CN102409011A - Bacillus flexus and application thereof - Google Patents
Bacillus flexus and application thereof Download PDFInfo
- Publication number
- CN102409011A CN102409011A CN2011103113807A CN201110311380A CN102409011A CN 102409011 A CN102409011 A CN 102409011A CN 2011103113807 A CN2011103113807 A CN 2011103113807A CN 201110311380 A CN201110311380 A CN 201110311380A CN 102409011 A CN102409011 A CN 102409011A
- Authority
- CN
- China
- Prior art keywords
- genus bacillus
- enzyme
- alkali
- crooked genus
- bacillus
- 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.)
- Granted
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a Bacillus flexus JN46 with the collection number of CCTCC (China Center for Type Culture Collection) NO:M2011231. Alkali amylase produced by the Bacillus flexus has higher alkali resistance and a wider applicable pH value range and can be used for efficiently degrading starch under an alkali condition. The alkali amylase produced by the Bacillus flexus is mainly used for the fields of spinning desizing, detergent additives, food, medicines and the like.
Description
Technical field
The present invention relates to a kind of crooked genus bacillus and application thereof, particularly a kind of crooked genus bacillus and application thereof of producing the alkali starch enzyme.
Background technology
AMS (EC 3.2.1.1) is a kind of important industrial enzymes of degraded starch, is mainly used in fields such as food, weaving, medicine, washing composition.Alkali starch enzyme optimal reaction pH should be higher than 8.0, and the alkali starch enzyme can be used for textiles destarch, detergent additives and makes viscosity modifier of sticker etc. with starch.Horikoshi (Horikoshi K. Production of alkaline enzymes by alkalophilic microorganisms. Agricultural and Biological Chemistry; 1971,35 (11): 1783-1791) at first reported in 1971 and originate from the alkali starch enzyme of having a liking for alkali bacterium A-40-2.Saxena (Saxena R, Voight BF, Lyssenko V in 2007; Burtt NP, de Bakker PIW, Chen H; Roix JJ, Kathiresan S, Hirschhorn JN; Daly MJ. Genome-wide association analysis identifies loci for type 2 diabetes and triglyceride levels. Science, 2007,316 (5829): 1331-1336) wait the people to screen the bacterial strain that a strain can be produced the alkali starch enzyme.Pancha (Pancha I, Jain D, Shrivastav A in 2010; Mishra S, Shethia B, Mishra S; VP M; Jha B. A thermoactive [alpha]-amylase from a Bacillus sp. isolated from CSMCRI salt farm. International Journal of Biological Macromolecules, 2010,47 (2): 288-291) wait the people to filter out a strain and produce the heatproof amylase strain.People such as the Lu Tao of Sichuan University (Lu Tao etc. high-temperature is produced the seed selection [J] of bacterial strain. Sichuan University's journal (natural science edition); 2002; 39 (6): 1131-1133) from Baxiu County, Damxung, Tibet hot spring soil, filter out the enhanced variant that alpha-amylase is secreted in a strain, be Bacillus licheniformis, the geographical environment of this bacteria growing is unique; Its hot properties is good, has obtained the active territory of a kind of pH broad, the higher glycase of thermostability through transforming.At present, domestic pre-treatment zymin market is monopolized by company of outstanding ability section of the Denmark Novozymes Company and the U.S. basically.A kind of alkali starch enzyme has been introduced by Novozymes Company, and the pH and the TR of broad arranged.Domestic and international industrialized alkali starch enzyme does not almost have at present, and therefore the strain excellent of screening product alkali starch enzyme is necessary very much.
Summary of the invention
The invention provides a kind of crooked genus bacillus (
Bacillus flexus) JN46, being preserved in Chinese typical culture collection center on July 1st, 2011, deposit number is CCTCC M 2011231; Classify basic phylogenetic tree as shown in Figure of description 1 with its 16SrDNA total order.
