Mannanase enzymes, genes coding for them and a method for isolating the genes, as well as a process for bleaching of lignocellulosic pulp

The present invention relates to a DNA sequence, which codes for endomannanase produced by fungi of the genus Trichoderma and transferred into a yeast or fungus strain induces that strain to produce endomannanase, as well as to a method for isolating genes coding for endomannanases. The invention also relates to vectors, yeast strains and fungal strains containing the DNA sequence. Furthermore, the invention provides an enzyme product containing at least one endomannanase, which contains at least one of the following endomannanases produced by fungi of the Trichoderma genus: an enzyme having mannanase activity and an isoelectric point (pI) of about 3.8, an enzyme having mannanase activity and a pI of about 4.1, an enzyme having mannanase activity and a pI of about 4.5, an enzyme having mannanase activity and a pI of about 5.4 and an enzyme having mannanase activity and a pI of about 6.5, the isoelectric points being determined by isoelectric focusing. The endomannanase enzyme and enzyme products according to the invention can be used for hydrolyzation of mannopolymers, in particular in connection with bleaching of lignocellulosic pulps.

This application has been filed under 35 U.S.C. .sctn.371 as the national 
stage application of PCT/FI93/00219, filed May 24, 1993, which claims 
priority from application Ser. No. 922373, filed May 22, 1992, and 
application Ser. No. 931193, filed Mar. 17, 1993, both of Finland. 
The present invention relates to a DNA sequence which codes for mannanase 
enzymes. The invention also concerns vectors, yeast and fungus strains 
containing said DNA sequence. Furthermore the invention relates to a 
method for isolating genes coding for 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 
method for preparing mannanase enzymes and enzyme products. Finally, the 
invention concerns a process for hydrolyzing mannopolymers and to a 
process for bleaching lignocellulosic pulps. 
The main components of wood are cellulose, lignin and hemicellulose. 
Softwood mainly comprises arabino-4-O-methylglucuronoxylan, whereas 
hardwood xylan consists of O-acetyl-4-O-methylglucuronoxylan. The 
glucomannan polymers are formed by a main chain consisting of glucose and 
mannanose units. The main chain is substituted with galactose and 
acetylunits. There are only small mounts (2-5%) of glucomannanes in 
hardwood and the glucomannan of hardwood trees differs structurally from 
that of softwood trees in the sense that it is not acetylated nor does it 
contain galactose side groups (Timell 1967). During chemical pulping the 
relative mounts of hemicelluloses is changed compared to that of the 
native tree. The main hemicellulose component of both softwood and 
hardwood kraft pulp is xylan (Sjostrom 1977). Softwood pulp contains also 
large mounts of glucomannan. During pulping, a pan of the hemicellulose of 
the wood is dissolved because of the very alkaline cooking liquor. When 
the cooking is continued, pH decreases and xylan with no or few side 
chains will start to precipitate on the surface of the cellulose fibers 
(Yllner and Enstrom 1956, Yllner et al. 1957). This xylan reprecipitation 
takes place simultaneously with the precipitation of dissolved lignin. The 
glucomannan behaves in alkaline cooking conditions in a different way 
compared to xylan. Softwood glucomannanes are subjected to a partial 
decomposition already at approx. 130.degree. C. and, thus, the relative 
proportion of glucomannan in pulp is smaller than in the original softwood 
(Sjostrom 1977). The glucomannan remaining in the fiber is, howvever, very 
stable (Simonson 1971). Glukomannan is believed to be evenly distributed 
within the fiber, wheras the xylan concentration is largest on the surface 
of the fiber (Luce 1964). 
After chemical pulping, the pulp will contain darkened residual lignin 
which is removed by bleaching. In conventional chlorine bleaching the 
lignin is dissolved using chlorine or chlorine dioxide. Nowadays oxygen 
bleaching, hydrogen peroxide and combinations of these and the former are 
also often used. 
It has been found out that the bleachability of the fibres can be improved 
by using hemicellulases, in particular xylanase, (Kantelinen et al 1988, 
Viikari et al 1991a). The mounts of enzymes needed for achieving bleaching 
are small and an enzymatic treatment can easily be combined with pulping 
processes. In the present applications, thus far enhanced bleachability of 
the fibres has been achieved by hydrolyzing the fibre xylan. According to 
the present belief, the xylanases act mainly on the xylan located on the 
surface of the pulp fibres (Kantelinen et al 1991). Hemicellulase 
(xylanase) treatments have been combined with various bleaching sequences. 
The use of enzymes in bleaching has been evaluated by peroxide 
delignification (Viikari et al. 1986, Viikari et al. 1987, Viikari et al. 
1990) and by various chlorine bleaching sequences carried out .on 
laboratory scale. Industrially enzyme treatments have been combined with 
different chlorine bleaching sequences and also with various multistage 
peroxide bleaching sequences (Viikari et al. 1991b). However, even if the 
peroxide bleaching is carried out as a multistage bleaching sequence, the 
brightness of pulp bleached entirely without chlorine chemicals remains 
lower than that of chlorine bleached pulp. 
The use of bacterial and fungal mannanases for pretreatment of pulp before 
bleaching has been studied (Clark et al 1990, Clark et al 1991). The 
mannanase preparations used have been produced by the organisms Bacillus 
subtilis, T. harzianum and Aspergillus niger. The products are reported 
slightly to improve bleaching but, because of the impurities which the 
products contain, the influence of the mannanase component has not been 
shown. 
