WO2015036000A1

WO2015036000A1 - Enzyme substrate

Info

Publication number: WO2015036000A1
Application number: PCT/DK2014/050283
Authority: WO
Inventors: William George Tycho WILLATS; Stjepan Kresimir KRACUN; Julia SCHÜCKEL
Original assignee: Københavns Universitet
Priority date: 2013-09-13
Filing date: 2014-09-12
Publication date: 2015-03-19

Abstract

The present invention relates to an enzyme substrate comprising an enzymatically degradable microparticulate plant material having a polysaccharide content in the range of 35%(w/w) to 95%(w/w), the microparticulate plant material having a releasably attached detectable marker, wherein enzymatic degradation of the microparticulate plant material results in release of the detectable marker and to an enzyme substrate composition comprising the enzyme substrate. The invention further relates to uses of the enzyme substrate and to a method for its preparation.

Description

Enzyme substrate

The work leading to this invention has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement n°32497.

Field of the invention

The present invention relates to an enzyme substrate and an enzyme substrate composition. The enzyme substrate allows screening for enzymes capable of degrading plant polysaccharides and lignin and it is suitable for use in the screening of enzymes for use in the provision of fermentable sugars in the production of biofuels. The invention also relates to a kit comprising the enzyme substrate or the enzyme substrate composition and methods of their production.

Prior art Biofuel production from agricultural raw materials has been presented as an alternative to fossil fuels. However, some current technologies generally rely on fermenting sugars and carbohydrates from plant material that can be used as a food source for humans; this is referred to as first generation ("1G") biofuel production. There is therefore an interest in providing technologies that can provide biofuels from plant material not considered to be a source of human food. Such plant materials comprise agricultural waste, such as straw, hay, sawdust, grass, wood waste etc., or plants may be grown mainly to provide biomass for subsequent production of biofuels. Production of biofuel from biomass not considered a source of food for humans is referred to as second generation ("2G") biofuel production. The starting point or 'feedstock' for 2G biofuel production can thus be polysaccharide-rich walls that surround each plant cell. Plant cell walls are the largest source of biomass on earth. The conversion of plant cell polysaccharides into biofuels depends upon the breakdown of the cell wall components into smaller molecules and sugars that can then be fermented into alcohols. However, this breakdown of polysaccharides is dependent on enzymatic degradation, for example by the action of glycosyl hydrolase (GH) enzymes. There is a considerable commercial incentive to produce GHs that are cheaper and more effective because this would decrease the cost of 2G biofuel production and might also allow more diverse feedstocks to be used for 2G production. Many microorganisms (fungi and bacteria) produce GHs and it is relatively easy to identify large numbers of genes that putatively encode GHs. It is also relatively easy to generically alter GHs with the aim of improving their activity. Thus, large numbers of variant and potentially useful GHs can be produced - but their activities have to be tested and this is currently a bottleneck in the overall process of producing and improving GHs.

Furthermore, the presence of lignins and lignin components typically has a negative effect of the fermentation and the removal of lignin may be of interest in the preparation of a substrate for production of e.g. alcohols by fermentation. The lignin may also constitute a significant proportion of the cell wall, and in addition to being a potential inhibitor in a fermentation process, the lignin and lignin components may constitute raw materials of interest.

There is therefore an ongoing interest in screening for enzymes capable of degrading plant materials for biofuel production and in these contexts enzyme substrates are needed for detecting and quantifying enzyme activities. Commonly employed enzyme substrates comprise a detectable marker or the like, such as a dye molecule, that allows detection of an enzyme activity. In the screening of new enzymes it is desirable to use an enzyme substrate that is as close as possible to the substrate in order to mimic the degradation of a raw material as it will occur in an industrial process. Enzyme substrates comprising cross-linked purified polysaccharides labelled with dye molecules are readily available, such as those provided by the company Megazyme (Wicklow, Ireland). The cross-linked purified polysaccharides are insoluble and when degraded by an appropriate enzyme the dye molecule is released into solution, e.g. as a part of a small, dyed oligosaccharide fragment which is excised from the insoluble substrate via the appropriate enzyme that also renders the fragment soluble. The released dyed oligosaccharides produces a coloured supernatant whose absorbance can be measured to reflect the enzyme activity. However, such substrates must be cross-linked, e.g. using bifunctional cross-linking agents such as 1,4-butanediol diglycidyl ether, epichlorohydrin or the like, in order to be insoluble. The cross- linking of the carbohydrate chain modify the chain to such an extent that it no longer sufficiently resembles the substrate to detect and correctly quantify an enzyme otherwise capable of degrading the carbohydrate chain. Also, purified polysaccharides do not closely resemble the real life substrates in plant cell walls which are typically composed of multiple mixed polymers.

A need for enzyme substrates allowing screening for enzymes capable of degrading plant materials therefore exists. In particular, there is a need for enzyme substrates more closely resembling the material encountered by an enzyme in its natural conditions so that a screening using the substrate will more closely reflect the natural situation a degrading microorganism meets.

Cotton is an example of a plant material, and cotton has been dyed since prehistoric times. At present cotton is commonly dyed by coupling reactive dye molecules, such as chloro triazine dyes, to available hydroxyl groups on the surface of cotton threads. However, while cotton consists of more than 90%, e.g. 95%, cellulose dyed cotton is not an appropriate enzyme substrate because the dying chemistry used precludes the effective release of detectable oligomers. Other plants, e.g. flax and hemp, have also been used to provide textile fibres which have been dyed. However, as with cotton, such fibres are inappropriate as enzyme substrates.

Lignin has also been considered as a source of biopolymer, which can be dyed. However, despite the abundance of lignin in plant material no lignin based enzyme substrate has been described.

WO 2000/34401 discloses a preparation of a paint based on lignin. The preparation can involve mixing a lignin solution with a phenol oxidising enzyme, and a dye or pigment, e.g. Berlin Blue. The product of WO 2000/34401 does not appear to be of use in an enzyme assay.

WO 2010/046542 discloses a method for modifying a lignocellulosic material by oxidising phenolic groups or phenoxy derivative groups of the lignocellulosic material to quinines, quinone derivatives or other carbonyl groups, and coupling the oxidised fibre material with a reactive nucleophilic group to form a covalent bond with the lignocellulosic material. The nucleophilic group may be an amine. The method allows modification of the properties of the lignocellulosic material, e.g. colour may be introduced. The method may comprise enzymatic treatment of the lignocellulosic material although the product itself does not function as an enzymatic substrate. There is no mention of using the product as an enzymatic substrate in an enzyme assay.