The invention provides a kind of alkali starch enzyme, this enzyme is CCTCC NO:M 2011231 production, optimal reaction pH8.0-10.5, especially 9.7.
The crooked genus bacillus of said product alkali starch enzyme is from alkaline soil, to screen; Its storage conditions is following: access the crooked genus bacillus of 3 rings to the shake-flask culture base from well-grown flat board; 30 ℃ of shaking tables (200 rpm) are cultivated 36 h; Get 0.65 mL immigration and contain in the glycerine pipe of the aseptic glycerine of 0.3 mL, put into-70 ℃ of Ultralow Temperature Freezers and preserve.
Said solid-state plate culture medium is: 1.0% W-Gum, 0.5% yeast powder, 1.5% peptone, 0.5% NaCl, 0.05% KH
2PO
4, 2% agar powder after the sterilization, is used Na
2CO
3Transfer pH 9.5-10.5.
Said liquid fermentation medium is: 1.5% Zulkovsky starch, 1.0% yeast powder, 1.0% peptone, 0.5% NaCl, 0.05% KH
2PO
4, after the sterilization, use Na
2CO
3Transfer pH 9.5-10.5.
The culture condition of said crooked genus bacillus is: the crooked genus bacillus of preservation glycerine pipe is inserted liquid fermentation medium, and 30 ℃, 200 rpm, shaking table cultivate 36 h.
With the fermented liquid behind the crooked fermentation of bacillus that produces the alkali starch enzyme, centrifugal 10 min of 10000 rpm, under pH 10.0 conditions, the enzyme that adopts the DNS method to measure the alkali starch enzyme enzyme of centrifugal secondary fermentation supernatant is lived.Under alkaline condition, enzyme is lived obviously.Under different alkaline pH values, measure the optimal reaction pH of alkali starch enzyme, the optimal reaction pH of alkali starch enzyme is 9.7, under alkaline environment, can efficient degradation starch.
The present invention in alkaline soil, filter out a strain produce the crooked genus bacillus of alkali starch enzyme (
Bacillus flexus) JN46, this bacterial strain can be produced the alkali starch of anti-alkaline enzyme, this alkali starch enzyme can be under the alkaline environment efficient degradation starch.With the alkali starch enzyme of the bacterial strain production fields such as destarch, washing composition interpolation, medicine, food that are applied to weave, can reach required purpose at efficient degradation starch under the alkaline environment.
Description of drawings
Fig. 1: the phylogenetic tree of crooked genus bacillus (Bacillus flexus) JN46
Fig. 2: the optimal reaction pH of the alkali starch enzyme that crooked genus bacillus produces
Fig. 3: the righttest stable pH of the alkali starch enzyme that crooked genus bacillus produces
Biological material specimens preservation statement:
Crooked genus bacillus (Bacillus flexus) JN46 that the present invention screening obtains is preserved in Chinese typical culture collection center on July 1st, 2011, and deposit number is: CCTCC NO:M 2011231, the preservation address is Chinese Wuhan Wuhan University.
Embodiment
Embodiment 1: the crooked genus bacillus screening method that produces the alkali starch enzyme
Get a certain amount of alkaline soil of taking; Put into the 250mL triangular flask that 100 mL sterilized waters and granulated glass sphere are housed; Vibration; Dilution different concns gradient, the sample separate application that the different concns gradient dilution is good are on 10.0 the solid medium flat board to the pH value that contains W-Gum, 30 ℃ of cultivation 36 h.Carry out plate streaking repeatedly and separate, triplicate obtains single bacterium colony of purifying, supplies next step to identify and uses.
Identify and mainly to pass through: 1) colony characteristics is observed: containing on the solid medium flat board of W-Gum, direct viewing with the naked eye has the bacterial strain of starch degradation transparent circle in periphery of bacterial colonies.2) individual morphology is observed: observe with opticmicroscope dyeing, provoke the bacterial strain of gemma.The bacterial strain that satisfies the evaluation condition is chosen glycerine guarantee the Tibetan.
