CN109486796A - A method of preparing dextranase enzyme preparation - Google Patents
A method of preparing dextranase enzyme preparation Download PDFInfo
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- CN109486796A CN109486796A CN201811483451.XA CN201811483451A CN109486796A CN 109486796 A CN109486796 A CN 109486796A CN 201811483451 A CN201811483451 A CN 201811483451A CN 109486796 A CN109486796 A CN 109486796A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
- C12N9/2454—Dextranase (3.2.1.11)
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- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01011—Dextranase (3.2.1.11)
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Abstract
The present invention provides a kind of method for preparing dextranase enzyme preparation, it is related to technical field of enzyme preparation field, this method method is the following steps are included: (1) Spawn incubation: preparing seed liquid culture medium, will generate the strain inoculated of the dextranase constant temperature incubation into the liquid culture medium；(2) fermented and cultured: preparing fermentation medium, is inoculated with strain ferment at constant temperature；(3) crude enzyme liquid separates and purifies to obtain enzyme preparation；The present invention can prepare high activity dextranase enzyme preparation, as sugar industry industrial additive, solve that sugar industry industry faced containing dextran the case where.
Description
Technical field
The present invention relates to technical field of enzyme preparation, and in particular to a method of prepare dextranase enzyme preparation.
Background technique
In sucrose and white granulated sugar production process, bacterium is that microorganism is main in syrup in soil, that is, air of raw material entrainment
Source.Common microbiological is mainly by yeast, mould and bacterium three classes.Part microorganism can secret out of invertase, by sugarcane
Sugar conversion saccharogenesis acid anhydride, wherein a large amount of sucrose that consume generate dextran the most serious is Leuconostoc mesenteroides.Dextran is white
The main component of granulated sugar acidity flocculate, dextran viscosity is high, will increase liquid glucose viscosity containing dextran in liquid glucose, influences
Sugar and separation process are boiled, later period lock out operation cost is increased；Meanwhile accumulation of the dextran in syrup can obscure pol,
It causes the purity falseness of syrup to rise, influences to produce analysis management；In addition, dextran makes to generate abnormal morphology when crystallization of sucrose
Crystal, increase sugar making cost.Dextran causes the loss late of sugar refining technology sucrose to be up to 0.07%, so eliminating preparation
The problem of dextran generated in technique is sugar industry urgent need to resolve.
Dextranase belongs to sugared acid anhydride hydrolase, and dextran can be hydrolyzed to low molecule carbohydrate, low molecule carbohydrate pair
Closed Circulation in Sugar Production influences little.In western India, the dextranase of 3 enzyme-activity units is added into 100ml sugarcane juice, is shaken at 40 DEG C
Reaction 20min is swung, 68.5% dextran can be removed, therefore, the addition of dextranase can improve sugar production process.
It is different according to the hydrolysis method of dextran, dextranase is divided into inscribe dextranase and circumscribed Dextranase two
Kind.Circumscribed dextranase mainly from the non-reducing end of dextranase chain degradation key, releases glucose.Inscribe dextran
Enzyme mainly hydrolyzes α (1-6) key inside dextran chain, low-molecular-weight polysaccharide in synthetic time series, most of inscribe dextrans
Enzyme only its catalytic action of competence exertion in the presence of multiple coherent α-(1-6) glucoside bonds, but also do not need multiple
Coherent α-(1-6) glucoside bond is reported in the presence of that can either play inscribe Dextranase, such as Wynter of hydrolysis etc.
One plant of anaerobic thermophilic bacterium Rt364 in road, the Dextranase that it is generated can hydrolyze α-single in starch (1-6) glucoside
Key.
Due to the high efficiency of Dextranase degradation dextran, the Dextranase for developing a kind of safe sources becomes close
Research hotspot over year.
Summary of the invention
(1) the technical issues of solving
In view of the deficiencies of the prior art, it the present invention provides a kind of method for preparing dextranase enzyme preparation, prepares
High activity dextranase enzyme preparation, as sugar industry industrial additive.
(2) technical solution
In order to achieve the above object, the present invention is achieved by the following technical programs:
A method of dextranase enzyme preparation is prepared, the described method comprises the following steps:
(1) Spawn incubation: preparing seed liquid culture medium, and concrete operations are to choose 250g sweet potato, peeled and washed, is cut into 1-
3cm square continues to boil 30-45min after adding water 1.5L, water to open, filters off sweet potato block, obtain filtrate and complement to 1.5L, to sweet potato
Carbon source, nitrogen source, source of iron, phosphorus source, magnesium source and potassium resource are added in filtrate, adjusting initial pH value is 5.0-6.0, then will generate dextrorotation
Liquid culture medium after inoculation is placed in 28 DEG C of constant incubators and cultivates into the liquid culture medium by the strain inoculated of sugared acid anhydride enzyme
20-30h；
(2) fermented and cultured: preparing fermentation medium, and concrete operations such as step (1) prepares sweet potato filtrate, and nitrogen source, carbon is added
Fermentation training is added in the liquid culture medium mixed liquor containing strain prepared in step (1) by source, phosphorus source, source of iron, magnesium source and potassium resource
Feeding base is inoculated with, and inoculum concentration is the 2-4% of fermentation medium liquid volume fraction；Adjusting initial pH value is 5.0-6.0, then
28 DEG C are placed in, is cultivated in the constant-temperature table that revolving speed is 200r/min, incubation time 85-100h；
(3) crude enzyme liquid separates: microbial fermentation solution being centrifugated using rotary drum vacuum suction filter, thallus is removed, obtains
To the supernatant rich in dextranase as crude enzyme liquid；
(4) enzyme preparation purifies: being mentioned using the crude enzyme liquid that polyacrylamide gel electrophoresis prepares step (4)
Pure, concrete operations are the gel slab that certain size is prepared with grease removal sheet frame electrophoresis apparatus, gum concentration 6-8%, injection volume 10-
15ml, electric current 24-32mA, the electrophoresis 4-5h when pH is 7.0, the adhesive tape of 3mm is respectively cut from gel slab two sides, is immediately placed on
It is dyed in 0.1% Coomassie brilliant blue G250 solution, after the appearance of blue region band, adhesive tape is rinsed well with deionized water and is put back to
It is in situ, remainder on gel slab is scaled off, is extracted with 4ml distilled water at 4 DEG C, is filtered to remove gel after standing a night, washes
Wash rear air drying, obtain dextranase enzyme preparation, by dextranase enzyme preparation deposit in 4-5 DEG C it is stored refrigerated.
