CN1416470A - method for producing monoglycosidated flavonoids - Google Patents
method for producing monoglycosidated flavonoids Download PDFInfo
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- CN1416470A CN1416470A CN01806409A CN01806409A CN1416470A CN 1416470 A CN1416470 A CN 1416470A CN 01806409 A CN01806409 A CN 01806409A CN 01806409 A CN01806409 A CN 01806409A CN 1416470 A CN1416470 A CN 1416470A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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Abstract
The invention relates to a method for producing monoglycosidated flavonoids by enzymatic hydrolysis of rutinosides, using an enzyme immobilized on a carrier for the enzymatic hydrolysis. The inventive method reduces the costs for the enzymes, and simultaneously provides for a high degree of automation associated and an optimized space/time yield.
Description
The present invention relates to prepare the method for monose glycosidation flavonoid by the enzymically hydrolyse rutinoside.In this operating process, the rhamanopyranosyl of enzymatic cleavage rutinoside.
For the purposes of the present invention, rutinoside is considered to contain the compound of the aglycon composition that is connected with formula (I) group by glycosidic link.
For example, rutinoside can be the flavonoid that contains bioside unit shown in the formula (I).Can obtain rhamnosyl and/or corresponding glycopyranoside from rutinoside.Owing to contain formula (I with aglycon composition bonding
*) group but not the group of formula (I), these glycopyranosides derive from rutinoside.
For example, can obtain rhamnosyl and different quercetin from violaguercitrin.
Rhamnosyl is the natural a kind of monose that is present in many places, but in most cases it only exists with a small amount of.The important source of rhamnosyl comprises the glucosides group of for example natural flavonoid such as violaguercitrin, can be from wherein obtaining rhamnosyl by removing glucosides.Rhamnosyl is extremely valuable, for example can be used as the initiator of preparation non-natural aromatoising substance such as furaneol.
Different quercetin is the monose glycosidation flavonoid with following structural formula (II)
(Latin: the flavu=Huang), it is the dyestuff that extensively is distributed in the plant to so-called flavonoid (flavonoids), is meant for example glucosides of flavones, and flavonoid and flavones have common flavones precursor structure (2-phenyl-4H-1-chromene-4-ketone).
The aglycon composition of flavonoid is so-called aglycone.For example, different quercetin is the glucosides of aglycone quercetin (2-(3, the 4-dihydroxy phenyl)-3,5,7-trihydroxy--4H-1-chromene-4-ketone) (itself and flavones difference are to exist 5 oh groups).In different quercetin, the hydroxyl bonding that carbohydrate group glucose and quercetin are the 3rd.For example, different quercetin is named as quercetin-3-O-β-D-glycopyranoside or 2-(3, the 4-dihydroxy phenyl)-3-(β-D-glucopyranosyl oxygen base)-5,7-dihydroxyl-4H-1-chromene-4-ketone.Yet it occurs with for example trade(brand)name Hirsutrin again.
Flavonoid or flavonoid mixture are used to for example foods and cosmetics industry, and wherein their importance increases day by day.Especially, the flavonoid of monose glycosidation such as different quercetin are being absorbed as feature well by human body.
Example with natural flavonoid of bioside unit is a violaguercitrin, and it has following structural formula (III):
The same with different quercetin, violaguercitrin is the glucosides of aglycone quercetin equally, and wherein the carbohydrate group rutinose is connected with the hydroxyl of the 3rd of quercetin.Carbohydrate group in the violaguercitrin is included in the 1st with 6 glucose units that are connected be combined in terminal rhamnosyl or 6-deoxymannose unit.For example, violaguercitrin is known as quercetin-3-O-β-D-rutinoside or 2-(3, the 4-dihydroxy phenyl)-3-{[6-O-(6-deoxidation-α-mannopyranose base)-β-D-glucopyranosyl] the oxygen base }-5,7-dihydroxyl-4H-1-chromene-4-ketone.But it also is known as for example sophorine, birutan, rutabion, taurutin, rutarin, melin or rutin (rutoside).
Violaguercitrin and three molecular crystal water form pale yellow to green spicule.Anhydrous violaguercitrin has weak acid character, becomes brown under 125 ℃ and 214-215 ℃ of decomposition.Violaguercitrin is present in many plant species--usually kind, the Chinese lime as ascorbic accompaniment-for example kind in the kind of Citrus, at yellow hybrid violet, Forsythia and Acacia, various Solanum and Nicotiana belongs to, comes in the plants such as lemon flower, Hypericum, tea, 1842 it from rue (Ruta graveolens), be split into.Violaguercitrin can also obtain from the leaf of Fagopyrum and East Asia dyestuff medicine Wie-Fa (Chinese scholartree, pulse family), and they contain the violaguercitrin of 13-27%.
May expect from natural matter as prepare rhamnosyl and monose glycosidation flavonoid from the flavonoid that contains bioside unit.For example, in the present context, it is significant with corresponding glycopyranoside that rutinoside is cracked into rhamnosyl.
Disclose the enzymatic preparation of rhamnosyl in the document.EP-A0 for example, 317,033 have described the method for preparing the L-rhamnosyl, wherein contain the rhamnosyl glycosidic bond of the glucosides of the rhamnosyl that is combined in terminal position by the hydrolysis of zymetology mode.This splitting action is in the substrate that exists with suspended substance in water-bearing media usually.Yet, the selectivity extreme difference of these reactions.For example, usually to cause two kinds of monose be the mixture of glucose and rhamnosyl to the bioside structure of the carbohydrate group in the violaguercitrin.And, can form most aglycone quercetin and other by product of not expecting usually.
And the enzymatic lysis of violaguercitrin also is described in JP-A0, in 121,3293, yet, in water-bearing media, carry out these reactions and equally often have very poor selectivity.
Above-mentioned these methods are used the enzyme in the solution, the i.e. enzyme of crude substance form.These methods relate to enzyme are directly added in the reaction soln.Although these methods can be carried out, can not carry out industrial application, because this enzyme can not recycling again from reaction soln on laboratory scale.Yet only using these expensive enzymes once is uneconomic on technical scale.
Known when with enzyme with they can be used for industrial application after upholder combines.This method is known as " immobilization ".Definition according to European biotechnology alliance (1983), term " in conjunction with (or immobilization) enzyme " comprises, " with what allow its state that utilizes is again existed " all enzyme (HelmutUhlig, Technische Enzyme and ihre Anwendung, Carl Hanser Verlag, 1991, the 198 pages of Munich/Vienna).Although this advantage is arranged, immobilization only adopts in limited scope thus to all enzymic process and not all suitable.Particularly, only two kinds of desmoenzymes use on commercial size: the fixed glucose isomerase and the immobilized penicillin acid amine enzyme that is used for the cracking penicillin G that are used for glucose isomerization.Usually the desmoenzyme method can not substitute resolvase or chemical process.Usually be that enzyme or reaction conditions are not suitable for carrying out immobilization.Therefore, there is not general process for fixation, all must does to consider separately each enzyme.
For example, when rutinoside during, for the suitability of enzyme and the aqueous system of wanting of overstating can cause solubility problem as the substrate of enzymic hydrolysis.Therefore should reaction preferably use the supersaturation substrate solution, promptly the rutinoside of aerosol form carries out.Yet wherein the supersaturated solution that exists with solid of substrate has stoped the use of process for fixation.Between starting material, product particle and immobilized enzyme, lack selectivity.
