WO1994028117A1 - Novel endoglucanase enzyme - Google Patents

Abstract

The present invention concerns a novel enzyme, EGV, having endoglucanase activity. The molecular weight of the enzyme is about 20 to 25 kDA and it is isolated from the fungus Trichoderma reesei. The invention also relates to a DNA sequence coding for the novel enzyme as well as vectors, yeast strains and fungal strains containing the DNA sequence. Furthermore, the invention concerns a method for isolating the DNA sequence coding for the novel enzyme and for constructing yeast and fungal strains which are capable of expressing endoglucanase. The invention also provides an enzyme product having endoglucanase activity and methods for enzymatically modifying lignocellulosic materials, in particular for modification or degradation of cellulose and/or β-glucan.

Description

NOVEL ENDOGLUCANASE ENZYME

Field of the invention

The present invention concerns a novel enzyme having endoglucanase activity. The enzyme is isolated from the fungus Trichoderma reesei. The invention also relates to an isolated and purified DNA sequence coding for the novel enzyme as well as vectors, yeast strains and fungal strains containing the DNA sequence. Furthermore, the invention concerns a method for isolating the DNA sequence coding for the novel enzyme and for constructing fungal strains which are capable of expressing endoglucanase. The invention also provides an enzyme product having endoglucanase activity and methods for enzymatically modifying cellulosic/lignocellulosic materials, in particular for modification or degradation of cellulose and/or 0-glucan.

Background of the invention

Many fungal species produce enzymes that degrade plant polymers into simple compounds like sugars. The fungus Trichoderma reesei is one of the most potent and most studied organisms degrading cellulose. It produces all the enzyme types needed for efficient break¬ down of crystalline cellulose, namely endo-l,4-β-D-glucanases (EC 3.2.1.4), cellobiohydro- lases (exo-l,4-β-D-glucanases, EC 3.2.1.91) and 1,4-β-D-glucosidases (EC 4.3.2.21). The number of enzymes belonging to each class is far from clear, but the existence of at least tw cellobiohydrolases, CBHI and CBHII, and two endoglucanases, EGI and EGII (formerly EGIII,) has been confirmed by cloning of the corresponding genes (Shoemaker et al. 1983, Teeri et al. 1983, Penttila et al. 1986, Chen et al. 1987, Teeri et al. 1987, van Arsdell et al. 1987, Saloheimo et al. 1988).

It is known in the art that the different types of cellulolytic enzymes mentioned above attack different parts of the cellulose molecule, and that cellulose hydrolyzation by a cellulase mixture is the result of synergy between its components. Therefore, if total hydrolysis of a cellulose substrate is aimed at, it is generally required that the cellulase mixture contain β- glucosidases, cellobiohydrolases as well as endoglucanases. As mentioned above, Trichoderma reesei produces such enzyme mixtures.

It is also known that the cellulase enzymes belonging to the same class are mutually differ as regards their activities towards some of the cellulosic substrates. For example, CBHII catalyze hydrolysis of 3-glucan whereas CBHI is inactive .toward that substrate.

The cellulase enzymes usually consist functionally of two different parts, viz. a core and a tail, which are interconnected by an intermittent part (known as the linker). The active cen of the enzyme is located in the core. The function of the tail consists mainly of its capabili to attach the enzyme to an insoluble substrate. Thus, if the tail is removed the activity of t enzyme toward macromolecular and crystalline substrates can be substantially decreased.

By way of a general definition, the name "endoglucanases" is assigned to enzymes that catalyze random hydrolysis of β- 1-4 glycosidic bonds between glucose units of cellulose polymers. The two major Trichoderma endoglucanases, EGI and EGII, contain about 500 t 600 amino acids and their molecular weights are about 50 to 60 kDa. Also the cellobiohyd lases are similar in size. These kinds or rather bulky molecules may have difficulties in penetrating some fibrous substrates whose adjacent polysaccharide chains are aligned and located close to each other. Such substrates are represented by fibrous materials of great economic values, such as cellulose pulp. Therefore, endoglucanases of a low molecular weight have been of an increasing interest during the last years.

Hakansson et al. (1978) have purified a small endoglucanase from culture filtrates of T. reesei. This enzyme has a size of about 20 kDa, a neutral pi and, unlike the major cellulases, it does not contain carbohydrate moieties. Hakansson et al. found the enzyme to be present in the culture medium in very small amounts. Small endoglucanases of similar properties have also been isolated by Gong et al. (1979) and Ulker and Sprey (1990). However, although the molecular weight of the endoglucanase isolated by Hakansson and partially sequenced by Stahlberg in 1991 is rather low, the molecular configuration of the enzyme is not advantageous as far as enzymatical applications are concerned. The molecule appears not to contain a linker domain and a cellulose binding domain (CBD) but only a co domain. The cellulose binding domain and a linker region, allowing for its flexible separati from the catalytic core, are essential features of true cellulases capable of efficient attachm to the substrate.

The PCT Application No. PCT/US91/07276 discloses an endoglucanase enzyme, called EGIII, derived from Trichoderma. The molecular size of the EGIII is 23 to 28 kDa, its p optimum is 5.5 to 6.0 and the pi 7.2 to 8.0. From the sequence data of EGIII, it is appare that said enzyme is the same as the one isolated by Hakansson and sequenced by Stahlberg and that it does not contain the linker and CBD domains.

Known in the art are also small endoglucanase enzymes isolated from other microorganism Thus, a gene coding for a polypeptide homologous to the short amino acid sequence availa from the protein described above has been isolated from the fungus Aspergillus aculeatus (Ooi et al. 1990). The PCT Patent Application No. PCT/DK91/00123 describes an endoglucanase derived from the fungus Humicola insolens. The size of the polypeptide molecule is 43 kDa and its isoelectric point is 5.1. The use of the enzyme for treatment of cellulose-containing fabrics is suggested.

Nothing has so far been reported on the existance of a small size, true Trichoderma endogl canase having cellulose binding regions. It has been a general conception that the cellulase system of Trichoderma consists of at least two CBH:s and two EGs and additionally of the EGm which lacks a CBD.

Isolation and manipulation of the cellulase genes is very important for the various commer¬ cial uses of enzymes and of the organisms producing them. Isolation of hydrolase genes fr eukaryotes has been a task demanding either extensive studies on the corresponding enzym or the laborous differential hybridization protocols.

Summary of the invention

It is an object of the present invention to provide a novel endoglucanase enzyme of low molecular weight and having a suitable configuration for enzymatical applications. This invention provides an endoglucanase enzyme derived from Trichoderma reesei which unglycosylated form) has a molecular weight of about 20 to 25 kDa and contains 242 amin acids (the mature protein contains less amino acids that that depending on the signal seque cleavage site), some 70 % of which are located in the core region, whereas roughly one si of the amino acids is in the linker, taking an extended conformation, and one sixth in the

CBD domain. This distribution of the amino acid residues within the molecule gives evide of it having an elongated, "wormish" form in comparison to other cellulases, which facilitates penetration between adjacent molecules of fibrous cellulosic substrates. Being different in structure and activity, the enzyme complements the cellulolytic enzyme mixtur acting in synergy, as the Examples below will show.

Another object of the invention is to provide a simple and rapid method for isolation of endoglucanase genes by function. In fact, the method described in more detail below, mak it possible to isolate any hydrolytic enzyme gene, such as genes coding for cellulases (for instance endoglucanases and cellobiohydrolases) and hemicellulases (for instance xylanases and mannanases), without previous knowledge of the corresponding proteins. In this connection it should be pointed out that before this invention there did not exist any data o protein level which would have suggested the existence of the novel endoglucanase describ herein. This fact is already indicative of the unusual properties resulting in its disregard in the biochemical characterization of the cellulase mixture produced by Trichoderma.

According to the present method, an expression cDNA library is made from the organism choice into a yeast expression vector. Yeast transformants are screened on plates containin the substrate of the desired activity. Using our earlier finding (Penttila et al. 1987, 1988) t yeast produces and secretes the major cellulases of T. reesei in active form, the enzymatic activities can be visualized on substrate plates.

In the following description of the present invention, the novel gene coding for the novel endoglucanase enzyme is characterized as is its transfer into, and the expression thereof, in suitable hosts, such as fungi of the genus Trichoderma, in particular various Trichoderma reesei strains, and yeasts, such as Saccharomyces cerevisiae. The invention also provides yeast and fungal strains transformed with the gene coding for novel endoglucanase enzyme. Finally, applications of the enzyme are suggested.

Brief description of the drawings

Fig. 1 shows the nucleotide sequence of the gene egl5 coding for the novel enzyme, EGV.

Fig. 2 A shows the cellulose binding domains and Fig. 2B linker regions of EGV compare with the same domains and regions of the other Trichoderma cellulases. In Fig 2B, the se and theronine residues have been boxed. Fig. 3 shows the endoglucanase gene egl5 integrated into plasmid pAJ401 resulting in plasmid pAS4.

Fig. 4 shows the endoglucanase gene egl5 integrated into plasmid pMLO16del5 resulting i plasmid pAS16.

Fig. 5 shows the structure of plasmid pMLOlό, Fig. 6 shows the structure of plasmid pMLO16del5(ll),

Figs. 7a to 7d depicts the construction of the egl5 expression plasmid pALK956, Fig. 7a a indicating the structure of plasmid pAS13,

Fig. 8 indicates the relative activity of the novel endoglucanase enzyme as a function of the pH, Fig. 9 shows the pH stability of the enzyme, and

Fig. 10 shows the introns and coding sequence of the eg 15 gene.

Detailed description of the invention

In the following description, the following abbreviations and definitions are used:

Abbreviations:

aa, amino acid(s); bp, base pair(s); CBD, cellulose-binding domain; CBH, cellobiohydrolas cbh, gene coding for CBH; CMC, carboxymethyl cellulose; EG, endoglucanase; egl, gene coding for EG; HCA, hydrophobic cluster analysis; HEC, hydroxyethyl cellulose; kb, kilo¬ base^); kDa, kilo dalton(s); MUC, 4-methyl-umbelliferyl β-D-cellobioside; MUL, 4-methy umbelliferyl β-D-lactoside; NMR, nuclear magnetic resonance; PCR, poiymerase chain reaction; PGK, 3-phosphoglycerate kinase gene of Saccharomyces cerevisiae; pi, isoelectric point.

Definitions:

Within the scope of the present invention, the term "cellulase" is used as a collective term which encompasses enzymes catalyzing reactions which participate in the degradation of insoluble cellulose or cellulosic substrates to soluble carbohydrate. "Cellulase" is known in the art to refer to such a group of enzymes. As mentioned above, for hydrolysis of cellulos to glucose, three cellulase enzymes (three types of cellulase enzyme activity) are needed: ra domly cleaving endoglucanases (l,4,- 3-D-glucan glucanohydrolase, EC 3.2.1.4) which usually attack substituted soluble substrates; cellobiohydrolase (l,4-_/8-D-glucan cellobiohydr lase, EC 3.2.1.91) which is capable of degrading crystalline cellulose but has no activity towards derivatized cellulose and 3-glucosidase (β-D-glucoside glycohydrolase, EC 3.2.1.2 which degrades cellobiose and cello-oligosaccharides to yield glucose. Each of the three ma types of enzymes listed above occurs in multiple forms. For example, two immunologically distinctive cellobiohydrolases, CBHI and CBHII are known. In addition, at least two distinc endoglucanases are known. Synergistic action between some of these enzymes has been demonstrated. "Cellulase activity" is synonymous with cellulolytic activity.

Enzymes having "endoglucanase activity" are, within the scope of the present invention, enzymes which will catalyse the hydrolysis of internal 3-1,4-linkages of cellulose.

By "enzyme preparation" is meant a composition containing enzymes which have been extracted from (either partially or completely purified from) the microorganisms (for instan the fungi) producing them. The term "enzyme preparation" is meant to include a compositio comprising medium used to culture such microorganisms and any enzymes which the micro organisms have secreted into such medium during the culture.