US 2011/094678 describes various ways to process biomass containing lignocellulosics. For example, it describes the step of mixing and reacting a lignocellulosic with an amino compound and water under increased temperature to produce an amino salt of lignin and amino-lignin-cellulose. US 2011/094678 describes reacting lignin with an amino group, and it is stated that the amino salt of lignin is water soluble. US 2011/094678 does not describe coupling of a dye molecule to the lignin, and there is no indication to use the amino salt of lignin as an enzyme substrate. US 6,426,189 discloses a method for screening of an active such as a biological compound or a nucleic acid sequence encoding a biological compound using a cellulose film comprising microfibrillated cellulose. The film may comprise a substance attached to the cellulose film, e.g. by covalent bonds, by ionic bonds or by hydrogen bonds. The cellulose film of US 6,426,189 mimics cellulose containing textile and may substitute textile or fabric when screening for compounds that interact with cellulose in textile or substances present on a textile surface, for example when searching for new cleaning agents. The microfibrillated cellulose is isolated and purified cellulose fibres. The material described in US 6,426,189 may thus be employed for screening enzymes. However, it is ill-suited for screening for enzymes capable of degrading plant material in a more complex form than the microfibrillated cellulose.

In light of the above it is an aim of the present invention to provide an enzyme substrate of a plant material that allows the facile detection and/or quantification of enzymes capable of degrading a plant materials.

A further aim of the present invention is to provide the enzyme substrate in a form that is convenient and appropriate for high throughput screening (HTS). HTS typically requires small amounts of multiple substrates and the handling of large numbers of different substrates is cumbersome for the end user, especially when only very small amounts are required for each sample to be analysed.

WO 2002/059372 describes a biochip having hydrogel cells attached to the top surface of a solid substrate in the form of an array. The hydrogel cells incorporate a multitude of different binding entities in an array format, which is suitable for high-throughput analysis of biomolecular interactions. The hydrogels are organic polymers that are capped with isocyanate-functional groups to provide isocyanate-functional hydrogels. The hydrogels are formed by polymerising a hydrophilic monomer in an aqueous solution; exemplary hydrogels are polyethylene glycol and polypropylene glycol hydrogels. The binding entity, which may be an enzyme substrate, may be incorporated in the hydrogel during the polymerisation of the hydrogel monomers but it is also possible to load the binding entity into the hydrogel cell after polymerisation of the hydrogel cell. WO 2002/059372 only specifies isocyanate-functional hydrogels and it is not clear how such hydrogels may affect an enzymatic process. In particular, WO 2002/059372 does not mention large carbohydrate or lignin molecules as enzyme substrates and the availability of such large molecules to enzymes may be hindered by the polyethylene glycol and polypropylene glycol hydrogels, especially when polymerisation is performed in situ.

It is the aim of the present invention to provide an enzyme substrate for screening enzymes capable of degrading plant material, e.g. for the provision of fermentable sugars for the production of biofuels.

Disclosure of the invention

The present invention relates to an enzyme substrate comprising an enzymatically degradable microparticulate plant material having a polysaccharide content in the range of 35%(w/w) to 95%(w/w), the microparticulate plant material having a releasably attached detectable marker, wherein enzymatic degradation of the microparticulate plant material results in release of the detectable marker. The enzyme substrate can be used in the screening of enzymes degrading cell wall and/or plant fibre contents, such as polysaccharides, e.g. cellulose, hemicellulose, pectin etc., or the polyphenolic compound lignin. The enzyme substrate is insoluble in water and therefore an enzyme capable of degrading a cell wall and/or plant fibre content to which the releasably attached detectable marker is attached can release the detectable marker to a solution or supernatant where the marker can be detected. Any type of enzyme capable of degrading cell wall and/or plant fibre material may be screened for using the enzyme substrate of the invention, such as for example glycosyl hydrolase (GH) enzymes, ligninase enzymes or laccase enzymes and proteases that degrade the protein moiety of cell wall proteoglycans. Highly purified polysaccharides have traditionally been employed in the prior art to provide enzyme substrates for screening some of these enzyme types. However, several disadvantages are involved in the production of such substrates. For example, purification of the polysaccharides is cumbersome and time consuming, and moreover it is necessary that the polysaccharides are cross-linked in order make the polysaccharide based substrate insoluble; insolubility is an important trait for polysaccharide based enzyme substrates since this allows that a detectable marker is released to a supernatant upon enzymatic degradation. The present inventors have now found that the for the enzyme substrate of the invention, e.g. based on native complex biomass types, no cross-linking is necessary in order to provide insolubility. It has furthermore surprisingly been found that when no cross-linking is present in the enzyme substrate the enzyme substrate provides a more accurate and sensitive screening of cell wall degrading enzymes since a higher level of detectable marker can be detected after enzymatic degradation. It is thus preferred that the enzyme substrate is not cross-linked.

The enzyme substrate of the invention comprises a microparticulate plant material having a polysaccharide content in the range of 35%(w/w) to 95%(w/w), such as up to 90%(w/w). Any plant material, e.g. with a polysaccharide content of up to 95%(w/w), may be used in the invention, and wheat straw, straw, hay, sugar cane bagasse, sawdust, grass and other biomass feedstock types are preferred plant materials. The polysaccharide contained in the plant material may be any polysaccharide found in plants and it is of particular interest that the enzyme substrate contains a polysaccharide that may be degraded by an enzyme to provide sugars, e.g. fermentable sugars. Polysaccharides of interest are i.a. cellulose, hemicellulose and pectin. In a specific embodiment the plant comprises up to 95%(w/w) cellulose, e.g. up to 90%(w/w), up to 85%(w/w), up to 80%(w/w), up to 75%(w/w) cellulose, up to 70%(w/w) cellulose, up to 65%(w/w) cellulose, up to 60%(w/w) cellulose, up to 55%(w/w) cellulose, up to 50%(w/w) cellulose, up to 40%(w/w) cellulose. The total polysaccharide content is not limited by the content of cellulose, and the plant material may for example have a polysaccharide content of up to 95%(w/w) with a cellulose content of up to e.g. 50%(w/w), such as up to 40%(w/w) with the remaining polysaccharides comprising hemicellulose and other polysaccharides. In certain embodiments, the plant material is selected based on the composition of the polysaccharide content and the cellulose content may thereby provide an indication of the contents of polysaccharides other than cellulose. Selecting a plant material, e.g. wheat straw, with a "low" cellulose content, e.g. up to 40%(w/w) or up to 35%(w/w), is thus especially useful for screening for enzymes since it provides a more complex substrate allowing different types of enzymes to act on the substrate and release detectable markers.

