CN1119212A - Process for preparing, separating and purifying acetolactate synthetase - Google Patents
Process for preparing, separating and purifying acetolactate synthetase Download PDFInfo
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Abstract
The process for preparing, separating and purifying acetolactate synthase (ALS) features that ALS is cultured in the plant such as Gramineae, Legume, Amaranthaceae, Chenopod, etc. By experiments, the ratio of raw material to homogenized solution is optimized. Its technological route comprises homogenizing, filtering, extraction, two-step centrifugal processing, two-step salting-out and chromatography or dialysis for desalting. Obtained ALS has the highest specific activity of 223 micrometers/mg.hr, half-life period of 7-8 days and storage period of 30 days. It serves as biocatalyst for screening agricultural chemical in molecular level.
Description
The present invention relates to the preparation of zymoprotein, isolation and purification, particularly be the preparation of acetolactate synthestase, isolation and purification.
A kind of new for setting up, the stripped pesticide screening test method of molecular level, selecting acetolactate synthestase (Acetolactate Synthase is hereinafter to be referred as the ALS enzyme) for use is catalyzer.The ALS enzyme is a kind of flavoprotein, belongs to 4 in classification of international system, and it is an acetylactis with the specificity of height and high catalytic efficiency catalysis pyruvic acid, and emits carbonic acid gas.Be present in the ALS enzyme in bacterium or the plant tissue, abundance is low, instability, and preservation period is short, does not all have standard substance both at home and abroad and sells.The present invention extracts the ALS enzyme from plant tissue.Through preliminary search, U.S. Pat 5,084,086 discloses the effect of weedicide antagonism crop, and US 5,206,135 disclose the oxygen sensitive electrode surveys the ALS enzymic activity, and two parts of patents relate to is that the generality of ALS enzyme is discussed and the problem of weeding resistance and variation aspect.Have with the akin document of the present invention: [1] Shaner D.L., Plant.Physiol., plant physiology 76,545-546 (1984) [2] Giuseppe Forlanl, Weed Science weeds science 39,553-557 (1991) [3] Oda Y., J.Gen.Microobiol., general microbiology magazine 128,1211-1216 (1982)
Document [1] with the preparation of ALS enzyme, separates in [2], and flow process and composition parameter that purifying is relevant are summarized as follows.1): raw material is 1 gram with the ratio of homogenizing solution: 5 milliliters; In the homogenizing solution, that counterfeit substrate is selected for use is disodium ethylene diamine tetraacetate (EDTA), and concentration is 1,000-5,000 μ M; The concentration of dipotassium hydrogen phosphate damping fluid is 50mM; What coenzyme was used is a word used in person's names propylhomoserin, leucine; The enrichment of saltouing is only carried out a step; The saturation ratio of used vitriol is 20-50%; Substrate pyruvic acid concentration 20mM in enzyme group's lysate; One step of centrifugation, 27,000 rev/mins.Unreasonable from above-mentioned each parameters relationship reflection raw material and homogenizing solution ratio, cause the ALS enzyme that contains in the homogenate few.Substrate content in homogenizing solution and the enzyme group lysate is excessive again, causes enzyme ' s reaction speeding quickening in the separation and purification process, has reduced the quality of ALS enzyme.Technical process is also unreasonable in addition, after homogenate is filtered, also has a lot of enzymes just not to be extracted in filter residue and directly carries out centrifugation.One step centrifugation, speed is very fast again, has a lot of thick enzymes to be contained in the filter residue and is discarded.Saltouing yet step only of enrichment, is not again to work under best salt point, and technical process is meticulous inadequately.Owing to above reason, cause the enzyme of acquisition of low quality, as the fragmentary record of document [3] and other document, obtaining the ALS enzyme than the value of living with general method is 1-2 μ M/mghr, and point out to store 24 hours down at 4 ℃, reduce by 46%, the record preservation period that also has only 12 hours-several days with original specific activity mutually, these parameters are all lower, can not satisfy the practical specification requirement of biological catalyst in the stripped pesticide screening test method of molecular level.
The objective of the invention is:
Use modern molecular enzymology theory and carry out serial scientific experiment, from Gramineae, pulse family, Amaranthaceae, Chenopodiaceae, polygonaceae, convolvulaceae, Cruciferae, Rubiaceae, composite family, Cyperaceae, Labiatae, Malvaceae, the acetolactate synthestase of cultivating in the Alismataceae plant is in preparation, during isolation and purification, the ratio of optimization optimum homogenizing solution is formed, concentration and the best techniqueflow of formulation, obtain the height ratio value of living, the best transformation period, the ALS enzyme of long storage life and good stability.This enzyme can satisfy new, in the stripped pesticide screening test method of molecular level as the practical specification requirement of biological catalyst.
