WO1994028117A1 - Nouvelle enzyme d'endoglucanase - Google Patents

Nouvelle enzyme d'endoglucanase Download PDF

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Publication number
WO1994028117A1
WO1994028117A1 PCT/FI1994/000234 FI9400234W WO9428117A1 WO 1994028117 A1 WO1994028117 A1 WO 1994028117A1 FI 9400234 W FI9400234 W FI 9400234W WO 9428117 A1 WO9428117 A1 WO 9428117A1
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WIPO (PCT)
Prior art keywords
endoglucanase
dna sequence
enzyme
host
trichoderma
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PCT/FI1994/000234
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English (en)
Inventor
Anu Saloheimo
Matti Siika-Aho
Merja Penttilä
Raija Lantto
Arja MÄNTYLÄ
Marja Paloheimo
Pirkko Suominen
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Oy Alko Ab
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Application filed by Oy Alko Ab filed Critical Oy Alko Ab
Priority to AU68461/94A priority Critical patent/AU6846194A/en
Publication of WO1994028117A1 publication Critical patent/WO1994028117A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase

Definitions

  • 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.
  • 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.
  • 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- ⁇ -D-glucanases (EC 3.2.1.4), cellobiohydro- lases (exo-l,4- ⁇ -D-glucanases, EC 3.2.1.91) and 1,4- ⁇ -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).
  • 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 capabilitiesi 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.
  • EGI and EGII Trichoderma endoglucanases
  • EGI and EGII Trichoderma endoglucanases
  • 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).
  • 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.
  • CDB cellulose binding 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.
  • PCT/US91/07276 discloses an endoglucanase enzyme, called EGIII, derived from Trichoderma.
  • EGIII endoglucanase enzyme
  • 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.
  • 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.
  • 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.
  • 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.
  • any hydrolytic enzyme gene such as genes coding for cellulases (for instance endoglucanases and cellobiohydrolases) and hemicellulases (for instance xylanases and mannanases)
  • 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.
  • 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.
  • suitable hosts such as fungi of the genus Trichoderma, in particular various Trichoderma reesei strains, and yeasts, such as Saccharomyces cerevisiae.
  • yeasts such as Saccharomyces cerevisiae.
  • yeasts such as Saccharomyces cerevisiae.
  • applications of the enzyme are suggested.
  • 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.
  • Fig. 2B shows 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 pMLOl ⁇
  • 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
  • Fig. 10 shows the introns and coding sequence of the eg 15 gene.
  • 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 ⁇ -D-cellobioside
  • MUL 4-methy umbelliferyl ⁇ -D-lactoside
  • NMR nuclear magnetic resonance
  • PCR poiymerase chain reaction
  • PGK 3-phosphoglycerate kinase gene of Saccharomyces cerevisiae
  • pi isoelectric point.
  • 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.
  • 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 ( ⁇ -D-glucoside glycohydrolase, EC 3.2.1.2 which degrades cellobiose and cello-oligosaccharides to yield glucose.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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 ⁇ - 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.
  • 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.
  • 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 ⁇ GV 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.
  • fungal strain e.g. Trichoderma
  • a culture medium typically contains cellulosic substrate, if endoglucanase production is aimed at.
  • 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
  • the endoglucanase gene is isolated by plating the expression library onto plates containing barley ⁇ -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.
  • the invention comprises the steps of
  • a fungal strain e.g. Trichoderma
  • 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
  • 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.
  • 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.
  • 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.
  • the predicted 17 aa signal peptide indicated in Figure 1 can be substituted by another suitable signal peptide possibly of a different length.
