WO2015091772A1

WO2015091772A1 - Method of determining the degradation of cellulosic materials

Info

Publication number: WO2015091772A1
Application number: PCT/EP2014/078430
Authority: WO
Inventors: Michael Adam NASH; Tobias VERDORFER; Klara MALINOWSKA
Original assignee: Ludwig-Maximilians-Universität München
Priority date: 2013-12-19
Filing date: 2014-12-18
Publication date: 2015-06-25

Abstract

The present invention relates to a method of determining the degradation of cellulosic materials in real-time, comprising: enzyme-mediated at least partial cleaving of the cellulosic materials, thus producing glucose monomers;reacting the glucose monomers with glucose oxidase, thus producing hydrogen peroxide;reacting the hydrogen peroxide with a Fenton reaction catalyst, thus producing hydroxyl radicals,wherein the reaction is performed at a pH of <5.0; andreacting the hydroxyl radicals with water-soluble monomer units having functional vinyl groups, thus producing a polymeric coating on the cellulosic materials; wherein the method does not comprise the external addition of glucose; and wherein the polymeric coating is produced in a spatially localized manner at the site of enzyme mediated cleavage of the cellulosic material. In particular embodiments, the method further comprises: quantifying the production of the polymeric coating by determining one or more physico- chemical parameters being indicative for the formation of the polymeric gel. The present invention also relates to the use of such a method for determining the cellulolytic enzyme activity during biomass conversion.

Description

METHOD OF DETERMINING THE DEGRADATION OF CELLULOSIC MATERIALS

FIELD OF THE INVENTION

The present invention is directed to a method for the quantitative determination of the degradation of cellulosic materials in real-time and in a spatially localized manner.

BACKGROUND OF THE INVENTION

Cellulose is a polymer of glucose moieties covalently bonded by p-1 ,4-linkages which can be cleaved inter alia by physical treatment (e.g., by heat or variation of the pH value) or enzymatically. For example, many microorganisms produce enzymes that hydrolyze β-linked glucans. Such enzymes include endoglucanases, exoglucanases, and β-glucosidases. Endoglucanases cleave cellulose at random locations, increasing the number of polysaacharide chains accessible to further attacks by exoglucanases that sequentially release soluble oligosaccharides (mainly cellobiose) from the ends of the cellulose polymer. β-glucosidases hydrolyze cellobiose to glucose.

The conversion of cellulose feedstocks into sugar moieties is of high impact with regard to various applications, in particular the production of biofuels such as ethanol. The production of ethanol from cellulose feedstocks has the advantages of the ready availability of large amount of starting material, the desirability of avoiding burning or land filling the materials, and the purity of the ethanol fuel. Wood, agricultural residues, crops, and municipal solid wastes have been considered as feedstocks for ethanol production. These materials primarily consist of cellulose, hemicellulose, and lignin. Once the cellulose is converted to glucose, the glucose is easily fermented by yeast into ethanol.

Detection systems for monitoring the conversion of cellulosic materials are thus of utmost importance in order to improve efficiency of biofuel production. Such systems need to be robust, rapid, cost effective and easily implementable to various applications. Furthermore, such systems should be characterized by high sensitivity and readily discernable yes/no signals.

However, existing glucose conversion systems often require high enzyme loadings (for example, up to 20 mg enzyme/g substrate) in order to achieve high conversion rates of the cellulosic materials into glucose monomers or short-chained oligosaccharides, and thus are costly and economically inefficient (cf. inter alia Klein-Marcuschamer, D. et al. (201 1 ) Biotechnol. Bioengineer. 109, 1083-1087). In order to make the process more efficient and affordable, research has focused on several areas, including development of pre-treatment methods that render the substrate more susceptible to enzymatic degradation (Kumar, P. et al. (2009) Ind. Eng. Chem. Res. 48, 3713-3729). Nevertheless, existing assay systems are still hampered by the unavailability of methods for the easy and accurate determination of the effectiveness of cellulolytic enzyme formulations on a range of substrates possessing variable composition, morphology, degrees of crystallinity and/or lignin content, and the like.

Cellulase assays have in the past been performed using a suite of biochemical methods (reviewed, e.g., in: Zhang, Y.H. et al. (2006) Biotechnol. Advances 24, 452-481 ; Dashtban, M. et al. (2010) Crit. Rev. Biotechnol. 30, 302-309). All these methods, despite being relatively straightforward to implement, have been unable to provide spatially resolved realtime information about where hydrolysis may preferentially be taking place on the substrate. Spatial localization of hydrolytic activity could provide important details on the mechanisms involved in cellulose degradation, and could allow correlation of digestability with substrate features such as fiber bundle size, degree of fiber branching, and crystal orientation.