The object of the present invention is achieved like this:
1. a kind of preparation is provided, and the method for isolation and purification acetolactate synthestase (Acetolactate Synthase, abbreviation ALS) mainly comprises following sequential steps:
(1) provides Gramineae, pulse family, Amaranthaceae, Chenopodiaceae, polygonaceae, convolvulaceae, Cruciferae, Rubiaceae, composite family, Cyperaceae, Labiatae, Malvaceae, any one bud and embryo that contains acetolactate synthestase is organized as raw material in the Alismataceae plant, with the substrate with the ALS enzyme, coenzyme, the homogenizing solution that stablizer and damping fluid are mixed with, mix to stir at normal temperatures and pressures and cut into tissue homogenate, substrate Sodium.alpha.-ketopropionate wherein, its concentration is 10-30 μ M, raw material is 1 gram with the ratio of homogenizing solution: 0.2-2.0 milliliters;
(2) tissue homogenate is filtered, filter residue leaches repeatedly repeatedly with homogenizing solution, and merging filtrate obtains thick enzyme;
(3) thick enzyme filtrate centrifugation in two steps, second step of centrifugal speed is higher than the first step, obtains centrifugal enzyme;
(4) centrifugal enzyme is saltoutd in two steps enrichment, per step all adds vitriol at low temperatures, and saturation ratio is 30-70%, precipitates centrifugal 30 minutes, and centrifugal speed 16, obtains centrifugal enzyme precipitation by 000-20,000 rev/min;
(5) with the substrate of ALS enzyme, coenzyme, damping fluid are made enzyme group lysate and are dissolved centrifugal enzyme precipitation, the precipitation enzyme lysate that obtains can be used chromatography, and the anti-dialysis method purifying of also available dialysis obtains highly purified purifying enzyme thus, substrate Sodium.alpha.-ketopropionate in enzyme group's lysate, its concentration are 1-5mM.
2. the preparation of raw material is with Gramineae, pulse family, Amaranthaceae, Chenopodiaceae, polygonaceae, convolvulaceae, Cruciferae, Rubiaceae, composite family, Cyperaceae, Labiatae, Malvaceae, after any one seed soaks in cold water in the Alismataceae plant, be placed on equably and be covered with in the culture plate of putting nylon wire on moist fine sand or the sawdust, cover gauze, at room temperature germinate, when just sprouting, add a cover the black cloth lucifuge and cultivate.
3. the coenzyme in the homogenizing solution is to select magnesium chloride for use, the fast cry of certain animals dinucleotide of thiaminpyrophosphate (TPP) and flavine gland (FAD), and the concentration of these three kinds of coenzyme is controlled at 0.3-1.0mM respectively, 0.3-1.0mM and 10-50 μ M scopes; Buffered soln is to select dipotassium hydrogen phosphate for use, and its concentration is controlled at 50-150mM scope; Add 10-30% glycerol again and make stablizer.
4. the first step centrifuge speeds is 6, and 000-8,000 rev/min, the second step centrifuge speeds is 14, and 000-20,000 rev/min, each centrifugation time is 20 minutes.
5. the vitriol when saltouing enrichment is ammonium sulfate, and best saturation ratio is 50%, and described low temperature is 0-4 ℃, precipitate centrifugal, centrifugal speed 16,000-20,000 rev/min.
6. the coenzyme in the enzyme group lysate is selected magnesium chloride for use, and concentration is 0.3-1.0mM; Buffered soln is selected dipotassium hydrogen phosphate for use, and concentration is 30-100mM; Sodium.alpha.-ketopropionate concentration is 1-5mM.
7. the enzyme group lysate of preparation can be used as leacheate in the chromatography and the dialyzate in the dialysis method.
According to the determined best-of-breed technology flow process of above-mentioned steps as shown in Figure 1.
As the enzyme of biological catalyst, have the sensitivity that high ratio value alive could improve catalyzed reaction, the best transformation period be arranged, the longest preservation period, use value is just arranged, and in use could keep sensitivity more stably, avoid the enzyme that extracts to cause damage with regard to inefficacy in several days.
