CN101619301B - Method for preparing alpha-amylase by high-temperature laceyella sacchari RHA1 virus strain and purification method thereof - Google Patents
Method for preparing alpha-amylase by high-temperature laceyella sacchari RHA1 virus strain and purification method thereof Download PDFInfo
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Abstract
The invention discloses a method for preparing high-temperature alpha-amylase by a heat resistant high-temperature laceyella sacchari RHA1 virus strain and a purification method thereof. The high-temperature laceyella sacchari RHA1 virus strain is white and transparent, has corrugations, dry surfaces, different positive colors and negative colors, ball shapes, laceyella sacchari gram-positive bacterium, no flagella or no pilus; and a large number of mycelium is produced when culturing liquid. The method for preparing the amylase by the high-temperature laceyella sacchari RHA1 comprises the following steps: carrying out ultrafilter concentration and ammonium sulfate precipitation on a fermentation supernatant through an ultrafilter envelope of 1KD to obtain crude enzyme liquid, and filtering by Superdex 75 10/300 gelatin to obtain pure heat resistant alpha-amylase. The molecular weight of the alpha-amylase obtained in the invention is only 11.9 KDa, and the alpha-amylase is a monomer protein and is an amylase with the minimal molecular weight reported recently. The alpha-amylase hydrolyzes amylose and soluble starch to obtain maltose and maltotriose, can resist high temperature and has wide action temperature range and application values in food processing, medicines, detergents and the like.
Description
Technical field:
The invention belongs to the using microbe field, particularly, relate to high temperature unwrapping wire sugar this bacterium of Lay (Laceyellasacchari) RHA1 bacterial strain, and the thermotolerance α-Dian Fenmei and the purification process thereof that therefrom obtain.
Background technology:
α-Dian Fenmei also claims liquefaction type amylase, β-1 in its energy hydrolyzed starch polysaccharide molecule, and the 4-glycosidic link produces monose and oligosaccharides.It is used to a plurality of fields always, for example, is used to the saccharification of cereal and potato in fermented liquid, is used as the starch paste remover in textile industry, is used as digestants in pharmacy industry, and is used to make the heavy-gravity malt syrup in grocery trade.
Because amylase generally will at high temperature make starch pasting in liquefaction, saccharifying, therefore, 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.
Therefrom isolated a variety of heat-staple amylase in warm type microorganism and the heat resistant type microorganism at present. the α-Dian Fenmei of different sources has many-sided different properties, thereby cause the range of application that differs from one another, thereby need constant exploitation new features α-Dian Fenmei better to satisfy industrial requirement.Thermophilic actinomycete RHA1 bacterial strain is not arranged in the prior art, and the thermotolerance α-Dian Fenmei that therefrom obtains and the report of purification process thereof.
Summary of the invention:
The present invention aims to provide a kind of this bacterium RHA1 bacterial strain of high temperature unwrapping wire sugar Lay that can efficiently produce the thermotolerance α-Dian Fenmei.
Another object of the present invention is to provide a kind of α-Dian Fenmei and purification process thereof that from this bacterium RHA1 bacterial strain of high temperature unwrapping wire sugar Lay, obtains.
In order to realize above-mentioned purpose of the present invention, the invention provides following technical scheme:
High temperature unwrapping wire sugar this bacterium of Lay (Laceyella sacchari) RHA1 bacterial strain, preserving number is: CGMCCNo.2639, it has the ability that produces α-Dian Fenmei.
Described this bacterium RHA1 bacterial strain of high temperature unwrapping wire sugar Lay, the bacterial strain colonial morphology is that white is transparent, and fold is arranged, surface drying, and positive and negative color difference, spherical, gram-positive microorganism, atrichia, no pili produces a large amount of mycelium when liquid culture.
The α-Dian Fenmei that from this bacterium RHA1 bacterial strain of high temperature unwrapping wire sugar Lay, obtains.
Described α-Dian Fenmei, its zymologic property is: optimal reactive temperature is 37 ℃, in 28 ℃ of-85 ℃ of relative broad ranges higher catalytic activity is arranged; Optimal pH is 6, under pH5.0-9.0, has high enzyme to live; Diastatic molecular weight is 11.9649KDa, is a monomeric protein.
