FI118264B - Mannanase enzyme and corresp. DNA - are produced in recombinant yeast or fungi; used for hydrolysis of manno-polymers, esp. in bleaching lignocellulose pulps - Google Patents

Abstract

DNA (I) encoding a Trichoderma enzyme having mannanase (MN) activity is new, which when transferred into a yeast or fungus strain induces that strain to produce MN. Also new are (a) a vector contg. (I), (b) a recombinant yeast or fungus strain contg. (I), (c) a process for producing a MN or enzyme mixt. by (i) treating a T. reesei culture medium with bentonite, (ii) subjecting the medium to first cation exchange then to anion exchange at pH 7-7.5, (iii) collecting the eluates and purifying by hydrophobic interaction chromatography, and (iv) opt. subjecting the prod. to additional purificn., and (d) an enzyme prepn. contg. at least 1 MN produced by Trichoderma with MN activity and an isoelectric point (pI) of about 3.8, 4.1, 4.5, 5.4 or 6.5, as determined by isoelectric focusing. Pref. (I) hybridises to the insert of vector pMAN1 (in specification), and the yeast is pref. Saccharomyces cerevisiae or Trichoderma species. In hydrolysis, the glucomannans of cellulose pulp are hydrolysed by MN and pref. of at least 1 xylan. The MN treatment (pref. with xylanase) is carried out before bleaching (chlorine-free or peroxide) of sulphite pulp.

Description

1 1 8264 '

The present invention relates to a DNA sequence encoding mannanase enzymes. The present invention relates to a DNA sequence encoding mannanase enzymes.

The invention also relates to vectors, yeast strains and mold strains containing said DNA sequence. The invention further relates to a method for the isolation of genes defining mannanases and to a method for constructing yeast strains capable of expressing mannanase. The invention also provides an enzyme product containing at least one mannanase and a process for preparing the mannanase enzyme and enzyme products. Finally, the invention relates to a process for hydrolyzing mannopolymers and a process for bleaching lignocellulosic pulp.

The main components of wood are cellulose, lignin and hemicellulose. Conifers mainly contain arabino-4-O-methylglucuronoxylan and hardwoodsilane is O-acetyl-4-O-methylglucuronoxylan. Glucomannans are formed by a backbone having glucose and mannose units and the backbone being substituted by galactose and acetyl. techniques. Deciduous tree contains little glucomannan (2-5%) and deciduous glucomannan- * · ·. ; 1; 20 ni differs in structure from coniferous glucomannan in that it is not acetylated and has no galactose side groups (Timell 1967). During the chemical pulp cooking process, the relative composition of hemicelluloses changes compared to native wood and xylan is the major hemicellulose in both softwood pulp 1 1 1 t and i (Sjöström 1977).

· · · 2: 118264 is believed to be evenly distributed in the fiber, whereas xylan concentration is highest on the fiber surface (Luce 1964).

In chemical pulping, pulp leaves residual darkened lignin in the pulp, which is removed from the pulp during bleaching. In conventional chlorine bleaching, chlorine or chlorine dioxide are used to dissolve the lignin. Oxygen bleaching, hydrogen peroxide, or combinations of these and the above are also frequently used today.

Hemicellulases, especially xylanase, have been found to provide fiber bleaching improvement (Kantelinen et al. 1988, Viikari et al. 1991a). The amount of enzyme required is small to achieve the bleaching effect and the enzyme treatment can easily be incorporated as part of the pulping process. In applications to date, hydrolysis of fiber xylan has been able to promote fiber bleaching. Thus, xylanases are currently thought to act primarily on the xylan on the pulp fiber (Kantelinen et al. 1991). The hemicellulases (xylanases) are linked to various bleaching sequences. On a laboratory scale, the use of enzymes in bleaching has been investigated by peroxide delignification (Viikari et al. 1986, Viikari et al. 1987, Viikari et al.

1990) and various chlorine bleaching sequences. On a factory scale, enzymes are present, linked to various chlorine bleaching sequences and also to multiphase peroxide derivatives. . *. 20 cosmic sequences (Viikari et al. 1991b). In spite of the multistep peroxide sequences, the whitened pulp, without chlorine-free chemicals, has a lower lightness than • ·: the chlorine-bleached pulp.

• · ·

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»· ·

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The use of bacterial and mold-based mannanases as a pre-treatment for bleaching has also been studied (Clark et al. 1990, Clark et al. 1991). These mannanase preparations have been produced with the aid of Bacillus subtilis, T. harzianum and Aspergillus niger. The products have · · V: also been reported to have a mild bleaching effect, but due to their impurities *: ···, the effect of the mannanase component has not been specifically demonstrated *: ··:.

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The object of the present invention is to eliminate the drawbacks associated with the prior art and to provide an entirely novel enzyme product which can be used e.g.

3 118264 for bleaching cellulosic pulp.

The present invention is based on the surprising discovery that mannanase enzymes produced by molds of the genus Trichoderma are particularly suitable for the hydrolysis of mannopolymers and, for example, for the treatment of cellulosic pulps.

5

It is known that mannanase production in several molds can be induced by using both cellulose (Lyr 1963) and mannan-containing components in the medium (Reese and Shibata 1964). It is also known that the Trichoderma reesei home is an efficient producer of enzymes that break down cellulose (e.g. Bisaria and Ghose 1981) and xylan (Poutanen et al. 1987). When grown naturally, Trichoderma reesei makes extensive use of plant material in addition to cellulose and its hemicellulose moiety.

In the research underlying the invention, one strain of Trichoderma reesei (Rut C-30, VTT D-86271) was found to produce high levels of mannanase activity in a fermentor grown on medium containing cellulose as carbon source and corn steep water as nitrogen source. In the context of the invention, a Trichoderma reesei strain is provided with an enzyme mixture containing at least one of the following enzymes: mannanase activity having an isoelectric point (pI) of about 3.8, mannanase activity, an enzyme having a pI of about 4.1, mannanase activity, whose pl is • · ·. . ·. 20 about 4.5, an enzyme with mannanase activity having a pI of about 5.4 and an enzyme with mannan ** # nanase activity having a pI of about 6.5. The isoelectric points are • ·; determined by isoelectric focusing with a precision of the order of ± 0.5 «·· · p1 units.

The above mannanase preparation is different in properties from the previously known mannanases and is significantly more efficient in the hydrolysis of mannopolymers than, for example, mannase isolated from Bacillus subtilis.

According to the invention, the desired enzyme (s) can also be produced by using other strains of Trichoderma reesei, other strains of the genus Trichoderma * ♦ ♦ * and other media. Therefore, in the context of the invention, genes encoding the above enzymes have also been isolated to allow the production of enzyme 118264 4, for example, by genetically or mutating improved Trichoderma reesei strains or other genetically modified production hosts (e.g., yeast cell coding genes). genes have been transferred. At the same time, a new method for isolating genes has been developed.

