WO2002098824A2

WO2002098824A2 - Improvements in enzyme stability

Info

Publication number: WO2002098824A2
Application number: PCT/AU2002/000746
Authority: WO
Inventors: Khawar Sohail Siddiqui; Ricardo Cavicchioli
Original assignee: Unisearch Limited
Priority date: 2001-06-07
Filing date: 2002-06-07
Publication date: 2002-12-12
Also published as: WO2002098824A3

Abstract

An enzyme for cleaving β-1,4 -glucosidic bonds of cellulose comprising a cellulase having an aromatic group linked to a side chain of an amino acid residue or to a terminal amino acid residue of the cellulase wherein the enzyme functions at an elevated temperature and/or has an extended half-life at elevated temperature compared to the corresponding unmodified cellulose.

Description

IMPROVEMENTS IN ENZYME STABILITY

FIELD OF THE INVENTION

The invention relates to chemical modification of an enzyme in order to improve the stability of the enzyme. In particular, the invention relates to cellulases which have improved temperature stability, and/or which have high activity at elevated temperature.

BACKGROUND OF THE INVENTION

All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

Cellulose is one of the most prevalent natural polymers in the world. It is a major constituent of plant matter, textiles and paper, and also constitutes a large proportion of the world's municipal waste. Thus, efficient and effective means of treating cellulose are required.

Cellulose is a polymer of glucose residues joined by β-1,4 linkages. Cellulases are enzymes, which cleave the β-l,4-glucosidic bonds of cellulose to form oligosaccharides and/or monosaccharides . These enzymes are used in many industrial processes including, for example, the textile industry for treating as additives (stone washing) , in household laundry detergents for improving fabric softness and brightness, in the processing of fruit juice, in baking, and in the efficient conversion of cellulosic biomass to soluble breakdown products. Thus, cellulases represent industrially important enzymes (Godfrey, T and West, S (1996) Industrial Enzymology. Macmillan Press, London) .

A significant problem with the industrial application of cellulases is that many processes in which cellulases would be useful are carried out at temperatures above that at which cellulases are functional. Thus, significant limitation applies to the use of cellulases in many industrial applications.

Enzyme thermostability and activity of cellulases at elevated temperature was generally believed to require a combination of a number of features including hydrophobic interactions, compact packing of residues, salt bridges, reduction of conformational flexibility, reduction of the entropy of unfolding, α-helix stabilization, hydrogen bonding, disulfide bridges, metal binding, surface loop stabilization and resistance to degradation. Previous studies by the inventor has shown that thermostability in water miscible organic solvent mixtures, but not aqueous solvents, is conferred on carboxymethylcellulase (CMCase) when the enzyme is double modified with dimethylamine and acetic anhydride to neutralise all charged surface groups.

The inventor has now found that by linking an aromatic group to a side chain of an amino acid residue of a cellulase, or to a terminal amino acid residue of a cellulase, the cellulase is then capable of cleaving the β- 1,4-glucosidic bonds of cellulose at an elevated temperature and/or has an extended half-life at an elevated temperature.

SUMMARY OF THE INVENTION In a first aspect the invention provides an enzyme for cleaving β-1,4 -glucosidic bonds of cellulose comprising a cellulase having an aromatic groups linked to a side chain of an amino acid residue of the enzyme or to a terminal amino acid residue of the enzyme wherein the enzyme functions at an elevated temperature and/or has an extended half-life at an elevated temperature compared to the corresponding unmodified cellulase.

In one embodiment, the enzyme has a maximum activity at a temperature that is higher than the temperature at which the corresponding unmodified cellulase has maximum activity.

Preferably the enzyme functions at an elevated temperature and/or has an extended half-life at elevated temperature compared to the corresponding unmodified cellulase at a pH of between 5.0 and 9.0, more preferably at a pH between 5.2 and 7.8, and even more preferably, at a pH between 5.2 and 6.8.

Preferably the enzyme functions at an elevated temperature and/or has an extended half-life at elevated temperature compared to the corresponding unmodified cellulase in an aqueous solvent. More preferably the enzyme functions at an elevated temperature and/or has an extended half-life at elevated temperature compared to the corresponding unmodified cellulase in aqueous solvent and in water- miscible organic solvents. The aromatic group may be any aromatic group that improves the capacity of the enzyme to cleave β-1,4 glucosidic bonds of cellulose at elevated temperature. In one embodiment the aromatic group is preferably a derivative of benzene. More preferably the aromatic group is an optionally substituted phenylalkylamino group, an optionally substituted aralkylamino group, or an optionally substituted benzoyl group. Even more preferably the aromatic group is selected from the group consisting of benzylamine, aniline, benzoic acid, phthalic acid, mellitic acid, pyromellitic acid and 3, 3', 4, 4'- benzophenone tetracarboxylic acid. In another embodiment the aromatic group is a heterocyclic amine. Preferably the heterocyclic amine is selected from the group consisting of adenine, adenosine, pyridine, cis-aconitic acid and 2, 3-pyridine carboxylic acid.

In one embodiment, the side chain is the side chain of an acidic amino acid. Preferably the acidic amino acid is aspartate or glutamate.

In another embodiment, the side chain is the side chain of a basic amino acid. Preferably the basic amino acid is lysine.

The aromatic group may be linked to the side chain by any means known in the art. Preferably the aromatic group is linked to the side chain of an amino acid, or to a terminal amino acid, by an amide bond.

The enzyme may further comprise groups other than aromatic groups linked to side chains of amino acid residues of the enzyme.

In various embodiments, the enzyme (a) comprises at least one cis-aconitic residue linked to the side chain of a lysine residue, or to an amino terminal amino acid residue of the enzyme, (b) comprises at least one 3, 3', 4, 4' benzophenone tetracarboxylic acid linked to the side chain of a lysine residue, or to an amino terminal amino acid residue of the enzyme .

^® comprises at least one 2,3 pyridine carboxylic acid residue linked to the side chain of a lysine residue, or to an amino terminal amino acid residue of the enzyme. (d) comprises at least one benzoic acid residue linked to the side chain of a lysine residue or to an amino terminal amino acid residue of the enzyme.

(e) comprises at least one pyromellitic acid residue linked to the side chain of a lysine residue, or to an amino terminal amino acid residue.

(f) comprises at least one adenosine residue linked to the side chain of an aspartate residue, a glutamate residue or a carboxy terminal residue.

(g) comprises at least one adenine residue linked to the side chain of an aspartate residue, a glutamate residue or a carboxy terminal amino acid residue.

(h) comprises at least one pyridine residue linked to the side chain of an aspartate residue, a glutamate residue or to an amino terminal amino acid residue, (i) comprises at least one phthalic acid residue linked to the side chain of a lysine residue or to an amino terminal amino acid residue.

(j) comprises two aniline residues. Preferably, at least one of the aniline residues is linked to an aspartate residue or a glutamate residue. (k) comprises a further aromatic group linked to a side chain of an amino acid residue of the enzyme, or linked to a terminal amino acid residue of the enzyme, the further aromatic group for improving the capacity of the enzyme to cleave β-1,4 glucosidic bonds of cellulose at an elevated temperature .

(1) comprises at least two aromatic groups, in which one is selected from the group consisting of benzylamine, aniline and pyridine, and one is a benzoyl group, (m) comprises a pyridine residue and a benzoic acid residue, wherein the pyridine residue and benzoic acid residue are linked to side chains of amino acid residues or to terminal amino acid residues of the enzyme. Preferably the pyridine residue is linked to an aspartate residue, a glutamate residue or to a carboxy terminal amino acid residue. Preferably the benzoic acid residue is linked to a lysine residue or to an amino terminal amino acid of the enzyme.

(n) further comprises at least one amino group for improving the capacity of the enzyme to cleave β-1,4 glucosidic bonds of cellulose at an elevated temperature, and the amino group is linked to the side chain of an amino acid residue of the enzyme or linked to the carboxyl-terminal amino acid residue of the enzyme. Preferably the amino group is linked to the side chain of an acidic amino acid residue. More preferably the acidic amino acid is aspartate or glutamate.

(o) comprises a pyromellitic residue linked to the side chain of a lysine residue or to an amino terminal amino acid residue, and an amino group linked to the side chain of an aspartate or glutamate residue or linked to the carboxyl-terminal amino acid residue of the enzyme.

(p) further comprises an aliphatic amine containing group linked to a side chain of an amino acid residue of the enzyme .

(q) further comprises an aliphatic amine containing group linked to the carboxyl terminal amino acid residue of the enzyme. Preferably the aliphatic amine containing group is selected from the group consisting of argininamide, arginine methyl ester, arginine ethyl ester, glycinamide, methylamine, dimethylamine and trimethylamine . (r) further comprises an arginine residue linked to a side chain of an amino acid residue of the enzyme, (s) In another embodiment, the enzyme further comprises an arginine residue linked to the carboxyl terminal amino acid residue of the enzyme. In one embodiment, the aromatic group is selected from the group consisting of benzoic, phthalic, mellitic and pyromellitic groups. Preferably the enzyme further comprises an argininamide, an arginine methyl ester or an arginine ethyl ester linked to a side chain of an amino acid residue of the enzyme.

In one embodiment, the enzyme comprises at least one arginine methyl ester residue and at least one pyromellitic acid residue, wherein the pyromellitic .acid residue is linked to the side chain of a lysine residue or to an amino terminal amino acid residue. Preferably the arginine methyl ester residue is linked to the side chain of an aspartate residue or a glutamate residue, or to a carboxyl terminal amino acid residue of the enzyme.

The enzyme may further include amino acid residues modified in a manner other than through linking a group to the amino acid side chain. For example, the enzyme may further comprise at least one homoarginine residue for improving the capacity of the enzyme to cleave β-1,4- glucosidic bonds of cellulose at an elevated temperature.