The purification process of the α-Dian Fenmei that obtains from this bacterium RHA1 bacterial strain of high temperature unwrapping wire sugar Lay comprises:
(1) ultrafiltration and concentration: the fermentation culture conditions of high temperature unwrapping wire sugar this bacterium of Lay (Laceyella sacchari) RHA1 bacterial strain is 50 ℃ of temperature, PH7.5 is incubation time 16-18 hour, centrifugal with fermented liquid, get supernatant liquor, utilize the ultra-filtration membrane bag of 10KDa to carry out ultrafiltration and concentration;
(2) ammonium sulfate precipitation: concentrate the saturation ratio that back liquid adds ammonium sulfate to 40%, the centrifuging and taking supernatant continues to add the saturation ratio of ammonium sulfate to 60%, the centrifugal supernatant of abandoning, and crude enzyme liquid is made in the last phosphate buffered saline buffer dissolving with PH7.0 of precipitation;
(3) Superdex 7510/300 gel permeation chromatography: the crude enzyme liquid that obtains is centrifugal, supernatant concentrates, be splined on and use 50mM, the Superdex 75 10/300 gel permeation chromatography posts that the phosphate buffered saline buffer pre-equilibration of PH7.0 is crossed, wash-out, collect the enzymic activity part, be the pure enzyme of α-Dian Fenmei.
High temperature unwrapping wire of the present invention sugar this bacterium of Lay (Laceyella sacchari) RHA1 bacterial strain on August 26th, 2008 in the common micro-organisms center preservation of Beijing China Committee for Culture Collection of Microorganisms, preserving number is: CGMCC No.2639.
High temperature unwrapping wire sugar this bacterium of Lay (Laceyella sacchari) RHA1 bacterial strain of the present invention separates from the Tengchong hot spring.The bacterial strain screening substratum is (g/L): (NH
4)
2SO
4, 1.3; Peptone, 1; Zulkovsky starch, 4; 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.This bacterium RHA1 bacterial strain of high temperature unwrapping wire sugar Lay, colonial morphology is that white cloth is transparent, and fold is arranged, surface drying, and positive and negative color difference.Get through gramstaining, the RHA1 bacterial strain is spherical, gram-positive microorganism, and by electron microscopic observation, the RHA1 strain cell is spherical, and diameter is about 800nm; Atrichia, no pili produces a large amount of mycelium when liquid culture, and the mycelium diameter is about 300nm.
The purification step of the α-Dian Fenmei of this bacterium RHA1 bacterial strain of high temperature unwrapping wire sugar Lay involved in the present invention is:
(1) ultrafiltration and concentration: the fermentation culture conditions of thermophilic actinomycete RHA1 bacterial strain is 50 ℃ of temperature, PH7.5, about 18 hours of incubation time.Fermented liquid is centrifugal, get supernatant liquor, utilize the ultra-filtration membrane bag of 10KDa to carry out ultrafiltration and concentration.
(2) ammonium sulfate precipitation: concentrate the saturation ratio that back liquid adds ammonium sulfate to 40%, the centrifuging and taking supernatant continues to add the saturation ratio of ammonium sulfate to 60%, the centrifugal supernatant of abandoning, and crude enzyme liquid is made in the last phosphate buffered saline buffer dissolving with PH7.0 of precipitation.
(3) Superdex 7510/300 gel permeation chromatography: the crude enzyme liquid that obtains is centrifugal, and supernatant concentrates, and is splined on to use 50mM, the Superdex 7510/300 gel permeation chromatography post that the phosphate buffered saline buffer pre-equilibration of PH7.0 is crossed, wash-out is collected the enzymic activity part, is pure enzyme.
This bacterium RHA1 α-Dian Fenmei that bacterial strain produces of high temperature unwrapping wire sugar Lay has following zymologic property:
1, the suitableeest enzyme temperature alive is 37 ℃, and at 28～85 ℃ the greater catalytic vigor is arranged all.
2, optimal pH is 6.0, all has high enzyme to live under PH5.0-9.0.
3, the metal ion dependency is: Na
+, Ca
2+, Mg
2+This enzyme there are certain activation, especially Mg
2+Act on the strongest, and Zn
2+, Fe
2+, Fe
3+,, Cu
2+, Ni
2+, it is had stronger restraining effect.
4, substrate specificity is: solubility amylopectin (100%)＞Zulkovsky starch (70.6%)＞amylose starch (50.3%).
5, the molecular weight of proteolytic enzyme is 11.9KDa.
