KR20030013371A - Method for producing monoglycosidated flavonoids - Google Patents

Abstract

The present invention relates to a method for producing monoglycoside flavonoids by enzymatic hydrolysis of lutinosides, wherein the enzymatic hydrolysis is carried out using immobilized enzyme on a support. . The method can avoid high enzyme costs while achieving a high degree of automation with high space-time yields.

Description

Method for producing monoglycosided flavonoids {METHOD FOR PRODUCING MONOGLYCOSIDATED FLAVONOIDS}

For the purposes of the present invention, lutinosides are regarded as compounds containing aglycosuric components in which the radicals of formula (I) are linked via glycoside bonds:

For example, lutinosides are flavonoids containing bisglycoside units illustrated in Formula (I). Rhamnose and / or the corresponding glucopyranoside can be obtained from lutinoside. Glucopyranosides are derived from lutinosides containing radicals of the general formula (Ia) attached to the non-sugar component instead of radicals of the general formula (I). For example, both rhamnose and isoquecetin can be obtained from the routine:

Rhamnose is a monosaccharide that occurs naturally in many places, but mainly in small amounts. Important sources of rhamnose include glycoside radicals of natural flavonoids, such as rutin, for example, in which rhamnose can be obtained by removal of glycosides. Rhamnose is important as a starting material for the production of non-natural aroma substances such as, for example, furaneol.

Isoquecetin is a monoglycosided flavonoid of the structure of formula II:

Flavonoids (latin: flabu = yellow), which are dyes widely distributed in plants, mean, for example, glycosides of flavones commonly having a parent structure of flavone (2-phenyl-4H-1-benzopyranone-4). do.

The non-sugar component of flavonoids is called aglycone. Isoquecetin is aglycone quercetin (2- (3,4-dihydrophenyl) -3,5,7-trihydroxy-4H-1- different from flavone, for example, by the presence of five hydroxyl groups. Benzopyranone-4) glycosides. In isoquircetin, the carbohydrate radical glucose is bound to the hydroxyl group at the 3 position of quercetin. Isoquecetin is, for example, quercetin-3-O-β-D-glucopyranoside or 2- (3,4-dihydroxyphenyl) -3- (β-D-glucopyranosiloxy) -5, It is shown as 7-dihydroxy-4H-1-benzopyranone-4. However, it is also known, for example, under the trade name Hisutrin.

Flavonoids or flavonoid blends are used, for example, in the food and cosmetic industry, where their importance is increasing. In particular, monoglycosided flavonoids such as isoquecetin have excellent absorbency in the human body.

An example of a naturally occurring flavonoid having a bisglycoside unit is a routine having the structure of Formula III:

Like isoquircetin, rutin is a glycoside of aglycon quercetin, in which the carbohydrate radical lutinose is linked to a hydroxyl group at the 3 position of quercetin. Carbohydrate radicals in rutin include glucose units linked at the 1 and 6 positions and rhamnose or 6-deoxymannose units linked at the ends. The routine is, for example, quercetin-3-O-β-D-lutinoside or 2- (3,4-dihydroxyphenyl) -3-{[6-O- (6-deoxy-α-only Nopyranosol) -β-D-glucopyranosyl] oxy} -5,7-dihydroxy-4H-1-benzopyranone-4. However, it is also known, for example, by the names sofolin, virutan, rutabion, taurutin, phytomeline, melin or rutoside.

Rutin forms a pale yellow to green acicular material with three crystalline water molecules. Anhydrous rutin has the properties of a weak acid, turns brown at 125 ° C. and degrades at 214 to 215 ° C. In many plant species, for example, citrus species, yellow pansy, forsythia and acacia species, various eggplant and tobacco species, oleander, lime, St. John vegetation, tea trees, etc. are often accompanied by vitamin C. The routine produced as a substance was separated in 1842 from Ruta graveolen. Rutin is also a medicinal product from Eastern Asian dyeers containing leaves of buckwheat, and 13-27% of Rutin (Sophora japonica, Fabaceae Can be obtained from)).

It is preferred to produce both rhamnose and monoglycosided flavonoids from natural raw materials such as flavonoids containing bisglycoside units. It is of interest here for example to decompose lutinoside to rhamnose and the corresponding glucopyranoside.

Enzymatic catalyzed preparation of rhamnose is disclosed in the literature. EP-A 0,317,033, for example, describes a process for the production of L-rhamnose in which the rhamnose bonds of glycosides containing rhamnose bound at the terminal positions are degraded by enzymatic hydrolysis. This decomposition is carried out on a substrate which is usually present as a suspension in an aqueous medium. However, this reaction is mostly poor selectivity. For example, the bisglycoside structure of the carbohydrate radical in the routine often results in the production of a mixture of two monosaccharides, glucose and rhamnose. In addition, aglycon quercetin and other undesired by-products are generally produced in high proportions.

In addition, the enzymatic catalytic degradation of rutin is also described in JP-A 0,121,3293. However, this reaction, which is carried out in an aqueous medium, is also generally poor in selectivity.

These methods described above use enzymes as natural substances in solution. The method involves adding the enzyme directly to the reaction solution. This method can be carried out on a laboratory scale but is not feasible for industrial use since the enzyme cannot be returned from the reaction solution for reuse. However, the use of such expensive enzymes only once is not economical on an industrial scale.

It is known that the enzyme can be used industrially when the enzyme is bound to a support. This method is called "immobilization". The term "bound (or immobilized) enzyme" includes all enzymes "existing in a state where they can be reused," according to The European Federation of Biotechnology (Helmut Uhlig). Technische Enzyme and ihre Anwendung, Carl Hanser Verlag, Munich / Vienna 1991, pp 198). However, despite these advantages, immobilization is not suitable for all enzymatic methods and has been applied to a limited extent up to now. Specifically, only two binding enzymes (immobilized glucose isomerase for glucose isomerization and immobilized penicillin amidase for penicillin-G degradation) are in use on a commercial scale. Often the binding-enzyme method cannot tolerate free-enzyme or chemical methods. It often happens that the enzyme or reaction conditions are not suitable for immobilization. Thus, there is no universal method of immobilization and each enzyme must be considered individually.

For example, aqueous systems that are important for the utility of enzymes cause solubility problems when lutinoside is used as the substrate for enzymatic hydrolysis. Therefore, this reaction is preferably carried out using a supersaturated substrate solution, ie in the form of a lutinoside suspension. However, supersaturated solutions in which the substrate is present as a solid material render the use of the immobilization method impossible. There is a lack of selectivity between raw materials, product particles and binding enzymes.