"Culture medium" denotes a medium previously used to culture a fungi ("spent" culture medium), such culture medium containing enzymes which the fungi have secreted into the medium during the culture. The culture medium can be used as such or as partially or completely purified, concentrated, dried or immobilized.

By "hybridization " are meant conditions, under which all the different forms of DNA sequences hybridize to the DNA sequence encoding for the Trichoderma enzyme having endoglucanase activity, the molecular weight of the unglycosylated form of said enzyme being about 20 to 25 kDa and containing 242 amino acids (the mature protein having less amino acids).

"Gene" denotes a DNA sequence containing a template for a RNA poiymerase. RNA that codes for a protein is termed messenger RNA (mRNA).

It is well known that mutations occur in genes and can cause changes in the amino acid sequence of the encoded polypeptide. Changes can also be introduced by genetic engineeri techniques. As used herein, the term egl5 gene includes all DNA sequences homologous w the sequence herein disclosed for egl5 and encoding polypeptides with the fuctional or strucmral properties of the about 20 to 25 kDa polypeptide. It is known in the art that cellulases lacking the linker and CBD regions still exhibit catalytic activity towards the β- 1,4-glucosidic linkage, and thus a smaller core polypeptide is also included in the denotion egl5. Sequences artificially derived from this gene but still encoding a polypeptide with the desired fuctional or strucmral properties are also included and encompassed by the express "functional equivalents".

A cloning vehicle or a vector is a plasmid or phage DNA or other DNA sequence (such as linear DNA) which provides an appropriate nucleic acid environment for the transfer of a gene of interest into a host cell. The cloning vehicles of the invention may be designed to replicate autonomously in prokaryotic and eukaryotic hosts. In Trichoderma, the cloning vehicles generally do not autonomously replicate and instead, merely provide a vehicle for the transport of the gene of interest into the Trichoderma host for subsequent insertion into the Trichoderma genome. The cloning vehicle may be further characterized by one or a s number of endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without loss of an essential biological function of the vehicle, and int which DNA may be spliced in order to bring about replication and cloning of such DNA. The cloning vehicle may further contain a marker suitable for use in the identification of cells transformed with the cloning vehicle. Markers, for example, are tetracycline resistanc or ampicillin resistance for E. coli and for example phleomycin resistance or acetamidase f Trichoderma. The word "vector" is sometimes used for "cloning vehicle. " Alternatively, such markers may be provided on a cloning vehicle which is separate from that supplying t gene of interest.

A vehicle or vector similar to a cloning vehicle but which is capable of expressing a gene o interest which has been cloned into it, after transformation into a desired host, is called an expression vector. In a preferred embodiment, such expression vehicle provides for an enhanced expression of a gene of interest which has been cloned into it, after transformatio into a desired host.

The gene of interest which is provided to a fungal host as part of a cloning or expression vehicle integrates into the fungal chromosome. Sequences which derive from the cloning vehicle or expression vehicle may also be integrated with the gene of interest during the integration process.

The gene of interest may preferably be placed under the control of (i.e., operably linked to) certain control sequences such as promoter sequences provided by the vector (which integra with the gene of interest). If desired, such control sequences may be provided by the fungal host's chromosome as a result of the locus of insertion.

A nucleic acid molecule, such as DNA, is said to be "capable of expressing" a polypeptide it contains expression control sequences which contain transcriptional regulatory informatio and such sequences are "operably linked" to the nucleotide sequence which encodes the polypeptide. Method of isolating genes

Bacterial cellulase genes have widely been isolated by transforming genomic libraries into coli and screening activities on cellulose-containing plates (reviewed by Beguin et al. 1987 This approach relies on the functionality of promoter sequences from other prokaryotes in coli and is not applicable to eukaryotes. Furthermore, eukaryotic genes, such as the T. re ΕGV described here, contain introns which cannot be excised in E. coli and thus disturb t reading frame. Moreover, the Trichoderma cellulases cannot generally be expressed in E. coli in active form even if expressed from cDNA coupled to bacterial expression signals. Traditionally fungal cellulase genes have been cloned using either differential hybridizatio antibodies raised against the corresponding enzymes or hybridization with oligonucleotide probes based on the protein sequence of the enzymes (Beguin et al. 1987). All these meth are laborous and demand a lot of time and previous knowledge of the corresponding enzymes.

With the method according to the present invention, genes coding for new activities can be easily isolated without any previous knowledge of the protein. According to the invention, fungal strain (e.g. Trichoderma) is cultivated on a culture medium which will induce enzy production. Such culture medium typically contains cellulosic substrate, if endoglucanase production is aimed at. After cultivation, the mRNA of the strain is isolated and the corresponding cDNA is formed. cDNA made from the organism of interest is cloned into yeast vector to construct an expression gene library in yeast, for instance Saccharomyces cerevisiae. The genes of the fungus are then expressed under any suitable promoter provid sufficient expression level, such as the yeast promoter PGK. The enzyme, e.g. endoglucanase, is extracellularly secreted and the colonies producing the desired enzymes, e.g. the endoglucanase, can be identified on the basis of their production of enzyme activit

Screening can be effected with activity plate assays. Thus, according to one preferred embodiment of the present invention, the endoglucanase gene is isolated by plating the expression library onto plates containing barley β-glucan as substrate. After growth the cel are washed away and the plates are stained with congo red to reveal the hydrolysis halos. to 50 % of the clones giving halos may contain endoglucanase. The genes coding for different endoglucanases can be identified by analyzing the clones.

The expression gene library can also be constructed by using some other yeast promoter which will provide a weaker level of expression. If it is to be expected that the enzyme is deleterions to the yeast, the inducible GALl promoter would be recommendable. It is also possible to use the endoglucanase 's own promoter and, for the purpose of isolating the ge a chromosomal gene library can, in some cases, be used. The gene library can also be constructed in a single copy plasmid. Also any other yeast strain with established trans¬ formation procedures can be used as a host, because their secretion capabilities are usually even higher than that of Saccharomyces.

In summary, the invention comprises the steps of

- enriching the mRNA pool of a fungal strain, e.g. Trichoderma, producing endoglucanase activity in respect of the mRNA of the endoglucanase by cultivating t strain in conditions which will induce the endoglucanase production of said strain,

- isolating mRNA from the strain,

- preparing cDNA corresponding to the isolated mRNA,

- placing the cDNA thus obtained in a vector under the control of a suitable promoter

- transforming the recombinant plasmids into a yeast strain which naturally does not produce significant amounts of the endoglucanase in order to provide an expression library,

- cultivating the yeast clones u us obtained on a cultivation medium in order to expres the expression library in the yeast,

- separating the yeast clones producing endoglucanase from the other yeast clones, - isolating the plasmid-DNA of said separated yeast clones, and,

- if desired, sequencing the DNA in order to determine the DNA sequence coding for the endoglucanase. Endoglucanase V and the gene esl5

The gene egl5 isolated according to d e above method was sequenced according to conven¬ tional methods. The DNA sequence of eg 15 is shown in Figure 1 and also indicated in SE ID NO. 1.

The gene eg 15 codes for a previously unknown protein of 242 amino acids, me amino acid sequence of which is depicted in SEQ ID NO. 2. Interestingly, this protein contains the tw conservative domains found in all Trichoderma cellulases, namely the cellulose-binding domain (CBD) and the linker region that connects the CBD to the catalytic core domain. T approximate regions comprising these domains are indicated in Figure 1, the linker region being the part of the sequence marked with the letter B, whereas the cellulose binding domain is marked with the letter A. The putative N-glycosylation site is marked with an asterisk. At the beginning of the protein a 17 amino acid long signal sequence (Met-Lys-Al Thr-Leu-Val-Leu-Gly-Ser-Leu-Ile-Val-Gly-Ala-Val-Ser-Ala), which is underlined in Figure can be predicted. If the signal sequence cleavage occurs at this position, die mature protein consists of 225 amino acids and has a calculated molecular weight of 22.799 KDa.

The core of the endoglucanase is separately depicted in SEQ ID NO. 3. It would appear th the core of the novel endoglucanase is primarily responsible for the cellulolytic activity of t novel enzyme. Thus, it is conceived that an endoglucanase enzyme product may in principl comprise the polypeptide of the core domain only. However, the surprising enzymatic properties described below are probably attributable to a combination of me above three regions and domains, and they will therefore best be obtained if the protein comprises all three parts.

It is believed that the predicted 17 aa signal peptide indicated in Figure 1 can be substituted by another suitable signal peptide possibly of a different length. Such a signal sequence should typically comprise a positively charged amino acid at the beginning followed by a stretch of hydrophobic amino acids. Depending on the signal sequence cleavage site in vivo and die possible proteolytic processing occurring frequently in cellulases, the molecular weight of the active polypeptide may vary somewhat and the novel endoglucanase is therefore referred to as having a molecular weight in unglycosylated form of about 20 to kDa.

In its O- and N-glycosylated form the enzyme can be significantly bigger having apparent molecular weights of 35 kDa or even much higher when produced in the yeast

Saccharomyces. Furthermore, cellulases frequently undergo drastic proteolytic cleavage which removes the CBD (and linker) regions and consequently the size of EGV in fungal culture medium can be even about 115 kDa in unglycosylated form.

While modelling protein conformations from first principles is not possible, the high sequence similarity between fungal CBDs warrants the construction of a homology model (Sali et al , 1990). The feasibility of modelling side chain conformations has been demonstrated in similar cases (Blundell et al , 1988, Heiner et al , 1993). Modelling of m EGV CBD revealed some interesting differences compared to the known structure of the CBHI CBD. This wedge-shaped domain seems less sharp in EGV and tiiere are some differences in main chain and side chain conformations and in hydrophobic properties in areas known to be important for binding of the CBHI CBD onto the cellulose surface or f the full activity of the CBHI enzyme against crystalline cellulose. Preliminary binding data indicate mat the EGV CBD is able to bind to cellulose.

The protein belongs to a new family K of cellulases together with the endoglucanase B of Pseudomonas fluorescens and die endoglucanase V of Humicola insolens as smdied by hyd phobic cluster analysis by Henrissat and Bairoch (1993). This strongly suggests that EGV structurally different from all Trichoderma cellulases characterized so far. Based on this, i would also appear that there are catalytic differences between the present enzyme and me other cellulases. The fact that EGV is a true endoglucanase was confirmed by Η-NMR spectroscopy, which showed that the internal 0-1,4-linkages were hydrolysed by EGV whe barley 3-glucan (a soluble glucose polymer containing 0-1,4- and /3-1,3-linkages) was used substrate.

Thus, for instance, as evidenced by Example 11 below, me novel endoglucanase appears t work synergetically with the known endoglucanase EGII on hydroxyethyl cellulose. Expression of the gene esl5

Once the vector or DNA sequence containing die construct(s) is prepared for expression, t DNA construct(s) is introduced into an appropriate host cell by any of a variety of suitable means, including transformation as described above. After the introduction of the vector, recipient cells are grown in a selective medium, which selects for the growth of transforme cells. Expression of the cloned gene sequence(s) results in the production of the desired protein, or in the production of a fragment of this protein. This expression can take place i a continuous manner in the transformed cells, or in a controlled manner.

Expression of the gene can be obtained in any fungus with developed transformation and expression methods.

Trichoderma is an especially useful and practical host for the syntiiesis of the enzyme preparations of the invention because Trichoderma is capable of secreting protein at large amounts, for example, concentrations as much as 40 g/L culture fluid have been reported; the homologous Trichoderma cbhl promoter provides a very convenient promoter for expression of genes-of-interest because it is a strong, single copy promoter which normally directs the synthesis of up to 60 % of the secreted protein from the Trichoderma host; the transformation system is highly versatile and can be adapted for any gene of interest; the Trichoderma host provides an "animal cell type" high mannose glycosylation pattern; and culture of Trichoderma is supported by previous extensive experience in industrial scale fermentation techniques. In addition, several promoters active on glucose medium can be used, which enable the production of the enzyme essentially free from other cellulases.