5

The features of the solution according to the invention will be more clearly apparent from the appended claims.

By "mannanase" is meant an enzyme capable of cleaving polyose chains (mannopolymers) containing mannose units. Mannanase thus refers to both endomannanases which cleave mannopolymers within the chain and exomanananases which cleave mannopolymers at the ends of the chain. Examples of mannopolyomers include glucomannan, galactoglucomannan and galactomannan.

The term "enzyme preparation" as used in this application is intended to mean any product containing at least one mannanase enzyme. Thus, the enzyme preparation may be e.g. mannanase or mannase-containing medium, isolated mannanase or a mixture of two or more mannanases.

• * · «**; *. "Hybridization" refers to conditions under which various forms of DNA sequences hybridize to DNA sequences encoding Trichoderma mannanase.

In this invention, mannanase was isolated from Trichoderma reesei growth medium per se · 4 V: by known protein purification methods. Anionic and cationic ion exchangers and hydrophobic interaction chromatography were used to separate the enzyme. Surprising was the simplicity and speed of enzyme separation. However, the invention does not limit: • V ·: this method of enzyme separation is possible, but it is possible to * *: * ·: also use other known methods.

♦ ·· ** · • · 30 The enzyme mannanase activity has been determined using a method of allowing a sample of the enzyme to affect John's bread mannan for a desired period of time, after which the sugars released from the solution are analyzed. The process proceeds as follows: 5118264

Prepare a 0.5% solution of John's Bread Marma (Sigma G-0753, locust bean gum) in 50 mM Na citrate buffer at pH 5.3 and centrifuge to remove insoluble solids from this mannan solution. To 0,80 ml of a suitably diluted enzyme sample is added to 1,80 ml of mannan solution which is allowed to act on mannan at + 50 ° C. Exactly 5 minutes after the addition of the enzyme sample, 3.00 ml of DNA reagent are added and the mixture is kept in boiling water for 5 minutes, after which the mixture is cooled with cold water. The color formed is measured with a spectrophotometer at 540 nm. Calculate the amount of reducing sugars in the sample by comparing the measured spectrophotometer reading, minus the effect of reducing sugars contained in the enzyme sample as such, with that obtained by treatment of a known strength of the mucus solution with that of the enzyme sample. One unit of activity (cat / mL) is the amount of enzyme contained in 1 mL of undiluted sample that releases 1 mole of reducing sugars per second under the assay conditions described above.

15

The invention further describes specific genes encoding Trichodemum mannanases. The DNA sequence of the invention is thus characterized in that it encodes a mold mannanase of the genus Trichoderma and, when introduced into a yeast or mold strain, obtains (mannanase produced by this strain.

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It should be noted that current biochemical and molecular biology data show that; :: A similar enzyme activity suitable for the applications may possibly be obtained by expression of the same enzyme. fragments of genes or naturally occurring nucleotide sequences

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V 1 different gene forms or synthetic genes. Thus, all forms of genes that resemble the genes described in this invention, i.e., which encode a similar mannanase activity, are also within the scope of the present invention.

The DNA sequence can be transformed into a vector, such as the yeast vectors described in Example 1, "pMAN1, pMAN2, pMAN3 or pMAN4. These vectors are stored in a yeast strain * 30 Saccharomyces. cerevisiae DBY746 to Deutsche Sammlung von Mikroor- *

i ': ganismen und Zellkulturen GmbH on 30 December 1992 under the number DSM 7363, DSM

f * · 7364, DSM 7366 and DSM 7365, respectively. The yeast strain used for cloning is described in more detail in Penttilä, M.E. et al., Cloning of Aspergillus niger genes in yeast. Expression of the gene coding Aspergillus / 3-glucosidase, Mol. Gen. Genet. 194 (1984), 494-499.

The present invention also provides a method for isolating the genes defining Trichoderma reesei mannanases. However, the method is also suitable for isolation of other mannase-determining genes of microorganisms. The method utilizes an expression gene bank constructed in a yeast, especially a strain of Saccharomyces cerevisiae. The mold genes are then expressed in yeast under the yeast pro-10 engine. Mannanase protein is secreted from the yeast into extracellular cells and yeast clones containing the mannanase gene can be found on the basis of the mannanase activity they produce, which can be detected by enzyme activity assay or plate assays. Yeast producing mannanase activity can also be found, for example, by means of an antibody corresponding to mannanase. A method based on enzyme activity is highly recommended for isolation of 15 mannanase genes, as this will find yeast producing the active mannanase enzyme and the gene can be isolated for characterization from these yeasts. The expression yeast bank may also be constructed using another yeast promoter, including a lower expression level. Optionally, the mannanase gene's own promoter may also be used, and the chromosomal gene bank may also be used to isolate the genes. The gene bank can also be constructed, for example, in a single copy · · «plasmid.

* · T • * · • · · • • * *

In view of the foregoing, the method of isolating the mannanase-determining genes in the general form may be defined by the following preferred actions.

25 measures: messenger RNA of a microorganism producing mannanase activity

The pool is first enriched for mannanase messenger RNA by growth of the microorganism. under conditions which induce the production of the microorganism mannanase. Mannanase * · · · production is induced, for example, by medium containing mannanase and / or cellulose. The messenger RNA is isolated from the microorganism and the corresponding cDNA is produced. The resulting? 30 cDNA is inserted into a vector under the yeast promoter and the resulting recombinant plasmids are transformed into a yeast strain which does not essentially produce the corresponding mannanase to provide an expression gene bank. The resulting yeast clones are then cultivated on a medium with which the expression gene bank is expressed in yeast. Mannanase producing yeast clones are distinguished from other yeast clones. The plasmid DNA of the former clones is isolated and, if desired, sequenced to determine the corresponding DN A sequence of mannanase.

5

As stated above, the process preferably uses mold as a microorganism producing mannanase, and particularly preferably a strain of the genus Trichoderma. According to a preferred embodiment, the mold is then grown on a substrate having a carbon source containing at least a mannan (e.g., galactomannan) and / or cellulose-containing substrate, and optionally acetylglucuronoxylan and / or xylan. As exemplified in Example 2 below, the medium may, for example, contain a combination of all of these substances, and further, as a nitrogen source, e.g.

In the next step of the method, the recombinant plasmids are preferably transferred to a yeast strain of the genus Saccha-15 romyces, most preferably Saccharomyces cerevisiae; strain grown to produce mannanase. The yeast promoter of the plasmid vector may be, for example, PGK or such a strong promoter, whereby yeast clones can be grown on various media, such as glucose and galactose. The medium is selected according to the promoter under which the gene is placed. According to the invention 20, it is also possible to use a yeast vector in which the promoter is * * · * ·;

«« · Γ \ with glucose. The promoter PGK has the advantage of having a strong level of expression, which is advantageous for enzymes with low catalytic activity. Its manipulation is also easy · · · * 'V. As more particularly illustrated in Example 1, for example, the vector pAJ401, prepared from pFL60 vector by cleaving it with restriction enzymes and inserting an adapter consisting of oligonucleotides, can be used as the plasmid vector * # ···. The cDNA synthesized from the microorganism messenger RNA is then inserted into the vector · · · · between the promoter and terminator of pAJ401, downstream of the promoter.