It is an aim of the present invention to eliminate the problems associated 
with the prior art and to provide a completely novel enzyme product which 
can be used, for instance, in bleaching of cellulose pulp. 
The present invention is based on the surprising finding that the mannanase 
enzymes produced by fungi of the genus Trichoderma are particularly well 
suited for hydrolyzing mannopolymers and, thus, for treatment of cellulose 
pulp, for instance. 
It is known in the art that the mannanase production of several fungi can 
be induced by using cellulose (Lyr 1963) and mannan-containing components 
(Reese and Shibata 1964) in the growth medium. It is also known that the 
fungus Trichoderma reesei is an efficient producer of enzymes which 
hydrolyze cellulose (for instance Bisaria and Chose 1981) and xylan 
(Poutanen et al. 1987). Trichoderma reesei growing in nature can utilize 
not only the cellulose of plant material but also the hemicellulose part. 
In the research work forming the basis of the invention it could be shown 
that a certain strain of Trichoderma reesei (Rut C-30, VTT D-86271) 
produces a lot of mannanase activity when cultivated in a fermentor on a 
culture medium containing cellulose as a carbon source and corn steep 
syrup as a nitrogen source. In connection with the invention, an enzyme 
mixture was obtained from the Trichoderma reesei strain which contained at 
least one selected from the group comprising an enzyme having mannanase 
activity and an isoelectric point (pI) of about 3.8, an enzyme having 
mannanase activity and a pI of about 4.1, an enzyme having mannanase 
activity and a pI of about 4.5, an enzyme having mannanase activity and a 
pI of about 5.4 and an enzyme having mannanase activity and a pI of about 
6.5. The isoelectric points indicated have been determined by using 
isoelectric focusing, the accuracy of the determination being on the order 
of .+-.0,5 pI units. 
The properties of the above mannanase preparation differ from those of the 
known mannanase and it is far more efficient in the hydrolysis of 
mannopolymers than, for instance, the mannanase isolated from the species 
Bacillus subtilis. 
According to the invention the desired enzyme(s) can also be produced by 
other Trichoderma reesei strains, by other strains belonging to the genus 
Trichoderma and by using other culture media. For this reason the genes 
coding for the above-mentioned enzymes have, in connection with the 
present invention, been isolated in order to provide the possibility to 
produce the enzyme by using, for instance, genetically or mutationally 
improved Trichoderma reesei strains or by using other genetically modified 
production hosts (such as yeast cells) to which the genes coding for the 
Trichoderma reesei mannanases have been transferred. 
A novel method for isolating the genes has, at the same time, been 
developed. 
The characteristic features of the invention are indicated in the attached 
claims. 
The term "mannanase" denotes an enzyme which is capable of cleaving polyose 
chains containing mannose units (mannopolymers). Mannanase therefore 
covers both endomannanases which internally cleave mannopolymers and 
exomannanases which cleave mannopolymers from the terminal ends of the 
chain. As examples of mannopolymers, glucamannan, galactoglucomannan and 
galactomannan may be mentioned. 
Within the scope of the present application, the term "enzyme preparation" 
designates any product which contains at least one mannanase enzyme. Thus, 
the enzyme preparation can, for instance, be a culture medium containing a 
mannanase or several mannanases, an isolated mannanase, or a mixture of 
two or more mannanases. 
By "hybridization" are meant conditions under which different forms of DNA 
sequences hybridize to DNA sequences coding for a Trichoderma mannanase. 
According to the present invention, the mannanase was isolated from the 
culture medium of Trichoderma reesei by using known methods for purifying 
proteins. For isolating the enzymes anionic and cationic ionexchangers 
were used as was hydrophobic interaction based chromatograpy, the 
isolation of the enzyme being surprisingly easy and fast. The invention is 
not, however, restricted to this enzyme isolation method, but it is 
possible to isolate the enzyme with other known methods. 
The mannanase activity of the enzyme has been determined by using a method 
in which an enzyme sample is allowed to act on the mannan of Locust bean 
for a desired period of time, after which the sugars released are 
analysed. The method is the following: 
A 0.5 % solution of Locust bean gum mannan (Sigma G-0753) is prepared in a 
50 mM sodium citrate buffer at pH 5.3, and the insoluble solid matter is 
removed from this mannan solution by centrifugation. To 1.80 ml of the 
mannan solution 0.200 ml of a suitably diluted enzyme sample is added and 
it is allowed to act on mannan at .+-.50.degree. C. Exactly 5 minutes 
after the addition of the enzyme sample 3.00 ml of DNS reagent is added 
and the mixture formed is heated for 5 min in boiling water. Then, the 
mixture is cooled with cold water. The colour formed is measured by using 
spectrophotometer at 540 nm. The amount of reducing sugars in the sample 
is determined by substracting from the value obtained by spectrophotometer 
the influence of the reducing sugars contained in the enzyme sample as 
such, and then comparing the value with values obtained when a mannose 
solution of known concentration is treated in the same way as the sample. 
One activity unit (cat/ml) corresponds to the amount of enzyme contained 
in 1 ml of undiluted sample, which releases 1 mole of reducing sugars per 
second under the experimental conditions described above. 