More recent attempts to introduce spatial resolution into cellulase activity determinations include atomic force microscopy imaging of substrate degradation (Ganner, T. et al. (2012) J. Biol. Chem. 287, 43215-43222). On the other hand, preparation of the requisite ultra-flat cellulose substrates in combination with long AFM scan times may limit application of this approach.

Thus, there is a need for improved methods for the accurate monitoring in real-time of the conversion of cellulosic materials into sugar moieties that overcome the above-referenced limitations. Accordingly, it is an object of the present invention to provide such methods. SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a method of determining the degradation of cellulosic materials in real-time, comprising: (a) enzyme-mediated at least partial cleaving of the cellulosic materials, thus producing glucose monomers; (b) reacting the glucose monomers with glucose oxidase, thus producing hydrogen peroxide; (c) reacting the hydrogen peroxide with a Fenton reaction catalyst, thus producing hydroxyl radicals, wherein the reaction is performed at a pH of < 5.0; and (d) reacting the hydroxyl radicals with water- soluble monomer units having functional vinyl groups, thus producing a polymeric coating on the cellulosic materials; wherein the method does not comprise the external addition of glucose; and wherein the polymeric coating is produced in a spatially localized manner at the site of enzyme mediated cleavage of the cellulosic material.

In preferred embodiments, the method further comprises: quantifying the production of the polymeric coating by determining one or more physico-chemical parameters being indicative for the formation of the polymeric gel. Particularly, the physic-chemical parameters are selected from the group consisting of fluorescence emission, optical opacity, solution viscosity, pH value, sensitivity to temperature, and electric field strength. In particularly preferred embodiments, the fluorescence emission is the auto-fluorescence emission of the cellulosic material.

In particularly preferred embodiments of the method, the quantification step comprises the use of labeled monomer units, particularly fluorescently labeled monomer units.

In specific embodiments of the method, step (a) comprises cleaving of the cellulosic materials by means of one or more exoglucanases, endoglucanases and β-glucosidases.

In specific embodiments of the method, the Fenton reaction catalyst is selected from the group consisting of Fe(ll), Cu(l), Ti(lll), Cr(ll), Co(ll), and iron oxidase nanoparticles. In further specific embodiments of the method, the water-soluble monomer units having functional vinyl groups are selected from the group consisting of acrylates, methacrylates, acrylamides, and methacrylamides.

In particular embodiments, the cellulosic material employed in the method is lignocellulosic biomass.

In yet further specific embodiments, method is performed under ambient atmospheric conditions and at a reaction temperature of 37°C.

In a further aspect, the present invention relates to the use of a method as described herein above for determining the cellulolytic enzyme activity during biomass conversion. Preferably, the biomass conversion is for the production of a biofuel.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 : Assay system for real-time imaging of cellulose hydrolysis according to the present invention, (a) Reaction scheme. Oligosaccharides produced by cellulolytic enzymes are converted into hydrogen peroxide via sequential reaction with β-glucosidase and glucose oxidase. Hydrogen peroxide undergoes a Fenton reaction with Fe²⁺, producing hydroxyl radicals which then initiate co-polymerization of monomers having functional vinyl groups. Formation of fluorescent hydrogel at the sites of cellulose hydrolysis can be monitored in real-time using total internal reflection fluorescence microscopy (TIRF) microscopy, (b) Photograph of filter paper that was partially immersed in the hydrogel reagent mixture for 30 minutes, (c) Chemical structures of the fluorescent monomer methacryloxyethyl thiocarbamoyl rhodamine B and the gel cross-linker poly(ethylene glycol) diacrylate.

FIGURE 2: Detection of soluble and insoluble cellulose hydrolysis using the assay system according to the present invention, (a) Carboxymethyl cellulose (CMC) was added to the assay system with varying amounts of endo-1 ,4-beta-D-glucanase while the monitoring absorbance at 650 nm. Initiation of polymerization via CMC hydrolysis and subsequent conversion to hydroxyl radicals resulted in an increase of absorbance due to light scattering from the gel. (b) Fluorescence intensity vs. time plot generated by applying the assay system and Trichoderma reesei enzyme cocktail to filter paper and rinsing at the given time points, (c) pH-dependence of the hydrogel formation reaction. Oxidation of Fe(ll) at a pH of greater than 5.0 quenches the production of hydroxyl radicals, (d) Various substrates were tested and found to provide high signal to noise ratios for 1 mg/ml T. reesei enzymes.