It should be noted that within the scope of the present invention, mannanase genes can be isolated in principle by any other known method, e.g., · · · in which an expression gene bank is constructed in E.coli e.g. in a lambda vector, the clone contained in the corresponding gene can be found, e.g., by means of an antibody corresponding to the protein. The gene can also be found, e.g., by using oligonucleotides corresponding to the N-terminal amino acid sequence of the mannanase protein as a probe in hybridization of the gene bank.

5

The invention describes the expression of T. manesease genes in yeast and the secretion of the active gene product into a yeast growth medium. Since S.cerevisiae does not naturally secrete mannanase activity, recombinant yeast producing T.reesei mannase can be used as such to produce a specific mannanase-containing enzyme preparation. Expression of any mannanase gene in a foreign host has not previously been described. Our invention demonstrates that mold-derived mannanase can be produced and secreted in yeast, and thus the invention also encompasses the production of the mannanase enzyme in other yeasts, e.g., Kluyveromyces, Pichia or Hansenula. The Mannanaa gene can be transferred to other molds, such as Aspergillus, Penicillium, Neurospora or Phanerochaete, by known methods and induced to release the active

Trichoderman mannanase. Also, the production of Trichodemum self-mannanases after gene isolation can be enhanced or modified by known genetic engineering means, e.g. * · · Where mold does not naturally produce mannanases. Thus, the invention includes organisms producing the said Trichoderma mannanases.

• * • · · • · «t · ·

The recombinant yeast or mold strain according to the invention is thus characterized in that it contains a DNA sequence encoding a mold mannanase of the genus Trichoderma.

Preferably, this yeast strain is a strain of Saccharomyces cerevisiae, such as S.c.mani, · · ·. * ”S.c.man2, S.c.man3 or S.c.man4 (see Example 1 below). The recombinant home * * "* strain is again preferably of the genus Trichoderma.

From the foregoing, there is also provided a method of constructing yeast strains V. which are capable of expressing mannanase. According to the method, DNA sequences encoding * · mannanase are isolated from a suitable mold, DNA. The yeast vector is formed from the 118264 sequence and transferred to a suitable yeast strain. The yeast vectors are then transferred to a yeast strain of, for example, Saccharomyces, Kluyveromyces, Pichia or Han-senula.

The invention further comprises a method of producing a mannanase enzyme, comprising isolating a DNA sequence encoding mannanase from a Trichoderma reesei homogen, followed by generating a vector containing said DNA sequence and transferring it to a suitable yeast or homebase to obtain a recombinant strain. The recombinant strain is then grown under conditions where the strain is capable of expressing man-10 nanoase. The mannanase produced is recovered, for example, by isolation from the culture medium.

The mannanase preparations produced according to the invention can be particularly advantageously used for the hydrolysis of mannopolymers, in particular in the bleaching of cellulose pulps. The mannanases isolated from T. reese (or otherwise produced as described above) are used in bleaching, either as a mixture, purified or as a growth medium.

Mannanases can be used in combination with xylanase. It has been found particularly advantageous to perform a combined mannanase / xylanase treatment in the context of bleaching using chlorine chemicals. Instead, chlorine-free bleaching methods (e.g., peroxide bleaching) achieve quite good results with mannanase enzyme alone. Enzyme treatment can further enhance the extraction of residual lignin in chemical bleaching and thus has a significant environmental and economic impact.

In particular, the mannanase treatment of the present invention can achieve a whiteness increase in purity which cannot be achieved by peroxide or other chemical agents alone, particularly in the completely chlorochemical bleaching sequences. to achieve the same low cost or ··· without losing the strength of the pulp. The mannanase treatment disclosed in the invention combined

• • «I

xylanase treatment promotes bleaching particularly advantageously in both conventional chlorine sequences and completely chlorine-free sequences. In traditional chlorine bleaching sequences, the pulp can be bleached at a higher dose of standard chemical to the final whitening, or alternatively, enzymes can reduce the chemical dose of * «· · · f in initial bleaching to achieve a certain brightness, with enzyme treatment.

Ik 118264 ίο environmentally friendly effect.

Thus, more efficient enzymes can also directly influence the type and amount of chemicals used to industrially extract lignin from the fiber and thus further improve environmentally beneficial low-chlorine or chlorine-free bleaching processes.

The invention will now be discussed in the light of a few non-limiting examples.

10

The examples refer to the accompanying drawings, in which Figure 1 shows the structure of the pAJ401 plasmid constructed from pFL60, Figure 2 shows the restriction enzyme cleavage sites of the insert encoding the yeast vector pMAN1, Figure 3 shows the restriction enzyme coding for the yeast vector pMAN2 Figure 4 shows the restriction enzyme cleavage sites of the yeast vector pMAN3 mannanase encoding insert and Figure 5 shows the restriction enzyme cleavage sites of the yeast vector pMAN4 mannanase coding insert.

* * * • * · tlf «* *

EXAMPLE 1 4 Isolation and Expression of Mannanase Genes in Yeast lichen Trichoderma reesei QM 9414 was grown in a fermentor for 42 h at medium containing m per liter. : 20 g Solca floc cellulose, 10 g Locust bean gum (Sigma), 5 g ·; * KHzPO 4, 5 g (NH 4) 2 SO 4. Subsequently, 1 g of lactose, 1 * * * 1 g of Birke 150 acetylglucuronoxylan and 1 g of Oat Spelt xylan were added to the medium, and the mold was further grown for 24 h. Mold was isolated from RNA by Chirgwin et al. (1979) and * * 30 of this was separated by poly A + mRNA oligo (dT) chromatography essentially as

.1. (Maniatis et al., 1982). mRNA was used to synthesize cDNA Stratagene ZAP cDNA

»I · synthesis and was inserted into the plasmid vector pAJ401. pAJ401 was prepared from 118264 11 vectors pFL60 (Minet & Lacroute, 1990) by digestion with EcoRI and XhoI and inserting the adapter obtained by combining the oligonucleotide 5'-tcgaagaattegagagactcgagt-3ct and 3'ctag The orientation of the EcoRI and XhoI cleavage sites was then changed in the plasmid. The synthesized cDNA 5 was inserted into the EcoRI and Xholt-cleaved pAJ401 vector between the PGK promoter and the terminator. In cloning, the 5 'side of the cDNA sits downstream of the PGK promoter. The ligation mixture was transformed into E. coli strain by PLK-F 'electroporation (Dower et al., 1988).

The structure of plasmid pAJ401 is shown in the figure.

