Multicomponent system for the enzymatic oxidation of substrates and method of enzymatic oxidation

 

The invention relates to biotechnology; multicomponent system for mediated mediated enzymatic oxidation includes (a) an oxidation catalyst chosen from the group margantsovistyh oxidase, (b) an oxidizing agent chosen from the group comprising oxygen and oxygen-containing compounds, b) the mediator from the group of compounds containing Mn ions. The method of oxidation of the substrate margaritavision oxidase occurs in the presence of oxygen ions and MP. The invention provides a simple and cheap processing oxidizable substrate. 2 S. and 9 C.p. f-crystals, 4 PL.

The invention relates to a multicomponent system for mediated mediated enzymatic oxidation of substrates, and to a method of enzymatic oxidation.

Multicomponent system for mediated mediated enzymatic oxidation of substrates and associated methods are generally known. In this case, as mediators, as a rule, apply such compounds that can be oxidized oxidoreductase, i.e. first of all the substrates of the enzymatic oxidation catalysts. The main difference between these mediators is that oxidized is a rule long life, so after interaction with the oxidoreductase to diffuse into the substrate itself oxidative system and to interact with him. In the interaction of activated mediator with the substrate latest oxidized mediator. As a result of oxidation of the substrate, the mediator may either be regenerated (catalytic oxidation system), or inaktivirovanie (stoichiometric oxidative system). If the mediator is regenerated, it can be used in a new cycle of catalysis. The electrons carried by the mediator from the substrate for the oxidoreductase, is transmitted, as in the direct enzymatic oxidation method, the final electron acceptors, such as oxygen or peroxide.

In methods in which the final electron acceptor is peroxide (introduced directly or released from predecessors), as oxidoreductases can be used only peroxidase. Despite the prominence of a number of such methods, they are all characterized by numerous disadvantages.

Technology of production of peroxidases is too expensive so they could be used in various processes, such as the bleaching of paper, or in detergents is imich for the effective implementation of the process, peroxide has the inactivating effect on the peroxidase. Many are described as mediators of peroxidase are not suitable from a technical point of view, as they have intense color, no safe in Toxicological or ecotoxicological respect, cannot be done at all or only poorly biodegradable and cannot be obtained in sufficient quantities or a relatively cheap method. Therefore, the methods in which as the final electron acceptor is peroxide, up to the present time were unsuitable for cost-effective industrial applications.

In methods in which as the final acceptor of electrons coming from the oxidized substrate is oxygen, as the oxidoreductase used oxidase. Described two groups oxidase, which can be used in mediated mediated enzymatic oxidation method, namely laccase (Boubonnais and Paice, FEBS LETTERS, vol. 267(1), pp. 99-102 (1990), WO 94/29510) and tyrosinase (WO 94/29510). Unlike peroxidases, in which the active redox center serves as a prosthetic hem-comprising group, in the case of lakas and tyrosines the electron transfer catalyzed by using four copper). However, the mediator shall be selected one after the other in a total of four electrons, which are then transferred using chetyrehletnego transfer to the oxygen molecule. While oxygen is reduced with the formation water, and the oxidase is regenerated and can be used in a new reaction cycle. A number of mediators, which catalyze the transfer of electrons from the oxidized substrate for the oxidase described, for example, in WO 94/29510, WO 97/06244, WO 96/10079 or WO 95/01426.

It is known that such mediated mediated enzymatic oxidation methods using oxidase and oxygen is used for coal liquefaction (WO 94/29510), for pulp bleaching (WO 94/29510), in organic synthesis (Potthast, etc., J. Org. Chem., volume 60, page 4320-4321 (1995)), as a bleaching system in detergents and means for washing dishes (WO 97/06244), for bleaching denim (WO 96/12846), to prevent re-staining when washing (WO 98/23716) or for the decomposition of polycyclic aromatic hydrocarbons (Johannes and others, Appl. Environ. Biotechnol., volume 46, pages 313-317 (1996)).