The plant material of the enzyme substrate is in a microparticulate form meaning that the plant material comprises particles with a largest dimension of about 500 m and any technology to reduce the particle sizes of a plant material, e.g . disrupt the plant material, is contemplated with the invention. It is preferred that the plant material has been treated to degrade or disrupt the cell walls of the plant material. In particular it is also preferred that plant fibres of the plant material are disrupted, i.e. reduced to have a largest dimension of about 500 pm. Disruption of the plant material will typically disrupt both the cell walls and the plant fibres. Disruption of the cell walls and the plant fibres provides access to the respective components, such as carbohydrates and lignin, so that an enzyme may degrade the carbohydrates and/or lignin allowing the microparticulate plant material to be used in the enzyme substrate. In particular, the accessibility of the enzymes to the plant material provided by the disruption allows effective release of the detectable marker, e.g. as detectable oligomers. Disruption of the plant material, e.g. cell walls and/or plant fibres, further allows removal of undesirable components, such as chlorophyll, pigments and proteins. It is further preferred that the plant material has been lyophilised, in particular that the plant material has been lyophilised prior to disrupting the plant material. Lyophilisation will improve the subsequent disruption of the cell walls and may thereby act synergistically to provide access to cell wall components and also the removal of undesirable components.

The enzyme substrate of the invention resembles the plant material from which it is prepared since it is based on a native complex biomass type. This provides that GH enzymes encounter an enzyme substrate very similar to the substrates the GH enzymes would encounter in nature so that the enzyme substrate will allow screening for GH enzymes, ligninase enzymes, laccase enzymes or the like that are expected to be active in the degradation of a plant material of interest, e.g. in the production of sugars for use in biofuel production. In contrast, an enzyme substrate based on purified polysaccharides, in particular cross-linked purified polysaccharides, or otherwise highly processed polysaccharides, e.g. microfibrillated cellulose or the like, do not resemble the substrates such enzymes will encounter. Inclusion of highly purified or processed polysaccharides in an enzyme substrate may provide an enzyme substrate yielding an incorrect reflection of the enzymatic activity of a sample with respect to degradation of native complex substrate. It is preferred that the enzyme substrate of the invention does not comprise purified or processed forms of polysaccharides present in the plant material, such as microfibrillated cellulose or purified cellulose fibres.

Any releasably attached detectable marker may be used in the invention.

Preferred detectable markers comprise a dye, e.g. with an absorbance in the UV- VIS range, a fluorescent moiety or a luminescent moiety. In particular, the releasably attached detectable marker may be any marker molecule or moiety that can be released from the enzyme substrate when the enzyme substrate is contacted with an enzyme capable of degrading the enzyme substrate to which the releasably attached detectable marker is attached. Thereby, the detectable marker will be released from the enzyme substrate allowing its detection and consequently detection of the enzymatic activity. Enzymatic degradation of the enzyme substrate may provide that the releasably attached detectable marker is attached to an enzyme degradation product after enzymatic degradation of the enzyme substrate. Regardless of the enzymatic activity release of the releasably attached detectable marker indicates that the enzyme substrate has been degraded. Detection of an enzyme activity is, in its broadest sense, achieved by monitoring the detectable marker. For example, the detectable marker may be released to a supernatant allowing its detection in the supernatant. Alternatively, the detectable marker present on the enzyme substrate may also be monitored so that a decrease in amount of detectable marker on the enzyme substrate indicates that the enzyme substrate has been degraded by an enzyme. When both the detectable marker present on the enzyme substrate and the detectable marker released to a supernatant are monitored an improved quantification of the enzyme activity can be obtained since a mass balance of the detectable marker can be calculated. However, even when the detectable marker present on the enzyme substrate is not monitored, monitoring the detectable marker in the supernatant alone provides a sensitive screening of enzymes since only small amounts of the detectable marker need to be detected in the supernatant in order to indicate an enzyme activity.

The releasably attached detectable marker may be attached to the microparticulate plant material using any type of interaction, such as covalent or non-covalent, ionic, or hydrophobic interaction or through a combination of such interactions. It is preferred that the releasably attached detectable marker is covalently attached to the plant material, e.g. to hydroxyl groups of a carbohydrate or hydroxyl or phenol groups of lignin. When a detectable marker is covalently attached e.g. to a hydroxyl group on a polysaccharide chain, enzymatic degradation of the polysaccharide chain can release the detectable marker from the insoluble polysaccharide chain and provide a degradation product comprising the detectable marker still covalently attached to an oligosaccharide degradation product. Release of the detectable marker will show that an enzymatic has acted on the polysaccharide chain and subsequent analysis of the cleaved off oligosaccharide with the attached detectable marker allows that the enzymatic activity is characterised. Oligosaccharide degradation products are typically soluble, e.g. in aqueous solvents, in contrast to the enzyme substrate. Thus, enzymatic degradation of an enzyme substrate to release an oligosaccharide degradation product with an attached detectable marker allows detection of the enzymatic reaction and also characterisation of the reaction. Covalent attachment of detectable markers to polysaccharide chains of a plant material will typically attach the detectable marker to a free hydroxyl group, e.g . on C2, C3 and C6 for amylose with alpha-l,4-linked glucose units, and enzymatic degradation resulting in release of an oligosaccharide with attached detectable marker will show the activity of a GH enzyme. The same considerations are relevant for lignin and lignin degrading enzymes.