Gene bank plasmids were isolated in one batch from approximately 42,000 bacterial colonies and transformed into Saccharomyces cerevisiae yeast strain DBY746 (Penttilä et al., 1984) by electroporation (Becker & Guarante 1990) by selecting transformants on Sc medium lacking uracil. Yeast colonies were scraped from plates and stored in a single batch on Se grade medium in 15% glycerol at -70 ° C.

For isolation of the mannanase genes, the gene bank mix was pipetted onto substrate plates (minimum yeast plates containing: 0.058 histidine, 0.082 g tryptophan, 0.262 g leucine and 0.1% Locust bean gum (0.5% stock solution 50 ♦ * »•« · MM in citrate buffer pH 5.3 A total of approximately 100,000 gene bank clones were tested for · · 1 week growth for mannanase activity by first replicating the colonies, rinsing the plates with water, and staining the plates with 0.1% Congo I « t • at ψ,:. Red solution. The yeast colonies giving the hydrolysis ring were picked from replica plates and their mannanase activity was re-tested by the plate assay.

· * · Four mannanase-positive yeast clones were named Saccharomyces cerevisiae mani, • * «·; S.C. man2, S.c. man3 and S.c. Man4. These positions were deposited in Deutsche *

Sammlung von Mikroorganismen und Zellkulturen GmbH of 30 December 1992 under Nos. DSM 7363, DSM 7364, DSM 7366 and DSM 7365 respectively.

i • · · »av VV»

The yeast clone S.c. mani (DSM 7363) contains the yeast vector pMAN1 consisting of plasmid * 828212 mmid pAJ401, which has a 0.7 kb insert at the opening site of the restriction enzymes EcoRI and XhoI.

The yeast clone S.c. man2 (DSM 7364) contains the yeast vector pMAN2, consisting of plasmid pAJ401, which has a 0.7 kb insert at the opening site of the restriction enzymes EcoRI and XhoI.

The yeast clone S.c. man3 (DSM 7366) contains the yeast vector pMAN3, consisting of plasmid pAJ401, which has a 1.4 kb 10 'mannanase coding insert at the EcoRI and Xho I site.

The yeast clone S.c. man4 (DSM 7365) contains the yeast vector pMAN4, consisting of plasmid pAJ401, which has a 1.0 kb mannanase coding insert at the EcoRI and Xho I site.

15

DNA was isolated from mannanase-positive yeast clones (Sherman et al. 1981), which transformed E.coli strain DH5a. Plasmid DNA was isolated from ampicillin positive transformants by conventional methods and digested with several restriction enzymes to determine which clones contained the same mannanase gene.

. Clones were sequenced by conventional methods to obtain the fullest possible · · ·: clone from each gene.

The cDNA sequence of the mannanase genes was determined by conventional methods.

*

In the Sequence Listing SEQ NO. 1 shows the nucleotide sequence of the insert of the vector pMAN1.

The chromosomal copy from a previously prepared gene library was also isolated from the clones (Vanhanen et al. 1989), and the 5 'end of the gene was determined by sequencing using the j: cDNA sequence. 5 'over specific primers. Sequence Loans • · · * ... · 30 in SEQ ID NO. 2 shows the nucleotide sequence *: V of the 5 'end of the insert of vector pMAN2 in SEQ ID NO. 3, respectively, its 3'-end nucleotide sequence.

!. *. {In SEQ ID NO. 4 shows the 5 'end of the insert of pMAN3 and 13-18-246 of SEQ ID NO. 5 corresponding to its 3'-end.

Example 2

Isolation of Mannanase Genes This example describes an alternative way to isolate mannanase genes from a gene bank mixture. Growth of Trichoderma reesei strain QM 9414, generation of gene bank plasmids, and expression of genes in yeast were done as described in Example 1.

10

For isolation of the mannanase genes, the gene bank mixture was pipetted into Sc-grout growth medium on microtiter plates so that each well of the microtiter plate received an average of 70 individual yeast bank clones, totaling approximately 75,000 clones. Microtiter plates were grown for 4 days and cells were centrifuged at the bottom of the wells. The supernatant was tested for man-15 nanoase activity using John's Bread as substrate (see above). A few wells contained mannanase activity, and the yeast cells contained in those wells were plated on Sc-groove plates as separate colonies. About 130 colonies per well were re-tested for mannanase activity to identify individual yeast clones containing the mannanase gene.

20

EXAMPLE 3 Cultivation and Characterization of Yeast Clones Example 3 This example illustrates an alternative way of characterizing the mannanase containing genes «♦ · • '.

**** 25 yeast clones.

• · · • · ·

The yeast clones Scmani, Scman2, Scman3, and Scman4, corresponding to the plasmid clones pMAN1, pMAN2, pMAN3 and pMAN4, ··· obtained according to Example 1, were grown in Sc-career medium for 2 days and the supernatant analyzed by Western blot. The 5 'end nucleotide sequence obtained from the clones • *. was also compared to the N-terminal sequence obtained from the purified mannanases. This revealed which isolates were isolated. The nanase genes correspond to previously purified mannanase proteins.

Example 4 Enzyme Isolation

For isolation of the enzyme, the growth medium of Trichoderma reesei (Rut C-30, VTT D-86271) was treated with bentonite as described by Zurbriggen et al. (1990). The solution was then concentrated by ultrafiltration and the concentrate dried on a spray dryer.

10

Enzyme isolation was initiated by dissolving the spray-dried growth medium in phosphate buffer. The insoluble material was separated by centrifugation and the enzyme solution was buffered by gel filtration to pH 7.2 (Sephadex G-25). At this pH, the enzyme solution was pumped onto a cation exchange chromatography column (CM-Sepharose FF) onto which some of the 15 sample proteins were trapped. The desired enzyme was collected through the column in non-bound fractions.

At this pH (pH 7.2), the enzyme solution was pumped onto an anion exchange chromatography column (DEAE-Sepharose FF) to which most of the proteins in the sample were adhered.

The desired enzyme was collected through the column in unbound fractions.

«· · • ·« »« * t «· ·

The enzyme-containing fractions were further purified by a hydrophobic interaction chromatography column (Phenyl Sepharose FF). The enzyme was bound * * * **. * E.m. material at a salt concentration of 0.3 M (NH 4) 2 SO 4. The · · · 25 enzyme trapped on the column was eluted with pH 6.5 buffer to form a decreasing linear gradient of · · · · · · (NH 4) 2 SO 4 between 0.3 and 0 M, followed by elution with pH 6.5 buffer. Mannanase enzyme was collected at the end of the gradient and thereafter. collected verses.

tl · * ··· · · · ·] 30 The enzyme solution was buffered by gel filtration to pH 4.3 (Sephadex G-25). Enzymatic. mi at this pH was bound to a cation exchange chromatography column (CM-Sepha- ··· * · * * rose FF), and some of the proteins adhered to the column (including the majority of the remaining 118264 cellulases) eluted with buffer pH 4.4. The mannanase enzyme was eluted with pH 4.3 buffer, to which sodium chloride was added to form a linear concentration gradient from 0 to 0.05 M. The purified enzyme was collected by gradient eluted fractions.

