EP1305431A2 - Mutant trichoderma reesei egiii cellulases, dna encoding such egiii compositions and methods for obtaining same - Google Patents

Abstract

The present invention relates to novel EGIII-like cellulase variants that have improved stability under oxidative conditions.

Description

NOVEL VARIANT EGIII-LIKE CELLULASE COMPOSITIONS

GOVERNMENT-SPONSOREDRESEARCHAND DEVELOPMENT Not applicable.

BACKGROUND OF THE INVENTION

Cellulases are enzymes capable of hydrolyzing the β-D-glucosidic linkages in celluloses. Cellulolytic enzymes have been traditionally divided into three major classes: endoglucanases, exoglucanases or cellobiohydrolases and β-glucosidases (Knowles, et al, TIBTECH 5:255-261 (1987)); and are known to be produced by a large number of bacteria, yeasts and fungi.

Because of its effectiveness in many industrial processes, there has been a trend in the field to search for specific cellulase compositions or components that have particularly effective performance profiles with respect to one or more specific applications. As possible sources of cellulases, practitioners have focused on fungi and bacteria. For example, cellulases produced by certain fungi such as Trichoderma spp. (especially Trichoderma reesei (longibrachiatum)) have been given much attention because a complete cellulase system capable of degrading crystalline forms of cellulose is readily produced in large quantities via fermentation procedures. This specific cellulase complex has been extensively analyzed to determine the nature of its specific components and the ability of those components to perform in industrial processes (see, Wood, et ah, METHODS IN ENZYMOLOGY 160:234 (1988)). U.S. Patent No. 5,475,101 (Ward, et al.) discloses the purification and molecular cloning of one particularly useful enzyme called endoglucanase III (EGIII) which is derived from Trichoderma reesei.

PCT Publication No. WO 94/14953 discloses endoglucanases that are encoded by a nucleic acid which comprises any one of a series of DNA sequences, each having 20 nucleotides.

Ooi, et al, Curr. Genet. 18:217-222 (1990) disclose the cDNA sequence coding for endoglucanase F 1 -CMC produced by Aspergillus aculeatus which contains the amino acid strings NNLWG, ELMIW and GTEPFT. Sakamoto, et al, Curr. Genet. 27:435- 439 (1995) discloses the cDNA sequence encoding the endoglucanase CMCase-1 From Aspergillus kawachii IFO 4308 which contains the amino acid strings ELMIW and GTEPFT. Ward, et al, discloses the sequence of EGIII having the amino acid strings NNLWG, ELMIW and GTEPFT. Additionally, two cellulase sequences, one from Erwinia carotovara and Rhodothermus marinus are disclosed in Saarilahti, et al, Gene 90:9-14 (1990) and Hreggvidsson, et al, Appl. Environ. Microb. 62:3047-3049 (1996) which contain the amino acid string ELMIW.

Despite knowledge in the art related to many cellulase compositions having applications in some or all ofthe above areas, there is a continued need for new cellulase compositions which have improved stability under conditions present in applications for which cellulases are useful, e.g., household and laundry detergents and textile treatment compositions.

SUMMARY OF THE INVENTION

According to the present invention, a variant EGIII or EGIII-like cellulase is provided wherein one or more amino acids are modified or deleted to confer improved performance, including stability in oxidative systems.

In one embodiment, an EGIII-like cellulase variant is provided wherein the variant comprises a substitution or deletion at a position corresponding to one or more of residues M79, M154 and/or M118 in EGIII from Trichoderma reesei. In a preferred embodiment, the variant comprises a substitution of methionines at positions 79, 118 and/or 154 with leucine, isoleucine, valine threonine serine or alanine.

In another aspect of this embodiment, the cellulase is derived from a fungus, bacteria or Actinomycete. In a preferred aspect, the cellulase is derived from a fungus. In a more preferred aspect, the fungus is a filamentous fungus. In a most preferred embodiment, the filamentous fungus belongs to Euascomycete, including but not limited to, Aspergillus spp., Gliocladium spp., Fusarium spp., Acremonium spp., Myceliophtora spp., Verticillium spp., Myrothecium spp., or Penicillium spp. In another aspect of this embodiment, the cellulase is an endoglucanase.

In another embodiment of this invention a DNA that encodes the EGIII-like cellulases of this invention. In a further embodiment, the DNA is contained in a vector. In yet a further embodiment, a host cell is transformed with the vector. In another embodiment, a method is provided for producing a cellulase of this invention. The method comprises the steps of culturing the host cell according to claim 12 in a suitable culture medium under suitable conditions to produce cellulase, obtaining said produced cellulase, and optionally purifying said cellulase to provide a purified cellulase product.

In another embodiment of this invention, a detergent is provided that comprises a variant EGIII or EGIII cellulase comprising a substitution or deletion at a position corresponding to one or more of residues M79 and/or Ml 18 in EGIII from Trichoderma reesei. In a preferred embodiment, the variant comprises a substitution of methionines at positions 79, 118 and/or 154 with leucine, isoleucine, valine threonine serine or alanine.

In another aspect of this embodiment, the detergent is a laundry detergent. In yet another aspect, the detergent is a dish detergent.

In another embodiment the variant EGIII or EGIII-like cellulases of this invention are used in the treatment of a cellulose-containing textile.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates the amino acid sequence of mature EGIII protein from Trichoderma reesei (SEQ ID NO:l) showing the residues described in accordance with the present invention.

Figure 2 illustrates the DNA (SEQ ID NO:2) sequence that encodes EGIII from T reesei.

Figure 3 is a schematic showing the alignment of amino acids (SEQ ID NO:3-24) in EGIII and EGIII-like cellulases.

DETAILED DESCRIPTION OF THE INVENTION

The Applicant has discovered that oxidation of T. reesei EGIII occurs at three specific sites in the enzyme, +79, +118 and +154. In the native cellulase, these sites are occupied by methionine. By substituting other amino acids for the native methionines, the Applicant has prepared oxidatively more stable enzymes. Because cellulases homologous to T. reesei EGIII are known and residues equivalent to the methionines found in EGIII are determinable by sequence comparison; the present invention, in addition, encompasses EGIII-like cellulases with amino acid modifications that change the performance ofthe EGIII-like cellulases under oxidative conditions.

Definitions 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. All references are incorporated by reference for all purposes. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing ofthe present invention, the preferred methods and materials are described. For purposes ofthe present invention, the following terms are defined below. "Cellulase" is a well-classified category of enzymes in the art and includes enzymes capable of hydrolyzing cellulose polymers to shorter cellooligosaccharide oligomers, cellobiose and/or glucose. Common examples of cellulase enzymes include exo- cellobiohydrolases and endoglucanases and are obtainable from many species of cellulolytic organisms, particularly including fungi and bacteria.

"EGIII" cellulase refers to the endoglucanase component described in U.S. Patent No. 5,475,101 and Proceedings on the Second TRICEL Symposium on Trichoderma Reesei Cellulases And Other Hydrolases, Suominen & Reinikainen eds., Espoo Finland (1993), pp. 153-158 (Foundation for Biotechnical and Industrial Fermentation Research, Vol. 8). As discussed therein, EGIII is derived from Trichoderma reesei (longibrachiatum) and is characterized by a pH optimum of about 5.8, an isoelectric point (pi) of about 7.4 and a molecular weight of about 25 kD. The enzyme commonly referred to as EGII from Trichoderma reesei has been previously referred to in the literature by the nomenclature EGIII by some authors, but that enzyme differs substantially from the enzyme defined herein as EGIII in terms of molecular weight, pi and pH optimum.

"EG-III like enzyme", "EGIII-like protein" or "EGIII-like cellulase" according to the present invention means enzymes that are related to EGIII by having certain amino acid strings in common with EGIII. Homologous cellulases are intended to be within the genus of EGIII-like cellulases. As used herein, EGIII-like cellulase is also intended to encompass EGIII from Trichoderma reesei. Thus an EGIII-like cellulase comprises an enzyme having cellulolytic activity which comprises an amino acid sequence comprising therein an amino acid string selected from the group consisting of one or more of:

1) Asn-Asn-(Leu/Phe/Lys/Ile)-Trp-Gly (SEQ ID NO:25)

2) Glu-(Leu/Phe/Ile)-Met-Ile-Trp (SEQ ID NO:26)

3) Gly-Thr-Glu-Pro-Phe-Thr (SEQ ID NO:27)

4) (Ser/Tyr/Cys/Trp/Thr/Asn/Lys/Arg)-(Val/Pro)-(Lys/Ala)-(Ser/Ala)- (Tyr/Phe) (SEQ ID NO:28); and

5) Lys-Asn-Phe-Phe-Asn-Tyr (SEQ ID NO:29).