It is preferred that the enzyme substrate is used in a hydrogel and in another aspect the invention relates to the enzyme substrate composition comprising the enzyme substrate of the invention in a hydrogel. Any hydrogel is appropriate in the present invention and the hydrogel may contain any buffers, cofactors, coenzymes or the like deemed necessary for detecting an enzyme activity. When the enzyme substrate is in a hydrogel, handling of the enzyme substrate is simplified compared to handling the enzyme substrate in another form, e.g. as a dry powder. For example, the enzyme substrate may be supplied in a reaction vessel, such as a plastic tube or a microtiter plate, and the sample suspected of containing an enzyme capable of degrading the plant material may be added directly to the reaction vessel with the enzyme substrate and following the reaction the detectable marker can be quantified directly in the supernatant, e.g. using an optical method, or the supernatant can be withdrawn from the reaction vessel and the detectable marker quantified in a detection vessel, e.g. a cuvette or the like. The hydrogel with the enzyme substrate advantageously removes the need to settle, e.g. centrifuge, the enzyme substrate before removing the supernatant or quantifying the detectable marker directly in the supernatant. A further advantage of having the enzyme substrate in a hydrogel is that an enzyme present in a sample will encounter the enzyme substrate in a form that is more accessible that when the enzyme substrate, e.g. as a dry powder, is suspended in an aqueous solution.

In another aspect the invention relates to the use of the enzyme substrate or the enzyme substrate composition of the invention in the screening for a plant material degrading enzyme, e.g. a cell wall degrading enzyme, such as a glycosyl hydrolase (GH) enzyme, a ligninase enzyme or a laccase enzyme. The enzyme substrate of the invention is generally used in an aqueous environment, for example the enzyme substrate may be suspended in an aqueous buffer or solution of e.g. cofactors, coenzymes, metal ions or the like and a sample suspected of containing an enzyme capable of degrading the enzyme substrate is then added to contact the sample with the enzyme substrate. When the enzyme degrades the enzyme substrate the detectable marker will be released from the enzyme substrate, e.g. to a supernatant where the detectable marker may be detected or quantified. The detectable marker may be detected in the supernatant, for example after separation of the supernatant from the suspended enzyme substrate or directly in the supernatant. It is further possible to also quantify the amount of detectable marker attached to the enzyme substrate both before and after contacting the sample with the enzyme substrate. It is particularly preferred that the enzyme substrate or the enzyme substrate composition is employed in a high throughput screening (HTS) format.

In yet another aspect the invention relates to a kit comprising a reaction vessel with the enzyme substrate composition or the enzyme substrate of the invention. Any appropriate reaction vessel may be included in the kit, and the kit may also contain a plurality of reaction vessels with the same or different enzyme substrates; in a preferred embodiment the reaction vessel contains a plurality of reaction compartments in a single substrate. In the context of the invention a reaction vessel is any entity capable of retaining the enzyme substrate or enzyme substrate composition in a specific location where the enzyme substrate or the enzyme substrate composition may be contacted with a sample allowing a reaction between the enzyme substrate and any enzymes present in the sample. In a specific embodiment, the reaction vessel may be a microtiter plate wherein the wells contain the enzyme substrate, optionally in a hydrogel. For example, a microtiter plate with 96 wells may contain enzyme substrates prepared from different plant materials or differently treated plant materials or a combination of these. In another embodiment, the reaction vessel is a filter paper or the like, which is impregnated with the enzyme substrate in defined areas. The kit may contain any number of positive or negative controls, and the kit may comprise any buffer, reagent, cofactor, coenzyme or the like.

The enzyme substrate, the enzyme substrate composition and the kit of the invention thus provide tools for screening of enzymes capable of degrading a plant of interest. For example, the enzyme substrate may be prepared from a plant of interest, e.g. a crop or waste material from a crop, and this enzyme substrate can be used in the screening of enzymatic activities from an unknown sample, e.g. from a bacterium or fungus or from another source. Following a positive reaction the sample, and optionally the actual enzyme substrate, may be analysed further to characterise the enzyme. The enzyme substrate is useful in the HTS of potential sources of enzymes since a large number of enzyme substrates may be prepared and supplied, e.g. in a single kit, allowing fast screening of samples against a large number of enzyme substrates derived from different plants.

In a further embodiment the invention relates to a method for the preparation of the enzyme substrate, which comprises the steps of:

-providing a plant material,

-optionally lyophilising the plant material,

-disrupting the plant material,

-optionally drying the disrupted plant material,

-reacting the disrupted plant material with a reactive marker to provide the enzyme substrate.

Any method of disrupting the plant material is contemplated for the method and it is preferred that the disruption of the plant material can disrupt the cell walls. The disrupted plant material may be washed with a solvent to remove components soluble in the solvent. The solvent may be any solvent capable of removing an undesired component from the plant material. Exemplary undesirable components are chlorophyll, pigments and proteins, and the plant material, which has been washed with the solvent may also be referred to as a "decolourised plant material". It is preferred that the solvent does not chemically react with the plant material, in particular it is preferred that the solvent does not oxidise or reduce the plant material, e.g. the polysaccharides or the lignin. In a preferred embodiment the solvent is a chlorophyll dissolving solvent, which may provide a decolourised plant material. Any chlorophyll dissolving solvent may be employed in the method. The chlorophyll dissolving solvent serves to remove undesirable components, such as coloured compounds, from the plant material and the step of washing the plant material with the chlorophyll dissolving solvent is particularly advantageous when detection of the detectable marker involves optical methods, e.g. when the detectable marker is a dye, a fluorescent moiety or a luminescent moiety. The reactive marker may be any molecule capable of attaching the marker to the plant material in order to provide the plant material with a releasably attached detectable marker. The reactive marker may carry any functionality or functional group allowing its attachment to the plant material, e.g. the reactive marker may be attached via a covalent attachment or via physical adsorption.

The invention also relates to a method for the preparation of an enzyme substrate composition, which comprises the steps of:

-providing a hydrogel precursor,

-providing an enzyme substrate of the invention, -mixing the enzyme substrate with the hydrogel precursor,

-forming a hydrogel with the enzyme substrate.

Any hydrogel may be employed in the method of the invention. It is preferred that the hydrogel does not comprise a carbohydrate expected or known to be present in the plant material employed to prepare the enzyme substrate. This will provide that enzymatic activity is detected with higher sensitivity since any enzymes present in a sample will not degrade the hydrogel but only the enzyme substrate.

All embodiments and variations described above for the enzyme substrate and the enzyme substrate composition, respectively, are relevant for the methods of preparation.

Brief description of the figures

In the following the invention will be explained in greater detail with the aid of examples and with reference to the drawings, in which

Figure 1 compares the activity of plant material degrading enzymes on enzyme substrates of the invention.