5 '

Example 5

Characterization of the Enzyme The protein properties of the purified enzyme preparation according to Example 4 were determined by conventional protein chemistry techniques. Molecular weights were determined by SDS-PAGE with an accuracy of about ± 10%.

The preparation contains two mannanase isoenzymes which were found to be nearly identical in terms of biochemical and functional properties. The isoenzymes are designated EM 3 and EM 4. The properties of these are described in Table 1.

• · · · · • • • * · · * - - - - - - - · -

t ♦ · • * ♦ »•« • • * · · · «« «« «« «« «« « · ·

♦ · I

• · · * IM • «* Φ • · · 118264 16

Table 1. Properties of Trichoderma reesei mannanase

Feature Unit EM 3 EM 4

Molecular weight denatokDa 51 53 5 under reflux conditions isoelectric point 4.5 5.4 relative specific% reference subactivity wire activity desta comparison * 100 100 10 - guar mannan 45 40 - peanut 7 9 insoluble mannan - peanut 126 145 soluble mannan 15 pH optimum 3-5.3 3-5.3 temperature optimum at 70 ° C under assay conditions soluble / 3-1,4-mannan M2, M3 M2, M3 20 hydrolysis products a: John's Bread mannan. M2 mannobiosis M, mannotriosis * · · «Il 4 4« • · \ * ·; N-terminal amino acid sequences have also been determined for the enzymes EM3 and EM4, and • ·: these are reported in SEQ ID NOs: 6, and SEQ ID NOs, respectively. 7.

«« · «·« · *>.

• * * * *

Example 6 0 • 0 0 * ·· * · 30 Mannanases produced by Trichoderma reesei in their growth medium ***

• 4 I

• * * *

Trichoderma reesei (VTT-D-86271) was grown in a fermentor for 5 days * on a nutrient medium containing cellulose and corn steep water as the main components. After this, cells were separated from the solution by centrifugation and the activity of the growth solution mannanase * * * * * * · * * * 118264 17 was characterized using isoelectric focusing, ion exchange chromatography and chromatofocusing.

The isoelectric focusing was performed on a Pharmacia-LKB Phast apparatus according to the manufacturer's instructions 5 on a gel supplied by the same manufacturer which developed a pH gradient from pH 3 to pH 9 after concentration. This isoelectric focusing run was used to transfer proteins to the staining gel containing John's bean. From this gel, the site of the mannanase proteins could be detected by Congo red staining, which was used to evaluate the isoelectric point of the mannanase enzymes.

10

The proportion of isoenzymes in total mannanase activity was estimated by running chromatofocusing on PBE 94 gel supplied by Pharmacia-LKB according to the manufacturer's instructions, and measuring the fractions obtained by mannanase activity and staining for activity as described above. In addition, the different 15 fractions of the purification procedure were subjected to activity staining in addition to the mannanase assay, if necessary. Using these methods, the proportion in the solution of the Example could be estimated to an accuracy of 10-20%.

Table 2 shows the isoelectric points and relative activities of the mannanase enzymes.

Table 1. Isoelectric points and • § · relative proportions of total mannanase activity of mannanases produced by Trichoderma reesei. · · V • 1 · * 1 1 • 1 · 1 · /

«M

: Γί 25 pl Estimated percentage of mannanase activity (%) * ::: emi 3.8 5-15 * 1 · - _. · • · · - - - EM 2 4,1 10-30 • ___ • 4 »» «- - EM 3 4,5 30-40

* · «·« —....................,, I I

EM 4 5.4 30 - 40 '· 2 1' 30 EM 5 6.5 Under 5 * 1 1 7 »2 •» · 118264 18

Example 7

Hydrolysis test of sulphate pulp.

Pine sulphate pulp, pine meal and glucomannan fractions isolated from them were hydrolyzed with T. reesei mannanase. The dose of enzyme used was 5000 nkat / g calculated for glucomannan. The treatment was performed in 50 mM citrate buffer pH 5.0. The treatment took 24 hours and the temperature was 45 ° C. After treatment, dissolved sugars were measured after acid hydrolysis. The result was expressed as% of the original glucomannan which had been hydrolyzed (Table 3).

10

Table 3. Relative proportion of hydrolysed glucomannans

Substrate% Glucomannan Hydrolysed 15 µm Pine Wood Flour 11 Pine Mannan Isolated 65 Pine Sulphate Pulp 10 Pine Sulphate Pulp Mannan 79 20 _; __________ from pine wood and from pine sulfate pulp i · », 1 / mannan isolated to produce glucomanno oligomers.

t $ ·

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Example 8, ·, Pulp Bleaching Test (Mannanase Pretreatment)

• t I

, Treatment of pine sulfate pulp (kappa 26.2) with T. reesei mannanase at 5% consistency, * ·. 30 2 h, pH 5, 45 ° C. After treatment, the pulp was washed (2x10x pulp solids) and • ·. was bleached with a 1-step peroxide sequence. After the peroxide step, the mass · · 1 * 1 1 1 '•' t 'was acidified and lightness, kappa and viscosity were measured. The results are given in Table 4. The chemical doses and conditions used were: H 2 O 2 3%, NaOH 1.5%, DTPA 0.2%, MgSO 4 0.5%, 80 ° C, 1h.

S Table 4. Bleaching of sulphate pulp with peroxide after mannanase treatment

Dose released kappa lightness viscosity nkat / g reducing% dm3 / kg sugars% ka 100 0.25 13.9 42.9 970 500 0.48 13.5 43.2 940 10 ref 0 17.6 41.0 980

With the mannanase treatment alone, the peroxide step kappa reduction was 23% compared to the reference treatment.

15

Example 9

Pulp bleaching test.

• «· •» · ·· * ψ ·! Λ! The procedure described in Example 5 was followed except that the softwood pulp t · * *: :: 20 was treated with mannanase and xylanase. The pulp was subjected to peroxide delignification, as in Example 4. The results are reported in Table 5.

* * · S * f * · · f • · t • »· 4. · · • a · · · • · ···» «* ♦ * ··» · 1 • · 9 «♦ * · 118264 20

Table 5. Bleaching of sulphate pulp with peroxide after mannanase / xylanase treatment

Ents. Annos Peik. sugars kappa lightness viscosity nkat / g% ka% dm3 / kg XYL + 100 0.45 12.8 45.0 990 5 MAN + 100 XYL + 100 0.9 11.8 44.8 950 MAN + 500 XYL 100 0.41 16.8 43.7 990 ref - 0 17.6 41.0 980 10 XYL = xylanase MAN = mannanase

Mannanase treatment in combination with xylanase treatment reduced the peroxide bleached mass by 15 · 32% compared to the reference treatment.

