CN101608169B - Thermophilic bacillus pumilus strain Tamy12 and high-temperature amylase produced by same - Google Patents
Thermophilic bacillus pumilus strain Tamy12 and high-temperature amylase produced by same Download PDFInfo
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- CN101608169B CN101608169B CN2009100946493A CN200910094649A CN101608169B CN 101608169 B CN101608169 B CN 101608169B CN 2009100946493 A CN2009100946493 A CN 2009100946493A CN 200910094649 A CN200910094649 A CN 200910094649A CN 101608169 B CN101608169 B CN 101608169B
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a bacillus pumilus novel strain Tamy12, high-temperature amylase obtained by utilizing the same through fermentation and a preparation method thereof. The colony of the Tamy12 strain is white, the surface is dry, and the edge is round; cells are in a short bar shape and are Gram positive bacteria, and the temperature range is 38-70 DEG C. Native-page analysis indicates that coarse enzyme liquid obtained by fermenting the Tamy12 contains amylase, and soluble starch can be resolved into glucose, maltose and oligosaccharide with 3-6 glucosyls. The related high-temperature amylase of the invention has higher operative temperature and wider pH adaptation range and has a certain application potential in application to the fields of the food brewing industry, the environmental protection industry, the energy utilization industry, the medicine industry and the like.
Description
Technical field
The invention belongs to the using microbe field.Particularly, relate to a kind of bacillus pumilus (Bacillus pumilus) Tamy 12 bacterial strains, and therefrom produce the alpha-amylase that obtains.
Background technology
Have a liking for this speech of atomic biology (Extremophiles) and put forward in 1974, be meant the general designation of the microbial population that in the extreme environment that general biology can't be survived (high temperature, cold, strong acid, highly basic, high salt, high pressure, high radiation etc.) can normal existence by MacElroy.Mainly comprise acidophilic microorganism, halophile, halophilic microorganism, marine microorganism, barophilic microorganisms, cryophile, thermophilic microorganism etc.
Thermophilic microorganism is that a class is lived in the microorganism in the hot environment, as crater and peripheral region thereof, hot spring, factory's hot wastewater discharge region etc.Derive from the extreme enzyme of thermophilic microorganism, under extreme environment, have with normal enzyme different vigor and stability are arranged.The preparation cost that Zimadzhunt L 340 has zymin is low, and kinetic reaction is fast, requires standard low to the reaction cooling system, reduces energy consumption, has improved advantages such as product purity, therefore contains certain application potential in various fields.
Amylase is to use maximum a kind of enzymes at present, and it has in starch industry the most widely uses, can hydrolysis and amyloclastic.α-Dian Fenmei (E.C.3.2.1.1) is an endo-type amylase, when it acts on starch is to cut α-1,4 glycosidic link arbitrarily from intramolecule, and starch molecule is degraded rapidly, loses the color reaction of viscosity and iodine, and the reducing power of hydrolyzate is increased.When being substrate with the amylose starch, the reaction generally be divided into two the step carry out, it cuts α-1,4 glycosidic link at first arbitrarily, make the rapid hydrolysis of amylose starch generate maltose, trisaccharide maltose and more macromolecular oligosaccharides, and then trisaccharide maltose and oligosaccharides slowly are hydrolyzed into maltose and glucose.In second step, when α-Dian Fenmei acted on amylopectin, it is hydrolyzing alpha-1 arbitrarily, 4 glycosidic links, but can not cut α-1,6 key of tapping point, near the α-1 can not the hydrolysis tapping point, 4 glycosidic links, but can cross α-1,6 key and cut inner α-1,4 key, therefore hydrolysate also contains a series of α-limit dextrin except that maltose, glucose.
Because amylase generally will at high temperature make starch pasting in liquefaction, saccharifying, so the conversion of starch needs heat-staple amylase (heat-stable amylase).The thermotolerance α-Dian Fenmei has good thermostability, has a wide range of applications in industrial production.
Alpha-amylase is screened, and its biological characteristics is analyzed, will provide foundation for alpha-amylase hot adaptability Mechanism Study and the applied research in industrial or agricultural thereof from now on.
So far, do not have the report of bacillus pumilus (Bacillus pumilus sp.) Tamy 12 bacterial strains in the prior art, do not have report yet by the alpha-amylase of its generation.
