WO2007104316A1

WO2007104316A1 - Coating of carbohydrate materials

Info

Publication number: WO2007104316A1
Application number: PCT/DK2007/000130
Authority: WO
Inventors: Peter Fojan; Oene Robert Veltman; Steffen Bjørn PETERSEN
Original assignee: Aalborg Universitet
Priority date: 2006-03-16
Filing date: 2007-03-16
Publication date: 2007-09-20

Abstract

The present invention relates to a suspension for coating a carbohydrate material and for reducing the degradation of a carbohydrate material. In particular the present invention relates to a suspension comprising a protective agent and an enzyme for coating a carbohydrate material and for reducing the degradation of a carbohydrate material.

Description

COATING OF CARBOHYDRATE MATERIALS

Technical field of the invention The present invention relates to a suspension for coating a carbohydrate material and for reducing the degradation of a carbohydrate material. In particular the present invention relates to a suspension comprising a protective agent and an enzyme for coating a carbohydrate material and for reducing the degradation of a carbohydrate material.

Background of the invention

Carbohydrate materials, such as wooden material, e.g. on houses, in a yard or a fence, will be exposed to rain water, a part of which will be retained by the wood due to its ability to transport water through the stem of a tree from the roots to the leaves. This increases the water activity in the carbohydrate material, such as wood, and will provide ideal growth conditions for growth of micro-organisms, such as fungi, on the surface of the wood, since micro-organisms, such as fungi, need a certain water activity as well as a substrate they can feed upon. Growth and activity of these micro-organisms may cause excretion of enzymes which degrade the carbohydrate materials, such as cellulose, hemi-cellulose and lignin

Different methods have been proposed in the prior art to protect the carbohydrate material from degrading. These methods describe the sequential use of enzymatic treatment of the carbohydrate material using enzymes having cellulolytic, hemicellulolytic, ligninolytic or pectinolytic activity in order to create an improved accessibility for the impregnation solution.

Hence, it is of interest for the public to obtain an improved suspension and an improved method for coating of carbohydrate materials with a protective agent, which is easy to handle, reproducible, environmental friendly and which display improved protective activity. Such a suspension and such a method have been provided by the present invention.

Summary of the invention Accordingly, it is an aspect of the present invention to provide a suspension comprising a protective agent and an enzyme. Another aspect of the present invention relates to a carbohydrate material coated with a protective agent.

Yet another aspect of the present invention is to provide a method for coating a carbohydrate material with a protective agent, said method comprising the step of treating the carbohydrate material with a suspension according to the present invention resulting in the carbohydrate material being coated with the protective agent.

Still another aspect of the present invention is to provide a method for delaying and/or reducing the degradation of a carbohydrate material by treating the carbohydrate material with a suspension according to the present invention resulting in the carbohydrate material being coated with a protective agent.

Brief description of the figures

Figure 1 shows a molecular representation of the β 1-4 linked D-glycopyranoside structure of cellulose, showing the internal hydro bonding network responsible for its crystalline appearance and relatively high chemical resistance for a carbohydrate.

Figure 2 shows a schematic representation of the β 1-4 linked structure of hemicellulose polysaccharide backbone and examples of branching carbohydrate groups. Due to the presence of the various branching groups the formation of a crystalline structure like cellulose is precluded. The open accessible structure of hemicelluloses leaves it substantially more accessible to enzymatic modification,

Figure 3 shows a schematic representation of the random molecular structure of the lignin polymer.

Figure 4 shows a schematic representation of the difference between a hydrolase catalysed enzymatic esterification reaction (A) and a hydrolase catalysed enzymatic transesterification reaction (B). The esterification reaction used an acyl acid as substrate (A), whereas the transesterification reaction used an acyl ester as substrate (B). In the invention the acyl acid and acyl ester represent e.g. the free lipid form of acypetacs (A) or the (vinyl) esterified variants of acypetacs. The depicted HO-R' group represents an accessible hydroxyl group of cellulose, hemicellulose or lignin. Figure 5 shows an overview of the synthesis pathway of the main natural building blocks of lignin, using phenylalanine as the basic ground substrate. Laccases and other peroxidises are involved in the final step of lignin polymer formation, and

Figure 6 shows structural representations of the building blocks of lignin. The invention proposes derived esters, where the methyl ester groups of these substrates are substituted by longer lipid side chains that might be branched and/or halogenated.

The present invention will now be described in more detail in the following.

Detailed description of the invention

The concept provided by the present invention relates to a suspension suitable for delaying and/or preventing degradation of a carbohydrate material by coating a protective agent to the surface of the carbohydrate material.

In the present context the term "coating" relates to the covalent attachment of the protective agent to the carbohydrate material. Furthermore, the covalent nature the attachment between the protective agents, such as antifungal chemicals agents, and the carbohydrate material will only be released upon hydrolytic attack of the carbohydrate material, such as wood.