Figure 2 shows degradation of an enzyme substrate of the enzyme substrate composition of the invention

Detailed description of the invention

The present invention relates to an enzyme substrate comprising an enzymatically degradable microparticulate plant material having a polysaccharide content in the range of 35%(w/w) to 95%(w/w), the microparticulate plant material having a releasably attached detectable marker, wherein enzymatic degradation of the microparticulate plant material results in release of the detectable marker. The invention further relates to an enzyme substrate composition comprising the enzyme substrate in a hydrogel. In other aspects the invention relates to uses of the enzyme substrate and the enzyme substrate composition and to methods of their preparation. The invention also relates to a kit comprising the enzyme substrate or the enzyme substrate composition.

In the context of the present invention, the term "enzyme substrate" refers to a material that may be degraded by an enzyme and allowing the activity of the enzyme to be detected and/or quantified. For example, and enzyme substrate may be a material, e.g. a carbohydrate chain or a lignin molecule, carrying a detectable marker, e.g. a dye molecule, which will be released from the substrate when the substrate is degraded by an appropriate enzyme. The enzyme substrate of the invention is insoluble, in particular the enzyme substrate of the invention is insoluble in water. Release of the marker from the substrate thus allows that the enzyme activity is detected. For example, the marker may be released into solution where it can be detected by appropriate means, such as measurement of the absorbance in case of a dye molecule, or the released marker may diffuse into a surrounding solid or gel-like material, such as a filter paper or a hydrogel where its presence allows detection of the enzyme activity.

In the context of the present invention the term "carbohydrate" refers to a compound comprising one or more sugar moieties. When a carbohydrate comprises two or more sugar moieties it may also be referred to as an "oligosaccharide" or "polysaccharide". For example, a carbohydrate comprising from two to about ten sugar moieties may be referred to as an "oligosaccharide" while a carbohydrate comprising more than about ten sugar moieties may be referred to as a "polysaccharide". The terms "carbohydrate" and "saccharide" are used interchangeably in this document.

The enzyme substrate comprises a detectable marker. In the context of the invention the "marker" may be any moiety, group or atom allowing the marker to be detected and quantified. For example, the marker may comprise a coloured, dyed, fluorescent, or luminescent moiety allowing detection by measuring colour or colour change, fluorescence or luminescence, respectively. The marker may also be a specific binding partner of a complementary binding partner allowing detection using the complementary binding partner, e.g. biotin may be used as a marker and biotin may be detected using streptavidin. Further exemplary markers are isotopically labelled moieties, e.g. radioactively labelled moieties or ¹³C-labelled moieties.

The detectable marker is releasably attached to the enzyme substrate. In the context of the invention "releasably attached" means that the marker can be cleaved off from the substrate by an appropriate enzyme allowing it to be detected in a supernatant above the enzymatically treated enzyme substrate. The detectable marker may be cleaved off from the enzyme substrate while being attached to a degradation product from the enzyme substrate or the detectable marker may be cleaved off as a detectable marker not attached to a degradation product. In both cases the detectable marker is considered to have been releasably attached.

In the context of the present invention, the term "plant material" refers to any plant material, and it may refer to whole plants, certain parts of plants, such as straw, hay, sawdust, grass, leaves, stems, roots, bark but also fruits, tubers, nuts, berries etc., or any mixtures of these. The present invention is not limited to a specific plant or group of plants, and material from any plant is relevant to the invention. Plant material is a complex mixture of soluble and insoluble material comprising proteins and non-protein components, such as carbohydrates, e.g. starch, pectin, cellulose and hemicellulose, minerals and other organic components. The plant material, e.g. after lyophilising, will be subjected to disruptive processing in the methods of the inventions. This disruptive processing (or "disruption" or derived forms of this term) may be any processing intended to reduce the size of parts or particles of the plant material, in particular the plant fibres, and typical disruptive processing involves cutting, pressing, chopping, milling, grinding, crushing, grating, shredding etc. In particular the disruption aims to degrade or disrupt the cell walls of the plant material to make the contents, e.g. the cell wall polysaccharides and lignin, of the cells accessible to enzymatic degradation. Furthermore, breakdown of the cell wall also allows that undesired cell contents are removed. These may comprise coloured components, e.g. chlorophyll, that can provide false positives if they are not removed; proteins that may react with a reactive marker molecule resulting in coupling of the marker to the protein instead of to the carbohydrates or lignin of the plant; or other loosely bound plant material, e.g. soluble sugars or primary or secondary metabolites.

The disrupted plant material may be washed with a solvent that can remove undesirable compounds such as chlorophyll, and preferably also other pigments and proteins, from the plant material. Relevant solvents may be polar or non-polar. For example, the solvent may be aqueous, e.g. water or mixtures of water and polar organic solvents, or the solvent may be non-polar. The solvent may also be referred to as a chlorophyll dissolving solvent. In the context of the invention a "chlorophyll dissolving solvent" is any solvent that can remove chlorophyll, and preferably also other undesirable compounds such as pigments and proteins, from the plant material. Exemplary chlorophyll dissolving solvents are alcohols, ketones, ethers, alkanes or aromatic hydrocarbons although it is preferred that the solvent, e.g. the chlorophyll dissolving solvent, is water miscible. The method may include any number of washes with the chlorophyll dissolving solvent, and in certain embodiments different solvents may be employed consecutively to promote removal of the undesired compounds. Alcohols and ketones are preferred as chlorophyll dissolving solvents, in particular water miscible alcohols, e.g. ethanol, methanol and propanol, and water miscible ketones, e.g. acetone are preferred. In a preferred embodiment the disrupted plant material is washed with ethanol, e.g. 70% in water, followed by methanol, e.g. 100%, and acetone. The ratio of disrupted plant material to chlorophyll dissolving solvent may be chosen freely, such as in the range of 1 : 100 to 100: 1, e.g. 1 : 10 to 10 : 1. Preferred ratios are in the range of about 1 : 50 to about 1 : 20. Each washing step will generally comprise mixing of the disrupted plant material and the chlorophyll dissolving solvent and will have a duration of up to 1 hour, e.g. 15 minutes, 10 minutes, 1 minute or less. It is, however, also possible to degrade, e.g. enzymatically, the chlorophyll before dissolving and removing it.