Preferably the enzyme has an amino acid sequence of a cellulase of an organism. In a preferred embodiment, the cellulase is from an organism selected from the group consisting of invertebrate, angiosperm, fungus, yeast, bacteria including archeaebacteria and eubacteria, and algae. In a preferred embodiment, the organism is a psychrophilic or a mesophilic organism. Preferably, the organism is a fungus. Preferably the fungus is selected from the group consisting of Aspergillus sp, Scopulariopsis sp. and Trichoderma sp. Preferably the fungus is Aspergillus niger or Trichoderma sp. In another embodiment, the enzyme has the amino acid sequence of a cellulase encoded by a recombinant nucleic acid molecule. In a preferred embodiment, the recombinant nucleic acid molecule is from an organism selected from the group consisting of invertebrate, angiosperm, fungus, yeast, bacteria including archeaebacteria and eubacteria, and algae. In a preferred embodiment, the recombinant nucleic acid molecule is from a psychrophilic or a mesophilic organism. Preferably, the organism is a fungus. Preferably the fungus is selected from the group Aspergillus sp, Scopulariopsis sp. and Trichoderma sp.

Preferably the fungus is Aspergillus niger or Trichoderma sp.

In a second aspect, the invention provides a process for producing an enzyme of the first aspect of the . invention, the process comprising the step of contacting an enzyme capable of cleaving a β-1,4-glucosidic bond of cellulose with a compound which comprises an aromatic group in conditions sufficient for linking the aromatic group to a side chain of an amino acid residue of the enzyme, or to a terminal amino acid residue of the enzyme. In one embodiment, the process comprises activating carboxyl groups of amino acid residues of the enzyme in the presence of an aromatic nucleophile. The aromatic nucleophile may be an amine containing derivative of benzene or a heterocyclic amine. Preferably, the aromatic nucleophile is adenine hydrochloride, adenosine hydrochloride, aniline hydrochloride, benzylamine hydrochloride or pyridine hydrochloride.

In another embodiment, the process comprises contacting the enzyme in the presence of an aromatic anhydride in conditions sufficient for linking the aromatic group to an amino group of a basic amino acid residue of the enzyme, or to the amino terminal amino acid residue of the enzyme. The aromatic anhydride may be any aromatic containing anhydride. Preferably the aromatic anhydride is selected from the group consisting of benzoic anhydride, pyromellitic dianhydride, mellitic trianhydride, trimellitic anhydride, phthalic anhydride, cis aconitic anhydride, 3, 3', 4, 4' benzophenone tetracarboxylic dianhydride and 2, 3 pyridine carboxylic anhydride .

The carboxyl groups may- be activated by any compound that provides sufficient conditions for an aromatic group to be linked to the side chain of an amino acid residue of the enzyme, or linked to the carboxy-terminal amino acid residue of the enzyme. In one embodiment, carboxyl groups are activated by carbodiimide. In a preferred embodiment, the carboxyl groups of the enzyme are activated by 1- ethyl-3 (3-dimethylaminopropyl) carbodiimide or l-(3- dimethylaminopropyl) -3 -ethyl carbodiimide methiodide. In one embodiment, the process comprises the further step of contacting the enzyme with an agent for controlling the linkage of the aromatic group to a side chain of an amino acid residue or a terminal amino acid residue located in a catalytic site of the enzyme. Preferably the agent is an inhibitor of the enzyme. Preferably the inhibitor is cellobiose. Alternatively the agent is a substrate of the enzyme. The substrate may be any oligomer of β-1,4 linked glucose residues. Preferably, the substrate is selected from the group cellotriose, cellotetriose and cellopentiose. In another embodiment, the process comprises the further step of guanidination of a lysine residue and the terminal amino group of the enzyme.

In one embodiment, the process comprises activating carboxyl groups of amino acid residues of the enzyme in the presence of an aromatic nucleophile for linking the aromatic group of the aromatic nucleophile to a carboxyl group of an acidic amino acid, and contacting the enzyme in the presence of an aromatic anhydride in conditions sufficient for linking the aromatic group of the anhydride to an amino group of a basic amino acid.

In one embodiment, the process comprises the further step of contacting the enzyme with an aliphatic amine- containing nucleophile under conditions sufficient for linking of an aliphatic amine-containing group to a carboxyl group of a side chain of an amino acid residue of the enzyme, or to the terminal carboxyl group of the enzyme. Preferably the aliphatic amine- containing group is linked to the carboxyl group of a side chain of an aspartate residue or a glutamate residue. More preferably the aliphatic amine-containing nucleophile is selected from the group argininamide dihydrochloride, arginine methyl ester dihydrochloride, arginine ethyl ester dihydrochloride, glycinamide hydrochloride, methylamine hydrochloride, dimethylamine hydrochloride, ethylenediamine dihydrochloride and trimethylamine hydrochloride . In a third aspect, the invention provides an enzyme which has improved capacity to cleave β-1, 4- glucosidic bonds of cellulose at an elevated temperature, comprising an amino acid sequence of a cellulase in which at least one lysine residue has been replaced by a homoarginine residue, and optionally

(a) an aromatic group linked to a side chain of an amino acid residue of the enzyme or to a terminal amino acid residue of the enzyme, and/or (b) an amino group linked to the side chain of an amino acid residue of the enzyme or linked to the carboxyl-terminal amino acid residue of the enzyme, wherein the enzyme functions at an elevated temperature and/or has an extended half-life at an elevated temperature compared to the unmodified cellulase.

Preferably the enzyme is a cellulase from Aspergillus niger. The lysine may be converted to homoarginine by chemical means, or it may be replaced in the amino acid sequence of the enzyme via recombinant methods.

In a fourth aspect, the invention provides a process for producing an enzyme of the third aspect, comprising the step of contacting an enzyme capable of cleaving a β-1, 4-glucosidic bond of cellulose with guanyl-3,5- dimethyl pyrazole under conditions sufficient to form at least one homoarginine residue.

In a fifth aspect, the invention provides a composition comprising an enzyme according to the first or the third aspect of the invention, together with an appropriate carrier.

In a sixth aspect, the invention provides a product produced by the process of the second or the fourth aspect of the invention.

In a seventhth aspect, the invention provides a use of an enzyme according to the first or the third aspect of the invention for cleaving the β-1, 4-glucosidic bonds of cellulose.

For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows an analysis of the effect of reaction time in a preferred method of the invention on extent of modification of carboxymethylcellulase by native enzyme mobility shift assay. Reaction times are as follows: lane 1: control (without modification), lane 2: 0.5 in, lane 3: 1 min, lane 4: 3 min, lane 5: 5 min, lane 6: 7 min, lane 7: 10 min and lane 8: 15 min.

Figure 2 shows the results of an analysis of a preferred enzyme according to the present invention. Illustrated is a plot of the number of modified carboxyl groups of the enzyme (E_nth) versus time. Figure 3 shows temperature optima of native carboxymethylcellulase and a preferred enzyme according to the present invention in aqueous medium (circles) or 40%(v/v) aqueous dioxan (triangles). Native CMCases are indicated by open circles and triangles, modified CMCase is indicated by closed circles and triangles.

Figure 4 shows the temperature optimum (T_opt) of a preferred enzyme according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION The practice of the present invention employs, unless otherwise indicated, conventional chemistry, protein chemistry, molecular, biological and enzymological techniques within the skill in the art. Such techniques are well known to the skilled worker, and are explained- fully in the literature See, for example, Coligan, Dunn, Ploegh, Speicher and Wingfield "Current protocols in Protein Science" (1999) Volume I and II (John Wiley & Sons Inc.); Sambrook and Russel "Molecular Cloning: A Laboratory Manual" (2001); Cloning: A Practical Approach," Volumes I and II; (D.N. Glover, ed., 1985); Bailey, J.E. and Ollis, D.F., Biochemical Engineering Fundamentals, McGraw-Hill Book Company, NY, 1986; Glazer, AN; DeLange, RJ; Sigman, DS (1975) Chemical modification of proteins. North Holland Publishing Company, Amsterdam; Lundblad, RL (1995) Techniques in protein modification. CRC Press, Inc. Boca Raton, FI . USA; Hirs, CHW; Ta asheff, SN, Eds. (1972) Methods in Enzymology, Vol XXV. Academic Press, New York.

Before the present methods are described, it is understood that this invention is not limited to the particular materials and methods described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and it is not intended to limit the scope of the present invention which will be limited only by the appended claims. It must be noted that as used herein and in the appended claims, the singular forms "a",

"an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "an enzyme" includes a plurality of such enzymes, and a reference to "an amino acid" is a reference to one or more amino acids. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any materials and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred materials and methods are now described.

All publications mentioned herein are cited for the purpose of describing and disclosing the protocols and reagents which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

As described herein, aromatic groups were linked to side chains of amino acid residues of an endo-β-1, 4-D- glucanase or linked to the terminal amino acid residues of the enzyme, and that enzyme was then observed to have activity at an elevated temperature. As used herein, "elevated temperature" refers to a temperature, which is elevated relative to the temperature at which the corresponding unmodified cellulase exhibits maximum activity. As used herein, the term "modified" refers to a cellulase having an aromatic group linked to a side chain of an amino acid or to a terminal amino acid of the enzyme, and the term "unmodified" refers to the corresponding cellulase not having an aromatic group linked to a side chain of an amino acid or to a terminal amino acid of the enzyme. As used herein, the expression "the enzyme functions at an elevated temperature" means that the enzyme has an extended half-life at elevated temperature compared to that of the corresponding unmodified cellulase, and may have a maximum activity (optimum temperature or T_opt) at an elevated temperature. The term "extended half-life" means that the half-life of the enzyme is longer compared that to that of the corresponding unmodified cellulase. In one embodiment, the enzyme functions at a temperature of between 65°C and 160°C, preferably between 65°C and 155°C. In another embodiment, the enzyme functions at a temperature of between 65°C and 95°C, preferably between 65°C and 85°C, more preferably between 65°C and 75°C. As described herein, aromatic groups were linked to side chains of amino acid residues of an endo-β-l,4-D-glucanase or linked to the terminal amino acid residue of the enzyme, and that enzyme was then observed to have activity at a temperature of more than 120°C. The temperature at which endo-β-1, 4-D- glucanase having aromatic groups linked to side chains of amino acid residues or linked to the terminal amino acid residue of the enzyme has maximum activity was observed to be elevated by as much as about 70°C relative to the temperature at which endo-β-l,4-D glucanase has maximum activity.