The invention further describes the specific genes encoding Trichoderma 
mannanase. Typically, the DNA sequence according to the invention codes 
for the mannanase of a fungus of the genus Trichoderma and transferred to 
a yeast or fungal strain induces that strain to produce mannanase. 
It should be noted that present knowledge on biochemistry and and molecular 
biology shows that an enzyme activity suitable for similar application can 
be provided also by expressing parts of the above-mentioned genes or by 
expressing gene forms or synthetic genes which differ from the native 
genes as far as their nucleotide sequences are concerned. Therefore the 
scope of the invention also covers all forms of genes which remind of the 
genes described in this invention and which code for a similiar kind of 
mannanase activity. 
Vectors, such as the yeast vectors pMAN1, pMAN2, pMAN3 or pMAN4 described 
in Example 1, can be formed from the DNA sequence. These vectors inserted 
into the yeast strain Saccharomyces cerevisiae DBY746 have been deposited 
with the collection Deutsche Sammlung von Mikroorganismen und Zellkulturen 
GmbH on 30 Dec. 1992 under the numbers DSM 7363, DSM 7364, DSM 7366 and 
DSM 7365, respectively. The yeast strain used for cloning is described in 
more detail in the article Penttila, M. E. et al., Cloning of Aspergillus 
niger genes in yeast. Expression of the gene coding Aspergillus 
.beta.-glucosidase, Mol. Gem Genet. 194 (1984), 494-499. 
The present invention also provides a method for isolating genes coding for 
mannanases of, in particular, Trichoderma reesei. The method is, however, 
suited for the isolation of genes coding for mannanases of other 
microorganisms, as well. The method utilizes an expression library 
constructed in yeast, in particular in Saccharomyces cerevisiae, the genes 
of the fungus being expressed in the yeast under a yeast promoter. The 
mannanase protein is extracellularly secreted by the yeast and the yeast 
clones containing the mannanase gene can be found on basis of their 
production of mannanase activity, which can be detected by enzyme activity 
tests or by plate assays. Alternatively, the yeast producing mannanase 
activity can be found, for instance, by using the antibody corresponding 
to the mannanase. The enzyme activity based method is a particularly 
preferred alternative for isolating mannanase genese because it makes it 
possible to find the yeasts producing the active mannanase enzyme and the 
genes can be isolated from these yeasts for characterization. The yeast 
expression library can be constructed using some other yeast promoter, 
even a promoter providing a lower expression level. It is possible also to 
use the mannanase gene's own promoter, and the genes can be isolated by 
using a chromosomal gene library. The gene library can be constructed 
also, for instance, in a single-copy plasmid. 
Based on the above, the method according to the invention for isolating 
genes coding for mannanases can be defined by the following preferred 
method steps: 
The messenger RNA pool of a microorganism producing mannanase activity is 
enriched in respect of the messenger RNA of the mannanase by culturing the 
microorganism in conditions which will induce the mannanase production of 
said microorganism. The mannanase production is induced by, e.g., 
mannan-containing and/or cellulose-containing culture media. Messenger RNA 
is isolated from the microorganism and the cDNA corresponding to the 
isolated mRNA is prepared. Then the cDNA thus obtained is placed in a 
vector under the control of a yeast promoter and the recombinant plasmids 
obtained are transformed into a yeast strain, which naturally does not 
produce the corresponding mannanase, in order to provide an expression 
library. The yeast clones thus obtained are then cultured on a culture 
medium in order to express the expression library in the yeast. The yeast 
clones producing the mannanase are separated from the other yeast clones, 
and the plasmid-DNAs of said separated yeast clones are isolated. If 
desired, the DNA is sequenced in order to determine the DNA sequence 
coding for the mannanase. 
As mentioned above, the mannanase-producing microorganism used in the 
method preferably comprises a fungus and in particular a fungal strain 
belonging to the genus Trichoderma. According to one preferred embodiment 
the fungus is cultivated on a culture medium comprising at least mannan- 
(e.g. galactomannan) and/or cellulose-containing substrate as carbon 
source and in addition possibly acetyl glucurono xylan and/or xylan. As 
Example 2 below will show, the culture medium can for instance contain a 
combination of all these substances and as nitrogen source, for instance, 
corn steep syrup. 
In the following step of the method the recombinant plasmids are preferably 
transferred to a yeast strain belonging to the genus Saccharomyces, in 
particular to the strain Saccharomyces cerevisiae, which is cultivated for 
producing mannanase. The yeast promoter of the plasmid vector can be, for 
instance, PGK or a similar promoter which is considered to be strong. The 
yeast clones can be cultivated on different substrates such as glucose and 
galactose. The culture medium is selected depending on under which 
promoter the gene is placed. According to the invention, it is also 
possible to use a yeast vector, wherein the promoter can be induced by 
galactose, the hydrolase genes being expressed by galactose and not by, 
for instance, glucose. The benefit of the promoter PGK is a strong level 
of expression which is advantageous as far as enzymes having a low 
catalytic activity are concerned. It is also easy to manipulate it. As 
described in more detail in Example 1, the plasmid vector used, can 
comprise, for instance, vector pAJ401, which is prepared from vector pFL60 
by cutting it with restriction enzymes and ligating to it an adapter 
consisting of oligonucleotides. The cDNA synthetized from the messenger 
RNA is then ligated between the promoter and terminator of vector pAJ401, 
downstream from the promoter. 