Since, however, the mediators have properties that are in conflict with the terms of the technical application, the enzymatic oxidation methods based on skin is estwe the most effective mediator, is not biodegradable (Amann, 9th international Symposium on chemistry of wood and technical cellulose (Int. Symp. on Wood and Pulp. Chem.), F4-1 to F4-5 (1997)); - many mediators, such as the peso and Vilanova acid in its active form inactivate used oxidase (Amann, 9th international Symposium on chemistry of wood and technical cellulose (Int. Symp. on Wood and Pulp. Chem. ), F4-1 to F4-5 (1997)), and therefore, when using the appropriate method is the high consumption of the enzyme; one of the groups of mediators, with a very high activity, characterized by the presence of the N-O and contains at least one N atom on the molecule mediator (WO 94/29510). Therefore, when the use of compounds containing the group N, there are known problems associated with toxicity and biological razlagaemogo. In addition, biological wastewater treatment in the pulp industry are often not adapted for processing additional N-containing products; - some mediators, such as ABTS, form radicals intense color, which leads to undesirable staining oxidizable substrate; the majority of mediators contain N - or S-atoms, and therefore is relatively the same time for oxidation of substrates.

At Archibald and Roy (Archibald and Roy, Appl. Environ. Environ., volume 58, pp. 1496-1499 (1992) described a method in which as a mediator for the interaction oxidase to oxidize the substrate serves as a redox cascade consisting of phenols and Mn2+/Mn3+. As the final electron acceptor is oxygen system. As the oxidase enzyme is laccase. First laccase oxidizes phenols such as meta-hydroxybenzoic acid or meta-cresol, serving as the primary substrate. Then in the presence of complexing agents, such as pyrophosphate ions, with the participation of oxidized phenols may occur secondary oxidation of Mn2+to Mn3+. However, this does not say that such a system can be used naftalene substrates. In addition, there is no evidence that using a system in which for the oxidation of lignin used only laccase, Mn2+and complexing agent, you can delignification. This is mediated by mediator system enzymatic oxidation of no significant technical advantages in relation to other mentioned systems, because as mediators used is of editorof prevent the application of this method for Toxicological reasons. In addition, the practical application of this system is also hampered by a prolonged reaction time of more than 15 hours of

Based on the foregoing, the present invention was based on the task of developing a multi-component system for mediated mediated enzymatic oxidation of substrates, which would not have the disadvantages inherent in the known multi-component systems, and which, moreover, would be simple and cheap processing the substrate being oxidized.

This problem is solved using a multi-component system comprising the oxidation catalyst, the oxidant and the mediator, characterized in that the oxidation catalyst is chosen from the group of manganese oxidase, (b) an oxidizer selected from the group including oxygen and oxygen-containing compounds, b) the mediator is chosen from the group of compounds containing manganese ions.

Multicomponent system according to the invention preferably includes a complexing substance selected from the group of complexing agents that can form complexes with ions of MP.

In the context of the present invention under the manganese oxidase understand such oxidase, which directly oxidizes MP of ILSI, which have the ability in the presence of oxygen and complexing agents to oxidize MP2+to MP3+.

Preferred are manganese oxidase, which as a redox catalytic groups contain copper ions.

Manganese oxidase can be obtained, for example, from known microorganisms. For this purpose, such microorganisms are cultured under conditions in which they produce manganese oxidase. In the composition according to the invention as manganese oxidase in the most simple case can be applied cell preparations obtained by mechanical, enzymatic or chemical means from the full microorganisms containing manganese oxidase. However, you can also apply containing manganese oxidase supernatant culture or dedicated manganese oxidase.

Manganese oxidase can be produced, for example, such microorganisms as Leptothrix discophora, Bacillus SG-1 and Pseudomonas sp. (Nealson and others , Metal ions and bacteria, p. 383-341, Ed. by Beveridge and Doyle, published by John Wiley and Sons, Inc., New York (1989)). Genes encoding manganese oxidase from Leptothrix discophora and Bacillus SG-1 were cloned and sequenced (Corstjens and others, Geomicrobiol. Journal, volume 14, pages 91-108 (1997) and van Waasber is siyasah blue copper binding copper sequence motif and their activity as manganese oxidase reinforce by adding copper.

Along with the mentioned producing manganese oxidase by microorganisms as a source of manganese oxidase can also be used and other microorganisms. So, for example, as a source of enzymes in the method according to the invention can be applied to microorganisms, in which the manganese oxidase deposited in disputes, such as, for example, Bacillus SG-1, and a dedicated disputes.

In addition, can be applied manganese oxidase who receive recombinant method. Under recombinant method of obtaining it should be understood that any methods by which genes encoding manganese oxidase, extracted from natural producers and then using the known methods is inserted into the corresponding producing strains, which, in particular, can also be a source producers of enzymes.