The enzyme of the invention may be capable of cleaving β-1, 4-glucosidic bonds at elevated temperature in both an aqueous solvent and a water iscible solvent. As used herein, "water miscible solvent" refers to an organic solvent that is miscible in the water. Water miscible organic solvents include, for example, dioxan, dimethylsulfoxide, ethanol or methanol. The water miscible organic solvent may be used neat, or preferably mixed with water. Preferably, the water miscible organic solvent is dioxan preferably mixed with water at a concentration of 40% v/v.

Preferably, the enzyme of the invention has a pH optimum that is decreased relative to the pH optimum of endo-β-l,4-D glucanase. Accordingly, the enzyme may be more efficient at hydrolysing endo-β-l,4-D glucosidic bonds of cellulose at lower pH than endo-β-l,4-D glucanase.

The first step in preparing the enzyme of the invention involves selecting the cellulase to which the aromatic group is to be linked. The cellulase may be any enzyme that is capable of cleaving the β-l,4-D glucosidic bonds of cellulose. The cellulase may be isolated, for example, from an organism selected from the group consisting of vertebrate, invertebrate, angiosperm, fungus, yeast, bacteria, archeae and algae. Preferably, the cellulase is isolated from an organism of bacterial or fungal origin. As many cellulases are related in structure (for example, see Wood, WA; Kellogg, ST. Eds. 1988 Methods in Enzymology, Vol 160 Academic Press, New York) , a person skilled in the art would readily appreciate that a number of cellulases may be capable of functioning at elevated temperature after linking an aromatic group to the side chain of an amino acid or to a terminal amino acid of the enzyme. Examples of organisms from which cellulases may be obtained which may be suitable include species such as Humicola, Coprinuc, Sporutrichum, Thielavia, Myceliopthora, Fusarium, Myceliophthora, Acremonium, Cephalosporium, Scytalidium, Penicillium or Aspergillus (see for example EP 458162) , Trichoderma, Bacillus, Streptomyces, Scopuloropsis. Examples of particular organisms and strains from which cellulases may be isolated include Humocola insolens (see for example US Pat. No. 4,435,307), Coprinus cinereus, Fusarium oxysprorum, Myceliophthora thermophila, Meripilus giganteus, Thielavia terrestris, Acremonium sp., Acremonium persicinum, Acremonium acremonium, Acremonium brachypenium, Acremonium dichromosporum, Acremonium obclavaturn, Acremonium pinkertoniae, Acremonium roseogriseum, Acremonium incoloratum, Acremonium furatum, Cephelosporium sp., Trichoderma viride, Trichoderma reesei, Trichoderma koningii, Bacillus sp. (see for example, US Pat. No. 3,844,890 and EP 458162), Streptomyces sp. (see for example EP 458162) . In one embodiment, the organisms and strains from which cellulases may be isolated include Arctonis spp., Scopuloropsis spp., Aspergillus niger, Aspergillus oryzae, Aspergillus nidulans . In one embodiment., the organism is Aspergillus spp., Scopulariopsis spp. or Trichoderma spp. Even more preferably, the organism is Trichoderma sp. or Aspergillus niger . The cellulase may be the product of a recombinant nucleic acid molecule, or in other words, the cellulase may have an amino acid sequence of a cellulase encoded by a recombinant nucleic acid molecule. As used herein, the expression "recombinant nucleic acid molecule" refers to a nucleic acid molecule that has been cloned or isolated and is expressed in a host organism that is different to the organism from which the nucleic acid molecule derives. The term "nucleic acid molecule" refers to deoxyribonucleic acid and ribonucleic acid in all their forms, ie. Single and double stranded DNA, cDNA, mRNA, and the like. The recombinant nucleic acid molecule may be obtained from any organism that is capable of producing cellulase enzyme. For example, the recombinant nucleic acid molecule may be obtained from any of the abovementioned organisms. The cellulase derived from recombinant nucleic acid may have an amino acid sequence that is the same as the organism from which it is derived. Alternatively, the recombinant nucleic acid may be a biologically active fragment of a cellulase. As used herein, the term "biologically active fragment" refers to a cellulase where one or more amino acid residues are added, deleted or substituted at the N- or C- terminus of, or within, the cellulase amino acid sequence. For example, the biologically active fragment may be a mutant cellulase gene which has been isolated or synthesized for desired properties such as, for example, improved activity under certain conditions such as, for example, temperature, pH, salt concentration etc.

Preferably, the cellulase is carboxymethylcellulase. Even more preferably, the cellulase is carboxymethyl cellulase from Aspergillus niger, Trichoderma sp. or Scopulariopsis sp. .

The cellulase which is used in the method of the present invention may be produced by fermentation of any of the organisms mentioned above on nutrient media containing suitable carbon and nitrogen sources and inorganic salts, using procedures known in the art such as that described in Bennett, J..W. and LaSure(Eds.) More Gene Manipulations in Fungi, Academic Press, CA, 1991. Temperature ranges and other conditions suitable for growth and cellulase production are also known in the art and are described in, for example, Bailey, J.E. and Ollis, D.F., Biochemical Engineering Fundamentals, McGraw-Hill Book Company, NY, 1986. As used herein, the term "fermentation" refers to any growth condition which results in production of cellulase by the organism(s) . It will be understood by persons skilled in the art that fermentation can refer to small or large scale fermentation and includes, for example, shake- flask cultivation, continuous, batch, fed-batch and solid state fermentation in laboratory or industrial fermenters .

The cellulases may be isolated by any method that is suitable for isolating active cellulase from growth medium. Suitable methods known in the art include, for example, centrifugation, filtration, spray drying, evaporation, precipitation, ion exchange chromatography, gel filtration chromatography, hydrophobic-interaction chromatography (HIC) , affinity chromatography or the like, and combinations thereof. An example of an isolation method is as follows: fermentation broth is separated from the culture medium by centrifugation at 8000rpm.

Cellulase is precipitated from the supernatant using a 65% saturated solution of ammonium sulphate. The precipitate is subsequently dissolved in 25mM phosphate buffer pH 7, 5mM EDTA. The solution is then applied to a Q-Sepharose FF (diameter 5cm, length 10cm) Anion Exchange column. The column is subsequently washed with 25mM phosphate buffer pH 7, 5mM EDTA until an absorbancy of 0.2 Absorbance Units. A gradient of 0 to 0.5M NaCl in 25mM phosphate buffer pH 7, 5mM EDTA is applied to the column in 80 minutes followed by a gradient from 0.5 to IM NaCl in 10 minutes. Elution may be performed in the first gradient.

The cellulase for use in the method of the invention may be a single isolated cellulase or a mixture of cellulases from different sources. For example, the cellulase may be those described in, for example, W091/17244 or WO-920609, or commercially available preparations such as, for example, Cellusoft L ™, Cellusoft Ultra ™, Aspergillus niger cellulase from Sigma or Trichoderma sp. cellulase from Megazyme.

Preferably, the cellulase is used as a single isolated cellulase. Alternatively, the cellulase may be a portion of a mixture of different enzymes or other compounds. For example, it is envisaged that the cellulase may be used in a crude form with contaminating compounds including other enzymes and proteins . In this circumstance, the cellulase may not be the only enzyme to which an aromatic group is linked, however the resulting mixture will retain the ability to cleave cellulose at elevated temperature because of the presence in the mixture of cellulase having an aromatic group linked to a side chain of an amino acid residue of the enzyme or to a terminal amino acid residue of the enzyme.

Once the cellulase is obtained as described above, an aromatic group is contacted with the amino acid side chain. As used herein, the term "contacted" refers to sufficient contact between the amino acid side chain and the aromatic group which permits the aromatic group to be linked to the amino acid side chain in conditions sufficient for linking the aromatic group to an amino acid side chain or a terminal amino acid of the enzyme. As used herein, the term "aromatic group" means any compound that comprises a benzene ring or is a heterocyclic compound and which improves the capacity of the enzyme to cleave β-1, 4-glucosidic bonds at elevated temperature. The aromatic group may be, for example, an unsubstituted, singly substituted or multiply substituted benzene ring or heterocyclic compound. Preferably, the aromatic group is selected from the group comprising aniline, pyridine, benzylamine, adenine, adenosine, cytosine, cytidine, benzoic acid, pyromellitic acid, mellitic acid, pthalic acid, cis-aconitic acid, benzophenone tetracarboxylic acid, and 2,3- pyridine carboxylic acid. The term "linked" refers to any linkage formed between a portion of the amino acid side chain and the aromatic group. It will be appreciated by those skilled in the art that following linkage of the aromatic group to the amino acid side chain, the amino acid side chain to which the aromatic group is linked will be altered and will differ from the amino acid side chains common to many proteins owing to the presence of the aromatic group linked to the side chain of the amino acid. The amino acid side chains "common to many proteins" will be understood by those skilled in the art to mean the side chains belonging to the amino acids alanine, asparagine, aspartate, arginine, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, lysine, leucine, methionine, phenyialanine, proline, serine, tyrosine, tryptophan, threonine and valine. The aromatic group may be linked to the amino acid side chain in any manner. In one embodiment, the aromatic group is linked to the amino acid side chain through one or more nitrogen atoms. Preferably, the aromatic group is linked to the amino acid side through an amide bond. In another embodiment, the aromatic group may be linked to the amino acid side chain through a linker. As used herein, a "linker" is a molecule which is not part of the aromatic group nor part of the amino acid side chain, but serves to link the aromatic group to the side chain of the amino acid.