It should be noticed that, within the scope of the present invention, the 
mannanase genes can, in principle, be isolated by all other commonly known 
methods, for instance by constructing an expression library into an E. 
coli strain bacterium, e.g. into a lambda vector, whereby the clone 
containing the gene corresponding to a certain protein can be found by, 
e.g. the antibody corresponding to the protein. The gene can also be found 
by using oligonucleotides corresponding to the N-terminal amino acid 
sequence of the mannanase protein as a probe in gene library 
hybridization. 
The present invention relates to the expression of mannanase genes of the 
fungus T. reesei in yeast and to the secretion of the active gene product 
into the culture medium of the yeast. Because S. cerevisiae normally does 
not produce mannanase activity, a recombinant yeast which produces T. 
reesei mannase can be used as such for producing an enzyme composition 
containing a specific mannanase. The prior art is completely silent about 
the expression of mannanase genes in foreign host organisms. Our invention 
shows that fungal mannanase can be produced in yeast, which means that the 
invention includes the production of mannanase enzyme in other yeast 
strains as well, for instance in yeast strains belonging to the genera 
Kluyveromyces, Pichia and Hansenula. The mannanase gene can be transferred 
to other fungi, such as strains of the genera Aspergillus, Penicillium, 
Neurospora and Phanerochaete, by methods known per se and the fungus can 
be made to secrete active Trichoderma mannanase. Even the mannanase 
production of Trichoderma itself can be improved or modified after gene 
isolation by known gene technical means, by, for instance, transferring 
several copies of the chromosomal mannanase gene into the fungus or by 
placing the gene under the (e.g. stronger) promoter of another gene and 
thus to provide mannanase expression under desired growth conditions, such 
as on culture media which natively do not produce mannanases. The 
invention covers the organisms producing said Trichoderma mannanases. 
The recombinant yeast or fungal strain according to the invention is 
therefore characterized in that it contains a DNA sequence which codes for 
a mannanase of a fungus of the genus Trichoderma. The yeast strain is 
preferably a strain of the species Saccharomyces cerevisiae, such as 
S.c.man1, S.c.man2, S.c.man3 or S.c.man4 (cf. Example 1 below). The 
recombinant fungal strain is preferably a strain belonging to the genus 
Trichoderma. 
Based on the above, the invention also provides a method for constructing 
yeast strains which are capable of expressing mannanase. According to the 
method DNA sequences coding for mannanase are isolated from a suitable 
fungus species, a yeast vector is formed from the DNA sequence and the 
yeast vector is transferred to a suitable yeast strain. For instance, the 
yeast vectors are transferred to a yeast strain selected from the group 
comprising strains of genera Saccharomyces, Kluyveromyces, Pichia or 
Hansenula. 
The invention also comprises a method for producing mannanase enzyme, which 
method comprises isolating the DNA sequence coding for mannanase from a 
fungus of the strain Trichoderma reesei, forming a vector containing said 
DNA sequence, which is then transferred to a suitable yeast or fungus 
strain for achieving a recombinant strain. Said recombinant strain is then 
cultivated under conditions which enable the strain to express mannanase. 
The mannanase produced is recovered, for instance, by isolating it from 
the culture medium. 
The mannanase preparations produced according to the invention can be 
advantageously used for hydrolysis of mannopolymers, in particular in 
connection with cellulosic pulp bleaching. The marmanses used in bleaching 
comprise mannanases isolated from T. reesei (or produced in any of the 
above-mentioned ways) either in the form of a mixture, in purified form or 
in culture medium. The mannanases can be used together with xylanase. 
According to a particularly preferred embodiment a combined 
mannanase/xylanase treatment is carried out in connection with bleaching 
employing chlorine chemicals. Chlorine free bleaching processes (e.g. 
peroxide bleaching) provide rather good results already by a mannanase 
enzyme preparation. The enzyme treatment makes it possible even further to 
increase the extraction of residual lignin in chemical bleaching, and the 
method is therefore as well environmentally as economically advantageous. 
By means of the mannanase treatment described in this invention it is 
possible to achieve an increase of the brightness produced by, in 
particular, totally chlorine free pulp bleaching sequences, which cannot 
be obtained with peroxide only or with any other chemical at so low costs 
or without impairing the strength of the pulp. The mannanase treatment 
according to the invention combined with a xylanase treatment improves 
brightness particularly when it is used in connection with both 
conventional chlorine-based bleaching sequences and totally chlorine 
chemical free bleaching sequences. In conventional chlorine bleaching, the 
pulp can be bleached with a standard dosage of the chemical to a higher 
final brightness or, alternatively, by adding enzymes it is possible to 
lower the chemical dosage in the initial stage of the bleaching when a 
certain brightness level is aimed at, which makes it possible to reduce 
the detrimental impact on the environment. 
The higher efficiency of the enzymes make it possible directly to affect 
the type and amount of the chemicals used for industrially extracting 
lignin from the fibres and, thus, to improve the low-chlorine or 
chlorine-free bleaching methods which are environmentally advantageous. 
The invention is described in more detail with the help of the following 
non-limiting examples.