The term disrupted plant material may refer to any part of a plant, a solid or liquid material, e.g. a liquid comprising suspended particles, obtained during disruptive processing or a mixture of these. Disruption of the plant material will provide the microparticulate plant material. In the context of the invention, "microparticulate" describes a material comprising particles with a largest dimension of about 500 μιη, such as about 400 μιη, about 300 μιη, about 200 μιτι, about 100 μιτι, about 50 μιτι, about 40 μιτι, about 30 μιτι, about 20 μιτι, about 10 μιτι, about 5 μιτι, about 1 μιη. The microparticulate material may also contain particles with a largest dimension above 500 μιη as long as the microparticulate material also comprises particle sizes below 500 μιη. In a certain embodiment the microparticulate plant material does not comprise particles above 500 μιη. It is preferred that the particle sizes of the microparticulate material is in the range of from about 0.1 μιη to about 100 μιτι, e.g. about 1 μιη to about 10 μιη. It is particularly preferred that the microparticulate plant material comprises plant cells with a disrupted cell wall.

The plant material may also be lyophilised, e.g. prior to disruption. Lyophilisation of the plant material generally improves a subsequent disruption process so that the plant material can more easily be brought to a microparticulate form, and moreover the cell walls will be more efficiently disrupted so that any effects obtained in the disruption are stronger when the plant material is lyophilised.

In the preparation of the enzyme substrate the plant material is reacted with a reactive marker. The reactive marker will have a reactive group allowing coupling with the carbohydrate or lignin as desired. Any reactivity may be employed to couple the reactive marker to the carbohydrate or lignin. It is preferred that the reactive group can be coupled covalently to the hydroxyl groups of carbohydrates and phenol or hydroxyl groups of lignin as appropriate. However, the coupling need not be covalent and e.g. ionic or hydrophobic interactions may also be employed for coupling the reactive marker to the carbohydrate or lignin. Exemplary coupling chemistries are nucleophilic substitution, e.g. using marker moieties carrying a chloro group or marker moieties carrying an epoxy group, or reaction via an activated double bond. Further appropriate chemistries are known from the preparation of chromatographic matrices and appropriate pairs of complementary reactivities include, e.g. nucleophile-epoxy, carboxylate-amine (via a carbodiimide compound), aldehyde-amine (optionally via a reducing agent), carbonyldiimidazole-amine etc. with modifications of the carbohydrate chains as appropriate. These and many more are described in detail by e.g. Scopes, R,K., Protein purification. Principles and practice. Third edition, Springer-Verlag, New York 1993, and Atherton, E. ; Sheppard, R.C. Solid phase peptide synthesis: A practical approach. Oxford, England : IRL press (1989), which are hereby incorporated by reference. In a preferred embodiment the reactive marker is a chloro derivatised triazine dye which can be coupled to carbohydrates or lignin via nucleophilic aromatic substitution. In general, the reaction conditions will be chosen to comply with the chemical or physical reaction involved. For example, reactions involving nucleophilic substitution, e.g. nucleophilic aromatic substitution, may be performed under basic conditions, e.g. pH> 10, at increased temperature, e.g. above 50°C, with appropriate mixing during the reaction time, which may be as desired, e.g. 1 hour or more. Reaction conditions are well known to the skilled person.

In a specific embodiment the enzyme substrate is formulated as an enzyme substrate composition in a hydrogel. In general, the enzyme substrate is mixed with a hydrogel precursor before forming a hydrogel with the enzyme substrate. In the context of the invention a "hydrogel precursor" is any compound or composition that may form a hydrogel under appropriate conditions. The hydrogel precursor may be in a dry form, e.g. as a powder or the like, or the hydrogel may be a liquid, e.g. an aqueous solution of a compound capable of forming a hydrogel. For example, the enzyme substrate may be mixed with a hydrogel precursor in dry form before dissolving the hydrogel precursor and suspending the enzyme substrate in water; hydrogel formation may be induced e.g. by lowering the temperature of the solution or by reacting, e.g. polymerising, the hydrogel precursor as appropriate. It is also possible to add the enzyme substrate to an aqueous solution of a hydrogel precursor, e.g. at high temperature, to suspend the enzyme substrate in the aqueous solution and the allowing formation of the hydrogel, e.g. by cooling the suspension. Appropriate hydrogels are, or are prepared from, carbohydrate polymers, carboxymethyl- cellulose or other cellulose derivatives, agar, carrageenan, gelatine, pectin, xanthan gum, gum arabic, guar gum, locust bean gum, alginate, acrylic acid, acrylates, acrylamides, ethylene glycol, propylene glycol, glycidyl ethers, alkyl halides. The hydrogels may be cross-linked and/or may be co-polymers of different monomer components. A preferred hydrogel is agar.

Example Example 1 - Preparation and testing of enzyme substrates comprising dyed alcohol-insoluble residue (AIR) plant samples

Preparation of alcohol-insoluble residue (AIR) from raw plant material A plant material of Arabidopsis thaliana was provided. The plant material comprised the arial parts of the plants, e.g. without root material. The plant material was frozen and lyophilised and then crushed into a fine powder using a Tissue Lyser by shaking the plant material together with a steel ball in a steel container for 2 minutes at a frequency of 30 Hz. The powder (1-2 grams) was collected into a 50 mL Falcon tube and washed 4 times with 70% ethanol and then 3 times with 100% methanol to decolourise the material from chlorophyll and other pigments and to remove proteins. The washing was performed by consecutive shaking (15 minutes), centrifugation and decanting. The last wash was done with 100% acetone and then the tubes containing AIR were left covered by perforated parafilm in the fume hood to dry.

Cross-linking of plant material

In order to compare the effect of cross-linking on the enzyme substrates aliquots of the prepared enzyme substrates were also cross-linked. In general, a cross-linking using epoxide functionalised cross-linking agents, such as 1,4- butanediol diglycidyl ether or epichlorohydrin, is based on a base-catalysed (e.g. 0.5 M or 2 M NaOH in water) epoxide ring opening and a nucleophilic attack of the terminal carbon in the epoxide ring on the hydroxyl groups in polysaccharides following an S_N2 mechanism. A similar reaction is relevant for the chloro group of epichlorohydrin. Both ends of the cross-linker terminal epoxides react with random groups on the polysaccharide chains and in this way cross-link the material making it more and more insoluble and eventually completely insoluble. The reaction may be conducted at room temperature making the cross-linking reaction relatively slow and during the 48 hours of standing the reaction gets slower and slower as the cross-linking makes the reaction mixture more and more dense and hence the movement of the molecules in the mixture becomes slower and slower.