The present invention relates to the delay and/or prevention of the degradation of carbohydrate material, such as wood, by enzymes from e.g. microbiological or fungal sources.

The present invention relates to a suspension suitable for this purpose and an environment friendly method to covalently attach a protective agent, such as short chain fatty acid lipids (acypetacs), to the enzyme accessible carbohydrates and lignin surfaces.

The invention further describes protecting or weather proofing parts of wood and timber towards wetting and/or fungal attack and degradation. Furthermore, the concept presented in the present invention may be by the skilled person to synthesize special fine- chemical derivatives using celluloses, hemicelluloses or lignin as a base.

An advantage of coating protective agents to the carbohydrate material may be that decreased amounts of the effective agents may be used. Furthermore, due to their covalent nature the protective agents, such as antifungal chemicals agents, will only be released upon hydrolytic attack of the carbohydrate material, such as wood. In conclusion this process will largely reduce the amount of environmental strain and significantly reduce the health risks for humans in handling and application.

The suspension In a preferred embodiment of the present invention a suspension comprising a protective agent and an enzyme may be provided.

In the present context the term "suspension" relates to the mixture of at least one protective agent and at least one enzyme. Preferably, the protective agent and the enzyme are substantially completely dissolved in the suspension. In the event the protective agent and the enzyme are substantially completely dissolved the suspension may be in the form of a solution.

In one embodiment of the present invention the mixture of at least one protective agent and at least one enzyme forms two (or more) phases.

In a further embodiment of the present invention the suspension may be an aqueous suspension, a suspension based on organic solvent(s) or a suspension based on oil.

The protective agent

Most protective agents used as preservative in carbohydrate materials, such as wood, are toxic. The following list indicates some of the toxic protective agents and their degree of toxicity expressed in LD50.

2-phenylphenol (LD50 = 2480) mild skin irritant, endocrine disruptor, toxic to fish

3-iodo-2-propynyl-n-butyl carbarmate (LD50 = 1470) cholinesterase inhibitor carbendazim (LD50 = 10000) endocrine disruptor copper sulphate (LD50 = 300) weight loss, kidney and liver damage cypermethrin (LD50 = 250) endocrine disruptor, mild skin and eye irritant, possible skin sensitizer lindane (LD50 = 76) very toxic orally, skin, eye and respiratory tract irritant, evidence of chronic disease, carcinogenicity and mutagenicity pentachlorophenol (LD50 = 80) foetotoxic, skin and eye irritant permethrin (LD50 = 500) endocrine disruptor, mild skin and eye irritant, skin sensitizer pirimiphos-methyl (LD50 = 2018) cholinesterase inhibitor, mild skin and eye irritant sodium fluoride (LD50 = 180) highly toxic tributyltin oxide (LD50 = 224) irritating to skin, eyes and respiratory tract, considered to be teratogenic acypetacs zinc (LD50 = 1935) azaconazole (LD50 = 308) benzalkonium (LD50 = 400) borax (boric acid, boric oxide, sodium tetraborate, disodium tetraborate) (LD50 = 4500) dichlofluanid (LD50 = 5000) furmecyclox (LD50 = 3780) oxine copper (LD50 = 10000) propiconazole (LD50 = 1520) tebuconazole (LD50 = 4000) tributyltin naphthenate (LD50 = 224)

The LD50 determines the dose of a chemical (in mg/kg) with the potency to kill 50% of a sample group of animals; in this case it applies to rats dosed orally.

The following list indicates other toxic protective agents, however the degree of toxicity of the protective agents is not known:

2-(thiocyanomethylthio) benzothiazole; 2-methyl-4-isothiazolin-3-one; 5-chloro-2-methyl- 4-isothiazolin- 3-one; acypetacs copper; alkylaryltrimethyl ammonium chloride; alkyltrimethyl ammonium chloride; ammonium bifluoride; arsenic pentoxide; chromium acetate; chromium trioxide; copper carbonate hydroxide; copper naphthenate; copper oxide; copper acypetacs (C8-C12); copper versatate; creosote / coal tar creosote; dialkyldimethyl ammonium chloride; disodium octaborate; disodium tetraborate decahydrate; dodecylamine lactate: dodecylamine salicylate; methylene bis(thiocyanate); pentachlorophenol laurate; potassium 2-phenylphenoxide; potassium dichromate; sodium dichromate; sodium pentachlorophenoxide; TC oil tri(hexylene glycol) biborate; tributyltin phosphate; zinc naphthenate; zinc octoate; and zinc versatate.

Some non-toxic protective agents may be found among certain fatty acids, such as C8-C10 fatty acids with saturated chains, which can be branched or unbranched, constitute a class of fatty acids that may display fungicide properties (acypetacs) (Fig. 5). Protective agents, such as acypetacs, presently used are in the form of copper and zinc acypetacs, but these are considered toxic. Benzamacril belongs to another class of substances displaying fungicide properties containing a free carboxylic acid group, which can be accessed by a lipase in an esterification reaction to modify the surface of the carbohydrate material, such as (hemi-) cellulose.