EXAMPLE 1 
Isolation of mannanase genes and expression in yeast 
Trichoderma reesei -strain QM 9414 was cultivated in a fermentor for 42 h 
on a cultivation medium containing (per liter): 20 g of Solca floc 
cellulose, 10 g of Locust bean gum galactomannan (Sigma), 5 g of KH.sub.2 
PO.sub.4, and 5 g of (NH.sub.4).sub.2 SO.sub.4. After the 42 hours of 
growth, 1 g of lactose, 1 g of Birke 150 acetylglucuronoxylan and 1 g of 
Oat Spelt xylan was added for each liter of cultivation medium, and the 
fungus was cultivated for further 24 h. The RNA was isolated from the 
fungus as described by Chirgwin et al. (1979), and the poly A+mRNA was 
isolated by chromatography through oligo(dT) essentially as described 
(Maniatis et al., 1982). cDNA was synthesized by using the ZAP-cDNA 
synthesis kit from Stratagene and it was ligated to the plasmid vector 
pAJ401. Plasmid pAJ401 was derived from plasmid pFL60 (Minet & Lacroute, 
1990) by cutting it with EcoRI and XhoI restriction enzymes and by 
ligating inbetween an adapter, which was obtained by combining the 
oligonukleotides 5'-tcgaagaattcgagagactcgagt-3' and 
3'-tcttaagctctctgagctcattaa-5'. The orientation of the restriction sites 
of enzymes EcoRI and XhoI was then reversed in the plasmid. The 
synthetized cDNA was ligated to the pAJ401 vector cut with EcoRI and XhoI 
between the PGK promoter and terminator. The 5' end of the cDNA is bound 
downstream from the PGK promoter during cloning. The ligating mixture was 
transformed into the E. coli strain PLK-F' by electroporation (Dower et 
al., 1988). 
The structure of plasmid pAJ401 is shown in the figure. 
The gene library plasmids were isolated as one batch from about 42,000 
bacterial colonies and transformed into the Saccharomyces cerevisiae yeast 
strain DBY746 (Penttila et al., 1984) by electroporation (Becker & 
Guarante 1990) while selecting the transformants on a Sc medium which is 
deficient in uracil. The yeast colonies were scraped from the plates and 
maintained as one batch in a Sc-ura-medium containing 15% glycerol at 
-70.degree. C. 
For isolating the mannanase genes the gene library mixture was pipeted onto 
substrate-containing plates (yeast minimal medium plates, containing for 
each 1000 ml: 0.058 g histidine, 0.082 g tryptophan, 0.262 g leucine and 
0.1% Locust bean gum (0.5 % stock solution in 50 mM citrate buffer at pH 
5.3. In all about 100,000 gene library clones were tested for mannanase 
activity after one week of cultivation by replicating first the colonies 
from the plates, then flushing the plates with water, and by staining the 
plates with a 0.1% solution of Congo Red. Yeast colonies producing 
hydrolysis halos were picked up from the replica plates and they were 
tested again for mannanase activity on plate assay. 
Four mannanase-positive yeast clones were called Saccharomyces cerevisiae 
man1, S.c. man2, S.c. man3 and S.c. man4. These strains were deposited 
into the collection Deutsche Sammlung von Mikroorganismen und Zellkulturen 
GmbH on the 30th of Dec. 1992 under the numbers DSM 7363, DSM 7364, DSM 
7366 and DSM 7365, respectively. 
The yeast clone S.c. man1 (DSM 7363) contains the yeast vector pMAN1, which 
consists of the plasmid pAJ401, having a 0.7 kb mannanase coding insert in 
the restriction sites of restriction enzymes EcoRI and XhoI. 
The yeast clone S.c. man2 (DSM 7364) contains the yeast vector pMAN2, which 
consists of the plasmid pal401, having a 0.7 kb mannanase coding insert in 
the restriction sites of restriction enzymes EcoRI and XhoI. 
The yeast clone S.c. man3 (DSM 7366) contains the yeast vector pMAN3, which 
consists of the plasmid pAJ401, having a 1.4 kb mannanase coding insert in 
the restriction sites of restriction enzymes EcoRI and XhoI. 
The yeast clone S.c. man4 (DSM 7365) contains the yeast vector pMAN2, which 
consists of the plasmid pAJ401, having a 1.0 kb mannanase coding insert in 
the restriction sites of restriction enzymes EcoRI and XhoI. 
The DNA of the mannanase positive yeast clones was isolated (Sherman et al 
1981), and used for transformation of the E. coli strain DH5.alpha.. From 
the ampicillin-positive transformants plasmid DNA was isolated by 
conventional methods and digested by several restriction enzymes, to 
determine which clones contained the same mannanase gene. The clones were 
sequenced by conventional methods to obtain a suitably long a clone from 
each gene. 
The cDNA sequence of the mannanase genes was determined by conventional 
methods. The nucleotide sequence of the insert of the vector pMAN1 is 
indicated in sequence list SEQ NO. 1. 
The chromosomal copies were also isolated from the clones using the 
previously prepared gene library (Vanhanen et al. 1989), and the 5' end of 
the gene was sequenced by utilizing primers specific for the 5' end 
designed on basis of the cDNA sequence. Sequence listing SEQ ID NO. 2 
shows the nucleotide sequence of the 5' end and sequence listing SEQ ID 
NO. 3 the nucleotide sequence of its 3' end. Sequence listing SEQ ID NO. 4 
indicates the 5' end of the insert of vector pMAN3 and sequence listing 
SEQ ID NO. 5 the 3' end thereof. 