In a specific example, 500 mg of the Arabidopsis AIR sample were weighed out and the cross-linking was done based on a modified procedure from literature (Semde, R., A.J. Moes, M.J. Devleeschouwer, and K. Amighi, Synthesis and enzymatic degradation of epichlorohydrin cross-linked pectins. Drug Development and Industrial Pharmacy, 2003. 29(2) : p. 203-213). The weighed out AIR sample was mixed with 150 microliters of epichlorohydrin diluted in 1.5 mL of 96% ethanol and then 0.5 mL of 5M aqueous NaOH were added and then additional 3 mL of 96% ethanol were added to facilitate easier mixing of the sample. The suspension was mixed at 200 rpm for 4 hours at 40°C. After the reaction, the suspension was allowed to cool down and then the sample was washed 2 times with MeOH and 2 times with water by consecutive shaking (2 minutes), centrifugation and decanting. After the last wash - the sample was frozen and lyophilised.

Dying of alcohol-insoluble residue

The treated plant material was reacted with chloro triazine dyes as reactive markers. The dyes used were monochloro- or dichloro-triazine dyes that in basic conditions (0.5M or 2M NaOH in water) and with elevated temperature (60°C) alkylate the hydroxyl groups in polysaccharides (either pure or part of a cell wall sample) and lignin with the loss of the chloride leaving group and nucleophilic substitution reaction on the triazine ring with a hydroxyl group via a S_NAr mechanism (nucleophilic aromatic substitution). To a sample of 500 mg of AIR, 1 gram of chloro triazine dye was added and then 10 mL of aqueous 0.5M NaOH. The mixture was vortexed thoroughly and then incubated for 4 hours at 60°C. The washing was performed with DI water by consecutive shaking (2 minutes), centrifugation and decanting. When there was no more dye present in the supernatant after centrifugation - the sample was considered ready for subsequent experiments.

Specifically, to the lyophilised AIR sample - 1 gram of the red dye (Cibacron Brilliant Red 3B-A) was added and then 10 mL of aqueous 0.5M NaOH. The mixture was vortexed thoroughly and then incubated for 4 hours at 60°C. The washing was performed with DI water by consecutive shaking (2 minutes), centrifugation and decanting. When there was no more dye present in the supernatant after centrifugation - the sample was considered ready for subsequent experiments. Comparison of enzyme substrates

The cross-linked and unlinked Arabidopsis AIR sample was diluted in isopropanol (around 12.5 mg/mL) and 50 μΙ_ of this solution was transferred in each well of a filter-plate. The substrate was washed twice with water. 150 μΙ_ buffer (with the preferred pH of each enzyme, see Table 1) and 5 μΙ_ enzyme solution (Table 1) was added to each well, so that the final enzyme concentration was 10 U/mL. Additionally, H₂0₂ (final concentration in the well : 0.01%) was added to Manganese peroxidase (MP) and Lignin Peroxidase (LP). The plates were sealed (Adhesive PCR plate seals, Thermo Scientific) and incubated rotating at room temperature for 1 hour. After centrifugation (4000 rpm, 15 min), the absorbance at 517 nm (red) was measured using a plate reader. The results are depicted in Figure 1, which shows the absorbance obtained from each enzyme incubated with the cross-linked Arabidopsis AIR enzyme substrate or the Arabidopsis AIR enzyme substrate that was not cross-linked.

Table 1 : List of enzymes (enzyme concentration : 31 U/ml, final enzyme concentration in well : 1 U/ml)

Code Name Activity PH enzyme buffer

(U/ml) (ML) (ML) eARAl enc/o-arabinase 100 4.0 93 207 eGAL2 endo- 1,4-/3- D-Ga lacta nase 506 4.0 30.6 470 ePOL2 enc/o-polygalacturonase 2100 4.0 7.4 493 eXYLl 3-xylanase,M4 (Aspergillus niger) 1040 4.5 14.9 485 eXYL4 er?c/o-l,4- 3-Xylanase M l (T. viride) 2300 4.5 6.74 495 eCELl enc/o-cellulase (EGII) (Trichoderma 540 4.5 28.7 472 longibrachiatum)

eGLCl er?c/o-l,3- 3-glucanase 50 4.5 186 114

PI Pectinase (Rhizopus sp.), 441201 5.0

Calbiochem

P2 Pecto lyase Y-23 (Aspergillus 5.5

japonicus), duchefa biochemie,

CAS:9033-35-6

P3 Pectolyase (Aspergillus japonicus), 5.5

Sigma P-3026

eXYL3 endo- l,4- 3-Xyla nase ( Cellvibrio 500 5.5 31 459 japonicus) Code Name Activity PH enzyme buffer

(U/ml) (ML) (ML) eXYL5 endo- l,4-/3-Xyla nase ( Cellvibrio 750 5.5 20.6 480 mixtus)

eXYL6 er?c/o-l,4-/3-Xylanase 10000 5.5 1.6 499

(Neocallimastix patriciarum)

eXGl Xyloglucanase (GH5) (Paenibacillus 1000 5.5 16 484 sp.)

ePOLl enc/o-polygalacturonase 5000 5.5 3.1 497

(Aspergillus niger) M2

eFERl Feruloyl Esterase 100 6.0 93 207 eMAN2 endo-1,4 3-Mannanase (Cellvibrio 5000 7.0 3.1 497 japonicus)

eXYL2 exo-l,4-/3-D-xylosidase (B. 75 7.5 124 176

Pumilus)

ePEC2 Pectate lyase (Aspergillus sp.) 120 8.0 77.5 222 ePECl Pectate lyase (C. japonicus) 500 10.0 31 469

LacT La cease Tra metes versicolor 4.5 100 100

(Sigma, 53739)

LacU Culture supernatant with unknown undiluted 4.5

laccasse from Jorn Dalgaard

Mikkelsen (DTU)

MP Manganese peroxidase from 4.5 100 100

Phanerochaete chrysosporium

(Sigma, 93014)

LP Lignin Peroxidase (Sigma, 42603) 3.0 100 100

Overall, it was shown, that the cross-linking did not preserve any polysaccharides (except possibly xyloglucan). The results showed the opposite effect, there was strikingly more colour release in the non-cross-linked AIR sample demonstrating that even though the dying of the AIR samples is performed in basic conditions (NaOH) and at elevated temperature - the conditions that are traditionally used for extraction of hemicelluloses from AIR samples - the dyed AIR sample performed well and hemicellulose-degradation- related colour release was readily detectable. The cross-linked-AIR control actually had lower values for virtually all polysaccharides demonstrating that cross-linking is not necessary and may even be detrimental when analysing dyed AIR samples.

