CN102409010B - Gamma-proteobacteria and application thereof - Google Patents
Gamma-proteobacteria and application thereof Download PDFInfo
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- CN102409010B CN102409010B CN2011103112607A CN201110311260A CN102409010B CN 102409010 B CN102409010 B CN 102409010B CN 2011103112607 A CN2011103112607 A CN 2011103112607A CN 201110311260 A CN201110311260 A CN 201110311260A CN 102409010 B CN102409010 B CN 102409010B
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- mycetozoan
- enzyme
- starch enzyme
- alkali starch
- alkali
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Abstract
The invention discloses a gamma-proteobacteria (gamma Proteobacterium) JN25 for producing alkaline amylase, the preservation number is CCTCC No: M 2011230. The alkaline amylase produced by the proteobacteria has high alkali resistance and wide applicable pH value; it can efficiently degrade starch under alkaline condition. The alkaline amylase produced by the proteobacteria is mainly applied to textile desizing, detergent additives, food, medicine and other fields.
Description
Technical field
The present invention relates to strain γ-mycetozoan and an application thereof, particularly γ-mycetozoan and the application thereof of alkali starch enzyme produced in a strain.
Background technology
α-amylase (EC 3.2.1.1) is a kind of important industrial enzymes of degraded starch, is mainly used in the 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 mlcroorganusms.Agricultural and Biological Chemistry, 1971,35 (11): 1783-1791) at first reported the alkali starch enzyme that originates from basophilic bacterium A-40-2 in 1971.Saxena (Saxena R in 2007, Voight BF, Lyssenko V, Burtt NP, de Bakker PIW, Chen H, Roix JJ, Kathiresan S.fiirschhorn JN, Daly MJ.Genome-wide association analysis identifies loci for type 2 diabetes and triglyceride levels.Science, 2007,316 (5829): 1331-1336) etc. the people screens 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) etc. the people filters out a strain and produces the heatproof amylase strain.The people such as the Lu Tao of Sichuan University (Lu Tao etc. high-temperatureα-amylase 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 amylase that the active territory of a kind of pH is wider, 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 wider 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 strain γ-mycetozoan (gamma Proteobacterium) JN25, be preserved in Chinese Typical Representative culture collection center, deposit number CCTCC NO:M 2011230 on July 1st, 2011; 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 2011230 production, and optimal reaction pH is 8.0-10.5, especially 10.0.
γ-the mycetozoan of described product alkali starch enzyme is to screen from alkaline soil, its storage conditions is as follows: access 3 ring γ-mycetozoans to the shake-flask culture base from well-grown solid-state plate culture medium, 30 ℃ of shaking tables (200rpm) are cultivated 36h, 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.
Described solid-state plate culture medium is (w/v): 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 (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.
The culture condition of described γ-mycetozoan is: the γ of preservation glycerine pipe-mycetozoan is accessed liquid fermentation medium, and 30 ℃, 200rpm, shaking table is cultivated 36h.
With the fermented liquid that produces after the γ of alkali starch enzyme-mycetozoan fermentation, 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 10.0, under alkaline environment, can efficient degradation starch.
The present invention filters out γ-mycetozoan (gamma Proteobacterium) JN25 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 at efficient degradation starch under the alkaline environment, reach required purpose.
Description of drawings
Fig. 1: the phylogenetic tree of γ-mycetozoan (gamma Picteobacteriuin) JN25
The optimal reaction pH of the alkali starch enzyme that Fig. 2: γ-mycetozoan produces
The suitableeest stable pH of the alkali starch enzyme that Fig. 3: γ-mycetozoan produces
Biological material specimens preservation statement:
γ-mycetozoan (gamma Proteobacterium) JN25 that the present invention's screening obtains, be preserved in Chinese Typical Representative culture collection center on July 1st, 2011, deposit number is: CCTCC NO:M 2011230, the preservation address is Wuhan, China Wuhan University.
Embodiment
Implement 1: the γ of product alkali starch enzyme-mycetozoan screening method
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 respectively the pH value that contains W-Gum, cultivates 36h for 30 ℃.Repeatedly carry out plate streaking and separate, triplicate obtains single bacterium colony of purifying, identifies for next step.
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: use observation by light microscope, provoke the bacterial strain of mycetozoan form.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.
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.
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 610.2U/mL, identify by 16SrDNA, sequential analysis as can be known this bacterial strain is γ-mycetozoan, be preserved in Chinese Typical Representative culture collection center on July 1st, 2011, deposit number is CCTCC M 2011230.
Embodiment 2: γ-mycetozoan 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 a small amount of water, add 0.5g phenol, dissolve again 0.075g S-WAT, 2.5g sodium hydroxide, 50g Seignette salt, 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/L1.0g/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.Take the concentration of maltose as X-coordinate, take light absorption value as ordinate zou, 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 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 (seeing Fig. 2).The optimal reaction pH of this alkali starch enzyme is 10.0, can efficient degradation starch under alkaline environment, be the alkali starch enzyme.
Embodiment 3: γ-mycetozoan 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 from the damping fluid of different pH, in 25 ℃ of water-baths, is incubated 24h, under different pH, adopt respectively among the embodiment 2 3) measure the method for alkali starch enzyme, measure the amount that the residual sugar enzyme is lived.Live initial value as the basis take enzyme, 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 (seeing Fig. 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 (7)
1. a strain γ-mycetozoan (Gamma Proteobacterium) is preserved in Chinese Typical Representative culture collection center on July 1st, 2011, and deposit number is: CCTCC NO:M 2011230.
2. γ-mycetozoan according to claim 1 is characterized in that this bacterium produces the alkali starch enzyme.
3. γ-mycetozoan claimed in claim 2 is characterized in that described amylase optimal pH is 8.0-10.5.
4. γ-mycetozoan according to claim 3 is characterized in that described amylase optimal reaction pH is 10.0.
5. the application of the arbitrary described γ-mycetozoan of claim 1-4 in producing the alkali starch enzyme.
6. application according to claim 5 is characterized in that the fermention medium component of producing the alkali starch enzyme 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. arbitrary described γ-mycetozoan is in the application of weaving destarch, washing composition, chemical industry, field of food according to claim 1-4.
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|---|---|---|---|
| CN2011103112607A CN102409010B (en) | 2011-10-14 | 2011-10-14 | Gamma-proteobacteria and application thereof |
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| CN2011103112607A CN102409010B (en) | 2011-10-14 | 2011-10-14 | Gamma-proteobacteria and application thereof |
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| CN102409010A CN102409010A (en) | 2012-04-11 |
| CN102409010B true CN102409010B (en) | 2013-01-30 |
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