Additionally, the esterified compounds belonging to the class of acyl-amino acid fungicides (e.g., benalaxyl, metalaxyl or furalaxyl) can be chemically bound to celluloses and hemicelluloses by means of a trans-esterification. In an embodiment of the present invention the protective agent does not include a metal ion, such as cupper, nickel, zinc or the like.

Cellulose material is a natural polymer and one of the main constituents of wood. Cellulose (and other carbohydrate materials) is composed of sugar units. These sugar units have several free hydroxyl groups, which can be the alcoholic reaction partner in an enzymatic catalysed (trans-)esterification reaction, resulting in novel modified cellulose materials (and other carbohydrate materials). The resulting novel cellulose materials (and other carbohydrate materials) will be hydrophobic due to the nature of the lipidic coat and thereby be water repellent. Furthermore since these lipids used for the modification of the cellulose do display fungicide properties this coat will prevent fungal growth on the surface of the material.

The invention concerns a method for binding protective agents, such as acypetacs and other microbicidal compounds, covalently to the carbohydrate material, such as wood, that need protection. Due to the covalent nature the invention and applications additionally are less environmentally taxing. Covalent modification may be expected to have distinct advantages over regular application of protective agents, such as acypetacs. Due to the fact that these protective agents, such as acypetacs may be covalently bound and not associated with copper and zinc they are significantly less toxic in handling. Furthermore, as the protective agents, such as fungicides may be covalently bound they will stick better to the matrix, as they cannot be removed by rain water. For application where the carbohydrate material, such as wood, may be applied in soil the covalent attachment, leaching into the ground water will be prevented.

Covalently bound protective agents, such as acypetacs, may help to increase the hydrophobicity of the carbohydrate material, such as wood, at its molecular surface and will create a hydrophobic water repelling layer that reduces the ability of water to penetrate the wood matrix and interact with the cellulose, hemi-cellulose and lignin constituents of the wood. Minimizing water activity in carbohydrate materials, such as wood, may be of great importance for preventing fungi colonization and subsequent invasion and breakdown of the wood.

Another advantage of optimising the surface characteristics might be that increased hydrophobicity will likely deteriorate the recognition, the binding affinity and subsequent attachment of the cellulose binding domain of cellulases, thus preventing or slowing down the breakdown of the cellulose. In an embodiment of the present invention the protective agent may be either a repellent agent or an antibiotic agent. Preferably the repellent agent may be a water repelling agent or a micro-organism repelling agent.

In a further embodiment of the present invention the antibiotic agent may be selected from the group consisting of a fungicide, a bactericide, a biocide, an insecticide, a molluscicide, a rodenticide, a nematicide, a virucide and an algicide.

In still a further embodiment of the present invention the protective agent may be selected from the group consisting of saturated fatty acids having from 8 to 10 carbon atoms (branched or unbranched), acypetacs, benzamacril compounds, acyl-amino acid compounds (such as benalaxyl, metalaxyl or furalaxyl) and alkyl-chains or esters having 4- 14 carbon atoms (in particular from 8-12 carbon atoms) optionally branched.

Preferably the alkyl-chains or esters having 4-14 carbon atoms may be substituted with a halogen, such as fluorine, chlorine or bromine.

In an embodiment of the present invention the protective agent comprises a carboxyl group.

In still an embodiment of the present invention the protective agent may be selected from unclassified fungicides, such as acypetacs or benzamacril, acyl-amide acid fungicides, such as metalaxyl, strobilurin fungicides, such as azoxystrobin or pyraciostrobin, or benzimidazole fungicides, such as carbendazim.

Preferably, the protective agent may not be obtained from the carbohydrate material or a derivative thereof.

In another embodiment of the present invention the protective agent is not a phenolic compound and/or does not comprise a phenolic compound.

A matter of concern might be photochemical (light induced) degradation of the coated antibiotic compounds (in this patent used in the widest definition, including fungicidal, insecticidal, bactericidal, algicidal, molluscicidal, rodenticidal, nematicidal and virucidal compounds as well as antifeedant - and repellent compounds) by mainly UV light. UV light interacts with a carbohydrate material, such as wood, e.g. via two main principles, scattering and absorption. By virtue of their crystalline nature, cellulose fibres of the carbohydrate material may scatter light deeper into the carbohydrate material, such as wood, whereas the poly-aromatic lignin molecules absorb UV light (Barsberg et al., 2003) and thus efficiently limit penetration of the UV light into the wood matrix. Excited double bonds become susceptible towards chemical modification: oxidation reactions, and thus might destroy aromatic antibiotic protectants. At the present, not much is known about the penetration depth of UV light into the bare carbohydrate material, such as wood. This penetration depth depends upon the density of carbohydrate material, such as wood, and the wavelength of the incoming light; its content of lignin and also its colour hence might vary considerably amongst different species of carbohydrate material, such as wood. Lignin due to its poly-aromatic compound composition efficiently absorbs UV light. However, lignin is also capable to act as a sensitizer, which means that UV exited lignin can create singlet oxygen that might result in photo-oxidation and photo-yellowing of the wood (Ross et al., 1998). However, this effect will due to the limited penetration dept of the UV light occur mostly at the surface of the carbohydrate material, such as wood.