Non-limiting representatives of EGIII-like cellulases can be found in Figure

"Variant" means a protein which is derived from a precursor protein (e.g., an EGIII-like cellulase) by addition of one or more amino acids to either or both the C- and N- terminal end, substitution of one or more amino acids at one or a number of different sites in the amino acid sequence, deletion of one or more amino acids at either or both ends ofthe protein or at one or more sites in the amino acid sequence, or insertion of one or more amino acids at one or more sites in the amino acid sequence. An "EGIII variant" means an EGIII enzyme modified as described above. The preparation of an enzyme variant is preferably achieved by modifying a DNA sequence which encodes for the native protein, transformation of that DNA sequence into a suitable host, and expression ofthe modified DNA sequence to form the derivative enzyme. The variant EGIII-like cellulase ofthe invention includes peptides comprising altered amino acid sequences in comparison with a precursor enzyme amino acid sequence wherein the variant EGIII-like cellulase retains the characteristic cellulolytic nature ofthe precursor enzyme but which may have altered properties in some specific aspect. For example, a variant EGIII-like cellulase may have increased stability under oxidative conditions but will retain its characteristic cellulolytic activity. However, the activity ofthe variant may be increased or decreased relative to the precursor enzyme. It is contemplated that the variants according to the present invention may be derived from a DNA fragment encoding a EGIII-like cellulase variant wherein the functional activity ofthe expressed cellulase variant is retained. For example, a DNA fragment encoding a cellulase may further include a DNA sequence or portion thereof encoding a hinge or linker attached to the cellulase DNA sequence at either the 5 ' or 3' end wherein the functional activity ofthe encoded cellulase domain is retained.

A residue in an EGIII-like cellulase which is "corresponding" or "equivalent" to a residue present in EGIII means a residue which exists in an equivalent position to that in EGIII, as indicated by primary sequence homology, tertiary structural homology (as shown by, e.g., crystal structure or computer modeling) or functional equivalence. A variant EGIII-like cellulase has an amino acid sequence that is derived from the amino acid sequence of a precursor EGIII-like cellulase. The precursor cellulases include naturally occurring cellulases and recombinant cellulases (as defined herein). The amino acid sequence ofthe EGIII-like cellulase variant is derived from the precursor EGIII- like cellulase amino acid sequence by the substitution, deletion or insertion of one or more amino acids ofthe precursor amino acid sequence. Such modification is ofthe precursor DNA sequence that encodes the amino acid sequence ofthe precursor cellulase rather than manipulation of the precursor cellulase enzyme per se. Suitable methods for such manipulation ofthe precursor DNA sequence include methods disclosed herein and in commonly owned US patent 4,760,025 and 5,185,258. Specific residues corresponding to the positions that are responsible for instability in the presence of surfactant are identified herein for substitution or deletion. The amino acid position number (e.g., +35) refers to the number assigned to the mature Trichoderma reesei EGIII sequence presented in Figure 1. The invention is directed to the mutation of EGIII-like cellulases that contain amino acid residues at positions which are equivalent to the particular identified residue in Trichoderma reesei EGIII. A residue (amino acid) of a precursor cellulase is equivalent to a residue of Trichoderma reesei EGIII if it is either homologous (i.e., corresponding in position in either primary or tertiary structure) or is functionally analogous to a specific residue or portion of that residue in Trichoderma reesei EGIII (i.e., having the same or similar functional capacity to combine, react, or interact chemically or structurally). As used herein, numbering is intended to correspond to that ofthe mature EGIII amino acid sequence as illustrated in Figure 1 unless otherwise indicated.

"Detergent composition" means a mixture that is intended for use in a wash medium for the laundering of soiled cellulose containing fabrics. In the context ofthe present invention, such compositions may include, in addition to cellulases and surfactants, additional hydrolytic enzymes, builders, bleaching agents, bleach activators, bluing agents and fluorescent dyes, caking inhibitors, masking agents, cellulase activators, antioxidants, and solubilizers. Such compositions are generally used for cleaning soiled garments and are not used during the manufacturing process, in contrast to stonewashing compositions. Detergent compositions comprising cellulase are described in, for example, U.S. Patent No. 5,290,474 and EP Publication No. 271 004, incorporated herein by reference.

"Expression vector" means a DNA construct comprising a DNA sequence that is operably linked to a suitable control sequence capable of effecting the expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome-binding sites on the mRNA, and sequences that control termination of transcription and translation. Different cell types are preferably used with different expression vectors. A preferred promoter for vectors used in Bacillus subtϊlis is the AprE promoter; a preferred promoter used in E. coli is the Lac promoter, a preferred promoter used in Saccharomyces cerevisiae is PGK1, a preferred promoter used in Aspergillus niger is glaA, and a preferred promoter for Trichoderma reesei is cbhl. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently ofthe host genome, or may, under suitable conditions, integrate into the genome itself. In the present specification, plasmid and vector are sometimes used interchangeably. However, the invention is intended to include other forms of expression vectors that serve equivalent functions and which are, or become, known in the art. Thus, a wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention. Useful expression vectors, for example, may consist of segments of chromosomal, non- chromosomal and synthetic DNA sequences such as various known derivatives of S V40 and known bacterial plasmids, e.g., plasmids from E. coli including col El, pCRl, pBR322, pMb9, pUC 19 and their derivatives, wider host range plasmids, e.g., RP4, phage DNAs e.g., the numerous derivatives of phage λ, e.g., NM989, and other DNA phages, e.g., M13 and filamentous single stranded DNA phages, yeast plasmids such as the 2μ plasmid or derivatives thereof, vectors useful in eukaryotic cells, such as vectors useful in animal cells and vectors derived from combinations of plasmids and phage DNAs, such as plasmids which have been modified to employ phage DNA or other expression control sequences. Expression techniques using the expression vectors ofthe present invention are known in the art and are described generally in, for example, Sambrook. Often, such expression vectors including the DNA sequences ofthe invention are transformed into a unicellular host by direct insertion into the genome of a particular species tlirough an integration event (see e.g., Bennett & Lasure, MORE GENE MANIPULATIONS IN FUNGI, Academic Press, San Diego, pp. 70-76 (1991) and articles cited therein describing targeted genomic insertion in fungal hosts, incorporated herein by reference).

"Host strain" or "host cell" means a suitable host for an expression vector comprising DNA according to the present invention. Host cells useful in the present invention are generally prokaryotic or eukaryotic hosts, including any transformable microorganism in which expression can be achieved. Preferred host strains include, but are not limited to, Bacillus subtilis, Escherichia coli, Trichoderma reesei, Saccharomyces cerevisiae or Aspergillus niger. Host cells are transformed or transfected with vectors constructed using recombinant DNA techniques. Such transformed host cells are capable of both replicating vectors encoding the variant EGIII-like enzymes or expressing the desired peptide product. In a preferred embodiment according to the present invention, "host cell" means both the cells and protoplasts created from the cells of Trichoderma sp.

"Signal sequence" means a sequence of amino acids bound to the N-terminal portion of a protein that facilitates the secretion ofthe mature form ofthe protein outside of the cell. This definition of a signal sequence is a functional one. The mature form ofthe extracellular protein lacks the signal sequence that is cleaved off during the secretion process.

"DNA vector" means a nucleotide sequence which comprises one or more DNA fragments or DNA variant fragments encoding an EGIII-like cellulase or variants described above which can be used, upon transformation into an appropriate host cell, to cause expression ofthe variant EGIII-like cellulase.

"Functionally attached to" means that a regulatory region, such as a promoter, terminator, secretion signal or enhancer region is attached to a structural gene and controls the expression of that gene.

T. reesei EGIII

The amino acid sequence of mature EGIII is known and is given in Figure 1 (SEQ ID NO:l). As can be seen, mature EGIII contains three methionines. Thus, to modify oxidative stability of EGIII, the methionines at these three positions are substituted with other amino acids. Preferably, the substitution is done by altering the DNA sequence that encodes EGIII rather than manipulation of EGIII per se. Suitable methods for such manipulation ofthe EGIII DNA sequence include methods disclosed herein and in commonly owned U.S. Patent Nos. 4,760,025 and 5 , 185,258, incorporated herein by reference.