Summary of the invention
The present invention aims to provide a kind of can at high temperature the generation and has the active high-temperature amylase strain of high enzyme, and promptly bacillus pumilus (Bacillus pumilus sp.) Tamy 12 bacterial strains produce the alpha-amylase that obtains, its preparation method by it.
In order to realize above-mentioned purpose of the present invention, the invention provides following technical scheme:
A kind of bacillus pumilus (Bacillus pumilus) Tamy12 bacterial strain, culture presevation CGMCCNo.2641, it has the ability that produces alpha-amylase.
Bacillus pumilus (Bacillus pumilus) Tamy12 bacterial strain bacteria colony white, surface drying, edge rounding; Cell is that rod-short produces gemma, gram-positive microorganism, and growth pH scope is 5.0-9.0, optimal pH is 7.5; Temperature range is 38-70 ℃, and optimum growth temperature is 55 ℃.
The present invention also provides the alpha-amylase that obtains from bacillus pumilus (Bacillus pumilus) Tamy 12 bacterial strains.
The zymologic property of alpha-amylase is: crude enzyme liquid can be decomposed into Zulkovsky starch the oligosaccharides of glucose, maltose and 3 to 6 glucosyl groups, and optimal pH is 5, and temperature of reaction is 45-80 ℃.
Mutant bacillus pumilus of the present invention (Bacillus pumilus) Tamy 12 bacterial strains on August 26th, 2008 in the common micro-organisms center preservation of Beijing China Committee for Culture Collection of Microorganisms, culture presevation number is: CGMCC No.2641.
This strains separation is in Tengchong heat sea.The bacterial strain screening solid medium is (g/L): (NH
4)
2SO
4, 1.3; Peptone, 1; Zulkovsky starch, 10; KH
2PO
4, 0.276; MgSO
47H
2O, 0.25; Yeast extract, 1; Na
2MoO
42H
2O, 0.000025; CuSO
4, 0.000016; MnSO
4H
2O, 0.0022; H
3BO
3, 0.0005; ZnSO
47H
2O, 0.0005; CoCl
26H
2O, 0.000046; CaSO
4H
2O, 0.06; FeSO
4, 0.099; Agar, 20; PH7.5.
Bacillus pumilus (Bacillus pumilus sp.) Tamy 12 bacterial strain bacteria colony whites, surface drying, edge rounding; Cell is that rod-short produces gemma, gram-positive microorganism, and growth pH scope is 5.0-9.0, optimal pH is 7.5; Temperature range is 38-70 ℃, and optimum growth temperature is 55 ℃.
Identify through morphology and physiological and biochemical property,, isolating thermophile bacteria is accredited as the bacterial strain that bacillus pumilus belongs to, called after Bacillus pumilus sp.Tamy12 in conjunction with 16S rRNA gene sequencing.
Bacillus pumilus (Bacillus pumilus sp.) Tamy12 bacterial strain institute produced high-temperature amylase has following zymologic property:
1, optimal reactive temperature is about 80 ℃, and activity is preferably all arranged in 45-80 ℃ of scope, and the activity about 30% is still arranged under 98 ℃.
2, Tamy12 crude enzyme liquid optimal reaction pH is 5.
3, amylase can be decomposed into Zulkovsky starch the oligosaccharides of glucose, maltose and 3 to 6 glucosyl groups in the crude enzyme liquid of Tamy12;
Description of drawings
The alpha-amylase Native-PAGE electrophoresis reactive coloration result that Fig. 1 produces for the Tamy12 bacterial strain, wherein the 1:Tamy12 bacterial strain produces crude enzyme liquid coomassie R250 coloration result; The 2:Tamy12 bacterial strain produces crude enzyme liquid starch active coloring result;
Fig. 2 is the product TLC thin-layer chromatography result of the high-temperature starch enzymic hydrolysis Zulkovsky starch that the Tamy12 bacterial strain produces, among the figure 1: glucose, 4: the Zulkovsky starch of bacterial strain Tamy12 crude enzyme liquid digestion, 5: indigested Zulkovsky starch, 6: maltose;
The suitableeest catalytic pH curve of alpha-amylase that Fig. 3 produces for the Tamy12 bacterial strain;
The suitableeest catalytic temperature curve of alpha-amylase that Fig. 4 produces for the Tamy12 bacterial strain.
