CN103232982A - Preparation method for high-activity beta-amylase - Google Patents
Preparation method for high-activity beta-amylase Download PDFInfo
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Abstract
The invention relates to a separation and purification method for amylase in a plant, and particularly relates to a method for industrially producing high-activity beta-amylase. The preparation method for the beta-amylase disclosed by the invention comprises the following steps of: collecting separation water; pre-treating; precipitating and dissolving out the beta-amylase; forming a composite precipitate from polyacrylic acid and the beta-amylase in the solution; dissolving the composite precipitate; and adjusting the pH to be greater than or equal to 6.0, and dissolving out the beta-amylase. The invention provides a preparation method for beta-amylase, which is less in resource consumption, high in yield (of about 75%), and high in activity (of 700000-900000 u/ml generally); and the obtained product is good in tolerance to temperature and pH value, and low in cost in case of being applied to maltose production.
Description
Technical field
The present invention relates to the diastatic separating and purifying method in the kind of plant, especially a kind of method of suitability for industrialized production high-activity beta-amylase.
Background technology
Beta-amylase (E.C3.2.1.2) is diastatic a kind of, claims 1 again ,-3-maltoside enzyme.Beta-amylase extensively is present in barley, wheat, and sweet potato in the plants such as beans, also has many microorganisms can produce beta-amylase.Beta-amylase as biological catalyst, can be applied to production and the manufacturing of maltose, beer, bread etc. in foodstuffs industry.Use beta-amylase can prevent age of starch in the heart in food store, make injection maltose at medicine industry, can with α-Dian Fenmei together as digestive pharmaceutical.
In recent years, make malt syrup with beta-amylase as biological catalyst, supplied the situation of China's sugar deficiency.Along with scientific-technical progress, people are to high maltose syrup, and superhigh maltose syrup research is very active.
But domestic by sweet potato, the vigor that wheat is produced beta-amylase low (500,000 u/ml), temperature tolerance poor (58 ℃) does not meet high malt sugar, the production requirement of superhigh maltose syrup.Though the high-activity beta-amylase of import can meet the production of high maltose syrup, but because its temperature tolerance is not as good as soya-beta amylase, the usage quantity of beta-amylase is big for the production of high malt sugar the time, and necessary Prolan enzyme dosage also is greater than soya-beta amylase (the narrow and Prolan enzyme pH of the use pH of barley beta-amylase inconsistent cause) when producing high malt sugar.
The process application of above-mentioned high vigor soya-beta amylase is in suitability for industrialized production, and use range is restricted, and the price height can not satisfy the demands.
Meanwhile, though domestic scientific research department has obtained preparation method's scientific payoffs of many beta-amylases, but most of energy consumption height, environmental pollution height (salt analysis method), making a low multiple use of raw material, cost height (raw material adopt low temperature soy meal, price is more than 3500 yuan/ton) (being called " preparation technology of soya-beta amylase ", the patent No. referring to name is 99102506 Chinese invention patent).
Summary of the invention
It is few to the purpose of this invention is to provide a kind of consumption of natural resource, yield height, vigor height, the beta-amylase preparation method that performance is good.The product that obtains according to this preparation method has heatproof, the acidproof advantage that is better than like product, and storage stability is good, is effect high-activity beta-amylase preferably.
The invention provides a kind of preparation method of beta-amylase, comprise the dried up collection of branch, pre-treatment, precipitation and stripping beta-amylase adopt the beta-amylase in polyacrylic acid and the solution to form the composite precipitation thing, dissolving composite precipitation thing, regulate pH more than or equal to 6.0 stripping beta-amylases, through purifying, concentrate, be equipped with stablizer, refining back obtains high-activity beta-amylase
In the aforesaid method, can adopt discarded plant extraction liquid as raw material.For example, in the preference of the present invention, divide dried up as dividing dried up raw material with soybean protein.
In the aforesaid method, can adopt the filter material of flocculation agent and screening to dividing the dried up solid-liquid separation of carrying out, make filtrate can not stop up ultra-filtration membrane.
Wherein, divide dried up collection after, adjust 10-50 ℃ of temperature, pH 3.2-5.0.
Adding concentration is the PAA(polyacrylic acid of 0.05-5%).Sediment separate out, neutralization precipitation thing stripping beta-amylase again behind the adding PAA.
In a preferred embodiment of the invention, above-mentioned PAAE(polyacrylic acid beta-amylase) the composite precipitation thing, being dissolved in water and adding calcium carbonate is neutralized to more than the pH6.0, promotes Ca
2+Can form the littler mixture precipitation of solubleness with PAA in the throw out, and it is soluble in water that zymoprotein is discharged.