In a preferred embodiment, an EGIII variant library is created. Contained within the library are DNA molecules that encode EGIII variants. The EGIII variants have amino acid substitutions or deletions at either or all of positions 79, 118 or 154. In a more preferred embodiment, the EGIII variants also have substitutions at positions that give the EGIII variant increased enzymatic activity. In one aspect of this embodiment, the library is randomized, e.g., the DNA molecules contain a codon that encodes one ofthe twenty naturally occurring amino acids at the critical positions. In a preferred aspect, however, the DNA molecules contain a codon that encodes an equivalent amino acid at each critical position. Equivalent amino acids are determined by aligning EGIII-like cellulases. The alignment can be based on structure or on function. For example, in Figure 3, it can be seen that a methionine resides at position 139 of T reesei EGIII (this corresponds to position 79 ofthe mature EGIII as shown in Figure 1). In eleven ofthe EGIII-like cellulases listed in Figure 3, an isoleucine is at position 139. Thus, isoleucine is an equivalent amino acid to methionine at position 79 ofthe mature EGIII.

As can be seen in Example 3, changing the methionine residues inactivates EGIII. Thus to restore activity, it may be necessary to further engineer the EGIII variant by the substitution, deletion or addition of amino acid residues. In one aspect of this embodiment, DNA shuffling or directed evolution techniques can be used. For a discussion of DNA shuffling or directed evolution, see U. S. Patent 5,830,721, which is incorporated by reference in its entirety. Briefly, a DNA library is created from an EGIII or EGIII variant template. If the EGIII sequence is used, the desired methionine substitution must be effected prior to shuffling. Introducing codon mutations into DNA coding sequences is well known in the art and can be found in Ausubel, for example. If an EGIII variant with the desired substitution is used, this step is not necessary. Once an EGIII variant with the desired methionine substitution is available, a library of mutants can be made. To create the library, the EGIII variant and other sequences to be shuffled are fragmented. Fragmentation of nucleic acid sequences is well known in the art and exemplary techniques can be found in U.S. Patent 5,830,721. The other sequences may include, but are not limited to, EGIII-like cellulases, including those listed in Figure 3. Preferably the fragmented sequences are double stranded. In a first step, the double stranded fragments are denatured. This can be accomplished by heating the mixture of nucleic acids to a temperature, which considering the conditions, is suitable for denaturation. Preferably, the temperature is between 80°C and 100°C.

After denaturing the nucleic acid to single strands, the strands are reannealed. The fidelity of annealing will depend on many factors, but in particular on the temperature. One of skill will recognize that relatively low temperatures, e.g., from 20°C to 30°C, will facilitate reannealing of sequences with low degrees of homology. Thus, if a variety of sequences is desired, it would be best to encourage cross-over events and reanneal the fragments at low temperatures. However, for most applications, a reannelaing temperature of 45°C to 65°C will be adequate. After the reannealing step or concurrently with reannealing, polymerase is added to assemble the fragments into complete coding regions. If the degree of homology is low and low temperatures are used, Klenow fragment can be used. However, if the degree of homology is high and a higher temperature is used for reannealing, Taq polymerase may be used to assemble the coding regions. After assembly, the nucleic acid fragments will exist in long DNA molecules comprising concatemeric nucleic acid fragments. This large nucleic acid may contain multiple copies of DNA that are the same size as the original EGIII variant template. Single copies ofthe EGIII variant and EGIII-like cellulase variant (if EGIII like cellulase coding regions are represented in the nucleic acid fragment mixture) are released from the large DNA molecule by restriction enzyme digestion or by other methods well known to those of skill. These single copies are then inserted into vectors for expansion, cloning and analysis. Those of skill will realize that PCR amplification may be utilized to increase the copy number of either the large DNA molecule prior to restriction digest, the single copies after restriction digest or the single copies after insertion into the vector of choice. In a preferred embodiment, the tertiary structure of EGIII is examined and modeled to determine which amino acid substitutions, deletions and/or additions will restore the necessary active sites to restore enzymatic activity. This may be done by determining homology at the level of tertiary structure for cellulases, including EGIII, whose tertiary structure has been determined by x-ray crystallography. Equivalent residues are defined as those for which the atomic coordinates of two or more ofthe main chain atoms of a particular amino acid residue of T. reesei EGIII and a cellulase (N on N, CA on CA, C on C and O on O) are within 0.13nm and preferably 0. lnm after alignment. Alignment is achieved after the best model has been oriented and positioned to give the maximum overlap of atomic coordinates of non-hydrogen protein atoms ofthe cellulase in question to the T reesei EGIII. The best model is the crystallographic model giving the lowest R factor for experimental diffraction data at the highest resolution available.

τ„\Fo(h)\-\Fc(h)\

^{Rfactor =} τ_h\Fo(k)\

In addition to structurally equivalent residues, functionally equivalent residues may be substituted. Equivalent residues which are functionally analogous to a specific residue of T. reesei EGIII are defined as those amino acids of a cellulase which may adopt a conformation such that they either alter, modify or contribute to protein structure, substrate binding or catalysis in a manner defined and attributed to a specific residue ofthe T. reesei EGIII. Further, they are those residues ofthe cellulase (for which a tertiary structure has been obtained by x-ray crystallography) which occupy an analogous position to the extent that, although the main chain atoms ofthe given residue may not satisfy the criteria of equivalence on the basis of occupying a homologous position, the atomic coordinates of at least two ofthe side chain atoms ofthe residue lie with 0.13nm ofthe corresponding side chain atoms of T. reesei EGIII. The crystal structure of T. reesei EGIII is presented The Protein Society, Fourteenth Symposium. San Diego, CA. August 5-9, 2000, the disclosure of which is incorporated by reference in its entirety. The coordinates of CelB of Streptomyces lividans, a homologous member ofthe Family 12 glycosyl hydrolases is provided in Sulzenbacher, et al, Biochemistry 36:6032 (1997) and in Sulzenbacher, et al, Biochemistry 38:4826 (1999). EGIII-like Cellulases

In another embodiment of this invention, cellulases similar to EGIII (EGIII- like cellulases) are the precursor proteins that are modified to improve oxidative stability. For example, from the alignment in Figure 3, it can be seen that all 21 EGIII-like cellulases listed have a methionine at position 181 (position 118 of mature EGIII). It is likely that, as with EGIII, an amino acid substitution at this position will modify the oxidative stability of the EGIII-like cellulases. Also, as with EGIII, activity ofthe EGIII-like cellulase variant may be restored or improved through engineering and substitution of other critical residues. In addition to the cellulases illustrated in Figure 3, this invention encompasses other enzymes with significant structural and/or sequence homology to EGIII. Thus, in one aspect of this embodiment ofthe invention, the enzyme has at least 30%, preferably at least 40% and most preferably at least 60% amino acid identity to EGIII. However, it should be recognized that homology alone is often not an appropriate measure for whether a particular enzyme identified by the methods described herein represents an EGIII-like enzyme. Similar enzymatic function with or without reduced homology may identify an EGIII-like cellulase. Accordingly, while homologous enzymes are indeed detected by the methods described and exemplified herein, the degree of homology should not be seen as limiting the scope ofthe invention.

Homologous proteins can be determined by using a "sequence comparison algorithm." Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'lAcad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection.

An example of an algorithm that is suitable for determining sequence similarity is the BLAST algorithm, which is described in Altschul, et al, J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive- valued threshold score T when aligned with a word ofthe same length in a database sequence. These initial neighborhood word hits act as starting points to find longer HSPs containing them. The word hits are expanded in both directions along each ofthe two sequences being compared for as far as the cumulative alignment score can be increased. Extension ofthe word hits is stopped when: the cumulative alignment score falls off by the quantity X from a maximum achieved value; the cumulative score goes to zero or below; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed ofthe alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)) alignments (B) of 50, expectation (E) of 10, M'5, N'-4, and a comparison of both strands.