• · · * ·· * t i * i • ·· «· ♦ · • · ♦ • · φ • ··»

Example 10 t · · · ♦

Chlorine Chemical Bleaching Test for Cellulose Pulp f 20 - Laboratory-cooked pine sulfate pulp (kappa 34.4) was treated with T. reesei iT: purified mannanase at 5% consistency at pH 5 and 45 ° C. After treatment, the pulp was washed and bleached with ED (D50 / C50).

t «25 ♦ Ψ ♦ f» t * f · W t f ♦ · 118264 21

Table 6. Bleaching of softwood pulp with chlorine chemicals after enzyme treatment

Ents. Dose Chlorination Spacer Lightness Viscosity nkat / g factor% dm3 / kg MAN 90 0.15 6.4 88.4 1050 5 XYL pI9 100 0.15 5.6 89.5 1080 MAN + 90 + 0.15 5.2 90.3 1060 XYL 100 ref - 0.15 6.5 88.1 1050 ref - 0.18 4.5 90.5 1050 10

Compared to xylanase treatment alone, the combined xylanase and mannanase treatment resulted in a significantly smaller spacer and higher final brightness.

Example 11 15 Bleaching Test of Mannanase Treated Sulphite Pulp. The coniferous sulphite pulp was treated with mannanase at 5% consistency at pH 5, 45 ° C for 2 h.

'· ·; After treatment, the pulp was washed and bleached with a 1-step peroxide sequence.

· * »} \ J After the peroxide step, the pulp was measured for brightness, kappa and viscosity. The chemical doses used were: 1.5% H 2 O 2, NaOH 1.2% and MgSO 4 0.05%. Duration 60 min, 80 ° C. (Enz = XYL 100, XYL 100 +500, MAN 500) • * * t * · • * ·

It was found that the mannanase treatment was able to promote the bleaching of the sulphite pulp '··'.

: «F * ¥ t ·. * '25 ♦» 4 # · f • t ♦ {«f ·· • * I' #« «» f · '.

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Example 12

Bleaching test for sulphate pulp treated with mannanase and xylan

Coniferous pulp (spruce / pine) was treated with mannanase and xylanase as in Example 7. After treatment, the pulp was washed and bleached with D (ED) DED. Bleaching conditions are shown in Table 7A.

Mannanase treatment resulted in better mass spacing and brightness than the reference mass. When mannanase treatment was combined with xylanase treatment, 10 spacers were significantly smaller and over 90% final brightness was achieved with 15% chemical saving 7B.

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References

Becker, D.M. & Guarante, L. (1990) High efficiency Transformation of yeast by electroporation. Meth. Enzymol. 194: 182-.

5

Beldman, G., Searle-van Leeuwen, M.F., Rombouts, F.M. and Voragen, F.G.J.

(1985) Cell Purification, Characterization and Comparison of Trichoderma Viride Purified Endoglucanases, Exoglucanases and O-Glucosidases. Eur. J. Biochem. 146: 301-308.

10

Bisaria, V.S. and Ghose, T.K. (1981) Biodegradation of cellulosic materials: substrates, microorganisms, enzymes and products. Enzyme Microb. Technol. 3: 90-104.

Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J., and Rutter, W.J. (1979) Isolation 15 of biologically active ribonucleic acid from sources enriched in ribonuclease.

Biochemistry 18: 5294-5299.

Clark TA, McDonald AG, Senior DJ, Mayers PR (1990), Abstr. 4th Int. Conf. in Biotechnology in the Pulp and Paper Industry, Raleigh 1989, p. 39-40.

. .-20 »I I • · · j. ·. Clark, T.A., Steward, D., Bruce, M.E., McDonald, A.G., Singh, A.P. and Senior, • »· j D.J. (1991) Proc. 45th APPITA Annual General Conference, Melbourne 1991, Vol. I, * * • pp. 193-200.

»· * · · *« · ·. * 1 * 25 Dower, W.J., Miller, J.F. & Ragsdale, C. W. (1988) High efficiency Transformation of * E.coli by high voltage electroporation. Nucl. Acids Res. 16: 6127-6145.

* »·« · ·: Kantelinen, A., Rättö, M., Sundquist, J., Ranua, M., Viikari, L. and Linko, M.

....: (1988) Hemicellulases and their potential role in bleaching. 1988 International Pulp ·; · 50 Bleaching Conference. Tape Proceedings, 1-9.

* • · · -.

«II •« ·

Kantelinen, A., Sundquist, J., Linko, M. and Viikari, L. (1991) The role of • 118264 26 reprecipitated xylan in the enzymatic bleaching of kraft pulp. Proc. 6th International Symposium on Wood and Pulping Chemistry, Melbourne 1991, Vol. I, p. 493-500.

Luce, J.E. (1964) Radial distribution of properties through a cell wall. Pulp and 5 Paper Magazine of Canada 65, 419-423.

Lyr, H. (1963) Occurence of mannanases in fungi. Z. Allgem. Microbiol. 3, 25-35.

Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning. A laboratory 10 manual. Cold Spring Harbor Laboratory, New York.

Minet & Lacroute (1990) Curr. Genet. 18: 287-291.

Penttilä, M.E., Nevalainen, K.M.H., Ray nai, A. & Knowles, J.K.C. (1984) Cloning 15 of Aspergillus niger genes in yeast. Expression of the gene coding for Aspergillus j3-glucosidase. Mol. Gen. Genet. 194: 494-499.

Poutanen, K. Rättö, M., Puls, J. and Viikari, L. (1987) Evaluation of different Microbial xylanolytic systems. J. Biotechnol. 6: 49-60.

.20 • · · * '! Reese, E.T. and Shibata, Y. (1965) 0-Mannanases of fungi. Can. J. Microbiol. 11: • · · 167-183.

• · t * ·· * ·.

· • «· • · ·

Sherman, F., Fink, G.R. and Hicks, J.B. (1981) Methods in Yeast Genetics. Cold * 1 1 1: 1: 125 Spring Harbor Laboratory, New York.

* ··· Simonson, R. (1971) The hemicellulose in the sulfate pulping process. Svensk • · · ·: T: Papperstidning 74, 691–700.

** ··· * · ... 30 Sjöström, E. (1977) The behavior of wood polysaccharides during alkaline Pulping .1. processes. Pin 60, 151-154.

• · 1 • · · ...

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Timell, T.E. (1967) Recent progress in chemistry of wood hemicelluloses. Wood Science and Technology 1, 45-70.

Vanhanen, S., Penttilä, M., Lehtovaara, P. & Knowles, J. (1989) Isolation and 5 characterization of the 3-phosphoglycerate kinase gene (pgk) from the filamentous fungus Trichoderma reesei. Curr. Genet. 15: 181-186.