FIGURE 3: Estimation of Michelis-Menten kinetic parameters for T. reesei enzymes. The light scattering due to gel formation was measured, and the initial velocity (V₀) determined for the hydrogel reagent system combined with 0.5 mg/ml T. reesei enzymes and variable amounts of a soluble CMC substrate. The activity vs. substrate concentration plot was fitted with the Michelis-Menten equation to quantify kinetic rate constants for the enzyme.

FIGURE 4: Time-lapse TIRF imaging of cellulose hydrolysis using the assay system according to the present invention. Algal Cladophora cellulose was labelled with a fluorescein derivative (i.e., 5-(4,6-dichlorotriazinyl)aminofluorescein, 5-DTAF), and patterned in lines onto a coverslip using PDMS microfluidic channels. Under blue illumination ('Fiber channel', image (a)), the patterned lines of cellulose were clearly visible at the top and bottom of the image. Next, the hydrogel reagent mixture, including 10 nM rhodamine-methacrylate, was added and images were collected over time under green illumination ('Hydrogel channel', images (b), (c), and (d)). Polymerization of the fluorescent hydrogel was observed and occurred preferentially at locations of deposited cellulose.

FIGURE 5: Atomic force microscopy (AFM) imaging of hydrogel formation, (a) AFM height image of algal Cladophora cellulose exposed to the hydrogel reagents and 1 mg/ml of T. reesei cellulase cocktail for 60 minutes, (b) Negative control showing no hydrogel formation on a fiber bundle exposed to the hydrogel reagents without the T. reesei enzymes.

FIGURE 6: Schematic illustration of an embodiment according to the present invention. Auto-fluorescence emitted from cellulose in the biomass is used as detection signal for determining cellulose hydrolysis cf. also FIG. 1 ). Polymerization of the hydrogel in response to enzymatic cleavage results in attenuation of cellulose auto-fluorescence. Hence, the methodological approach of the present invention can be used on any natural biomass substrate without requiring costly dye labeling of the water-soluble monomer units. FIGURE 7: Examples of the pre-treated biomass substrates used in connection with the present invention. Cylindrical disks 6 mm in diameter were produced from napier grass and miscanthus perennial grass that had been pre-treated by mechanical comminution and exposure to diluted acid.

FIGURE 8: Proof-of-concept for new measurement method. Various concentrations from 0-100 μg ml of an enzyme cocktail from Trichoderma reese/ were tested on pretreated napier grass substrates. (Top) The normalized auto-fluorescence ("Norm. Fluorescence") of napier grass was monitored during the saccharification reaction. A decrease in the normalized fluorescence signal indicates gel polymerization. Data points represent mean (n=5) ± standard deviation (one-sided errors shown for clarity). (Bottom) The rate of change of the normalized fluorescence produces a clear peak at the point of polymerization. The height and onset time of the peak are indicative of the saccharification strength of the enzyme cocktail.

FIGURE 9: The auto-fluorescence attenuation method was used to monitor saccharification efficiency of Trichoderma reesei enzyme cocktails on pre-treated miscanthus perennial grass. (Top) Normalized auto-fluorescence ("Norm. Fluorescence") of miscanthus grass was monitored during the saccharification reaction for a range of enzyme concentrations from 0-100 μg ml. (Bottom) The rate of change of the normalized fluorescence produces a clear peak at the point of polymerization. The height and onset time of the peak are indicative of the saccharification strength of the enzyme cocktail.

FIGURE 10: The auto-fluorescence attenuation method described in FIG. 6 was used to monitor endo-/exoglucanase synergy. (Top) Normalized auto-fluorescence of pretreated miscanthus grass was monitored during enzymatic hydrolysis. A fixed amount of exoglucanase (1 μΜ) was used while adding endoglucanase across a range from 0-0.1 μΜ. (Bottom) Rate of change of normalized auto-fluorescence from top plot. The height of the peak and onset time can be used to quantify the synergism between these two enzymes.

FIGURE 11 : The auto-fluorescence attenuation method described in FIG. 6 was used to monitor endo-/exoglucanase synergy. (Top) Normalized auto-fluorescence of pretreated napier grass was monitored during enzymatic hydrolysis. A fixed amount of exoglucanase (1 μΜ) was used for all samples while adding endoglucanase in a range of 0-0.1 μΜ. (Bottom) Rate of change of normalized auto-fluorescence from top plot. The height of the peak and onset time can be used to quantify the synergism between these two enzymes.