The BLAST algorithm then performs a statistical analysis ofthe similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat 'I. Acad. Sci. USA 90:5873- 5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication ofthe probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, an amino acid sequence is considered similar to a protease if the smallest sum probability in a comparison ofthe test amino acid sequence to a protease amino acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

Isolation of DNA encoding EGIII-like cellulases

It is contemplated the EGIII-like cellulases ofthe invention may be found in many organisms which produce cellulases. However, likely sources of EGIII-like cellulase include those derived from a bacterium or fungus, and more particularly, from an Actinomycete, a Bacillus or a filamentous fungus. In a preferred embodiment, the cellulase is derived from the filamentous fungal family Metazoa, preferably Euascomycetes. Within Metazoa, fungal phylogenetic classifications that produce EGIII-like cellulases include the mitosporic Pyrenomycetes (including Acremonium), Sordariales (including Thielavia), Hypocreales (including Nectriaceae such as Fusarium, Necitia, Verticillium, Myrothecium and Gliocladium; and Hypocrea) and Eurotiales (including mitosporic Trichocomaceae such as Aspergillus and Penicillium). The Euascomycete preferably belongs to Diaporthales, Halosphaeriales, Microascales, Ophiostomatales, Phyllachorales, Sordariales or Xylariales. Also preferably, the Eusacomycete belongs to Hypocreales comprising Clavicipitaceae, Melanosporaceae, Necfriaceae, Niessliaceae or Mitosporic Hypocreales. Further preferably, the Euascomycete belongs to Hypocreaceae, wherein said Hypocreaceae does not comprise Trichoderma. Most preferably, the Euascomycete is Gliocladium spp., Fusarium spp., Acremonium spp., Myceliophtora spp., Verticillium spp., Myrothecium spp., Penicillium spp., Chaetomium spp., Emercella spp., and Phanerochaete spp. Specific organisms which are contemplated as possessing EGIII-like cellulases include Chaetomium thermophilum var. therm., Chaetomium atrobrunneum, Chaetomium brasiliense, Chaetomium globosum, Chaetomium vitellium, Paecilomyces lilacinus, Chaetomium thermophilum var. dissitum, Humicola insolens, Humicola brevis, Memnoniella echinata, Fusarium equiseti, Fusarium oxysporum, fusarium stilboides, Myceliophthora thermophila, Fusarium javanicum, Humicola grisea var. thermoidea, Stibella thermophila, Melanocarpus albomyces, Arthrobotrys superba, Myceliophthora hinunilea, Chaetomium pachypodiodes, Myrothecium verrucaria, Penicillium crysogenum, Malbranchea sulfurea, Lunulospora curvula, Emericella desertorum, Acremonium strictum, Cylindrocarpon heteronema, and Ulocladium chartarum. Within the Actinomycetes, Streptomyces appears to possess EGIII-like cellulases. EGIII-like cellulases according to the invention may be obtained according to the following methods. Degenerate DNA primers are constructed which encode an amino acid sequence selected from the group consisting of one or more of:

1) Asn-Asn-(Leu/Phe/Lys/ϊle)-Trp-Gly (SEQ ID NO:25)

2) Glu-(Leu/Phe/Ile)-Met-Ile-Trp (SEQ ID NO:26) 3) Gly-Thr-Glu-Pro-Phe-Thr (SEQ ID NO:27)

4) (Ser/Tyr/Cys/Trp/Thr/Asn/Lys/Arg)-(Val/Pro)-(Lys/Ala)-(Ser/Ala)- (Tyr/Phe) (SEQ ID NO:28); and

5) Lys-Asn-Phe-Phe-Asn-Tyr (SEQ ID NO:29) and used to clone DNA, and genes, encoding enzymes having cellulolytic activity according to established methods. Techniques for obtaining DNA using degenerate primers are well known in the art and can be found in Sambrook et al. MOLECULAR CLONING - A LABORATORY MANUAL (2ND ED.) VOL. 1-3, Cold Springs Harbor Publishing (1989) ("Sambrook"); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel et α .(eds.), Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1997 Supplement) ("Ausubel"). In addition, the EGIII ofthe invention may be obtained by other methods conventional in molecular biology, e.g., library screening with labeled probes, expression screening and PCR cloning, using one ofthe cellulase backbones identified herein as an EGIII-like cellulase.

The degenerate primers can be used as hybridization probes against a genomic library obtained from a target organism to analyze whether a given fragment correlates to a similar sequence in the target organism. A useful hybridization assay is as follows: Genomic DNA from a particular target source is fragmented by digestion with a restriction enzyme(s), e.g., EcoR I, Hind III, Bam HI, Cla I, Kpn I, Mlu I, Spe I, Bgl II, Nco I, Xba I, Xho I and Xma I (supplied by New England Biolabs, Inc., Beverly, MA and Boehringer Mannheim) according to the manufacturer's instructions. The samples are then electrophoresed through an agarose gel (such as, for example, 0.7% agarose) so that separation of DNA fragments can be visualized by size. The gel may be briefly rinsed in distilled H₂O and subsequently depurinated in an appropriate solution (such as, for example, 0.25M HCl) with gentle shaking followed by denaturation for 30 minutes (in, for example, 0.4 M NaOH). A renaturation step may be included in which the gel is placed in 1.5 M NaCl, IM Tris, pH 7.0 with gentle shaking for 30 minutes. The DNA is then be transferred onto an appropriate positively charged membrane, for example the Maximum Strength

Nytran Plus membrane (Schleicher & Schuell, Keene, N.H.), using a transfer solution (such as, for example, 6XSSC (900 mM NaCl, 90 mM trisodium citrate). After the transfer is complete, generally at about 2 hours or greater, the membrane is rinsed (in, for example, 2X SSC[2X SSC = 300 mM NaCl, 30 mM trisodium citrate]) and air dried at room temperature. The membrane is then be prehybridized, (for approximately 2 hours or more) in a suitable prehybridization solution (such as, for example, an aqueous solution containing per 100 mL: 30-50 mL formamide, 25 mL of 20X SSPE (IX SSPE = 0.18 M NaCl, 1 mM EDTA, 10 mM NaH₂PO₄, pH 7.7), 2.5 mL of 20% SDS, and 1 mL of 10 mg/ml sheared herring sperm DNA). A DNA probe corresponding to the primer sequences above is be isolated by electrophoresis in an agarose gel, the fragment excised from the gel and recovered from the excised agarose. This purified fragment of DNA is then labeled (using, for example, the Megaprime labeling system according to the instructions ofthe manufacturer to incorporate P³² in the DNA (Amersham International PLC, Buckinghamshire, England)). The labeled probe is denatured by heating to 95° C for 5 minutes and immediately added to the prehybridization solution above containing the membrane. The hybridization reaction should proceed for an appropriate time and under appropriate conditions, for example, 18 hours at 37 °C with gentle shaking. The membrane is rinsed (for example, in 2X SSC/0.3% SDS) and then washed with an appropriate wash solution and with gentle agitation. The stringency desired will be a reflection ofthe conditions under which the membrane (filter) is washed. Specifically, the stringency of a given reaction (i.e., the degree of homology necessary for successful hybridization) will largely depend on the washing conditions to which the filter from the Southern blot is subjected after hybridization. "Low-stringency" conditions as defined herein will comprise washing a filter from a Southern blot with a solution of 0.2X SSC/0.1% SDS at 20° C for 15 minutes. Standard-stringency conditions comprise a further washing step comprising washing the filter from the Southern blot a second time with a solution of 0.2X SSC/0.1% SDS at 37 °C for 30 minutes.

In a preferred embodiment according to this aspect ofthe invention, degenerate primers are prepared corresponding to one or more ofthe above peptides. The primers are combined with a genomic DNA from a target organism (i.e., the organism in which the EGIII-like cellulase is sought) under conditions suitable to initiate a standard PCR reaction. In this embodiment, it is advantageous to select degenerate primers corresponding to peptides (a) and or (d) plus primers corresponding to (c) and/or (e) and amplify DNA with those primers. After the PCR reaction has been performed, the resulting DNA is run on a polyacrylamide gel and bands corresponding in size to the EGIII fragment comprising peptides (a) and/or (d) in addition to (c) and/or (e), i.e., those in the 400-1000 base pair range, are selected. These fragments are pooled and reamplified using primers corresponding to peptides (a) and/or (d) plus primers corresponding to peptide (b) or, alternatively, using primers corresponding to peptide (c) and/or (e) plus primers corresponding to peptide (b). Strong bands ofthe expected size (in the case of EGIII-like cellulases, the bands will correspond to approximately 250-500 base pair) are excised and sequenced. The isolated sequences are then used to design primers and these primers are used via, e.g., rapid amplification of genomic DNA ends (RAGE), to obtain the full length gene, see e.g., Mizobuchi, et al, BioTechniques 15:215-216 (1993).