Expression of the protein in the Trichoderma hosts requires the use of regulatory regions functional in such hosts. A wide variety of transcriptional and translational regulatory sequences can be employed, since Trichoderma generally recognize eukaryotic host transcriptional controls, such as, for example, those of other filamentous fungi. Such contr regions may or may not provide an initiator methionine (AUG) codon, depending on whem the cloned sequence contains such a methionine. Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis in the host cell. According to the invention the DNA sequence encoding EGV can be transformed into Trichoderma and expressed, for example under the strong cbhl promoter, as described in EP-A 244,234 and US 5,298,405, or other promoter functional in Trichoderma. The DN sequence coding for EGV can be integrated into the general expression vector pAHMHO. The transformation can be done as a cotransformation using two circular plasmids, me selection marker being located in one of the plasmids and me DNA sequence encoding egl in the other, or the selection marker and the DNA sequence encoding the eg 15 can be loca in the same plasmid, or linear fragments can be used in me transformation. Possible selec markers are, for instance, trpC or argB from Aspergillus nidulans or argB or pyr4 from reesei or amdS from A. nidulans or trpl from Neurospora crassa or phleomycine or hygr mycine resistance markers from bacterial origin (EP-A 244,234, US 5,298,405, and EP-B 539,395 and Ulhoa et al., 1992, Transformation of Trichoderma species with dominant selectable markers, Curr. Genet 21:23-26) or other selection marker shown to function in Trichoderma in future (Karhunen et al. 1993, High frequency one-step gene replacement i Trichoderma reesei I, Endoglucanase I overproduction, MGG, 241: 515-522, and Suomin et al., 1993, High frequency one-step gene replacement in Trichoderma reesei II, Effects deletions of individual cellulase genes. MGG, 241: 523-530.

To construct a Trichoderma strain producing endoglucanase V as die main cellulolytic enz me it is possible to construct Trichoderma strains diat do not produce die endoglucanases I and II or all other cellulolytic enzymes: endoglucanase I and II and cellobiohydrolase I an II. The desired cellulolytic genes can be made deficient (EP-A 244,234, US 5,298,405, Karhunen et al. (1993) and Suominen et al. 1993). If genes are expressed under the cbhl promoter the expression is repressed by glucose and thus the strains must be grown on cellulose-containing medium.

Alternatively, it is possible to construct Trichoderma strains expressing EGV under glucos promoter. This means that the Trichoderma strains expressing EGV can be grown on gluc containing medium. Possible glucose promoters are, for example, glucose derepressed cbh promoter of the plasmid pMLO16del5(ll) (et al. , 1992) and the promoter of the cDNAl gene (Nakari et al , 1992) or other glucose promoters. According to the invention, there is also provided a method for producing in fungal and y hosts, such as the yeast Saccharomyces and filamentous fungi, such as Trichoderma, an enzyme preparation having an endoglucanase activity stemming from an endoglucanase enzyme, die molecular weight of which (in unglycosylated form) is 20 to 25 kDa.

Further, if desired activities are present in more than one recombinant host, such preparations can be isolated from the appropriate hosts and combined prior to use in the method of the invention.

Enzyme preparations

To obtain die enzyme preparations of the invention, containing elevated levels of the EGV, the recombinant hosts described above having the desired properties (that is, hosts capable expressing the novel endoglucanase enzyme) are cultivated under suitable conditions (cf. above), die desired enzymes are secreted from the host into me culture medium, and die enzyme preparation is recovered from said culture medium by metiiods known in the art.

As mentioned above, me enzyme preparation can be produced by cultivating the fungal stra in conditions where the regulatory regions directing endoglucanase expression are operatin such as on a glucose-containing medium if me yeast PGK or Trichoderma glucose promote are used. Thus, if endoglucanase V is expressed under glucose promoter, the Trichoderma strains can be grown on, e.g., glucose minimal medium (Penttila et al, 1987) or other glucose containing medium, for example Bacto-Peptone 5 g/1, Yeast extract 1 g/1, KH₂PO₄ g/1, (NH₄)₂SO₄ 4 g/1, MgSO₄ 0,5 g/1, CaCl₂ 0,5 g/1 and trace element FeSO₄"7H₂O 5 mg/1, MnSO₄.H₂O 1,6 mg/1, ZnSO₄.7H₂O 1,4 mg/1 and CoCl₂.6H₂O 3.7 mg/1, pH 5.0 - 6.0.

The enzyme can be produced also in other conditions, such as on Solca floe cellulose, if th Trichoderma cbhl promoter is used, or on a galactose-containing medium, if the yeast galactose- inducible promoter is used. The cellulose-containing cultivation medium may, for instance, comprise, 6 % Solca floe cellulose (BW40, James River Corporation, Hackensac NJ), 3 % distiller's spent grain, 0.5 % KH₂PO₄, 0.5 % (NH4)₂SO₄, and 0.1 % struktol as antifoaming agent (struktol SB 2023, Schill & Seilacher, Hamburg, FRG). Trichoderma strains are sensitive to glucose repression and require an inducer (cellulose, lactose or sophorose). The pH should preferably be kept at approximately pH 5 to 6 by the addition phosphoric acid or ammonia and the temperature at 30 °C during d e cultivation.

The enzyme preparation is recovered from the culture medium by using memods well kno in the art. However, the enzyme preparations of the invention may be utilized directiy fro the culture medium with no further purification. If desired, such preparations may be lyop lized, immobilized or the enzymatic activity otherwise concentrated and/or stabilized for storage.

If desired, the expressed endoglucanase protein may be further purified in accordance with conventional conditions, such as extraction, precipitation, chromatography, affinity chroma graphy, electrophoresis, or the like.

Applications of the novel enzyme

The catalytic core of the novel enzyme is the smallest of fungal or bacterial cellulases characterized. Therefore the enzyme and die enzyme preparations according to die inventio have application in the treatment of pulp and paper and in the textile industry. Furthermor the enzyme can be used in the fodder industry. The properties of the novel endoglucanase unexpected for a endoglucanase on basis of general knowledge.

Being a 3-glucanase, the novel enzyme can be used for hydrolyzation of the 3-glucan of barley. As a result, the viscosity of the fodder is lowered and the nutritional value of d e fodder is improved.

As evidenced in Example 8, the pH optimum of die enzyme is higher than tiiose of the oth endoglucanases produced by strains of the species Trichoderma. This favorable pH range c be utilized in many ways. One preferred application is for removing colour from denim jeans; in acidic pH, reabsorption of the colour occurs, but at neutral pH there is much less reabsorption. Another preferred embodiment comprises deinking. Normally, the pH of a slurry of water and newsprint is about 5.5 to 6.0 and therefore the novel enzyme can be us without any need for adjustment of the pH. On the other hand, coated paper contains filler and pigments which will raise the pH of an aqueous paper slurry formed therefrom. If the pH of the slurry is lowered by adding mineral acid, at least some of the suspended or dissolved fillers and pigments may precipitate, e.g. in die form of calcium sulphate.

The small size and d e advantageous pH range of the novel enzyme make it possible to use for treating recycled fibre in order to improve the technical properties thereof. The enzyme also applicable for improving pulp drainage.

The invention is described in more detail with the aid of the following non-limiting examples.

In the examples, the following strains and vectors were employed: E. coli strains PLK-F', pBluescript SK^", and XL- 1 -Blue (Stratagene) were used as hosts for plasmids and PLK-F' a host for the cDNA library. The following plasmids were used: pASll, pAS13, pALK487 and pALK183. The T. reesei strain QM9414 was used as a source of RNA for cDNA preparation and Northern analysis. T reesei ALKO2221 and ALKO3524 were used as host for ΕGV expression. S. cerevisiae strain DBY746 ( his3 1 leu2-3 leu2-112 ura3-52 trp 1-2 cyh^r cir⁺) was used as a host for the expression library. Strain MD40-4c ( ura2 trpl leu2- leu2-112 his3-ll his3-15) was used as a host for the plasmids ρMP311, pMS3, pMPll and pMP29 carrying egll, egl2, cbhl and cbh2 genes of T. reesei, respectively (Penttila et al. 1987, 1988). The yeast expression vector pFL60 (Minet and Lacroute 1990) containing the constitutive yeast PGK promoter and terminator, URA3 marker gene and me 2 micron plasmid replication origin was kindly provided by Dr. M. Minet, Centre de Genetique Moleculaire, C.N.R.S., France. Example 1

Isolation of endoglucanase gene by expression in yeast and hydrolytic properties of the yeast

T. reesei strain QM9414 was cultivated in a 10 liter fermentor at 28 °C and pH 4.0 for 42 hours. The cultivation medium used to induce hydrolytic enzyme production contained 2 % Solka floe cellulose, 1 % distiller's spent grain, 0.2 % Locust bean gum -galactomannan (Serva), 0.5 % KH₂PO₄ and 0.5 % (NH₄)₂SO₄. After 42 hours of growth, lactose (Sigma), Birke 150 acetylglucuronoxylan and Oat spelt arabinoxylan were added in an amount of 0.1 % each and the cultivation was continued for further 24 hours.

Total RNA from the T. reesei strain was isolated as described by Chirgwin et al. (1979), a the poly (A) ⁺ fraction was separated by chromatography through oligo(dT)-cellulose (BRL). cDNA, synthesized by die ZAP-cDNA synthesis kit (Stratagene), was ligated to d e EcøRI- Xhol cut plasmid pAJ401. Plasmid pAJ401 was derived from plasmid pFL60 (Minet and Lacroute 1990) by changing die two cloning sites EcoRI and Xhol between die yeast PGK promoter and terminator into die reverse orientation. Transformation of E. coli strain PLK- by electroporation (Bio-Rad) according to die manufacturer's instructions yielded a library 3.5 x IO⁴ independent clones. Plasmids were isolated from the pool of E. coli transformants and transformed into S. cerevisiae strain DBY746 by electroporation (Bio-Rad) according t the manufacturer's instructions. Electroporation with 7 μg of plasmid DNA yielded a librar of 8 x 10⁴ yeast transformants.

1.2 x 10⁵ yeast cells were plated on barley β-glucan-containing plates to a density of 2000 colonies / 85 mm plate and grown at 30 °C for 3 days. Colonies were replicated and die original plates stained witii Congo Red. Unstained areas around yeast colonies indicate hydrolysis of the substrate to oligosaccharides. Colonies showing activity were picked up from the replica plates and purified on new activity plates. Plasmids were recovered from t purified clones and analysed by restriction enzyme digestions. 20 clones gave a similar pattern of bands which was clearly different from the earlier isolated cellulase genes of T. reesei. Transformation of the plasmids back to yeast confirmed that the activities were caused by cDNA inserts. One of these plasmids, pAS4 (cf. Figure 3), was smdied further. The inser the pAS4 plasmid was named eg 15 and die corresponding protein EGV.

egl5 cDNA was sequenced from both strands of the original pAS4 plasmid using the Sang dideoxynucleotide method, T7 DNA poiymerase (Pharmacia) and sequence specific primer

The sequence obtained is shown in SEQ ID NO.

The chromosomal egl5 gene was isolated from a T. reesei cosmic library (Mantyla, A. et a Curr. Genet. 1992, 21 All-All) by using the egl5 cDNA as a probe. About 6 kb Hindlll fragment was subcloned to pBluescript SK^", resulting in plasmid pAS13 (Fig. 7a). The introns and coding sequence of egl5 gene are shown in Figure 10 (SEQ ID NO. 11).

The activities of the yeast strain DBY746 carrying the pAS4 plasmid were smdied by plate assays and they were compared widi the activities of the yeast strains producing CBHI, CBHII, EGI and EGII.