Therefore, the purpose of this invention is to provide the method that preparation can be used for plant-scale monose glycosidation enzyme, realize the automatization of height, high space-time output and high productivity and selectivity simultaneously to avoid high enzyme loss.
This purpose is achieved by the method that the enzymically hydrolyse rutinoside prepares monose glycosidation flavonoid, and the enzyme that wherein is used for enzymically hydrolyse is the enzyme that has been fixed on the upholder.
We are surprised to find, although the dissolving of rutinoside is slight, it also is possible using immobilized enzyme to carry out enzymically hydrolyse.Immobilization makes can be continuously or implement this method in batches, has high efficiency with the reacting phase that uses natural enzyme to carry out than this.The place that the inventive method is especially different is that it makes and the increasingly automated possibility that becomes of whole procedure comprises the feed back of solvent and the monitoring of enzymic activity.
Fig. 1 example the continuous preparation of different quercetin from violaguercitrin, as the example of the inventive method.
The suitable rutinoside that is used for the inventive method is to contain as those of the following parent material of aglycon composition or aglycone, described parent material comprises 2-phenyl-4H-1-chromene-4-ketone, its 3rd portable type (I) group and its phenyl (except the 3rd) can by-OH or-O (CH
2)
n-H carries out single or polysubstituted, and wherein n from 1 to 8.N preferably represents 1.
-OH and/or-O (CH
2)
n-H preferably occurs on the 5th, 7,3 ' and/or 4 ' the replacement of parent material 2-phenyl-4H-1-chromene-4-ketone.The particularly preferred rutinoside that is to use formula (A):
Wherein R represents H, OH or OCH
3
Wherein on behalf of the compound of H, R be known as the kaempferol rutinoside; Wherein R represents OCH
3Rutinoside be known as the isorhamnetin rutinoside.Wherein R is that the compound of OH is called violaguercitrin.Therefore, method of the present invention can produce rhamnosyl and kaempferol glucosides, produce rhamnosyl and different quercetin, produces rhamnosyl and isorhamnetin glucosides from the isorhamnetin rutinoside from violaguercitrin from the kaempferol rutinoside.
Especially preferably use the rutinoside violaguercitrin.
The initiator that is used for the inventive method can be the pure rutinoside or the mixture of rutinoside.This rutinoside also can be mixed with other flavonoid of not negative impact reaction or the residue of producing from rutinoside.
The enzyme that is used for the enzymically hydrolyse rutinoside can be the routine hydrolysis enzyme that can go out the rhamnosyl group from the rutinoside cracking.The preferred lytic enzyme that uses available from Penicillium decumbens bacterial strain.Especially preferably use alpha-L-Rhamnosidase, because they show the selectivity of height to the hydrolysis of rhamanopyranosyl.Suitable alpha-L-Rhamnosidase for example has hesperidinase, naringinase and is described in (1973) J.Biochem. such as Kurosawa, the 73rd volume: those among the 31-37.Very preferably use hesperidinase.
The rutinoside and the enzyme that are used for the inventive method all can obtain with commercial form.Also can separate or prepare this initiator and enzyme by well-known process.
This enzyme is fixed on the suitable upholder.For this purpose, can use conventional upholder, for example silica gel such as commercial spherical silica gel or commercial broken silica gel are as Lichrosorb
, Lichroprep
, Lichrospher
, and Trisoperl
And commercial poly upholder, as Eupergit
, Fractogel
, Fractogel epoxy especially
And Fractoprep
Silica gel can be considered to preferred support material.
Perhaps, can use magnetic-particle as upholder.Preferably these are the support material with magnetic core.This core is sealed with inorganic oxide usually.This inorganic oxide is silica gel preferably.The example of these magnetic supports comprise MagneSilTM (Promega Corp., Madison, Wisconsin, US), MagPrep
TM(Merck) and AGOWAmag
TM(AGOWA GmbH, Berlin, DE).Perhaps, the used magnetic upholder can be magnetic glass particle suspensions (for example, MPG (CPG Inc., Lincoln Park, New Jersey, USA)) and pigment (the Microna Matte for example that contains magnetite, Mica Black, Colorona Blackstar (all from Merck)).That especially be fit to is atresia magnetic-particle (for example MagPrep), does not cause enzymic activity to be subjected to violent destruction because they can not cause hole plug.
The enzyme upholder has following feature usually: the granular size of upholder is preferably from 0.005 to 1mm, more preferably from 0.01 to 0.5mm.Bore dia is usually from 10 to 4000nm, especially preferred 30 to 100nm bore dia.Enough big bore dia will guarantee that this enzyme can be loaded on the upholder and do not lose activity.The particle surface area preferably from 40 to 100m
2/ g, and pore volume is preferably selected from 0.5 to 3mL/g scope.In some cases, the very big bore dia of from 2 to 20 μ m may be suitable.
Enzyme can carry out combination by covalent linkage or absorption.Usually, preferably covalently bonding.The example of covalent coupling comprises that epoxidation, carbodlimide method, silanization, von Braum reaction, glutaraldehyde cross-linking or xylyl chlorine method are (referring to bio-transformation and enzyme reaction, A.S.Bommarius, Biotechnology (the 2nd edition), the 3rd volume, the 427-465 page or leaf, G.Stephanopoulos compiles, VCH Weinheim, Germany 1993; D.R.Walt etc., Trends in Analytical Chemistry, the 13rd the 10th phase of volume, 1994; N.H.Park, H.N.Chang, J.Ferment.Technol. the 57th volume (4), 310-316,1979; M.Puri etc., Enz.Microb.Technol., 18,281-285,1996; And H.-Y.Tsen, J.Ferment.Technol.62 (3), 263-267,1984).In order to implement present method, must do the surface to upholder with suitable functional group and modify.Can transform by the functions of use monomer copolymerizable or by base polymer functional group is added on the upholder.Especially preferred use amino, aldehyde radical or oxirane ring or glycol carry out finishing.Then can be with enzyme and these group covalent bondings.
This enzymically hydrolyse carries out in suitable reactor.Commercial tower especially is fit to carrying out continuously of the inventive method.When working, can use the tower that for example is used to preparation property HPLC with small-scale.Reactor, especially tower should show high hydraulic pressure efficient.This can quantize by the quantity of theoretical stage.Therefore guarantee advantageously that fixture surface and material solution have tight the contact so that effectively utilize enzyme and obtain high productivity.Above-mentioned preparation HPLC post satisfies these requirements, can also equip suitable technical tool and peripherals (pump, valve, control tool) to it.And advantageously developed detection means such as UV or the RI detection means that is used for this purpose, if so that can measure and control the degree of conversion that reaction reaches automatically during expectation.
If continuous operation mode uses the magnetic support material, then usually use design to make magnetic-particle keep the tubular reactor of stable suspersion thing form, for example in flow duct, produce the solenoid in the magnetic line of force of the magnetic flux basic homogeneous magnetic field (helmholtz magnetic field) parallel with flow direction.In the stable fluidized-bed reactor of this magnetic (magnetic stabilization fluid bed (MSFB)), than in conventional fluidized-bed or fluidisation column (also being applicable to these purposes), obtaining higher in fact flow velocity.This technology also can be used for promoting the catalyzed reaction of adhesive reaction medium.
