CN103232982B - Preparation method for high-activity beta-amylase - Google Patents
Preparation method for high-activity beta-amylase Download PDFInfo
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- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 abstract description 27
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- MYPRUMQOPMOOSZ-UHFFFAOYSA-N 1-(diaminomethylidene)-3-(3-sulfamoylphenyl)urea;hydrochloride Chemical compound Cl.NC(N)=NC(=O)NC1=CC=CC(S(N)(=O)=O)=C1 MYPRUMQOPMOOSZ-UHFFFAOYSA-N 0.000 description 1
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- Enzymes And Modification Thereof (AREA)
- Separation Using Semi-Permeable Membranes (AREA)
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 a 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 one, also known as 1 ,-3-maltoside enzyme.Beta-amylase is extensively present in barley, wheat, sweet potato, in the plants such as beans, also has many microorganisms to produce beta-amylase.Beta-amylase, in the food industry as biological catalyst, can be applied to production and the manufacture of maltose, beer, bread etc.Use beta-amylase to prevent age of starch in the heart in food store, medicine industry manufactures injection maltose, can as digestive pharmaceutical together with α-amylase.
In recent years, manufacture malt syrup using beta-amylase as biological catalyst, supply 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 low (500,000 u/ml) of beta-amylase produced by wheat, and temperature tolerance difference (58 DEG C), does not meet high malt sugar, the production requirement of superhigh maltose syrup.Although 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, large in the usage quantity for the production of beta-amylase during high malt sugar, and when producing high malt sugar, necessary Prolan enzyme dosage is also greater than soya-beta amylase (the use pH of barley beta-amylase is narrow and Prolan enzyme pH is inconsistent caused).
The process application of above-mentioned high vigor soya-beta amylase is in suitability for industrialized production, and use range is restricted, and price is high, can not satisfy the demands.
Meanwhile, although Domestic Scientific Research department achieves preparation method's scientific payoffs of many beta-amylases, but most of energy consumption is high, environmental pollution high (salting-out method), making a low multiple use of raw material, cost high (raw material adopts low temperature soy meal, price more than 3500 yuan/ton) (be called " preparation technology of soya-beta amylase " see name, the patent No. is the Chinese invention patent of 99102506).
Summary of the invention
The object of this invention is to provide a kind of consumption of natural resource few, yield be high, vigor is high, beta-amylase preparation method that performance is good.The product obtained according to this preparation method has heatproof, the acidproof advantage being better than like product, and storage stability is good, is the good high-activity beta-amylase of effect.
The invention provides a kind of preparation method of beta-amylase, comprise Separation of Water to collect, pre-treatment, precipitation is stripping beta-amylase also, adopts the beta-amylase in polyacrylic acid and solution to form composite precipitation thing, dissolve composite precipitation thing, pH is regulated to be more than or equal to 6.0 stripping beta-amylases, through purification, concentrated, be equipped with stablizer, after refining, obtain high-activity beta-amylase
In aforesaid method, discarded plant extraction liquid can be adopted as raw material.Such as, in preference of the present invention, using soybean protein Separation of Water as Separation of Water raw material.
In aforesaid method, the filter material of flocculation agent and screening can be adopted to carry out solid-liquid separation to Separation of Water, make filtrate block ultra-filtration membrane.
Wherein, after Separation of Water is collected, adjustment temperature 10-50 DEG C, pH 3.2-5.0.
Add the PAA(polyacrylic acid that concentration is 0.05-5%).Add sediment separate out after PAA, then neutralization precipitation thing stripping beta-amylase.
In a preferred embodiment of the invention, above-mentioned PAAE(polyacrylic acid beta-amylase) composite precipitation thing, being dissolved in water and adding calcium carbonate is neutralized to more than pH6.0, promotes Ca
2+can form the less complex precipitate of solubleness with PAA in throw out, and it is soluble in water that zymoprotein is discharged.
In aforesaid method, after stripping beta-amylase, also comprise the coacervation removal of impurity, the step of ultrafiltration.