According to modern molecular enzymology theory, enzyme-to-substrate is like " induced fit " relation, that is, for the activity that shows enzyme must have the existence of substrate and coenzyme, the selection of concentration of substrate had both influenced the activity of enzyme, related to the preservation of enzyme again.Enzymatic reaction also never stops slowly taking place, till enzymic activity completely loses, so concentration of substrate should not be too big under non-suitable condition.Concentration of substrate is big, and enzyme ' s reaction speeding is fast, and the loss of activity of enzyme is fast, and preservation period is also just short.Therefore suitable concentration of substrate is to obtain the height ratio value of living, an important factor of the best transformation period and long storage life.Moreover enzymatic reaction is to carry out under the synergy of coenzyme.Coenzyme is little non-protein molecular and metal ion.The present invention selects thiaminpyrophosphate (TPP) for use, and flavin adenine dinucleotide (FAD), magnesium ion are coenzyme.If select ZnSO for use
4, MnSO
4Though, can prepare enzyme, effect is bad.TPP can make the pyruvic acid decarboxylation, and the necessary intermediate product of activation condensation reaction.FAD can prevent that hydroxyethyl-TPP intermediate product is protonated, and magnesium ion can make enzyme all activate, and makes ALS enzyme and TPP form reversible network and thing slowly.Tested below from three kinds of different homogenizing solution of forming and extracted the influence that the contrast of ALS enzyme is lived, seen Table 1.
Table 1. extracts the influence that the contrast of ALS enzyme is lived from three kinds of different homogenizing solution of forming
| Form and concentration | Measurement result | ||||||||
| K 2HPO 4mM | Sodium.alpha.-ketopropionate mM | ?MgCl 2?mM | ?TPP ?mM | ?FAD ?μM | Glycerol V/V% | Enzyme μ M/ml alive | Albumen mg/ml | Than μ M/mghr alive | |
| ?A ?B ? | 100 100 100 | 1 5(EDTA) 0 | ?0.5 ?0.5 ?0 | ?0.5 ?0.5 ?0 | ?10 ?10 ?0 | 10 10 10 | 8.39 2.67 2.18 | 12.45 9.78 12.26 | 0.673 0.273 0.177 |
By table 1 explanation, the existence of coenzyme and a small amount of substrate or counterfeit substrate is extremely important to keeping enzymic activity.Thus, determined that by a large amount of experiments the present invention is separating and optimum composition of solution and concentration during purifying.
The present invention selects for use ammonium sulfate solids adding mode to carry out two and goes on foot the enrichment of saltouing.Raising with concentration of salt solution according to the solubleness of enzyme under low salt concn increases, after this, when the salt concn enzyme solubleness that slowly rises again begins to descend, the characteristics separated out of precipitation then, the best salt point of having determined enzyme is that the vitriol saturation ratio is 50%.
Raw materials used and the reagent of the present invention all can be bought on market.
Advantage that the present invention compared with prior art has and unusual effect:
1. the advantage of the inventive method:
1. the measures of when the plant breeding is cultivated, taking controlling plant germination effectively, not mouldy, without putrefaction, and can obtain the most high-load ALS enzyme.
2. optimize optimum extraction solution composition and concentration.Show that mainly the ALS enzyme substrates chooses suitable concentration, the preparation of coenzyme, the adding of stablizer, the preparation and the concentration of the rational proportion of raw material and homogenizing solution and enzyme group lysate.These all are to improve the activity of enzyme and the effective measure of preservation period.
3. the formulation of hand work flow process, as the leaching repeatedly of filter residue, two steps are centrifugal, and the first step centrifugal speed was lower than for second step, the two step enrichments of saltouing, these all are to prevent that enzyme is mixed in the slag and loses.And enzyme is precipitated maturely separate out, obtain high reactivity, the effective measure of high density enzyme.
2. the unusual effect that brings of the inventive method:
1. because in preparation, the isolation and purification stage has been taked above various measures, therefore use the inventive method can obtain the high-quality enzyme, show as:
The A.ALS enzyme is higher than the value of living.Centrifugal enzyme is 10-12 μ M/mghr than measured value alive, can reach 223.44 μ M/mghr behind the purifying.The ratio work of enzyme is an index of its specification of quality.Higher than living, the catalytic efficiency that the meaning enzyme has is also high.
The long preservative period of B.ALS enzyme can be preserved 30 days.After the ALS enzyme exsomatized, because its inherent ununiformity, unstable was difficult to storage.To such an extent as to complete deactivation in 12 hours to several days can't be provided for the stripped pesticide screening test of molecular level.The present invention in the extraction separation process, create one with ALS enzyme similar environment in live body.Suitable concentration of substrate is arranged between preservation period, and so active centre that can stabilized enzyme can reduce enzyme ' s reaction speeding again, to greatest extent long preservative period.