  • a signal sequence should typically comprise a positively charged amino acid at the beginning followed by a stretch of hydrophobic amino acids.
  • 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.
  • the enzyme in its O- and N-glycosylated form can be significantly bigger having apparent molecular weights of 35 kDa or even much higher when produced in the yeast
  • 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.
  • EGV is a true endoglucanase
  • ⁇ -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.
  • t DNA construct(s) is introduced into an appropriate host cell by any of a variety of suitable means, including transformation as described above.
  • 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.
  • several promoters active on glucose medium can be used, which enable the production of the enzyme essentially free from other cellulases.
  • Trichoderma hosts require 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.
  • 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.
  • 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.
  • Trichoderma strains expressing EGV under glucos promoter.
  • 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.
  • 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.
  • the recombinant hosts described above having the desired properties that is, hosts capable expressing the novel endoglucanase enzyme
  • suitable conditions that is, hosts capable expressing the novel endoglucanase enzyme
  • die desired enzymes are secreted from the host into me culture medium
  • die enzyme preparation is recovered from said culture medium by metiiods known in the art.
  • 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.
  • 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, KH 2 PO 4 g/1, (NH 4 ) 2 SO 4 4 g/1, MgSO 4 0,5 g/1, CaCl 2 0,5 g/1 and trace element FeSO 4 "7H 2 O 5 mg/1, MnSO 4 .H 2 O 1,6 mg/1, ZnSO 4 .7H 2 O 1,4 mg/1 and CoCl 2 .6H 2 O 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, Ralphensac NJ), 3 % distiller's spent grain, 0.5 % KH 2 PO 4 , 0.5 % (NH4) 2 SO 4 , 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • E. coli strains PLK-F', pBluescript SK " , and XL- 1 -Blue 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 ⁇ GV expression.
  • 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 Moleisme, C.N.R.S., France.
  • pFL60 Minet and Lacroute 1990
  • 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 % KH 2 PO 4 and 0.5 % (NH 4 ) 2 SO 4 .
  • 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.
  • 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 Ec ⁇ RI- 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 IO 4 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 ⁇ g of plasmid DNA yielded a librar of 8 x 10 4 yeast transformants.
  • 1.2 x 10 5 yeast cells were plated on barley ⁇ -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 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.
  • EGV protein showed a clear activity against ⁇ -glucan but the activity was lower than t activities of die strains producing EGI, CBHII or EGII (Table).
  • EGI hydroxyethyl cellulose
  • 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).
  • Trichoderma reesei The extent of hydrolysis of the substrate was estimated visually and is indicated by 4- .
  • the vector pMLOl ⁇ ( 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 (S ⁇ Q 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).
  • 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 .XTz ⁇ l 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 DH5 ⁇ _ (BRL) by the method described in (Hanahan D, 1983) and die deletion end points were sequenced by using standard methods.
  • 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 Sm ⁇ l-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).
  • the promoter of the cDNAl gene (Nakari et al, 1992) was also used to direct the synthes of the ⁇ GV 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 (S ⁇ Q ID NO. 7) and the 3 'primer GAT GCA
  • 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 (S ⁇ Q ID NO. 9) and the 3 'primer ATC GTG GAT CCA TTA TTA ACA CTT CGG TGG (S ⁇ Q NO. 10).
  • the underlined CCGCGG in the 5 'primer is a Kspl site.
  • 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 %, KH 2 PO 4 3 %, K 2 PO 4 0.8 %, (NH 4 ) 2 SO 4 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 ⁇ - 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.
  • the expression plasmid pALK956 (Figs. 7d) contains:
  • E. coli hph hygromycin B phosphotransferase; Gritz and Davies, 1983
  • T. reesei pki pyruvate kinase; Schin- dler et al, 1993
  • pALK956 The construction of pALK956 is shown in detail in Figs. 7a - 7d.
  • 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 ex 30 (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 S ⁇ cII 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 Bam ⁇ l 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.