Viikari, L., Ranua, M., Kantelinen, A., Linko, M. and Sundquist, J. (1986) Bleaching with enzymes. Biotechnology in the Pulp and Paper Industry, Proc 3rd Int. Conf., 10 Stockholm, p. 67-69.

Viikari, L., Ranua, M., Kantelinen, A., Linko, M. and Sundquist, J. (1987) Application of enzymes in bleaching. Proc. 4th Int. Symp. Wood and Pulping Chemistry, Paris, Vol. 1, p. 151-154.

15

Viikari, L., Kantelinen, A., Poutanen, K. and Ranua, M. (1990) Characterization of pulps treated with hemicellulolytic enzymes prior to bleaching. In: Kirk, K. T. and Chang, H.-M. (eds), Biotechnology in Pulp and Paper Manufacture, Butterworth-

Heinemann, Boston, p. 145-151.

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• § · «·; · 12-22.

• · · · • · · 1:: 25

Viikari, L., Sundquist, J. and Kettunen, J. (1991b) Bleaching of Xylanase enzymes to promote pulp. Paper and Timber 73, 384-389.

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• · · • · · · · · * ··. ·· *. Yllner, S., Ostberg, K. and Stockman, L. (1957) A study of removal

II

118264 28 constituents of pine wood in the sulphate process using a continuous liquor flow method. Svensk Papperstidning 60, 795-802.

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5 Biotechnol. 13: 267-278.

Zurbriggen, B.D., Penttilä, M.E., Viikari, L. & Bailey, M.J. (1991). Pilot scale production of fungal endo- / 3-glucanase by brewer’s yeast. J. Biotechnol. 17: 133-146.

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Sequence Listing

118264 (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: National Technical Research Center (B) STREET: Tekniikantie 12 (C) CITY: Espoo (E) COUNTRY: Finland (F) ZONE: 02150 (G) PHONE: +358 0 4561 (ii) TITLE OF THE INVENTION: Mannanase Enzymes, Genes Encoding Them, and Method for Isolating Them, and Method for Bleaching a Lignocellulosic Mass (lii) MAY 19922 (A) APPLICATION NUMBER! EN 931193 (B) DATE OF APPLICATION: MARCH 17, 1993 (2) SEQ ID NO: l DETAILS: * (i) SEQUENCE FEATURES: <(j) · (·) LENGTH: 536 base pairs • t '·· (B) TYPE: Nucleic Acid I «*« (C) DRAINAGE: Single strand ***. (D) TOPOLOGY: linear * ** ··

ij · (ii) TYPE OF MOLECYL: cDNA

(vi) ORIGIN: ··· * ··· (A) ORGANISM: Trichoderma reesei (B) STRAIN: QM9414 »···· • · · ♦ * ·· * • * • * l ···.

* • * · • · * · * · 118 264 (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 1: GAATTCGGCA CGAGAGTCTC TCCTTTTTTT CTCACTATAT CTTTCCTTTG TCCATGTTGC 60 TGTTGTCGTC GTTGTTCTTG AGACTATGCT GGCCTGGTTG GTCCGGATGA ACTGGTGGTT 120 GGGATGGCTG GAGCCTGTGG AAGCGGGCTC GCCGTGGCGT TGGTCAACCG ATTGTATATC 180 AGTCTATGCC TCTGTACACT TCGTCTCTAG CGAAGAGGAG TTGAATACAA ATCTGTAAAA 240 CACTTGACTG TGTCTTCTAG CTATGAGACT CCCTTGCCTA CTGGAGCCTT CAAGATACTT 300 TGGTACTGTA TGAGACCACG CCTACCTCGA CTTCATGTTT GAAACCAGTC AGTAATTCTC 360 TATGAACATG AAACAACACA TTGATCTCTG TAACATCTCA TTGCATAGTA AACCTTCTTA 420 CATTGATTAC TGGCTATGAA CAAAGGTTGT AGGGTAGGTA ACGAAAAAAA aaaaaaaaaa 480 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa CTCGAG 536 (2) SEQ ID NO: 2 INFORMATION: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH :: 243 base pairs (B) TYPE: nucleic acid (C) MOBILITY: single strand (D) TOPOLOGY: linear * «· • ·» * ««

, (ii) MOLECYLYL TYPE: cDNA

* I ♦ 4 «« »· '· * i (vi) ORIGIN: ·, ·; (A) ORGANISM: Trichoderma reesei (B) STRAIN: QM9414 f ♦ ♦ * * r * * · * (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: GAATTCGGCA CGAGCTCTTA ACCAACCACC AAACTACAGC CACCCACCAT GTCC • · * * · * 'GACTACTACG TCCAGCGCCT CCGGAGGCAG CAGCAACGGT TATCCTCCTC AGCAGTACCA 120 f «r« * · * * *. CCAGCAGCAG CAACAGCCAT ACGGCCAGCA GCAGCCAGGG TATGACCAGC AGTACCCCGG 180 »» * · * • »CGCCGGAGTC GATGGAGGCC CCGACGGAGA GCGCGGCCTC GGCGCGACCC TCGTCGGCGG 240» '* * 4GGGGGGGGCCGGGGGGCGG 240 »» * * 4GGGGGGGCGG 3 DETAILS: 3i 1 1 8 2 6 4 (i) SEQUENCE FEATURES: (A) LENGTH: 289 bp (B) TYPE: Nucleic Acid (C) DRAINAGE: Single strand (D) TOPOLOGY: Linear (ii) MOLECYLITY ( vi) ORIGIN: (A) ORGANISM: Trichoderma reesei (B) STRAIN: QM9414 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: CTGCAGCCGC CGGAGCCATT GGGGCGAATC TCATTGAGAA CGCATACAAG AAGCACAAGG 60 AGGAAAAGAT GTACAATGAT GGCGGACACC ATGGCCATCA CCACCATTCG CATCATCAAT 120 AGCACTAGAA TGATTTGATG AAGAATATGA CATTGCATGC CTGCTACATA CGTAGATTAT 180 GATTGGGGGA GGGGCGTTTT TGATGAATAT ATATATAT CAATAAAAAA AAAAAAAAAA 240 AAAAAAAAAA AAAAAAAAAAAAAAAAAAA AAAAAAAAAA AAACTCGAG 289 * I · Φ • · ·

«« I

, (2) SEQ ID NO: 4 DETAILS: * 1 ·> · '1i (i) SEQUENCE FEATURES: jj: (A) LENGTH: 865 base pairs (B) TYPE: nucleic acid "ΐ (C) JURITY: single strand t »·: 1 * · 1 1 (D) TOPOLOGY: linear

(ii) MOLECYLYL TYPE: cDNA

9 • f I »• · f« Il v 1 (vi) ORIGIN: · "»! (A) ORGANISM: Trichoderma reesei • f. (B) STRAIN: QM9414 * 4 «» · ·> · »9 1 f V. · F · 9Ψ 9 ...