Description of drawings:
Fig. 1 is the temperature of reaction and the relative enzyme relation curve alive of the α-Dian Fenmei of RHA1 bacterial strain generation;
Fig. 2 is the reaction PH and the relative enzyme relation curve alive of the α-Dian Fenmei of RHA1 bacterial strain generation;
The product TLC of the high-temperature hydrolysis Zulkovsky starch that Fig. 3 RHA1 bacterial strain produces analyzes,
Wherein 1: be Marker: glucose, Fructus Hordei Germinatus disaccharides, trisaccharide maltose; 2: direct-connected starch, 3 amylopectin, 4 Zulkovsky starches.
Embodiment:
Further specify essentiality content of the present invention below in conjunction with embodiment, but do not limit the present invention with this.
Embodiment 1:
The separation screening and the evaluation of this bacterium RHA1 bacterial strain of high temperature unwrapping wire sugar Lay:
Thermophilic actinomycete RHA1 bacterial strain separates from the Tengchong hot spring.The bacterial strain screening substratum is (g/L): (NH
4)
2SO
4, 1.3; Peptone, 1; Zulkovsky starch, 4; 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.Culture condition is 50 ℃ of temperature, 150rpm shaking culture, about 18 hours of incubation time.
Adopt the bacterial system authentication method that this bacterium RHA1 bacterial strain of high temperature unwrapping wire sugar Lay is identified.The morphological feature of this bacterium is as follows: white cloth is transparent, and fold is arranged, surface drying, and positive and negative color difference, gram-positive microorganism.By electron microscopic observation, the RHA1 strain cell is spherical, and diameter is about 800nm; Atrichia, no pili, growth PH scope is 5.0-9.0, optimal pH is 7.5; Growth temperature range is 25～78 ℃, and optimum temperuture is 50 ℃.
Embodiment 2:
The purifying of α-Dian Fenmei:
(1) ultrafiltration and concentration: the fermentation culture conditions of thermophilic actinomycete RHA1 bacterial strain is 50 ℃ of temperature, pH7.5, about 18 hours of incubation time.Fermented liquid is centrifugal, get supernatant liquor, utilize the ultra-filtration membrane bag of 10KDa to carry out ultrafiltration and concentration.
(2) ammonium sulfate precipitation: concentrate the saturation ratio that back liquid adds ammonium sulfate to 40%, the centrifuging and taking supernatant continues to add the saturation ratio of ammonium sulfate to 60%, the centrifugal supernatant of abandoning, and precipitation is last with the dissolving of PH7.0 phosphate buffered saline buffer, makes crude enzyme liquid.
(3) Superdex 7510/300 gel permeation chromatography: the crude enzyme liquid that obtains is centrifugal, and supernatant concentrates, and is splined on to use 50mM, the Superdex 7510/300 gel permeation chromatography post that the phosphate buffered saline buffer pre-equilibration of PH7.0 is crossed, wash-out is collected the enzymic activity part, is pure enzyme.Flow velocity is 0.4ml/min, collects elution peak, detects protein concentration and enzymic activity part, and is concentrated in 4 ℃ of centrifugal collections with the evaporating pipe of 10KDa, is pure enzyme, freezing preservation.Spissated sample is carried out SDS-P B GE electrophoretic analysis, obtain single band, it is pure that the α-Dian Fenmei that shows purifying has reached electrophoresis, and molecular weight is 11.9649KDa.
The mensuration of alpha-amylase activity: employing iodine-starch colorimetry (Zhou Jingxiang etc. proteolytic enzyme and amylase activity detect. Chinese feed, 2001,23 (11)).Amylase activity is defined as: 100ml enzyme liquid is at 55 ℃ and substrate starch storage crop 30min, and it is 1 liquefon that hydrolysis produces 10mg starch.
The detection of α-Dian Fenmei purity: adopt SDS-P B GE vertical gel electrophoresis (Laemmli U K, 1970).The mensuration of protein content: adopt Bradford detection method (Marion M.Bradford, 1976).
Embodiment 3:
The characteristic of α-Dian Fenmei:
1. the suitableeest enzyme temperature alive
Enzyme liquid is added in the phosphate buffered saline buffer (50mM phosphate buffered saline buffer, PH 7.0), as substrate, is under 1% the condition, to measure the work of the enzyme under the differing temps in 28-98 ℃ of scope of RHA1 amylase that bacterial strain produces with Zulkovsky starch at concentration of substrate.The result shows that the suitableeest enzyme temperature alive of this α-Dian Fenmei is 37 ℃ as shown in Figure 1.