The C.ALS half life of enzyme is long: value is high because the ratio of ALS enzyme is lived, long preservative period, so the transformation period reach 7-8 days.Thereby improved actual utility value, the inhibitor screening method added value height of being created by the ALS enzyme simultaneously.
2. in the initiative novel pesticide, the exploitation of novel herbicide is an importance.To this, ALS enzyme provided by the invention is a kind of important practical enzyme.Owing to adopt the ALS enzyme of present method preparation to reach in the screening method practical specification requirement, that is: there is the high ratio value of living to improve sensitivity based on the measuring method of rate of catalysis reaction as biological catalyst; Long half time and long preservative period just can provide actual use.Therefore provide a favorable guarantee for the screening of ALS enzyme inhibitors.Practice has also proved, makees target with the ALS enzyme of this law preparation, in screening classes of compounds process, has found that two kinds have an active new lead compound of equal inhibition with chlorine sulphur is grand.(this point has detailed description in the process for screening inhibitor of acetolactate synthetase application case.)
3. the flow process and the top condition of enriching and purifying enzyme are provided, and the purifying enzyme that makes acquisition is than living up to 223 μ M/mghr, and this has created condition for further the ALS enzyme being done further investigation.
The invention will be described further below by way of embodiments and drawings: Fig. 1. the technology of the present invention schema; The wash-out spectrogram of Fig. 2 .Sephadex G-25 gel chromatography column; Fig. 3. the preservation period of enzyme and transformation period.
Embodiment 1:
The best-of-breed technology flow process of determining according to the present invention is introduced, as shown in Figure 1:
(1) cultivation of ALS enzyme:
Do fabaceous representative with pea.The breeding pea was soaked in cold water 24 hours, evenly be placed on the moist fine sand that is covered with 1 cm thick or the sawdust and put in the culture plate of nylon wire, seed loam cake gauze, at room temperature germinate, when just sprouting, add a cover the black cloth lucifuge and cultivate, get the 1-3 centimetre high bud that contains acetolactate synthestase and the raw material of embryo tissue conduct extraction ALS enzyme.
(2) preparation of ALS enzyme:
At first water preparation homogenizing solution wherein contains K
2HPO
4100mM; Substrate Sodium.alpha.-ketopropionate 10 μ M, coenzyme magnesium chloride 0.5mM, thiaminpyrophosphate (TPP) 0.5mM, flavin adenine dinucleotide 10 μ M; Stablizer glycerol 15%.Press bud (gram): the relation of solution (milliliter)=1: 0.5, take by weighing the homogenizing solution of 200 gram raw materials and 100ml, normal temperature and pressure is mixing down.In processing machine, stir to cut after 5 minutes and make tissue homogenate.
(3) isolation and purification of ALS enzyme:
Through 10 layers of gauze, 2 layers of nylon cloth filter with tissue homogenate.Filter residue after the filtration leaches 3 times repeatedly with homogenizing solution, merges with above-mentioned filtrate, obtains thick enzyme solution, and filter residue discards.
Obtain centrifugal enzyme after the thick enzyme filtrate centrifugation in two steps.The first step centrifugal speed is 7,000 rev/mins, and the second step centrifugal speed is 15,000 rev/mins, and each centrifugal 20 minutes, the filter residue after centrifugal discarded.
With the enrichment of saltouing in two steps of centrifugal enzyme, obtain centrifugal enzyme precipitation.Operate under 0-4 ℃ and carry out, ammonium sulfate solids adds, and saturation ratio is 50%.ALS zymoprotein constantly precipitation is separated out, leave standstill 2 hours after, with 20,000 rev/mins speed centrifugation 30 minutes, solution discarded again.The purpose of preliminary realization this moment concentration and separation purifying.
(4) being further purified of ALS enzyme:
At first water preparation enzyme is rolled into a ball lysate, wherein contains the substrate Sodium.alpha.-ketopropionate 1mM of ALS enzyme, coenzyme magnesium chloride 0.5mM, damping fluid dipotassium hydrogen phosphate 50mM.
Dissolve centrifugal enzyme precipitation with enzyme group lysate, sulfate ion is removed in the desalination of dissolved enzyme require again, and desalting technology can be used chromatography, and the anti-dialysis method of also available dialysis realizes, obtains highly purified enzyme thus.