In the aforesaid method, behind the stripping beta-amylase, also comprise the coacervation removal of impurity, the step of ultrafiltration.
In the ultra-filtration process, can adopt the filter material of 10K-30K Dalton molecular weight to concentrate.
Described filter material can be organic membrane, mineral membrane or metallic membrane.
Ultrafiltration is adjusted pH and flocculation agent and is made residual protein and colloidalmaterial precipitate after certain multiple.
Ultrafiltration can divide two sections to carry out, and first section is carried out to concentrated solution that coacervation is handled and adjust pH, makes that the non-enzyme material that contains obtains precipitation in the concentrated solution; After filtering, second section product to first section acquisition carry out secondary concentration again.
Ultrafiltration divides secondary to carry out, and at first crude enzyme liquid is carried out the coacervation processing and adjusts pH4.5-5.5, makes non-enzyme material obtain precipitation, and the crude enzyme liquid after the filtration carries out ultrafiltration and concentration again.Thereby obtain highly active beta-amylase thereby improved cycles of concentration effectively.
In preference of the present invention, added enzyme stabilizers (containing salt, dextrin, sodium-acetate), with the performance of further lifting gained soya-beta amylase.
In the aforesaid method, can adopt flocculating aids to carry out solid-liquid separation or microorganism is made with extra care and removed to microporous membrane
Preparation method of the present invention is raw material with the discarded plant extracting solution, not only turns waste into wealth, and environmental protection, and can significantly reduce soy-protein factory and divide dried up middle COD.
Chemical oxygen demand COD (Chemical Oxygen Demand) is chemically to measure the amount that needs oxidized reducing substances in the water sample.Water sample is index with the amount of the oxygenant that reducing substances was consumed in the oxidation 1 premium on currency sample under certain condition, be converted to every premium on currency sample all oxidized after, the milligram number of the oxygen that needs is represented with mg/L.It has reflected the degree that polluted by reducing substances.This index is also as one of overall target of organism relative content.
Utilize preparation method of the present invention, produce 1 ton of high-activity beta-amylase can save 10 surplus ton dregs of beans, can also reduce simultaneously soy-protein factory divide dried up in the about 20-30% of COD.
On the other hand, the present invention also provides the soya-beta amylase of producing according to above-mentioned preparation method, and its characteristic is optimum temperature 60-63 ℃, 40-65 ℃ of operative temperature scope, the suitableeest action pH 5.5, action pH scope 3.8-7.0.The beta-amylase of the present invention's preparation, its enzyme the highest 1,000,000 u/ml(1u that can reach alive are 1 enzyme activity unit, it is to specify under the suitable reaction condition, per hour produces the enzyme amount of 1 milligram of maltose).
Because this product possesses above-mentioned excellent specific property, guarantee the sugaring high-level efficiency, strengthened again and resisted the harm that microorganism pollutes saccharification liquid.
Fig. 1 shows that the soya-beta amylase that preparation method of the present invention produces is in higher temperature, and not only active not reduction slightly promotes on the contrary.And under the same temperature, the activity of barley beta-amylase just descends rapidly.Fig. 2 shows, under the identical situation of other conditions, barley beta-amylase does not almost have activity in pH≤4.5 o'clock, and after pH was greater than 4.5, activity progressively improved, and reached the climax when pH=5.5, immediately descended; And the soya-beta amylase that preparation method of the present invention produces is activity stabilized in wideer pH scope.
High-activity beta-amylase preparation method excellent effect of the present invention:
Adopt discarded soybean protein parting liquid to extract beta-amylase and reach comprehensive utilization, turn waste into wealth, can reduce soy-protein factory and divide dried up middle COD, play fine effect to improving environment.
It is simple to adopt the PAA precipitate and separate to obtain the beta-amylase method, and the yield height adopts ultrafiltration and concentration can obtain high-activity beta-amylase.
The more important thing is, use the present invention to obtain the more excellent product performance of beta-amylase that extracts than other plant, the high-level efficiency that guarantees to refine sugar has strengthened again resists the harm that microorganism pollutes saccharification liquid.
High-activity beta-amylase of the present invention is suitable for the manufacturing of above-mentioned Industrial products.Particularly make high maltose syrup, superelevation maltose syrups and prevent the preferred product of age of starch.
Comparison test shows that when producing high malt sugar, the usage quantity of soya-beta amylase is compared barley beta-amylase can reduce 20%, and the consumption of general Luo Lan enzyme also can reduce 20%.
Description of drawings
Fig. 1 is that soya-beta amylase and barley beta-amylase are to the tolerance comparison diagram of temperature.