The DNA that hybridizes with the DNA primers outlined above and thus identified by this method a corresponding EGIII encoding gene may be isolated by routine methods and used to express the corresponding EGIII-like cellulase according to routine techniques. Upon obtaining the cloned gene, routine methods for insertion ofthe DNA into a vector that can then be transformed into a suitable host cell are used. Culturing the transformed host cell under appropriate conditions results in production ofthe EGIII-like cellulase that can be obtained, purified and prepared as necessary for a particular application.

Manufacture and Purification of EGIII-like Cellulases and Variants

The EGIII-like cellulases ofthe invention are preferably isolated or purified. In the context ofthe present invention, purification or isolation generally means that the EGIII-like cellulase is altered from its natural state by virtue of separating the EGIII-like cellulase from some or all ofthe naturally occurring substituents with which it is associated in nature, e.g., the source organism or other cellulases or enzymes expressed by the source organism in conjunction with the EGIII cellulase. Similarly, the EGIII-like cellulases ofthe invention may be combined with other components that are not naturally present in the natural state. Isolation or purification may be accomplished by art recognized separation techniques such as ion exchange chromatography, affinity chromatography, hydrophobic separation, dialysis, protease treatment, ammonium sulfate precipitation or other protein salt precipitation techniques, centrifugation, size exclusion chromatography, filtration, microfiltration, gel electrophoresis or separation on a gradient to remove whole cells, cell debris, impurities, extraneous proteins, or enzymes undesired in the final composition.

The present invention relates to the expression, purification and/or isolation and use of variant EGIII-like cellulases. These enzymes are preferably prepared by recombinant methods utilizing the gene identified and isolated according to the methods described above. However, enzymes for use in the present invention may be obtained by other art-recognized means such as purification from natural isolates.

The microorganism to be transformed for the purpose of expressing an EGIII-like cellulase according to the present invention may advantageously comprise a strain derived from Trichoderma sp. Thus, a preferred mode for preparing EGIII-like cellulases according to the present invention comprises transforming a Trichoderma sp. host cell with a DNA construct comprising at least a fragment of DNA encoding a portion or all ofthe EGIII-like cellulase detected as described above. The DNA construct will generally be functionally attached to a promoter. The transformed host cell is then grown under conditions so as to express the desired protein. Subsequently, the desired protein product is purified to substantial homogeneity.

However, the best expression vehicle for a given DNA encoding a variant EGIII-like cellulase may differ from its natural host. Thus, it may be that it will be most advantageous to express a protein in a transformation host that bears phylogenetic similarity to the source organism for the variant EGIII-like cellulase. For example, in an alternative embodiment, Aspergillus niger can be used as an expression host. See, WO 98/31821 for a description of transformation into A. niger.

Accordingly, the present description of a Trichoderma spp. expression system is provided for illustrative purposes only and as one option for expressing the variant EGIII-like cellulase ofthe invention. One of skill in the art, however, may be inclined to express the DNA encoding variant EGIII-like cellulase in a different host cell if appropriate and it should be understood that the source ofthe variant EGIII-like cellulase should be considered in determining the optimal expression host. Additionally, the skilled worker in the field will be capable of selecting the best expression system for a particular gene through routine techniques utilizing the tools available in the art.

In one embodiment, the strain comprises T. reesei, which is a useful strain for obtaining overexpressed protein. For example, RL-P37, described by Sheir-Neiss, et al, Appl. Microbiol. Biotechnol. 20:46-53 is known to secrete elevated amounts of cellulase enzymes. Functional equivalents of RL-P37 include Trichoderma reesei strain RUT-C30 (ATCC No. 56765) and strain QM9414 (ATCC No. 26921). It is contemplated that these strains would also be useful in overexpressing EGIII-like cellulases.

Where it is desired to obtain the EGIII-like cellulase in the absence of potentially detrimental native cellulolytic activity, it is useful to obtain a Trichoderma host cell strain which has had one or more cellulase genes deleted prior to introduction of a DNA construct or plasmid containing the DNA fragment encoding the EGIII-like cellulase. Such strains may be prepared by the method disclosed in U.S. Patent No. 5,246,853 and WO 92/06209, which are hereby incorporated by reference. By expressing an EGIII-like cellulase in a host microorganism that is missing one or more cellulase genes, the identification and subsequent purification procedures are simplified. Any gene from Trichoderma sp. which has been cloned can be deleted, for example, the cbhl, cbh2, egll, and egl3 genes as well as those encoding EGIII and/or EGV protein (see e.g., U.S. Patent No. 5,475,101 and WO 94/28117, respectively).

Gene deletion may be accomplished by inserting a form ofthe desired gene to be deleted or disrupted into a plasmid by methods known in the art. The deletion plasmid is then cut at an appropriate restriction enzyme site(s), internal to the desired gene coding region, and the gene coding sequence or part thereof replaced with a selectable marker. Flanking DNA sequences from the locus ofthe gene to be deleted or disrupted, preferably between about 0.5 to 2.0 kb, remain on either side ofthe selectable marker gene. An appropriate deletion plasmid will generally have unique restriction enzyme sites present therein to enable the fragment containing the deleted gene, including flanking DNA sequences, and the selectable marker gene to be removed as a single linear piece. A selectable marker must be chosen so as to enable detection ofthe transformed microorganism. Any selectable marker gene that is expressed in the selected microorganism will be suitable. For example, with Trichoderma sp., the selectable marker is chosen so that the presence ofthe selectable marker in the transformants will not significantly affect the properties ofthe fungus. Such a selectable marker may be a gene that encodes an assayable product. For example, a functional copy of a Trichoderma sp. gene may be used which if lacking in the host strain results in the host strain displaying an auxotrophic phenotype.

In a preferred embodiment, apyr4^~ derivative strain of Trichoderma sp. is transformed with a functional pyr4 gene, which thus provides a selectable marker for transformation. Apyr4^~ derivative strain may be obtained by selection of Trichoderma sp. strains that are resistant to fluoroorotic acid (FOA). The pyr4 gene encodes orotidine-5'- monophosphate decarboxylase, an enzyme required for the biosynthesis of uridine. Strains with an intact pyr4 gene grow in a medium lacking uridine but are sensitive to fluoroorotic acid. It is possible to select pyr4^~ derivative strains that lack a functional orotidine monophosphate decarboxylase enzyme and require uridine for growth by selecting for FOA resistance. Using the FOA selection technique it is also possible to obtain uridine-requiring strains which lack a functional orotate pyrophosphoribosyl transferase. It is possible to transform these cells with a functional copy ofthe gene encoding this enzyme (Berges & Barreau, Curr. Genet. 9:359-365 (1991)). Selection of derivative strains is easily performed using the FOA resistance technique referred to above, and thus, the pyr4 gene is preferably employed as a selectable marker.

To transformer^^" Trichoderma sp. so as to be lacking in the ability to express one or more cellulase genes, a single DNA fragment comprising a disrupted or deleted cellulase gene is then isolated from the deletion plasmid and used to transform an appropriate pyf Trichoderma host. Transformants are then identified and selected based on their ability to express the pyr4 gene product and thus compliment the uridine auxotrophy of the host strain. Southern blot analysis is then carried out on the resultant transformants to identify and confirm a double crossover integration event that replaces part or all ofthe coding region ofthe genomic copy ofthe gene to be deleted with the pyr4 selectable markers. Although the specific plasmid vectors described above relate to preparation of pyf transformants, the present invention is not limited to these vectors. Various genes can be deleted and replaced in the Trichoderma sp. strain using the above techniques. In addition, any available selectable markers can be used, as discussed above. In fact, any Trichoderma sp. gene that has been cloned, and thus identified, can be deleted from the genome using the above-described strategy.

As stated above, the host strains used are derivatives of Trichoderma sp. that lack or have a nonfunctional gene or genes corresponding to the selectable marker chosen. For example, if the selectable marker of pyr4 is chosen, then a specific pyr4^~ derivative strain is used as a recipient in the transformation procedure. Similarly, selectable markers comprising Trichoderma sp. genes equivalent to the Aspergillus nidulans genes amdS, argB, trpC, niaD may be used. The corresponding recipient strain must therefore be a derivative strain such as argB^', trpC, niaD^', respectively.

DNA encoding the EGIII-like cellulase is then prepared for insertion into an appropriate microorganism. According to the present invention, DNA encoding an EGIII- like cellulase comprises the DNA necessary to encode for a protein that has functional cellulolytic activity. The DNA fragment or DNA variant fragment encoding the EGIII-like cellulase or derivative may be functionally attached to a fungal promoter sequence, for example, the promoter ofthe cbhl or egll gene.