The protective agent, such as antibiotic compounds, that have double bonds or aromatic moieties aimed for wood protection are all, to varying degrees, sensitive towards ambient UV light. Antibiotic compounds like aliphatic acypetacs that do not contain double bonds are almost insensitive towards ambient UV light, and can be applied easily onto the surface of carbohydrate material, such as wood. The more UV sensitive antibiotic compounds are more sensibly applied using impregnation techniques, where due to the higher penetration depths the antibiotic compounds are coated also on inside and hence are protected from the UV light by the carbohydrate material, such as wood. The combination of varying antibiotic compounds can largely increase the effectiveness of the protective capabilities. Impregnating wood with a combination of mentioned compounds, which have varying photochemical stabilities, will provide a way of applying light sensitive chemicals and still efficiently protect carbohydrate materials, such as wood, against biologic degradation. The light sensitive compounds will protect the carbohydrate material, such as wood, on the inside while more light stable compounds will still effectively protect the carbohydrate material, such as wood, on the outside.

In another embodiment of the present invention cocktails comprising two or more protective agents may be provided. Preferably, the cocktail comprises 3 or more protective agents, such as 4 or more protective agents, e.g. 5 or more protective agents. The cocktails may provide a possibility for broadening protection towards different classes of plagues and, additionally, will significantly lower the chance that an organism will develop a resistance or way of evading the toxic effect of an applied antibiotic.

UV light induced degradation of the antibiotic compounds may virtually be absent, when the compounds are applied as a colour pigment based paint, which effectively shields the sensitive compounds from light induced degradation. In an embodiment of the present invention it is proposed to the use of longer aliphatic (C4-C14, especially in the range of C8-C12) alkyl chain variants to substitute for the methyl ether groups of Coniferyl alcohol and/or Sinapyl alcohol (or homologue phenolic compounds) as substrates to be used for a covalently bound hydrophobic chemical coating of carbohydrate material, such as wood. These aliphatic chain variants may be linear or branched (e.g., Acypetacs like) or alternatively substituted with halogenated (Fluorinated, chlorinated or brominated). Halogenated variants are more resistant to microbial degradation, and in case of fluorination, are more hydrophobic than regular lipid tails. Some reactions catalysed by laccases are facilitated by using a reaction mediator. A reaction mediator improves on the reaction efficiency of laccase enzyme. Using a mediator, laccase oxidises the mediator, followed by the oxidation of the substrate (lignin) by the mediator. Alternatively, the reaction mediator (e.g., hydroxy benzotrizole) can be applied as a separate compound, but, preferably, the applied coating substrate intrinsically has the mediator capability.

The enzyme

In an embodiment of the present invention the enzyme may be capable of catalysing the covalent attachment of the protective agent to the carbohydrate material. Preferably, the enzyme may be capable of catalysing an esterification process, a trans-esterification process and/or an enzymatic oxidation reduction of a carbohydrate material.

In another embodiment of the present invention the enzyme may be a hydrolase, an esterase, a lipase, a proteinase, a peptidase or an oxidoreductase, such as laccase or hydrogen peroxidases.

In still an embodiment of the present invention the enzyme may not be obtained from the carbohydrate material.

The carbohydrate material

Carbohydrates materials are molecules with many hydroxyl groups, usually one on each carbon atom that may act as a functional group. Carbohydrates are the most abundant biological molecules, and fill numerous roles in living things, such as the storage and transport of energy and structural components in plants and trees.

Furthermore, carbohydrate materials may form basis for many different purposes, e.g. wood for building houses and for the interior of houses, cellulose for bandages, diapers or tampons. In a preferred embodiment of the present invention a carbohydrate material coated with a protective agent may be provided.

In a further embodiment of the present invention the carbohydrate material may be cellulose, hemi-cellulose and/or lignin.

In yet an embodiment of the present invention the cellulose, hemi-cellulose and/or lignin is in the form of wood, timber, tampons, diapers, bandages, implants or coatings.

The coating process

The present invention relates to a protective coat for a carbohydrate material, which fe may be achieved by a specific surface modification of the carbohydrate material through an enzymatic esterification or trans-esterification process (see Fig. 4) or by an enzymatic oxidation reduction of the carbohydrate material.

In addition this treatment of the carbohydrate material, such as wood, by applying pressure the protective agent may also penetrate into the deeper layers than just the surface of the carbohydrate material, such as wood, and modify the cellulose contents inside the carbohydrate material and thereby protecting it from fungal rot. In a way this novel coat may provide a dual protection against fungal attacks, by repelling water from the surface and thereby reducing the water activity and the protected agent itself, attached to the carbohydrate material, may display antifungal properties.