FIGURE 12: (Top) Multi-enzyme complexes (i.e., mini-cellulosomes) comprising 3 copies of Cel8A-dockerin assembled onto a scaffold protein containing 3 copies of cohesin were assayed using the auto-fluorescence attenuation method described in FIG. 6. A decrease in the normalized fluorescence signal indicates gel polymerization, caused by mini- cellulosomes containing or lacking a carbohydrate binding module (CBM). Data represent mean (n = 5) ± standard deviation (shaded). (Bottom) Rate of change of normalized autofluorescence from top plot. The rate of change of the normalized auto-fluorescence produces a clear peak at the point of polymerization. The height of the peak and the onset time are indicative of the saccharification strength of the mini-cellulosomes with or without CBM contained within the scaffold.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is related to the unexpected finding that a rather simple reagent system enables localized real-time observation of the complex reactions involved in cellulose hydrolysis on the nanometer length scale. The enzyme-mediated hydrogelation system according to the present invention uses cascade enzyme signaling and a Fenton reagent to amplify sugar signals produced by cellulolytic enzymes and initiate polymerization of a fluorescent hydrogel coating on the cellulose substrate that can be readily detected and allows for an easy and accurate determination of the degradation of cellulosic materials in situ. In addition, the system does neither require the external addition of glucose nor necessarily supplementation with any labeled components, such as fluorescent dyes.

The present invention will be described in the following with respect to particular embodiments and with reference to certain drawings but the invention is to be understood as not limited thereto but only by the appended claims. The drawings described are only schematic and are to be considered non-limiting. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. For the purposes of the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising". If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group, which preferably consists only of these embodiments.

Where an indefinite or definite article is used when referring to a singular noun e.g. "a", "an" or "the", this includes a plural of that noun unless specifically stated otherwise.

In case, numerical values are indicated in the context of the present invention the skilled person will understand that the technical effect of the feature in question is ensured within an interval of accuracy, which typically encompasses a deviation of the numerical value given of ± 10%, and preferably of ± 5%.

Furthermore, the terms first, second, third, (a), (b), (c), and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

Further definitions of term will be given in the following in the context of which the terms are used. The following terms or definitions are provided solely to aid in the understanding of the invention. These definitions should not be construed to have a scope less than understood by a person of ordinary skill in the art.

In one aspect, the present invention relates to a method of determining the degradation of cellulosic materials in real-time, comprising:

(a) enzyme-mediated at least partial cleaving of the cellulosic materials, thus producing glucose monomers;

(b) reacting the glucose monomers with glucose oxidase, thus producing hydrogen peroxide; (c) reacting the hydrogen peroxide with a Fenton reaction catalyst, thus producing hydroxyl radicals, wherein the reaction is performed at a pH of < 5.0; and

(d) reacting the hydroxyl radicals with water-soluble monomer units having functional vinyl groups, thus producing a polymeric coating on the cellulosic materials;

wherein the method does not comprise the external addition of glucose; and wherein the polymeric coating is produced in a spatially localized manner at the site of enzyme mediated cleavage of the cellulosic material.

The term "cellulosic materials", as used herein, relates to any substrate material comprising polymers of glucose moieties covalently bonded by p-1 ,4-linkages. The term includes cellulose molecules being solely composed of glucose moieties as well as hemicellulose molecules being composed of several other sugar moieties, in addition to glucose, such as xylose mannose, galactose, rhamnose, and arabinose. The cellulose and/or hemicellulose may be derived from any origin such as bacteria (e.g. from Acetobacter xylinum), green algae (e.g., from Cladophora spec), or from plants. The cellulose and/or hemicellulose may be provided in natural occurring form or in manufactured form, for example, as filter paper (e.g., Whatman type I; GE Healthcare) or microcrystalline cellulose (e.g., Avicel PH; Sigma Aldrich). The cellulosic materials to be employed may be provided in purified form, in semi- purified or enriched form or as a raw material, such as a plant feedstock, that is, organic material derived from lignin, cellulose and hemicellulose (i.e. lignocellulosic biomass), such as wood, plants, and organic wastes (e.g., alfalfa, wheat straw, com stover, wood fibers, and the like).

In a particular embodiment of the method, the cellulosic material employed is lignocellulosic biomass.

The term "determining in real-time", as used herein, is to be understood as monitoring any reaction parameters that are measured immediately after collection and in a continuous manner over a given period of time.