It is also contemplated that more than one copy of DNA encoding a EGIII- like cellulase may be recombined into the strain to facilitate overexpression. The DNA encoding the EGIII-like cellulase may be prepared by the construction of an expression vector carrying the DNA encoding the cellulase. The expression vector carrying the inserted DNA fragment encoding the EGIII-like cellulase may be any vector which is capable of replicating autonomously in a given host organism or of integrating into the DNA ofthe host, typically a plasmid. In preferred embodiments two types of expression vectors for obtaining expression of genes are contemplated. The first contains DNA sequences in which the promoter, gene-coding region, and terminator sequence all originate from the gene to be expressed. Gene truncation may be obtained where desired by deleting undesired DNA sequences (e.g., coding for unwanted domains) to leave the domain to be expressed under control of its own transcriptional and translational regulatory sequences. A selectable marker is also contained on the vector allowing the selection for integration into the host of multiple copies ofthe novel gene sequences.

The second type of expression vector is preassembled and contains sequences required for high-level transcription and a selectable marker. It is contemplated that the coding region for a gene or part thereof can be inserted into this general-purpose expression vector such that it is under the transcriptional control ofthe expression cassettes promoter and terminator sequences. For example, pTEX is such a general-purpose expression vector. Genes or part thereof can be inserted downstream ofthe strong cbhl promoter.

In the vector, the DNA sequence encoding the EGIII-like cellulase ofthe present invention should be operably linked to transcriptional and translational sequences, i.e., a suitable promoter sequence and signal sequence in reading frame to the structural gene. The promoter may be any DNA sequence that shows transcriptional activity in the host cell and may be derived from genes encoding proteins either homologous or heterologous to the host cell. The signal peptide provides for extracellular production ofthe EGIII-like cellulase or derivatives thereof. The DNA encoding the signal sequence is preferably that which is naturally associated with the gene to be expressed, however the signal sequence from any suitable source, for example an exo-cellobiohydrolase or endoglucanase from Trichoderma, is contemplated in the present invention.

The procedures used to ligate the DNA sequences coding for the EGIII-like cellulase ofthe present invention with the promoter, and insertion into suitable vectors are well known in the art.

The DNA vector or construct described above may be introduced in the host cell in accordance with known tecliniques such as transformation, transfection, microinjection, microporation, biolistic bombardment and the like.

In the preferred transformation technique, it must be taken into account that the permeability ofthe cell wall to DNA in Trichoderma sp. is very low. Accordingly, uptake ofthe desired DNA sequence, gene or gene fragment is at best minimal. There are a number of methods to increase the permeability ofthe Trichoderma sp. cell wall in the derivative strain (i.e., lacking a functional gene corresponding to the used selectable marker) prior to the transformation process. The preferred method in the present invention to prepare Trichoderma sp. for transformation involves the preparation of protoplasts from fungal mycelium. The mycelium can be obtained from germinated vegetative spores. The mycelium is treated with an enzyme that digests the cell wall resulting in protoplasts. The protoplasts are then protected by the presence of an osmotic stabilizer in the suspending medium. These stabilizers include sorbitol, mannitol, potassium chloride, magnesium sulfate and the like. Usually the concentration of these stabilizers varies between 0.8 M and 1.2 M. It is preferable to use about a 1.2 M solution of sorbitol in the suspension medium.

Uptake ofthe DNA into the host Trichoderma sp. strain is dependent upon the calcium ion concentration. Generally, between about 10 mM CaCl₂ and 50 mM CaCl₂ is used in an uptake solution. Besides the need for the calcium ion in the uptake solution, other items generally included are a buffering system such as TE buffer (10 Mm Tris, pH 7.4; 1 mM EDTA) or 10 mM MOPS, pH 6.0 buffer (morpholinepropanesulfonic acid) and polyethylene glycol (PEG). It is believed that the polyethylene glycol acts to fuse the cell membranes thus permitting the contents ofthe medium to be delivered into the cytoplasm of the Trichoderma sp. strain and the plasmid DNA is transferred to the nucleus. This fusion frequently leaves multiple copies ofthe plasmid DNA tenderly integrated into the host chromosome. Usually a suspension containing the Trichoderma sp. protoplasts or cells that have been subjected to a permeability treatment at a density of 10⁸ to 10⁹/ml, preferably 2 x 10⁸/ml are used in transformation. A volume of 100 microliters of these protoplasts or cells in an appropriate solution (e.g., 1.2 M sorbitol; 50 mM CaCl₂) are mixed with the desired DNA. Generally a high concentration of PEG is added to the uptake solution. From 0.1 to 1 volume of 25% PEG 4000 can be added to the protoplast suspension. However, it is preferable to add about 0.25 volumes to the protoplast suspension. Additives such as dimethyl sulfoxide, heparin, spermidine, potassium chloride and the like may also be added to the uptake solution and aid in transformation. Generally, the mixture is then incubated at approximately 0°C for a period of between 10 to 30 minutes. Additional PEG is added to the mixture to further enhance the uptake ofthe desired gene or DNA sequence. The 25% PEG 4000 is generally added in volumes of 5 to 15 times the volume ofthe transformation mixture; however, greater and lesser volumes may be suitable. The 25% PEG 4000 is preferably about 10 times the volume ofthe transformation mixture. After the PEG is added, the transformation mixture is then incubated at room temperature before the addition of a sorbitol and CaCl₂ solution. The protoplast suspension is then further added to molten aliquots of a growth medium. This growth medium permits the growth of transformants only. Any growth medium can be used in the present invention that is suitable to grow the desired transformants. However, if Pyr⁺ transformants are being selected it is preferable to use a growth medium that contains no uridine. The subsequent colonies are transferred and purified on a growth medium depleted of uridine.

At this stage, stable transformants may be distinguished from unstable transformants by their faster growth rate and the formation of circular colonies with a smooth, rather than ragged outline on solid culture medium lacking uridine. Additionally, in some cases a further test of stability may be made by growing the transformants on solid non-selective medium (i.e. containing uridine), harvesting spores from this culture medium and determining the percentage of these spores which will subsequently germinate and grow on selective medium lacking uridine. In a particular embodiment ofthe above method, the EGIII-like cellulases or derivatives thereof are recovered in active form from the host cell after growth in liquid media either as a result ofthe appropriate post translational processing ofthe novel EGIII- like cellulase or derivatives thereof.

The expressed EGIII-like cellulase may be recovered from the medium by conventional techniques including separations ofthe cells from the medium by centrifugation, filtration, and precipitation ofthe proteins in the supernatant or filtrate with a salt, for example, ammonium sulphate. Additionally, chromatography procedures such as ion exchange chromatography or affinity chromatography may be used. Antibodies (polyclonal or monoclonal) may be raised against the natural purified EGIII-like cellulase, or synthetic peptides may be prepared from portions ofthe EGIII-like cellulase molecule and used to raise polyclonal antibodies.

Uses of EGIII-like Cellulases and Variants

The cellulases of this invention have many uses, e.g., in textile treatments. Treatment of textiles according to the present invention contemplates textile processing or cleaning with a composition comprising a cellulase. Such treating includes, but is not limited to, stonewashing, modifying the texture, feel and/or appearance of cellulose containing fabrics or other techniques used during manufacturing or cleaning/reconditioning of cellulose containing fabrics. Additionally, treating within the context of this invention contemplates the removal of "immature" or "dead" cotton, from cellulosic fabric or fibers. Immature cotton is significantly more amorphous than mature cotton and results in a lesser quality fabric when present due to, for example, uneven dyeing. It is believed the enzymes contemplated in the present invention would be particularly useful in washing soiled manufactured cellulose containing fabrics. For example, the cellulase may be used in a detergent composition for washing laundry. Detergent compositions useful in accordance with the present invention include special formulations such as pre-wash, pre-soak and home-use color restoration compositions. Such treating compositions, as described herein, may be in the form of a concentrate which requires dilution or in the form of a dilute solution or form which can be applied directly to the cellulose containing fabric. General treatment techniques for cellulase treatment of textiles are described in, for example, EP Publication No. 220 016 and GB Application Nos. 1,368,599 and 2,095,275.