The "conditions sufficient" for linking the aromatic group to a side chain of an amino acid residue or a terminal amino acid residue may be any conditions which allow a reaction to occur between the amino acid side chain and the aromatic group which results in linkage of the aromatic group to the amino acid side chain. In one embodiment, the carboxyl groups of amino acid side chains and/or the carboxy terminus of the enzyme are activated in the presence of an aromatic nucleophile. Preferably, the amino acid side chain is the side chain of aspartate and/or glutamate residues. As used herein, the term "activated" means to render the carboxyl groups reactive with an aromatic nucleophile. Preferably, carboxyl groups of the enzyme may be activated by incubating the enzyme with a carbodiimide. Methods for the use of carbodiimide in the activation of carboxyl groups are provided in, for example, Carraway, K.L. and Koshland, D.E. Jr, Carbodiimide modification of proteins. In: Methods in Enzymology (Hirs, C.H.W. and Timasheff, S.N., Eds.) Academic Press, New York, 1972, XXV, 616-623, Sheehan and Hess, J. Am. Chem. Soc. 77:1067, 1955 and Khorana, Chem.Ind. 1087, 1995. The reaction is preferably a condensation of the carboxyl with a substituted carbodiimide to form an active O-acylisoourea intermediate. Nucleophilic substitution with the aromatic nucleophile forms a stable amide with elimination of the substituted urea. The carbodiimide may be, for example, l-ethyl-3 (3 -dimethylaminopropy1) carbodiimide or l-(3- dimethyla inopropyl) -3 -ethyl carbodiimide methiodide. The term "aromatic nucleophile" refers to any nucleophile comprising an aromatic group. Preferably, the aromatic nucleophile is an amine containing derivative of benzene or a heterocyclic amine. For example, aromatic nucleophiles may include aniline hydrochloride, pyridine hydrochloride, benzylamine hydrochloride, adenine hydrochloride or adenosine hydrochloride. The carboxyl groups of the amino acid may be activated with carbodiimide prior to adding the aromatic nucleophile to the reaction. Preferably, the carboxyl groups of the amino acid side chains are activated with carbodiimide in the presence of the aromatic nucleophile. Preferably, the nucleophile is dissolved in an appropriate buffer such as, for example, K2HPO4/KH₂PO₄ buffer at a pH of preferably between 3.0 and 7.0, more preferably between 4.0 and 6.0. The buffer may optionally contain a cellulase inhibitor. Suitable inhibitors may be, for example, cellobiose, cellotetriose, cellόtriose, cellopentiose, or any other substrate of cellulase which is capable of protecting the active site of cellulase from modification. Cellulase is added to the solution either as a dried preparation or as a solution. The reaction is initiated by the addition of carbodiimide to a final concentration of preferably between 30mM and 200mM, more preferably between 40mM and lOOmM. It will be appreciated by persons skilled in the art that optimum times for allowing the reaction to proceed will vary depending on factors such as the concentration of reagents, the source of reagents, temperature conditions etc, and may be determined empirically. Preferably, the enzyme is further purified using techniques known in the art such as, for example, dialysis, centrifugation, filtration, spray drying, evaporation, precipitation, ion exchange chromatography, gel filtration chromatography, hydrophobic-interaction chromatography, affinity chromatography or the like, or combinations thereof . In another embodiment, an aromatic group may be linked to the amine group of a side chain of an amino acid or to the amino terminus of the enzyme by contacting the enzyme with an aromatic anhydride. Preferably, the amine group of an amino acid side chain is the amine group of side chains of lysine residues. As used herein, an aromatic anhydride is an anhydride, which comprises an aromatic group. Aromatic anhydrides may include, for example, benzoic anhydride, pryromellitic dianhydride, mellitic trianhydride, trimellitic anhydride, phthalic anhydride, cis aconitic anhydride, 3, 3', 4, 4' benzophenone tetracarboxylic dianhydride or 2,3 pyridine carboxylic anhydride. Preferably, the cellulase is dissolved or diluted in a buffer, preferably between pH 7.0 and 12, more preferably between pH 8.0 and 11.0. Optionally, a cellulase inhibitor may be included as mentioned above.

The aromatic anhydride is preferably added to the enzyme solution to begin the reaction. The resulting solution is thereafter incubated for an amount of time that can readily be determined by those skilled in the art. The aromatic anhydride may be added to the enzyme solution in a single application or as a plurality of smaller aliquots. Preferably, the enzyme is further purified using techniques known in the art such as, for example, dialysis, centrifugation, filtration, spray drying, evaporation, precipitation, hydrophobic-interaction chromatography, ion exchange chromatography, gel filtration chromatography, affinity chromatography or the like, or combinations thereof.

Also contemplated are enzymes comprising two or more different aromatic groups linked to side chains of amino acids of the enzyme. In preparing these enzymes, the aromatic groups may be linked, for example, by incubating the enzyme with carbodiimide in the presence of two or more different aromatic nucleophiles, or by incubating the enzyme in, for example, the presence of two or more different aromatic anhydrides.

In one embodiment, the enzyme may comprise an aromatic group linked to a carboxyl group and an aromatic group linked to an amino group. In preparing an enzyme of this type, both of the above reactions may be applied to the enzyme. For example, firstly, the enzyme may be reacted with a carbodiimide and an aromatic nucleophile, and subsequently reacted with an aromatic anhydride. Alternatively, the enzyme may be reacted with an aromatic anhydride followed by reaction with a carbodiimide and an aromatic nucleophile.

It is also envisaged that the enzyme of the invention may comprise additional groups that are not aromatic groups. For example, aliphatic amino containing nucleophiles may be used to link aliphatic amine containing groups to carboxyl groups on amino acid side chains or the carboxy terminus of the enzyme using the methods described herein. Aliphatic amine containing nucleophiles may include, for example, argininamide dihydrochloride, arginine methyl ester dihydrochloride, arginine ethyl ester dihydrochloride, glycinamide hydrochloride, methylamine hydrochloride, dimethylamine hydrochloride, ethylenediamine dihydrochloride and trimethylamine hydrochloride .

Also contemplated are compositions comprising the enzyme according to the present invention. In a preferred embodiment, the compositions comprise the enzyme according to the invention as the major enzymatic component. Alternatively, the composition may comprise multiple enzymatic activities, such as an aminopeptidase, an amylase, a carbohydrase, a carboxypeptidase, a catalase, a chitinase, a cutinase, a deoxyribonuclease, an esterase, an alpha-galactosidase, a beta-galactosidase, a glucoamylase, an alpha-amylase, an alpha-glucosidase, a beta-glucosidase, a haloperoxidase, an invertase, a laccase, a lipase, a mannosidase, a mutanase, an oxidase, a pectinolytic enzyme, a peroxidase, an iso erase, a phytase, a decarboxylase, a dehydrogenase, a polyphenoloxidase, a proteolytic enzyme, a ribonuclease, or a xylanase.

The composition may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition. For instance, the composition may be in the form of a granulate or a microgranulate. The additional enzymes to be included in the composition may be stabilized in accordance with methods known in the art. The dosage of the polypeptide composition of the invention and other conditions under which the composition is used may be determined on the basis of methods known in the art.

The enzyme according to the present invention and compositions comprising the enzyme may be applied in industrial processes conventionally involving the action of cellulases. Major applications for cellulases are found in the detergent industry, in the textile industry, in paper pulp processing industry, and in the food and feed industry. In preferred embodiments the enzyme preparation of the invention may be used for degradation or modification of plant material, e.g. cell walls, for the treatment of fabric or textile, preferably for preventing backstaining, for bio-polishing or "stone-washing" cellulosic fabric, in the treatment of paper pulp, preferably for debarking, defibration, fibre modification, enzymatic de-inking or drainage improvement or for degradation of cellulose into glucose for the production of products such as ethanol or fructose. An embodiment of the invention is now described in the following Examples which will be understood to merely exemplify and not to limit the scope of the invention.

EXAMPLE 1 CHEMICAL MODIFICATION OF CARBOXYMETHYLCELLULASE

To link aromatic groups to side chains of amino acid residues arranged on the surface of the enzyme, surface carboxyl groups were activated using l-ethyl-3(3- dimethylaminopropyl) carbodiimide (EDC) in the presence of aniline-hydrochloride as nucleophile and cellobiose as competitive inhibitor for the protection of active-site residues. Aniline hydrochloride (25 mM) was added to 9 ml of 20 mM K₂HPO₄/KH₂PO₄, pH5.25 buffer containing 20 mM cellobiose as competitive inhibitor, the pH was readjusted to 5.25 with 5 M NaOH and the volume was made up to 10 ml. Purified freeze dried Scopulariopsis sp. carboxymethylcellulase powder was dissolved in the above mentioned nucleophilic solution at a concentration of 10 mg ml^"1 (2 U ml^"1) and allowed to equilibrate at 30°C for 30 min. The coupling reaction was initiated by adding solid EDC to a final concentration of 50 mM. Aliquots were withdrawn at different time intervals, added to equal amount of 0.5 M sodium acetate, pH6 buffer to quench the reaction and subjected to native enzyme mobility shift assay, NEMSA as described in Rashid et al . (1997) using high resolution in situ inhibited native (HiRISIN) -PAGE as described in Afzal et al . (2000) containing 1.5% (w/v) carboxymethylcellulose in the resolving gel and subsequently stained for cellulase activity for the determination of extent of modification. Aniline linked carboxymethylcellulase, which was modified for three minutes was subjected to G-25 desalting chromatography on Pharmacia Fast Protein Liquid Chromatography system for the removal of excess reagents and subsequent characterization.