Summary of the Invention

Accordingly, it is an object of the present invention to provide a method for producing monoglycosidase which can be used on an industrial scale, avoiding high enzyme costs while achieving a high degree of automation, high space-time yield and high productivity and selectivity. .

This object is achieved by a method for producing monoglycosided flavonoids by enzymatic hydrolysis of lutinosides, wherein the enzyme used for enzymatic hydrolysis is immobilized on a support.

Surprisingly, despite the low solubility of lutinosides, it has been found that enzymatic hydrolysis with binding enzymes is possible. Immobilization is highly efficient compared to reactions involving natural enzymes and allows the process to be carried out continuously or batchwise. The process of the invention is particularly outstanding in that it enables a high degree of automation of the overall process, including the feedback of solvents and the monitoring of enzyme activity.

The present invention relates to a process for preparing monoglycosided flavonoids by enzymatic hydrolysis of lutinosides. During this process, the rhamnose radicals of lutinosides are degraded by enzymes.

1 illustrates a method for the continuous production of isoquecetin from a routine as an example of the method of the present invention.

Suitable lutinosides for use in the process of the present invention are those having -A or -O (CH ₂ ) _n -H wherein the phenyl group is at the 3 position and the phenyl group is , Preferably 1), containing as parent sugar component or aglycone a parent substance comprising 2-phenyl-4H-1-benzopyranone-4 which may be mono- or polysubstituted.

Substitution of the parent material 2-phenyl-4H-1-benzopyranone by -OH and / or -O (CH ₂ ) _n -H preferably takes place at the 5, 7, 3 'and / or 4' positions.

Particular preference is given to using lutinosides of the formula A:

Wherein R represents H, OH or OCH ₃ .

Compounds in which R represents H are known as camphorol lutinosides, and lutinosides in which R represents OCH ₃ are known as isoramnetine lutinosides. Compounds in which R represents OH are known as rutin. Thus, the process of the invention allows the production of rhamnose and camphorol glucoside from camperol lutinoside, rhamnose and isoquecetin from rutin, and rhamnose and isoramnetine glycoside from isoramnetine lutinoside.

Particular preference is given to using lutinoside routines.

The starting materials used in the process of the invention may be pure lutinosides or optionally mixtures of lutinosides. Lutinosides may also be contaminated with residues or other flavonoids from the lutinoside preparation without negatively affecting the reaction.

The enzyme used for enzymatic hydrolysis of lutinoside may be a conventional hydrolase capable of separating rhamnose groups from lutinoside. Preferably, hydrolases obtained from the strain Penicillium decumben are used. Particular preference is given to using α-L-rhamnosidase as the enzyme, since it exhibits a high selectivity for hydrolysis of the rhamnose group. Suitable α-L-rhamnosidases are described, for example, in hesperidinase, naringinase and Kurasawa et al., J. Biochem. , Vol. 73: 31-37. It is highly desirable to use hesperidinase enzyme.

Both lutinosides and enzymes used in the methods of the invention can be obtained as commercially available products. It is also possible to isolate or prepare starting materials and enzymes by well known methods.

The enzyme is immobilized on a suitable support. Conventional supports such as silica gels for this purpose, such as commercially available spherical or commercially available broken silica gels (eg, Lichrosorb ^R , Lichroprep ^R , Lichrospher ^R and Trisoperl ^R ) and commercially available polymeric supports (eg, Eupergit ^R , Fractogel ^R , in particular Fractogel epoxy ^R and Fractoprep ^R ) can be used. Silica gel can be considered as a preferred support material.

Alternatively, magnetic powder may be used as the support. It is preferably a support material having a magnetic core. This core is largely encased in inorganic oxides. The inorganic oxide is preferably silica gel. Examples of such magnetic supports include Magnesil® (MagneSil) (obtained from Promega Corp., Madison, WI), MagPrep® (MagPrep) (from Merck). Obtained) and Agowamag (registered trademark, AGOWAmag) (obtained from AGOWA GmbH, Berlin, Germany). Magnetic supports used are alternatively magnetic glass particles (e.g. MPG (CPG Inc., Lincoln Park, NJ)), and pigments containing magnetite (e.g. Microna Matte, Mica Black, Colorona Blackstar (all of which are available from Merck), non-porous magnetic powders (eg Magprep) may be enzymatically active. It is particularly well suited as it can not cause pores occlusion that leads to extreme deterioration.

Enzyme supports generally have the following properties: The particle size of the support is preferably 0.005 to 1 mm, more preferably 0.01 to 0.5 mm. Pore diameters generally range from 10 to 4000 nm, with pore diameters of 30 to 100 nm being particularly preferred. Suitably the large pore size will ensure that the enzyme can be accommodated on the support without loss of activity. The surface area of the particles is preferably 40 to 100 m ² / g, and the pore volume is preferably selected in the range of 0.5 to 3 ml / g. In some cases very large pore diameters of 2-20 μm may be suitable.

Enzymes can be linked by covalent bonding or adsorption. In general, covalent bonding would be preferred. Examples of covalent bonding methods include epoxidation, carbodiimide method, silanization, bromocyanogen method, glutaric acid dialdehyde crosslinking or dicresyl chloride method (AS Bommarius et al. Biotransformations and Enzyme Reactions, Biotechnology (2nd Edition), Vol. 3, pp 427-465, edited by G. Stephanopoulos, VCH Weinheim, Germany 1993], DR Walt et al., Trends in Analytical Chemistry, Vol. 13 , No. 10, 1994], NH Park, HN Chang, J. Ferment. Technol., Vol. 57 (4), 310-316, 1979, M. Puri et al. Compare Enz. Microb. Technol., 18, 281-285, 1996 and HY Tsen, J. Ferment. Technol., 62 (3), 263-267, 1984. It is necessary to surface-modify the support with a suitable functional group to carry out the method. The functional groups can be applied to the support by copolymerization with functional monomers or by polymer-like conversion. Particular preference is given to surface modification or diol modification using amino groups, aldehyde groups or epoxide rings. Enzymes may be covalently bound to these groups.

Enzymatic hydrolysis takes place in a suitable reactor. Commercial towers are particularly suitable for the continuous execution of the process of the present invention. For small scale runs, for example, the tower used for preparative HPLC can be used. Reactors, in particular towers, should exhibit high hydraulic efficiency. This can be quantified by the theoretical number of plates. Therefore, in order to utilize the enzyme effectively and to obtain a high production rate, it is desirable to ensure that it is a raw material solution and in intimate contact with the surface of the immobilisate. The preparative HPLC column described above meets this need and is equipped with suitable technical means and peripherals (pumps, valves, control means). It is also desirable to develop detection means, for example UV or RI detection means, for this purpose so that the measurement and control of the degree of conversion achieved by the reaction can be automated if desired.