Preferably, nitrogen source additional amount is the 0.75% of sweet potato filtrate quality in above-mentioned steps (1), and carbon source addition is sweet potato
The 1.53% of filtrate quality, phosphorus source additional amount are the 0.06% of sweet potato filtrate quality, and magnesium source additional amount is sweet potato filtrate quality
0.011%, source of iron additional amount is the 0.0005% of sweet potato filtrate quality, and potassium resource additional amount is the 0.03% of sweet potato filtrate quality；
Nitrogen source additional amount is the 1.2% of sweet potato filtrate quality in above-mentioned steps (2), and carbon source addition is sweet potato filtrate quality
2.67%, phosphorus source additional amount is the 0.13% of sweet potato filtrate quality, and magnesium source additional amount is the 0.021% of sweet potato filtrate quality, iron
Source additional amount is the 0.0011% of sweet potato filtrate quality, and potassium resource additional amount is the 0.06% of sweet potato filtrate quality.
Preferably, above-mentioned nitrogen source is yeast extract, and the carbon source is glucose, and above-mentioned phosphorus source is potassium dihydrogen phosphate, above-mentioned magnesium
Source is magnesium sulfate, and the source of iron is ferric sulfate, and above-mentioned potassium resource is potassium chloride.
Preferably, above-mentioned strain is thin beautiful superior strain of the hair shell through mutagenesis screening.
Preferably, in above-mentioned steps (1) strain inoculum concentration be seed liquid culture substrate amount 1-3%；Above-mentioned steps (2)
Middle inoculum concentration is the 2-4% of fermentation medium volume fraction.
Preferably, centrifugal force is 6000-7000g in above-mentioned steps (4), and the centrifugally operated time is 15-25min.
(3) beneficial effect
The present invention provides a kind of methods for preparing dextranase enzyme preparation.This method is sieved through meticulous beautiful hair shell through mutagenesis
As dextranase zymophyte, prepare activity by Spawn incubation, fermentation, centrifugation and purification is the superior strain of choosing
The dextranase enzyme preparation of 20000-50000u/g.The present invention is to thin beautiful cupreum bacterium culture medium and fermentation medium nitrogen
Source, carbon source, phosphorus source, source of iron, the substance classes and dosage of magnesium source and potassium resource preferably, carry out initial pH value preferably, to bacterium
Kind and fermentation time carry out preferably, obtaining the optimal carefully beautiful cupreum kind of activity；By being carried out preferably to separation and purifying technique,
Finally obtain the very high dextranase enzyme preparation of activity.
Detailed description of the invention
Fig. 1 is dextranase enzyme preparation hydrolysis effect figure.
Specific embodiment
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with the embodiment of the present invention,
Technical scheme in the embodiment of the invention is clearly and completely described, it is clear that described embodiment is the present invention one
Divide embodiment, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not making
Every other embodiment obtained, shall fall within the protection scope of the present invention under the premise of creative work.
Embodiment 1:
A method of dextranase enzyme preparation is prepared, the described method comprises the following steps:
(1) Spawn incubation: preparing seed liquid culture medium, and concrete operations are to choose 250g sweet potato, peeled and washed, is cut into 1-
3cm square continues to boil 35min after adding water 1.5L, water to open, filters off sweet potato block, obtain filtrate and complement to 1.5L；To sweet potato filtrate
Middle addition glucose, yeast extract, ferric sulfate, potassium dihydrogen phosphate, magnesium sulfate and potassium chloride, yeast extract additional amount are sweet potato filtrate matter
The 0.75% of amount, glucose additional amount are the 1.53% of sweet potato filtrate quality, and potassium dihydrogen phosphate additional amount is sweet potato filtrate quality
0.06%, magnesium sulfate additional amount is the 0.011% of sweet potato filtrate quality, and ferric sulfate additional amount is sweet potato filtrate quality
0.0005%, potassium chloride additional amount is the 0.03% of sweet potato filtrate quality；Adjusting initial pH value is 5.5；It then will thin beautiful hair shell
Superior strain through mutagenesis screening is inoculated into the liquid culture medium, and inoculum concentration is the 2% of seed liquid culture substrate amount；It will connect
Liquid culture medium after kind, which is placed in 28 DEG C of constant incubators, cultivates 20h.