(ⅰ) gel chromatography:
With diameter is 1 centimetre, and height is that the glass column of 100 centimetres band water jacket is done chromatography column, does column packing with the SephadexG-25 sephadex.
Adorn post with drainage pattern again.Do the leacheate balance with 100 milliliters enzyme group lysate and wash post.The precipitation enzyme lysate that the capital sample introduction is 3 milliliters with 25 droplets/minute flow velocity drip washing, slowly separates the ALS enzyme by gel chromatography column.Collecting live elutriant when the highest of enzyme from post lower end outlet, is exactly the purifying enzyme that the present invention will prepare, and sees Fig. 2.000 are depicted as the albumen curve among the figure; ● ● ● be depicted as enzyme curve alive; ▲ ▲ ▲ is depicted as the sulfate ion curve.Albumen is measured with the Folin method; The sulfate ion ion-chromatographic determination.
With 100 ml water wash-out pillars, sulfate radical can all wash out, and pillar is treated to use next time.
(ⅱ) the anti-dialysis method of dialysing
Select dialysis value 12,000-14,000, flat wide 1.0 inches dialysis tubing intercepts 35 cm long, soaks 24 hours in the water, and 50 milliliters of the precipitation of packing into enzyme solvent solns are tightened for two and sealed.Put into 1000 milliliters of dialyzates.What dialyzate was selected for use is enzyme group lysate.Dialyse four times each 5 hours down at 0-4 ℃.Taking-up is placed on anti-dialysis the among polyoxyethylene glycol (PEG) 6000, see when dialysis tubing dwindles till.Enzyme in the bag is exactly the purifying enzyme that the present invention will prepare.
Be prepared according to method of the present invention, isolation and purification, what obtain various enzymes sees Table 2 than measurement result alives, and its height ratio work value is 223.44 μ M/mghr.
The ratio of the various enzymes of table 2. is lived
| Kind | Enzyme (μ M/ml) alive | Than live (μ M/mghr) |
| The thick centrifugal enzyme purification enzyme of enzyme (chromatography column) purifying enzyme (dialysis tubing) | ????82.04 ????116.98 ????19.44 ????47.75 | ????6.49 ????10.77 ????223.44 ????11.43 |
The preservation period of ALS enzyme is monitored from the centrifugal enzyme from extracting, and obtaining long storage life is 30 days, and the best transformation period is 7 days, and it the results are shown in Figure 3, and A% is residual % alive among the figure.
Embodiment 2:
Selecting barley for use is the grass representative.
Embodiment 3:
Selecting corn for use is the grass representative.
Embodiment 4:
Select for use the amaranth grass to be amaranth, lamb's-quarters, knotweed, japanese bearbind, cruciate flower, madder, chrysanthemum, nutgrass flatsedge, lip, the representative of each section plant (farmland weed) of high mallow and rhizoma alismatis.
The preparation of above-mentioned 2,3,4 three examples, the method for isolation and purification and the techniqueflow of foundation are all identical with embodiment 1, and the composition of compound used therefor is also identical, and the concentration that each is formed and the related parameter difference is arranged sees Table 3.
