CN102409011B - Bacillus flexus and application thereof - Google Patents
Bacillus flexus and application thereof Download PDFInfo
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- CN102409011B CN102409011B CN 201110311380 CN201110311380A CN102409011B CN 102409011 B CN102409011 B CN 102409011B CN 201110311380 CN201110311380 CN 201110311380 CN 201110311380 A CN201110311380 A CN 201110311380A CN 102409011 B CN102409011 B CN 102409011B
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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
α-Dian Fenmei (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 binding agent 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 in 2007, Voight BF, Lyssenko V, Burtt NP, de Bakker PIW, Chen H, Roix JJ, Kathiresan S, Hirschhorn JN, Daly MJ.Genome-wide association analysis identifies loci for type 2diabetes 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 in 2010, Jain D, Shrivastav A, 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 uniqueness of this bacteria growing, its hot properties is good, has obtained the amylase that the active territory of a kind of pH is wideer, thermostability is higher through transformation.At present, domestic pre-treatment zymin market is monopolized by company of outstanding energy section of Denmark Novozymes Company and the U.S. substantially.A kind of alkali starch enzyme has been introduced by Novozymes Company, and wideer pH and temperature range are 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, be preserved in Chinese typical culture collection center on July 1st, 2011, deposit number is CCTCC M 2011231; Classify the phylogenetic tree on basis 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 described product alkali starch enzyme is to screen from alkaline soil, its storage conditions is as follows: access the crooked genus bacillus of 3 rings to the shake-flask culture base from well-grown flat board, 30 ℃ of shaking tables (200rpm) are cultivated 36h, get the 0.65mL immigration and contain in the glycerine pipe of the aseptic glycerine of 0.3mL, put into-70 ℃ of Ultralow Temperature Freezers and preserve.
Described 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.
Described 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 described crooked genus bacillus is: the crooked genus bacillus of preservation glycerine pipe is inserted liquid fermentation medium, and 30 ℃, 200rpm, shaking table is cultivated 36h.
With the fermented liquid behind the crooked fermentation of bacillus of product alkali starch enzyme, the centrifugal 10min of 10000rpm, under pH 10.0 conditions, the enzyme that adopts the DNS method to measure the alkali starch enzyme enzyme of centrifugal secondary fermentation supernatant liquor 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 filters out crooked genus bacillus (Bacillus flexus) JN46 that the alkali starch enzyme is produced in a strain in alkaline soil, this bacterial strain can be produced the alkali starch enzyme of anti-highly basic, this alkali starch enzyme can be under the environment of highly basic 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 suitableeest 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 100mL sterilized water and granulated glass sphere are housed, vibration, dilution different concns gradient, it is on 10.0 the solid medium flat board that the sample that the different concns gradient dilution is good is applied to the pH value that contains W-Gum respectively, cultivates 36h for 30 ℃.Carry out plate streaking repeatedly and separate, triplicate obtains single bacterium colony of purifying, identifies for next step 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 the preservation of glycerine pipe.
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 the 36h of 30 ℃ of shaking tables cultivations, 10000rpm, 4 ℃ of centrifugal 10min.After the centrifugal end, get supernatant liquor, adopt the DNS method
[5], under alkaline condition, measure the enzyme of alkali starch enzyme and live.
By 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, the alkali starch enzyme enzyme that produces live and to be 767.5U/mL, analyzing as can be known by 16SrDNA, 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 2.5g 3, the 5-dinitrosalicylic acid is dissolved in the less water, add 0.5g phenol, dissolve 0.075g S-WAT, 2.5g sodium hydroxide, 50g Seignette salt again, it is changed in the 500mL volumetric flask shake up constant volume, be stored in brown bottle and be placed in 4 ℃ of refrigerators stand-by.
2) making of maltose typical curve: the maltose solution of preparation 0.2g/L-1.0g/L different concns.Get the maltose of 1mL different concns and mix with DNS solution with volume, put into boiling water bath, water-bath 10min.With the cold water cooling, be settled to 10mL, A
540Measure light absorption value.Concentration with maltose is X-coordinate, is ordinate zou with the light absorption value, the production standard curve.
3) Zulkovsky starch with 2mL 1% joins in the test tube, adds the damping fluid of 1mL, mixing, and 55 ℃ of preheating 5min add 0.4mL and dilute good enzyme liquid, reaction 5min.Get the 1ml reaction solution with the DNS reagent mixing of volume, boiling water bath boils 10min, with the cold water cooling, is settled to 10ml, behind the mixing, there not to be enzyme-added liquid but add equivalent deionized water reaction system in contrast, measure A
540Light absorption value.
The mensuration of alkali starch enzyme optimal reaction pH value
Adopt glycine-sodium hydrate buffer solution, prepare the damping fluid (7.0,7.5,8.0,8.5,9.0,9.5,10.0,10.5,11.0) of different pH, with damping fluid and Zulkovsky starch mixing, adopt 3) method mensuration 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 10.0,55 ℃ of temperature produce the needed enzyme amount of 1 μ g reducing substance (calculating with maltose) at 1min degraded Zulkovsky starch, are 1 enzyme unit (U) alive.
Embodiment 3: crooked genus bacillus produces the mensuration of the suitableeest stable pH of alkali starch enzyme
Adopt glycine-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 (500U/mL) is mixed in test tube with the damping fluid of different pH, in 25 ℃ of water-baths, is incubated 24h, under different pH, adopt among the embodiment 23 respectively) measure the method for alkali starch enzyme, measure the amount that the residual sugar enzyme is lived.Based on enzyme initial value alive, all enzyme work divided by initial value, be multiply by 100%, obtain alkali starch enzyme enzyme activity along with the variation of different pH, the situation that vigor is residual.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 amylase is alkali resistance amylase.
Be understandable that, for 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 (4)
1. a crooked genus bacillus (Bacillus flexus) is preserved in Chinese typical culture collection center on July 1st, 2011, and deposit number is: CCTCC NO:M2011231.
2. the application of the described crooked genus bacillus of claim 1 in producing amylase.
3. the described application of claim 2 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 pH9.5-10.5.
4. the described crooked genus bacillus of claim 1 is in the application of chemical industry, field of food.
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| CN102409011B true CN102409011B (en) | 2013-08-28 |
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| CN103333825B (en) * | 2013-06-24 | 2014-12-10 | 贵州珍酒酿酒有限公司 | Bacillus flexus 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 |
| 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 |
| 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 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
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| 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 (4)
| 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》.2004,1369–1375. * |
| 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);赵建等;《微生物学报》;20080604;第48卷(第6期);750-756 * |
| 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》.2008,第48卷(第6期),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》.2007,第16卷(第2期),226-229. * |
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