(2) fermented and cultured: preparing fermentation medium, and concrete operations are that such as step (1) prepares sweet potato filtrate, and yeast is added
Cream, glucose, potassium dihydrogen phosphate, ferric sulfate, magnesium sulfate and potassium chloride, yeast extract additional amount are the 1.2% of sweet potato filtrate quality,
Glucose additional amount is the 2.67% of sweet potato filtrate quality, and potassium dihydrogen phosphate additional amount is the 0.13% of sweet potato filtrate quality, sulphur
Sour magnesium additional amount is the 0.021% of sweet potato filtrate quality, and ferric sulfate additional amount is the 0.0011% of sweet potato filtrate quality, potassium chloride
Additional amount is the 0.06% of sweet potato filtrate quality；The liquid culture medium mixed liquor containing strain prepared in step (1) is added
Fermentation medium is inoculated with, and inoculum concentration is the 2% of fermentation medium liquid volume fraction；Adjusting initial pH value is 5.0, then
28 DEG C are placed in, is cultivated in the constant-temperature table that revolving speed is 200r/min, incubation time 85h；
(3) crude enzyme liquid separates: using rotary drum vacuum suction filter in centrifugal force for 6000g microbial fermentation solution, is centrifuged
Operating time is that 15min is separated, and removes thallus, obtains the supernatant rich in dextranase as crude enzyme liquid.
(4) enzyme preparation purifies: being mentioned using the crude enzyme liquid that polyacrylamide gel electrophoresis prepares step (4)
Pure, concrete operations are the gel slab that certain size is prepared with grease removal sheet frame electrophoresis apparatus, gum concentration 6-8%, injection volume 10-
15ml, electric current 24-32mA respectively cut the adhesive tape of 3mm from gel slab two sides, set immediately when pH is 7.0 electrophoresis 4-5 hours
It is dyed in 0.1% Coomassie brilliant blue G250 solution, after the appearance of blue region band, adhesive tape is rinsed well with deionized water and is put
Original position is gone back to, remainder on gel slab is scaled off, is extracted with 4ml distilled water at 4 DEG C, is filtered to remove gel after standing a night,
Air drying after washing obtains dextranase enzyme preparation, by dextranase enzyme preparation deposit in 4-5 DEG C it is stored refrigerated.
It is 35654u/g by the dextrorotation acid anhydride enzyme enzyme preparation activity prepared.It is 10000u/ that enzyme preparation, which is diluted to enzyme activity,
G adds 10ppm enzyme solution in the sugarcane juice containing 1000ppm dextran concentration, hydrolyzes Fig. 1 in situation such as Figure of description
It is shown.
Embodiment 2:
A method of dextranase enzyme preparation is prepared, the described method comprises the following steps:
(1) Spawn incubation: preparing seed liquid culture medium, and concrete operations are to choose 250g sweet potato, peeled and washed, is cut into 1-
3cm square continues to boil 45min after adding water 1.5L, water to open, filters off sweet potato block, obtain filtrate and complement to 1.5L；To sweet potato filtrate
Middle addition glucose, yeast extract, ferric sulfate, potassium dihydrogen phosphate, magnesium sulfate and potassium chloride, yeast extract additional amount are sweet potato filtrate matter
The 0.75% of amount, glucose additional amount are the 1.53% of sweet potato filtrate quality, and potassium dihydrogen phosphate additional amount is sweet potato filtrate quality
0.06%, magnesium sulfate additional amount is the 0.011% of sweet potato filtrate quality, and ferric sulfate additional amount is sweet potato filtrate quality
0.0005%, potassium chloride additional amount is the 0.03% of sweet potato filtrate quality；Adjusting initial pH value is 6.0；It then will thin beautiful hair shell
Superior strain through mutagenesis screening is inoculated into the liquid culture medium, and inoculum concentration is the 3% of seed liquid culture substrate amount；It will connect
Liquid culture medium after kind, which is placed in 28 DEG C of constant incubators, cultivates 30h.
(2) fermented and cultured: preparing fermentation medium, and concrete operations are that such as step (1) prepares sweet potato filtrate, and yeast is added
Cream, glucose, potassium dihydrogen phosphate, ferric sulfate, magnesium sulfate and potassium chloride, yeast extract additional amount are the 1.2% of sweet potato filtrate quality,
Glucose additional amount is the 2.67% of sweet potato filtrate quality, and potassium dihydrogen phosphate additional amount is the 0.13% of sweet potato filtrate quality, sulphur
Sour magnesium additional amount is the 0.021% of sweet potato filtrate quality, and ferric sulfate additional amount is the 0.0011% of sweet potato filtrate quality, potassium chloride
Additional amount is the 0.06% of sweet potato filtrate quality；The liquid culture medium mixed liquor containing strain prepared in step (1) is added
Fermentation medium is inoculated with, and inoculum concentration is the 4% of fermentation medium liquid volume fraction；Adjusting initial pH value is 5.0, then
28 DEG C are placed in, is cultivated in the constant-temperature table that revolving speed is 200r/min, incubation time 100h；
(3) crude enzyme liquid separates: using rotary drum vacuum suction filter in centrifugal force for 7000g microbial fermentation solution, is centrifuged
Operating time is that 25min is separated, and removes thallus, obtains the supernatant rich in dextranase as crude enzyme liquid.
(4) enzyme preparation purifies: being mentioned using the crude enzyme liquid that polyacrylamide gel electrophoresis prepares step (4)
Pure, concrete operations are the gel slab that certain size is prepared with grease removal sheet frame electrophoresis apparatus, gum concentration 8%, injection volume 15ml,
Electric current is 32mA, and when pH is 7.0 electrophoresis 5 hours, the adhesive tape of 3mm is respectively cut from gel slab two sides, is immediately placed on 0.1% and examines
It is dyed in Mas bright blue G250 solution, after the appearance of blue region band, adhesive tape is rinsed well with deionized water and puts back to original position, will be coagulated
Remainder scales off on offset plate, is extracted with 4ml distilled water at 4 DEG C, is filtered to remove gel after standing a night, room temperature is dry after washing
It is dry, obtain dextranase enzyme preparation, by dextranase enzyme preparation deposit in 5 DEG C it is stored refrigerated.