Hydrolytic activities produced by recombinant yeast cells were detected on SD plates con- taining 0.1 % barley 0-glucan (3-D-l,3-l,4-glucan, viscosity 20-30 c.s.; Biocon, UK,

Sherman 1991) or hydroxyethyl cellulose (HEC, Fluka, Switzerland, product 54290). After growth the plates were stained witii Congo Red (Merck) as described by Penttila et al. (1987) to reveal the hydrolysis halos. Xylanase activity plates containing 0.2 % of a Remaz Brilliant Blue-dyed derivative of xylan (RBB-xylan, Sigma) needed no further treatment. Activities against synd etic substrates, 4-methylumbelliferyl 3-D-cellobioside (MUC; Koch-

Light, UK) or 4-methylumbelliferyl β-D-lactoside (MUL; Lambda Probes & Diagnostics, Austria) were detected as described by Penttila et al. (1987).

The EGV protein showed a clear activity against β-glucan but the activity was lower than t activities of die strains producing EGI, CBHII or EGII (Table). However, the expression levels and the secretion efficiencies of foreign proteins in yeast may vary and dius it is not possible to draw any definite conclusions concerning die level of enzyme activity. Also, the pH on the plates is not optimal for EGV function. EGV shows some activity against hydroxyethyl cellulose (HEC) in plate assays. No activity was detected on plate assays towards RBB-xylan or the small synthetic substrates, methylumbelliferyl cellobioside (M or methylumbelliferyl lactoside (MUL).

Table 1 Hydrolytic activities of the yeast strains carrying the cellulase genes of

Trichoderma reesei. The extent of hydrolysis of the substrate was estimated visually and is indicated by 4- .

EGV EGI EGII CBHI CBHII 3-glucan + + + + + + + + + + - + + + +

HEC + + + + + + + - -

MUL - + + + - + + -

MUC - + + + - 4- -

RBB-xylan - + + + - - -

Example 2

Construction of endoglucanase expression vectors with truncated fragments of the chb promoter

The vector pMLOlό (Figure 5) contains a 2.3 kb cbhl promoter fragment (SEQ ID 4) starting at 5' end from the EcoRI site, isolated from chromosomal gene bank of Trichoder reesei (Teeri et al, 1983), a 3.1 kb BamHI fragment of the lacZ gene from plasmid pAN9 21 (van Gorcom et al., 1985) and a 1.6 kb cbhl terminator (SΕQ ID 5) starting from 84 b upstream from the translation stop codon and extending to a BamHI site at the 3' end (Sho maker et al. 1983; Teeri et al., 1983). These pieces were linked to a 2.3 kb long EcoRI- RvwII region of pBR322 (Sutcliffe, J.G. , 1979) generating junctions as shown in Figure 5. The exact in frame joint between the 2.3 kb cbhl promoter and the 3.1 kb lacZ gene was constructed by using an oligo depicted in Figure 5. A poly linker shown in Figure 5 was cloned into e single internal Xbal site in the chbl promoter for the purpose of promoter deletions. A short Sail linker shown in Figure 5 was cloned into the joint between the pBR322 and cbhl promoter fragments so that die expression cassette can be released from the vector by restriction digestion witii Sail and Sphl. Progressive unidirectional deletions were introduced to the cbhl promoter by cutting the vector with Kpnl and .XTzøl and using d Erase-A-Base System (Promega, Madison, USA) accordign to manufacturer's instructions. Plasmids obtained from different deletion time points were transformed into ie E. coli strai DH5α_ (BRL) by the method described in (Hanahan D, 1983) and die deletion end points were sequenced by using standard methods.

Example 3

Construction of vectors for expression of EGV in Trichoderma in glucose-containing medium

In order to produce EGV protein in Trichoderma reesei QM9414 strain essentially free of other cellulases in a medium containing glucose, die plasmid pAS16 (Fig. 4) was construc¬ ted. There, the egl5 cDNA was cloned under the truncated, glucose derepressed cbhl prom ter of the plasmid pMLO16del5(ll), generated as explained in Example 2. The plasmid contained a 1110 bp deletion in d e cbhl promoter beginning from the promoter internal polylinker and ending 385 bp before the translation initiation site (Fig. 5). The sequence of tiiis truncated promoter is provided as SEQ ID NO. 6. Plasmid pMLO16del5(ll) was diges ted witii the restriction enzymes Kspl and Smal. The vector part containing me glucose- derepressed cbhl promoter, the cbhl terminator and die pBR322 sequence was blunt-ended witii the Mung bean nuclease, dephosphorylated with Calf intestin alkaline phosphatase and ligated to the eg 15 cDNA fragment.

The yeast expression plasmid pAS4 was digested witii EcoRI and partially with Xhol to isolate the full-length egl5 cDNA. The ends of the cDNA were filled-in with the Klenow poiymerase enzyme and the fragment was ligated into me Smøl-cleaved vector pSP73 (Pro- mega). The resulting plasmid pASll was digested witii EcoRI and Xbal, filled-in with the Klenow poiymerase and ligated to the vector part of the expression vector pMLO16del5(ll). Twenty micrograms of the pAS16 plasmid were digested with EcoRI and Sphl, phenol- extracted, precipitated and transformed into Trichoderma reesei QM9414 together with thr micrograms of the plasmid p3SR2 (Hynes et al, 1983) containing the acetamidase gene according to Penttila et al, (1987).

The promoter of the cDNAl gene (Nakari et al, 1992) was also used to direct the synthes of the ΕGV protein on glucose-containing medium.

The promoter of the cDNAl gene was cloned from the chromosomal DNA by PCR using 5 'primer GGT CTG AAG GAC GTG GAA TGA TGG (SΕQ ID NO. 7) and the 3 'primer GAT GCA

TCG ATC GTC CGC GGG TTG AGA GAA GTT GTT GGA TTG ATC AAA AAG (SΕQ ID NO. 8). The underlined ATCGAT in the 3 'primer is a Clal site and the CCGCGG a Kspl site.

The egl5 cDNA and the cbhl terminator were cloned as one fragment from the plasmid pAS16 by PCR using the 5'primer GAG AGA CCG CGG TGA TCT TCC ATC TCG TGT CTT GCT AAC (SΕQ ID NO. 9) and the 3 'primer ATC GTG GAT CCA TTA TTA ACA CTT CGG TGG (SΕQ NO. 10). The underlined CCGCGG in the 5 'primer is a Kspl site.

Eight micrograms of both of the fragments were digested with the Kspl enzyme, purified fr agarose gel and ligated. The ligation mixture was extracted with phenol, precipitated and us instead of a plasmid in the Trichoderma transformation together with three micrograms of t p3SR2 plasmid.

The Amd⁺ transformants from the pAS 16 transformation were streaked twice onto plates containing acetamide (Penttila et al, 1987), and then cultivated on Potato Dextrose Agar plates (Difco) from which spore suspensions were made. EGV production was tested from 5 ml shake flask cultures carried out in minimal medium according to Penttila et al, (1987) except that the amount of glucose was 4 %, KH₂PO₄ 3 %, K₂PO₄ 0.8 %, (NH₄)₂SO₄ 0.2 % and the medium was supplemented with 0.2 % peptone. Glucose was added as 15 % solutio when necessary to keep the level above 1 % during the whole four days of the cultivation.

The culture supernatants of 55 transformants were analyzed for activity against barley β- glucan by the DNS-method (Zurbriggen et al, 1990). The spore suspensions of the three best EGV-producing clones (numbers 101, 79 and 19) were purified to single spore cultures on Potato Dextrose Agar plates. EGV production was analyzed again from these purified clones as described above. The best producing transfor- mant 101c was analysed by Southern blotting using conventional methods and the presence the expression casette in the genomic DNA was confirmed. Northern analysis showed that t egl5 gene was expressed from the constructs on glucose medium.

Example 4 Construction of EGV expression plasmid pALK956

The expression plasmid pALK956 (Figs. 7d) contains:

1) T. reesei egl5 gene fused to the cbhl promoter. A fragment containing the cbhl terminato was included after egl5 to ensure stop in the transcription.

2) E. coli hph (hygromycin B phosphotransferase; Gritz and Davies, 1983) as a marker gene for transformation. The gene was expressed from the T. reesei pki (pyruvate kinase; Schin- dler et al, 1993) promoter.

3) Elongated cbhl terminator as a flanking region to ensure stop in pki transcription and to target the expression cassette, together with the cbhl promoter fragment, to the cbhl locus.

The construction of pALK956 is shown in detail in Figs. 7a - 7d. For the construction, the plasmids pASl l, pAS13, pALK487 and pALK183 were used. The plasmid pASl l contains the egl5 cDNA (Fig. 1) and pAS13 contains the chromosomal egl5 gene (Fig. 10). The plasmid pALK487 contains the T. reesei cbhl promoter (the 2.2 kb Stwl - S cII fragment originally from the plasmid pAMHl lO; Nevalainen et al, 1991) and cbhl terminator (the 0. kb Avail fragment starting 113 bp before the stop codon of the cbhl gene; for the cbhl sequence, see Shoemaker et al, 1983). The plasmid pALK183 contains hph gene under the control of t e pki promoter. It was constructed from pRLM_ex30 (Mach et al, 1994) by changing the cbh2 terminator to 1.6 kb cbhl elongated terminator (Avail - BamHI fragment).

The exact fusion of the egl5 gene to the cbhl promoter was done by PCR. The 5 '-primer contained the last 26 nucleotides of the cbhl promoter including the SαcII site and the first nucleotides of the coding sequence of eg/5 (5'-CAATAGTCAACCGCGGACTGCGCATCA GAAGGCAACTCTGGTT; the Sacll site is underlined, egl5 sequence is bolded). The 3'- primer contained 21 nucleotides of egl5 sequence, including the BamHI site about 0.7 kb fr the beginning of egl5 sequence (5' -GGGCGTGGGATCCGTCTCTTG; the BamΑl site is underlined). The plasmid pAS13 was used as a template in the PCR reaction.

The 0.7 kb PCR fragment (filled in with DNA poiymerase I Klenow fragment and cut with BamHI), containing the exact link between the cbhl promoter and the egl5 gene, was ligate to RvwII - BamHI digested pASl 1 to obtain pALK951. The fusion and the PCR fragment were sequenced to ensure that no mistakes had occurred in the PCR amplification. Plasmid pALK955, containing the fusion of the egl5 to the cbhl promoter, was obtained by ligating EcoRI/Klenow - SαcII fragment from pALK951 between the cbhl promoter and terminator the plasmid pALK487 (RαmHI/Klenow - SαcII). The hph marker gene (under the control o the pki promoter) and die cbhl 3 '-flanking region (elongated terminator) were ligated to Stw cut pALK955 from pALK952 (Xhol - Hindlll fragment / Klenow) to construct pALK956.

The plasmid pALK952 was constructed from pALK183 by shortening the pki promoter compared to the promoter used in pALK183 and pRLM_ex30 (Notl/partial - .ΛTrøl, Klenow).

The 7.4 kb expression cassette from pALK956 can be removed with NotI digestion.

Thus, in summary, in the expression plasmid pALK956, the egl5 gene is fused to the cbhl promoter. The E. coli hph (hygromycin B phosphotransferase) gene is used as a marker for the transformations. The cbhl 3 '-flanking region (elongated terminator) is included to ensur stop in the pki transcription and to target the expression cassette, together with the promoter fragment, to the cbhl locus.

Example 5 Expression of EGV under the cbhl promoter in cellulase-inducing medium

The EGV expression plasmid, pALK956, was digested witii NotI, and the 7.4 kb fragment was purified from agarose gel. 2-3 μg of the linear fragment was transformed into T. reese strains ALKO2221 and ALKO3524 according to Penttila et al. (1987) with the modificatio described in Karhunen et al. (1993). ALKO2221 is a low protease mutant from T. reesei VTT-D-79125 (Bailey and Nevalainen, 1981), prepared in our laboratory (A. Mantyla). ALKO3524 is a strain derived from VTT-D-79125, where the cbh2, egl2 and egll genes h been deleted using the A. nidulans trpC (Yelton et al., 1984), A. nidulans amdS (Kelly an Hynes, 1985) and Streptoalloteichus hindustanus phleo^r (Mattern et al., 1987) marker genes respectively. The method of one-step gene replacement with a linear fragment and flanking regions of the corresponding cellulase locus is described in Suominen et al. (1993).