In ultra-filtration process, the filter material of 10K-30K Dalton molecular weight can be adopted to concentrate.
Described filter material can be organic membrane, mineral membrane or metallic membrane.
After ultrafiltration to certain multiple, adjustment pH and flocculation agent make residual protein and colloidalmaterial precipitation.
Ultrafiltration can divide two sections to carry out, and first paragraph carries out coacervation process to concentrated solution and adjusts pH, and the non-enzyme material that contains in concentrated solution is precipitated; Second segment carries out secondary concentration after filtering the product that first paragraph obtains again.
Ultrafiltration divides secondary to carry out, and first carries out coacervation process to crude enzyme liquid and adjusts pH4.5-5.5, and non-enzyme material is precipitated, and the crude enzyme liquid after filtration carries out ultrafiltration and concentration again.Thus effectively improve cycles of concentration thus obtain highly active beta-amylase.
In preference of the present invention, add enzyme stabilizers (containing salt, dextrin, sodium-acetate), to promote the performance of gained soya-beta amylase further.
In aforesaid method, flocculating aids can be adopted to carry out solid-liquid separation or microporous membrane is refined, and remove microorganism
Preparation method of the present invention, with discarded plant extracting solution for raw material, not only turns waste into wealth, environmental protection, and significantly can reduce COD in soy-protein factory Separation of Water.
Chemical oxygen demand COD (Chemical Oxygen Demand) chemically measures in water sample the amount needing oxidized reducing substances.Water sample under certain condition, to be oxidized the amount of the oxygenant that reducing substances consumes in 1 premium on currency sample for index, be converted to every premium on currency sample all oxidized after, the milligram number of the oxygen of needs, represents with mg/L.It reflects in water by the degree that reducing substances pollutes.This index is also as one of the overall target of organism relative content.
Utilize preparation method of the present invention, produce 1 ton of high-activity beta-amylase and can save more than 10 ton dregs of beans, can also reduce COD in soy-protein factory Separation of Water is about 20-30% simultaneously.
On the other hand, present invention also offers the soya-beta amylase produced according to above-mentioned preparation method, its characteristic is optimum temperature 60-63 DEG C, operative temperature scope 40-65 DEG C, the suitableeest action pH 5.5, action pH scope 3.8-7.0.Beta-amylase prepared by the present invention, the work of its enzyme is the highest can reach 1,000,000 u/ml(1u i.e. 1 enzyme activity unit, and it is under specifying suitable reaction condition, the enzyme amount of generation per hour 1 milligram of maltose).
Because this product possesses above-mentioned excellent specific property, ensure sugar-making high-efficiency rate, in turn enhance the harm resisted microorganism and saccharified liquid is polluted.
Fig. 1 shows, and 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 at same temperature, the activity of barley beta-amylase just declines rapidly.Fig. 2 shows, and when other conditions are identical, barley beta-amylase, when pH≤4.5, does not almost have activity, and after pH is greater than 4.5, activity progressively improves, and reaches climax, immediately decline when pH=5.5; And the soya-beta amylase that preparation method of the present invention produces is within the scope of wider pH, activity stabilized.
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, COD in soy-protein factory Separation of Water can be reduced, fine effect is played to environmental protect.
Adopt PAA precipitate and separate to obtain beta-amylase method simple, yield is high, adopts ultrafiltration and concentration to obtain high-activity beta-amylase.
The more important thing is, use the present invention to obtain the product performance more excellent than the beta-amylase of other plant extraction, ensure that sugar-making high-efficiency rate in turn enhances the harm resisted microorganism and pollute saccharified liquid.
High-activity beta-amylase of the present invention is suitable for the manufacture of above-mentioned Industrial products.Particularly manufacture high maltose syrup, superelevation maltose syrups and prevent the preferred product of age of starch.
Comparison test shows, and when producing high malt sugar, the usage quantity of soya-beta amylase is compared barley beta-amylase and can be reduced 20%, and the consumption of general Luo Lan enzyme also can reduce 20%.