The parameters of using among table 3. embodiment 1-4 (adopting raw material 100-500 grams)
| Material name | Homogenizing solution | Raw material: solution g: ml | |||||
| K 2HPO 4mM | Sodium.alpha.-ketopropionate μ M | ?MgCl 2?mM | ?TPP ?mM | FAD μM | Glycerol % | ||
| Pea barley | 100 50 150 120 | 10 20 30 25 | ?0.5 ?0.3 ?1.0 ?0.8 | ?0.5 ?0.3 ?1.0 ?0.8 | ?10 ?30 ?50 ?40 | 15 10 30 20 | ?1∶0.5 ?1∶1.0 ?1∶1.5 ?1∶0.3 |
Table 3. is continuous
| Material name | One step | Two steps | Saltout | Enzyme group's lysate | ||
| Centrifugal rev/min | Centrifugal rev/min | Enrichment % | K 2HPO 4mM | Sodium.alpha.-ketopropionate mM | MgCl 2mM | |
| Pea barley corn amaranth grass | 7000 6000 8000 7500 | 15000 14000 20000 18000 | 50 30 70 40 | 50 30 100 80 | 1 3 5 2 | 0.5 0.3 1.0 0.8 |
Claims (7)
1. one kind prepares, and the method for isolation and purification acetolactate synthestase (Acetolactate Syn-thase, abbreviation ALS) is characterized in that mainly comprising following sequential steps:
(1) provides Gramineae, pulse family, Amaranthaceae, Chenopodiaceae, polygonaceae, convolvulaceae, Cruciferae, Rubiaceae, composite family, Cyperaceae, Labiatae, Malvaceae, in the Alismataceae plant any one contain acetolactate synthestase bud and embryo be organized as raw material, with the substrate with the ALS enzyme, coenzyme, the homogenizing solution that stablizer and damping fluid are mixed with, mix to stir at normal temperatures and pressures and cut into tissue homogenate, substrate Sodium.alpha.-ketopropionate wherein, its concentration is 10-30 μ M, raw material is 1 gram: 0.2-2.0ml with the ratio of homogenizing solution;
(2) tissue homogenate is filtered, filter residue leaches repeatedly repeatedly with homogenizing solution, and merging filtrate obtains thick enzyme;
(3) thick enzyme filtrate centrifugation in two steps, second step of centrifugal speed is higher than the first step, obtains centrifugal enzyme;
(4) centrifugal enzyme is saltoutd in two steps enrichment, per step all adds vitriol at low temperatures, and saturation ratio is 30-70%, precipitates centrifugal 30 minutes, and centrifugal speed 16, obtains centrifugal enzyme precipitation by 000-20,000 rev/min;
(5) with the substrate of ALS enzyme, coenzyme, damping fluid are made enzyme group lysate and are dissolved centrifugal enzyme precipitation, the precipitation enzyme lysate that obtains can be used chromatography, and the anti-dialysis method purifying of also available dialysis obtains highly purified purifying enzyme thus, substrate Sodium.alpha.-ketopropionate in enzyme group's lysate, its concentration are 1-5mM.
2. preparation according to claim 1, the method for isolation and purification acetolactate synthestase, the preparation that it is characterized in that raw material is with Gramineae, pulse family, Amaranthaceae, Chenopodiaceae, polygonaceae, convolvulaceae, Cruciferae, Rubiaceae, composite family, Cyperaceae, Labiatae, Malvaceae, after any one seed soaks in cold water in the Alismataceae plant, be placed on equably and be covered with in the culture plate of putting nylon wire on moist fine sand or the sawdust, cover gauze, at room temperature germinate, when just sprouting, add a cover the black cloth lucifuge and cultivate.
3. preparation according to claim 1, the method of isolation and purification acetolactate synthestase, it is characterized in that the coenzyme in the homogenizing solution is to select magnesium chloride for use, thiaminpyrophosphate (TPP), with flavin adenine dinucleotide (FAD), the concentration of these three kinds of coenzyme is controlled at 0.3-1.0mM respectively, 0.3-1.0mM and 10-50 μ M scopes; Buffered soln is to select dipotassium hydrogen phosphate for use, and its concentration is controlled at 50-150mM scope; Add 10-30% glycerol again and make stablizer.
4. preparation according to claim 1, the method for isolation and purification acetolactate synthestase is characterized in that the first step centrifuge speeds is 6,000-8,000 rev/min, the second step centrifuge speeds is 14,000-20,000 rev/min, each centrifugation time is 20 minutes.
5. preparation according to claim 1, the method for isolation and purification acetolactate synthestase, the vitriol when it is characterized in that saltouing enrichment is ammonium sulfate, best saturation ratio is 50%, and described low temperature is 0-4 ℃, precipitates centrifugal, centrifugal speed 16,000-20,000 rev/min.
6. preparation according to claim 1, the method for isolation and purification acetolactate synthestase is characterized in that the coenzyme in the enzyme group lysate is selected magnesium chloride for use, concentration is 0.3-1.0mM; Buffered soln is selected dipotassium hydrogen phosphate for use, and concentration is 30-100mM; Sodium.alpha.-ketopropionate concentration is 1-5mM.
7. preparation according to claim 1, the method for isolation and purification acetolactate synthestase, the enzyme that it is characterized in that preparing group lysate can be used as leacheate in the chromatography and the dialyzate in the dialysis method.
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|---|---|---|---|---|
| CN101175849B (en) * | 2005-05-09 | 2011-04-06 | 组合化学工业株式会社 | Method for transformation using mutant acetolactate synthase gene |
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| CN101175849B (en) * | 2005-05-09 | 2011-04-06 | 组合化学工业株式会社 | Method for transformation using mutant acetolactate synthase gene |
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