In the composition according to the invention as a mediator ions are manganese. They can be used in the process with any degree of oxidation.

Preferably used manganese ions with oxidation state +2 or +3.

Manganese with oxidation state +2 is preferably used in the form of sulfate Marga is th mn containing complex. Examples of such mn containing complex is manganese/formate, manganese/lactate, manganese and/or manganese oxalate/malonate.

The mediator (e.g., ion MP3+) accepts an electron from oxidizable substances. As a result, the substance oxidized, and the ion MP is restored (for example, to ion MP2+). In this form ion MP transfers electron accepted for manganese oxidase, and gives it again oxidized to the original oxidation state (for example, to ion MP3+). For manganese oxidase oxidant is oxygen, which is thus the ultimate electron acceptor.

Oxygen can be formed directly during the reaction using known chemical or enzymatic produce oxygen systems. It may also be formed as a result of electrohydraulic water or you can add it to the environment directly in gaseous or liquid form.

Preferably the oxygen is used either directly in gaseous form or liquid form or in the form of oxygen-containing gas mixture, for example air. Especially preferably the oxygen from oxygen-containing gas mixture such as air.

Direct abraspungo substances.

As complexing agents preferably used such compounds that form complexes with Mn3+and as a result gain stability.

Preferably used complexing agents, which do not contain nitrogen, readily biodegradable and toxicologically safe. Such complexing agents are, for example, formate, lactate, malonate or oxalate.

The system according to the invention has, compared with the known systems the following advantages: - when using the composition according to the invention does not occur the problem of inactivation of oxidoreductases, as is, for example, when using peroxide as the electron acceptor, as in this case, the oxidant and the final electron acceptor is oxygen;
- adding oxygen in sufficient quantities is technologically simple operation;
manganese oxidase unlike peroxidases and manganese peroxidases do not require for their activation, the presence of prosthetic groups, such as hem-comprising groups. Therefore, manganese oxidase can be obtained without difficulty in the industrial maccready or painted or poorly biodegradable, or toxic not safe mediators. As a redox mediator serve only ions of manganese, which change two oxidation States, preferably the oxidation States +2 and +3. In contrast to inactivation of known mediators, for example, the chemical reaction does not occur inactivation of this manganese mediator.

The composition according to the invention allows to carry out the oxidation of various substrates. Preferably it should be used for the oxidation of these substrates, which can oxidize Mn ions. Especially preferably it should be used for the oxidation of these substrates that can be oxidized ions of Mn3+.

Multicomponent system according to the invention can be used, for example, as a teeth whitening system in detergents and means for washing, bleaching of pulp for bleaching denim, wastewater, organic synthesis, to prevent re-staining when washing or for the decomposition of polycyclic aromatic hydrocarbons. Another possible application is, for example, the liquefaction of coal. Corresponding methods are described in the prior art to be mediated mediadevicecache known method presented in this description of the components and conditions of the implementation process, to offer the composition can be applied in the above order.

In addition, the present invention relates to a method for oxidation of the substrate, characterized in that the manganese oxidase in the presence of oxygen and, if necessary, compleksoobrazutee substances capable of forming complex ions with MP, forms MP3+by direct oxidation MP2+, ion MP3+oxidizes the substrate, in this case, he is restored to MP2+and again can be used for the direct oxidation of manganese oxidase.

In the method according to the invention, the substrate preferably used in the form of an aqueous solution, mixture or suspension.

In the method according to the invention is preferably used from 0.001 to 50 mg of active manganese oxidase per liter of volume of the reaction mixture.

Manganese oxidase preferably contribute to the reaction mixture in the form of granules, in the form of a solution, in the form of a suspension or in combination with a carrier.

Particularly preferably manganese oxidase contribute to the reaction mixture in the form of a suspension which contains from 0.5 to 50 wt.% enzyme in nonionic surface-active substance.

In the method according to the invention as oxidation in the/sup>. Ions with manganese oxidation States of 2+ or 3+ can be applied directly in the form of ions with a degree of oxidation, or they can be obtained in the reaction of manganese ions with other oxidation States such as Mn4+or Mn7+.

Manganese with oxidation state +3 is preferably added to the reaction mixture in the form of soluble mn containing complex such as manganese/formate, manganese/lactate, manganese and/or manganese oxalate/malonate.