It is 45346u/g by the dextrorotation acid anhydride enzyme enzyme preparation activity prepared.
Embodiment 3:
A method of dextranase enzyme preparation is prepared, the described method comprises the following steps:
(1) Spawn incubation: preparing seed liquid culture medium, and concrete operations are to choose 250g sweet potato, peeled and washed, is cut into 1-
3cm square continues to boil 35min after adding water 1.5L, water to open, filters off sweet potato block, obtain filtrate and complement to 1.5L；To sweet potato filtrate
Middle addition glucose, yeast extract, ferric sulfate, potassium dihydrogen phosphate, magnesium sulfate and potassium chloride, yeast extract additional amount are sweet potato filtrate matter
The 0.75% of amount, glucose additional amount are the 1.53% of sweet potato filtrate quality, and potassium dihydrogen phosphate additional amount is sweet potato filtrate quality
0.06%, magnesium sulfate additional amount is the 0.011% of sweet potato filtrate quality, and ferric sulfate additional amount is sweet potato filtrate quality
0.0005%, potassium chloride additional amount is the 0.03% of sweet potato filtrate quality；Adjusting initial pH value is 6.5；It then will thin beautiful hair shell
Superior strain through mutagenesis screening is inoculated into the liquid culture medium, and inoculum concentration is the 2% of seed liquid culture substrate amount；It will connect
Liquid culture medium after kind, which is placed in 28 DEG C of constant incubators, cultivates 22h.
(2) fermented and cultured: preparing fermentation medium, and concrete operations are that such as step (1) prepares sweet potato filtrate, and yeast is added
Cream, glucose, potassium dihydrogen phosphate, ferric sulfate, magnesium sulfate and potassium chloride, yeast extract additional amount are the 1.2% of sweet potato filtrate quality,
Glucose additional amount is the 2.67% of sweet potato filtrate quality, and potassium dihydrogen phosphate additional amount is the 0.13% of sweet potato filtrate quality, sulphur
Sour magnesium additional amount is the 0.021% of sweet potato filtrate quality, and ferric sulfate additional amount is the 0.0011% of sweet potato filtrate quality, potassium chloride
Additional amount is the 0.06% of sweet potato filtrate quality；The liquid culture medium mixed liquor containing strain prepared in step (1) is added
Fermentation medium is inoculated with, and inoculum concentration is the 3% of fermentation medium liquid volume fraction；Adjusting initial pH value is 5.5, then
28 DEG C are placed in, is cultivated in the constant-temperature table that revolving speed is 200r/min, incubation time 90h；
(3) crude enzyme liquid separates: using rotary drum vacuum suction filter in centrifugal force for 6200g microbial fermentation solution, is centrifuged
Operating time is that 20min is separated, and removes thallus, obtains the supernatant rich in dextranase as crude enzyme liquid.
(4) enzyme preparation purifies: being mentioned using the crude enzyme liquid that polyacrylamide gel electrophoresis prepares step (4)
Pure, concrete operations are the gel slab that certain size is prepared with grease removal sheet frame electrophoresis apparatus, gum concentration 6-8%, injection volume 10-
15ml, electric current 24-32mA respectively cut the adhesive tape of 3mm from gel slab two sides, set immediately when pH is 7.0 electrophoresis 4-5 hours
It is dyed in 0.1% Coomassie brilliant blue G250 solution, after the appearance of blue region band, adhesive tape is rinsed well with deionized water and is put
Original position is gone back to, remainder on gel slab is scaled off, is extracted with 4ml distilled water at 4 DEG C, is filtered to remove gel after standing a night,
Air drying after washing obtains dextranase enzyme preparation, by dextranase enzyme preparation deposit in 4-5 DEG C it is stored refrigerated.
It is 29876u/g by the dextrorotation acid anhydride enzyme enzyme preparation activity prepared.
Embodiment 4:
A method of dextranase enzyme preparation is prepared, the described method comprises the following steps:
(1) Spawn incubation: preparing seed liquid culture medium, and concrete operations are to choose 250g sweet potato, peeled and washed, is cut into 1-
3cm square continues to boil 40min after adding water 1.5L, water to open, filters off sweet potato block, obtain filtrate and complement to 1.5L；To sweet potato filtrate
Middle addition glucose, yeast extract, ferric sulfate, potassium dihydrogen phosphate, magnesium sulfate and potassium chloride, yeast extract additional amount are sweet potato filtrate matter
The 0.75% of amount, glucose additional amount are the 1.53% of sweet potato filtrate quality, and potassium dihydrogen phosphate additional amount is sweet potato filtrate quality
0.06%, magnesium sulfate additional amount is the 0.011% of sweet potato filtrate quality, and ferric sulfate additional amount is sweet potato filtrate quality
0.0005%, potassium chloride additional amount is the 0.03% of sweet potato filtrate quality；Adjusting initial pH value is 5.5；It then will thin beautiful hair shell
Superior strain through mutagenesis screening is inoculated into the liquid culture medium, and inoculum concentration is the 2% of seed liquid culture substrate amount；It will connect
Liquid culture medium after kind, which is placed in 28 DEG C of constant incubators, to be cultivated for 24 hours.