When present method is undertaken by batch mode, conventional containers, the conventional containers that is preferably provided with agitator is fit to.Therefore, the round-bottomed flask of being furnished with agitator can be used in the time of on a small scale, stirred pot can be used when extensive.
Before reaction, in the usual way fixture is packed in the reactor.
Rutinoside to be transformed is added reactor with the form of solution or suspended substance usually, and for example post or tower are as fixed bed column.If reactor used is fixed-bed reactor, then this rutinoside solution should not contain solid matter fully.Advantageously in jar, rutinoside is dissolved in the solvent in advance, preferably by stirring and/or heating and realize, so that obtain best solubleness.If required, can also pre-filtering solution so that remove any solid matter.Solvent preferably aqueous system so that guarantee enzymic activity and sex change that prevention is possible.In order to guarantee the dissolving of rutinoside, can add other solvent.Method preferably of the present invention is carried out in the solvent mixture of water and at least a organic solvent.
The organic solvent that replenishes comprise can with water miscible and with water can not be miscible organic solvent.
The appropriate solvent that is used for the inventive method has nitrile such as acetonitrile, amides such as dimethyl formamide, ester class such as acetic ester, especially methyl acetate or ethyl acetate, alcohols such as methyl alcohol or ethanol, ethers such as tetrahydrofuran (THF) or methyl tertiary butyl ether and hydrocarbon such as toluene.
The inventive method is preferably carried out when one or more organic solvent such as ethyl acetate, methyl alcohol, ethanol, methyl tertiary butyl ether or toluene exist.The inventive method is very preferably carried out in the presence of one or more ethyl esters especially methyl acetate and water.
For the inventive method, the suitable proportion of water and organic solvent is 1: 99 to 99: 1 a volume ratio.The inventive method is preferably used 20: 80 to 80: 20 water and organic solvent volume ratio, and especially 50: 50 to 70: 30 volume recently carries out.
The rutinoside amount that is present in the inventive method in solvent or the solvent mixture is controlled by the solubleness of rutinoside in this solvent or solvent mixture.When rutinoside dissolved easily, the inventive method can obtain best enforcement.Reason preferably uses supersaturated solution to operate for this reason.The amount of rutinoside is from 0.001 to 5g/L in usual solvents or the solvent mixture, preferably from 0.05 to 2g/L, more preferably from 0.1 to 1.5g/L.
The ratio of rutinoside and fixture or enzyme depends on life-span and during immobilization form its activity of this enzyme in tower or post.
Reaction is carried out under 15 ℃ to 80 ℃ temperature usually.Preferably from 30 ℃ to 60 ℃ temperature, 40 ℃ to 50 ℃ temperature is particularly advantageous in to be avoided may destroying the high-dissolvability that guarantees rutinoside simultaneously again to any of enzyme.
Spend when low when reaction temperature, the enzymic activity of reduction can cause reaction to carry out with unsuitable deferred reaction speed.In addition, the solubleness of rutinoside also can be reduced to a certain degree, so that needs unnecessary a large amount of solvents.On the other hand, if temperature of reaction is too high, sex change and so inactivation can take place as protein in enzyme.
When the inventive method is at high temperature carried out, can be equipped with temperature-control device to reactor.Temperature-control device commonly used contains heating coil system or double-jacket.And, advantageously rutinoside to be transformed, especially rutinoside solution are being entered advance trip temperature control of reactor.For this purpose, common taking-up rutinoside solution from remain on the temperature required controlled temperature tower down of reaction.Perhaps, can make solution to be added by the heating flexible tubing so that make its temperature before entering reactor reach expected value.Described heating can also prevent the rutinoside crystallization.
The suitable pH that is used for the inventive method is pH3 to 8.Preferably, the inventive method is carried out under pH3 to 7, especially pH3 to 6.Yet preferred pH can change in specific limit according to used enzyme.For example, when using hesperidinase, very preferred 3.8 to 4.3 pH.
Preferably, present method is carried out in the mode of using buffering system to regulate pH.In theory, all buffering systems commonly used that are suitable for being provided with above-mentioned pH all are available.Yet, preferably use the aqueous citric acid buffering salt.
Can put into the reactor that contains fixture with the rutinoside mixture that solution or aerosol form exist, so that carry out enzymically hydrolyse.This reaction can be carried out continuously or in batches.
If reaction is undertaken by batch mode, then usually the rutinoside suspended substance is put into reactor.Degree of conversion is decided by the amount of rutinoside and fixture.Usually, the ratio of rutinoside and fixture is 100: 1 to 1: 1000, preferably from 10: 1 to 1: 100, more preferably from 1: 1 to 1: 20.The ratio of fixture and suspended substance cumulative volume is generally 1: 1000 to 1: 1, preferably from 1: 100 to 1: 2, more preferably from 1: 50 to 1: 5.The residence time in the reactor normally is 1 hour to 10 days, preferred 8 hours to 4 days, and more preferably 1 to 2 day.
When reaction is carried out continuously, by suitable pump rutinoside solution is transported consistently by reactor usually, especially tower or MSFB reactor.By flow velocity suitably is set, can obtain any desired degree of conversion.Under the normal circumstances, based on the blank pipe cross section of tower or post, used flow velocity is 0.001 to 1mm/s.
The activity of enzyme is found along with the time reduces in this system.Therefore, must regularly completely or partially change fixture.In order to remedy the loss of enzymic activity, advantageously detect and estimate degree of conversion by UV or RI, forming with box lunch can be by pumping control so that this is eliminated when changing.
After reaction soln leaves reactor, can separate obtained product.Reaction is when finishing, and reaction mixture mainly is made up of the additive such as the buffer substance of the monose glycosidation flavonoid of solvent, unconverted rutinoside, rhamnosyl, expectation and possible other.Usually precipitation takes place and little by little assembles to be solid in monose glycosidation flavonoid when reaching solubility limit.
Using the magnetic support material to carry out under the situation of batch operation, can after finishing, reaction from the product suspended substance, isolate desmoenzyme in simple mode by the magnetic resolution device.On laboratory scale, the strong permanent magnet of plate-like form can be used for this purpose.Yet existing exploitation is used for the big separator of very various industrial application, and their great majority are pressed HGMS principle (high gradient magnetic separation) operation.This equipment can be made up of the perpendicular flow pipe that for example contains thin stainless steel metal tow.Suitably the solenoid of placing produces the high magnetic flux gradient along these wires, and mode can be separated even the nano sized particles of nanometer scale very effectively whereby.If magnetic-particle is super paramagnetic, promptly when not having the external magnetic field to exist, do not show residual magnetization, then after closing magnetic field, they easily and fully can be removed from separator by the water repetitive scrubbing.
The reaction product of separating expectation by the common method that relates to the routine inspection device.
Preferably, by the concentrating and precipitating product.If it is solvent comprises the solvent mixture that contains at least a organic solvent, then preferred by this organic solvent of distillation removal under reduced pressure.Crystalline monose glycosidation flavonoid, as siphon under reduced pressure or filtration or by centrifugal to sedimentary crystal, is separated from remaining reaction mixture usually by the Solid-Liquid Separation method.Then, preferably wash this solid with water, dry then.
