | A process for screening microorganisms producing glucose-2-oxidase |
| CN102382773A (en) * | 2011-10-26 | 2012-03-21 | 江南大学 | Aspergillus niger strain capable of producing glucose oxidase and application thereof |
| CN103409340A (en) * | 2012-03-07 | 2013-11-27 | 昆明金泽实业有限公司 | Microbial strain and application of bactericide thereof in biological treatment of phenolic wastewater |
| CN102925362A (en) * | 2012-09-17 | 2013-02-13 | 中国农业科学院农业环境与可持续发展研究所 | Screening method of fusarium oxysporum and application thereof |
| CN106434393A (en) * | 2016-09-09 | 2017-02-22 | 南京百斯杰生物工程有限公司 | Recombinant Aspergillus niger expression strain |
Non-Patent Citations (3)
| Title |
|---|
| 刘峰等: "从土壤中快速筛选葡萄糖氧化酶产生菌及发酵工艺的优化 ", 《生物技术》 * |
| 解玉红等: "土壤和空气中纤维素酶高产菌株的筛选 ", 《生物技术通讯》 * |
| 韩建春等: "产葡萄糖氧化酶菌株的筛选及发酵培养基的优化 ", 《食品科学》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110066849A (en) * | 2019-04-04 | 2019-07-30 | 大连大学 | A kind of screening technique producing cholesterol oxidase bacterial strain |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103865797B (en) | A kind of rich selenium subtilis zymolyte and preparation method thereof | |
| CN104928202A (en) | Fermentation culture method of bacillus | |
| CN103614447B (en) | A kind of Ferment of DM production method utilizing cane molasses Substitute For Partial W-Gum | |
| CN104630166A (en) | Method for producing low-temperature glucose oxidase by virtue of microbial fermentation | |
| CN103614428A (en) | Method for fermentation production of L-tryptophan with high efficiency | |
| CN104630167A (en) | Method for producing low-temperature glucose oxidase by fermentation of marine microorganisms | |
| CN104561140B (en) | A kind of method of preparation of citric acid by fermentation | |
| CN109097435A (en) | A method of screening malaga carbohydrate oxidase strain from air | |
| CN101514328B (en) | Alkaline pectase high-yielding strain and method for producing alkaline pectase by fermenting | |
| CN102268379B (en) | Trametes trogii and method for producing cellulase by using same | |
| CN101638674B (en) | Method for manufacturing citric acid by utilizing cane sugar hydrolysate fermentation method | |
| CN104131042B (en) | Method for production of L-lactic acid by control of growth form of rhizopus oryzae | |
| CN101638675B (en) | Method for manufacturing citric acid by cane sugar fermentation method | |
| CN104673767A (en) | Method for producing feruloyl esterase | |
| CN105154360B (en) | A kind of cultural method of Comamonas testosteroni HY-08D | |
| CN108587923B (en) | Method for improving malic acid fermentation performance | |
| CN105624213B (en) | A method of 2,3- butanediol is produced using microalgae for raw material | |
| CN106754829A (en) | A kind of method of utilization bacillus HS17 fermenting and producing chitosan enzymes and its application | |
| CN107988085B (en) | A kind of microorganism Aspergillus aculeatus bacterial strain of high yield acid pectase and its application | |
| CN105969811B (en) | The method for preparing pyrogallic acid using lactobacillus plantarum fermentation gallic acid | |
| CN105505787A (en) | Aspergillus niger mutant strain for producing transglucosidase | |
| CN110106095A (en) | The aspergillus niger and its construction method and application that one plant of calcium channel CchA gene inactivates | |
| CN105441367A (en) | Serratia marcescens and application thereof | |
| CN116286513B (en) | Lactobacillus johnsonii FR-1012 and method for industrially producing gamma-aminobutyric acid by same | |
| CN102134581A (en) | Method for fermenting lactic acid bacteria |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20181228 |