In the method according to the invention is preferably used with manganese oxidation States +2. Particularly preferably used manganese sulfate or manganese chloride.

In the method according to the invention is used manganese ions in concentrations of from 0.005 mm to 50 mm. Preferably used manganese ions in concentrations of from 0.05 mm to 5 mm and most preferably in concentrations of from 0.1 mm to 1 mm.

Depending on the particular application desired manganese may already be contained in the oxidizable substrate. An example of a possible application of the method according to the invention, in which, as a rule, do not require the addition of manganese ions from the outside, is the bleaching of pulp. Wood and from the pulp often already in a natural comp>/p>In the method according to the invention oxygen is preferably added at a partial pressure of 0.05-5 bar. Particularly preferably oxygen is added at a partial pressure of 0.1 to 2.5 bar. Most preferably the oxygen is added at a partial pressure of 0.2 to 1 bar.

In the method according to the invention these complexing substances are preferably used in concentrations from 1 mm to 500 mm. More preferably the corresponding complex-forming substances used in concentrations from 5 mm to 100 mm and particularly preferably in concentrations from 10 mm to 50 mm.

The oxidation method according to the invention can be used to oxidize all compounds that can be oxidized ions of Mn3+. The proposed method can be applied, for example, for the oxidation of lignin in the production of paper and cellulose, as oxidative systems in detergents and means for washing dishes, for enzymatic bleaching of dyed fabrics for enzymatic bleaching of textiles, as the system prevent re-staining when washing, for specific oxidation of organic target molecules in organic synthesis, for the oxidative treatment of wastewater for the decomposition of chlorinated preteenie illustrated in the examples.

Example 1
Selection of microorganisms producing manganese oxidase
1.A. The selection of microorganisms that oxidize manganese
Oxidizing the manganese microorganisms, especially fungi and bacteria were isolated on indicator plates according to the method of Krumbein and Altmann (Krumbein and Altmann,wiss. Meeresunters., volume 25, pages 347-356 (1973)).

In the presence of manganese ions with oxidation state of Mn3+-Mn7+barelyevil blue at pH 4-7 is a dye having an intense blue color. At elevated concentrations in berbelanja blue in the presence of the ions Mn is formed is colored blue oxidation product with pH value to 10.

For screening on tablets nutrient medium was supplemented with colorless barelyevil blue (N, N'-dimethylamino-n,n'-triphenylmethane-o-sulfonic acid). At concentrations of 10-4-10-5g barelyawake blue to 100 ml of the nutrient medium is not yet inhibiting the growth of bacteria.

To highlight heterotrophic oxidizing manganese bacteria used the following environment:
3.5 g/l bacto-peptone Difco,
0.8 g/l MnSO4H2O,
100 mg/l FeSO47H2O,
pH of 7.6.

Such indicator plate inflicted containing bacteria samples, such as seawater samples, sediment samples, soil samples, samples from ore dumps, etc. in appropriate dilutions or concentrations so that on one indicator plate grew 100-300 individual colonies.

The bacteria after incubation for 5-10 days on such flat plates formed colonies of blue color (i.e., activity against the oxidation of manganese is associated with cells) or around the colonies in which the supernatant liquid was formed blue halos (i.e., oxidizing manganese enzymes were allocated in the supernatant of the culture), were subjected to further analysis.

1. B. Confirmation of the presence of manganese oxidase in microorganisms oxidizing barelyevil blue
The microorganisms that were isolated as described in 1.and the method can produce manganese oxidase, however, the blue color can also be caused by manganese peroxidase or oxidation of Fe2+. The activity of manganese oxidase was identified according to the method described Boogerd and de Vrind (Boogerd and de Vrind, J. Bacteriol., volume 169(2), pages 489-494 (1987)):
First, the studied microorganisms were grown to the click of the cells were separated by centrifugation (15 min, 10000g, 4oC). Cellular debris derived from cells that formed colonies of blue to the one described in 1.indicator plates were subjected to gel electrophoresis according to the method described below. For cells, which are flat plates formed halos of blue, not containing cell supernatant liquid was concentrated to one-fiftieth of the original volume by ultrafiltration using UV membrane, retaining particles with molecular weight of more than 10,000 Da. Concentrate or cellular debris was mixed with an equal volume of buffer (0.125 M Tris/Cl, pH 6.8, 20% glycerol, 2% LTOs, 10% 2-mercaptoethanol, 0.01% of bromophenol blue). Aliquots of 50 μl of these mixtures were separated by electrophoresis on 10% polyacrylamide gel. After electrophoresis the gel was washed four times for 15 min with deionized water. Then the gel was incubated for 2 h in 100 mm MnCl2in 10 mm Hepes-buffer, pH 7.5. The result of this procedure in samples containing manganese oxidase, in the field, where after electrophoresis the gel was localized manganese oxidase, was formed precipitate of oxide of manganese brown. Discovered in this way the formation of oxide Mar tsotsitaal and in the sample had not attended by other ions, other than Mn2+.