Accompanying drawing explanation
Fig. 1 is that soya-beta amylase and barley beta-amylase are to the tolerance comparison diagram of temperature.
Wherein, X-coordinate is temperature (degree Celsius), and ordinate zou is that relative enzyme is lived.SBA is soya-beta amylase, and BBA is barley beta-amylase.Fig. 1 shows, and 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 at same temperature, the activity of barley beta-amylase just declines 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 pH value, and ordinate zou is that relative enzyme is lived.SBA is soya-beta amylase, and BBA is barley beta-amylase; Show in figure, when other conditions are identical, barley beta-amylase, when pH≤4.5, does not almost have activity, and after pH is greater than 4.5, activity progressively improves, and reaches climax, immediately decline when pH=5.5; And the soya-beta amylase that preparation method of the present invention produces is within the scope of wider pH, activity stabilized.
The soya-beta amylase of Tu3Shi330Ge unit (330u/g starch) and the difference of each concentration Prolan enzyme.
Reaction conditions: pH4.5 62 DEG C.
The barley beta-amylase of Tu4Shi330Ge unit (330u/g starch) and the difference of each concentration Prolan enzyme.
Reaction conditions: pH4.5 62 DEG C.
Fig. 5 is under general Luo Lan enzyme adds 0.5ASPU situation, and barley beta-amylase and soya-beta amylase generate the performance of maltose.
Fig. 6 is under general Luo Lan enzyme adds 1.0ASPU situation, and barley beta-amylase and soya-beta amylase generate the performance of maltose.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, the condition usually conveniently described in condition, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number calculate by weight.
Unless otherwise defined, all specialties used in literary composition and scientific words and this area is skilled, personnel the same meaning be familiar with.In addition, any method similar or impartial to described content and material all can be applicable in the present invention.The use that better implementation method described in literary composition and material only present a demonstration.
Embodiment 1 is preparation example 1 preferably
Get the active 4125u/ml of Separation of Water (waste water after albumen sepn) 100L(), adjustment temperature 45 C, pH3.4 adds 0.1%PAA and obtains precipitation, adds 100g calcium carbonate through dissolving, in and pH6.30, through 300cm
2metre filter, obtains filtrate and uses hollow fiber membrane concentration, is equipped with enzyme stabilizers (salt, dextrin, sodium-acetate), refines to obtain 71.64 ten thousand u/ml finished product 416ml.
Embodiment 2 is preparation example 2 preferably
Get the active 4380u/ml of collection Separation of Water 1500L(), adjustment temperature 44 DEG C, pH3.3 adds 0.2%PAA and obtains precipitation, and the 1:5 that adds water dissolves, and adds in 1.1kg calcium carbonate and pH6.05, uses 1m
2plate Filtration obtains filtrate, with 4 cun of rolled film ultrafiltration and concentration, is equipped with enzyme stabilizers (salt, dextrin, sodium-acetate), refines and obtains 74.58 ten thousand u/ml finished product 6589ml.
Embodiment 3 is preparation example 3 preferably
Get collection Separation of Water 10M
3(active 4217u/ml) adjusts temperature 44 DEG C, in and pH3.4 add 0.50%PAA obtain precipitation, 1:5 water dissolution is added through dissolving, add in 10kg calcium carbonate and pH6.18, with 8 cun of rolled film ultrafiltration and concentration, 91.64 ten thousand u/ml finished product 34787ml are obtained after enzyme stabilizers (salt, dextrin, sodium-acetate) is refining.
Embodiment 4 is preparation example 4 preferably
Get collection Separation of Water 1000L(4530u/ml), adopt the diatomite filtration process of screening, filtrate surpasses worry with film and is concentrated to 20 times, first time concentrated solution polymerize aluminum chloride (PAC) 0.03% coacervation also adjusts pH to 4.0-6.0, and the albumen in concentrated solution and colloidalmaterial are precipitated, and filters and removes impurity, filtrate carries out second ultrafiltration, secondary concentration liquid is refined through enzyme stabilizers (salt, dextrin, sodium-acetate) and is obtained 900,000 u/ml finished product 4836ml.