The extent of coupling was followed by native enzyme mobility shift assay, NEMSA as shown in Figure 1. The presence of a single band in each lane shows the absence of appreciable amounts of heterogeneity during chemical modification (Figure 1) . Figure 2 illustrates a plot of the number of modified carboxyl groups of the enzyme (E_nth) versus time, showing kinetics of aniline coupling. E_nth = dB_nth/(Δ_n+ι - Eo) where E_n,,, = corresponding carboxymethylcellulase in which n^th number of carboxyl groups are modified at that time, dB_nth = distance of any n^th band at any time in the ladder, E₀ = dBn/Δ_n+ι = distance a band migrates upwards in NEMSA when a single carboxyl is modified (Δ_n+ι = dB _n+1 - dB _n) where dB _n is the distance of unmodified or native band from the tracking dye front and dB _n+ι = distance of next band in the ladder from tracking dye front.

As indicated in figure 2, in three minutes of chemical modification, approximately two aromatic groups were linked to the carboxymethylcellulase molecule (Figure 1, lane 4 and Figure 2) .

EXAMPLE 2 ENZYMATIC ACTIVITY OF MODIFIED

CARBOXYMETHYLCELLULASE AT ELEVATED TEMPERATURE To determine the effect of modification of the enzyme, native and modified enzyme were assayed by incubating appropriate amount of the enzyme in 1 ml of 50 mM MES, pH 6 buffer containing 1.5% (w/v) carboxymethylcellulose at different temperatures ranging from 20 to 98°C. For temperatures between 100 and 152°C, the enzyme assays were carried out in the presence of increasing amount of glycerol (20-50%, v/v) for the elevation of boiling point. To assay the enzyme in water- miscible organic solvent, native and aniline linked carboxymethylcellulose were assayed in the presence of 40%

(v/v) aqueous dioxan. Appropriate controls were included at higher temperatures for the non-enzymatic hydrolysis of the substrate as described in Bauer et al. (1999). After 10-15 min, 3 ml of dinitrosalicylic acid- (DNS) reagent was added, the solution boiled for 10 min and A₅₅₀ was determined. One unit of carboxymethylcellulase activity is defined as μmol glucose equivalents liberated min^"1.

The aniline linked carboxymethylcellulase enzyme was prepared thrice to check the reproducibility of modification. The ratio (U ml^"1) 9o_°c/ (U ml^"1)₄₀<'c of aniline linked carboxymethylcellulase was measured every time native enzyme was modified and found to be 3.365± 0.312 with concomitant coupling of two anilines, indicating that hyper thermostabilization character of aniline linked carboxymethylcellulase is reproducible. The native carboxymethylcellulase did not show any activity at 90°C. As shown in Figures 3 and 4, aniline linked carboxymethylcellulase displayed a dramatically increased temperature optimum of 122°C. The addition of glycerol (for the determination of thermostability at temperatures >100°C) had no stabilizing effect on aniline linked carboxymethylcellulase as is apparent from the identical T_βq of 90°C (Table 1) . Thus, the difference in temperature optimum between native and aniline linked carboxymethylcellulase enzyme is 73°C (Figures 2A and 2B) . The results (Table 1) clearly show that aniline linked carboxymethylcellulase is extremely stable in presence of the substrate in aqueous solvent as compared with the native carboxymethylcellulase (Δτ_opt=73°C) .

In water-miscible organic solvent, the T_opt of aniline linked carboxymethylcellulase was 65°C higher than that of the native carboxymethylcellulase in 40% aqueous dioxan (Figure 3, triangles and Table 1). Accordingly, aniline linked carboxymethylcellulase is simultaneously stable in aqueous as well as water-miscible organic solvent. This is an unexpected result because the changes which make an enzyme more stable in organic solvent results in the enzyme being less stable in water and vica versa.

EXAMPLE 3 THERMAL INACTIVATION IN THE ABSENCE OF SUBSTRATE

To determine the irreversible thermal inactivation of native and aniline linked carboxymethylcellulase in the absence of substrate, solutions of the enzyme in 50 mM MES, pH 6.5 buffer were incubated at different temperatures. The aliquots withdrawn at different time intervals were cooled in ice and assayed for residual enzyme activity as described in example 2.

The study of irreversible thermostability in aqueous solvent showed that aniline linked carboxymethylcellulase (T_βq=85°C) was 25°C more stable than native carboxymethylcellulase (T_eq=60°C) . This is a substantial improvement in thermostability of aniline linked carboxymethylcellulase in the absence of substrate but not as much as has been shown in the presence of the substrate (Table 1) . The T_opt of aniline linked carboxymethylcellulase was 65°C higher than that of the native carboxymethylcellulase in 40% aqueous dioxan (Figure 3, triangles and Table 1) . Accordingly, aniline linked carboxymethylcellulase is simultaneously stable in aqueous as well as water-miscible organic solvent. This is an unexpected result because the changes which make an enzyme more stable in organic solvent results in the enzyme being less stable in water and vica versa.

EXAMPLE 4 CALCULATION OF Vmx AND K_MG OF NATIVE AND

MODIFIED CARBOXYMETHYLCELLULASE To determine V^_ax and K^, native and aniline linked carboxymethylcellulase were assayed at 40°C as described in Example 2 with the modification that the assay mixture contained varying amounts of carboxymethylcellulose. The results are shown in Table 1.

The results indicate that the V,-^ of aniline linked carboxymethylcellulase was found to be 120 fold less than the native enzyme at 40°C (Table 1) . TABLE 1

PROPERTIES OF NATIVE AND ANILINE LINKED CARBOXYMETHYLCELLULASE

Properties Native Modified

1- V_πax (μmol min'mg^"1) 545 3.55 2. K,- (% w/v) 9.35 0.61

3 • _πax/K-, 58 6

4. Temperature optimum, T_opt (°C) : a. Aqueous (for assays <100°C) 50 <98 b. Glycerol (for assays >100°C) nd 122 c. Dioxan, 40% (v/v) 25 90

5. Irreversible thermostability: a . Aqueous , Temperature of equivalent 60 85 half-life (1.2 min), T_eq b. Aqueous, Temperature of equivalent nd 90 half-life (0.9 min), T_eq c. Glycerol, Temperature of equivalent nd 90 half-life (0.9 min), T_eq d. Dioxan, % residue activity left after 8.5 (25°C) 7(60°C) 10 min incubation

4b: increasing amounts of glycerol (20-50% v/v) were used in buffer for the elevation of boiling point.

5a,b,c: T_eq = temperature at which two forms of enzyme have similar half-lives .

5c: [glycerol] = 25% (v/v).

5b, c: T_eq of aniline linked carboxymethylcellulase in the presence and absence of 50% (v/v) glycerol was identical indicating that glycerol per se has no stabilization effect on the enzyme. EXAMPLE 5 MODIFICATION OF TRICHODERMA SP. CELLULASE WITH ADENOSINE To link adenosine groups to the side chains of amino acids of cellulase from Trichoderma sp., the nucleophile adenosine hydrochloride (25mM or 50mM as indicated in

Table 2) was added to 40mM K₂HPO₄/KH₂PO₄, pH4.24 or 5.24 (as indicated in Table 2) with or without cellobiose (as indicated in Table 2), and pH was readjusted to pH 4.24 with 2M NaOH. Ammonium sulfate suspended Trichoderma cellulase was first dialyzed against distilled water and then added to the above-mentioned nucleophilic solution to give a final concentration of 40-50 U ml^"1. The coupling reaction was initiated by adding solid EDC to a final concentration of 50 mM. After 20 or 30 min (as indicated) , the reaction was stopped by adding equal amount of 100 mM sodium acetate, pH 7 buffer and the modified enzyme was dialysed against 50 mM K₂HP0₄/citric acid, pH 5 buffer.

Native and adenosine-linked (modified) enzyme were then assayed for half-life at elevated temperature as indicated. Results of assays are shown in Table 2.

As can be seen from the results in Table 2, linking of adenosine groups to amino acid side chains of Trichoderma sp. cellulase results in a 2 -fold increase in enzyme half-life at 65°C.

EXAMPLE 6 MODIFICATION OF TRICHODERMA SP. CELLULASE WITH ADENINE To link adenine groups to the side chains of amino acids of cellulase from Trichoderma sp. , the nucleophile adenine hydrochloride (lOOmM) was added to 40mM K₂HPO₄/KH₂PO4, pH5.2 with cellobiose, and pH was readjusted to pH 5.2 with 2M NaOH. Ammonium sulfate suspended Trichoderma cellulase was first dialyzed against distilled water and then added to the above-mentioned nucleophilic solution at a concentration of 40-50 U ml^"1. The coupling reaction was initiated by adding solid EDC to a final concentration of 50 mM. After 20 min the reaction was stopped by adding equal amount of 100 mM sodium acetate, pH 7 buffer and the modified enzyme was dialysed against 50 mM K₂HP0₄/citric acid, pH 5 buffer. Native and adenine-linked (modified) enzyme were then assayed for half-life at elevated temperature as indicated. Results of assays are shown in Table 2.

As can be seen from the results in Table 2, linking of adenine groups to amino acid side chains of Trichoderma sp. cellulase results in a 1.45-fold increase in enzyme half-life at 65°C.

EXAMPLE 7 MODIFICATION OF TRICHODERMA CELLULASE WITH PYROMELLITIC DIANHYDRIDE To link pyromellitic groups to the side chains of amino acids of cellulase from Trichoderma sp., Ammonium sulfate suspended Trichoderma cellulase was first dialyzed against distilled water and then diluted (25μl/ml buffer, -10 U ml^"1) in 100 mM NaH₂Pθ₄/Na₂HPθ₄, pH8.3 or 40 mM sodium borate/NaOH, pH 9.4 (as indicated in Table 2) buffer containing 100 mM sodium acetate. A pyromellitic dianhydride solution in dimethylsulfoxide solvent was prepared and aliquots of the resulting solution of pyromellitic dianhydride was added to 4 ml of enzyme solution to give a final concentration of 7.5 mM pyromellitic dianhydride. To attempt greater modification, 3 successive aliquots of a pyromellitic dianhydride solution were added to the enzyme solution to each provide a concentration of 6.3 mM. A modification was also carried out with 3 aliquots of 12.6 mM in K₂HP0₄/P0₄ buffer at pH 9.4.