Measure-70 ℃ of glycerine pipe preservative fluids with 3% inoculation and be inoculated in the fermention medium, 30 ℃, the 200rpm shake-flask culture.Get the fermented liquid of 36 h of 30 ℃ of shaking tables cultivations, 10000 rpm, 4 ℃ of centrifugal 10 min.After the centrifugal end, get supernatant, adopt the DNS method
[5], under alkaline condition, measure the enzyme of alkali starch enzyme and live.
Through utilizing the DNS method alkali starch enzyme is measured; But filter out that a strain has stronger alkaline-resisting ability and the bacterial strain of efficient degradation starch under alkaline environment; To produce the work of alkali starch enzyme enzyme be 767.5 U/mL; Can know that through the 16SrDNA analysis this bacterial strain is crooked genus bacillus, be preserved in Chinese typical culture collection center in 2011, deposit number is CCTCC M 2011231.
Embodiment 2: crooked genus bacillus produces the mensuration of alkali starch enzyme optimal reaction pH
The DNS method is measured alkali starch enzyme enzyme and is lived
1) configuration of DNS reagent: take by weighing 2.5 g 3; The 5-dinitrosalicylic acid is dissolved in the less water; Add 0.5 g phenol; Dissolve 0.075 g S-WAT, 2.5 g sodium hydroxide, 50 g Seignette salts again, it is changed in the 500 mL volumetric flasks shake up constant volume, be stored in brown bottle and be placed in 4 ℃ of refrigerators for use.
2) making of SANMALT-S typical curve: the maltose solution of preparing 0.2 g/L-1.0 g/L different concns.Get the SANMALT-S of 1 mL different concns and mix, put into boiling water bath, water-bath 10 min with DNS solution with volume.With the cold water cooling, be settled to 10 mL, A
540Measure light absorption value.Concentration with SANMALT-S is X-coordinate, is ordinate zou with the light absorption value, the production standard curve.
3) Zulkovsky starch with 2 mL 1% joins in the test tube, adds the damping fluid of 1 mL, mixing, and 55 ℃ of preheating 5 min add the good enzyme liquid of 0.4 mL dilution, react 5 min.Get 1 ml reaction solution with the DNS reagent mixing of volume, boiling water bath boils 10 min, with the cold water cooling, is settled to 10 ml, behind the mixing, as contrast, measures A with the reaction system that do not have enzyme-added liquid but add normal deionized water
540Light absorption value.
The mensuration of alkali starch enzyme optimal reaction pH value
Adopt glycocoll-sodium hydrate buffer solution, prepare the damping fluid (7.0,7.5,8.0 of different pH; 8.5,9.0,9.5,10.0; 10.5,11.0), with damping fluid and Zulkovsky starch mixing, adopt 3) method measure alkali starch enzyme optimal reaction pH (see figure 2).The optimal reaction pH of this alkali starch enzyme is 9.7, can efficient degradation starch under alkaline environment, be the alkali starch enzyme.
The enzyme activity unit definition: at pH 10.0, temperature 55
oC produces the needed enzyme amount of 1 μ g reducing substance (calculating with SANMALT-S) at 1 min degraded Zulkovsky starch, is 1 enzyme unit (U) alive.
Embodiment 3: crooked genus bacillus produces the mensuration of the righttest stable pH of alkali starch enzyme
Adopt glycocoll-sodium hydrate buffer solution, dispose different gradient pH damping fluids (7.0,7.5,8.0,8.5,9.0,9.5,10.0,10.5,11.0).Respectively same concentrations enzyme liquid (500 U/mL) is mixed in test tube with the damping fluid of different pH, insulation 24 h in 25 ℃ of water-baths adopt among the embodiment 23 respectively under different pH) measure the method for alkali starch enzyme, measure residual sugar enzyme amount alive.Be the basis with enzyme initial value alive, all enzyme work divided by initial value, multiply by 100%, obtain of the variation of alkali starch enzyme enzyme activity, the situation that vigor is residual along with different pH.The result shows that in the pH value was the scope of 8.0-10.5, the alkali starch enzyme was stablized (see figure 3), proved absolutely that this glycase is alkali resistance glycase.