EXAMPLE 2 
Isolation of mannanase genes 
This example illustrates an alternative way of isolating mannanase genes 
from a gene library mixture. Trichoderma reesei strain QM 9414 was 
cultivated, the gene library plasmids were formed and the genes were 
expressed in yeast as described in Example 1. 
The gene library mixture was pipated into the Sc-ura cultivation medium on 
microtiter plates so that each well of the microtiter plates contains an 
average of 70 different yeast library clones, in total about 75,000 
clones. The microtiter plates were cultivated for 4 days and the cells 
were centrifuged to the bottom of the wells. The supernatants were tested 
for their mannanase activity by using Locust bean gum as a substrate. (cd. 
above). Some of the wells contained mannanase activity, and the yeast 
cells contained in said wells were plated onto Sc-ura-plates as separate 
colonies. About 130 separate colonies of each well were retested for their 
mannanase activity in order to identify single yeast clones containing the 
mannanase gene. 
EXAMPLE 3 
Cultivation and characterization of the yeast clones 
This example describes an alternative embodiment for characterization of 
yeast clones containing mannanase genes. 
Yeast clones S.c.man1, S.c.man2, S.c.man3 and S.c.man4, which correspond to 
plasmid clones pMAN1, pMAN2, pMAN3 and pMAN4, were obtained by following 
the procedure described in Example 1. The clones were cultivated on a 
Sc-ura cultivation medium for 2 days in a flask and the supernatant was 
analysed by the Western blot -method using antibodies prepared against 
purified mannanases obtained from T. reesei. The 5' end of the nucleotide 
sequence obtained from the clones was also compared with the N-terminal 
sequence from the purified mannanases. It was thus ascertained which 
isolated mannanase genes correspond to the earlier purified mannanase 
proteins. 
EXAMPLE 4 
Isolation of the enzyme 
In order to isolate the enzyme, the culture medium of Trichoderma reesei 
(Rut C-30, VTT D-86271) was first treated with bentonite, as described by 
Zurbriggen et al. (1990). Then the solution was concentrated by 
ultrafiltration and the concentrate was dried by spray drying. 
The isolation of the enzyme was started by dissolving the spray dried 
culture medium in a 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). The enzyme solution was pumped at this pH 
through a cation exchange chromatography column (CM-Sepharose FF), to 
which a part of the proteins of the sample were bound. The desired enzyme 
was collected in the fractions eluted through the column. 
At said pH (pH 7.2) the enzyme solution was pumped to an anion exchange 
chromatography column (DEAE-Sepharose FF), to which most of the proteins 
of the sample were bound. The desired enzyme was collected in the fraction 
eluted through the column. 
The enzyme-containing fractions were further purified by using hydrophobic 
interaction chromatography (Phenyl Sepharose FF). The enzyme was bound to 
said material at a salt concentration of 0.3M (NH.sub.4).sub.2 SO.sub.4. 
The bound enzyme was eluted with a buffer at pH 6.5, so as to form a 
decreasing linear concentration gradient of (NH.sub.4).sub.2 SO.sub.4 from 
0.3 to 0M. After this, elution was continued with the buffer of pH 6.5. 
The mannanase enzyme was collected at the end of the gradient and in the 
fractions collected after that. 
The enzyme solution was buffered by gel filtration to pH 4.3 (Sephadex 
G-25). The enzyme was bound at this pH to a cation exchange chromatography 
column (CM-Sepharose FF), and a part of the proteins bound to the column 
(i.a. most of the remaining cellulases) were eluted with a buffer, pH 4.4. 
The mannanase enzyme was eluted with a buffer, pH 4,3, to which sodium 
chloride was added in order to form a linear concentration gradient of 
sodium chloride from 0 to 0.05M. The purified enzyme was collected in the 
fractions eluted by the gradient. 
EXAMPLE 5 
Enzyme characterization 
The protein properties of the enzyme preparation purified according to 
Example 1 were determined by methods known per se in protein chemistry. 
The molecular weights were determined by the SDS-PAGE-method, the accuracy 
of which is about .+-.10%. 
The preparation contains two mannanase isoenzymes, which biochemically and 
functionally proved to be almost identical. The isoenzymes are named EM 3 
and EM 4. Their properties are described in Table 1. 
TABLE 1 
______________________________________ 
Properties of the Trichoderma reesei mannanase 
Property Unit EM 3 EM 4 
______________________________________ 
Molecular weight under 
kDa 51 53 
denaturating conditions 
Isoelectric point 4.5 5.4 
Relative specific activity 
percentage of the 
activity of the 
reference 
substance 
reference.sup.a 100 100 
guar mannan 45 40 
Insoluable mannan of 7 9 
Corozo nut 
Soluable mannan of 126 145 
the Corozo nut 
Optimum pH 3-5.3 3-5.3 
Optimum temperature at 
.degree.C. 70 70 
for activity testing 
Hydorlysation products of M.sub.2, M.sub.3 
M.sub.2, M.sub.3 
.beta.-1,4-mannan 
______________________________________ 
.sup.a : Locust bean gum 
M.sub.2 : mannobiose 
M.sub.3 : mannotriose 
The N-terminal amino acid sequences of enzymes EM3 and EM4 have also been 
determined and they are indicated in sequence listings SEQ ID NO. 6 and 
SEQ. ID NO. 7, respectively. 