Example 2
Receipt and allocation of manganese oxidase
As described in example 1 method can be selected Bacillus isolates that their disputes have deposits of manganese oxidase. For technical applications such containing manganese oxidase disputes used directly or containing manganese oxidase membrane dispute is first subjected to processing, and then used. Such controversy or dispute shell was prepared as follows.

Cultivation
To obtain containing manganese oxidase dispute the appropriate strain of Bacillus cultivated under aerobic conditions at 25oWith the following environment:
2 g/l peptone (Difco company), 0.5 g/l yeast extract (Difco company), 10 µg/l Fe-adtc, 100 μg/l, sterilized by filtration MnCl24H2O in 50 mm Tris 80% natural sea water, pH 7.0.

After culturing for 10 days, more than 95% of all cells form spores.

Processing of dispute
Spores were collected by centrifugation (30 min, l0000g, 4oC), washed with deionized water and suspended in 10 mm Tris/Cl, pH 7.0 (0.1 g/ml). To obtain membranes dispute this product was subjected to [daln] is but disputes then they were subjected to further processing as follows.

To the suspension was added 50 μg/ml of lysozyme, the product was incubated for 30 min at 37oS, after which the spores were washed in 1 M NaCl, 0.15 M NaCl, 0.1% LTOs and five times with deionized water. Treated thus spores were stored in deionized water at 4oTo apply according to the invention.

Getting shells dispute
The entire procedure, unless otherwise stated, were carried out at room temperature. With the exception of a 1% solution of LTOs and used for rinsing deionized water all solutions contained 10 mm add, pH 7.5 and phenylmethylsulfonyl (PMSF) (0.3 wt%). Purified spores suspended according to the above-described method in 10 mm Tris, pH 7.0 (0.1 g/ml) and thereto was added an equal volume of glass beads (diameter of 10-50 μm). Then the suspension for 15 minutes with 30-second intervals at 0oWith irradiated by ultrasound with a maximum amplitude in the ultrasonic device for cell disruption (firm Sonifier). After treatment, the suspension is incubated for 5 min on ice and then the supernatant was separated from the added glass beads. Thereafter, the glass beads washed twice with 10 mm Tris pH 7.0 in an amount equal VCI, and centrifuged for 15 min at 15000g. Then separated the supernatant, the residue suspended in 10 mm Tris, pH 7.5, and within 30 minutes was treated at 37oWith lysozyme (100 µg/ml). The product is again centrifuged for 15 min at 15000g. The precipitate containing shell dispute, washed with 1 M NaCl, To 0.14 M NaCl, 1% LTOs and 5 times with deionized water.

2.B. Manganese oxidase from Leptothrix discophora
As described in example 1 method can be obtained isolates forming case (vagina) of microorganisms, such as Leptothrix discophora that produce manganese oxidase. Such isolates may be obtained from commercial collections strains (Leptothrix discophora ATCC 51168, Leptothrix discophora ATCC 51169). Especially suitable for the production of manganese oxidase are strains of Leptothrix discophora, which have lost the ability to form a pouch. Such strains arise spontaneously, when forming the plastic strains continuously for a long time cultivated in laboratory conditions (Adams and Ghiorse, Arch. Environ., volume 145, pages 126-135 (1986)). Such derivatives that have lost the ability to form the case, secrete manganese oxidase in the culture medium.

In the next primers lost the ability to form plastic strain Leptothrix discophora, deposited according to the Budapest Treaty in the German collection of microorganisms and cell cultures (DSMZ GmbH, D-38124, Braunschweig) under the registration number DSMZ 12667.