If the magnetic support material is used for a continuous mode of operation, a device which generally maintains the magnetic particles in a stable suspension, eg a substantially uniform magnetic field (helmholtz magnetic field in which the magnetic flow lines are parallel to the flow direction in the flow tube) A tubular reactor with an electromagnetic coil is produced. In such magnetically stable fluidized bed reactors (magnetically stable fluidized bed (MSFB)), significantly higher flow rates can be achieved in conventional fluidized bed or fluidized bed columns, which are also suitable for this purpose. The technique can also be advantageously used for catalysis in viscous reaction media.

If the method is carried out batchwise, conventional receptacles, preferably receptacles equipped with stirrers, are suitable. Thus, round scale flasks equipped with stirrers for small scales and stirred tanks for large scales may be used.

The immobilized product is charged to the reactor in a conventional manner before the reaction.

The lutinoside to be converted is fed to the reactor, eg a column or tower (eg a fixed bed column), usually in the form of a solution or suspension. If the reactor used is a fixed bed reactor, the lutinoside solution should be completely free of solid material. In order to achieve optimum solubility, it is advantageous to pre-dissolve the lutinosides in the tank, preferably with a solvent while stirring and / or heating. If necessary, prefiltration of the solution may be further performed to remove any solid material. The solvent is preferably an aqueous system to ensure enzymatic activity and prevent possible denaturation. Additional solvents may be added to ensure dissolution of the lutinosides. Preferably, the process of the invention is carried out in the presence of a solvent mixture of water and one or more organic solvents.

Auxiliary organic solvent (s) include water miscible and water immiscible organic solvents.

Suitable solvents for use in the process of the invention include nitrile (eg acetonitrile), amides (eg dimethylformamide), esters (eg acetate, in particular methyl acetate or ethyl acetate), alcohols (Eg, methanol or ethanol), ethers (eg tetrahydrofuran or methyl-t-butyl ether) and hydrocarbons (eg toluene).

The process of the invention is preferably carried out in the presence of at least one organic solvent, ethyl acetate, methanol, ethanol, methyl-t-butyl ether or toluene. The process of the invention is very preferably carried out in addition to water in the presence of at least one acetate, in particular methyl acetate.

Suitable ratios of water to organic solvents for the purposes of the present invention are in a volume ratio of 1:99 to 99: 1. The process of the invention is preferably carried out using a volume ratio of water to organic solvent of 20:80 to 80:20, in particular 50:50 to 70:30.

The amount of lutinoside present in the solvent or solvent mixture in the process of the invention depends on the solubility of the lutinoside in the solvent or solvent mixture. Optimal implementation of the process of the invention is obtained when the lutinosides are readily dissolved. For this reason, it is preferable to carry out using a saturated solution. In general, the amount of lutinoside in the solvent or solvent mixture is from 0.001 to 5 g / L, preferably from 0.05 to 2 g / L, more preferably from 0.1 to 1.5 g / L.

The ratio of lutinoside to immobilized or enzyme depends on the lifetime of the enzyme in the tower or column and its activity in the immobilized form.

The reaction is usually carried out at a temperature of 15 to 80 ° C. Temperatures of 30 to 60 ° C. are preferred and temperatures of 40 to 50 ° C. are particularly preferred in order to avoid the possibility of any breakdown of enzymes while ensuring high solubility of lutinosides.

If the reaction temperature is too low, the enzyme activity is reduced causing the reaction to occur at an inappropriately slow reaction rate. In addition, the solubility of lutinosides is reduced to the extent that an unnecessarily large amount of solvent is required. On the other hand, if the reaction temperature is too high, the enzyme, which is a protein, is denatured and deactivated.

If the process of the invention is carried out at elevated temperatures, temperature-control means can be provided in the reactor. Typical temperature-control means include a heating coil system or a double jacket. It is further advantageous if the lutinoside to be converted, especially the lutinoside solution, is temperature controlled before being introduced into the reactor. For this purpose, the lutinoside solution is recovered from a temperature-controlled tank, which is generally maintained at the temperature required for the reaction. Alternatively, the solution supplied can be passed through a heated flexible conduit to adjust the temperature to the desired value prior to introduction into the reactor. Such heating may interfere with the crystallization of lutinosides.

Suitable pH for use in the process of the invention is 3 to 8. Preferably, the process of the invention is carried out at pH 3 to 7, in particular at pH 3 to 6. However, more preferred pH may vary within certain ranges depending on the enzyme used. For example, when using hesperidinase enzyme, a pH of 3.8 to 4.3 is very preferred.

Preferably, the method is carried out in a manner in which the pH is adjusted with the aid of a buffer system. In theory, any commonly used buffer system suitable for adjusting the aforementioned pH can be used. However, aqueous citrate buffers are preferably used.

The lutinoside mixture, which may be present in solution or suspension form, is placed in a reactor containing a fixative to effect enzymatic hydrolysis. This reaction can be carried out continuously or batchwise.

If the reaction is carried out batchwise, the lutinoside suspension is usually placed in the reactor. The degree of conversion is determined by the amount of lutinoside and immobilized. In general, the ratio of lutinoside to immobilized product is from 100: 1 to 1: 1000, preferably from 10: 1 to 1: 100, more preferably from 1: 1 to 1:20. The ratio of the total volume of the immobilized to suspension is generally 1: 1000 to 1: 1, preferably 1: 100 to 1: 2, more preferably 1:50 to 1: 5. The residence time in the reactor is usually in the range of 1 hour to 10 days, preferably 8 hours to 4 days, more preferably 1 to 2 days.

If the reaction is carried out continuously, the lutinoside solution is generally transported constantly through the reactor, preferably the tower or MSFB reactor, by means of a suitable pump. By suitably adjusting the flow rate, any desired degree of conversion can be achieved. Typically, the flow rate used is from 0.001 to 1 mm / s based on the cross section of the empty tube of the column or column.

Enzyme activity in the system was found to decrease with time. Therefore, it is necessary to replace the fixed cargo completely or partially at regular intervals. In order to compensate for the loss of activity of the enzyme, it is desirable to assess the degree of conversion by UV or RI detection so that, if there is a composition change, this can be counteracted by control over the flow of the pump.

If the reaction solution remains in the reactor, the resulting product can be separated. At the completion of the reaction, the reaction mixture consists mainly of possible additional additives such as solvents, unconverted lutinosides, rhamnose, the desired monoglycosided flavonoids and buffers. When the solubility limit is reached, monoglycosided flavonoids are usually precipitated and gradually accumulate as solids.