The term "enzyme-mediated at least partial cleaving", as used herein, concerns any catalytic process involving the at least partial degradation of the cellulosic materials into glucose monomers or other soluble oligosaccharides by exposing the cellulosic materials to one or more enzymes being capable of cleaving β-1 ,4-glycosidic bonds. In specific embodiments of the method, at least 20% (w/w), at least 40% (w/w), at least 60% (w/w), at least 80% (w/w) or at least 90% (w/w) of the cellulosic material initially present are converted into glucose monomers. In one specific embodiment, the entire material initially present is converted into glucose monomers. In case of an only partial conversion of the cellulosic materials into glucose monomers, other sugar monomers (e.g., xylose, arabinose or galactose) and/or sugar dimers or oligomers (e.g., cellobiose or saccachose) may be present as well.

The one or more enzymes being capable of cleaving β-1 ,4-glycosidic bonds (herein also referred to as "cellulolytic enzymes") may be selected from the group consisting of exoglucanases, endoglucanases, cellobiohydrolases, and β-glucosidases. All these enzymes are well known in the art and may be derived from any suitable source, for example, from bacteria, yeasts or fungi. Examples of such enzyme-producing microorganisms include Trichoderma reesei, Aspergillus awamori, Clostridium thermocellum, and Thermoanaerobacterium saccharolyticum. The enzymes may be provided in purified form, either individually or as a mixture. If applicable, the one or more enzymes may be added to the cellulosic materials sequentially in a certain order. Alternatively, two or more enzymes may be added simultaneously. Instead of adding purified (or semi-purified) enzymes it is also possible to contact the cellulosic materials with an extract from an enzyme-producing microorganism.

In specific embodiments of the method, the cellulosic materials are at least partially cleaved by means of one or more exoglucanases, endoglucanases (both degrading cellulose and/or hemicellulose into dimeric or oligomeric sugar moieties) and β-glucosidases (degrading the sugar moieties into glucose monomers).

In particular embodiments, the method does not comprise the external addition of glucose monomers. Rather, the entire glucose monomers employed are generated intrinsically from the cellulosic materials.

Subsequently, the glucose monomers are converted into gluconolactone by means of glucose oxidase. Thereby, the enzyme is reduced. The subsequent reaction with molecular oxygen regenerates the enzyme and produces hydrogen peroxide. The glucose oxidase employed in the method may be added in purified form and be derived from any source, such as the fungi Aspergillus niger and Penicillium amagasakiense.

The hydrogen peroxide is then reacted with a Fenton reaction catalyst at a pH of < 5.0 and converted into hydroxyl radicals through classical Fenton's chemistry (Fenton, H.J.H. (1894) J. Chem. Soc. Trans. 65, 899-91 1 ). A preferred example of an appropriate Fenton reaction catalyst is Fe(ll), also referred to as Fe²⁺ (e.g., provided in form of iron(ll)sulfate. Ferrous iron(ll) (i.e. Fe²⁺) is oxidized by hydrogen peroxide to ferric iron (III), a hydroxyl radical, and a hydroxyl anion. Iron (III) (i.e. Fe³⁺) is then reduced back to iron (II), a superoxide radical, and a proton by the same hydrogen peroxide. The net effect is a disproportionation of hydrogen peroxide in order to generate two different oxygen radical species, with water as a byproduct. Further particularly suitable Fenton reaction catalyst that can be used include inter alia Cu(l), Ti(lll), Cr(ll), Co(ll), and iron oxidase nanoparticles (cf. also Goldstein, S. et al. (1993) Free Radical Biol. Med. 15, 435-445)

Finally, the hydroxyl radicals are reacted with water-soluble monomer units having functional vinyl groups, thus producing a polymeric coating on the cellulosic materials, that is, the reaction is performed in situ (i.e. exactly in place where it occurs). The polymeric coating is typically a gel-like matrix, preferably a hydrogel.

The term "vinyl group", as used herein, is also referred to as ethenyl group (i.e., -CH=CH₂). Vinyl groups are capable of forming polymers with the aid of free radicals as polymerization initiators.

In further specific embodiments of the method, the water-soluble monomers having functional vinyl groups are selected from the group consisting of acrylates, methacrylates, acrylamides, and methacrylamides. All these monomers are well known in the art. The hydroxyl radicals may be reacted with only one type of water-soluble monomers (e.g., acrylamides) or with a mixture of two or more different types of water-soluble monomers (e.g., acrylamides and methacrylates).

In preferred embodiments, the method further comprises quantifying the production of the polymeric coating by determining one or more physico-chemical parameters being indicative for the formation of the polymeric gel. In specific embodiments, the physico-chemical parameters are selected from the group consisting of fluorescence emission, optical opacity, solution viscosity, pH value, sensitivity to temperature, and electric field strength, with fluorescence emission, optical opacity, and solution viscosity being preferred.