Embodiment 5 soya-beta amylase and barley beta-amylase generate the Performance comparision of maltose
The soya-beta amylase obtained by embodiment 3 and at present China generally barley beta-amylase to make comparisons the application manufactured in maltose.
Prolan enzyme (the GCI OPTIMAX L-1000) activity used is 1000 ASPU/g.The soya-beta amylase used is close with the activity of barley beta-amylase, is respectively soybean β-amylase 705870U/ml, barley β-amylase 708840u/ml.
The condition of mashing test is set as follows:
Substrate concn: Japanese starch 100,33%W/V concentration (DE=11 is suitable)/50mM Glacial acetic acid-sodium acetate buffer.
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 DEG C; Barley beta-amylase is pH5.5,58.5 DEG C.
Saccharification time: 0 ~ 48 hour.
Saccharification target: more than 70%.
According to the most applicable condition of soya-beta amylase and barley beta-amylase, make comparisons with regard to saccharification capability.
In table 1, soya-beta amylase is when reducing by 20% addition than barley beta-amylase, and saccharification can reach identical saccharification result after 40 hours equally.
In addition due to the applicable pH of barley beta-amylase and the blue enzyme of Pu Luo inconsistent, therefore interdependence (interpolation is greater than 2ASPU) is had to general Luo Lan enzyme, and soya-beta amylase is consistent with the applicable pH of the blue enzyme of Pu Luo, therefore little to the accretion of maltose after general Luo Lan enzyme adds 0.75ASPU to, so the addition of general Luo Lan enzyme can reduce 20% when producing high malt sugar.When this illustrates that soya-beta amylase of the present invention manufactures maltose, advantage of lower cost.
Note: G1=glucose, G2=maltose, G3=Fructus Hordei Germinatus three pool, G4=tetrose.
The saccharification comparison test of table 1 soya-beta amylase and barley beta-amylase
Claims (8)
1. a preparation method for beta-amylase, comprises Separation of Water and collects, pre-treatment, adopt the beta-amylase in polyacrylic acid and solution to form composite precipitation thing to precipitate and stripping beta-amylase, it is characterized in that, adopt soybean protein Separation of Water as Separation of Water raw material, and it comprises step: to dissolve this composite precipitation thing, pH is regulated to be more than or equal to 6.0 and to be less than or equal to 6.30, stripping beta-amylase, through purification, concentrated, be equipped with stablizer, the refining beta-amylase obtaining high vigor.
2. preparation method according to claim 1, is characterized in that, after Separation of Water is collected, and adjustment temperature 10-50 DEG C, 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, after stripping beta-amylase, also comprises ultrafiltration and deimpurity step.
5. preparation method according to claim 4, is characterized in that, ultrafiltration adopts the organic membrane of 10K-30K Dalton molecular weight, mineral membrane or metallic membrane to concentrate.
6. preparation method as claimed in claim 4, is characterized in that, after ultrafiltration to certain multiple, adjustment pH and flocculation agent make residual protein and colloidalmaterial precipitation.
7. preparation method as claimed in claim 4, it is characterized in that, ultrafiltration divides two sections to carry out, and first paragraph carries out coacervation process to concentrated solution and adjusts pH, and the non-enzyme material that contains in concentrated solution is precipitated; Second segment carries out secondary concentration after filtering the product that first paragraph obtains again.
8. preparation method according to claim 1, is characterized in that employing flocculating aids carries out solid-liquid separation or microporous membrane carries out refining and removing microorganism.
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|---|---|---|---|---|
| FR3022257B1 (en) * | 2014-06-16 | 2018-03-30 | Roquette Freres | PROCESS FOR PRODUCING A STABLE AQUEOUS SOLUTION OF BETA-AMYLASE, AQUEOUS SOLUTION OBTAINED AND USES THEREOF |
| 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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| CN103232982A (en) | 2013-08-07 |
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