For batch operation involving the use of a magnetic support material, the binding enzyme can be separated from the suspension of the product upon completion of the reaction in a simple manner with the aid of a magnetic separation device. On a laboratory scale, a strong permanent magnet in the form of a plate can be used for this purpose. However, there are huge separators developed for a wide variety of industrial applications and operating mainly under the HGMS principle (high gradient magnetic separation). Such a plant may, for example, consist of a vertical flow tube containing fine stainless steel wire packing. Properly placed electromagnetic coils create a high magnetic flow gradient along the wire, which allows for very efficient separation of even the smallest particles, such as nanometers in size. If the magnetic particles do not exhibit paramagnetic, ie, residual magnetization in the absence of an external magnetic field, they can be easily removed completely from the separator by switching off the magnetic field and repeatedly washing with water.

Separation of the desired reaction product is carried out by a commonly used method involving conventional reaction finishing equipment.

Preferably, the product is precipitated by concentration. If the solvent consists of a solvent mixture containing at least one organic solvent, it is preferred to remove the organic solvent by distillation under reduced pressure. Crystallized monoglycosided flavonoids are usually separated from the residual reaction mixture by solid-liquid separation methods such as siphoning or filtration under reduced pressure, or centrifugation of precipitated crystals. The solid material is then preferably washed with water and then dried.

Alternatively, the entire reactor contents can be filtered first. The filter cake containing the product is then treated with a solvent or buffered solvent mixture in which the product is dissolved. During the process the reaction product is extracted from the filter cake.

In a batch process, the mixture remains insoluble catalyst, i.e., a fixed product. Essentially no solvent or buffer solvent mixture should have a deleterious effect on the enzyme. Binding enzymes, such as naringinase or hesperidinase, have no additional activity or only a fraction of the original activity in certain buffer solvent mixtures or under weakly alkaline conditions, but the enzyme is buffered in a pH range of 4-6 It has been found that the activity can be restored substantially completely when carefully rinsed with; Thus, the loss of activity is only temporary and is not equivalent to the irreversible denaturation of the enzyme.

In this procedure, a very suitable extractant is a tetrahydrofuran buffer mixture, preferably a tetrahydrofuran buffer mixture having a tetrahydrofuran content of 10 to 25%, especially when used at slightly elevated temperatures. Other suitable extraction components are, for example, 1-propanol, 2-propanol, 1,4-dioxane and methyl acetate. The product can be recovered very easily from the extract by distilling under reduced pressure to remove the solvent and then cooling the aqueous solution containing the product to 0-10 ° C. The reaction product is crystallized from the mother liquor in a very high purity state.

As the reaction product has phenolic OH groups which are deprotonated in a weakly basic medium, as an alternative to the solvent / buffer mixture, dilute ammonia or soda solutions can be used as extractant; The anion of the reaction product shows a relatively good solubility, but is also very susceptible to oxidation, as can be seen from the fact that the extract gradually discolors from yellow to brown. Therefore, the modification must be carried out very quickly, ie the extraction should be completed within 10 minutes to 6 hours, preferably 20 minutes to 2 hours. The process is preferably carried out under a blanket of protective gas.

In addition, treatment with a weakly basic extractant such as an aqueous solution of an alkali-metal or ammonium salt of acetic acid, oxalic acid, citric acid, phosphoric acid, boric acid or carbonic acid, or an aqueous solution of alkylamine, piperidine or pyridine does not result in a loss of enzyme activity. The reaction product can be reprecipitated by acidifying the extract and cooling to 0-10 ° C.

The purity of the resulting monoglycosided flavonoids when using pure lutinosides is typically higher than 94%. To achieve further purification, the final product can be recrystallized, for example, from a suitable solvent, for example from water or a solvent mixture comprising toluene and methanol, or from water and methyl acetate.

The solvent remaining after the reaction is preferably recovered in order to maintain the economic value of the process of the invention. The recycling is usually, continuously and automatically. Commercial evaporation plants with suitable control means can be used for this purpose. If the solvent to be used is a solvent mixture of water and at least one organic solvent, it is usually not possible to immediately reuse the distillate in the process since the solvent ratio is changed by distillation of the organic solvent. By performing automatic quality control and correction, it is possible to properly recycle the solvent to reset the desired proportions of the solvent.

In addition, concentration may involve membrane processing or nanofiltration. In these processes, the solvent mixture is separated without changing its composition.

The following examples are intended to illustrate the invention. However, they are never considered to be limiting.

Example 1 . Immobilization of Hesperidinase Enzyme on Silica Gel Supports

(1) Condition of surface of support before immobilization

1.1) Properties of Support Material

Silica gel LiChrospher; Diameter: 15-40 탆; Pore diameter: 300 mm 3; Particle surface area: 80 m ² / g; Pore volume: 0.73 ml / g; Density: 2 g / ml.

1.2) Activation of Silica Gel

In a 1 L flask, 250 g of silica gel is mixed with sufficient HCl (7%) and allowed to stand overnight to wet the silica gel.

The silica gel suspension is then washed with demineralized water until there is no chloride. To this end, the supernatant should be tested with nitric acid and silver nitrate after each wash. Due to the nature of the silica gel particles, the washing is carried out in a ceramic funnel having a diameter of about 24 cm.

1.3) surface modification by amino groups

In a three-neck flask with a capacity of 2 L and equipped with a reflux condenser and a dropping funnel, the acid-treated silica gel is mixed with sufficient water to make it stirrable. With complete mixing at room temperature, 1 mmol / g of support comprising 3-aminopropyltrimethoxysilane is added dropwise to the silica gel suspension at a rate of about 5 drops per second (135 ml of solution is required for 250 g of silica gel). The suspension is then stirred at 90 ° C. for 2 hours. The suspension is then cooled with ice.

Supernatants should be taken for possible residues of 3-aminopropyltrimethoxysilane by taking pH readings. The suspension of beads is washed with demineralized water until the pH is kept constant.

1.4) Glutaric Acid Dialdehyde Coating

To the resulting silica gel suspension is added glutaric acid dialdehyde (GDA) at a concentration of 1 mmol / g support (13 ml of a 50% concentration of GDA solution is required for 250 g of support). The suspension (with a small amount of water) is shaken in a flask with a volume of 1 L for 2 hours at room temperature. The suspension is initially yellow in color and dark red at the end of the procedure.

The supernatant obtained after each wash is examined for residues of glutaric acid dialdehyde by precipitation reaction with dinitrophenylhydrazine. Carefully wash the suspension until the test is negative.