Cultivation:
To obtain manganese oxidase Leptothrix discophora cultivated in an environment that includes the following components per liter of deionized water:
0.25 g/l Difco peptone,
0.25 g/l yeast extract Difco,
0.25 g/l glucose,
0.6 g/l MgSO47H2O,
0.07 g/l CaCl2H2O,
0.015 g/l MgSO44H2O.

Before autoclaving the medium pH value was brought to 7.6 with 1 M NaOH solution.

To obtain manganese oxidase 45 l describes the environment to infect two liters of pre-culture Leptothrix discophora in the same environment. The cultivation was carried out for 40 h at 26oWhen the transmission of air (0.2 volume of air/volume of medium/minute).

Obtaining the enzyme from the supernatant culture:
After 40 h of cultivation was stopped. Cells Leptothrix discophora was separated from the supernatant liquid culture by centrifugation. Containing manganese oxidase supernatant, which was absent clay with molecular weight of more than 10,000 Da. Concentrated supernatant was used as source of manganese oxidase in the examples below.

Example 3
Quantitative evaluation of the activity of manganese oxidase using N,N,N', N'-tetramethyl-para-phenylenediamine (TMPD)
Ions of Mn3+have the ability to oxidize colorless compound CMPD. The oxidation product, "Worcester blue", it has an intense blue colour, the strengthening of this painting can be estimated by photometric at a wavelength of 610 nm.

Measurement procedure:
For each dimension were prepared fresh mother liquor TMPD, representing a 2.1 mm solution TMPD in distilled water. Designed for the assessment of enzyme products were diluted in 10 mm HEPES (N-2-hydroxyethylpiperazine-N'-2-econsultancy acid) (pH 7.5) so that the optical density (OP), measured at a wavelength of 610 nm for 10-minute period using the procedures below assessment activity was in the range of 0.5 to 1.5.

As a source of manganese served 10 mm solution MnSO4in distilled water.

To determine the activity of manganese oxidase in samples of enzymes in parallel, each time mixing 3 ml of the diluted solution Fe is ulali 10 ál stock solution MnSO4. Both samples were mixed and incubated for 10 min at 40oC. After 10 min the samples were centrifuged in a short period of time (15 s, 5000 rpm), after which he determined OP in the sample of the supernatant liquid volume of 1 ml containing MnSO4. As control served as a comparative sample not containing MnSO4. The increased absorbance at 610 nm of 1.0 corresponds to the formation of 100 μm "Worcester blue". For 1 unit (1 u) manganese oxidase took that amount of enzyme, which results in over 1 min to the formation of 1 μm "Worcester blue" from TMPD.

For measurements, described below, was applied each time a concentrated containing manganese oxidase, adosados liquid culture of example 2. b and 50 μl of the suspension shells dispute from example 2.A. The following results were obtained:
The enzyme from OP
Leptothrix discophora - 1,03
Bacillus - 0,85
Example 4
Oxidation in homogeneous condition of the substrate, representing fertility alcohol, depending on different concentrations of manganese and complexing substances
Procedure:
Of the substrate, representing veratility alcohol (3,4-dimethoxybenzyl alcohol (Aldrich company)) cook the AI. To do this each time 22,23 ml of solutions containing complexing agent (50 mm formate, lactate, malate or oxalate; in all cases, the pH value of 7.5, was mixed with MnSO4(0,00, of 0.05, 0.50 mm MnSO4). Then each time added on 0,268 ml stock solution vertiligo alcohol. The mixture was allowed to come to equilibrium within 10 min at 45oWith, then started the reaction by adding 2.5 ml of an enzyme solution (manganese oxidase from Leptothrix discophora, 10 u/ml).

After 8 h the reaction mixture was analyzed using GHUR on the subject of education vertiligo aldehyde. For this purpose, 0.8 ml of test mixture was mixed with 0.4 ml of a solution of H2SO4(0,5 mol/l). 20 µl of this sample was made in the separation column LiChospher 60 RP Select B (Merck 50940) and suirable a mixture of N2O/Meon (65:35). The speed of the flow was 1 ml/min. the Product in the eluate was detected using a UV detector at a wavelength of 275 nm.

In table. 1 presents the number (%) vertiligo alcohol, which according to the method according to the invention in the following conditions were oxidized for 8 h to vertiligo aldehyde.