It is understandable that, concerning those of ordinary skills, can be equal to replacement or change according to technical scheme of the present invention and inventive concept thereof, and all these changes or replacement all should belong to the protection domain of the appended claim of the present invention.
Claims (7)
1. a crooked genus bacillus is preserved in Chinese typical culture collection center on July 1st, 2011, and deposit number is: CCTCC NO:M 2011231.
2. the described crooked genus bacillus of claim 1 is characterized in that this bacterium produces the alkali starch enzyme.
3. the described crooked genus bacillus of claim 2 is characterized in that said glycase ph optimum is 8.0-10.5.
4. the described crooked genus bacillus of claim 3 is characterized in that said glycase optimal reaction pH is 9.7.
5. the arbitrary described crooked genus bacillus of claim 1-4 is used to produce glycase.
6. the described application of claim 5 is characterized in that producing diastatic fermention medium component and is (w/v): 1.5% Zulkovsky starch, 1.0% yeast powder, 1.0% peptone, 0.5% NaCl, 0.05% KH
2PO
4, after the sterilization, use Na
2CO
3Transfer pH 9.5-10.5.
7. the arbitrary described crooked genus bacillus of claim 1-3 is in the application of weaving destarch, washing composition, chemical industry, field of food.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110311380 CN102409011B (en) | 2011-10-14 | 2011-10-14 | Bacillus flexus and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110311380 CN102409011B (en) | 2011-10-14 | 2011-10-14 | Bacillus flexus and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102409011A true CN102409011A (en) | 2012-04-11 |
| CN102409011B CN102409011B (en) | 2013-08-28 |
Family
ID=45911362
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110311380 Expired - Fee Related CN102409011B (en) | 2011-10-14 | 2011-10-14 | Bacillus flexus and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102409011B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103333825A (en) * | 2013-06-24 | 2013-10-02 | 邱树毅 | Bacillus flexus and application thereof |
| CN104630091A (en) * | 2014-12-13 | 2015-05-20 | 河南农业大学 | Tobacco plant growth promoting bacterium YC4 and application thereof |
| CN105238717A (en) * | 2015-10-21 | 2016-01-13 | 江南大学 | Bacillus flexus with high yield of beta-amylase and application of bacillus flexus |
| CN114015589A (en) * | 2020-12-10 | 2022-02-08 | 山东省果树研究所 | Biocontrol microbial agent containing campylobacter TA-12 and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1954076A (en) * | 2004-04-06 | 2007-04-25 | 梅坦诺米克斯有限公司 | Process for the production of fine chemicals |
-
2011
- 2011-10-14 CN CN 201110311380 patent/CN102409011B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1954076A (en) * | 2004-04-06 | 2007-04-25 | 梅坦诺米克斯有限公司 | Process for the production of fine chemicals |
Non-Patent Citations (3)
| Title |
|---|
| SURESH K.，ET AL.: "Bacillus indicus sp. nov., an arsenic-resistant bacterium isolated from an aquifer in West Bengal, India", 《INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY》, 31 December 2004 (2004-12-31), pages 1369 - 1375 * |
| 赵建等: "一株碱性淀粉酶产生菌Bacillus flexus XJU-3 的分离鉴定及酶学特性分析(英文)", 《微生物学报》, vol. 48, no. 6, 4 June 2008 (2008-06-04), pages 750 - 756 * |
| 邓钢桥，等: "中药中耐辐射微生物的分离与鉴定", 《激光生物学报》, vol. 16, no. 2, 30 April 2007 (2007-04-30), pages 226 - 229 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103333825A (en) * | 2013-06-24 | 2013-10-02 | 邱树毅 | Bacillus flexus and application thereof |
| CN103333825B (en) * | 2013-06-24 | 2014-12-10 | 贵州珍酒酿酒有限公司 | Bacillus flexus and application thereof |
| CN104630091A (en) * | 2014-12-13 | 2015-05-20 | 河南农业大学 | Tobacco plant growth promoting bacterium YC4 and application thereof |