Wherein, X-coordinate be temperature (degree centigrade), ordinate zou is that relative enzyme is lived.SBA is soya-beta amylase, and BBA is barley beta-amylase.Fig. 1 shows that the soya-beta amylase that preparation method of the present invention produces is in higher temperature, and not only active not reduction slightly promotes on the contrary.And under the same temperature, the activity of barley beta-amylase just descends rapidly.
Fig. 2 is that soya-beta amylase and barley beta-amylase are to the tolerance comparison diagram of potential of hydrogen.
Wherein, SBA=Soy β-amylase (soya-beta amylase), BBA=Barley β-amylase(barley beta-amylase);
X-coordinate is the pH value, and ordinate zou is that relative enzyme is lived.SBA is soya-beta amylase, and BBA is barley beta-amylase; Show among the figure that under the identical situation of other conditions, barley beta-amylase does not almost have activity in pH≤4.5 o'clock, after pH was greater than 4.5, activity progressively improved, and reached the climax when pH=5.5, immediately descended; And the soya-beta amylase that preparation method of the present invention produces is activity stabilized in wideer pH scope.
Fig. 3 is the difference of soya-beta amylase He each concentration Prolan enzyme of 330 units (330u/g starch).
62 ℃ of reaction conditions: pH4.5.
Fig. 4 is the difference of barley beta-amylase He each concentration Prolan enzyme of 330 units (330u/g starch).
62 ℃ of reaction conditions: pH4.5.
Fig. 5 adds under the 0.5ASPU situation at general Luo Lan enzyme, and barley beta-amylase and soya-beta amylase generate the performance of maltose.
Fig. 6 adds under the 1.0ASPU situation at general Luo Lan enzyme, and barley beta-amylase and soya-beta amylase generate the performance of maltose.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to the described condition of normal condition, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and this area are skillfully in the literary composition, personnel were familiar with.In addition, any method similar or impartial to described content and material all can be applicable among the present invention.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
Embodiment 1 preferred preparation example 1
Get the active 4125u/ml of branch dried up (waste water after the albumen sepn) 100L(), adjust 45 ℃ of temperature, pH3.4 adds 0.1%PAA and obtains precipitation, adds 100g calcium carbonate through dissolving, in and pH6.30, through 300cm
2Strainer filters, and obtains filtrate and concentrates with the mesopore tunica fibrosa, is equipped with enzyme stabilizers (salt, dextrin, sodium-acetate), refining 71.64 ten thousand u/ml finished product 416ml.
Embodiment 2 preferred preparation examples 2
Getting collection divides dried up 1500L(active 4380u/ml), adjust 44 ℃ of temperature, pH3.3 adds 0.2%PAA and obtains precipitation, adds water 1:5 dissolving, adds in the 1.1kg calcium carbonate and pH6.05, uses 1m
2Plate Filtration obtains filtrate, with 4 cun rolled film ultrafiltration and concentration, is equipped with enzyme stabilizers (salt, dextrin, sodium-acetate), the refining 74.58 ten thousand u/ml finished product 6589ml that obtain.
Embodiment 3 preferred preparation examples 3
Get collection and divide dried up 10M
3(active 4217u/ml) adjusts 44 ℃ of temperature, in and pH3.4 add 0.50%PAA and obtain precipitation, add the 1:5 water dissolution through dissolving, add in the 10kg calcium carbonate and pH6.18, with 8 cun rolled film ultrafiltration and concentration, after enzyme stabilizers (salt, dextrin, sodium-acetate) is refining, obtain 91.64 ten thousand u/ml finished product 34787ml.
Embodiment 4 preferred preparation examples 4
Get collection and divide dried up 1000L(4530u/ml), adopt the diatomite filtration of screening to handle, filtrate is concentrated to 20 times with the super worry of film, for the first time concentrated solution makes albumen and colloidalmaterial in the concentrated solution obtain precipitation, filtration removal impurity with polymerize aluminum chloride (PAC) 0.03% coacervation and adjust pH to 4.0-6.0, filtrate is carried out second ultrafiltration, secondary concentration liquid is through the refining 900,000 u/ml finished product 4836ml that obtain of enzyme stabilizers (salt, dextrin, sodium-acetate).
The performance that embodiment 5 soya-beta amylases and barley beta-amylase generate maltose compares
The soya-beta amylase that obtains with embodiment 3 and at present China generally make comparisons at the barley beta-amylase of usefulness and make application aspect the maltose.
Employed Prolan enzyme (GCI OPTIMAX L-1000) activity is 1000 ASPU/g.Employed soya-beta amylase and barley beta-amylase active close is respectively soybean βDian Fenmei 705870U/ml, barley βDian Fenmei 708840u/ml.