Perhaps, can at first leach the whole reactor content.Dissolved solvent or buffer solvent mixture process contain the filter cake of product therein with product then.Extracting goes out reaction product from filter cake in this operating process.
In batch operation, be left insoluble catalyzer, i.e. fixture in this mixture.For solvent or buffer solvent mixture necessity is that this enzyme is not had destruction.For example naringinase or hesperidinase no longer have activity or only have the part original activity in some buffer solvent mixture or under weak basic condition to have been found that desmoenzyme, if but in the buffered soln of pH4 to 6, carefully washed this enzyme afterwards, in fact this activity could be recovered completely; Therefore, loss of activity herein only is temporary transient, is not equivalent to the irreversible denaturation of enzyme.
For this method, when especially implementing this method under high slightly temperature, very the extractant of Shi Heing is the tetrahydrofuran (THF) buffer mixture, preferably has those of tetrahydrofuran (THF) of 10-25% content.Other suitable extracting composition has for example 1-propyl alcohol, 2-propyl alcohol, 1,4-diox and methyl acetate.Product can remove the aqueous solution that will contain product then of desolvating by the distillation of reducing pressure down and be cooled to 0 ℃-10 ℃ and very easily reclaim from extract and obtain.This reaction product in mother liquor with the extreme high purity crystallization.
As substituting of solvents/buffer mixture, can use weak ammonia or soda solution as extractant because reaction product have can deprotonation in weak alkaline medium phenolic hydroxyl group; The anionic reactive product shows goodish solubleness, but it also very is easy to oxidation, and it is yellow certainly to brown variable color gradually that this shows as extract apparently.Therefore, the method for this variation must be operated very apace, promptly should finish this extracting in preferred 20 minutes to the 2 hours time at 10 minutes to 6 hours.This operation is preferably carried out under the protective gas layer.
In addition, use weakly alkaline extractant such as the basic metal of acetate, oxalic acid, citric acid, phosphoric acid, boric acid or carbonic acid or the aqueous solution of ammonium salt, or the aqueous solution of alkyl amine, piperidines or pyridine is handled the forfeiture that can not cause enzymic activity.Reaction product can and be cooled to 0-10 ℃ by the acidifying extract and precipitate once more.
When using pure rutinoside, the purity of the monose glycosidation flavonoid that obtains is generally more than 94%.In order to be further purified, can be for example from appropriate solvent, for example from water or comprise toluene and the solvent mixture of methyl alcohol or water and methyl acetate the recrystallization end product.
In order to keep the economic worth of the inventive method, preferably reclaim reacted residual solvent.This recirculation is carried out in continuous and automatic mode usually.Commercial evaporation unit with suitable control device can be used for this purpose.If solvent for use is the solvent mixture of water and at least a organic solvent, it is impossible then immediately distillment being used for present method once more usually, because the ratio regular meeting of solvent changes because of the distillation of organic solvent.Utilize automated quality control and proofread and correct, can be by the circulating solvent solvent ratios of establishing expectation once more again suitably.
And the concentrated film that can comprise is handled or Nano Filtration.In these methods, can isolate solvent mixture and not change its composition.
Following examples are intended to illustrate the present invention.Yet they never are construed as limiting.Embodiment 1 fixing hesperidinase on the silica gel upholder
1) adjusting of fixing preceding support surface
1.1) character of support material
Silica gel LiChrospher
Diameter=15-40 μ m
Bore dia=300
Granule surface area=80m
2/ g
Pore volume=0.73mL/g
Density=2g/mL
1.2) activation of silica gel
250g silica gel and competent HCl (7%) are mixed in capacity is the flask of 1L, place and spend the night so that wetting silica gel.
Wash the silica gel suspended substance up to no longer containing muriate with softening water then.For this reason, must be in each washing back with nitric acid and Silver Nitrate test supernatant liquor.Because the character of silica gel particle, washing is carried out in having the ceramic funnel of about 24scm diameter.
1.3) carry out finishing with amino
Capacity 2L and be furnished with reflux exchanger and the three-necked flask of dropping funnel in, acid-treated silica gel and competent water are mixed to reach the degree that can stir.After thoroughly mixing under the room temperature, comprise the 1mmol/g upholder (250g silica gel needs 135mL solution) of 3-TSL 8330 with the speed dropping of 5 of about per seconds to the silica gel suspended substance.90 ℃ of stirring suspension things are 2 hours then.Use ice-cooled this suspended substance then.
Must check the remaining 3-TSL 8330 of possibility in the supernatant liquor by reading pH.Keep constant with softening water washing suspended particle up to pH.
1.4) with glutaraldehyde bag quilt
Concentration by the 1mmol/g upholder adds glutaraldehyde (GDA) (the 250g upholder needs 13mL 50%GDA strong solution) in obtaining silica gel suspended substance.In the flask of capacity 1L, under room temperature, shook this suspended substance (adding small amount of water) 2 hours.Suspended substance becomes yellow during beginning, and it is a garnet during EP (end of program).
By precipitin reaction, the supernatant liquor that each washing back is obtained carries out remaining glutaraldehyde detection with dinitrophenylhydrazine.Careful washing suspended substance is negative up to test result.
2) fixing
2.1) hesperidinase
Proteinic first kind of interpolation
In 500mL Citrate trianion/phosphate buffered liquid mixture (pH6.0), stir the 3.8g hesperidinase.In order to increase dissolving, add 300 μ l tensio-active agents (Tween20).Filter this enzyme solution then.
In the flask of capacity 1L, will about 230g press 1.4) the silica gel suspended substance of described acquisition mixes with this enzyme solution.Shook enzyme upholder suspended substance then under the room temperature about 40 hours.
Proteinic second kind of interpolation
Stir about 0.76g hesperidinase (Amano) in the 120mL Citrate trianion that contains 60 μ l tensio-active agents/phosphate buffered liquid mixture (pH6.0), subsequent filtration.
In the flask with the above-mentioned capacity 1L of this enzyme solution impouring, shake this enzyme solution under the room temperature.
2.2) BSA (being used for separation detection)
In 100mL Citrate trianion/phosphate buffered liquid mixture (pH6.0), stir 0.3BiomexBSA (bovine serum albumin powder).In the flask of capacity 0.5L, will about 20g press 1.4) the silica gel suspended substance of described acquisition therewith protein soln mix, and add 60 μ l ProClin300.
3) measure proteinic amount and activity
3.1) proteinic amount (the protein mg number of every mL)
Measure Protein content in the solution by the Bradford method of testing.Carry out standard test.This by with 20 μ l sample mix in 1mL Bradford dye reagent (1: 5 dilution), 595nm reads that photometric quantity carries out after 15 minutes then.
Minimum protein concn need use micromethod.This comprises that with the 0.8mL sample mix 595nm reads photometric quantity after 15 minutes then in 0.2mL Bradford dye reagent (spissated).
3.2) activity
By the reaction of solution and alternative substrate, measure the activity of solution.
For each duplicate samples, use:
88 μ l Citrate trianions/phosphate buffered liquid mixture (pH=4.0)
100 μ l samples
20 μ l samples
20 μ l substitute substrate: p-nitrophenyl-alpha-L-rhamnoside (rhamnosyl
The glycosides enzymic activity)
P-nitrophenyl-α-L-glucoside (glucosidase activity)
This 1mL solution is mixed in the Eppendorf reaction tubes.40 ℃ of difference incubations mix per 100 μ l reaction mixtures after 2 minutes and 5 minutes in the flask with 1mL 1M soda solution.Then in the concentration of 400nm by the photometer measurement p-NP.Change calculated activity from time per unit p-NP concentration.