HygB4- transformants were selected on plates containing T. reesei minimal medium (Penttil et al., 1987) with 100 μg hygromycin/ml. Transformants were purified by single spore selection on selective medium and then cultivated on Potato Dextrose Agar. Purified transformants were grown in shake flasks in a medium containing 4 % whey, 1.5 % compl nitrogen source derived from grain, 1.5 % KH₂PO₄ and 0.5 % (NH₄)₂S0₄. Cultures were maintained at 30 °C and 250 rpm for 7 days. The culture supernatants were analyzed for activity against barley β-glucan at pH 6.3 by the DNS-method (Zurbriggen et al., 1990). Soluble protein was assayed by the method of Lowry et al. (1951) using bovine serum albumin as standard. The detection of the 67 kDa CBHI protein was done in SDS-PAGE followed by Coomassie Brilliant Blue staining. The results from the best EGV transformant and the corresponding host strains are shown in Table 2. In the EGV-transformants the β-glucanase activity measured at the optimum pH of EGV was enhanced about twofold.

Table 2. Expression of EGV under the cbhl promoter in cellulase-inducing medium

Strain Protein β-glucanase CBHI

(mg/ml) activity (BU/jnl) (+/-)

ALKO2221 7.1 1945 +

EGV/ALKO2221/11 5.2 3262 +

EGV/ALKO2221/47 5.5 3236 -

EGV/ALKO2221/31 3.5 3757 -

EGV/ALKO2221/68 3.9 3479 +

ALKO3524 9.3 3338 +

EGV/ALKO3524/27 5.3 6770 +

EGV/ALKO3524/28 7.6 7588 +

EGV/ALKO3524/31 7.0 6622 +

Example 6.

Enzyme preparation containing EGV protein obtained from yeast where egl5 gene was expressed.

Saccharomyces cerevisiae DBY 746 containing the pAS4 plasmid was grown in a bioreactor (Chemap LF 20, working volume 16 1) on a standard YPD medium. The inoculum (5 times 200 ml) was grown in shake flasks in selective synthetic complete medium without uracil. Cultivation conditions were: temperature 30 °C, pH controlled between 5.2 and 5.9, aeration about 15 1 min^'1 and cultivation time 45 h. The yeast cells were separated from the medium by centrifugation and the culture supernatant was concentrated 4-fold by ultrafiltration (PCI ES 625 membranes).

The enzymatic activity in the concentrate was assayed by standard methods using appropriat incubation times for the enzyme reaction against β-glucan (Zurbriggen et al., 1990a) and hydroxyethyl cellulose, HEC (IUPAC, 1987). The β-glucanase activity was 0.7 nkat ml^"1 and endoglucanase (HEC) activity less than 0.4 nkat ml^'1. No endoglucanase activity could be detected in culture filtrates of control cultivations of yeast missing the EG V gene (S. cerevisiae DBY 746 carrying the plasmid pAJ 401).

The purified EGV preparation can be obtained from the ultrafiltration concentrate by standa protein chromatography methods. The EGV protein can be bound to an anion exchanger res (e.g. Mono Q columns or DEAE Sepharose FF, Pharmacia) in low ionic strength buffer and at appropriate pH. The protein can be eluted out of the column using increasing gradient of NaCl (e.g. from 0 to 0.5 M in the buffer of binding). Alternatively, the impurities from the preparation of EGV can be removed by binding them in anion exchange resin at appropriate pH and ionic strength where EGV is not bound to the resin. Cation exchanger resins (e.g. Mono S columns or CM Sepharose FF, Pharmacia) can be used in analogous way by selecti buffers of appropriately low pH (e.g. pH 4 - pH 6). EGV can also be purified by gel permeation chromatography where it can be separated due to its small molecular size. The columns of various materials (e.g. Sephacryl S-100 HR or various types of Sepharose and

Superose, Pharmacia; Fractogel TSK HW-55, Merck) in e.g. phosphate or acetate buffers containing e.g. 0.05 - 0.5 M NaCl can be used. Hydrophobic interaction chromatography an various affinity chromatography methods may also be used.

Example 7

Enzyme preparation containing EG V protein obtained from Trichoderma reesei grown on glucose.

One of the best producing T. reesei QM9414 transformants (number QM/lOlc) was grown i a bioreactor (Chemap LF 20, working volume 16 1) on a medium of Mandels and Weber (1969) where Solka floe cellulose (10 g l^'1) was replaced by 20 g 1^"' of glucose and where the concentrations of other nutrients were correspondively doubled. The inoculum (5 times 200 ml) was grown in shake flasks in a medium containing 40 g l^"1 glucose and the adequate mineral salts for nutrients and buffering of the medium. Cultivation conditions were: temper ture 29 °C, pH controlled between 4.0 and 5.0, aeration about 15 1 min^"1 and cultivation time 93 h. During the cultivation glucose concentration in the fermentor was maintained above 5 l^"1 by adding continuously sterile glucose (40 g l^'1) solution. The mycelium was separated from the medium by centrifugation and the culture supernatant was concentrated 1.6 times ultrafiltration (PCI ES 625 membranes).

The clarified supernatant was first fractionated by hydrophobic interaction chromatography.

The pH of the sample was was adjusted to pH 6.0 and conductivity of the sample to the va corresponding to 10 mM sodium phosphate buffer, pH 6.0, containing 1.25 mol l^"1 (NH₄)₂ O₄. The sample was applied to a column (113 x 110 mm) of Phenyl Sepharose FF (Pharma cia), previously equilibrated with 10 mM sodium phosphate buffer, pH 6.0. containing 1.25 mol l^"1 (NH₄)₂SO₄. Elution was started by the equilibrating buffer followed by a linear dec¬ reasing gradient of ammonium sulphate from 1.25 M to 0 M. Fractions (each 450 ml) whic contained the major endoglucanase activity were combined, eluted at the end of the decreas gradient and by 10 mM phosphate buffer. The other adsorbed proteins were eluted by distil water and the column was washed with 6 M urea.

The enzyme preparation obtained in the first chromatographic step was equilibrated to 4 m sodium phosphate, pH 7.2 by gel filtration (Sephadex G-25 coarse). The equilibrated protei solution was applied to a column (113 x 190 mm) of DEAE Sepharose FF (Pharmacia), pre equilibrated with the same buffer. Elution was performed first with the equilibrating buffer remove unadsorbed proteins and thereafter by stepwise additions of sodium chloride to con¬ centration of 200 mM. Fractions (each 900 ml) which contained the endoglucanase and which eluted by 200 mM NaCl were collected and the fraction with the highest activity was concentrated by ultrafiltration (Amicon PM-10 membranes). The specific activity of the preparate was 360 nkat mg^"1 protein and purification factor of ca. 30 was obtained when compared to the supernatant of the fermentor liquid.

The preparate was further characterized by isoelectric focusing on PBE-94 anion exchange material (Pharmacia). The column was equilibrated by 25 mM imidazole-HCl buffer, pH 7. and elution was carried out by Polybuffer 74 (Pharmacia) - HCl buffer, pH 4.0 according to the manufacturer's instructions. EGV, measured by β-glucanase activity, eluted from the column at pH 6.6 - 7.2. Example 8 pH-Optimum of EGV

1.0 % solutions of barley β-glucan (Megazyme, Australia) was prepared in Mcllvaine buffer diluted one to four (corresponding ca. 50 mM citrate-phosphate buffer), in pH range from 3. to 8.0. Activity of an enzyme sample prepared as described in Example 7 was assayed using as substrate the β-glucan solutions prepared in the varying pH-values. The assay procedure was otherwise similar to the procedure of endoglucanase assay (IUPAC, 1987). Incubation time in the assay was 10 min at 50 °C, after which the enzyme reaction was terminated by boiling. Reducing sugar groups formed in the reaction were measured by DNS-reaction.

The pH-optimum of EGV was 6.0 - 6.5 (Figure 8).

Example 9 pH-Stability of EGV

An EGV sample was prepared as described in Example 7, except that the last concentration by ultrafiltration was omitted (activity 48 nkat ml, assayed at pH 6.3 against barley β-glucan analogously to endoglucanase assay, IUPAC, 1987). This sample was diluted (1 part per 2 parts of buffer) by 100 mM buffers of sodium acetate and sodium phosphate, prepared in different pH values. The diluted samples were incubated at 40 °C for 20 h, and the activity was assayed as described earlier. The pH of incubation was measured after the incubation.

More than 80 % of the original activity was observed in the samples incubated at pH range from ca. pH 5.4 to ca. pH 6.8. The relative recovered activity is presented in Figure 9.

Example 10 Hydrolysis of insoluble cellulosic substrates by EGV.

Avicel (Serva 14204) which is mainly crystalline cellulose and phosphoric acid-swollen amorphous (Walseth, 1952) cellulose were hydrolysed by the new endoglucanase enzyme (EGV) alone and in combinations with the previously known cellobiohydrolases of T. rees The preparation of EGV was obtained as described in Example 7. CBHI and CBHII were purified from culture filtrates of Trichoderma reesei and were pure proteins as judged by SDS-PAGE. As a control, the cellobiohydrolases were incubated without the addition of E

The reducing sugars liberated in the treatments were assayed using the DNS method and th reaction products were analysed by HPLC.

The substrates for the hydrolysis were prepared in 50 mM sodium citrate buffer, pH 5.8 in concentration of 10 g l^"1. EGV was dosed on the basis of activity against β-glucan at pH 5. (500 or 2000 nkat g^"1 substrate) and cellobiohydrolases (CBHI and CBHII) on the basis of protein (1.0 or 4 mg g^"1 substrate). The reaction mixtures were incubated for 20 h at pH 5. 40 °C after which the hydrolysis was terminated by boiling. The values for reducing sugars glucose assayed from the reaction mixture are presented in Tables 3 and 4. The enzyme dosage was 500 nkat g^"1 substrate for EGV and 1.0 g g^"1 substrate for CBHI and CBHII

(Table 3), and 2000 nkat g^"1 substrate for EGV and 4.0 g g^"1 substrate for CBHI and CBHI (Table 4). Duration of the hydrolysis was 20 h in both cases.

The major hydrolysis product of EGV was cellobiose but also cellotetraose was detected in the hydrolysate by HPLC. The strong synergy of EGV with CBHI in the hydrolysis of thes substrates can be clearly seen. Even though EGV released only small amounts of soluble sugars the enhancing effect on the cellulose hydrolysis by CBHI was remarkable.

Table 3. Reducing sugars liberated by cellulases from T. reesei in the hydrolysis of crystalline cellulose (Avicel) and amorphous (Walseth) cellulose

enzymes reducing sugars as glucose (mg ml^'1)

Avicel Walseth

EGV alone 0.00 0.01 (*) CBHI alone 0.09 0.16 CBHII alone 0.23 0.67

CBHI and EGV 0.15 0.25

CBHII and EGV 0.23 0.74

(*) determined by HPLC

Table 4. Reducing sugars liberated by cellulases from T. reesei in the hydrolysis of crystal ne cellulose (Avicel) and amorphous (Walseth) cellulose

enzymes reducing sugars as glucose (mg ml^"1)

Avicel Walseth

EGV alone 0.03 (*) 0.05

CBHI alone 0.35 0.49

CBHII alone 0.43 1.44

CBHI and EGV 0.43 0.71

CBHII and EGV 0.47 1.67

EGV (larger dosage 0.06 0.13 (•*)

(*) determined by HPLC

(**) dosed activity 5000 nkat g^"1 substrate

Example 11

Hydrolysis of soluble cellulosic substrates by EGV

HEC (hydroxyethyl cellulose, Fluka 54290) which is a soluble substituted cellulose polymer and barley β-glucan (Megazyme, Australia) were hydrolysed by the new endoglucanase enzyme (EGV) alone and in combinations with two previously known endoglucanases of T. reesei. The preparation of EGV was obtained as described in Example 7. Endoglucanases E and EGII were purified from culture filtrates of Trichoderma reesei and were pure proteins judged by SDS-PAGE. As a control, the endoglucanases were incubated without the additio of EGV. The reducing sugars liberated in the treatments were assayed using the DNS meth

The substrates for the hydrolysis were prepared in 50 mM sodium acetate buffer, pH 5.8 in concentration of 10 g l^"1. Endoglucanases were dosed on the basis of activity against β-gluc (dosage for each: 100 nkat g^"1 substrate) The reaction mixtures were incubated at 40 °C aft which the hydrolysis was terminated by boiling. The values for reducing sugars as glucose assayed from the reaction mixture for HEC are presented in Table 5, and for β-glucan in Table 6. The enhancing effect of EGV on the hydrolysis of HEC and especially β-glucan c clearly be seen.