EXAMPLE 6 
Mannanases secreted by Trichoderma reesei into its culture medium 
The fungus Trichoderma reesei (VTT-D-86271) was cultivated in a fermentor 
for 5 days on a culture medium, whose main components were cellulose and 
corn steep syrup. After the cultivation the cells were separated from the 
solution by centrifugation and the mannanase activity of the culture 
medium was characterized by isoelectric focusing, ionexchange 
chromatography and chromatofocusing. 
The isoelectric focusing was made by Phast equipment (Pharmacia-LKB) 
according to the instructions by the manufacturer on a gel supplied by the 
same manufacturer. A pH gradient from pH 3 to pH 9 was formed in the gel 
during focusing. After the isoelectric focusing time the proteins were 
transferred from this gel to a staining gel which contained Locust bean 
gum mannan. By staining with Congo red, the location of the mannanase 
proteins could be detected on this gel, which made it possible to estimate 
the isoelectric point of the mannanase enzymes. 
Each isoenzyme's relative portion of the total mannanase activity isoenzyme 
was estimated by running chromatofocusing on a PBE 94 gel supplied by 
Pharmacia-LKB according to the instructions of the manufacturer. The 
mannanase activity of the fractions obtained was measured and they were 
then subjected to an activity staining as described above. Furthermore the 
different fractions of the purification procedure were, if needed, 
subjected to activity staining in addition to mannanase determination. 
These methods made it possible to make an estimation of the relative 
amounts of the enzymes in the solution of the Example with an accuracy of 
10 to 20%. 
Table 2 indicates the isoelectric points of the mannanase enzymes and their 
relative activities. 
TABLE 2 
______________________________________ 
Isoelectric points of the Trichoderma reesei mannanases and their 
relative 
portions of the total mannanase activity 
Estimated portion of the 
pI mannanase activity (%) 
______________________________________ 
EM 1 3.8 5-15 
EM 2 4.1 10-30 
EM 3 4.5 30-40 
EM 4 5.4 30-40 
EM 5 6.5 &lt;5 
______________________________________ 
EXAMPLE 7 
Hydrolysis of cellulosic pulp 
Pine kraft pulp, pine saw dust and glucomannan fractions isolated from them 
were hydrolysated with T. reesei mannanase. The enzyme dosage was 5000 
nkat/g calculated on basis of the glucomannan. The treatment was carried 
out in 50 mM citrate buffer at pH 5.0. The treatment lasted for 24 h and 
the temperature was 45.degree. C. After the treatment the dissolved sugars 
were determined after acid hydrolysis. The result is indicated in percent 
of the original glucomannan which was hydrolyzed. (Table 3). 
TABLE 3 
______________________________________ 
The relative amounts of hydrolyzed glucomannans 
Substrate percentage of hydrolyzed glucomannan 
______________________________________ 
pine saw dust 11 
mannan isolated from pine tree 
65 
pine kraft pulp 10 
mannan isolated from pine kraft 
79 
pulp 
______________________________________ 
Thus, the mannanase are efficient in hydrolyzing mannan isolated from both 
pine tree and pine kraft pulp. As a result of the hydrolysis, 
glucomanno-oligomers are formed. 
EXAMPLE 8 
Bleaching of cellulose pulp (mannanase pretreatment) 
Pine kraft pulp (kappa 26.2) was treated with T. reesei mannanase at 5% 
consistency for 2 h at pH 5 and 45.degree. C. After the treatment the pulp 
was washed (2.times.10.times.dry matter of the pulp) and bleached with a 
1-stage peroxide sequence. Subsequent to the peroxide stage the pulp was 
acidified and its brightness, kappa and viscosity were determined. The 
results are indicated in Table 4. The chemical dosages and conditions 
were: H.sub.2 O.sub.2 3 %, NaOH 1.5 %, DTPA 0.2 %, MgSO.sub.4 0.5%, 
80.degree. C., 1 h. 
TABLE 4 
______________________________________ 
Peroxide bleaching of kraft pulp subsequent to mannanase treatment 
Released reducing Bright- 
Dose sugars in % of ness, Viscosity 
nkat/g dry matter Kappa % dm.sup.3 /kg 
______________________________________ 
100 0.25 13.9 42.9 970 
500 0.48 13.5 43.2 940 
ref 0 17.6 41.0 980 
______________________________________ 
After a simple mannanase treatment, the kappa reduction during the peroxide 
stage was 23% better than in the reference test. 
EXAMPLE 9 
Bleaching of cellulose pulp 
The procedure described in Example 5 was followed with the difference that 
coniferous pulp was treated with mannanase and xylanase. The pulp was 
subjected to peroxide bleaching similiar to the one of Example 4. The 
results appear from Table 5. 
TABLE 5 
______________________________________ 
Bleaching with peroxide of kraft pulp after mannanase/xylanase treatment 
Reducing 
Dose sugars in % Bright- 
Viscosity 
Enz. nkat/g of dry matter 
Kappa ness, % 
dm.sup.3 /kg 
______________________________________ 
XYL + 100 0.45 12.8 45.0 990 
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 
______________________________________ 
XYL = xylanase 
MAN = mannanase 
Combination of the mannanase treatement with a xylanase treatment lowered 
the kappa of the peroxide bleached pulp by 32% compared to the reference 
treatment. 