The enzymic activity unit of being expressed as (U) (the μ mol number of the substrate that=per minute transforms)
4) result
Protein content and activity value
(free hesperidinase)
1Hsp0: proteinic first kind is added Hsp1: proteinic second kind is added sample 1-6: supernatant liquor
| Sample 1 | Proteinic amount (standard test) | Active (substituting substrate) | |||
| ????mg | The mg/ml sample | ????U | The U/ml sample | U/mg albumen | |
| ????Hesp0 | ????740 | ????1.48 | ????4800 | ????96 | ????65 |
| ????1 | ????15 | ????0.03 | ????7 | ????0.014 | ????0.47 |
| ????2 | ????20 | ????0.04 | ????4 | ????0.008 | ????0.2 |
| ????Hesp1 | ????326 | ????2.72 | ????11400 | ????95 | ????35 |
| ????3 | ????56 | ????0.09 | ????1450 | ????2.33 | ????26 |
| ????4 | ????28 | ????0.045 | ????361 | ????0.58 | ????13 |
| ????5 | ????22 | ????0.035 | ????95 | ????0.15 | ????4.3 |
| ????6 | ????20.5 | ????0.033 | ????11 | ????0.018 | ????0.54 |
Protein content and activity value
(fixed hesperidinase)
Embodiment 2 uses fixture to produce different quercetin by enzymically hydrolyse from violaguercitrin
| *The protein content ≈ 1 of upholder, 045g bonded protein *Be positioned on the upholder for 4.7mg protein/g upholder *2% protein that adds does not have combination |
At capacity 4.5m
3Heating and stirring tank in (1) put into 3200L softening water and 800L 1-propyl alcohol.By steam-in (2) with mixture heating up to about 50-60 ℃.In solution, add 8000g violaguercitrin, DAB under the vigorous stirring.Stir the mixture and dissolve fully up to violaguercitrin.Monitor pH by recycle pump and online pH instrument (3a) then, and where necessary pH is set in 4.0-4.5 (use H
3PO
4And NaOH).Can take sample to be used to check the measurement of purpose and concentration by manual valve (4).
Add this solution by bag filter (5) and pipe filter (6) to piston-type dosing pump (7), with initial action.Bag filter is responsible for stoping the not solvent components of the overwhelming majority, and clean this solution to the 0.2 μ m fineness of pipe filter filter.
The flexible pipe transportation solution of piston-type dosing pump (7) by adding, the solution temperature when it will enter pillar by thermometer is adjusted to 40 ℃, and (100 * 400mm) speed is 1L/min to flow into pillar (9).Pillar contains the 1.5kg fixture.Because electrically heated flexible pipe can not make solution cooling, therefore the temperature in the stirred pot (1) is set to certain value, cause and be no more than 40 ℃ temperature so that under the pump delivery rate of maximum, flow to the cooling that takes place in the process on the way of pump.
Can take sample from solution by manual valve (10) after the solution diafiltration is by pillar, means can the off-line measurement temperature and the degree of conversion that reaches of reaction whereby.If the degree of conversion of measuring is lower than requirement, then suitably reduce the output of pump.
Finish fully by the pillar afterreaction at solution, so that this solution can be passed to collection tube (11).Solution reduces about 10-20% by condenser (12) volume there.By this mode, the content of propyl alcohol reduces considerably, this means that the solubleness of different quercetin also suddenly descends.Cooling subsequently makes solubleness further descend so that the product precipitation also can be separated in bag filter (13).From then on, product being passed to loft drier (14) dewaters.Mother liquor and in stirred pot (1), use again through distillatory condensation product recirculation together.
Embodiment 31. uses aldehyde group modified silica gel particles and fixing naringinase on this particle
In the container that can seal, 400mL 10% dense HCl is poured onto 250g silica gel (LiChrosper Si300 for example, Merck, Darmstadt) on, make this container degassing 10 minutes by ultrasonic afterwards, and placed room temperature 24 hours.Leach then silica gel and with several rise softening water washing up to pH greater than 4.5 and in filtrate, no longer can detect chlorion (use AgNO
3Acetic acid solution carry out spot reaction).
Acid-treated wetting silica gel placed capacity 4L and be furnished with the three-necked flask of precession glassed agitator, reflux exchanger and 100ml dropping funnel, stir pulping together with the 3L softening water therein.Stir down through 15 minutes by dropping funnel add the 100mL TSL 8330 (ABCR, Karlsruhe).Heat suspended substance then and stirred 90 minutes at 90 ℃.Filter refrigerative suspended substance and the washing of the each 1L of use softening water 8 times.
Capacity 4L and be furnished with the precession glassed agitator and the three-necked flask of 100ml dropping funnel in, amino activatory silica gel is suspended in by in the ultrasonic 3L water that has outgased; Add several 2M acetate pH is reduced to 8.0.Dripped through 1 hour then 100ml 50% glutaraldehyde strong solution (Merck, Darmstadt), this suspended substance of room temperature restir 2.5 hours.Filter activatory silica gel once more, and wash up in wash water, no longer detecting glutaraldehyde (using the sulphuric acid soln of 2,4 dinitrophenyl hydrazine to carry out spot reaction) with ice-cold softening water.
Using the precession glassed agitator to stir in the flask of capacity 4L is suspended in aldehyde group modified silica gel in the 500ml softening water.(Sigma Deisenhofen) is dissolved in the 2.5L0.25M phosphate buffered saline buffer (pH8.0) with the 13g naringinase.Add this enzyme solution to the silica gel suspended substance, and stirring at room 96 hours.Leach fixture then and at first use 50mM citrate buffer (pH4.0) washing then for several times with the 0.2M sodium chloride solution.(Sigma Deisenhofen) measures the rhamnosidase activity of fixture as substrate to use p-nitrophenyl-L-α-pyrans rhamnoside by the Kurasawa method.2. hesperidinase is fixed on the EupergitTMC
(Rohm Weiterstadt) mixes with 300mL 0.8M potassium phosphate buffer (pH8.5), and placed 30 minutes with 50g Eupergit in having the 500ml vial of screw-cap.Add 5.0g hesperidinase (Amano) then, on impeller, stirred this batch material 120 hours under the room temperature.Leach Eupergit by sintered glass filter, and at first for several times, use 0.1M citrate buffer (pH4.0) washed twice of each 1L afterwards with the washing of 0.2M sodium chloride solution.(Sigma Deisenhofen) measures the rhamnosidase activity of fixture as substrate to use p-nitrophenyl-L-α-pyrans rhamnoside by the Kurasawa method; It is 15U/g based on dried fixture, or is 4.2U/g based on wetting fixture.3. in agitator tank reactor, use the hesperidinase that is fixed on the Eupergit that violaguercitrin is converted into different quercetin, use tetrahydrofuran (THF)/buffer solution mixture extract product subsequently
In the round-bottomed flask of capacity 2000ml, 40 ℃ use the precession glassed agitator stir together 1000ml 50mM citrate buffer (pH4.0), 100g (weight in wet base) be fixed on the naringinase of the active 4.2U/g on the Eupergit and 10g violaguercitrin (Merck, Darmstadt); Analyze the METHOD FOR CONTINUOUS DETERMINATION degree of conversion by HPLC.After 96 hours, leach reactor content altogether by the Buchner filter.Filter cake is reloaded in this round-bottomed flask, and 40 ℃ were stirred 30 minutes in the mixture of 400ml 50mM citrate buffer (pH4.0) and 100ml tetrahydrofuran (THF), most different quercetin dissolvings in this operating process.This mixture of heat filtering and with 500mL damping fluid/tetrahydrofuran compound extracting filter cake 30 minutes once more.After the filtration, with twice different quercetin extract and for the first time filtrate merge, remove tetrahydrofuran (THF) by rotatory evaporator then.In order to precipitate this product fully, this moisture different quercetin solution is cooled to 4 ℃.Filtration also obtains 5.8g product output after the drying in moisture eliminator, it is made up of 98% different quercetin and 2% violaguercitrin.