The condition enactment of mashing test is as follows:
Substrate concn: Japanese starch 100,33%W/V concentration (DE=11 is suitable)/50mM Glacial acetic acid-sodium acetate buffer solution.
The addition of enzyme: the combination according to table 1 and table 2 is added.
Saccharification pH temperature: soya-beta amylase is pH4.5,62.0 ℃; Barley beta-amylase is pH5.5,58.5 ℃.
Saccharification time: 0～48 hour.
Saccharification target: more than 70%.
According to the most suitable condition of soya-beta amylase and barley beta-amylase, make comparisons with regard to saccharification capability.
Soya-beta amylase is when reducing by 20% addition than barley beta-amylase in the table 1, and saccharification can reach identical saccharification result after 40 hours equally.
In addition because the blue enzyme of the applicable pH of barley beta-amylase and Pu Luo is inconsistent, therefore general Luo Lan enzyme there is interdependence (adding greater than 2ASPU), and soya-beta amylase is consistent with the applicable pH of the blue enzyme of Pu Luo, therefore little to the accretion of maltose when general Luo Lan enzyme adds 0.75ASPU to after, so the addition of general Luo Lan enzyme can reduce 20% during the production high malt sugar.This illustrates that cost was relatively low when soya-beta amylase of the present invention was manufactured maltose.
Annotate: G1=glucose, G2=maltose, G3=Fructus Hordei Germinatus three pools, G4=tetrose.
The saccharification comparison test of table 1 soya-beta amylase and barley beta-amylase
Claims (10)
1. the preparation method of a beta-amylase, comprise the dried up collection of branch, pre-treatment, precipitation and stripping beta-amylase, it is characterized in that, it comprises step: adopt the beta-amylase in polyacrylic acid and the solution to form the composite precipitation thing, dissolving composite precipitation thing is regulated pH more than or equal to 6.0 stripping beta-amylases, through purifying, concentrate, be equipped with stablizer, the refining beta-amylase that obtains high vigor.
2. preparation method according to claim 1 is characterized in that, divide dried up collection after, adjust 10-50 ℃ of temperature, pH 3.2-5.0.
3. preparation method according to claim 1 is characterized in that, adding polyacrylic final concentration is 0.05-5%.
4. preparation method according to claim 1 is characterized in that, behind the stripping beta-amylase, also comprises ultrafiltration and deimpurity step.
5. preparation method according to claim 4 is characterized in that, ultrafiltration adopts organic membrane, mineral membrane or the metallic membrane of 10K-30K Dalton molecular weight to concentrate.
6. preparation method as claimed in claim 4 is characterized in that, ultrafiltration is adjusted pH and flocculation agent and made residual protein and colloidalmaterial precipitate after certain multiple.
7. preparation method as claimed in claim 4 is characterized in that, ultrafiltration divides two sections to carry out, and first section is carried out to concentrated solution that coacervation is handled and adjust pH, makes that the non-enzyme material that contains obtains precipitation in the concentrated solution; After filtering, second section product to first section acquisition carry out secondary concentration again.
8. preparation method according to claim 1 is characterized in that adopting flocculating aids to carry out solid-liquid separation or microorganism is made with extra care and removed to microporous membrane.
9. preparation method according to claim 1 is characterized in that, it is dried up as dividing dried up raw material to adopt soybean protein to divide.
10. the beta-amylase that obtains of preparation method according to claim 1.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106459943A (en) * | 2014-06-16 | 2017-02-22 | 罗盖特公司 | Method for manufacturing a stable aqueous solution of [beta]-amylase, aqueous solution obtained and uses thereof |
| CN109112121A (en) * | 2018-08-30 | 2019-01-01 | 山东禹王生态食业有限公司 | A method of soya-beta amylase is prepared using the pea green water of soybean protein |
| CN110863023A (en) * | 2019-12-04 | 2020-03-06 | 双桥（厦门）有限公司 | Preparation method of syrup for manioc sugar |
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Cited By (4)
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| CN106459943A (en) * | 2014-06-16 | 2017-02-22 | 罗盖特公司 | Method for manufacturing a stable aqueous solution of [beta]-amylase, aqueous solution obtained and uses thereof |
| CN109112121A (en) * | 2018-08-30 | 2019-01-01 | 山东禹王生态食业有限公司 | A method of soya-beta amylase is prepared using the pea green water of soybean protein |
| CN109112121B (en) * | 2018-08-30 | 2020-04-07 | 山东禹王生态食业有限公司 | Method for preparing soybean β -amylase by using soybean protein bean green water |
| CN110863023A (en) * | 2019-12-04 | 2020-03-06 | 双桥（厦门）有限公司 | Preparation method of syrup for manioc sugar |
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|---|---|
| CN103232982B (en) | 2015-03-04 |
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