After 30-60 min, the modified enzyme was dialysed against 50 mM K₂HP0₄/citric acid, pH 5 buffer to remove reagents.

Native and pyromellitic acid-linked enzyme (modified) were then assayed for half-life at elevated temperatre. Results of the assays are shown in Table 2 below.

As can be seen from the results in Table 2, linking of pyromellitic groups to amino acid side chains of Trichoderma sp. cellulase results in a 28- fold increase in enzyme half-life at 70°C, a 54-fold increase in enzyme half-life at 65°C, and as high as a 6-fold increase in enzyme half-life at 80°C.

EXAMPLE 8 DOUBLE MODIFICATION OF TRICHODERMA SP.

CELLULASE WITH PYRIDINE AND BENZOIC ANHYDRIDE To link pyridine groups to the side chains of amino acids of cellulase from Trichoderma sp., a pyridine HC1 solution was made (200mM) in 40mM KH₂PO₄/K₂HPO₄, pH5.5 buffer. The pH was adjusted to 5.15 with 2M KOH. To 1ml of this solution was added cellobiose (50mM) and lOOμl (40- 50 U) dialysed enzyme was added. The reaction was initiated by the addition of O.Olg EDC/ml (carbodiimide) [50mM] . The reaction was stopped after 5 min with 1ml of lOOmM sodium acetate, pH 7 buffer. The modified enzyme was repeatedly dialysed against 5 mM NaCl to remove reagents .

Following dialysis:

To link benzoic acid groups to the side chains of amino acids of the pyridine linked enzyme, 2ml of pyridine linked enzyme was mixed with an equal amount of 0.2M K₂HPO₄/KH₂PO₄, pH 7.2 buffer containing 200mM sodium acetate. IM Benzoic anhydride solution was made in DMSO (dimethylsulfoxide) , and 25μl of the Benzoic anhydride solution was added during vigorous vortexing. The double modified enzyme was put for repeated dialysis against 50mM K₂HP0₄/citric acid, pH 5 buffer.

Native and pyridine/benzoic acid-linked (modified) enzyme were then assayed for half-life at elevated temperature. Results of the assays are shown in Table 2 below.

As can be seen from the results in Table 2, linking of pyridine and benzoic acid groups to Trichoderma sp. cellulase results in a 1.7-fold increase in enzyme half- life at 80°C (see no. 9 Table 2).

EXAMPLE 9 DOUBLE MODIFICATION OF TRICHODERMA SP.

WITH ARGININE METHYL ESTER AND PYRMELLITIC DIANHYDRIDE

To link arginine methyl ester groups to the side chains of amino acids of cellulase from Trichoderma sp., arginine methyl ester solution was made (IM) in 40mM KH₂PO₄/K2HPO4, pH5.2 buffer. The pH was readjusted to 5.2 with 2M KOH. To 1ml of the arginine methyl ester solution was added cellobiose (50mM) and lOOμl (40-50 U) dialysed enzyme was added. The reaction was initiated by the addition of O.Olg EDC/ml (carbodiimide) [50mM] .

The reaction was stopped after 60 min with 1ml of lOOmM sodium acetate, pH 7 buffer followed by treatment with hydroxylamine [0.5 M] .

The modified enzyme was repeatedly dialysed to remove reagents against 5mM NaCl.

Following dialysis: To link pyromellitic acid groups to the arginine methyl ester linked enzyme, 2ml of modified enzyme was mixed with an equal amount of 2ml of 0.2M K2HPO4/KH2PO4, pH 8.4 buffer containing 200mM sodium acetate. Three aliquots of 25 μl pyromellitic dianhydride (7.5mM) was added, shaking between each addition of aliquot.

The double modified enzyme was put for repeated dialysis against 50mM K₂HP0₄/citric acid, pH 5 buffer. Native and arginine methyl ester/pyromellitic acid- linked enzymes were then assayed for half-life at elevated temperature. Results of the assays are shown in Table 2 below.

As can be seen from the results in Table 2, linking of arginine methyl ester and pyromellitic acid groups to Trichoderma sp. cellulase results in a 3.5-fold increase in enzyme half-life at 70°C (see no. 8 Table 2) .

TABLE 2

HALF-LIFE AND SPECIFIC ACTIVITY OF MODIFIED TRICHODERMA

CELLULASE

N = native, M = modified, CI = competitive inhibitor (50 mM Cellobiose) , DM = Double modification, tι_/2 = half life, t_d = doubling time or activation.

Total enzyme is determined by Bradford protein estimation method. The specific activity is determined as Activity Absorbance Units divided by Absorbance units by Bradford assay using same amount of enzyme A₅₄₀/A₅₉₅.

The enzyme activity is determined by Reducing Sugar Assay using dinitrosalicylic acid Reagent. Appropriate amounts of CMCase solution (20-100 μl) were added to 1 ml of 1.5% (w/v) carboxymethylcellulose (CMC) solution in 50 mM Na₂HP0₄/citric acid, pH 5 buffer and incubated at 45 °C. After 15 min the reaction was stopped by adding 1 ml of Dinitrosalicylic acid reagent and boiled for 5 min. The mixture is cooled and A₅₄₀ is determined against reagent blank.

Half-lives (irreversible thermal denaturation) were determined by heating (20-100 μl) of CMCase at a certain temperature (65, 70 or 80 °C) in 50 mM K₂HP0₄/citric acid, pH 5 buffer. Aliquots were taken at various time intervals, cooled in ice and residual activity determined by assaying the enzyme at 45 °C for 15 min. pH optimum was determined by assaying CMCases in buffers of various pH's at 45 °C for 15 min.

EXAMPLE 10 MODIFICATION OF ASPERGILLUS NIGER CELLULASE (ANC) WITH CIS-ACONITIC ANHYDRIDE To link cis-aconitic acid groups to the side chains of amino acids of cellulase from Aspergillus niger, ANC was diluted (50 μl/ml buffer from 100 mg/ l powder) in 100 mM NaH₂Pθ₄/Na₂HPθ₄ pH8.3 or 40 mM sodium borate/NaOH, pH 9.4 or 10.2 buffer (as indicated in Table 3) containing 100 mM sodium acetate. A cis-aconitic anhydride solution was prepared in dimethylsulfoxide solvent and 3 aliquots of the resulting solution were added to 4 ml of the enzyme solution such that each aliquot contributed a concentration of 6.3 mM of anhydride to the final solution. After 30-60 min, modified enzyme from each pH treatment was dialyzed against 50 mM K₂HP0₄/citric acid, pH 5 buffer to remove reagents.

Native and cis-aconitic acid-linked (modified) enzyme were then assayed for half-life at elevated temperature. Results of the assays are shown in Table 3 below. As can be seen from the results in Table 3, linking of cis-aconitic acid groups to amino acid side chains of ANC results in up to a 3.4-fold increase in enzyme half- life at 70°C.

EXAMPLE 11 MODIFICATION OF ASPERGILLUS AIGER CELLULASE

(ANC) WITH 3, 3', 4, 4' BENZOPHENONE

TETRACARBOXYLIC DIANHYDRIDE

To link 3,3' ,4,4' benzophenone tetracarboxylic acid groups to the side chains of amino acids of cellulase from Aspergillus niger, ANC was diluted (50 ^'μl/ml buffer from'.

100 mg/ml powder) in 100 mM NaH₂Pθ4/Na₂HPθ4, pH 8.3 or 40 mM sodium borate/NaOH, pH 9.4 or 10.2 buffers (as indicated in Table 3) buffer containing 100 mM sodium acetate. A 3, 3', 4, 4' benzophenone tetracarboxylic dianhydride solution was prepared in dimethylsulfoxide solvent and 3 aliquots of the resulting solution were added to 4 ml of the enzyme solutions such that each aliquot contributed a concentration of 6.3 mM of anhydride to the final solution. After 30-60 min, modified enzyme from each pH treatment was dialyzed against 50 mM K₂HP0₄/citric acid, pH 5 buffer to remove reagents. Native and 3, 3', 4, 4' benzophenone tetracarboxylic acid- linked (modified) enzyme were then assayed for half- life at elevated temperature. Results of the assays are shown in Table 3 below.

As can be seen from the results in Table 3, linking of 3, 3', 4, 4' benzophenone tetracarboxylic acid groups to amino acid side chains of ANC results in up to a 9.7 -fold increase in enzyme half-life at 70°C.

EXAMPLE 12 MODIFICATION OF ASPERGILLUS NIGER CELLULASE (ANC) WITH 2, 3 PYRIDINE CARBOXYLIC ANHYDRIDE

To link 2,3 pyridine carboxylic acid groups to the side chains of amino acids of cellulase from Aspergillus niger, ANC was diluted (50 μl/ml buffer from 100 mg/ml powder) in 100 mM NaH₂Pθ₄/Na₂HP0₄, pH 8.3 or 40 mM sodium borate/NaOH, pH 9.4 or 10.2 buffers (as indicated in Table 3) containing 100 mM sodium acetate. A 2, 3 Pyridine carboxylic anhydride solution was prepared in dimethylsulfoxide solvent and 3 aliquots of the resulting solution were added to 4 ml of the enzyme solutions such that each aliquot contributed a concentration of 6.3 mM of anhydride to the final solution. After 30-60 min, modified enzyme from each pH treatment was dialyzed against 50 mM K₂HP0₄/citric acid, pH 5 buffer to remove reagents .

Native and 2,3 pyridine carboxylic acid-linked (modified) enzyme were then assayed for half-life at elevated temperature. Results of the assays are shown in Table 3 below.