Mode of transesterification In the enzymatic (trans-)esterification of carbohydrate materials, such as cellulose (see Fig. 1), hemicelluloses (see Fig. 2), the reaction may occur most efficiently at the most accessible and most acidic hydroxyl groups of the sugar residues. Due to the β 1-4 bound D-glucopyranoside dimeric structure of cellulose, its hydroxyl groups are not that solvent accessible from the plane of the cellulose backbone (Fig. 1), which results in relatively low (trans-)esterification of its hydroxyl groups.

In contrast, hemicelluloses such as D-Xyloglucan and D-Xylan can be (trans-)esterified more efficiently, due to the exposed nature of its hydroxyl groups of the sugar molecules that are branched of from the cellulose backbone (Fig. 2). These branched sugar moieties comprise mainly of D.-Xylopyranose (D-Xyloglucan), D-Mannans and D-Galactans.

Alternatively, lignin (see Fig. 3) may be enzymatically coupled to lipid. It would also increase on the surface area. Additionally it gives an opportunity to have an additional or backup option in case the efficiency of esterification is too low. The lignin component of lignocellulose consists of a large polymer of inter cross-linked aromatic alcohol units (Fig. 3). The main three aromatic alcohols building blocks used are p-Coumaryl alcohol, Coniferyl alcohol (p-Coumaryl mono-methylether) and Sinapyl alcohol (p-Coumaryl di-methylether) (Fig. 6, 7). In addition, grasses and dicotyl lignin also contain substantial amounts of aromatic acids (e.g., ferulic and p-coumaric acid) that are esterified to the alcohol groups of each other and other alcohols such as mentioned aromatic alcohols.

Laccases (benzenediol: oxygen oxidoreductases) are capable of using the aromatic alcohol substrates to further attach additional units lignin polymer (Mayer & Staples, 2002). Laccases belong to a family of multi-copper oxidases capable of performing one-electron oxidations on a variety of aromatic substrates.

In a preferred embodiment of the present invention a method may be provided for coating a carbohydrate material with a protective agent, said method comprising the step of treating the carbohydrate material with a suspension according to claims 1-9 resulting in coating the carbohydrate material with the protective agent.

In another preferred embodiment of the present invention a method may be provided for delaying and/or reducing the degradation of a carbohydrate material by coating the carbohydrate material with a suspension according to any one of claims 1-9 resulting in coating the carbohydrate material with a protective agent.

Preferebly, the treatment may be conducted under pressure, preferably using the "Bethell process". The "Bethell process" is known for the person skilled in the art.

In an embodiment of the present invention the protective agent and an enzyme may not originally be present in the carbohydrate material or being derived from the carbohydrate material.

The enzymatic activity (and thus the coating process) may be stopped by increasing the temperature.

Further embodiments

In an embodiment the suspension according to the present invention may be used for coating cellulose, hemi-cellulose and/or lignin, such as wood, timber, tampons, diapers, bandages, implants or coatings. Preferably, the wood may be either for indoor use, such as for basements, attics, saunas etc. or for outdoor use, such as for houses, yards or fences. In another embodiment the suspension according to the present invention may be used for reducing degradation of cellulose, hemi-cellulose and/or lignin, such as wood, timber, tampons, diapers, bandages, implants or coatings. Preferably, the wood may be either for indoor use, such as for basements, attics, saunas etc. or for outdoor use, such as for houses, yards or fences.

A preferred embodiment of the present invention

In one preferred embodiment of the invention, the carbohydrate material, such as wood, may be dried and residual moisture within the wood cells and cavities is evacuated by applying a vacuum it in a barometric chamber according to the "Bethell process" wood preservation method. In the following step, the carbohydrate material, such as wood, may be treated with a concentrated mixture of the enzyme, such as lipase, and protective agent or a cocktail of protective agents, such as Acypetacs or/and alternative fungicide ester mixture, in water or organic solvent while pressurising the chamber. This forces the suspension on and into the wood, increasing the efficiency of the impregnation process and enzymatic activity within the carbohydrate material, such as wood. Then the carbohydrate material, such as wood, with suspension may be incubated at optimal working temperature of the enzyme yielding the most efficient (trans-)esterification activity of the enzyme, such as lipase. After the enzymatic process step has covalently bound the antifungal agents on the carbohydrate material, such as wood, a second vacuum step may be performed to evacuate the carbohydrate material, such as wood, internally from any introduced water or organic solvent. The increased hydrophobicity due to the protective agent, such as acypetacs, may diminish the uptake of water, and thus largely reduce the water activity within the carbohydrate material, such as wood.

In an alternative embodiment, the present enzyme, such as lipase, may be inactivated after ending the (trans-) esterification process by increasing the temperature to thermally denature the enzyme in the impregnation chamber in combination with or before applying the second vacuum step.