(2) fermented and cultured: preparing fermentation medium, and concrete operations are that such as step (1) prepares sweet potato filtrate, and yeast is added
Cream, glucose, potassium dihydrogen phosphate, ferric sulfate, magnesium sulfate and potassium chloride, yeast extract additional amount are the 1.2% of sweet potato filtrate quality,
Glucose additional amount is the 2.67% of sweet potato filtrate quality, and potassium dihydrogen phosphate additional amount is the 0.13% of sweet potato filtrate quality, sulphur
Sour magnesium additional amount is the 0.021% of sweet potato filtrate quality, and ferric sulfate additional amount is the 0.0011% of sweet potato filtrate quality, potassium chloride
Additional amount is the 0.06% of sweet potato filtrate quality；The liquid culture medium mixed liquor containing strain prepared in step (1) is added
Fermentation medium is inoculated with, and inoculum concentration is the 3% of fermentation medium liquid volume fraction；Adjusting initial pH value is 5.5, then
28 DEG C are placed in, is cultivated in the constant-temperature table that revolving speed is 200r/min, incubation time 95h；
(3) crude enzyme liquid separates: using rotary drum vacuum suction filter in centrifugal force for 6400g microbial fermentation solution, is centrifuged
Operating time is that 20min is separated, and removes thallus, obtains the supernatant rich in dextranase as crude enzyme liquid.
(4) enzyme preparation purifies: being mentioned using the crude enzyme liquid that polyacrylamide gel electrophoresis prepares step (4)
Pure, concrete operations are the gel slab that certain size is prepared with grease removal sheet frame electrophoresis apparatus, gum concentration 6-8%, injection volume 10-
15ml, electric current 24-32mA respectively cut the adhesive tape of 3mm from gel slab two sides, set immediately when pH is 7.0 electrophoresis 4-5 hours
It is dyed in 0.1% Coomassie brilliant blue G250 solution, after the appearance of blue region band, adhesive tape is rinsed well with deionized water and is put
Original position is gone back to, remainder on gel slab is scaled off, is extracted with 4ml distilled water at 4 DEG C, is filtered to remove gel after standing a night,
Air drying after washing obtains dextranase enzyme preparation, by dextranase enzyme preparation deposit in 4-5 DEG C it is stored refrigerated.
It is 28769u/g by the dextrorotation acid anhydride enzyme enzyme preparation activity prepared.
Embodiment 5:
A method of dextranase enzyme preparation is prepared, the described method comprises the following steps:
(1) Spawn incubation: preparing seed liquid culture medium, and concrete operations are to choose 250g sweet potato, peeled and washed, is cut into 1-
3cm square continues to boil 40min after adding water 1.5L, water to open, filters off sweet potato block, obtain filtrate and complement to 1.5L；To sweet potato filtrate
Middle addition glucose, yeast extract, ferric sulfate, potassium dihydrogen phosphate, magnesium sulfate and potassium chloride, yeast extract additional amount are sweet potato filtrate matter
The 0.75% of amount, glucose additional amount are the 1.53% of sweet potato filtrate quality, and potassium dihydrogen phosphate additional amount is sweet potato filtrate quality
0.06%, magnesium sulfate additional amount is the 0.011% of sweet potato filtrate quality, and ferric sulfate additional amount is sweet potato filtrate quality
0.0005%, potassium chloride additional amount is the 0.03% of sweet potato filtrate quality；Adjusting initial pH value is 5.0；It then will thin beautiful hair shell
Superior strain through mutagenesis screening is inoculated into the liquid culture medium, and inoculum concentration is the 2% of seed liquid culture substrate amount；It will connect
Liquid culture medium after kind, which is placed in 28 DEG C of constant incubators, cultivates 26h.
(2) fermented and cultured: preparing fermentation medium, and concrete operations are that such as step (1) prepares sweet potato filtrate, and yeast is added
Cream, glucose, potassium dihydrogen phosphate, ferric sulfate, magnesium sulfate and potassium chloride, yeast extract additional amount are the 1.2% of sweet potato filtrate quality,
Glucose additional amount is the 2.67% of sweet potato filtrate quality, and potassium dihydrogen phosphate additional amount is the 0.13% of sweet potato filtrate quality, sulphur
Sour magnesium additional amount is the 0.021% of sweet potato filtrate quality, and ferric sulfate additional amount is the 0.0011% of sweet potato filtrate quality, potassium chloride
Additional amount is the 0.06% of sweet potato filtrate quality；The liquid culture medium mixed liquor containing strain prepared in step (1) is added
Fermentation medium is inoculated with, and inoculum concentration is the 3% of fermentation medium liquid volume fraction；Adjusting initial pH value is 5.5, then
28 DEG C are placed in, is cultivated in the constant-temperature table that revolving speed is 200r/min, incubation time 90h；Inoculum concentration is fermentation medium
Volume fraction 3%.
(3) crude enzyme liquid separates: using rotary drum vacuum suction filter in centrifugal force for 6800g microbial fermentation solution, is centrifuged
Operating time is that 20min is separated, and removes thallus, obtains the supernatant rich in dextranase as crude enzyme liquid.
(4) enzyme preparation purifies: being mentioned using the crude enzyme liquid that polyacrylamide gel electrophoresis prepares step (4)
Pure, concrete operations are the gel slab that certain size is prepared with grease removal sheet frame electrophoresis apparatus, gum concentration 7%, injection volume 10-
15ml, electric current 24-32mA respectively cut the adhesive tape of 3mm from gel slab two sides, set immediately when pH is 7.0 electrophoresis 4-5 hours
It is dyed in 0.1% Coomassie brilliant blue G250 solution, after the appearance of blue region band, adhesive tape is rinsed well with deionized water and is put
Original position is gone back to, remainder on gel slab is scaled off, is extracted with 4ml distilled water at 4 DEG C, is filtered to remove gel after standing a night,
Air drying after washing obtains dextranase enzyme preparation, by dextranase enzyme preparation deposit in 4-5 DEG C it is stored refrigerated.