Embodiment
Embodiment 1:
The separation screening and the evaluation of bacillus pumilus (Bacillus pumilus sp.) Tamy12 bacterial strain:
The Tamy12 bacterial strain separates from Yunnan Province's Tengchong heat sea.The earth in Tengchong heat sea in the bacterial strain screening liquid nutrient medium 55 ℃, the short period of time shake-flask culture, be coated on the bacterial strain screening solid medium, cultivated 1 ~ 2 day for 55 ℃, choose the bacterial strain dibbling on the bacterial strain screening solid medium, 55 ℃ to cultivate after 3 ~ 4 days iodine liquid painted, observes the transparent circle size, and diastatic Tamy12 bacterial strain is produced in screening.The bacterial strain screening liquid nutrient medium is (g/L): (NH
4)
2SO
4, 1.3; Peptone, 1; KH
2PO
4, 0.276; MgSO
47H
2O, 0.25; Yeast extract, 1; Na
2MoO
42H
2O, 0.000025; CuSO
4, 0.000016; MnSO
4H
2O, 0.0022; H
3BO
3, 0.0005; ZnSO
47H
2O, 0.0005; CoCl
26H
2O, 0.000046; CaSO
4H
2O, 0.06; FeSO
4, 0.099; PH7.5.The bacterial strain screening solid medium is (g/L): (NH
4)
2SO
4, 1.3; Peptone, 1; Zulkovsky starch, 10; KH
2PO
4, 0.276; MgSO
47H
2O, 0.25; Yeast extract, 1; Na
2MoO
42H
2O, 0.000025; CuSO
4, 0.000016; MnSO
4H
2O, 0.0022; H
3BO
3, 0.0005; ZnSO
47H
2O, 0.0005; CoCl
26H
2O, 0.000046; CaSO
4H
2O, 0.06; FeSO
4, 0.099; Agar, 20; PH7.5.
Adopt the bacterial system authentication method that (Bacillus pumilus sp.) Tamy12 bacterial strain is identified.The morphological feature of this bacterium is as follows: bacterium colony is a white, surface drying, edge rounding; Cell is a rod-short, gram-positive microorganism; Temperature range is 38-70 ℃, and optimum growth temperature is 55 ℃.Identify through morphology and physiological and biochemical property,, isolating thermophilic bacteria is accredited as a bacterial strain of bacillus pumilus kind, name sp.Tamy12 into Bacillus pumilus in conjunction with 16S rRNA gene sequencing.
Embodiment 2:
In 50ml bacterial strain screening liquid nutrient medium, 55 ℃ of shaking tables are cultivated 24h with bacillus pumilus (Bacillus pumilus sp.) the Tamy12 inoculation of embodiment 1.Be seeded in the 500ml bacterial strain screening liquid nutrient medium in 1: 10 ratio, 55 ℃ of shaking tables were cultivated 4 days.Fermented liquid is in 4 ℃, and the centrifugal 10min of 6500rpm collects fermented supernatant fluid, uses twice of filter paper suction filtration again.With 10, the 000MWCO film carries out ultrafiltration and concentration to 50ml, 4 ℃ of preservation concentrated solutions.
Embodiment 3:
The Native-PAGE of embodiment 2 concentrated solutions:
1. Native-PAGE: in the discontinuous electro-phoresis, resolving gel concentration is 12% (add final concentration in the separation gel be 0.1% starch solution), and concentrated gum concentration is 5%.Deposition condition: behind the application of sample 15 μ l, voltage is adjusted to 130V, carries out under 4 ℃, and electrophoresis time is 3h.
2. after electrophoresis finishes, a semi-gelled is immersed in the 30%Tris-Tcl damping fluid, 55 ℃ of incubation 6h use iodine staining.According to the character of iodine-starch colour developing, around behind the amylorrhexis, have tangible hydrolysis circle generation.
3. gel-colored, decolouring: second half gel is dyeed, decolours.The steps include:
A. gel places plastics casing, adds coomassie brilliant blue R250 staining fluid (submergence gel), and 30min dyes on decolorization swinging table.
B. remove the coomassie brilliant blue R250 dye liquor, use the rinsed with deionized water gel, add 100ml destainer (25ml methyl alcohol then, the 10ml glacial acetic acid adds water and is settled to 100ml), the 30min that dyes on decolorization swinging table removes destainer, again add new destainer, liquid decoloured.
C. the protein band on gel is clear, when not having background color substantially, stops decolouring, removes destainer, uses the rinsed with deionized water gel.