Wash this wetting Eupergit once with cold tetrahydrofuran (THF) buffer solution mixture, use 50mM citrate buffer (pH4.0) repeated washing then, up to the smell that only can faintly differentiate tetrahydrofuran (THF).Enzymic activity still has 3.6U/g, and this has equaled to lose 14% activity.4. in agitator tank reactor, use the hesperidinase that is fixed on the Eupergit that violaguercitrin is converted into different quercetin, use the ealkaline buffer extract product subsequently
In the round-bottomed flask of capacity 2000ml, 40 ℃ use the precession glassed agitator stir together 1000ml 50mM citrate buffer (pH4.0), 100g (weight in wet base) be fixed on the naringinase of the active 4.2U/g on the Eupergit and 10g violaguercitrin (Merck, Darmstadt); Analyze the METHOD FOR CONTINUOUS DETERMINATION degree of conversion by HPLC.After 96 hours, leach reactor content altogether by the Buchner filter.Wet filter cake is reloaded in this round-bottomed flask, and stirring at room is 5 minutes in 300ml 50mM citrate buffer (pH10.0), produces deep yellow in this operating process middle part differentiation quercetin dissolving.Filter suspended substance and immediately with carbonate buffer solution this filter cake of extracting once more.After 7 extractings circulated altogether, essentially no color of Eupergit and different quercetin were dissolved in fact fully.Extract is merged, and the hcl acidifying of careful usefulness dilution near 3, is cooled to mixture 4 ℃ up to pH then.Filter and after the drying, obtain the product of output 4.9g in moisture eliminator, it comprises 98% different quercetin and 2% violaguercitrin.
Wash this wetting Eupergit twice with the 50mM citrate buffer, can be used for other reaction once more then.Enzymic activity still has 3.9U/g, and this has equaled to lose 7% activity.
Embodiment 41. uses aldehyde group modified magnetic silica particle and naringinase is fixed on this particle
At capacity 1L and be furnished with in the three-necked flask of precession glassed agitator, dropping funnel and reflux exchanger, put into 30g magnetic silica particle (MagPrep, Merck, Darmstadt) suspended substance in 600mL water.Under agitation dripped through 30 minutes the 20mL aminopropyltriethoxywerene werene (ABCR, Karlsruhe) and the mixture of 20ml Virahol.Stirred 1 hour with mixture heating up to 85 ℃ and under this temperature then.After the cooling, suspended substance is placed beaker, make particle aggregation at container bottom, then supernatant liquor is inclined to gently by strong permanent magnet.Use these particles of softening water repeated washing to keep constant up to the pH of washing lotion.Then these particles are suspended in the 600ml water again, and add several acetate the pH value is adjusted to about 8; After adding 24ml 50% glutaraldehyde strong solution, the stirring suspension thing is 4 hours under the room temperature, then with the softening water washing granule up in washing lotion, no longer detecting glutaraldehyde (sulphuric acid soln of use 2,4 dinitrophenyl hydrazine carries out spot reaction).
In the round-bottomed flask of capacity 1L, aldehyde radical deutero-particle is suspended in the 600mL 0.2M potassium phosphate buffer (pH9) again.(Sigma behind 100mL50mM sodium chloride solution Deisenhofen), used precession glassed agitator stirring at room mixture 2 days to add the 1g naringinase.Also at first use 50mM citrate buffer (pH4.0) repeated washing then by the permanent magnet separating particles then with the 0.2M sodium chloride solution.By Kurasawa method (Kurosawa, Ikeda, Egami, J.Biochem.73,31-37 (1973): (Sigma Deisenhofen) measures the rhamnosidase activity of fixture as substrate the alpha-L-Rhamnosidase of angle Turbo cornulus (Turbo eornutus) liver and Aspergillusniger (aspergillus niger)) to use p-nitrophenyl-L-α-pyrans rhamnoside; It is 162U/g.2. use oxirane ring modified magnetic silica dioxide granule and fixing naringinase on this particle
At capacity 1L and be furnished with in the three-necked flask of precession glassed agitator, dropping funnel and reflux exchanger, put into 30g magnetic silica particle (MagPrep, Merck, Darmstadt) suspended substance in the 600mL50mM sodium acetate solution.Stir dripped through 30 minutes down 20mL (3-epoxypropoxy) Trimethoxy silane (ABCR, Karlsruhe) and the mixture of 20ml Virahol.Stirred 1 hour with mixture heating up to 85 ℃ and under this temperature then.After the cooling, suspended substance is placed beaker, make particle aggregation at container bottom, then supernatant liquor is inclined to gently by strong permanent magnet.Use these particles of softening water repeated washing to keep constant up to the pH of washing lotion.In order to measure the amount of oxirane ring, have the sample of about this material of 0.5g with the methyl alcohol repeated washing, then in loft drier about 70 ℃ be dried to constant weight.By Pribyl method (Pribyl, Fresenius Z.Anal.Chem.303,113-116 (1980): Bestimmung of Epoxydendgruppen in modifiziertenchromatographischen Sorbentien and Gelen) measure this oxirane ring, obtain the value of 250 μ mol/g.
Dense suspended substance with 150ml 20% (w/v) epoxide deutero-magnetic-particle in the round-bottomed flask of capacity 1L mixes with 350mL 1M potassium phosphate buffer (pH9.0).(Sigma behind 15mL 50mM sodium chloride solution Deisenhofen), uses the precession glassed agitator to stir the mixture 16 hours for 40 ℃ to add the 1.5g naringinase.Then by the permanent magnet separating particles, and at first use 50mM citrate buffer (pH4.0) repeated washing then with the 0.2M sodium chloride solution.(Sigma Deisenhofen) measures the rhamnosidase activity of fixture as substrate to use p-nitrophenyl-L-α-pyrans rhamnoside by the Kurasawa method; It is 102U/g.3. use carboxyl modified magnetic silica particle and fixing naringinase on this particle
At capacity 1L and be furnished with in the three-necked flask of precession glassed agitator, dropping funnel and reflux exchanger, put into 30g magnetic silica particle (MagPrep, Merck, Darmstadt) suspended substance in 600mL water.Under agitation dripped through 30 minutes 28mL 3-(triethoxysilyl) propyl group succinyl oxide (ABCR, Karlsruhe) and the mixture of 28ml Virahol, then by dripping the pH regulator to 9.0 of 10% soda-lye with reaction mixture.Stirred 2 hours with mixture heating up to 80 ℃ and under this temperature then.Controlling this pH in this operating process at regular intervals also proofreaies and correct by adding alkali when needed.After the cooling, suspended substance is placed beaker, make particle aggregation at container bottom, then supernatant liquor is inclined to gently by strong permanent magnet.Use softening water to wash these particles three times, use the washing of 2M acetic acid solution to use the softening water repeated washing to keep constant once, then up to the pH of washing lotion.