It is 46783u/g by the dextrorotation acid anhydride enzyme enzyme preparation activity prepared.
Embodiment 6:
A method of dextranase enzyme preparation is prepared, the described method comprises the following steps:
(1) Spawn incubation: preparing seed liquid culture medium, and concrete operations are to choose 250g sweet potato, peeled and washed, is cut into 1-
3cm square continues to boil 38min after adding water 1.5L, water to open, filters off sweet potato block, obtain filtrate and complement to 1.5L；To sweet potato filtrate
Middle addition glucose, yeast extract, ferric sulfate, potassium dihydrogen phosphate, magnesium sulfate and potassium chloride, yeast extract additional amount are sweet potato filtrate matter
The 0.75% of amount, glucose additional amount are the 1.53% of sweet potato filtrate quality, and potassium dihydrogen phosphate additional amount is sweet potato filtrate quality
0.06%, magnesium sulfate additional amount is the 0.011% of sweet potato filtrate quality, and ferric sulfate additional amount is sweet potato filtrate quality
0.0005%, potassium chloride additional amount is the 0.03% of sweet potato filtrate quality；Adjusting initial pH value is 6.0；It then will thin beautiful hair shell
Superior strain through mutagenesis screening is inoculated into the liquid culture medium, and inoculum concentration is the 1% of seed liquid culture substrate amount；It will connect
Liquid culture medium after kind, which is placed in 28 DEG C of constant incubators, cultivates 30h.
(2) fermented and cultured: preparing fermentation medium, and concrete operations are that such as step (1) prepares sweet potato filtrate, and yeast is added
Cream, glucose, potassium dihydrogen phosphate, ferric sulfate, magnesium sulfate and potassium chloride, yeast extract additional amount are the 1.2% of sweet potato filtrate quality,
Glucose additional amount is the 2.67% of sweet potato filtrate quality, and potassium dihydrogen phosphate additional amount is the 0.13% of sweet potato filtrate quality, sulphur
Sour magnesium additional amount is the 0.021% of sweet potato filtrate quality, and ferric sulfate additional amount is the 0.0011% of sweet potato filtrate quality, potassium chloride
Additional amount is the 0.06% of sweet potato filtrate quality；The liquid culture medium mixed liquor containing strain prepared in step (1) is added
Fermentation medium is inoculated with, and inoculum concentration is the 2% of fermentation medium liquid volume fraction；Adjusting initial pH value is 6.0, then
28 DEG C are placed in, is cultivated in the constant-temperature table that revolving speed is 200r/min, incubation time 100h；
(3) crude enzyme liquid separates: using rotary drum vacuum suction filter in centrifugal force for 6900g microbial fermentation solution, is centrifuged
Operating time is that 20min is separated, and removes thallus, obtains the supernatant rich in dextranase as crude enzyme liquid.
(4) enzyme preparation purifies: being mentioned using the crude enzyme liquid that polyacrylamide gel electrophoresis prepares step (4)
Pure, concrete operations are the gel slab that certain size is prepared with grease removal sheet frame electrophoresis apparatus, gum concentration 6-8%, injection volume 10-
15ml, electric current 24-32mA respectively cut the adhesive tape of 3mm from gel slab two sides, set immediately when pH is 7.0 electrophoresis 4-5 hours
It is dyed in 0.1% Coomassie brilliant blue G250 solution, after the appearance of blue region band, adhesive tape is rinsed well with deionized water and is put
Original position is gone back to, remainder on gel slab is scaled off, is extracted with 4ml distilled water at 4 DEG C, is filtered to remove gel after standing a night,
Air drying after washing obtains dextranase enzyme preparation, by dextranase enzyme preparation deposit in 4-5 DEG C it is stored refrigerated.
It is 47238u/g by the dextrorotation acid anhydride enzyme enzyme preparation activity prepared.
Embodiment 7:
A method of dextranase enzyme preparation is prepared, the described method comprises the following steps:
(1) Spawn incubation: preparing seed liquid culture medium, and concrete operations are to choose 250g sweet potato, peeled and washed, is cut into 1-
3cm square continues to boil 40min after adding water 1.5L, water to open, filters off sweet potato block, obtain filtrate and complement to 1.5L；To sweet potato filtrate
Middle addition glucose, yeast extract, ferric sulfate, potassium dihydrogen phosphate, magnesium sulfate and potassium chloride, yeast extract additional amount are sweet potato filtrate matter
The 0.75% of amount, glucose additional amount are the 1.53% of sweet potato filtrate quality, and potassium dihydrogen phosphate additional amount is sweet potato filtrate quality
0.06%, magnesium sulfate additional amount is the 0.011% of sweet potato filtrate quality, and ferric sulfate additional amount is sweet potato filtrate quality
0.0005%, potassium chloride additional amount is the 0.03% of sweet potato filtrate quality；Adjusting initial pH value is 5.5；It then will thin beautiful hair shell
Superior strain through mutagenesis screening is inoculated into the liquid culture medium, and inoculum concentration is the 1% of seed liquid culture substrate amount；It will connect
Liquid culture medium after kind, which is placed in 28 DEG C of constant incubators, cultivates 28h.