(2) immobilization

2.1) Hesperidinase

Primary protein addition

3.8 g of hesperidinase is stirred in 500 ml of citrate / phosphate buffer mixture, pH 6.0. To improve dissolution, 300 μl of surfactant (Tween 20) is added. The enzyme solution is then filtered.

In a flask with a capacity of 1 L, about 230 g of the silica gel suspension obtained as described in 1.4) is mixed with the enzyme solution. The suspension of enzyme support is then shaken for about 40 hours at room temperature.

Secondary protein addition

About 0.76 g of hesperidinase (Amano) is stirred in 120 ml of citrate / phosphate buffer mixture (pH 6.0) containing 60 μl of surfactant and subsequently filtered.

The enzyme solution is poured into the aforementioned flask with a capacity of 1 L and the enzyme solution is shaken at room temperature.

2.2) BSA (for separation test)

0.3 g Biomax BSA (bovine serum albumin powder) is stirred in 100 ml citrate / phosphate buffer mixture, pH 6.0. In a flask with a volume of 0.5 L, about 20 g of the silica gel suspension obtained as described in 1.4) is mixed with the protein solution and 60 μl of ProClin 300 is added.

(3) measuring the amount and activity of protein

3.1) Amount of protein (mg of protein per ml)

The protein content of the solution is measured by the Bradford test. Perform standard assays. The assay is performed by mixing 20 μl of sample in 1 ml of Bradford dye reagent (diluted 1: 5) and taking a photometric reading at 595 nm 15 minutes later.

Very low protein concentrations should be used for microanalysis. The microanalysis involves mixing 0.8 ml of sample in 0.2 ml of Bradford dye reagent (concentrated) and taking a photometric reading at 595 nm 15 minutes later.

3.2) Active

The activity of the solution is measured by reaction with a substituted substrate.

For each sample the following is used: 88 μl of citrate / phosphate buffer mixture (pH 4.0); 100 μl of sample; 20 μl of sample; 20 μl of substitution substrate: p-nitrophenyl-α-L-rhamnoside (rhamnosidase activity), p-nitrophenyl-α-L-glucoside (glucosidase activity).

1 ml of this solution is mixed in an Eppendorf reaction vessel. After 2 and 5 minutes of incubation period at 40 ° C., respectively, in a stirrer, every 100 μl of reaction mixture is mixed with 1 ml of 1 M soda solution. Subsequently, the concentration of p-nitrophenol is measured by photometry at 400 nm. Activity is calculated from the change in concentration of p-nitrophenol per unit of time.

The activity of the enzyme is expressed in units (U) (= micromoles of substrate converted per minute).

(4) results

Protein content and activity value (unimmobilized hesperidinase) Sample ¹ Protein amount (standard analysis) Active (Substitute Substrate) Mg Mg / sample (ml) U U / sample (ml) U / protein (mg) Hesperidinase0 740 1.48 48000 96 65 One 15 0.03 7 0.014 0.47 2 20 0.04 4 0.008 0.2 Hesperidinase 1 326 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 ¹ Hesperidinase 0: primary protein addition Hesperidinase 1: secondary protein addition samples 1 to 6: supernatant

Protein content and activity value (immobilized hesperidinase) * Protein content of the support: about 1,045 g of bound protein. * 4.7 mg of protein / support (g) present on the support. * 2% of added protein is not bound.

Example 2 . Production of Isoquecetin from Rutin by Enzymatic Hydrolysis with Immobilized Goods

In a heated stirred tank 1 having a capacity of 4.5 m ³ , 3200 L of demineralized water and 800 L of 1-propanol are charged. The mixture is heated to about 50-60 ° C. via steam inlet 2. 8000 g of routine, DAB, is added to the solution with vigorous stirring. Stir the mixture until the routine is completely dissolved. The pH is then monitored via a circulation pump and in-line pH meter 3a and if necessary adjust the pH to 4.0 to 4.5 (using H ₃ PO ₄ and NaOH). Samples can be taken via manual valve 4 for inspection purposes and concentration measurements.

To initiate the reaction, the solution is fed to the piston type dosing pump 7 through a bag filter 5 and a tube filter 6. The bag filter resists most of the undissolved components, while the tube filter purifies the solution to a fineness of 0.2 μm.

The piston type dosing pump 7 transports the solution through a heatable flexible tube, which adjusts the temperature of the solution by means of a thermometer to 40 ° C. at the column inlet, and the column 9 (100 × 400 mm). The flow rate to the furnace is 1 l / min. The column contains 1.5 kg of fixative. Since electrically heated flexible tubes cannot cool the solution, the temperature in the agitation tank 1 is adjusted to a value such that the cooling that occurs while reaching the pump provides a temperature of 40 ° C. or less at maximum pump delivery.

By means of a manual valve 10 which allows the degree of conversion achieved by temperature and reaction to be measured off-line, a sample can be taken from the solution after perfusion through the column. If the measured conversion is lower than necessary, the outflow of the pump is appropriately reduced.

When the solution has passed through the column, the reaction is completely finished so that the solution can be transferred to the collection vessel 11. The solution is reduced by about 10-20% in volume by the condenser 12. Due to this, the content of propanol is considerably reduced, which means that the solubility of isoquecetin is drastically reduced. Subsequent cooling further lowers the solubility so that the product can precipitate and separate in the bag filter 13. From this, the product is transferred to a drying oven 14 for drying. The mother liquor and distilled condensate are recycled together for reuse in the stirred tank (1).

Example 3

1.Modification of Silica Gel Particles by Aldehyde Groups and Immobilization of Naringinase on the Particles

400 ml of 10% concentration of HCl is poured over 250 g of silica gel (e.g., LiChrospher Si 300, Merck, Darmstadt, Germany) in a container that can be sealed, and then the vessel is ultrasonically Degassed for 10 minutes and allowed to stand at room temperature for 24 hours. The silica gel was then filtered and washed with several liters of demineralized water until the pH was greater than 4.5 and chloride ions could no longer be detected in the filtrate (drop reaction with a solution of AgNO ₃ in acetic acid).

The acid-treated wet silica gel was placed in a three-necked flask with a capacity of 4 L and equipped with a precision glass stirrer, reflux condenser and a 100 ml dropping funnel, and stirred with 3 L of demineralized water in it. 100 mL of aminopropyltrimethoxysilane (ABCR, Karlsruhe) was added via dropping funnel with stirring for 15 minutes. The suspension was then heated and stirred at 90 ° C. for 90 minutes. The cooled suspension was filtered and washed eight times with 1 L of demineralized water each time.