In separate preferred embodiment of the invention, carbohydrate material, such as wood, may be dried and residual moisture within the carbohydrate material, such as wood, and cavities is evacuated by applying a vacuum it in a barometric chamber according to the "Bethell process" wood preservation method. In the following step, the carbohydrate material, such as wood, may be treated with a concentrated mixture of the enzyme, such as laccase, and the protective agent or a cocktail of protective agents, such as aliphatic sinapyl alcohol homologues or alternative fungicide ester mixture, while pressurising the chamber. This forces the suspension on and into the carbohydrate material, such as wood, increasing the efficiency of the impregnation process and enzymatic activity within the carbohydrate material, such as wood. Then the carbohydrate material, such as wood, with suspension may be incubated at optimal working temperature of the enzyme yielding the most efficient covalent oxidation-reduction modification of the enzyme, such as laccase. After the enzymatic process step has covalently bound the protective agent, such as hydrophobic antifungal agents, on the carbohydrate material, such as wood, a second vacuum step might be performed to evacuate the wood internally from any introduced water or solvent. The increased hydrophobicity due to the long aliphatic sinapyl homologues will diminish the uptake of water, and thus largely reduce the water activity within the carbohydrate material, such as wood.

In an alternative embodiment, the present enzyme, such as laccase, may be inactivated after ending the oxidation-reduction modification process by increasing the temperature to thermally denature the enzyme in the impregnation chamber in combination with or before applying the second vacuum step.

In a separate embodiment the protective agent and the enzyme may be applied in a water-, organics solvent- or oil based suspension. Preferably, this suspension may be used as maintenance or refreshment coat onto the impregnated wood or timber.

In another embodiment, the suspension may be applied as part of regular water or oil based paint where the covalent binding of the protective agent, such as hydrophobic acypetacs, increases the interaction between the hydrophobic the hydrophobic paint components and the wood, simultaneously reducing the possibility of water to move up between the paint layer and the wood by means of capillary suction.

In separate embodiment the suspension may be applied as a water-based suspension that can be painted onto naked wood. Preferably, said naked wood may be used in the house to prevent the attack of wood by fungi applied indoors in damp areas (e.g., basements, attics, saunas, the insides of wooden garden sheds etc.)

In separate embodiment the suspension may be applied as an oil-based suspension that can be painted onto naked wood. Preferably, said naked wood may be used in the house to prevent the attack of wood by fungi applied indoors in damp areas (e.g., basements, attics, saunas, the insides of wooden garden sheds etc.)

In separate embodiment the (trans-)esterification and/or the enzymatic oxidation reduction modification specific cellulose - or hemicellulose-esters for the use of fine chemicals and/or polymer and/or membranes may be generated. Cellulose derivatives may be used as in cellulose lacquers and paints, or as carrier matrix in separation technology where the invention can be applied to modify the matrix with special carrier groups yielding new possibilities in affinity, hydrophobic interaction, or other kinds of chromatography.

In a separate embodiment the (trans-)esterification and/or the enzymatic oxidation reduction modification inventions may be used to produce materials and surfaces for medically relevant applications. Preferably, the surfaces are antiseptic -, bactericidal or fungicidal (wound) bandages, implant coatings etc.

Items of the present invention

In an embodiment of the present invention a method may be provided for the enzymatic esterification or transesterification of carbohydrate materials, such as carbohydrate oligomers or polymers, more specifically the carbohydrate oligomers or polymers belonging to the class of cellulose and/or hemicellulose(s), and cellulose and/or hemicellulose(s) containing products, methyl-, ethyl- or propyl esterified derivatives thereof or materials.

In a further embodiment of the present invention a method may be provided for the enzymatic esterification or transesterification of lignin bound hydroxyl groups and/or polyoles or hydroxyl group containing materials or surfaces, including methyl-, ethyl- or (iso-)propyl esterified derivatives thereof.

In yet an embodiment of the present invention the esterification or transesterification may use enzymes, more specifically belonging to the class of hydrolases, more specifically being esterases, more specifically from the classes of lipases or proteinases or peptidases.

In another embodiment of the present invention a method may be provided for the enzymatic oxidation-reduction modification of lignin and/or carbohydrate oligomers or polymers, and/or phenolic materials and surfaces, more specifically the carbohydrate polymers belonging to the class of oligomeric or polymeric cellulose and/or oligomeric or polymeric hemicellulose(s), and cellulose and/or hemicellulose(s) containing products or materials. Preferably, the covalent oxidation-reduction modification use enzymes more specifically belonging to the group of oxido-reductases, more specifically from the classes of laccases or hydrogen peroxidases.

In a further embodiment of the present invention two or more coating process may be used simultaneously or sequentially. In an embodiment of the present invention the affinity and/or attachment and/or catalytic activity of degrading enzymes (such as cellulases, cellobiases, hemicellulases, glucanases, xyloglucanases, xylanases and/or laccases) aimed at degrading said carbohydrate materials may be reduced and/or prevented and/or inhibited,.