As can be seen from the results in Table 3, linking of 2,3 pyridine carboxylic acid groups to amino acid side chains of ANC results in up to a 3 -fold increase in enzyme half-life at 70°C.

EXAMPLE 13 MODIFICATION OF ASPERGILLUS NIGER CELLULASE WITH PYRIDINE. To link pyridine groups to the side chains of amino acids of cellulase from Aspergillus niger, pyridine hydrochloride (25mM) was added to 40mM K₂HP0₄/KH₂P0₄, pH5.16 or 5.22 (as indicated in Table 3), and pH was readjusted to pH 5.16 or 5.22 with 2M NaOH. Dialyzed Aspergillus niger cellulase [100 mg/ml] was diluted in the above mentioned nucleophilic solutions at a concentration of 10 mg ml^"1. The coupling reaction was initiated by adding solid EDC to a final concentration of 50 mM. After 20 min or 5 min (as indicated) , the modified enzyme was dialysed against 50 mM K₂HP0₄/citric acid, pH 5 buffer.

Native and pyridine-linked (modified) enzyme were then assayed for half-life at elevated temperature. Results of the assays are shown in Table 3 below.

As can be seen from the results in Table 3, linking of pyridine groups to amino acid side chains of. ANC results in an increase in enzyme half-life at 65°C. EXAMPLE 14 MODIFICATION OF ASPERGILLUS NIGER CELLULASE WITH BENZOIC ANHYDRIDE To link benzoic acid groups to the side chains of amino acids of cellulase from Aspergillus niger, ANC was diluted (50 μl/ml buffer from 100 mg/ml powder) in 100 mM NaH₂P04/Na₂HP0₄, pH 8.3 or 40 mM sodium borate/NaOH, pH 9.4 or 10.2 buffers containing 100 mM sodium acetate. A benzoic anhydride solution was prepared in dimethylsulfoxide solvent and 1 aliquot of the resulting solution were added to 4 ml of the enzyme solution such that each aliquot contributed a concentration of 6.3 mM of anhydride to the final solution. After 30-60 min, modified enzyme from each pH treatment was dialyzed against 50 mM K₂HP0₄/citric acid, pH 5 buffer to remove reagents.

Native and benzoic acid-linked (modified) enzyme were then assayed for half-life at elevated temperature. Results of the assays are shown in Table 3 below.

As can be seen from the results in Table 3, linking of benzoic acid groups to amino acid side chains of ANC results in an increase in enzyme half-life at 70°C.

EXAMPLE 15 MODIFICATION OF ASPERGILLUS NIGER CELLULASE WITH PHTHALIC ANHYDRIDE To link pthalic acid groups to the side chains of amino acids of cellulase from Aspergillus niger, ANC was diluted (50 μl/ml buffer from 100 mg/ml powder) in 100 mM NaH₂P04/Na₂HP0₄, pH 8.3 or 40 mM sodium borate/NaOH, pH 9.4 or 10.2 buffers (as indicated in Table 3), buffer containing 100 mM sodium acetate. A phthalic anhydride solution was prepared in dimethylsulfoxide solvent and 3 aliquots of the resulting solution were added to 4 ml of the enzyme solution such that each aliquot contributed a concentration of 6.3 mM of anhydride to the final solution. After 30-60 min, modified enzyme from each pH treatment was dialyzed against 50 mM K₂HP0₄/citric acid, pH 5 buffer to remove reagents.

Native and pthalic acid -linked (modified) enzyme were then assayed for half-life at elevated temperature. Results of the assays are shown in Table 3 below.

As can be seen from the results in Table 3, linking of pthalic acid groups to amino acid side chains of ANC results in up to a 7.8-fold increase in enzyme half-life at 70°C.

EXAMPLE 16 MODIFICATION OF ASPERGILLUS NIGER CELLULASE WITH GUANYL-3, 5-DIMETHYL PYRAZOLE .

To convert lysine residue of cellulase of Aspergillus niger to homoarginine residues, ANC was diluted- (100 μl/ml) directly in 0.5M guanyl-3 , 5-dimethyl pyrazole, pH 9.5 solution. A guanyl-3, 5-dimethyl pyrazole solution (0.5M) was prepared in H₂0 and pH was adjusted to 9.5 with 2 M

NaOH. After 26.5 hours at 1°C, modified enzyme from each pH treatment was dialyzed against 50 mM K₂HP0₄/citric acid, pH

5 buffer buffer to remove reagents.

Native and guanyl-3, 5-dimethyl pyrazole - modified enzyme were then assayed for half-life at elevated temperature. Results of the assays are shown in Table 3 below.

As can be seen from the results in Table 3 , converting lysine residues of ANC to homoarginine results in up to a 4-fold increase (2^nd phase of inactivation) in- - enzyme half-life at 70°C. EXAMPLE 17 MODIFICATION OF ASPERGILLUS NIGER CELLULASE WITH PYROMELLITIC DIANHYDRIDE.

To link pyromellitic acid groups to the side chains of amino acids of cellulase from Aspergillus niger, ANC was diluted (50 μl/ml buffer from 100 mg/ml powder) in 100 mM NaH₂P0₄/Na₂HP0₄, pH 8.3 or 40 mM sodium borate/NaOH, pH 9.4 or 10.2 buffers (as indicated in Table 3) containing 100 mM sodium acetate. A pyromellitic dianhydride solution was prepared in dimethylsulfoxide solvent and 3 aliquots of the resulting solution were added to 4 ml of the enzyme solution such that each aliquot contributed a concentration of 6.3 mM of anhydride to the final solution. After 30-60 min, modified enzyme from each pH treatment was dialyzed against 50 mM K₂HP0₄/citric acid, pH 5 buffer to remove reagents.

Native and pyromellitic acid-linked (modified) enzyme were then assayed for half-life at elevated temperature. Results of the assays are shown in Table 3 below.

As can be seen from the results in Table 3, linking of pyromellitic acid groups to amino acid side chains of ANC results in up to a 23-fold increase in enzyme half- life at 70°C, and up to a 2.3-fold increase in enzyme half- life at 80°C.

EXAMPLE 18 DOUBLE MODIFICATION OF ASPERGILLUS NIGER

CELLULASE WITH PYRIDINE AND BENZOIC ACID To link pyridine groups to the side chains of amino acids of cellulase from Aspergillus niger, pyridine HCl solution was made (200mM) in 40mM KH₂PO₄/K2HPO₄, pH5.5 buffer. The pH was adjusted to 5.15 with 2M KOH. To 1ml of pyridine HCl solution was added cellobiose (50mM) and lOOμl (10 mg) dialysed enzyme was added. The reaction was initiated by the addition of O.Olg EDC/ml (carbodiimide) [50mM] .

The reaction was stopped after 5 min with 1ml of lOOmM sodium acetate, pH 7 buffer.

The modified enzyme was repeatedly dialysed to remove reagents against distilled water.

Following dialysis:

To link benzoic acid groups to the side chains of amino acids of the pyridine linked enzyme, 2ml of pyridine linked enzyme was mixed with an equal amount of 0.2M

K₂HPO₄/KH2PO₄, pH 7.2 buffer containing 200mM sodium acetate.

IM Benzoic anhydride solution was made in DMSO (dimethyl sulfoxide) . 25μl of Benzoic anhydride solution was added during vigorous vortexing.

The double modified enzyme was put for repeated dialysis against 50mM K₂HP0₄/citric acid.

Native and pyridine/benzoic acid-linked enzyme (modified) were then assayed for half-life at elevated temperature. Results of the assays are shown in Table 3 below.

As can be seen from the results in Table 3, linking of pyridine and benzoic acid groups to amino acid side chains of ANC results in an increase in enzyme half-life at 70°C.

TABLE 3

CHEMICAL MODIFICATION OF ASPERGILLUS NIGER CELLULASE

N = native, M = modified,

Total enzyme is determined by Bradford protein estimation method. The specific activity is determined as Activity Absorbance Units divided by Absorbance units by Bradford assay using same amount of enzyme A₅₄₀/ ₅₉₅.

Half-lives (irreversible thermal denaturation) were determined by heating (20-100 μl) of CMCase at a certain temperature (65, 70 or 80 °C) in 50 mM K₂HP0₄/citric acid, pH 5 buffer. Aliquots were taken at various time intervals, cooled in ice and residual activity determined by assaying the enzyme at 45 °C for 15 min. pH optimum was determined by assaying CMCases in buffers of various pH's at 45 °C for 15 min. REFERENCES

Afzal, A.J., Bokhari, S.A., Ahmad, W., Rashid, M.H., Rajoka, M.I., Siddiqui K.S. Two simple and rapid methods for the detection of polymer degrading enzymes on high resolution alkaline cold in situ native (HiRACIN) -PAGE and high resolution in situ inhibited native (HiRISIN) -PAGE. Biotechnol. LeH. 2000, 22, 957-960.

Rashid, M.H., Naj us Saqib, A. ., Rajoka M.I., Siddiqui K.S. Native enzyme mobility shift assay (NEMSH) : a new method for monitoring carboxyl group modification of carboxymethylcellulose from Aspergillus niger. Biotechnol, Techniques, 1997, 11, 245-247.

Bauer,M.W., Driskill,L.E. , Callen, W. , Snead,M.A., Mathur, E.J. , Kelly, R.M. An endoglucanase, EglA, from the hyperthermophilic archaeon Pyrococcus furiosus hydrolyses b-1,4 bonds in mixed linkage (1-3) (1-4) -b-D-glucans and cellulose. J.Bacteriol.1999, 181, 284-290.

Claims

1. An enzyme for cleaving β-1,4 -glucosidic bonds of cellulose comprising a cellulase having an aromatic group linked to a side chain of an amino acid residue or to a terminal amino acid residue of the cellulase wherein the enzyme functions at an elevated temperature and/or has an extended half-life at elevated temperature compared to the corresponding unmodified cellulase.