The aminoactivated silica gel was suspended in 3 L of water and degassed by ultrasound in a 3-necked flask having a capacity of 4 L and equipped with a precision glass stirrer and a 100 ml dropping funnel; A few drops of 2M acetic acid lowered the pH to 8.0. Then, 100 ml of a 50% concentration of glutaric acid dialdehyde solution (Merck, Darmstadt, Germany) was added dropwise for 1 hour, and the suspension was further stirred at room temperature for 2.5 hours. Activated silica gel is refiltered and washed with ice cold demineralized water until glutaraldehyde can no longer be detected in the wash water (drop reaction with a solution of 2,4-dinitrophenylhydrazine in sulfuric acid).

The silica gel modified with the aldehyde group was suspended in 500 ml of demineralized water in a flask with a capacity of 4 L by stirring using a precision glass stirrer. 13 g of naringinase (Sigma, Daisenhofen, Germany) was dissolved in 2.5 L 0.25 M phosphate buffer (pH 8.0). The enzyme solution was added to the silica gel suspension and stirred for 96 hours at room temperature. The immobilized was then filtered and washed several times with 0.2 M sodium chloride solution first, followed by 50 mM citrate buffer, pH 4.0. The rhamnosidase activity of the immobilized product was measured using p-nitrophenyl-L-α-lamnopyranoside (Sigma, Daisenhofen, Germany) as a substrate by the Kurosawa method; The activity was 120 U / g.

2. Immobilization of Hesperidinase on Eupergit C

50 g of Eufergit (Rohm, Weiterstadt, Germany) was mixed with 300 ml of 0.8 M potassium phosphate buffer (pH 8.5) in a 500 ml glass bottle with screw lid and allowed to stand for 30 minutes. 5.0 g of hesperidinase (Amano) was then added and the batch was stirred on a rotary mixer for 120 hours at room temperature. The hoppers were filtered using a sinter-glass filter and first washed several times with 0.2 M sodium chloride solution and then twice with 1 L of 0.1 M citrate buffer (pH 4.0) each time. The rhamnosidase activity of the immobilized product was measured by p-nitrophenyl-L-α-rhamnopyranoside (Sigma, Daisenhof, Germany) as a substrate by the Kurosawa method; The value was 15 U / g based on dry fixative, or 4.2 U / g based on wet fixative.

3. Extraction of the product by tetrahydrofuran / buffer mixture after conversion of the routine to isoquecetin using a hesperidinase immobilized on a jug in a stirred-tank reactor

In a round-bottom flask with a volume of 2000 ml, using a precision glass stirrer, 1000 g of 50 mM citrate buffer (pH 4.0), 100 g of naringinase immobilized on a jujit and having an activity of 4.2 U / g ( Wet weight), and 10 g of Rutin (Merck, Darmstadt, Germany) were stirred together at 40 ° C; Conversion was measured continuously by HPLC analysis. After a total of 96 hours, the reactor contents were filtered through a Buchner filter. The filter cake was again placed in a round bottom flask and stirred at 40 ° C. in a mixture of 400 ml of 50 mM citrate buffer (pH 4.0) and 100 ml of tetrahydrofuran for 30 minutes, during which time most of the isoquecetin was dissolved. The mixture was filtered hot and the filter cake was reextracted with 500 ml buffer / tetrahydrofuran mixture for 30 minutes. After filtration, the two isoquecetin extracts were mixed with the first filtrate and the tetrahydrofuran was removed using a rotary evaporator. In order to precipitate the product completely, the aqueous solution of isoquecetin was cooled to 4 ° C. After filtration and drying in a drier, a 5.8 g yield of product was obtained, which consisted of 98% isoquecetin and 2% rutin.

The wet hoppers were washed once with cold tetrahydrofuran buffer mixture and then repeatedly washed with 50 mM citrate buffer (pH 4.0) until the smell of tetrahydrofuran was only weakly recognized. The activity of the enzyme was still 3.6 U / g, which is equivalent to a 14% loss of activity.

4. Extraction of the product by alkaline buffer after conversion of the routine to isoquecetin using hesperidinase immobilized on a jug in a stirred-tank reactor.

In a round-bottom flask with a volume of 2000 ml, using a precision glass stirrer, 1000 g of 50 mM citrate buffer (pH 4.0), 100 g of naringinase immobilized on a jujit and having an activity of 4.2 U / g ( Wet weight), and 10 g of Rutin (Merck, Darmstadt, Germany) were stirred together at 40 ° C; Conversion was measured continuously by HPLC analysis. After a total of 96 hours, the reactor contents were filtered through a Buchner filter. The wet cake was again placed in a round bottom flask and stirred at room temperature in 300 ml of 50 mM sodium carbonate buffer (pH 10.0) for 5 minutes, during which the portion of isoquecetin dissolved to a dark yellow color. The suspension was filtered and the filter cake was immediately reextracted with carbonate buffer. After a total of seven extraction cycles, the judgment was substantially colorless and the isoquecetin was substantially completely dissolved. The extracts were combined and carefully acidified with dilute hydrochloric acid until the pH was about 3, then the mixture was cooled to 4 ° C. After filtration and drying in a drier, a 4.9 g yield of product was obtained, which consisted of 98% isoquecetin and 2% rutin.

The wet hoppers were washed twice with 50 mM citrate buffer and then again prepared for use in further reactions. The activity of the enzyme was still 3.9 U / g, which is comparable to loss of activity of 7%.

Example 4

1.Modification of Magnetic Silica Particles by Aldehyde Groups and Immobilization of Naringinase on the Particles

In a 3-necked flask with a capacity of 1 L and equipped with a precision glass stirrer, a dropping funnel and a reflux condenser, a suspension of 30 g of magnetic silica particles (Magpref, Merck, Darmstadt, Germany) in 600 ml water was charged. A mixture of 20 mL of aminopropyltriethoxysilane (ABCR, Karlsruhe) and 20 mL of isopropanol was added dropwise over 30 minutes with stirring. The mixture was then heated to 85 ° C. and stirred at this temperature for 1 hour. After cooling, the suspension was placed in a beaker, particles were collected at the bottom of the vessel using a strong permanent magnet, and the supernatant was decanted. The particles were washed repeatedly with demineralized water until the pH of the wash solution was kept constant. The particles are then resuspended in 600 ml of water and the pH adjusted to a value of about 8 with a few drops of acetic acid; After addition of 24 ml of 50% concentration of glutaric acid dialdehyde solution, the suspension is stirred for 4 hours at room temperature and then until no more glutaraldehyde can be detected in the wash solution (2,4-di in sulfuric acid). Stationary reaction with nitrophenylhydrazine solution).