In still an embodiment of the present invention the covalent application of (trans-) esterifyable carboxylic acid group containing and/or phenolic group attached toxic antibiotic or biocidic agents belonging to the groups of; fungicides or insecticides, or bactericides, or algicides, or molluscicides, or rodenticides, or nematicides, or virucides, or antifeedants, or alternatively, the application of agents repelling organisms belonging to the group of insects, bacteria, algae, rodents, molluscs, nematodes or viruses (repellents), or alternatively compounds may have an inhibiting effect on carbohydrate materials degrading enzymes, such as wood degrading enzymes.

Preferably, (trans-)esterifyable carboxylic acid group containing substrates and compounds, which are water repelling and/or organism repelling and/or killing agents such as acypetacs and/or benzamacril and/or metalaxyl and/or similar or homologous compounds belonging to the same respective chemical classes, and/or alternatively the use of saturated and/or unsaturated aliphatic, and/or more specifically short branched lipids (preferably in the length of C4-C14) or lipid-esters (preferably in the length of C4- C14) that may or may not be halogenated (more specifically being either fluorinated, chlorinated and/or brominated) may be used as protective agents.

The suspension according to the present invention may be applied as paint or as impregnation.

The methods of the present invention may be used in combination with pressure cycling impregnation using the "Bethel process" or processes derived there from or altered variants thereof with varying pressure, application cycles, steps, temperature and time.

The efficiency of (trans-) esterification may be equal to or better than 0.001% or 0.01% or 0.1 % or 1% or 5% or 10% or 20% or 30% or 40% or 50% or 60% or 70% or 80% or 90% of possibly available esterifyable sites based on either location or area or volume or weight.

The efficiency of the enzymatic oxidation-reduction based modification may be equal to or better than 0.001% or 0.01% or 0.1 % or 1% or 5% or 10% or 20% or 30% or 40% or 50% or 60% or 70% or 80% or 90% of possibly available oxido-reductive sites based on either location or area or volume or weight It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

The invention will now be described in further details in the following non-limiting examples.

Examples

Example 1

Modification of cellulose and hemicelluloses using lipases.

Pure cellulose obtained from cotton wool and isolated hemicellulose fractions will be applied to organic suspensions of a set of lipases or alternatively to test the efficiency of esterification vs. trans-esterification using a model substrate lipid and its vinyl-ester variant.

Compare which matrix is the most efficient for enzymatic coating, cellulose or hemicelluloses. Additionally, the feasibility of (trans-)esterifying lignin will be investigated as part of the lignin matrix is covered with carboxylic acid groups.

Additionally will be investigated with lipid kind from a mixture will be most efficiently

(trans-)esterified. The most efficient coating conditions for lipid mixtures like acypetacs will be investigated and parameters will subsequently be optimised. This might be investigated using reverse phase chromatography analysis of the lipid fraction released from the substrate matrix, after being treated with strong base to hydrolyse the ester bonds. The amount and kind of lipid released gives insight into which enzyme to use, time and temperature of enzymatic incubation, which lipid substrate (mixture) and which solvent work best.

Example 2

Modification of lignin using laccases.

Commercially available lignin kinds will be incubated with a small set of laccases together with some commercially obtainable laccase substrates. Investigate to in what degree laccases are capable to coat cellulose and hemicelluloses with the laccase substrate coat. Investigated will be solvents, solvent mixtures, compositional concentration, reaction temperatures, incubation times, and if there is any need for a mediator molecule in covalently binding of the substrate.

The analysis of lignin coating needs to be investigated a little further. But depending on the used substrate one could think of UV or fluorescence spectroscopy methods or gravimetry or reverse phase HPLC as methods for analysis.

Example 3

Research the possibility of combining both the lipid and oxidation reduction substrate enzyme systems into one.

A more qualitative approach will be done in analysing if both methods can be applied in one step. The effect might be counteractive, neutral or ideally synergistic in application of the substrates. Coating efficiency will be analysed using the methods described in the previous points.

Example 4

Pressure cycling application (industrial scale) as a means for analysing the effectiveness in the impregnation of wood.

The working coating protocols will be applied by to small flat sticks of wood. The efficiency of pressure/ vacuum cycling will be compared to regular or heat dried wood strips in chemically binding of the coat towards the wood matrix.

In this way the optimal method for pre-treating the targeted wood (e.g., heat, organic solvent washing, UV irradiation activation) and for applying the enzymatic coat will be investigated.

Claims

1. A suspension comprising a protective agent and an enzyme.

2. The suspension according to claim 1 wherein the enzyme is capable of catalysing an 5 esterification process, a trans-esterification process and/or an enzymatic oxidation reduction of a carbohydrate material.