Example 2 - Enzyme activity assay using chromogenic cross-linked polysaccharide substrates in 96 well plates

Chromogenic substrates were transferred into a 96 well plate (filter plate MAHAS4510, Millipore or polypropylene microplate, V-bottom, grainer bio-one) and washed with water to remove free dye. 150 μΙ_ buffer (100 mM, sodium acetate or potassium phosphate buffer, pH dependent on the individual enzyme) was added to each well. Enzyme was added to each well at a final concentration which varied between 0 - 2 U/mL. The plate was sealed using Adhesive PCR plate seals (Thermo Scientific) and incubated usually for 1 h rotating at room temperature. Then the solutions were spun down at 4000 rpm for 5 min and the supernatant collected in a plate (96 microwell, Nunc, VWR) when using filter plates or 100 μΙ_ supernatant of each well transferred to the collection plate (96 microwell, Nunc, VWR) when using polypropylene microplates for the reaction. The absorbance of the reaction product was detected at 404 nm (for yellow), 517 nm (red), 595 nm (blue) and 630 nm (green) as well as the spectrum using an ELISA plate reader. Spectra were detected in steps of 1 nm for the range of 350 to 700 nm.

Different dyes (dyed hydrogels) display distinctly different absorption spectra within the scanned range. Mixtures of polysaccharides labelled with different dyes and spectra of mixed colours enable linear regression analysis enabling multiplexed usage of the hydrogel enzyme substrates to an extent where a spectrum scan together with a calibration curve can reveal the extent of digestion of either substrate (2-substrate mixtures have been tested) with an error of 5%.

Example 3 - Enzyme activity assay using chromogenic cross-linked polysaccharide substrates in agar plates

The agar plate was poured using 23 mM Britton-Robinson-Buffer (pH 6.0) containing 1% agar and 0.01% NaN₃. Holes were made with a metal cylinder (diameter: 6 mm). The enzyme substrates were ground using a tissue lyser (30 Hz for 15 min) to create a fluid solution : to 300 mg gel-substrate was added 500 μΙ_ water. 60 μΙ_ substrate mixture of 16%(w/w) R-pachyman, 25%(w/w) B- galactomannan, 34%(w/w) Y-xylan and 0.75%(w/w) agar were transferred in each hole. 20 μΙ_ 10 U/ml enzyme (endo- l,4- 3-Xylanase M l (Trichoderma viride), endo-l,4-/3-mannanase (Cellvibrio japonicus), er?c/o-l,3-p-glucanase (Trichoderma sp.); all from Megazyme) in 100 mM sodium acetate buffer (pH 4.5) were added and the plate incubate overnight at 30°C. Coloured halos appeared where active enzyme has degraded the polysaccharide and soluble oligosaccharides linked to the dye diffused into the agar (Figure 2).

Figure 2 shows how the detectable markers were released from the enzyme substrates in the hydrogel. The released detectable markers diffused into the surrounding gel to show that the hydrogel did not hinder the activity of the enzymes. Thus, the enzyme substrate composition of the invention provides a convenient enzyme substrate that is easy to handle; the enzyme substrate composition is preferable supplied as a kit, e.g. in the wells of a microtiter plate.

Claims

P A T E N T C L A I M S

1. An enzyme substrate comprising an enzymatically degradable microparticulate plant material having a polysaccharide content in the range of 35%(w/w) to 95%(w/w), the microparticulate plant material having a releasably attached detectable marker, wherein enzymatic degradation of the microparticulate plant material results in release of the detectable marker.

2. The enzyme substrate according to claim 1, wherein the plant material comprises up to 95%(w/w) cellulose.

3. The enzyme substrate according to claim 1 or 2, wherein the plant material comprises lignin.

4. The enzyme substrate according to any one of claims 1 to 3, wherein the microparticulate plant material is not cross-linked.

5. The enzyme substrate according to any one of claims 1 to 4, wherein the detectable marker is covalently attached to a carbohydrate or lignin.

6. The enzyme substrate according to any one of claims 1 to 5, wherein the reactive marker comprises a dye, a fluorescent moiety or a luminescent moiety.

7. The enzyme substrate according to any one of claims 1 to 6, wherein the microparticulate plant material comprises particles with a size in the range of from 0.1 m to 500 μιη.

8. The enzyme substrate according to any one of claims 1 to 7, wherein the enzyme substrate does not comprise microfibrillated cellulose or purified cellulose fibres.

9. A method for the preparation of an enzyme substrate according to any one of claims 1 to 8, the method comprising the steps of:

-providing a plant material,

-optionally lyophilising the plant material,

-disrupting the plant material,

-optionally drying the disrupted plant material,

10. The method of claim 9, further comprising the step of washing the disrupted plant material with a solvent to remove components soluble in the solvent.

11. The method of claim 10, wherein the solvent is water miscible.

12. The method of claim 10, wherein the solvent is selected from water, alcohols, ketones, ethers, alkanes or aromatic hydrocarbons.

13. The method according to any one of claims 10 to 12, wherein the solvent does not chemically react with the plant material.

14. An enzyme substrate composition comprising the enzyme substrate according to any one of claims 1 to 8 in a hydrogel.

15. The enzyme substrate composition according to claim 14, wherein the hydrogel is or is prepared from carbohydrate polymers, carboxymethyl-cellulose or other cellulose derivatives, agar, carrageenan, gelatine, pectin, xanthan gum, gum arabic, guar gum, locust bean gum, alginate, acrylic acid, acrylates, acrylamides, ethylene glycol, propylene glycol, glycidyl ethers, alkyl halides.

16. A method of preparing an enzyme substrate composition according to claim 14 or 15, the method comprising the steps of:

-providing a hydrogel precursor,

-providing an enzyme substrate according to any one of claims 1 to 8,

-mixing the enzyme substrate with the hydrogel precursor,

-forming a hydrogel with the enzyme substrate.

17. Use of an enzyme substrate according to any one of claims 1 to 8 or an enzyme substrate composition according to claim 14 or 15 in the screening for plant material degrading enzyme, such as a glycosyl hydrolase (GH) enzyme, a ligninase enzyme, a laccase enzyme or a protease capable of degrading the protein moiety of a cell wall proteoglycan.