Table 5. Reducing sugars liberated by endoglucanases from T. reesei in the hydrolysis of hydroxyethyl cellulose.

enzymes hydrolysis products as reducing sugars (mg ml^'1)

2 h hydrolysis 20 h hydrolysis

EGV alone 0.00 0.00

EGI alone 0.14 0.26

EGII alone 0.18 0.29

EGI and EGV 0.16 0.27

EGII and EGV 0.24 0.29

Table 6. Reducing sugars liberated by endoglucanases from T. reesei in the hydrolysis of barley β-glucan. Duration of hydrolysis 2 h.

enzymes hydrolysis products as reducing sugars (mg ml^"1)

EGV alone 0.96

EGI alone 1.1

EGII alone 0.90

EGI and EGV 2.4 EGII and EGV 2.0

Example 12 Modelling of the cellulose-binding domain of EGV

Sequences of the five cellulose-binding domains (CBD) of the T. reesei cellulases were aligned to define conserved regions using the computer program MALIGN (Johnson et al, 1993; Johnson and Overington, 1993), which is suitable for this purpose because the percen ge identity between the five CBDs is ~60 %. The construction of a 3-D model of the EGV

CBD was performed using the COMPOSER method (Sutcliffe et al, 1987a,b; Blundell et a 1988; Sali et al, 1990), which is based on rules derived from known three-dimensional structures. These rules can be used to define a conserved core for the model, to select appro priate fragments for the variable regions and to replace the side chains. The NMR-based structure of the CBHI CBD (Kraulis et al, 1989) was used as a basis for the EGV model.

The computer program CHARMm ver. 22 (Brooks et al, 1983) was used to soak the compl ted model in a 35 A cubic box of water and to refine the model through energy minimizatio and molecular dynamics simulation of 100 ps under periodic boundary conditions.

The sequence alignment shows that the CBDs of T. reesei are highly conserved except for o insertion and one deletion of a single aa in EGV. Therefore most parts of the 3-D structure the CBHI CBD, determined by NMR (Kraulis et al, 1989), could be used as a conserved c for modelling of the EGV CBD by the computer program COMPOSER. The CBHI CBD is wedge-shaped domain having two flat surfaces. One of these is predominantly hydrophilic a contains three tyrosine residues that have been shown by chemical modification to be impor tant for the binding of the enzyme to cellulose (Claeyssens and Tomme, 1989). Tyr⁴⁹² locat at the tip of the wedge has also been demonstrated by site.-directed mutagenesis to be invol in substrate binding (Reinikainen et al, 1992). This residue is replaced by a tryptophan (Trp²³⁶) in the EGV CBD (Fig 3B), an amino acid substitution also seen in many other fung CBDs. Both tyrosine and tryptophan residues interact readily with carbohydrates.

The backbones of the CBHI and EGV CBDs are very similar. Two disulfide bridges in identical positions stabilize the structures. The insertion and the deletion in EGV are situate in a single loop and thus compensate each other, maintaining the loop backbone unchanged compared with that of CBHI. However, there is an interesting difference in the backbone conformation at the other, more hydrophobic, flat face. A substantial change in torsion angl was observed at position Gly²²⁰ of EGV, where the φ-angle of the glycine residue changes from negative to positive during the refinement simulation. This causes the loop at region 217-221 to be pushed outwards. Interestingly, the corresponding loop in the CBHI CBD contains a proline residue (Pro⁴⁷⁴), mutation of which reduces the activity of CBHI against crystalline cellulose (Reinikainen et al, 1992). Simulations of the other CBDs from T. reese show that this region is the most flexible region of the CBD (A.-M. Hoffren, T.T. Teeri, an O. Teleman, submitted).

There are also moderate differences in the backbone regions, in which proline (Pro⁴⁹¹) and serine (Ser⁴⁸²) residues of the CBHI CBD are replaced by glutamine (Gin²³⁵) and proline (Pro²²⁷) residues in the EGV CBD, respectively. Moderate differences are also found in side chain conformations, but these changes are within the range of fluctuation occurring during simulation. One of the tyrosines (Tyr²¹⁰) forming the hydrophilic face of EGV CBD points more upwards than its counterpart (Tyr⁴⁶⁶) in CBHI. This difference in orientation is signifi¬ cant, but the flexibility allows Tyr²¹⁰ in EGV to occupy the same position as Tyr⁴⁶⁶ in CBHI Thus the difference in orientation is unlikely to affect substantially the affinity for cellulose.

Overall, the EGV CBD seems to be less wedge-shaped and the hydrophobic surface more rounded than that of the CBHI CBD. REFERENCES

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Beguin, P., Gilkes, N.R., Kilburn, D.G., Miller, R.C.Jr., O'Neill, G. and Warren, R.A.J. (1987), Cloning of cellulase genes. CRC Crit. Rev. Biotechnol. 6: 129-162.

Blundell, T.L., Carney, D., Gardner, S., Hayes, F., Howlin, B., Hubbard, T., Overington, J. Singh, D.A., Sibanda, B.L., and Sutcliffe, M. (1988), Knowledge-based protein modelling design. Eur J Biochem 172: 513-520.

Brooks, B.R., Bruccoleri, R.E., Olafson, B.D., Sates, D.J., Swaminathan, S., and Karplus, (1983), CHARMm: A program for macromolecular energy, minimization, and dynamics calculations. J. Comp. Chem. 4: 187-217.

Chen, CM., Gritzali, M., and Stafford, D.W. (1987), Nucleotide sequence and deduced primary structure of cellobiohydrolase II from Trichoderma reesei. Bio/Technology 5: 274- 278.

Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J., and Rutter, W.J. (1979), Isolation of biologically active ribonucleic acid from sources rich in ribonuclease. Biochem J 18: 5294- 5299.

Claeyssens, M., and Tomme, P. (1989), Structure-function relationships of cellulolytic proteins from Trichoderma reesei. In Trichoderma reesei Cellulases: Biochemistry, Genetics Physiology and Application. Kubicek, C.P. et al (eds.). Cambridge: Royal Society of Chemistry, pp. 1-11.

Gong, C.-S., Ladisch, M.R. and Tsao, G.T. (1979), Biosynthesis, purification, and mode of action of cellulases of Trichoderma reesei. Adv. Chem. Ser. 181: 261-287.

Gritz, L. and Davies, J. (1983), Gene 25: 179 - 188.

Hanahan, H. (1983), J. Mol. Biol. 166: 557-580.

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IUPAC (International Union of Pure and Applied Chemistry) (1987) Pure Appl. Chem. 59: 257-268.

Karhunen et al. (1993) High frequency one-step gene replacement in Trichoderma reesei I, Endoglucanase I overproduction, Mol Gen Genet 241: 515-522.

Kraulis, P.J., Clore, G.M., Nilges, M., Jones, T.A., Pettersson, G., Knowles, J. and Gronen- born, A.M. (1989) Biochemistry 28: 7241-7257.

Mach, R.L., M. Schindler and C.P. Kubicek. (1994), Curr. Genet. 25:567-570.

Mandels, M. and Weber, J. (1969), Production of cellulases. Adv Chem. Ser. 95: 391-413.

Minet, M. and Lacroute, F. (1990), Cloning and sequencing of a human cDNA coding for a multifunctional polypeptide of the purine pathway by complementation of the ade2-101 mutant in Saccharomyces cerevisiae. Curr. Genet. 18: 287-291.

Nakari, T.. Alatalo, E. and Penttila, M. (1992) Poster presentation at ECFG 1, Nottingham, England, 1992 Nevalainen, K.M.H., Penttila, M.E., Harkki, A., Teeri, T.T. and Knowles J. (1991), In: Leong, S.A. and Berka, R.M. (eds.) Mol. Ind. Mycology, Marcel Dekker, Inc., New York, 129 - 148.

Ooi, T., Shinmyo, A., Okada, H., Hara, S., Ikenaka, T., Murao, S., and Arai, M. (1990)

Cloning and sequence analysis of a cDNA for cellulase (FI-CMCase) from Aspergillus aculeatus. Curr. Genet. 18: 217-222.

Penttila, M., Lehtovaara, P., Nevalainen, H., Bhikhabhai, R. and Knowles, J. (1986) Homology between cellulase genes of Trichoderma reesei: complete nucleotide sequence of the endoglucanase I gene. Gene 45: 253-263.

Penttila, M.E., Andre, L., Saloheimo, M., Lehtovaara, P. and Knowles, J.K.C (1987a) Expression of two Trichoderma reesei endoglucanases in the yeast Saccharomyces cerevisia Yeast 3: 175-185.

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Reinikainen, T., Ruohonen, L., Nevanen, T., Laaksonen, L., Kraulis, P., Jones, T.A., Know les, J.K.C. and Teeri, T.T. (1992), Investigation of the function of mutated cellulose-bindin domains of Trichoderma reesei cellobiohydrolase I. PROTEINS 14: 475-482.

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Thesis, Inst. of Biochemistry, University of Uppsala, Sweden, 1991.

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SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: OY ALKO AB

(B) STREET: Salmisaarenranta 7 H

(C) CITY: Helsinki (E) COUNTRY: Finland

(F) POSTAL CODE: FIN-00180

(ii) TITLE OF INVENTION: Novel Endoglucanase Enzyme (iii) NUMBER OF SEQUENCES: 10

(iv) COMPUTER READABLE FORM

(A) MEDIUM TYPE: Floppy disc

(B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Releae #1.0, Version #1.25 (EPO)

(v) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: FI 932521 (B) FILING DATE: 2-JUNE-1993

(2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 884 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Trichoderma reesei

(B) STRAIN: QM9414

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 40..765

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

GATCTTCCAT CTCGTGTCTT GCTTGTAACC ATCGTGACC ATG AAG GCA ACT CTG 54

Met Lys Ala Thr Leu 1 5

GTT CTC GGC TCC CTC ATT GTA GGC GCC GTT TCC GCG TAC AAG GCC ACC 102

Val Leu Gly Ser Leu lie Val Gly Ala Val Ser Ala Tyr Lys Ala Thr 10 15 20

ACC ACG CGC TAC TAC GAT GGG CAG GAG GGT GCT TGC GGA TGC GGC TCG 150

Thr Thr Arg Tyr Tyr Asp Gly Gin Glu Gly Ala Cys Gly Cys Gly Ser 25 30 35 AGC TCC GGC GCA TTC CCG TGG CAG CTC GGC ATC GGC AAC GGA GTC TAC 198 Ser Ser Gly Ala Phe Pro Trp Gin Leu Gly lie Gly Asn Gly Val Tyr 40 45 50 ACG GCT GCC GGC TCC CAG GCT 'CTC TTC GAC ACG GCC GGA GCT TCA TGG 246 Thr Ala Ala Gly Ser Gin Ala Leu Phe Asp Thr Ala Gly Ala Ser Trp 55 60 65 TGC GGC GCC GGC TGC GGT AAA TGC TAC CAG CTC ACC TCG ACG GGC CAG 294 Cys Gly Ala Gly Cys Gly Lys Cys Tyr Gin Leu Thr Ser Thr Gly Gin 70 75 80 85

GCG CCC TGC TCC AGC TGC GGC ACG GGC GGT GCT GCT GGC CAG AGC ATC 342 Ala Pro Cys Ser Ser Cys Gly Thr Gly Gly Ala Ala Gly Gin Ser lie