  • 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 ex 30 (Notl/partial - . ⁇ Tr ⁇ l, Klenow).
  • the 7.4 kb expression cassette from pALK956 can be removed with NotI digestion.
  • 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 is included to ensur stop in the pki transcription and to target the expression cassette, together with the promoter fragment, to the cbhl locus.
  • the EGV expression plasmid, pALK956, was digested witii NotI, and the 7.4 kb fragment was purified from agarose gel. 2-3 ⁇ g 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 ⁇ g 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 % KH 2 PO 4 and 0.5 % (NH 4 ) 2 S0 4 . Cultures were maintained at 30 °C and 250 rpm for 7 days.
  • the culture supernatants were analyzed for activity against barley ⁇ -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 ⁇ -glucanase activity measured at the optimum pH of EGV was enhanced about twofold.
  • Enzyme preparation containing EGV protein obtained from yeast where egl5 gene was expressed 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 ⁇ -glucan (Zurbriggen et al., 1990a) and hydroxyethyl cellulose, HEC (IUPAC, 1987).
  • the ⁇ -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).
  • 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.
  • CM Sepharose FF Mono S columns or CM Sepharose FF, Pharmacia
  • 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
  • Enzyme preparation containing EG V protein obtained from Trichoderma reesei grown on glucose.
  • 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.
  • 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 (NH 4 ) 2 O 4 .
  • 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 (NH 4 ) 2 SO 4 . Elution was started by the equilibrating buffer followed by a linear dec ⁇ reasing gradient of ammonium sulphate from 1.25 M to 0 M.
  • 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.
  • 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 ⁇ -glucanase activity, eluted from the column at pH 6.6 - 7.2.
  • 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 ⁇ -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.
  • 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 ⁇ -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
  • HEC hydroxyethyl cellulose, Fluka 54290
  • EGV new endoglucanase enzyme
  • 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 ⁇ -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 ⁇ -glucan in Table 6. The enhancing effect of EGV on the hydrolysis of HEC and especially ⁇ -glucan c clearly be seen.
  • 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 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).
  • Tyr 492 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 (Trp 236 ) 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 Gly 220 of EGV, where the ⁇ -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.
  • the corresponding loop in the CBHI CBD contains a proline residue (Pro 474 ), 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).
  • Penttila M., Nevalainen, K.M.H., Ratt ⁇ , M., Salminen, E. and Knowles, J.K.C. (1987b), G 61: 155-164.
  • ORGANISM Trichoderma reesei
  • GATCTTCCAT CTCGTGTCTT GCTTGTAACC ATCGTGACC ATG AAG GCA ACT CTG 54
  • Val Leu Gly Ser Leu lie Val Gly Ala Val Ser Ala Tyr Lys Ala Thr 10 15 20
  • 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
  • ACATTCAAGG AGTATTTAGC CAAGGGATGCTTGAGTGTATC GTGTAAGGAG GTTTGTCTGC 1740

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Abstract

L'invention concerne une nouvelle enzyme, l'EGV, présentant une activité d'endoglucanase. Ladite enzyme qui présente une masse molaire d'environ 20 à 35 kDa est isolée du champignon Trichoderma reesei. L'invention porte également sur une séquence d'ADN codant la nouvelle enzyme ainsi que sur des vecteurs, des souches de levure et fongiques contenant ladite séquence d'ADN. L'invention se rapporte, de plus, à un procédé d'isolement de la séquence d'ADN codant la nouvelle enzyme et de construction de souches de levure et fongiques capables d'exprimer l'endoglucanase. Un produit enzymatique présentant une activité d'endoglucanase et des procédés de modification enzymatique de matières lignocellulosiques, et plus particulièrement de modification et de dégradation de la cellulose et/ou du β-glucane sont également décrits.
PCT/FI1994/000234 1993-06-02 1994-06-02 Nouvelle enzyme d'endoglucanase WO1994028117A1 (fr)

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AU68461/94A AU6846194A (en) 1993-06-02 1994-06-02 Novel endoglucanase enzyme

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FI932521A FI932521A0 (fi) 1993-06-02 1993-06-02 Nytt endoglukanasenzym

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