A concentrated cellulase composition can be prepared for use in the methods described herein. Such concentrates contain concentrated amounts ofthe cellulase composition described above, buffer and surfactant, preferably in an aqueous solution. When so formulated, the cellulase concentrate can readily be diluted with water so as to quickly and accurately prepare cellulase preparations having the requisite concentration of each constituent. When aqueous concentrates are formulated, these concentrates can be diluted so as to arrive at the requisite concentration ofthe components in the cellulase solution as indicated above. As is readily apparent, such cellulase concentrates will permit facile formulation ofthe cellulase solutions as well as permit feasible transportation ofthe composition to the location where it will be used. The treating concentrate can be in any art recognized form, for example, liquid, emulsion, gel, or paste. Such forms are well known to those skilled in the art.

When a solid cellulase concentrate is employed, the cellulase composition may be a granule, a powder, an agglomerate or a solid disk. The granules can be formulated so as to contain materials to reduce the rate of dissolution ofthe granules into the wash medium. Such materials and granules are disclosed in U.S. Patent No. 5,254,283, which is incorporated herein by reference in its entirety.

In a preferred embodiment ofthe present invention, the cellulase ofthe invention may be employed in a detergent composition. The detergent compositions according to the present invention are useful as pre-wash compositions, pre-soak compositions, or for cleaning during the regular wash or rinse cycle. Preferably, the detergent composition ofthe present invention comprises an effective amount of cellulase, a surfactant, and optionally includes other ingredients described below. In particular, the cellulases of this invention will be particularly useful in detergents containing oxidants, for example, bleach.

An effective amount of cellulase employed in the detergent compositions of this invention is an amount sufficient to impart the desirable effects known to be produced by cellulase on cellulose containing fabrics, for example, depilling, softening, anti-pilling, surface fiber removal, anti-graying and cleaning. Preferably, the cellulase in the detergent composition is employed in a concentration of from about 10 ppm to about 20,000 ppm of detergent. The concentration of cellulase enzyme employed in the detergent composition is preferably selected so that upon dilution into a wash medium, the concentration of cellulase enzyme is in a range of about 0.01 to about 1000 ppm, preferably from about 0.02 ppm to about 500 ppm, and most preferably from about 0.5 ppm to about

250 ppm total protein. The amount of cellulase enzyme employed in the detergent composition will depend on the extent to which the detergent will be diluted upon addition to water so as to form a wash solution. The detergent compositions ofthe present invention may be in any art recognized form, for example, as a liquid, in granules, in emulsions, in gels, or in pastes.

Such forms are well known to the skilled artisan. When a solid detergent composition is employed, the cellulase is preferably formulated as granules. Preferably, the granules can be formulated so as to additionally contain a cellulase protecting agent. The granule can be formulated so as to contain materials to reduce the rate of dissolution ofthe granule into the wash medium. Such materials and granules are disclosed in U.S. Patent No. 5,254,283, which is incorporated herein by reference in its entirety.

The detergent compositions of this invention employ a surface-active agent, e.g., a surfactant, including anionic, non-ionic and ampholytic surfactants well known for their use in detergent compositions. In addition to the cellulase composition and the surfactant(s), the detergent compositions of this invention can optionally contain one or more ofthe following components:

Treatment of a cellulosic material according to the present invention further contemplates the treatment of animal feed, pulp and/or paper, food and grain for purposes known in the art. For example, cellulase is known to increase the value of animal feed, improve the drainability of wood pulp, enhance food products and reduce fiber in grain during the grain wet milling process or dry milling process.

In order to further illustrate the present invention and advantages thereof, the following specific examples are given with the understanding that they are being offered to illustrate the present invention and should not be construed in any way as limiting its scope.

EXAMPLES Example 1: Preparation of Genomic DNA Encoding EGIII-Like Cellulases

Genomic DNA was prepared for several different microorganisms for the purpose of undertaking a PCR reaction to determine whether EGIII-like cellulases are encoded by the DNA of a particular organism. Genomic DNA was obtained from Acremonium brachypenium deposit no. CBS 866.73; Chaetomium brasillience deposit no. CBS 140.50; Chaetomium vitellium deposit no. CBS 250.85; Emericella desertoru deposit no. CBS 653.73; Fusarium equiseti deposit no. CBS 185.34; Gliocladium roseum deposit no. CBS 443.65; Humicola grisea var. thermoidia deposit no. CBS 225.63; Myceliopthora thermophila deposit no. ATCC 48102-48104; Penicillium notatum deposit no. ATCC 9178, 9179; and Phanerochaete chrysosporium deposit no. ATCC 28326 and isolated according to standard methods.

PCR was performed on a standard PCR machine such as the PCT- 150 MicroCycler from MJ Research Inc. under the following conditions: 1) 1 minute at 98°C for 1 cycle;

2) 1 minute at 94°C, 90 seconds at 40°C, 1 minute at 72°C

3) repeat step 2 for 30 cycles, 4) 7 minutes at 72°C for 1 cycle, and

5) lower temperature to 15°C for storage and further analysis. The following DNA primers were constructed for use in amplification of EGIII-like genes from the libraries constructed from the various microorganisms. All symbols used herein for protein and DNA sequences correspond to IUPAC IUB Biochemical Nomenclature Commission codes.

BOX1: primers coding for (N/Q)NLWG (SEQ ID NO:30) forward primer FRG001 : AAY AAY YTN TGG GG (SEQ ID NO:31) forward primer FRG002: CAR AAY YTN TGG GG (SEQ ID NO:32)

BOX1': primers coding for NNN(F/L/Y/I/L/N/K)WG (SEQ ID NO:33) forward primer FRG010: AAY AAY AAY HWI TGG GG (SEQ ID NO:34)

BOX2: primers coding for ELMIW ((SEQ ID NO:35) forward primer FRG003: GAR YTN ATG ATH TGG (SEQ ID NO:36) reversed primer FRG004: CCA DAT CAT NAR YTC (SEQ ID NO:37)

BOX2': primers coding for YELMIW (SEQ ID NO:38) forward primer FRG011 : TAY GAR YTI ATG ATH TGG (SEQ ID NO:39) reversed primer FRG012: CCA DAT CAT IAR YTC RTA (SEQ ID NO:40)

BOX3: primers coding for GTE(P/C)FT (SEQ ID NO:41) reversed primer FRG005 : GTR AAN GGY TCR GTR CC (SEQ ID NO:42) reversed primer FRG006: GTR AAN GGY TCR GTY CC (SEQ ID NO:43) reversed primer FRG007: GTR AAN GGY TCY GTR CC (SEQ ID NO:44) reversed primer FRG008: GTR AAN GGY TCY GTY CC (SEQ ID NO:45) reversed primer FRG009: GTR AAR CAY TCN GTN CC (SEQ ID NO:46)

PCR conditions were as follows: 10 μL of 10X reaction buffer (10X reaction buffer comprising lOOmM Tris HCl, pH 8-8.5; 250 mM KC1; 50 mM (NH₄)₂SO₄; 20 mM MgSO₄); 0.2 mM each of dATP, dTTP, dGTP, dCTP (final concentration), 1 μL of 100 ng/μL genomic DNA, 1 μL of PWO polymerase (Boehringer Mannheim, Cat # 1644-947) at 1 unit per μL, 500 mM primers (final concentration) and water to 100 μL. The solution was overlaid with mineral oil.

The PCR strategy was as follows: forward primers for BOX1 (SEQ ID NO:31 and 32, respectively) and Boxl' (SEQ ID NO:34) were combined with reversed primers from BOX3 (SEQ ID NO:42-46) in a mixture with the desired genomic DNA sample and run on a gel to obtain fragments in the 400-1000 base pair range. The fragments so obtained were pooled and the pool split into two approximately equal portions. The first pool was combined with the forward primers from BOX1 (SEQ ID NO:31 and 32, respectively) and BOX1 ' (SEQ ID NO:34) along with the reversed primer from BOX2 (SEQ ID NO:37). The second pool was combined with the forward primer from BOX2 (SEQ ID NO: 36) along with the reversed primers from BOX3 (SEQ ID NO:42-46).

Fragments having the approximate size relative to an EGIII-like cellulase considering the location ofthe primers within the gene, in this case corresponding to those between 250-500 base pairs, were isolated and sequenced.