The method according to the present invention is characterized in that the polymeric coating is produced in a spatially localized manner at the site of enzyme mediated cleavage of the cellulosic material, that is, in situ on the substrate. The formation of the polymeric coating on the cellulosic materials is monitored in real-time by determining at least one physico- chemical property of the coating that can be readily detected.

In preferred embodiments of the method, the quantification step comprises the use of labeled water-soluble monomer units having functional vinyl units. The term "labeled", as used herein, relates to the presence of any detectable markers which directly or indirectly generate a detectable compound or signal in a chemical, physical or enzymatic reaction. The labels may be selected inter alia from enzyme labels, colored labels, fluorescent labels, chromogenic labels, luminescent labels, radioactive labels, haptens, biotin, metal complexes, metals, and colloidal gold. All these types of labels are well established in the art. Fluorescent labels (e.g., employing rhodamine or fluorescein dyes) are particularly preferred. Fluorescently labeled monomer units are also commercially available from various suppliers.

An example for a physical reaction that is mediated by such labels is the emission of fluorescence or phosphorescence upon irradiation or excitation or the emission of X-rays when using a radioactive label. Alkaline phosphatase, horseradish peroxidase, β- galactosidase, and β-lactamase are examples of enzyme labels, which catalyze the formation of chromogenic reaction products.

Signal detection and quantification may be performed by employing standard methodology, such as inter alia fluorescence microscopy or atomic force microscopy, all well established in the art.

In specific embodiments, the method is performed under ambient atmospheric conditions and at a reaction temperature in the range between room temperature (i.e. about 20°C) and 40°C. Preferably, the reaction temperature is in the range between 25°C and 37°C, with 37°C being particularly preferred.

The invention is further described by the figures and the following examples, which are solely for the purpose of illustrating specific embodiments of this invention, and are not to be construed as limiting the claimed subject matter in any way.

EXAMPLES

The assay system according to the present invention, herein also referred to as "hydrogel reagent signaling system" (HyRES system), provides for a spatially resolved imaging of cellulose hydrolysis using a cellulase-mediated polymerization mechanism. The reagent system employed relies on an Fe(ll) Fenton reagent that is oxidized by hydrogen peroxide with concomitant stoichiometic production of a reactive hydroxyl radical which initiates polymerization. Since its discovery in the late 19^th century, Fenton chemistry has found use in many industrial applications (reviewed, for example, in Neyens, E. and Baeyens, J. (2003) J. Hazardous Materials 98, 33-50).

Herein, Fenton reagents (i.e. Fenton reaction catalysts), in particular Fe(ll), were used in combination with glucose oxidase, in a glucose detection system and shown to be capable of spatially-resolved imaging of cellulose hydrolysis in real-time using a simple fluorescent readout. Figure 1 shows a schematic overview of the HyRES system. The system combines the synergistic endo- and exoglucanase activities of a Trichoderma reesei (Tr) enzyme cocktail with the cellobiase activity of almond beta-glucosidase. Beta-glucosidase is frequently supplemented into cellulolytic enzyme mixtures to convert cellobiose to glucose, thus removing a primary inhibitor of exoglucanases contained in the cocktail. Beta- glucosidase is responsible for production of glucose which is further oxidized by glucose oxidase, resulting in production of hydrogen peroxide (H₂0₂) which directly participates in the Fenton reaction (Figure 1 a). A representative gel film that polymerized onto a piece of filter paper after immersion for 30 minutes into the HyRES system containing 1 mg/ml Tr enzyme cocktail is shown in Figure 1 b. The mixture of the assay system comprises cellulolytic enzymes (e.g., Tr enzymes), almond beta-glucosidase, glucose oxidase (GOX), Fe(ll) sulfate, PEG-diacrylate, and methacryl-rhodamine B, all combined in sodium acetate buffer at pH 4.5. Gel formation proceeded via hydroxyl radical initiated polymerization of the PEG- diacrylate in the mixture (Figure 1 c).

A schematic illustration of an embodiment according to the present invention being based on the attenuation of auto-fluorescence emitted from the cellulosic materials in the biomass in shown in Figure 6. Polymerization of the hydrogel in response to enzymatic cleavage results in attenuation of cellulose auto-fluorescence. Hence, the methodological approach of the present invention can be used on any natural biomass substrate without requiring costly dye labeling of the water-soluble monomer units.

Initially, the ability of the HyRES system was tested in order to detect endoglucanase activity using the soluble cellulose substrate carboxymethyl cellulose (CMC). The results from an exemplary experiment are shown in Figure 2a. Varying amounts of beta-1 ,4 endoglucanase from the thermophilic fungus Talaromyces emersonii were added to solutions of CMC and the reagents of the HyRES system at a reaction temperature of 37°C. The solution absorbance at 650 nm was then measured as a function of time. Since gel turned the solution turbid as it polymerized, the absorbance signal increased proportionally to the CMCase activity of the enzyme.