3. The suspension according to any one of claims 1 or 2, wherein the enzyme is a hydrolase, an esterase, a lipase, a proteinase, a peptidase or an oxidoreductase, such as

10 laccase or hydrogen peroxidases.

4. The suspension according to any one of claims 1-3, wherein the protective agent is selected from the group consisting of a fungicide, a bactericide, a biocide, an insecticide, a molluscicide, a rodenticide, a nematicide, a virucide and an algicide.

15

5. The suspension according to any one of claims 1-4, wherein the protective agent is selected from the group consisting of saturated fatty acids having from 8 to 10 carbon atoms (branched or unbranched), benzamacril compounds, acylamino acid compounds (such as benalaxyl, metalaxyl or furalaxyl) and alkyl-chains or esters having 4-14 carbon

20 atoms (in particular from 8-12 carbon atoms) optionally branched.

6. The suspension according to claim 5, wherein the alkyl-chains or esters having 4-14 carbon atoms is substituted with a halogen, such as fluorine, chlorine or bromine.

25 7. The suspension according any one of the preceding claims, wherein the protective agent comprises a carboxyl group.

8. The suspension according to any one of claims 1-7, wherein the carbohydrate material is cellulose, hemi-cellulose and/or lignin.

30

9. The suspension according to any one of claims 1-8, wherein the suspension is an aqueous suspension, a suspension based on organic solvent(s) or a suspension based on oil.

35 10. A carbohydrate material coated with a protective agent.

11. The carbohydrate material according to claim 10, wherein the carbohydrate material is cellulose, hemi-cellulose and/or lignin.

12. The carbohydrate material according to claim 11, wherein the cellulose, hemi-cellulose and/or lignin is in the form of wood, timber, tampons, diapers, bandages, implants or coatings.

5 13. The carbohydrate material according to any one of claims 10-12, wherein the protective agent is selected from the group consisting of a fungicide, a bactericide, a biocide, an insecticide, a molluscicide, a rodenticide, a nematicide, a virucide and an algicide.

10 14. The carbohydrate material according to any one of claims 10-13, wherein the protective agent is selected from the group consisting of saturated fatty acids having from 8 to 10 carbon atoms (branched or unbranched), benzamacril compounds, acylamino acid compounds (such as benalaxyl, metalaxyl or furalaxyl), alkyl-chains, or esters having 4-14 carbon atoms (in particular from 8-12 carbon atoms) optionally branched.

15

15. The carbohydrate material according to claim 14, wherein the alkyl-chains or esters having 4-14 carbon atoms is substituted with a halogen, such as fluorine, chlorine or bromine.

20 16. A method for coating a carbohydrate material with a protective agent, said method comprising the step of treating the carbohydrate material with a suspension according to claims 1-9 resulting in coating the carbohydrate material with the protective agent.

17. The method according to claim 16, wherein the method involves an esterification 25 process, a trans-esterification process and/or an enzymatic oxidation reduction.

18. The method according to any one of claims 16 or 17, wherein the carbohydrate material is cellulose, hemi-cellulose and/or lignin.

30 19. The method according to claim 18, wherein the cellulose, hemi-cellulose and/or lignin is in the form of wood, timber, tampons, diapers, bandages, implants or coatings.

20. The method according to any one of claims 16-19, wherein the treatment is conducted under pressure, preferably using the "Bethell process".

35

21. The method according to any one of claims 16-20, wherein the protective agent or the enzyme are not originally present in the carbohydrate material.

22. A method for delaying and/or reducing the degradation of a carbohydrate material by coating the carbohydrate material with a suspension according to any one of claims 1-9 resulting in coating the carbohydrate material with a protective agent.

5 23. The method according to claim 22, wherein the carbohydrate material is cellulose, hemi-cellulose and/or lignin.

24. The method according to claim 23, wherein the cellulose, hemi-cellulose and/or lignin is in the form of wood, timber, tampons, diapers, bandages, implants or coatings.

10

25. The method according to any one of claims 22-24, wherein the treatment is conducted under pressure, preferably using the "Bethell process".

26. The method according to any one of claims 22-25, wherein the protective agent and 15 the enzyme are not originally present in the carbohydrate material.

27. The method according to any one of claims 22-26, wherein the method involves an esterification process, a trans-esterification process and/or an enzymatic oxidation reduction.

20

28. Use of the suspension according to any one of claims 1-9 for coating cellulose, hemi- cellulose and/or lignin, such as wood, timber, tampons, diapers, bandages, implants or coatings

25 29. The use according to claim 28, wherein the wood is either for indoor use, such as for basements, attics, saunas etc. or for outdoor use, such as for houses, yards or fences.

30. Use of the suspension according to any one of claims 1-9 for reducing degradation of cellulose, hemi-cellulose and/or lignin, such as wood, timber, tampons, diapers, bandages,

30 implants or coatings.

31. The use according to claim 30, wherein the wood is either for indoor use, such as for basements, attics, saunas etc. or for outdoor use, such as for houses, yards or fences.

35