| CN104630091B (en) * | 2014-12-13 | 2018-04-17 | 河南农业大学 | A kind of tobacco rhizosphere Promoting bacteria YC4 and its application |
| CN105238717A (en) * | 2015-10-21 | 2016-01-13 | 江南大学 | Bacillus flexus with high yield of beta-amylase and application of bacillus flexus |
| CN105238717B (en) * | 2015-10-21 | 2019-01-11 | 江南大学 | A kind of Bacillus flexus of high yield beta amylase and its application |
| CN114015589A (en) * | 2020-12-10 | 2022-02-08 | 山东省果树研究所 | Biocontrol microbial agent containing campylobacter TA-12 and application thereof |
| CN114015589B (en) * | 2020-12-10 | 2022-07-12 | 山东省果树研究所 | Biocontrol microbial agent containing campylobacter TA-12 and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102409011B (en) | 2013-08-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Saxena et al. | Amylase production by solid-state fermentation of agro-industrial wastes using Bacillus sp. | |
| CN102409011B (en) | Bacillus flexus and application thereof | |
| Yin et al. | Bioproperties and purification of xylanase from Bacillus sp. YJ6 | |
| Mo et al. | Identification and characterization of a novel xylanase derived from a rice straw degrading enrichment culture | |
| Silva et al. | Effect of the culture conditions on the production of an extracellular protease by thermophilic Bacillus sp and some properties of the enzymatic activity | |
| CN102389022B (en) | Method for preparing ayfivin compound protein feed additive by degrading feather with bacillus licheniformis | |
| Singh | Optimization of culture conditions for thermostable chitinase production by Paenibacillus sp. D1 | |
| CN102311934B (en) | Alkalophilic bacillus for producing alkaline amylase and application thereof | |
| CN102816831A (en) | Kit for separating and identifying campylobacter rectus as well as preparation and application for kit | |
| CN113005054B (en) | Bacillus amyloliquefaciens SS-ZC-26 and preparation method and application thereof | |
| Kacagan et al. | Characterization of a xylanase from a thermophilic strain of Anoxybacillus pushchinoensis A8 | |
| Natsir et al. | Isolation and identification of chitinolytic bacteria of pohara river of South East Sulawesi and the optimization production of chitinase enzyme | |
| CN102409010B (en) | Gamma-proteobacteria and application thereof | |
| CN112410247A (en) | Pseudomonas defensins and application thereof in down feather detergent | |
| CN105132304B (en) | The life spore deficiency Bacillus clausii of one plant of high yield alkali protein | |
| CN105018373B (en) | A kind of Bacillus clausii of high yield alkali protein | |
| CN101942401B (en) | Strain for producing chitinase and method for producing chitinase in high yield | |
| CN101649309B (en) | Method for producing cellulase through solid-state fermentation of raw materials | |
| CN101182513A (en) | Method for mutagenesis breeding neuter protease high yield bacterial strain by hypophrenia N+ion injection technology | |
| CN107201382B (en) | A kind of detergent and its preparation method and application | |
| CN102191235A (en) | Method for producing beta-mannanase through fermentation by utilizing konjac flour microorganisms | |
| Li et al. | Antagonistic mechanism of Fusarium oxysporum of soybean root rot by Bacillus subtilis | |
| CN102604861B (en) | Pullulanase, pullulanase producing strain and application of pullulanase | |
| CN105969694A (en) | Providencia for synthesizing ethanol-resistant urethanase and urease | |
| CN108130347A (en) | A kind of method for producing chitin oligo saccharide |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130828 Termination date: 20141014 |
|
| EXPY | Termination of patent right or utility model |