(Sigma, 150mL 50mM sodium chloride solution Deisenhofen) mixes with the dense suspended substance of 150ml 20% (w/v) carboxyl deutero-magnetic-particle and 300mL 0.4M potassium phosphate buffer (pH5.0) and 1.5g naringinase in the round-bottomed flask of capacity 1L.Adding 8ml EDC (N-ethyl-N '-(3-dimethylaminopropyl) carbodiimide hydrochloride, Merck, Darmstadt) after 1% strong solution in water, this mixture of stirring at room 20 hours.Also at first use 50mM citrate buffer (pH4.0) repeated washing then by the permanent magnet separating particles then with the 0.2M sodium chloride solution.(Sigma Deisenhofen) measures the rhamnosidase activity of fixture as substrate to use p-nitrophenyl-L-α-pyrans rhamnoside by the Kurasawa method; It is 71U/g.4. use aldehyde group modified magnetic pigments, mica and fixing hesperidinase on these particles
At capacity 1L and be furnished with in the three-necked flask of precession glassed agitator, dropping funnel and reflux exchanger, put into 30g magnetic pigments, mica (" Colorona Blackstar Green ", Merck, darmstadt) suspended substance in 300mL water.Under agitation dripped through 30 minutes the 20mL aminopropyltriethoxywerene werene (ABCR, Karlsruhe) and the mixture of 20ml Virahol.Stirred 1 hour with mixture heating up to 852 and under this temperature then.After the cooling, suspended substance is placed beaker, make particle aggregation at container bottom, then supernatant liquor is inclined to gently by strong permanent magnet.Use these particles of softening water repeated washing to keep constant up to the pH of washing lotion.Then these particles are suspended in the 300ml water again, and add several acetate the pH value is adjusted to about 8; After adding 25ml 50% glutaraldehyde strong solution, the stirring suspension thing is 4 hours under the room temperature, then with the softening water washing granule up in washing lotion, no longer detecting glutaraldehyde (sulphuric acid soln of use 2,4 dinitrophenyl hydrazine carries out spot reaction).
In the round-bottomed flask of capacity 1L, 30g aldehyde radical deutero-pigments, mica " ColoronaBlackstar Green " is suspended in the 300mL 0.2M potassium phosphate buffer (pH7.5) again.After adding the 20mL 0.2M potassium phosphate buffer (pH7.5) of 1g hesperidinase (Amano), used precession glassed agitator stirring at room mixture 3 days.Also at first use 50mM citrate buffer (pH4.0) repeated washing then by the permanent magnet separating particles then with the 0.2M sodium chloride solution.(Sigma Deisenhofen) measures the rhamnosidase activity of fixture as substrate to use p-nitrophenyl-L-α-pyrans rhamnoside by the Kurasawa method; It is 10U/g.5. in agitator tank reactor, use the fixed naringinase violaguercitrin to be converted into different quercetin and to use permanent magnet to separate the magnetic bio catalyzer
In the multiple wall stirred reactor of capacity 500mL, 40 ℃ stir together 400mL 50mM citrate buffer (pH5.0), 20g be fixed on the naringinase of the active 102U/g on the magnetic silica particle and 10g violaguercitrin (Merck, Darmstadt); Analyze the periodic measurement degree of conversion by HPLC.After 24 hours, pump into reaction content in the beaker and use board-like magnet (Bakker 200mT) is collected in container bottom with catalyzer.Leach supernatant liquor by pump in a vacuum immediately, use the damping fluid of each 100mL to wash these magnetic-particles for several times then, so that wash the different quercetin of the viscous solid of last remnants.Filter the different quercetin of collecting, and with the washing of a small amount of frozen water for several times, dry in moisture eliminator then.Output is 6.5g.HPLC analyzes the composition that provides 96% different quercetin, 2% quercetin and 2% violaguercitrin.The activity of fixture still has 92U/g after conversion.This has equaled to lose 10% activity.6. in agitator tank reactor, use the fixed naringinase violaguercitrin to be converted into different quercetin and to use electromagnetic separator to separate magnetic bio catalyzer (Fig. 1)
In the multiple wall stirred reactor of capacity 500mL, 40 ℃ stir together 300mL 50mM citrate buffer (pH5.0), 10g be fixed on the naringinase of the active 102U/g on the magnetic silica particle and 5g violaguercitrin (Merck, Darmstadt); Analyze the continuously measured degree of conversion by HPLC.After 24 hours, the flow velocity that produces 25mL/min by peristaltic pump makes reactor content pass through electromagnetism HGMS device, mode is separated to the (technical data of tripping device: Glass tubing interior diameter 20mm, long 200mm, capacity 65mL on the metal wire base fully with magnetic-particle whereby, SS alloyed metal tow weight 15g, 4 placed in-line coils, strength of current 6A, the magneticstrength 25mT of Helmholtz field).The citrate buffer pump that activates magnetic field and twice each 100mL is measured is crossed pillar, so that the washing magnetic-particle.Filter the product suspended substance that merges, and it is dry in moisture eliminator then to wash different quercetin with frozen water.Output 3.1g.HPLC analyzes the composition that provides 97% different quercetin, 2% quercetin and 1% violaguercitrin.
In order to reclaim catalyzer, make the magnetic field inactivation and 100mL citrate buffer (pH5.0) recycle pump is crossed this tripping device 10 minutes with the flow velocity of 100mL/min, wherein change flow direction for several times.Then with in the catalyzer suspended substance blowback stirred pot and twice wash-out of citrate buffer that reuses each 100mL still be present in residual content catalyzer in the settling vessel.The activity that transforms the back fixture also has 94U/g.This equals to lose 8% activity.7. in agitator tank reactor, use the hesperidinase that is fixed on the mica particles violaguercitrin to be converted into different quercetin and to use board-like magnet to separate the magnetic bio catalyzer
In the multiple wall stirred reactor of capacity 500mL, 40 ℃ stir together 400mL 50mM citrate buffer (pH5.0), 30g be fixed on the hesperidinase of the active 10U/g on " Colorona Blackstar " and 5g violaguercitrin (Merck, Darmstadt); Analyze the continuously measured degree of conversion by HPLC.After 24 hours, pump into reactor content in the beaker and use board-like magnet (Bakker 200mT) is collected in container bottom with catalyzer.Leach supernatant liquor by pump in a vacuum immediately, use the damping fluid of each 100mL to wash these magnetic-particles for several times then, so that wash the different quercetin of the viscous solid of last remnants.Filter the different quercetin of collecting, and with the washing of a small amount of frozen water for several times, dry in moisture eliminator then.Output is 3.3g.HPLC analyzes the composition that provides 96% different quercetin and 4% violaguercitrin.The activity of fixture still has 9.7U/g after conversion.This has equaled to lose 3% activity.8. in the MSFB reactor, use the naringinase that is fixed on the magnetic silica gel particle that violaguercitrin is converted into different quercetin
40 ℃ of mixtures that stir 5g violaguercitrin, 900mL 50mM citrate buffer (pH5.0) and 100mL methyl acetate in receiving flask are up to the nearly homogeneous suspended substance that obtains not contain remarkable condensation product.This reactor has assembled temperature-control device.Add methyl acetate so that increase solubleness and dissolved speed again under the room temperature, and prevent to form quercetin.Simultaneously, the suspended substance of naringinase in 60mL 50mM citrate buffer (pH5.0) that 6g is fixed on the active 162U/g on the magnetic silica particle pumps in the pipe of MSFB reactor of magnetic field inactivation.Magnetic field is set in 20mT, at first upwards introduces fresh citrate buffer with the 5mL/min flow velocity then, in this magnetic field, reach steady state up to particle by the piston pump that is designed for accurate measurement.Room temperature was crossed the MSFB reactor through 3.5 hours with violaguercitrin suspended substance pump then.At first in rotary film evaporator, from product mixtures, remove methyl acetate, leach product then, with the frozen water washing for several times, dry in moisture eliminator then.Product output 2.9g; Product comprises 86% different quercetin and 14% violaguercitrin.