From the sequenced fragments, it was possible to use the RAGE technique (rapid amplification of genomic ends) to rapidly obtain the sequence ofthe full-length gene. Full-length genes have been obtained and are provided with several additional EGIII-like cellulase sequences in Fig. 3. As shown in Fig. 3, full length genes isolated from Hypocrea schweinitzii (SEQ ID NO:4), Aspergillus aculeatus (SEQ ID NO:5), Aspergillus kawachii (1) (SEQ ID NO:6), Aspergillus kawachii (2) (SEQ ID NO:7), Aspergillus oryzae (SEQ ID NO:8), Humicola grisea (SEQ ID NO:9), Humicola insolens (SEQ ID NO: 10), Chaetomium brasilliense (SEQ ID NO: 11), Fusarium equiseti (SEQ ID NO: 12), Fusarium javanicum (1) (SEQ ID NO: 13), Fusarium javanicum (2) (SEQ ID NO: 14), Gliocladium roseum (1) (SEQ ID NO: 15), Gliocladium roseum (2) (SEQ ID NO: 16), Gliocladium roseum (3) (SEQ ID NO: 17), Gliogladium roseum (4) (SEQ ID NO: 18), Memnoniella echinata (SEQ ID NO: 19), Actinomycete 11AG8 (SEQ ID NO:21), Streptomyces lividans CelB (SEQ ID NO:22), Rhodothermus marinus (SEQ ID NO:23), Emericella desertoru (SEQ ID NO:20), and Erwinia carotovara (SEQ ID NO:24) all comprised significant homology to EGIII from Trichoderma reesei.

Example 2: EGIII Variants

Site-directed mutagenesis was performed to incorporate amino acid substitutions in T. reesei EGIII. The following primers were used to produce the substitutions in EGIII from T. reesei. PCR was performed according to well-known tecliniques.

Table 1: PCR primers

Briefly, DNA that encodes T reesei EG III was amplified from a cDNA clone (Ward, et al, Proc. ofthe Tricel Symposium on "Trichoderma reesei cellulases and other hydrolases. " Espoo, Finland 1993 Ed. Suominen, P. and Reinikanen, T. Foundation for Biotechnical and Industrial Research. 8, ppl53-158; and U.S. Patent No. 5,475,101) using PCR primers that introduced a Bgl II restriction endonuclease site at the 5' end ofthe egl3 gene (immediately upstream ofthe first ATG codon) and an Xba I site at the 3' end (immediately downstream ofthe "stop" codon). The amplified fragment was then digested with Bgl II and Xba I, and ligated into pUC19 digested with Bgl II and Xba I. Variants were made in this plasmid using the QuikChange™ mutagenesis methods (Strategene). The variant genes were then subcloned into the Aspergillus expression vector pPGPT-pyrG (Berka and Barnett, Biotech.Adv. 7: 127 (1989)). Vectors carrying the variant genes were then transformed into A.niger var. awamori and the resultant strains grown in shake-flask cultures (WO 98/31821).

EG III variants were then purified from cell-free supernatants of these cultures by column chromatography. Briefly, approximately 1 mL of Pharmacia Butyl Sepharose (Fast Flow) resin per 10 mg of EGIII was loaded into a disposable drip column with 0.5 M. ammonium sulfate. The column was then equilibrated with 0.05 M Bis Tris Propane and 0.05 M ammonium acetate at pH 8.

The EGIII-like cellulase containing supernatants were treated overnight with 0.18 mg/mL of endoglucanase H at 37°C. Ammonium sulfate was added to the treated supernatants to a final concentration of approximately 0.5 M. After centrifugation, the supernatant was loaded onto the column. The column was then washed with 3 volumes equilibration buffer and then eluted with 2x1 volumes of 0.05 M Bis Tris Propane and 0.05 M ammonium acetate, pH 8. Each volume of flow through was collected as a separate fraction with the EGIII-like cellulase appearing in the second fraction.

Example 3: Specific Activity of EGIII-like Cellulases To assay for specific activity, a NPC hydrolysis assay was used. In a microtiter plate, 100 μl 50 mM sodium acetate, pH 5.5 and 20 μl 25 mg/mL o-NPC (o- Nitrophenyl o-D-Cellobioside (Sigma N 4764)) in assay buffer was added. The plate was incubated for 10 minutes at 40°C.

Once equilibrated, 10 μL EGIII-like cellulase was added and the plate incubated at 40°C for another 10 minutes. To quench the hydrolysis and stop the reaction, 70 iL of 0.2 M glycine, pH 10.0 was added. The plate was then read in a microtiter plate reader at 410 nm. As a guide, lOμL of a O.lmg/ml solution of T.reesei EGIII provided an OD of around 0.3.

The concentration of EGIII-like cellulase was determined by absorbance at 280 nm where the extinction coefficient was 78711 M^"1 cm^"1 or 3.352 g/L^"1 experimentally determined by the method of Edelhoch as described in Pace, et al, Pro. Sci. 4:2411 (1995). The melting point ofthe EGIII variants was determined by equilibrium CD. Experiments were performed on an Aviv 62DS or 62ADS spectrophotometer, equipped with a 5 position thermoelectric cell holder supplied by Aviv. Buffer conditions were 50 mM bis-tris propane and 50 mM ammonium acetate adjusted to pH 8.0 with acetic acid. The final protein concentration for each experiment was in the range of 5-30 mM. Data was collected in a 0.1 cm path length cell.

Spectra were collected from 265—210 nm. Thermal denaturations were performed at 217 nm from 30 to 90 °C with data collected every two degrees. The equilibration time at each temperature was 0.1 minutes and data was collected for 4 seconds per sample.

The remainder ofthe pH 8.0 sample was divided into 5 x 400 uL aliquots. Two samples were adjusted to pH 5 and 7 with acetic acid and two others were adjusted to pH 9 and 10 with sodium hydroxide. Thermal denaturations of all five samples were performed simultaneously as described above. The melting points were determined according to the methods of Luo, et al, Biochemistry 34:10669 and Gloss, et al, Biochemistry 36:5612.

Table 2: Specific Activity of EGIII-like Cellulases

As can be seen from Table 2, substitution with other amino acids significantly decreased specific activity ofthe EGIII variants. However, substitution of other amino acids into the EGIII variants may restore the specific activity ofthe variants.

Claims

1 1. An EGIII-like cellulase variant, wherein said variant comprises

2 a substitution or deletion at a position corresponding to one or more of residues M79,

3 Ml 54 and/or Ml 18 in EGIII from Trichoderma reesei.

1 2. The variant of claim 1 , wherein said variant comprises an

2 amino acid selected from the group consisting of leucine, isoleucine, vlaine,

3 threonine, serine or alanine substitution at a position corresponding to one or more of

4 residues M79, M154 and/or Ml 18 in EGIII.

1 3. The cellulase according to claim 1, said cellulase being derived

2 from a fungus, bacteria or Actinomycete.

1 4. The cellulase according to claim 3, wherein said cellulase is

2 derived from a fungus.

1 5. The cellulase according to claim 4, wherein said fungus is a

2 filamentous fungus.

1 6. The cellulase according to claim 5 wherein said filamentous

2 fungus belongs to Euascomycete.

1 7. The cellulase according to claim 6 wherein said Euascomycete

2 is Aspergillus spp., Gliocladium spp., Fusarium spp., Acremonium spp.,

3 Myceliophtora spp., Verticillium spp., Myrothecium spp., or Penicillium spp.

1 8. The cellulase according to claim 1, wherein said cellulase is an

2 endoglucanase. l 9. A DNA encoding the cellulase according to claim 1. l 10. A vector comprising the DNA of claim 9. l

11. A host cell transformed with the vector of claim 10.

1 12. A method of producing a cellulase comprising the steps of:

2 (a) culturing the host cell according to claim 12 in a

3 suitable culture medium under suitable conditions to produce cellulase; and

4 (b) obtaining said produced cellulase.

13. A detergent composition comprising a surfactant and a cellulase, wherein said cellulase comprises a variant EGIII-like cellulase comprising a substitution at an oxidatively sensitive residue.

14. The detergent of claim 13, wherein said variant EGIII or EGIII cellulase comprises a substitution or deletion at a position corresponding to one or more of residues M79 and/or Ml 18 in EGIII from Trichoderma reesei.

15. The detergent of claim 14, wherein said variant comprises an amino acid selected from the group consisting of leucine, isoleucine, vlaine, threonine, serine or alaninesubstitution at a position corresponding to one or more of residues M79, Ml 54 and/or Ml 18 in EGIII..

16. The detergent according to claim 13, wherein said detergent is a laundry detergent.

17. The use ofthe variant EGIII or EGIII-like cellulase according to claim 1 in the treatment of a cellulose containing textile.