Aldehyde-derived particles were resuspended in 600 ml of 0.2 M potassium phosphate buffer (pH 9) in a round bottom flask with a capacity of 1 L. After addition of a solution of 1 g of naringinase (Sigma, Daisenhofen, Germany) in 100 ml of 50 mM sodium chloride solution, the mixture was stirred at room temperature using a precision glass stirrer for 2 days. The particles were then separated using a permanent magnet and washed repeatedly first with 0.2 M sodium chloride solution and then with 50 mM citrate buffer, pH 4.0. Rhamnosidase activity of the immobilized product was measured by p-nitrophenyl-L-α-rhamnopyranoside (Sigma, Daisenhofen, Germany) as a substrate by the Kurosawa method [Kurosawa, Ikeda, Egami, J. Biochem , 73 , 31-37, 1973: α-L-rhamnosidase of the liver of Turbo cornutus and Aspergillus niger ; The activity was 162 U / g.

2. Modification of Magnetic Silica Particles by Epoxide Ring and Immobilization of Narginases on the Particles

30 g of magnetic silica particles in 600 ml of 50 mM sodium acetate solution (Merprev, Darmstadt, Germany) Suspension was added. A mixture of 20 ml of (3-glycidoxypropyl) trimethoxysilane (ABCR, Karlsruhi) and 20 ml of isopropanol was added dropwise with stirring for 30 minutes, then the mixture was heated to 85 ° C. and at this temperature 1 Stir for hours. After cooling, the suspension was placed in a beaker, particles were collected at the bottom of the vessel using a strong permanent magnet, and the supernatant was decanted. The particles were washed repeatedly with demineralized water until the pH of the wash solution was kept constant. To quantify the epoxide ring, a sample of about 0.5 g of material was washed repeatedly with methanol and then dried to constant weight at about 70 ° C. in a drying oven. Pribyl method [Pribyl, Fresenius Z. Anal. Chem ., 303 , 113-116, 1980: Bestimmung of Epoxydengruppen in modifizierten chromatographischen Sorbentien and Gelen] measured the epoxide ring to obtain a value of 250 micromoles / g.

150 ml of a 20% concentration (w / v) suspension of epoxy-derived magnetic powder was mixed with 350 ml of 1 M potassium phosphate buffer (pH 9.0) in a 1 liter round bottom flask. After addition of a solution of 1.5 g of naringinase (Sigma, Daisenhofen, Germany) in 15 ml of 50 mM sodium chloride solution, the mixture was stirred at 40 ° C. for 16 hours using a precision glass stirrer. The particles were then separated using a permanent magnet and washed repeatedly first with 0.2 M sodium chloride solution and then with 50 mM citrate buffer, pH 4.0. Rhamnosidase activity of the immobilized product was measured by p-nitrophenyl-L-α-rhamnopyranoside (Sigma, Daisenhofen, Germany) as a substrate by the Kurosawa method; The activity was 102 U / g.

3. Modification of Magnetic Silica Particles by Carboxyl Groups and Immobilization of Narginases on the Particles

In a 3-necked flask with a capacity of 1 L and equipped with a precision glass stirrer, a dropping funnel and a reflux condenser, a suspension of 30 g of magnetic silica particles (Magpref, Merck, Darmstadt, Germany) in 600 ml water was charged. A mixture of 28 ml of 3- (triethoxysilyl) propylsuccinyl anhydride (ABCR, Karlsruhi) and 28 ml of isopropanol was added dropwise with stirring for 30 minutes, followed by the dropwise addition of 10% sodium hydroxide solution The pH of the mixture was adjusted to 9.0. The mixture was heated to 80 ° C. and stirred at this temperature for 2 hours. During this process, the pH was controlled at regular intervals and, if necessary, corrected by the addition of alkali. After cooling, the suspension was placed in a beaker, particles were collected at the bottom of the vessel using a strong permanent magnet, and the supernatant was decanted. The particles were washed three times with demineralized water, once with 2M acetic acid solution and then with demineralized water until the pH of the wash solution remained constant.

150 ml of a 20% concentration of carboxyl-derived magnetic powder (w / v) in a round-bottom flask with a capacity of 1 L was prepared in 300 ml of 0.4 M potassium phosphate buffer (pH 5.0), and 1.5 in 150 ml of 50 mM sodium chloride solution. mixed with a solution of g naringinase (Sigma, Daisenhofen, Germany). After addition of 8 ml of a 1% concentration (w / v) solution of EDC (N-ethyl-N '-(3-dimethylaminopropyl) carbodiimide hydrochloride in water, Merck, Darmstadt, Germany) The mixture was stirred at rt for 20 h. The particles were then separated using a permanent magnet and washed repeatedly with 0.2 M sodium chloride solution and then with 50 mM citrate buffer (pH 4.0). Rhamnosidase activity of the immobilized product was measured by p-nitrophenyl-L-α-rhamnopyranoside (Sigma, Daisenhofen, Germany) as a substrate by the Kurosawa method; The activity was 71 U / g.

4. Modification of Magnetic Mica Pigments by Aldehyde Groups and Immobilization of Hesperidinase on the Particles

In a 3-necked flask with a capacity of 1 liter and equipped with a precision glass stirrer, a dropping funnel and a reflux condenser, 30 g of magnetic mica pigment in 300 ml water ("Colorona Blackstar Green", Darmstadt, Germany Merck) was added. A mixture of 20 mL of aminopropyltriethoxysilane (ABCR, Karlsruhe) and 20 mL of isopropanol was added dropwise over 30 minutes with stirring. The mixture was then heated to 85 ° C. and stirred at this temperature for 1 hour. After cooling, the suspension was placed in a beaker, particles were collected at the bottom of the vessel using a permanent magnet, and the supernatant was decanted. The particles were washed repeatedly with demineralized water until the pH of the wash solution was kept constant. The particles are then resuspended in 300 ml of water and the pH is adjusted to a value of about 8 with a few drops of acetic acid; After addition of 25 mL of 50% concentration of glutaric acid dialdehyde solution, the suspension is stirred at room temperature for 4 hours, and then until no more glutaraldehyde can be detected in the wash water (2,4- in sulfuric acid). Stationary reaction with dinitrophenylhydrazine solution).

30 g of aldehyde-derived mica pigment, "Colorona Blackstar Green", was resuspended in 300 ml of 0.2 M potassium phosphate buffer (pH 7.5) in a round bottom flask with a capacity of 1 L. After addition of a solution of 1 g hesperidinase (Amano) in 20 mL 0.2 M potassium phosphate buffer, pH 7.5, the mixture was stirred at room temperature using a precision glass stirrer for 3 days. The particles were then separated using a permanent magnet and washed repeatedly with 0.2 M sodium chloride solution and then with 50 mM citrate buffer, pH 4.0. Rhamnosidase activity of the immobilized product was measured by p-nitrophenyl-L-α-rhamnopyranoside (Sigma, Daisenhofen, Germany) as a substrate by the Kurosawa method; The activity was 10 U / g.