Claims (9)
1. the method for preparing monose glycosidation flavonoid by the enzymically hydrolyse rutinoside wherein uses the enzyme that is fixed on the upholder to carry out described enzymically hydrolyse.
2. the process of claim 1 wherein that used rutinoside is the rutinoside of formula (A)
Wherein R represents H, OH or OCH
3
3. claim 1 or 2 method, wherein used rutinoside is a violaguercitrin.
4. the method for each of claim 1 to 3, wherein used enzyme is an alpha-L-Rhamnosidase.
5. the method for each of claim 1 to 4, wherein used enzyme is a hesperidinase.
6. the method for each of claim 1 to 5, wherein used enzyme is fixed on the silica gel.
7. the method for each of claim 1 to 5, wherein enzymically hydrolyse carries out under the situation of the solvent mixture that has water and at least a organic solvent.
8. the method for each of claim 1 to 7 wherein is reflected under 15 ℃ to 80 ℃ the temperature of reaction and carries out.
9. the method for each of claim 1 to 8 wherein is reflected under the pH3 to 8 and carries out.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE10006147A DE10006147A1 (en) | 2000-02-11 | 2000-02-11 | Process for the preparation of monoglycosidated flavonoids |
| DE10006147.8 | 2000-02-11 |
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| CN1416470A true CN1416470A (en) | 2003-05-07 |
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| EP (1) | EP1259632A1 (en) |
| JP (1) | JP2003522532A (en) |
| KR (1) | KR20030013371A (en) |
| CN (1) | CN1416470A (en) |
| AU (1) | AU2001231726A1 (en) |
| BR (1) | BR0108273A (en) |
| CA (1) | CA2400014A1 (en) |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101605905A (en) * | 2007-01-19 | 2009-12-16 | 三得利控股株式会社 | The glucosides method of flavonoid class |
| CN101100683B (en) * | 2006-09-08 | 2010-05-12 | 颜廷和 | Glucoside type flavone biological transformation and purification technique |
| CN102286576A (en) * | 2011-09-14 | 2011-12-21 | 江苏科技大学 | Medium engineering method for synthesizing isoquercitrin by enhanced enzyme method |
| CN101670010B (en) * | 2009-10-10 | 2012-06-20 | 杭州富春食品添加剂有限公司 | Method for extracting flavonoid glycoside substance |
| CN105779473A (en) * | 2014-12-19 | 2016-07-20 | 上海交通大学 | Synthesis gene cluster of rhamnose isoflavone and applications thereof |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US20060099690A1 (en) * | 2002-09-23 | 2006-05-11 | Her Majesty The Queen In Right Of Canada, As Repre | Extraction, purification and conversion of flavonoids from plant biomass |
| KR20080033705A (en) * | 2006-10-13 | 2008-04-17 | (주)아모레퍼시픽 | Process for preparing kaempferol-3-o-rutinoside |
| KR101105846B1 (en) * | 2009-08-17 | 2012-01-13 | 주식회사 젬백스앤카엘 | Filter Material Easy To Exchange Absorbent |
| KR102528136B1 (en) * | 2018-01-02 | 2023-05-02 | 최병국 | Method for preparation of isoquercetin and α-glycosylisoquercetin |
| KR102615753B1 (en) * | 2022-11-30 | 2023-12-19 | 주식회사 다인소재 | Method of preparing isoquercetin having excellent antibacterial and antioxidant activity. |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB8727223D0 (en) * | 1987-11-20 | 1987-12-23 | Unilever Plc | Preparing l-rhamnose |
| US4971812A (en) * | 1989-08-31 | 1990-11-20 | National Science Council | Immobilized penicillium sp. naringnase and its use in removing naringin and limonin from fruit juice |
| EP0599159B1 (en) * | 1992-11-27 | 2001-09-19 | Aventis Research & Technologies GmbH & Co. KG | Heterogeneous protein mixture with alpha-L-rhamnosidase activity, process for preparation and use. |
-
2000
- 2000-02-11 DE DE10006147A patent/DE10006147A1/en not_active Withdrawn
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2001
- 2001-02-09 KR KR1020027010449A patent/KR20030013371A/en active IP Right Grant
- 2001-02-09 WO PCT/EP2001/001447 patent/WO2001059143A1/en not_active Application Discontinuation
- 2001-02-09 AU AU2001231726A patent/AU2001231726A1/en not_active Abandoned
- 2001-02-09 BR BR0108273-6A patent/BR0108273A/en not_active IP Right Cessation
- 2001-02-09 CA CA002400014A patent/CA2400014A1/en not_active Abandoned
- 2001-02-09 JP JP2001558479A patent/JP2003522532A/en active Pending
- 2001-02-09 EP EP01903733A patent/EP1259632A1/en not_active Withdrawn
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101100683B (en) * | 2006-09-08 | 2010-05-12 | 颜廷和 | Glucoside type flavone biological transformation and purification technique |
| CN101605905A (en) * | 2007-01-19 | 2009-12-16 | 三得利控股株式会社 | The glucosides method of flavonoid class |
| CN101605905B (en) * | 2007-01-19 | 2013-10-16 | 三得利控股株式会社 | Method for glycosylation of flavonoid |
| CN101670010B (en) * | 2009-10-10 | 2012-06-20 | 杭州富春食品添加剂有限公司 | Method for extracting flavonoid glycoside substance |
| CN102286576A (en) * | 2011-09-14 | 2011-12-21 | 江苏科技大学 | Medium engineering method for synthesizing isoquercitrin by enhanced enzyme method |
| CN105779473A (en) * | 2014-12-19 | 2016-07-20 | 上海交通大学 | Synthesis gene cluster of rhamnose isoflavone and applications thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| DE10006147A1 (en) | 2001-08-16 |
| EP1259632A1 (en) | 2002-11-27 |
| CA2400014A1 (en) | 2001-08-16 |
| KR20030013371A (en) | 2003-02-14 |
| BR0108273A (en) | 2003-03-05 |
| JP2003522532A (en) | 2003-07-29 |
| WO2001059143A1 (en) | 2001-08-16 |
| AU2001231726A1 (en) | 2001-08-20 |
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