Example 5
The pulp bleaching
Subjected to delignification with oxygen Kraft pulp from wood is salad buffer (pH 7.5) was brought to the concentration of the cellulose 5%. After this was added MnSO4(0.5 mm). The suspension is homogenized for 60 s using a suitable stirrer. Then in two parallel sample was added each time 5 IU manganese oxidase (Leptothrix, Bacillus) per 1 g of cellulose. The third sample, which was not added to the enzyme served as control. The samples again homogenized for 60 s and were placed in a steel autoclave, which can be maintained at a certain temperature and which can be vented gas. The autoclave was closed, set the oxygen pressure at 3 bar and incubation was performed in an autoclave for 4 hours at 45oC. Then unloaded the reaction mixture. The cellulose was washed with 10 volumes of running water with a temperature of 50oWith, cellulose 70oUsing aqueous alkaline solution of NaOH was extracted fragments of lignin. For the extraction of used 20 g of NaOH per 1 kg of pulp. After extraction, the pulp is again washed with 10 volumes of running water with a temperature of 50oC. Then according to the standard method SCAN-C 1: 77 Nordic Council of wood mass and the paper was determined by the content of lignin in the cellulose, characterized by the so-called Kappa value (see tab.2).

On Traunstein delignification 10.9-27,2%.

Example 6
Preventing re-coloring
Experiments to confirm it is possible to prevent re-staining using an oxidizing system according to the invention is carried out in a heated glass beakers (volume 100 ml). As control was used a mixture of representing a solution of 0.25 g of a commercially available detergent in 50 ml of water. Experiments with manganese oxidase was performed in 50 ml of 30 mm Na-malonate buffer (pH 7.5) containing 0.5 mm nSO4. A mixture of 50 ml volume were first heated to 45oAnd then in these mixtures containing buffer was added, if necessary, each time by 0.5 u/ml manganese oxidase. In the mixtures were placed samples of cotton fabrics (each 5 g), and then each mixture was added 10 ml of pre-heated to 45oWith a solution containing 15 μm Cibacron Marine C-B (firm Ciba) as a test of the dye. After soaking for 1 h at 45oWith the tissue samples were removed from the mixture and washed each time for 1 l of tap water at a temperature of 50oC.

Then tissue samples were washed and dried. After this spectroscopic method was evaluated by the grace of the original samples and the treated samples.

In table. 3 shows a comparison of the density treatments is stout about what is the oxidation system according to the invention can prevent the transfer of the deposited particles of the dye to the samples of cotton fabric. This effect is qualitatively comparable with the effect of commercially available detergents.

Example 7
Removing stains (use as a detergent)
To demonstrate the effectiveness of the proposed method for removing stains conducted experiments by washing. For this purpose, samples of cotton fabric in white (7,57.5 cm) created the standard (normalized) pollution by applying a droplet with a volume of 0.3 ml of an aqueous solution containing 100 frequent. /million dye Evans blue (obtained from Wako Pure Chemical Industry Co., Osaka). Then these tissue samples in glass beakers (volume 100 ml) with heating, under stirring (magnetic stirrer) was treated according to the invention manganese oxidase, MnSO4and the corresponding complex-forming substance in the presence of oxygen. As control served as solutions in which (a) was used only malonty buffer or that b) is not contained MnSO4or in which) was added manganese oxidase.

The experiments were carried out in 50 ml of 30 mm Na-malonate buffer (Agrawal 45oSince, then, the solutions that were supposed to contain manganese oxidase was added to each 1 IU/ml manganese oxidase from Bacillus, respectively, of Leptothrix. In the solutions contributed contaminated samples of cotton fabric. After soaking for 1 h at 45oWith samples of textile fabric was removed from the solutions and each washed with 1 l of tap water at a temperature of 50oC. Then the samples of the textile fabric was dried.

To determine differences in the coloration of the samples compared with the control sample tested without the addition of manganese oxidase and MnSO4using a differential colorimeter (type CR-200, firm Minolta) was measured Y values, and x air-dried samples. For evaluation of the whitening effect achieved by the method according to the invention, to calculate the value of the ratio Z=(1-x-y)Y/y).

In table.4 presents the characteristics of the degree of whiteness tested samples compared with the characteristics of the degree of whiteness of control samples without enzyme and MnSO4.