Next, hydrolysis of a solid substrate was measured using fluorescence detection. Cellulose- containing filter paper (Whatman #1 ) was cut into 6 mm disks and placed into the wells of a 96-well plate. The reagents of the HyRES system including a fluorescent rhodamine- methacrylate monomer were added to the FP disks, along with 1 mg/ml of Tr enzymes. At given time points, the wells were washed to remove unreacted dye molecules, and fluorescence was determined. After 120 minutes a pink-colored gel was formed that conformally coated the filter paper, observable by eye with macroscopic dimensions (several mm thick). When the reagent system was added in the absence of the hydrolytic enzymes (Figure 2b, "Without T reesei enzymes"), background fluorescence remained low indicating the hydrogel assay was specific for cellulose hydrolysis products. The pH-dependence of the assay was investigated by preparing the HyRES system in sodium acetate buffer at various pH values in the range between 4.5 to 7.5. The results from an exemplary experiment are shown in Figure 2c. A pH of 5.0 or less was necessarily required for the reaction to occur, a result attributable to base catalyzed oxidation of Fe(ll) to Fe(lll) at higher pH values and consequent quenching of the reaction. The performance of the HyRES system when employing various cellulose substrates was tested, as shown in Figure 2d. The HyRES system with fluorescent detection was found to provide high signal to noise ratios on every cellulose substrate we tested, including CMC, avicel, μ-crystalline cellulose powder (purchased from Sigma-Aldrich), dilute acid pretreated hay, filter paper, and algal Cladophora cellulose. The system has a wide applicability and will effectively provide signals in response to sugars produced by hydrolytic enzymes. Non-specific binding was not found to limit the applicability of the assay system for any of the substrates tested.

Based on kinetic data at varying substrate concentrations, the Michelis-Menten constants of cellulolytic enzymes could be evaluated by using the HyRES system according to the present invention. Figure 3 depicts a representative Michelis-Menten plot that was generated for Tr enzyme cocktails using a CMC substrate. Monitoring the absorbance vs. time plots allows calculation of the V₀ (initial reaction velocity). By measuring V₀ at variable substrate concentrations and fitting to the Michelis-Menten equation, k_m and V_max could be calculated. For the Tr mixture, a k_m = 1.58 mg/ml and a V_max = 9.3 E-3 absorbance units/sec could be determined.

Detection of the fluorescent polymer on solid substrates (e.g., filter paper) required rinsing away unreacted dye molecules and measuring the amount of dye that was incorporated into the gel coating. In order to facilitate real-time imaging total internal reflection fluorescence microscopy (TIRF microscopy) was used. Since TIRF only samples molecules within an evanescent field extending away from the glass surface to a distance of a few hundred nanometers, nM quantities of the rhodamine-methacrylate dye could be used while simultaneously rejecting the fluorescent background and performing real-time live imaging of the cellulose hydrolysis reaction. A time-resolved image series of the polymerization reaction is shown in Figure 4. Atomic force microscopy (AFM) was additionally used to image the locations of the polymeric gel adhering to the cellulose fibers (Figure 5). The applicability of the method illustrated in Figure 6, which based on the attenuation of auto-fluorescence emitted from the cellulosic materials was tested with different pre-treated biomass substrates (cf. Figure 7). Cylindrical disks 6 mm in diameter were produced from napier grass and from miscanthus perennial grass that had been pre-treated by mechanical comminution and exposure to diluted acid.

In brief, pre-treatment was accomplished by cutting the biomass samples (napier grass or miscanthus grass) into small pieces and processing them in a blender in order to produce a coarse powder, which was left in 0.1 M NaOH at 80°C for 12h. It was then filtered through Whatman filter paper using a vacuum pump, washed with water and immersed in 0.05 M HCI at room temperature for 12h. The biomass was filtered again, and the fine powder was washed thoroughly until a neutral pH was obtained. Following filtration, a damp layer of biomass with a thickness of 0.3 cm was obtained. The biomass film was then dried under pressure at 37°C overnight, e.g. with a heavy bottle on it. Finally, small disks with a diameter of 6 mm were punched out of the papery biomass using a hold punch.

The standard hydrogel mixture employed in these analyses consisted of 20 mM MES buffer, pH 4.5, 1 mg/ml glucose oxidase, 69.5 μg ml iron(ll) sulfate, 44.0 μg ml ascorbic acid, 150 mg/ml PEG-diacrylate and, in some experiments, 1 mg/ml β-glucosidase. Cellulolytic enzyme compositions were varied as indicated.