2. The enzyme according to claim 1, wherein the enzyme functions at an elevated temperature and/or has an extended half-life at elevated temperature compared to the corresponding unmodified cellulase at a pH of between 5.0 and 9.0.

3. The enzyme according to claim 1 wherein the enzyme functions at an elevated temperature and/or has an extended half-life at elevated temperature compared to the corresponding unmodified cellulase at a pH between 5.2 and 7.8.

4. The enzyme according to claim 1 wherein the enzyme functions at an elevated temperature and/or has an extended half-life at elevated temperature compared to the corresponding unmodified cellulase at a pH between 5.2 and 6.8.

5. The enzyme according to any one of claims 1 to 4, wherein the enzyme has the capacity to cleave β-1,4 glucosidic bonds of cellulose in an aqueous solvent.

6. The enzyme according to any one of claims 1 to 4, wherein the enzyme has the capacity to cleave β-1,4 glucosidic bonds of cellulose in aqueous solvents and water-miscible organic solvents

7. The enzyme according to any one of claims 1 to 6, wherein the aromatic group is a derivative of benzene.

8. The enzyme according to any one of claims 1 to 6, wherein the aromatic group is selected from the group consisting of benzylamine, aniline, benzoic acid, phthalic acid, mellitic acid, pyromellitic acid and 3, 3', 4, 4' benzophenone tetracarboxylic acid.

9. The enzyme according to any one of claims 1 to 6, wherein the aromatic group is a heterocyclic amine.

10. The enzyme according to claim 9, wherein the heterocyclic amine is selected from the group consisting of adenine, adenosine, pyridine, cis-aconitic acid and 2, 3 pyridine carboxylic acid.

11. The enzyme according to any one of claims 1 to 10, wherein the side chain is the side chain of an acidic amino acid.

12. The enzyme according to any one of claims 1 to 10, wherein the side chain is the side chain of a basic amino acid.

13. The enzyme according to any one of claims 1 to 12, wherein the aromatic group is linked to the side chain of an amino acid, or to a terminal amino acid, by an amide bond.

14. The enzyme according to claim 13, wherein the enzyme comprises at least one aromatic group selected from the group consisting of cis-aconitic acid, 3, 3', 4, 4' benzophenone tetracarboxylic acid, 2,3 pyridine carboxylic acid, benzoic acid, pyromellitic acid, phthalic acid, linked to the side chain of a lysine residue, or to a terminal amino acid residue of the enzyme.

15. The enzyme according to claim 13 wherein the enzyme comprises at least one aromatic group selected from the group consisting of adenosine, aniline, adenine and pyridine linked to the side chain of an aspartate residue, a glutamate residue or a carboxy terminal residue.

16. The enzyme according to any one of claims 1 to 15, wherein the enzyme further comprises an additional aromatic group linked to a side chain of an amino acid residue of the enzyme, or linked to a terminal amino acid residue of the enzyme.

17. The enzyme according to any one of claims 1 to 15, wherein the enzyme- comprises at least two aromatic groups, of which one is selected from the group consisting of benzylamine, aniline and pyridine, and one is a benzoyl group .

18. The enzyme according to claim 17, wherein the enzyme comprises a pyridine residue and a benzoic acid residue, and the pyridine residue and benzoic acid residue are linked to side chains of amino acid residues or to terminal amino acid residues of the enzyme.

19. The enzyme according to claim 18, wherein the pyridine residue is linked to an aspartate residue, a glutamate residue or to a carboxy terminal amino acid residue.

20. The enzyme according to claim 18 or 19 wherein the benzoic acid residue is linked to a lysine residue or to an amino terminal amino acid of the enzyme.

21. The enzyme according to any one of claims 1 to 20, wherein the enzyme further comprises at least one amino group for improving the capacity of the enzyme to cleave β-1,4 glucosidic bonds of cellulose at an elevated temperature, and amino group is linked to the side chain of an amino acid residue of the enzyme or linked to the carboxyl-terminal amino acid residue of the enzyme.

22. The enzyme according to claim 21, wherein the amino group is linked to the side chain of an acidic amino acid residue.

23. The enzyme according to claim 21 or 22, wherein the enzyme comprises a pyromellitic residue linked to the side chain of a lysine residue, and an amino group linked to the side chain of an aspartate or glutamate residue or linked to the carboxyl-terminal amino acid residue of the enzyme .

24. The enzyme according to any one of claims 1 to 20, wherein the enzyme further comprises; an aliphatic amine-containing group linked to a side chain of an amino acid residue or to the carboxyl terminal amino acid residue of the enzyme.

25. The enzyme according to claim 24, wherein the aliphatic amine-containing group is selected from the group consisting of argininamide, arginine methyl ester, arginine ethyl ester, glycinamide, methylamine, dimethylamine and trimethylamine.

26. The enzyme according to any one of claims 1 to 20, wherein the enzyme further comprises an arginine, an argininamide, an arginine methyl ester or an arginine ethyl ester residue linked to a side chain of an amino acid residue or to the carboxyl terminal amino acid residue of the enzyme.

27. The enzyme according to any one of claims 1 to 20, wherein the enzyme comprises at least one arginine methylester residue and at least one pyromellitic acid residue, and the pyromellitic acid residue is linked to the side chain of a lysine residue or to an amino terminal amino acid residue.

28. The enzyme according to claim 27, wherein the arginine methylester residue is linked to the side chain of an aspartate residue, a glutamate residue or to a carboxyl terminal amino acid residue of the enzyme.

29. The enzyme according to any one of claims 1 to 28, wherein the enzyme further comprises at least one homoarginine residue.

30. The enzyme according to any one of claims 1 to 29 wherein the cellulase is from an organism selected from the group consisting of invertebrate, angiosperm, fungus, yeast, bacteria including archeaebacteria and eubacteria, and algae.

31. The enzyme according to any one of claims 1 to 30, wherein the enzyme is a cellulase from a psychrophilic or a mesophilic organism.

32. The enzyme according to any one of claims 1 to 31, wherein the organism is a fungus selected from the group consisting of Aspergillus sp, Scopulariopsis sp. and Tri choderma sp.

33. The enzyme according to claim 32, wherein the fungus is Aspergillus niger or Trichoderma sp.

34. A process for producing the enzyme of any one of claims 1 to 33, comprising the step of contacting an enzyme capable of cleaving a β-1, 4-glucosidic bond of cellulose with a compound which comprises an aromatic group in conditions sufficient for linking the aromatic - group to a side chain of an amino acid residue of the enzyme, or to a terminal amino acid residue of the enzyme.

35. The process of claim 34, comprising the step of activating carboxyl groups of amino acid residues of the enzyme in the presence of an aromatic nucleophile.

36. The process of claim 35, wherein the aromatic nucleophile is selected from the group consisting of adenine hydrochloride, adenosine hydrochloride, aniline hydrochloride, benzylamine hydrochloride and pyridine hydrochloride .

37. The process of claim 34, wherein the process comprises the step of contacting the enzyme in the presence of an aromatic anhydride in conditions sufficient for linking the aromatic group to an amino group of a basic amino acid residue of the enzyme, or to the amino terminal amino acid residue of the enzyme.

38. The process of claim 37, wherein the aromatic anhydride is selected from the group consisting of benzoic anhydride, pyromellitic dianhydride, mellitic trianhydride, trimellitic anhydride, phthalic anhydride, cis aconitic anhydride, 3, 3', 4, 4' benzophenone tetracarboxylic dianhydride and 2, 3 pyridine carboxylic anhydride .

39. The process of claim 35 or 36, wherein the carboxyl groups are activated by carbodiimide. -

40. The process of any one of claims 34 to 39, further comprising the step of contacting the enzyme with an agent for controlling the linkage of the aromatic group to a side chain of an amino acid residue or a terminal amino acid residue located in a catalytic site of the enzyme .

41. The process of claim 40, wherein the agent is an inhibitor of the enzyme.

42. The process of claim 40 wherein the agent is a substrate of the enzyme.

43. The process according to any one of claims 34 to 42, wherein the process comprises the further step of guanidination of a lysine residue and the terminal amino group of the enzyme.

44. The process of any one of claims 34 to 43, wherein the process comprises activating carboxyl groups of amino acid residues of the enzyme in the presence of an aromatic nucleophile, thereby to link the aromatic group of the aromatic nucleophile to a carboxyl group of an acidic amino acid, and contacting the enzyme in the presence of an aromatic anhydride in conditions sufficient for linking the aromatic group of the anhydride to an amino group of a basic amino acid.

45. The process of any one of claims 34 to 44, wherein the process comprises the further step of contacting the enzyme with an aliphatic amine containing compound under conditions sufficient for linking of the aliphatic amine containing compound to a carboxyl group of a side chain of an amino acid residue of the enzyme, or to the terminal carboxyl group of the enzyme.

46. The process of claim 45, wherein the aliphatic amine containing compound is selected from the group argininamide hydrochloride, arginine methyl ester hydrochloride, arginine ethyl ester hydrochloride, glycinamide hydrochloride, methylamine hydrochloride, dimethylamine hydrochloride, ethylenediamine hydrochloride and trimethylamine hydrochloride.

47. An enzyme which has improved capacity to cleave β-1, 4-glucosidic bonds of cellulose at an elevated temperature, comprising an amino acid sequence of a cellulase in which at least one lysine residue has been replaced by a homoarginine residue.

48. A process for producing an enzyme of claim 47, comprising the step of contacting an enzyme capable of cleaving a β-1, 4-glucosidic bond of cellulose with guanyl- 3,5-dimethyl pyrazole in conditions sufficient to form at least one homoarginine residue.

49. An enzyme produced by the process of any one of claims 34 to 46 or 48.

50. A composition comprising an enzyme according to the any one of claims 1 to 33 or claim 47, together with an industrially-acceptable carrier.

51. A use of an enzyme according to any one of claims 1 to 34 or claim 47.