The result shows that certain albumen has amylase activity in the crude enzyme liquid as shown in Figure 1.
Embodiment 4:
(1) with 115 ℃ of oven activated 30min of silica gel G plate
(2) point sample: get silica gel thin sheet, at interval uniform point sample on the 1.5cm sea line of distance base, wherein select the treatments of the sample liquid of Marker (containing glucose, maltose and trisaccharide maltose), process 16h hydrolysis 1% Zulkovsky starch respectively, point of sample dries up with blower.
(3) exhibition layer: pour the degree of depth in the chromatography cylinder into and be developing agent about 1cm (propyl carbinol, glacial acetic acid, water, 5: 3: 2, V/V/V), thin layer plate is put into chromatography cylinder open up layer,, thin plate is taken out when developing agent forward position during from thin plate top 1-2cm, airing carries out secondary exhibition layer.Behind the secondary exhibition layer thin plate is put into 65 ℃ of baking oven 30min.
(4) colour developing: spray developer (4g pentanoic, 4ml aniline and 20ml 85% phosphoric acid are dissolved in 200ml acetone), in 85 ℃ of baking ovens, toast 10-20min then.
The result shows that (Bacillus pumilus sp.) Tamy12 bacterial strain produces amylase as shown in Figure 2.
Embodiment 5:
The mensuration of amylase activity in embodiment 2 concentrated solutions: the enzyme activity determination method is that the method for DNS survey reducing sugar is carried out amylase activity mensuration (Rick W, 1974).The hydrolysis unit of activity of alpha-amylase is defined as: at a certain temperature, per minute hydrolysis Zulkovsky starch produces the required enzyme amount of 1 μ g glucose.
The mensuration of protein concn: adopt the LOWRY method to measure (LOWRY.OH, 1951).
1. the suitableeest diastatic enzyme pH that lives in embodiment 2 concentrated solutions.
Preparation 0.1M citric acid-Na
2HPO
4Damping fluid (pH3,4); 0.1M acetate buffer solution (pH5,6); 0.1MTris-HCl damping fluid (pH7,8); 0.1M glycine-NaOH damping fluid (pH9,10).Add final concentration respectively and be 1% Zulkovsky starch.
Get the above-mentioned damping fluid 50 μ l of different pH, add 50 μ l concentrated broths respectively, estimate the activity of enzyme under condition of different pH with above-mentioned DNS method.
The result shows that the suitableeest enzyme of this amylase pH alive is 5. as shown in Figure 3
2. diastatic optimal reactive temperature in embodiment 2 concentrated solutions.
Get 50 μ l concentrated broths and 50 μ l starch substrates damping fluids, be put into 13 ℃, 28 ℃, 37 ℃, 45 ℃, 55 ℃, 65 ℃, 80 ℃, 98 ℃ incubation 30min respectively, estimate the activity of enzyme under condition of different temperatures with above-mentioned DNS method.
The result shows that this enzyme optimal reactive temperature is about 80 ℃ as shown in Figure 4.
Claims (2)
1. a bacillus pumilus (Bacillus pumilus) Tamy12 bacterial strain, culture presevation CGMCC No.2641, it has the ability that produces alpha-amylase.
2. the described Tamy12 bacterial strain of claim 1 is characterized in that the bacterial strain bacteria colony white, surface drying, edge rounding; Cell is that rod-short produces gemma, gram-positive microorganism, and growth pH scope is 5.0-9.0, optimal pH is 7.5; Temperature range is 38-70 ℃, and optimum growth temperature is 55 ℃.
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| CN104087529B (en) * | 2014-06-29 | 2016-08-31 | 新疆农业科学院微生物应用研究所 | bacillus pumilus (Bacillus pumilus) JY2 and application thereof |
| CN109504623B (en) * | 2018-11-20 | 2020-12-01 | 江南大学 | Bacillus brevis thermophilus for producing amylase and application thereof |
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| CN101412982A (en) * | 2008-11-24 | 2009-04-22 | 昆明理工大学 | Bacillus amyloliquefaciens P17 bacterial strain, low temperature pectic enzyme obtained thereform, and separation and purification method thereof |
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| CN101412982A (en) * | 2008-11-24 | 2009-04-22 | 昆明理工大学 | Bacillus amyloliquefaciens P17 bacterial strain, low temperature pectic enzyme obtained thereform, and separation and purification method thereof |
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