5. Conversion of Rutin to Isoquecetin by Fixed Naringinase in Stirred Tank Reactor and Isolation of Magnetic Biocatalyst by Permanent Magnet

In a double-walled stirred reactor with a capacity of 500 ml, 400 ml of 50 mM citrate buffer (pH 5.0), 20 g of naringinase immobilized on magnetic silica particles and having activity of 102 U / g, and 10 g of rutin ( Merk, Darmstadt, Germany) are stirred together at 40 ° C; Conversion was measured intermittently by HPLC analysis. After 24 hours, the reactor contents were charged to the beaker and the catalyst was collected at the bottom of the vessel using a flat plate magnet (Bakker, 200 mT). The supernatant was immediately filtered under vacuum using a pump and washed several times with 100 ml of buffer each time to rinse off the final residue of adhesive solid isoquecetin. The collected isoquecetin was filtered off, washed several times with a small amount of ice water and dried in a drier. Yield 6.5 g. HPLC analysis showed a composition of 96% isoquecetin, 2% quercetin and 2% rutin. After conversion, the activity of the fixative was still 92 U / g. This is equivalent to 10% loss of activity.

6. Conversion of rutin to isoquecetin by fixed naringinase in a stirred tank reactor and separation of magnetic biocatalyst by electromagnetic separator (FIG. 1)

In a double-walled stirred reactor with a capacity of 500 ml, 300 ml of 50 mM citrate buffer (pH 5.0), 10 g of naringinase immobilized on magnetic silica particles and having activity of 102 U / g, and 5 g of rutin ( Merk, Darmstadt, Germany) are stirred together at 40 ° C; Conversion was measured continuously by HPLC analysis. After 24 hours, the reactor contents were passed through an electromagnetic HGMS plant using a peristaltic pump providing a flow rate of 25 ml / min, whereby the magnetic particles were completely separated onto the wire matrix (technical data for the separation plant: 20 mm Glass pipe with internal diameter and length of 200 mm, capacity of 65 ml, wire packing weight of 15 g of SS alloy, four series-connected coils, current strength 6 A, magnetic field strength of Helmholtz 25 mT). The magnetic field was activated and the citrate buffer was pumped twice through the column in an amount of 100 ml each time to rinse the magnetic powder. The suspensions of the products were combined and filtered, the isoquecetin was washed with ice water and dried in a drier. The yield was 3.1 g. HPLC analysis showed a composition of 97% isoquecetin, 2% quercetin and 1% rutin.

To regenerate the catalyst, the magnetic field was deactivated and 100 ml of citrate buffer (pH 5.0) was pump-circulated through the separation plant at a flow rate of 100 ml / min for 10 minutes, the direction of the flow being changed several times. . The catalyst suspension was then fed back to the stir tank and the remaining amount of catalyst in the precipitator was rinsed again twice with 100 ml of citrate buffer each time. After conversion, the activity of the fixative was still 94 U / g. This is equivalent to 8% loss of activity.

7. Conversion of Rutin to Isoquecetin by Hesperidinase Immobilized on Mica Particles in a Stirred Tank Reactor and Isolation of Magnetic Biocatalyst Using Flat Magnets

In a double-walled stirred reactor with a capacity of 500 ml, 400 ml of 50 mM citrate buffer (pH 5.0), 30 g of hesperidinase fixed on a "Colorona Blackstar" and having an activity of 10 U / g, And 5 g of Rutin (Merck, Darmstadt, Germany) are stirred together at 40 ° C; Conversion was measured continuously by HPLC analysis. After 24 hours, the reactor contents were charged to a beaker and the catalyst was collected at the bottom of the vessel using a flat plate magnet (barker, 200 mT). The supernatant was immediately filtered under vacuum using a pump and washed several times with 100 ml of buffer each time to rinse off the final residue of adhesive solid isoquecetin. The collected isoquecetin was filtered off, washed several times with a small amount of ice water and dried in a drier. Yield 3.3 g. HPLC analysis showed a composition of 96% isoquecetin and 4% rutin. After conversion, the activity of the fixative was still 9.7 U / g. This is equivalent to a 3% loss of activity.

8. Conversion of rutin to isoquecetin by naringinase immobilized on magnetic silica gel particles in MSFB reactor

In a receiving flask, a mixture of 5 g routine, 900 mL 50 mM citrate buffer (pH 5.0) and 100 mL methyl acetate was stirred at 40 ° C. until a roughly homogeneous suspension was produced which did not contain distinct aggregates. . The reactor was equipped for temperature control. Methyl acetate was added to increase the solubility and redissolution at room temperature and to prevent the production of quercetin. Meanwhile, a suspension of 6 g of Naringinase immobilized on magnetic silica particles and having 162 U / g activity in 60 ml of 50 mM citrate buffer (pH 5.0) was fed to the tube of the MSFB reactor with the magnetic field deactivated. It was. The magnetic field was adjusted to 20 mT and fresh citrate buffer was first introduced upwards using a piston pump designed for precise metering at a flow rate of 5 ml / min until the particles reached a stable state in the magnetic field. The routine suspension was then fed through an MSFB reactor at room temperature for 3.5 hours. Methyl acetate was first removed from the mixture of products in a rotary film evaporator, then the product was filtered, washed several times with ice water and dried in a dryer. Product yield was 2.9 g; The product consisted of 86% isoquecetin and 14% rutin.

Claims (9)

A method for producing monoglycoside flavonoids by enzymatic hydrolysis of lutinosides, wherein the enzymatic hydrolysis is carried out using an immobilized enzyme on a support. The method of claim 1, The method in which the lutinoside used is the lutinoside of formula (A): Formula A Where R represents H, OH or OCH ₃ . The method according to claim 1 or 2, The lutinoside used is a routine. The method according to any one of claims 1 to 3, The enzyme used is α-L-lamnosidase. The method according to any one of claims 1 to 4, The enzyme used is hesperidinase. The method according to any one of claims 1 to 5, Enzyme immobilized on silica gel. The method according to any one of claims 1 to 5, The enzymatic hydrolysis is carried out in the presence of a solvent mixture of water and at least one organic solvent. The method according to any one of claims 1 to 7, The reaction is carried out at a reaction temperature of 15 to 80 ℃. The method according to any one of claims 1 to 8, The reaction is carried out at pH 3-8.