32 118264 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: GAATTCGGCA CGAGTTTTTT TTTTTTTTTT TACAGTATAC GTTAGGTGTA TTCGAACGAA 60 AGCCTGAAAT AGGTACAAAT AGCCACAACA GAAACGATTG TCTGACCTCC ACTGAGAAAA 120 TCCCCCTCGT CCCCCAGACA GGTCTGATCA AACACCAGCT ATCTACCTGC AAATGCCCCA 180 TATGTAGGTC ACACAGTAAG GAGTGGATTC GCTATATACA TCGGGTACGT TCGCATCACC 240 CCATGGCAAG GGAGGTGACT TAAGCAAAAC CGCCACTAAC CACAAAGCTC AACTGCATAG 300 TATCGACTTC AAGGAAAACA CGGACAAATA ATCATCATGG TTGCCTTTTG CAGCCTCATC 360 TGCGCTCTCA CGAGGCATCG CCAGTACTCT GGCGATGCCC ACAGGCTCGA GCCTGAGAGC 420 AGTGTCAACG TCACAGAGCG TGGCATGTAC GACTTTGTTC TTGGAGCTCA CAATGATCAT 480 CGCCGTCGTG CTAGCATCAA CTACGACCAA AACTACCAAA CTGGCGGACA AGTCAGCTAT 540 TCGCCTTCCA ACACTGGCTT CTCAGTGAAC TGGAACACTC AAGATGACTT TGTTGTGGGC 600 GTTGGTTGGA CGACTGGATC TTCTGCTCCC ATCAACTTTG GCGGCTCTTT TAGTGTCAAC 660 AGCGGAACTG GCCTGCTTTC CGTCTATGGC TGGAGCACCA ACCCACTGGT TGAGTACTAC 720 ATCATGGAGG ACAACCACAA CTACCCAGCA CAGGGCACCG TCAAGGGAAC CGTCAACAGC 780:: ί GACGGGGCAC TTACACCATC TTGGGGGA TT ACCGTGTAAC GAGCCTTCCA TCCAGGGTAC 840 * * * * * * · • · AGCGCCTTCA ACCGTACATT TCCGT 865 • · · · · 4 * • ·: (2) SEQUENCE ID NO: 5 DETAILS: ♦ · · ”··· (i) SEQUENCE FEATURES: • · ♦ (A) LENGTH: 319 base pairs (B) TYPE: Nucleic acid · ♦ · (C) EMBROIDERY: single strand (D) TOPOLOGY: linear il *.

• ·

·; ”(Ii) TYPE OF MOLECYL: cDNA

(vi) ORIGIN: · "* · (A) ORGANISM: Trichoderma reesei 4 * (B) STRAIN: QM9414 • · •« • · * (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: 118264 CAGATGAACT ACCCAGGTTG TCGCTGTCGA AAG 60 AGAGTGTCAG CAACTAGGTT CTGTTGATGT TGACTTGGAG TGGATGAGGG GTTTGAGCTG 120 GTATGTAGTA TTGGGGTGGT TAGTGAGTTA ACTTGACAGA CTGCACTTTG GCAACAGAGC 180 CGACGATTAA GAGATTGCTG TCATGTAACT AAAGTAGCCT GCCTTTGACG CTGTATGCTC 240 ATGATACATG CGTGACATCG AAATATATCA GCCAAAGTAT CCGTCCGGCG aaaaaaaaaa 300 aaaaaaaaaa AAACTCGAG 319 (2) SEQ ID NO: 6 INFORMATION: (i) SEQUENCE CHARACTERISTICS: (A ) LENGTH :: 15 amino acids (B) TYPE: amino acid (C) YARNITY: single strand (D) TOPOLOGY: linear (ii) MOLECYL TYPE: protein (iv) FRAGMENT TYPE: N-terminal (xi) SEQ ID NO: 6 :

Xaa Xaa Xaa Phe Vai Thr Ile Ser Xaa Thr Gin Xaa Xaa Ile Asp 1 5 10 15 * · · • * ·. ·. : (2) SEQ ID NO: 7 DETAILS: ♦ · * * · • ·: (i) SEQUENCE FEATURES: *; · (A) LENGTH: 15 amino acids (B) TYPE: amino acid * · * (c) DURABILITY: simple thread (D) TOPOLOGY: linear «· ·

•, · I

···· '.

(ii) TYPE OF MOLECYL: Protein * * * *. (iv) FRAGMENT TYPE: N-terminal * ** # ♦ · • · (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: • ·

Xaa Xaa Xaa Xaa Xaa Thr Ile Ser Gly Thr Gin Xaa Xaa Ile Asp. ···. 1 5 10 15 * · * * *

Claims (12)

1. An enzyme preparation, characterized in that it contains an elevated amount of one or more mannanase enzymes endogenous to Trichoderma reesei, produced by one or more microorganisms of the genus Trichoderma, and which is higher than that of wild-type Trichoderma. the growth substrate. An enzyme preparation according to claim 1, characterized in that it contains at least one of the following mannanases: an enzyme having mannanase activity having an isoelectric point (pI) of about 3.8, an enzyme having mannanase activity having a pI of about 4.1, an enzyme having mannanase activity having a p1.5 of about 4.5, an enzyme having mannanase activity of about 5.4, and an enzyme having mannanase activity of about 6.5, wherein the isoelectric points are determined by 15 isoelectric focusing. An enzyme preparation according to claim 1 or 2, characterized in that the preparation contains an elevated amount of EM3 and EM4 mannanases. A process for hydrolyzing mannopolymers, characterized in that. the enzyme preparation according to claim 1 is used in the hydrolysis. • * * • t The method according to claim 4, characterized in that the method ·. hydrolyzing glucomannans of the cellulosic pulp. ··· · 25 • · * *; Process according to claim 4 or 5, characterized in that the mannanase t is used in combination with at least one xylanase. ·· • · • »· A process for bleaching a cellulosic pulp, characterized in that the enzyme preparation according to claim 1 is used for hydrolysis. • «· ** • · * · *« · Process according to Claim 7, characterized in that the mannanases * * * · \. the treatment is carried out before bleaching. • · * * · 35 1 1 8264 Process according to Claim 8, characterized in that the mannanase treatment is carried out before the chlorine-free bleaching. The process according to claim 9, characterized in that the mannanase-5 treatment is carried out before the peroxide bleaching. 11. A method according to claim 8, characterized in that the mannanase treatment is combined with a xylanase treatment. Process according to one of Claims 7 to 11, characterized in that the mannanase treatment is carried out in conjunction with bleaching of the sulphite pulp. a * i »• · · * * * a ·« • · · • aa * · • · · aa a * a • aa • * • • aa a *. aa aa a aa • • aa • a • a • aa • • a • aa '• aa aa ·. aa · • a • a • a a • a • aa a a • a a a a • a 36 1 1 8 2 6 4