From the data presented in the table shows that the degree of whiteness of the samples using manganese oxidase and MnSO41.6 and 2.7 points higher than 30 samples without enzyme and Ben, the possibility of using the method according to the invention for the directed synthesis of organic compounds.

a) oxidation of the alcohol group to an aldehyde
In 22 ml of a 30 mm solution of malonate, pH 7.5, containing 0.5 mm MnSO4was added at 45oWith 269 mg (1.6 mmole) of 3,4-dimethoxybenzyl alcohol in 1 ml of ethanol. After exposure for 10 min was added manganese oxidase (40 units). After the reaction for 24 h, the reaction solution was extracted with chloroform and was investigated using NMR spectroscopy. In the case when I attended manganese oxidase from Leptothrix, the yield of 3,4-dimethoxybenzaldehyde was 32%, and in the case when I attended manganese oxidase from Bacillus, the yield of 3,4-dimethoxybenzaldehyde was 27%.

b) Oxidation of alcohols to ketones
In 22 ml of a 30 mm solution of malonate, pH 7.5, containing 0.5 mm nS4was added at 45oWith 196 mg (1.6 mmole) of 1-phenylethanol. After exposure for 10 min was added manganese oxidase (40 units). After the reaction for 24 h, the reaction solution was investigated using GHUR. In the case when I attended manganese oxidase from Leptothrix, the yield of acetophenone was 17%, and in the case when I attended manganese oxidase from Bacillus, the yield of acetophenone was 19%.

In both examples, it has been successful primeneniye serve only as examples to demonstrate the oxidative capabilities of the method and do not limit the range of the synthesized products.

Example 9
Bleaching denim
Cut a square piece dyed denim cloth (9 g/160 cm2) kept at 45oWith in a closed Cup with a volume of 500 ml total liquid volume of 11.5 ml manganese oxidase, 0.2 mm MnSO4and 15 mm solution of complexing substances (oxalate). The pH value of the mixture was 7.0 (15 mm oxalate). 1 g of tissue was added to a 10 IU manganese oxidase. After exposure for 4 h pieces of fabric were washed in running water until until the washing water became colorless. The cloth was dried flat in the dryer, then smoothed and evaluated using an optical spectrophotometer.

The degree of whitening was determined using a spectrophotometer type CM-3700d (firm Minolta) according to the manufacturer's instructions. The measurements were carried out without the gloss and without UV. The grace of the samples was estimated as the percentage of the total reflectivity compared to the standard white (R 457). The value of L* is a measure of lightness (white=100, black=0).

The data for the treated tissue compared with the data for the untreated control fabric. Changing luminance (L*) samples compared with untreated controls rasschitat is 0
Leptothrix - 23,14
Bacillus - of 28.72
Change LordshipsL*approximately 5, may already be 20 determined visually, i.e. the use of both manganese oxidase allows to achieve a significant whitening effect.


Claims

1. Multicomponent system for mediated mediated enzymatic oxidation, comprising the oxidation catalyst, the oxidant and the mediator, characterized in that the oxidation catalyst is chosen from the group margantsovistyh oxidase, (b) an oxidizer selected from the group including oxygen and oxygen-containing compounds, b) the mediator is chosen from the group of compounds containing ions MP.

2. Multicomponent system under item 1, characterized in that it further comprises a complexing substance selected from the group of complexing agents, which are able to form complexes with ions of MP.

3. Multicomponent system under item 1, characterized in that the mediator ions are MP2+or MP3+

4. Multicomponent system under item 2 or 3, characterized in that the complexing substance does not contain nitrogen, easily biodegradable and is executlve either directly in the gaseous state, either in the form of liquid oxygen, or oxygen-containing gas mixture.

6. The method of oxidation of the substrate, wherein the substrate is treated margaritavision oxidase in the presence of oxygen ions and MP.

7. The method according to p. 6, wherein the treatment is carried out in the presence of complexing substances capable of forming complex ions with MP.

8. The method according to p. 6, characterized in that the substrate used in the form of an aqueous solution, mixture or suspension.

9. The method according to p. 6 or 8, characterized in that the ions of manganese is used in concentrations of 0.005 to 50 mm.

10. The method according to PP.6, 7 or 8, characterized in that oxygen is added at a partial pressure of 0.05-5 bar.

11. The method according to any of paragraphs.6-9, characterized in that the complexing substances used in concentrations from 1 to 500 mm.

 

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