A black 96-well polypropylene plate with flat bottom (purchased from Greiner Bio One) was first cleaned with isopropyl alcohol (2-propanol) and washed with millipore water. The 6 mm biomass disks were carefully placed into the bottoms of the plate wells. The hydrogel mixture and the plate were then preheated to 37°C. The wells were then filled with 200 μΙ of the hydrogel mixture, and the plate was put into a multi-well plate reader (Infinite M1000 Pro, Tecan). The temperature was set to 42°C and fluorescence intensity was measured using a time-resolved kinetic cycle. The fluorescence intensity module was set as follows: excitation wavelength = 365 nm, emission wavelength = 430 nm, bandwidth = 5 nm, mode = top and multiple reads per well are used with filled circle, size = 4 x 4, border = 500 μηη. Mode 1 [400 Hz] is set to 50, the settle time to 20 ms and the z-position to 20.000 μηη. The results obtained for the monitoring of the saccharification efficacy of an enzyme cocktail from Trichoderma reesei on pre-treated napier grass and miscanthus perennial grass substrates are shown in Figure 8 and Figure 9, respectively. Corresponding analyses for testing the endo-/exoglucanase synergy using these substrates are shown in Figure 10 and Figure 11 , respectively. Finally, Figure 12 illustrates the results obtained for the detection of a carbohydrate binding module in a mini-cellulosome.

In conclusion, the HyRES system according to the present invention, comprising a mixture of cellulolytic enzymes, beta-glucosidase, GOX, Fe(ll), PEG-diacrylate, and rhodamine methacrylate, was shown to be capable of producing quantitative kinetic data reporting on the efficiency of cellulose hydrolysis by cellulolytic enzymes. We have characterized Michelis-Menten constants of Trichoderma reesei enzyme cocktails using an absorbance- based detection method in combination with a soluble cellulose substrate (i.e., CMC). Furthermore, the HyRES imaging system was established as an appropriate reagent mixture for use in combination with TIRF-microscopy to provide real-time observation of cellulose hydrolysis with high spatial resolution. It is tempting to speculate that when combined with superresolution image processing methods to localize positions of fluorophores that are stochastically switched into a dark state, the HyRES system can potentially provide nanoscale resolution of the hydrolysis process with high time resolution. These results taken together establish the HyRES system as a cellulase assay platform with added advantage of spatially resolved localized chemical imaging.

The present invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by embodiments and optional features, modifications and variations of the inventions embodied therein may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

1 . Method of determining the degradation of cellulosic materials in real-time, comprising:

(b) reacting the glucose monomers with glucose oxidase, thus producing hydrogen peroxide;

(c) reacting the hydrogen peroxide with a Fenton reaction catalyst, thus producing hydroxyl radicals, wherein the reaction is performed at a pH of < 5.0; and

2. The method of claim 1 , further comprising:

quantifying the production of the polymeric coating by determining one or more physico-chemical parameters being indicative for the formation of the polymeric gel.

3. The method of claim 2, wherein the physic-chemical parameters are selected from the group consisting of fluorescence emission, optical opacity, solution viscosity, pH value, sensitivity to temperature, and electric field strength.

4. The method of claim 3, wherein the fluorescence emission is the auto-fluorescence emission of the cellulosic material.

5. The method of claim 2 or 3, wherein the quantification step comprises the use of labeled water-soluble monomer units, particularly fluorescently labeled water-soluble monomer units.

6. The method of any one of claims 1 to 5, wherein step (a) comprises cleaving of the cellulosic materials by means of one or more exoglucanases, endoglucanases and β- glucosidases.

7. The method of any one of claims 1 to 6, wherein the Fenton reaction catalyst is selected from the group consisting of Fe(ll), Cu(l), Ti(lll), Cr(ll), Co(ll), and iron oxidase nanoparticles.

8. The method of any one of claims 1 to 7, wherein the water-soluble monomer units having functional vinyl groups are selected from the group consisting of acrylates, methacrylates, acrylamides, and methacrylamides.

9. The method of any one of claims 1 to 8, wherein the cellulosic material is lignocellulosic biomass.

10. The method of any one of claims 1 to 9, wherein the method is performed under ambient atmospheric conditions and at a reaction temperature of 37°C.

1 1 . Use of a method of any one of claims 1 to 10 for determining the cellulolytic enzyme activity during biomass conversion.

The use of claim 1 1 , wherein the biomass conversion is for the production of biofuel.