90 95 100

ATC GTC ATG GTG ACC AAC CTG TGC CCG AAC AAT GGG AAC GCG CAG TGG 390 lie Val Met Val Thr Asn Leu Cys Pro Asn Asn Gly Asn Ala Gin Trp 105 110 115

TGC CCG GTG GTC GGC GGC ACC AAC CAA TAC GGC TAC AGC TAC CAT TTC 438 Cys Pro Val Val Gly Gly Thr Asn Gin Tyr Gly Tyr Ser Tyr His Phe 120 125 130

GAC ATC ATG GCG CAG AAC GAG ATC TTT GGA GAC AAT GTC GTC GTC GAC 486 Asp lie Met Ala Gin Asn Glu lie Phe Gly Asp Asn Val Val Val Asp 135 140 145 TTT GAG CCC ATT GCT TGC CCC GGG CAG GCT GCC TCT GAC TGG GGG ACG 534 Phe Glu Pro lie Ala Cys Pro Gly Gin Ala Ala Ser Asp Trp Gly Thr 150 155 160 165

TGC CTC TGC GTG GGA CAG CAA GAG ACG GAT CCC ACG CCC GTC CTC GGC 582 Cys Leu Cys Val Gly Gin Gin Glu Thr Asp Pro Thr Pro Val Leu Gly

170 175 180

AAC GAC ACG GGC TCA ACT CCT CCC GGG AGC TCG CCG CCA GCG ACA TCG 630 Asn Asp Thr Gly Ser Thr Pro Pro Gly Ser Ser Pro Pro Ala Thr Ser 185 190 195

TCG AGT CCG CCG TCT GGC GGC GGC CAG CAG ACG CTC TAT GGC CAG TGT 678

Ser Ser Pro Pro Ser Gly Gly Gly Gin Gin Thr Leu Tyr Gly Gin Cys 200 205 210

GGA GGT GCC GGC TGG ACG GGA CCT ACG ACG TGC CAG GCC CCA GGG ACC 726

Gly Gly Ala Gly Trp Thr Gly Pro Thr Thr Cys Gin Ala Pro Gly Thr

215 220 225 TGC AAG GTT CAG AAC CAG TGG TAC TCC CAG TGT CTT CCT TGAGAAGGCC 775 Cys Lys Val Gin Asn Gin Trp Tyr Ser Gin Cys Leu Pro 230 235 240

CAAGATAGCC ATGTCTCTCT AGCATTCTTC CGGCGTCAGT CTGATCTGCC TATTTAATCA 835 GGTCAGTCAA TATGTATCCA GAGATAATAA ATTATGTATA TTATAGCAG 884

(2) INFORMATION FOR SEQ ID NO: 2

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 166 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Lys Ala Thr Leu Val Leu Gly Ser Leu lie Val Gly Ala Val Ser 1 5 10 15 Ala Tyr Lys Ala Thr Thr Thr Arg Tyr Tyr Asp Gly Gin Glu Gly Ala 20 25 30

Cys Gly Cys Gly Ser Ser Ser Gly Ala Phe Pro Trp Gin Leu Gly He 35 40 45

Gly Asn Gly Val Tyr Thr Ala Ala Gly Ser Gin Ala Leu Phe Asp Thr 50 55 60 Ala Gly Ala Ser Trp Cys Gly Ala Gly Cys Gly Lys -Cys Tyr Gin Leu 65 70 75 80

Thr Ser Thr Gly Gin Ala Pro Cys Ser Ser Cys Gly Thr Gly Gly Ala 85 90 95

Ala Gly Gin Ser He He Val Met Val Thr Asn Leu Cys Pro Asn Asn 100 105 110

Gly Asn Ala Gin Trp Cys Pro Val Val Gly Gly Thr Asn Gin Tyr Gly 115 120 125

Tyr Ser Tyr His Phe Asp He Met Ala Gin Asn Glu He Phe Gly Asp 130 135 140 Asn Val Val Val Asp Phe Glu Pro He Ala Cys Pro Gly Gin Ala Ala 145 150 155 160

Ser Asp Trp Gly Thr Cys Leu Cys Val Gly Gin Gin Glu Thr Asp Pro 165 170 175

Thr Pro Val Leu Gly Asn Asp Thr Gly Ser Thr Pro Pro Gly Ser Ser 180 185 190

Pro Pro Ala Thr Ser Ser Ser Pro Pro Ser Gly Gly Gly Gin Gin Thr 195 200 205

Leu Tyr Gly Gin Cys Gly Gly Ala Gly Trp Thr Gly Pro Thr Thr Cys 210 215 220 Gin Ala Pro Gly Thr Cys Lys Val Gin Asn Gin Trp Tyr Ser Gin Cys

225 230 235 240

Leu Pro

(2) INFORMATION FOR SEQ ID NO: 3

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 166 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 :

Tyr Lys Ala Thr Thr Thr Arg Tyr Tyr Asp Gly Gin Glu Gly Ala Cys 1 5 10 15

Gly Cys Gly Ser Ser Ser Gly Ala Phe Pro Trp Gin Leu Gly He Gly 20 25 30 Asn Gly Val Tyr Thr Ala Ala Gly Ser Gin Ala Leu Phe Asp Thr Ala 35 40 45

Gly Ala Ser Trp Cys Gly Ala Gly Cys Gly Lys Cys Tyr Gin Leu Thr 50 55 60

Ser Thr Gly Gin Ala Pro Cys Ser Ser Cys Gly Thr Gly Gly Ala Ala 65 70 75 80 Gly Gin Ser He He Val Met Val Thr Asn Leu Cys Pro Asn Asn Gly

85 90 95

Asn Ala Gin Trp Cys Pro Val Val Gly Gly Thr Asn Gin Tyr Gly Tyr 100 105 110

Ser Tyr His Phe Asp He Met Ala Gin Asn Glu He Phe Gly Asp Asn 115 120 125

Val Val Val Asp Phe Glu Pro He Ala Cys Pro Gly Gin Ala Ala Ser 130 135 140

Asp Trp Gly Thr Cys Leu Cys Val Gly Gin Gin Glu Thr Asp Pro Thr 145 150 155 160 Pro Val Leu Gly Asn Asp

165

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2211 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

GAATTCTCAC GGTGAATGTA GGCCTTTTGT AGGGTAGGAA TTGTCACTCA AGCACCCCCA 60

ACCTCCATTA CGCCTCCCCC ATAGAGTTCC CAATCAGTGA GTCATGGCAC TGTTCTCAAA 120

TAGATTGGGG AGAAGTTGAC TTCCGCCCAG AGCTGAAGGT CGCACAACCG CATGATATAG 180 GGTCGGCAAC GGCAAAAAAG CACGTGGCTC ACCGAAAAGC AAGATGTTTG CGATCTAACA 240

TCCAGGAACC TGGATACATC CATCATCACG CACGACCACT TTGATCTGCT GGTAAACTCG 300

TATTCGCCCT AAACCGAAGT GCGTGGTAAA TCTACACGTG GGCCCCTTTC GGTATACTGC 360

GTGTGTCTTC TCTAGGTGCA TTCTTTCCTT CCTCTAGTGT TGAATTGTTT GTGTTGGGAG 420

TCCGAGCTGT AACTACCTCT GAATCTCTGG AGAATGGTGG ACTAACGACT ACCGTGCACC 480 TGCATCATGT ATATAATAGT GATCCTGAGA AGGGGGGTTT GGAGCAATGT GGGACTTTGA 540

TGGTCATCAA ACAAAGAACG AAGACGCCTC TTTTGCAAAG TTTTGTTTCG GCTACGGTGA 600

AGAACTGGAT ACTTGTTGTG TCTTCTGTGT ATTTTTGTGG CAACAAGAGG CCAGAGACAA 660

TCTATTCAAA CACCAAGCTT GCTCTTTTGA GCTACAAGAA CCTGTGGGGT ATATATCTAG 720

AGTTGTGAAG TCGGTAATCC CGCTGTATAG TAATACGAGT CGCATCTAAA TACTCCGAAG 780 CTGCTGCGAA CCCGGAGAAT CGAGATGTGC TGGAAAGCTT CTAGCGAGCG GCTAAATTAG 840

CATGAAAGGC TATGAGAAAT TCTGGAGACG GCTTGTTGAA TCATGGCGTT CCATTCTTCG 900 ACAAGCAAAG CGTTCCGTCG CAGTAGCAGG CACTCATTCC CGAAAAAACT CGGAGATTCC 960

TAAGTAGCGA TGGAACCGGA ATAATATAAT AGGCAATACA TTGAGTTGCC TCGACGGTTG 1020

CAATGCAGGG GTACTGAGCT TGGACATAAC TGTTCCGTAC CCCACCTCTT CTCAACCTTT 1080

GGCGTTTCCC TGATTCAGCG TACCCGTACA AGTCGTAATC ACTATTAACC CAGACTGACC 1140

GGACGTGTTT TGCCCTTCAT TTGGAGAAAT AATGTCATTG CGATGTGTAA TTTGCCTGCT 1200

TGACCGACTG GGGCTGTTCG AAGCCCGAAT GTAGGATTGT TATCCGAACT CTGCTCGTAG 1260

AGGCATGTTG TGAATCTGTG TCGGGCAGGA CACGCCTCGA AGGTTCACGG CAAGGGAAAC 1320 CACCGATAGC AGTGTCTAGT AGCAACCTGT AAAGCCGCAA TGCAGCATCA CTGGAAAATA 1380

CAAACCAATG GCTAAAAGTA CATAAGTTAA TGCCTAAAGA AGTCATATAC CAGCGGCTAA 1440

TAATTGTACA ATCAAGTGGC TAAACGTACC GTAATTTGCC AACGCGTTGT GGGGTTGCAG 1500

AAGCAACGGC AAAGCCCACT TCCCACGTTT GTTTCTTCAC TCAGTCCAAT CTCAGCTGGT 1560

GATCCCCCAA TTGGGTCGCT TGTTTGTTCC GGTGAAGTGA AAGAAGACAG AGGTAAGAAT 1620 GTCTGACTCG GAGCGTTTTG CATACAACCA AGGGCAGTGA TGGAAGACAG TGAAATGTTG 1680

ACATTCAAGG AGTATTTAGC CAAGGGATGCTTGAGTGTATC GTGTAAGGAG GTTTGTCTGC 1740

CGATACGACG AATACTGTAT AGTCACTTCT GATGAAGTGG TCCATATTGA AATGTAAGTC 1800

GGCACTGAAC AGGCAAAAGA TTGAGTTGAA ACTGCCTAAG ATCTCGGGCC CTCGGGCTTC 1860

GGCTTTGGGT GTACATGTTT GTGCTCCGGG CAAATGCAAA GTGTGGTAGG ATCGACACAC 1920 TGCTGCCTTT ACCAAGCAGC TGAGGGTATG TGATAGGCAA ATGTTCAGGG GCCACTGCAT 1980

GGTTTCGAAT AGAAAGAGAA GCTTAGCCAA GAACAATAGC CGATAAAGAT AGCCTCATTA 2040

AACGAAATGA GCTAGTAGGC AAAGTCAGCG AATGTGTATA TATAAAGGTT CGAGGTCCGT 2100

GCCTCCCTCA TGCTCTCCCC ATCTACTCAT CAACTCAGAT CCTCCAGGAG ACTTGTACAC 2160

CATCTTTTGA GGCACAGAAA CCCAATAGTC AACCGCGGAC TGCGCATCAT G 2211

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1627 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

GGCGGTATTG GCTACAGCGG CCCCACGGTC TGCGCCAGCG GCACAACTTG CCAGGTCCTG 60

AACCCTTACT ACTCTCAGTG CCTGTAAAGC TCCGTGCGAA AGCCTGACGC ACCGGTAGAT 120 TCTTGGTGAG CCCGTATCAT GACGGCGGCG GGAGCTACAT GGCCCCGGGT GATTTATTTT 180