(2) fermented and cultured: preparing fermentation medium, and concrete operations are that such as step (1) prepares sweet potato filtrate, and yeast is added
Cream, glucose, potassium dihydrogen phosphate, ferric sulfate, magnesium sulfate and potassium chloride, yeast extract additional amount are the 1.2% of sweet potato filtrate quality,
Glucose additional amount is the 2.67% of sweet potato filtrate quality, and potassium dihydrogen phosphate additional amount is the 0.13% of sweet potato filtrate quality, sulphur
Sour magnesium additional amount is the 0.021% of sweet potato filtrate quality, and ferric sulfate additional amount is the 0.0011% of sweet potato filtrate quality, potassium chloride
Additional amount is the 0.06% of sweet potato filtrate quality；The liquid culture medium mixed liquor containing strain prepared in step (1) is added
Fermentation medium is inoculated with, and inoculum concentration is the 4% of fermentation medium liquid volume fraction；Adjusting initial pH value is 5.5, then
28 DEG C are placed in, is cultivated in the constant-temperature table that revolving speed is 200r/min, incubation time 98h；
(3) crude enzyme liquid separates: using rotary drum vacuum suction filter in centrifugal force for 6100g microbial fermentation solution, is centrifuged
Operating time is that 18min is separated, and removes thallus, obtains the supernatant rich in dextranase as crude enzyme liquid.
(4) enzyme preparation purifies: being mentioned using the crude enzyme liquid that polyacrylamide gel electrophoresis prepares step (4)
Pure, concrete operations are the gel slab that certain size is prepared with grease removal sheet frame electrophoresis apparatus, gum concentration 6-8%, injection volume 10-
15ml, electric current 24-32mA respectively cut the adhesive tape of 3mm from gel slab two sides, set immediately when pH is 7.0 electrophoresis 4-5 hours
It is dyed in 0.1% Coomassie brilliant blue G250 solution, after the appearance of blue region band, adhesive tape is rinsed well with deionized water and is put
Original position is gone back to, remainder on gel slab is scaled off, is extracted with 4ml distilled water at 4 DEG C, is filtered to remove gel after standing a night,
Air drying after washing obtains dextranase enzyme preparation, by dextranase enzyme preparation deposit in 4-5 DEG C it is stored refrigerated.
It is 27684u/g by the dextrorotation acid anhydride enzyme enzyme preparation activity prepared.
Embodiment 8:
A method of dextranase enzyme preparation is prepared, the described method comprises the following steps:
(1) Spawn incubation: preparing seed liquid culture medium, and concrete operations are to choose 250g sweet potato, peeled and washed, is cut into 1-
3cm square continues to boil 42min after adding water 1.5L, water to open, filters off sweet potato block, obtain filtrate and complement to 1.5L；To sweet potato filtrate
Middle addition glucose, yeast extract, ferric sulfate, potassium dihydrogen phosphate, magnesium sulfate and potassium chloride, yeast extract additional amount are sweet potato filtrate matter
The 0.75% of amount, glucose additional amount are the 1.53% of sweet potato filtrate quality, and potassium dihydrogen phosphate additional amount is sweet potato filtrate quality
0.06%, magnesium sulfate additional amount is the 0.011% of sweet potato filtrate quality, and ferric sulfate additional amount is sweet potato filtrate quality
0.0005%, potassium chloride additional amount is the 0.03% of sweet potato filtrate quality；Adjusting initial pH value is 5.0；It then will thin beautiful hair shell
Superior strain through mutagenesis screening is inoculated into the liquid culture medium, and inoculum concentration is the 2% of seed liquid culture substrate amount；It will connect
Liquid culture medium after kind, which is placed in 28 DEG C of constant incubators, cultivates 20-30h.
(2) fermented and cultured: preparing fermentation medium, and concrete operations are that such as step (1) prepares sweet potato filtrate, and yeast is added
Cream, glucose, potassium dihydrogen phosphate, ferric sulfate, magnesium sulfate and potassium chloride, yeast extract additional amount are the 1.2% of sweet potato filtrate quality,
Glucose additional amount is the 2.67% of sweet potato filtrate quality, and potassium dihydrogen phosphate additional amount is the 0.13% of sweet potato filtrate quality, sulphur
Sour magnesium additional amount is the 0.021% of sweet potato filtrate quality, and ferric sulfate additional amount is the 0.0011% of sweet potato filtrate quality, potassium chloride
Additional amount is the 0.06% of sweet potato filtrate quality；The liquid culture medium mixed liquor containing strain prepared in step (1) is added
Fermentation medium is inoculated with, and inoculum concentration is the 3% of fermentation medium liquid volume fraction；Adjusting initial pH value is 5.5, then
28 DEG C are placed in, is cultivated in the constant-temperature table that revolving speed is 200r/min, incubation time 98h；
(3) crude enzyme liquid separates: using rotary drum vacuum suction filter in centrifugal force for 6900g microbial fermentation solution, is centrifuged
Operating time is that 20min is separated, and removes thallus, obtains the supernatant rich in dextranase as crude enzyme liquid.
(4) enzyme preparation purifies: being mentioned using the crude enzyme liquid that polyacrylamide gel electrophoresis prepares step (4)
Pure, concrete operations are the gel slab that certain size is prepared with grease removal sheet frame electrophoresis apparatus, gum concentration 6-8%, injection volume 10-
15ml, electric current 24-32mA respectively cut the adhesive tape of 3mm from gel slab two sides, set immediately when pH is 7.0 electrophoresis 4-5 hours
It is dyed in 0.1% Coomassie brilliant blue G250 solution, after the appearance of blue region band, adhesive tape is rinsed well with deionized water and is put
Original position is gone back to, remainder on gel slab is scaled off, is extracted with 4ml distilled water at 4 DEG C, is filtered to remove gel after standing a night,
Air drying after washing obtains dextranase enzyme preparation, by dextranase enzyme preparation deposit in 4-5 DEG C it is stored refrigerated.