2. thermostability
Enzyme liquid is added in the phosphate buffered saline buffer (50mM phosphate buffered saline buffer, PH 7.0), and in the water bath with thermostatic control of differing temps, is incubated,, measure enzyme according to the enzyme activity determination method of standard then and live in sampling of different time (0-90min, the sampling interval is 15min).Temperature is expressed as the per-cent that residual enzyme shared original enzyme alive is lived behind certain hour to the influence of enzyme stability, and this amylase also has 14.6% vigor behind 98 ℃ of insulation 45min, show that this amylase has thermostability preferably.
3. optimal pH
With the different damping fluid dilution enzyme liquid of PH3.0 to PHl1.0, add 1% the Zulkovsky starch of corresponding PH respectively, measure enzyme activities down at 55 ℃, the result shows that α-Dian Fenmei enzyme when PH6.0 that the RHA1 bacterial strain is produced lives the highest as shown in Figure 2.
4. to the dependency of metal ion
The dilution enzyme liquid that is added with metal ion is incubated 10h in 55 ℃ after, quantitatively measures residual enzyme and live, compared with the control, obtain relative enzyme activity, the result is as shown in table 1, shows Na
+, Ca
2+, Mg
2+Enzyme there are certain activation, especially Mg
2+Enzyme had stronger activation
+And Cd
2+, Ni
2+, K
+, Mn
2+Enzyme there is certain restraining effect, all the other ion Zn
2+, Fe
2+, Fe
3+, Cu
2+, Ni
2+, etc. enzyme is had stronger restraining effect (seeing Table 1).
The enzyme activity of the α-Dian Fenmei that table 1RHA1 bacterial strain produces is subjected to different metal ionic influence degree
5. Substratspezifitaet research
Be 0.5% various substrates with pure enzyme and concentration at 55 ℃ down after the reaction 10min, measure the OD660 light absorption value.The result is as shown in table 2, shows that this enzyme has best katalysis to the substrate Zulkovsky starch, take second place for the katalysis of amylopectin substrate, and for the katalysis of amylopectin substrate the most weak (seeing Table 2).
The α-Dian Fenmei that table 2RHA1 bacterial strain produces is to the specificity of different substrates
Claims (2)
1. high temperature unwrapping wire sugar this bacterium of Lay (Laceyella sacchari) RHA1 bacterial strain, preserving number is: CGMCC No.2639, it has the ability that produces α-Dian Fenmei.
2. method of utilizing the described RHA1 bacterial strain of claim 1 purifying α-Dian Fenmei comprises:
(1) ultrafiltration and concentration: the fermentation culture conditions of this bacterium RHA1 bacterial strain of high temperature unwrapping wire sugar Lay is 50 ℃ of temperature, PH7.5, and incubation time 16-18 hour, fermented liquid is centrifugal, get supernatant liquor, utilize the ultra-filtration membrane bag of 10KDa to carry out ultrafiltration and concentration;
(2) ammonium sulfate precipitation: concentrate the saturation ratio that back liquid adds ammonium sulfate to 40%, the centrifuging and taking supernatant continues to add the saturation ratio of ammonium sulfate to 60%, the centrifugal supernatant of abandoning, and crude enzyme liquid is made in the last phosphate buffered saline buffer dissolving with PH7.0 of precipitation;
(3) Superdex 75 10/300 gel permeation chromatographies: the crude enzyme liquid that obtains is centrifugal, supernatant concentrates, be splined on and use 50mM, the Superdex 75 10/300 gel permeation chromatography posts that the phosphate buffered saline buffer pre-equilibration of PH7.0 is crossed, wash-out, collect the enzymic activity part, be the pure enzyme of α-Dian Fenmei.
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|---|---|---|---|
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|---|---|---|---|
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| CN106916772B (en) * | 2017-04-26 | 2019-10-25 | 陕西枫丹百丽生物科技有限公司 | One plant of acid and alkali-resistance, the thermoactinomyces sacchari of ethanol-tolerant quick composting vinasse and its application |
| CN106906168B (en) * | 2017-04-26 | 2019-07-19 | 陕西枫丹百丽生物科技有限公司 | A kind of spirit stillage decomposing agent and its preparation method and application |
| CN113817650B (en) * | 2021-10-26 | 2023-05-30 | 贵州大学 | Pyrazine-producing high-temperature actinomycetes, separation screening and application |
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