TTTTGTATCT ACTTCTGACC CTTTTCAAAT ATACGGTCAA CTCATCTTTC ACTGGAGATG 240

CGGCCTGCTT GGTATTGCGA TGTTGTCAGC TTGGCAAATT GTGGCTTTCG AAAACACAAA 300

ACGATTCCTT AGTAGCCATG CATCGGGATC CTTTAAGATA ACGGAATAGA AGAAAGAGGA 360 AATTAAAAAA AAAAA AAAA CAAACATCCC GTTCATAACC CGTAGAATCG CCGCTCTTCG 420

TGTATCCCAG TACCACGGCA AAGGTATTTC ATGATCGTTC AATGTTGATA TTGTTCCCGC 480

CAGTATGGCT GCACCCCCAT CTCCGCGAAT CTCCTCTTCT CGAACGCGGT AGTGGCGCGC 540

CAATTGGTAA TGACCATAGG GAGACAAACA GCATAATAGC AACAGTGGAA ATTAGTGGCG 600

CAATAATTGA GAACACAGTG AGACCATAGC TGGCGGCCTG GAAAGCACTG TTGGAGACCA 660

ACTTGTCCGT TGCGAGGCCA ACTTGCATTG CTGTCAAGAC GATGACAACG TAGCCGAGGA 720

CCGTCACAAG GGACGCAAAG TTGTCGCGGA TGAGGTCTCC GTAGATGGCA TAGCCGGCAA 780 TCCGAGAGTA GCCTCTCAAC AGGTGGCCTT TTCGAAACCG GTAAACCTTG TTCAGACGTC 840

CTAGCCGCAG CTCACCGTAC CAGTATCGAG GATTGACGGC AGAATAGCAG TGGCTCTCCA 900

GGATTTGACT GGACAAAATC TTCCAGTATT CCCAGGTcAC AGTGTCTGGC AGAAGTCCCT 960

TCTCGCGTGC ANTCGAAAGT CGCTATAGTG CGCAATGAGA GCACAGTAGG AGAATAGGAA 1020

CCCGCGAGCA CATTGTTCAA TCTCCACATG AATTGGATGA CTGCTGGGCA GAATGTGCTG 1080 CCTCCAAAAT CCTGCGTCCA ACAGATACTC TGGCAGGGGC TTCAGATGAA TGCCTCTGGG 1140

CCCCCAGATA AGATGCAGCT CTGGATTCTC GGTTACNATG ATATCGCGAG AGAGCACGAG 1200

TTGGTGATGG AGGGACAGGA GGCATAGGTC GCGCAGGCCC ATAACCAGTC TTGCACAGCA 1260

TTGATCTTAC CTCACGAGGA GCTCCTGATG CAGAAACTCC TCCATGTTGC TGATTGGGTT 1320

GAGAATTTCA TCGCTCCTGG ATCGTATGGT TGCTGGCAAG ACCCTGCTTA ACCGTGCCGT 1380 GTCATGGTCA TCTCTGGTGG CTTCGTCGCT GGCCTGTCTT TGCAATTCGA CAGCAAATGG 1440

TGGAGATCTC TCTATCGTGA CAGTCATGGT AGCGATAGCT AGGTGTCGTT GCACGCACAT 1500

AGGCCGAAAT GCGAAGTGGA AAGAATTTCC CGGNTGCGGA ATGAAGTCTC GTCATTTTGT 1560

ACTCGTACTC GACACCTCCA CCGAAGTGTT AATAATGGAT CCACGATGCC AAAAAGCTTG 1620

TGCATGC 1627

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1137 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

GAATTCTCAC GGTGAATGTA GGCCTTTTGT AGGGTAGGAA TTGTCACTCA AGCACCCCCA 60

ACCTCCATTA CGCCTCCCCC ATAGAGTTCC CAATCAGTGA GTCATGGCAC TGTTCTCAAA 120 TAGATTGGGG AGAAGTTGAC TTCCGCCCAG AGCTGAAGGT CGCACAACCG CATGATATAG 180

GGTCGGCAAC GGCAAAAAAG CACGTGGCTC ACCGAAAAGC AAGATGTTTG CGATCTAACA 240

TCCAGGAACC TGGATACATC CATCATCACG CACGACCACT TTGATCTGCT GGTAAACTCG 300

TATTCGCCCT AAACCGAAGT GCGTGGTAAA TCTACACGTG GGCCCCTTTC GGTATACTGC 360 GTGTGTCTTC TCTAGGTGCA TTCTTTCCTT CCTCTAGTGT TGAATTGTTT GTGTTGGGAG 420

TCCGAGCTGT AACTACCTCT GAATCTCTGG AGAATGGTGG ACTAACGACT ACCGTGCACC 480

TGCATCATGT ATATAATAGT GATCCTGAGA AGGGGGGTTT GGAGCAATGT GGGACTTTGA 540

TGGTCATCAA ACAAAGAACG AAGACGCCTC TTTTGCAAAG TTTTGTTTCG GCTACGGTGA 600

AGAACTGGAT ACTTGTTGTG TCTTCTGTGT ATTTTTGTGG CAACAAGAGG CCAGAGACAA 660

TCTATTCAAA CACCAAGCTT GCTCTTTTGA GCTACAAGAA CCTGTGGGGT ATATATCTAG 720

TGGCCAGAAT GCCTAGGTCA CCTCTAGAGA GTTGAAACTG CCTAAGATCT CGGGCCCTCG 780 GGCTTCGGCT TTGGGTGTAC ATGTTTGTGC TCCGGGCAAA TGCAAAGTGT GGTAGGATCG 840

ACACACTGCT GCCTTTACCA AGCAGCTGAG GGTATGTGAT AGGCAAATGT TCAGGGGCCA 900

CTGCATGGTT TCGAATAGAA AGAGAAGCTT AGCCAAGAAC AATAGCCGAT AAAGATAGCC 960

TCATTAAACG AAATGAGCTA GTAGGCAAAG TCAGCGAATG TGTATATATA AAGGTTCGAG 1020

GTCCGTGCCT CCCTCATGCT CTCCCCATCT ACTCATCAAC TCAGATCCTC CAGGAGACTT 1080 GTACACCATC TTTTGAGGCA CAGAAACCCA ATAGTCAACC GCGGACTGCG CATCATG 1137

(2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GGTCTGAAGG ACGTGGAATG ATGG 24

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 51 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: GATGCATCGA TCGTCCGCGG GTTGAGAGAA GTTGTTGGAT TGATCAAAAA G 51

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 42 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

GAGAGACCGC GGTGATCTTC CATCTCGTGT CTTGCTTGTA AC 42 (2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: ATCGTGGATC CATTATTAAC ACTTCGGTGG 30

Claims

Claims:

1. An isolated DNA sequence, characterized in that it codes for a Trichoderma enzyme having endoglucanase activity, the molecular weight of the unglycosylated form of said enzyme being 20 to 25 kDa and said enzyme comprising a core domain, a linker regio and a cellulose binding domain, and functional parts thereof.

2. The DNA sequence according to claim 1, wherein the DNA sequence hybridizes to the DNA sequence of SEQ ID NO.1 or to the DNA sequence of SEQ ID NO.11.

3. The DNA sequence according to claim 1, wherein the DNA sequence codes for the ami acid sequence of SEQ ID NO.2.

4. The DNA sequence according to claim 1, wherein the DNA sequence is the DNA sequence of SEQ ID NO.1 or to the DNA sequence of SEQ ID NO.11.

5. A DNA sequence, which codes for a polypeptide having endoglucanase activity, said sequence coding for the sequence of SEQ ID NO.3 or functional equivalents thereof.

6. A vector construction, characterized in that it comprises the DNA sequence o any one of claims 1 to 5.

7. A microorganism host, characterized in that it has been transformed with the DNA sequence of any one of claims 1 to 5 or with a vector construction of claim 6 and is able to express said DNA sequence.

8. The host according to claim 7, wherein said host is a fungal or yeast host.

9. The host according to claims 7 or 8, wherein said host is Trichoderma.

10. The host according to claims 7 or 8, wherein said host is Saccharomyces.

11. A culture medium, c h a r a c t e r i z e d in that is comprises the enzymes secreted from the host of claims 7 to 10.

12. A product derived from the culture medium of claim 11 by purifying, concentrating, drying or immobilizing said culture medium.

13. A method for isolating a DNA sequence coding for Trichoderma enzyme having endogl canase activity, c h a r a c t e r i z e d by - enriching the mRNA pool of a Trichoderma strain producing endoglucanase activity respect of the mRNA of the endoglucanase by culturing the Trichoderma strain in conditions which will induce the endoglucanase production of said strain,

- isolating mRNA from the strain,

- preparing cDNA corresponding to the isolated mRNA, - placing the cDNA thus obtained in a vector under the control of a yeast promoter,

- transforming the recombinant plasmids into a yeast strain which naturally does not produce the endoglucanase in order to provide an expression library,

- cultivating the yeast clones thus obtained on a culture medium in order to express the expression library in the yeast, - separating the yeast clones producing the endoglucanase from the other yeast clones,

- isolating the plasmid-DNA of said separated yeast clones, and,

- if desired, sequencing the DNA in order to determine the DNA sequence coding for t endoglucanase.

14. The method according to claim 13, wherein the recombinant plasmids are transformed i a strain of the yeast Saccharomyces cerevisiae.

15. The method according to claim 13, wherein the yeast clones are cultivated on a culture medium containing at least one substrate selected from the group comprising β-glucan, hydr xyethyl cellulose, methylumbelliferyl lactoside and methylumbelliferyl cellobioside.

16. A method for constructing a Trichoderma host capable of expressing an endoglucanase enzyme, characterized in that it comprises a) isolating the DNA sequence coding for an endoglucanase, the molecular weight o which in unglycosylated form is 20 to 25 kDa, or parts thereof, from a suitable d strain, b) constructing a vector carrying said DNA sequence and c) transforming the vector obtained into a Trichoderma host.

17. A method for producing a Trichoderma endoglucanase enzyme, characterize in that it comprises the steps of a) isolating the DNA sequence coding for an endoglucanase, the molecular weight o which in unglycosylated form is 20 to 25 kDa, or functional parts thereof, from a suitable donor strain b) constructing a vector carrying said DNA sequence, c) transforming the vector obtained to a Trichoderma host to obtain a recombinant h strain, d) cultivating said recombinant host strain under conditions permitting expression of said endoglucanase, and e) recovering said endoglucanase.

18. A method for constructing a Saccharomyces host capable of expressing an endoglucan enzyme, characterized in that it comprises a) isolating the DNA sequence coding for an endoglucanase, the molecular weight o whose unglycosylated form being 20 to 25 kDa, and functional parts thereof, fro suitable donor strain, b) constructing a vector carrying said DNA sequence and c) transforming the vector obtained into a Saccharomyces host.

19. A method for producing a Trichoderma endoglucanase enzyme, characterize by a) isolating the DNA sequence coding for an endoglucanase, the molecular weight o which is 20 to 25 kDa in unglycosylated form, or functional parts thereof, from a suitable donor strain b) constructing a vector carrying said DNA sequence, c) transforming the vector obtained to a Saccharomyces host to obtain a recombinant host strain, d) cultivating said recombinant host strain under conditions permitting expression of said endoglucanase, and e) recovering said endoglucanase.

20. An enzyme preparation, characterized in that it contains an endoglucanase enzyme having the amino acid sequence of SEQ ID NO.2 or functional derivatives thereof.

21. An enzyme preparation, characterized in that it contains elevated levels of a endoglucanase enzyme having in unglycosylated form a molecular weight in the range from 20 to 25 kDa, or functional parts thereof, and exhibiting catalytic activity towards the substrates β-glucan.

22. The enzyme preparation according to claim 22, wherein the endoglucanase enzymes exhibits activity towards crystalline cellulose substrate.

23. A method for enzymatically modifying a cellulosic substrate, character- i z e d by contacting said substrate with an enzyme preparation according to any one of claims 20 to 22.

24. The method according to claim 23, wherein the cellulosic substrate is fibrous.