It is 43562u/g by the dextrorotation acid anhydride enzyme enzyme preparation activity prepared.
It should be noted that, in this document, relational terms such as first and second and the like are used merely to a reality
Body or operation are distinguished with another entity or operation, are deposited without necessarily requiring or implying between these entities or operation
In any actual relationship or order or sequence.Moreover, the terms "include", "comprise" or its any other variant are intended to
Non-exclusive inclusion, so that the process, method, article or equipment including a series of elements is not only wanted including those
Element, but also including other elements that are not explicitly listed, or further include for this process, method, article or equipment
Intrinsic element.In the absence of more restrictions, the element limited by sentence "including a ...", it is not excluded that
There is also other identical elements in process, method, article or equipment including the element.
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although with reference to the foregoing embodiments
Invention is explained in detail, those skilled in the art should understand that: it still can be to aforementioned each implementation
Technical solution documented by example is modified or equivalent replacement of some of the technical features；And these modification or
Replacement, the spirit and scope for technical solution of various embodiments of the present invention that it does not separate the essence of the corresponding technical solution.
Claims (6)
1. a kind of method for preparing dextranase enzyme preparation, which is characterized in that the described method comprises the following steps:
(1) Spawn incubation: preparing seed liquid culture medium, and concrete operations are to choose 250g sweet potato, peeled and washed, is cut into 1-3cm
Square continues to boil 30-45min after adding water 1.5L, water to open, filters off sweet potato block, obtain filtrate and complement to 1.5L, to sweet potato filtrate
Middle addition carbon source, nitrogen source, source of iron, phosphorus source, magnesium source and potassium resource, adjusting initial pH value are 5.0-6.0, then will generate dextran
Liquid culture medium after inoculation is placed in 28 DEG C of constant incubators into the liquid culture medium and cultivates 20- by the strain inoculated of enzyme
30h；
(2) fermented and cultured: preparing fermentation medium, and concrete operations such as step (1) prepares sweet potato filtrate, and nitrogen source, carbon source, phosphorus is added
Fermentation medium is added in the liquid culture medium mixed liquor containing strain prepared in step (1) by source, source of iron, magnesium source and potassium resource
It is inoculated with, inoculum concentration is the 2-4% of fermentation medium liquid volume fraction, and adjusting initial pH value is 5.0-6.0, is subsequently placed in
28 DEG C, revolving speed is is cultivated in the constant-temperature table of 200r/min, incubation time 85-100h；
(3) crude enzyme liquid separates: microbial fermentation solution being centrifugated using rotary drum vacuum suction filter, thallus is removed, obtains richness
Supernatant containing dextranase is as crude enzyme liquid；
(4) enzyme preparation purifies: being purified, is had using the crude enzyme liquid that polyacrylamide gel electrophoresis prepares step (4)
Gymnastics is as the gel slab for preparing certain size with grease removal sheet frame electrophoresis apparatus, gum concentration 6-8%, injection volume 10-15ml,
Electric current is 24-32mA, and the electrophoresis 4-5h when pH is 7.0 respectively cuts the adhesive tape of 3mm from gel slab two sides, is immediately placed on 0.1%
It is dyed in Coomassie brilliant blue G250 solution, after the appearance of blue region band, adhesive tape is rinsed well with deionized water and puts back to original position, it will
Remainder scales off on gel slab, is extracted with 4ml distilled water at 4 DEG C, is filtered to remove gel, room temperature after washing after standing a night
It is dry, obtain dextranase enzyme preparation, by dextranase enzyme preparation deposit in 4-5 DEG C it is stored refrigerated.
2. the method for preparing dextranase enzyme preparation as described in claim 1, which is characterized in that nitrogen in the step (1)
Source additional amount is the 0.75% of sweet potato filtrate quality, and carbon source addition is the 1.53% of sweet potato filtrate quality, and phosphorus source additional amount is
The 0.06% of sweet potato filtrate quality, magnesium source additional amount are the 0.011% of sweet potato filtrate quality, and source of iron additional amount is sweet potato filtrate matter
The 0.0005% of amount, potassium resource additional amount are the 0.03% of sweet potato filtrate quality；Nitrogen source additional amount is sweet potato filter in the step (2)
The 1.2% of liquid quality, carbon source addition are the 2.67% of sweet potato filtrate quality, and phosphorus source additional amount is sweet potato filtrate quality
0.13%, magnesium source additional amount is the 0.021% of sweet potato filtrate quality, and source of iron additional amount is the 0.0011% of sweet potato filtrate quality,
Potassium resource additional amount is the 0.06% of sweet potato filtrate quality.
3. the method for preparing dextranase enzyme preparation as described in claim 1 or 2, which is characterized in that the nitrogen source is
Yeast extract, the carbon source are glucose, and phosphorus source is potassium dihydrogen phosphate, and the magnesium source is magnesium sulfate, and the source of iron is sulfuric acid
Iron, the potassium resource are potassium chloride.
4. the method for preparing dextranase enzyme preparation as described in claim 1, which is characterized in that the strain is thin beautiful hair
Superior strain of the shell through mutagenesis screening.
5. the method for preparing dextranase enzyme preparation as described in claim 1, which is characterized in that bacterium in the step (1)
Kind inoculum concentration is the 1-3% of seed liquid culture substrate amount；Inoculum concentration is fermentation medium volume fraction in the step (2)
2-4%.
6. as described in claim 1 prepare dextranase enzyme preparation method, which is characterized in that in the step (4) from
Mental and physical efforts are 6000-7000g, and the centrifugally operated time is 15-25min.
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