EXAMPLE 10 
Bleaching test carried out for cellulose pulp 
Pine kraft pulp cooked in a laboratory (kappa 34.4) was treated with 
mannanase purified from T. reesei at a consistency of 5%, at pH 5 and at a 
temperature of 45.degree. C. After the treatment the pulp was washed and 
bleached with the sequence (D50/C50) EDED. 
TABLE 6 
______________________________________ 
Bleaching with chlorine chemicals of coniferous pulp treated with 
enzymes 
Dose Chlorination Brightness, 
Viscosity 
Enz. nkat/g factor Int. kappa 
% dm.sup.3 /kg 
______________________________________ 
MAN 90 0.15 6.4 88.4 1050 
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 
______________________________________ 
In comparison to a simple xylanase treatment, a combined xylanase and 
mannanase treatment provided a considerably much smaller intermittent 
kappa and a higher final brightness. 
EXAMPLE 11 
Bleaching of mannanase-treated sulphite pulp 
Coniferous sulphite pulp was treated with mannanase at 5% consistency at pH 
5 and a temperature of 45.degree. C. for 2 h. After the treatment the pulp 
was washed and bleached with a one-stage peroxide sequence. After the 
peroxide stage the brightness, kappa and viscosity of the pulp were 
determined. The chemical dosages were: 1.5% H.sub.2 O.sub.2, 1.2% NaOH 
1,2% and 0.05% MgSO.sub.4. The bleaching time was 60 rain at 80.degree. C. 
(Enz=XYL 100, XYL 100+500, MAN 500) 
It was found that the mannanase treatment made it possible to improve the 
bleachability of sulphite pulp. 
EXAMPLE 12 
Bleaching test of kraft pulp treated with mannanase and xylanase 
Coniferous kraft pulp (spruce/pine) was treated with mannnase and xylanase 
as in Example 7. After the treatment the pulp was washed and bleached by 
the sequence D(ED)DED. The bleaching conditions are shown in Table 7A. 
After the mannanase treatment the intermittent kappa and the brightness 
were better than in the reference pulp. When the mannanase treatment was 
combined with a xylanase treatment the intermediate kappa was much lower 
and an over 90% final brightness was obtained with a 15% saving in 
chemical consumption (Table 7B). 
TABLE 7A 
______________________________________ 
Bleaching conditions. Bleaching sequence: D(EP)DED). 
Chlorination factors 0.15 and 0.20. 
Consis- T Duration Act. Cl 
Stage 
tency % .degree.C. 
min. Final pH 
% P NaOH 
______________________________________ 
D0 3 60 60 2.0-1.9 
3.3.sup.1,3) 
% % 
4.3.sup.2) 
EP 10 80 60 10.4-10.5 0.3 1.7.sup.1,3) 
2.1.sup.2) 
D1 10 70 180 3.4-3.6 
2.5.sup.1,2) 
3.0.sup.3) 
E2 10 70 60 10.8-10.9 0.9 
D2 10 70 180 3.8-4.1 0.5-1.0.sup.1,3) 
0.7 
______________________________________ 
.sup.1) Enzyme treated pulp, chlorination factor 0.15 
.sup.2) Chlorination factor 0.20 
.sup.3) Reference, chlorination factor 0.15 
TABLE 7B 
__________________________________________________________________________ 
Chlorine bleaching of mannanase and xylanase treated pulps. 
SequenceD(EP)DED. Initial kappa of pulp: 21.6. 
Reducing 
sugars, Chemicals 
Dosage, 
% of dry 
Chlorination 
Intermediate 
D-1 brightness 
Final brightness 
Viscosity 
consumption 
Enzyme nkat/g 
matter 
factor 
kappa % % dm.sup.3 /kg 
% act. 
__________________________________________________________________________ 
Cl 
MAN 500 0.40 0.15 6.2 80.1 89.8 940 6.7 
XYL 100 0.38 0.15 5.5 82.5 90.2 980 6.4 
MAN + XYL 
500 + 100 
0.70 0.15 5.2 83.5 90.7 960 6.3 
ref -- -- 0.15 6.5 79.8 89.7 970 7.1 
ref -- -- 0.20 5.1 82.8 90.1 970 7.4 
__________________________________________________________________________ 
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__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 7 
(2) INFORMATION FOR SEQ ID NO: 1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 536 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Trichoderma reesei 
(B) STRAIN: QM9414 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: 
GAATTCGGCACGAGAGTCTCTCCTTTTTTTCTCACTATATCTTTCCTTTGTCCATGTTGC60 
TGTTGTCGTCGTTGTTCTTGAGACTATGCTGGCCTGGTTGGTCCGGATGAACTGGTGGTT120 
GGGATGGCTGGAGCCTGTGGAAGCGGGCTCGCCGTGGCGTTGGTCAACCGATTGTATATC180 
AGTCTATGCCTCTGTACACTTCGTCTCTAGCGAAGAGGAGTTGAATACAAATCTGTAAAA240 
CACTTGACTGTGTCTTCTAGCTATGAGACTCCCTTGCCTACTGGAGCCTTCAAGATACTT300 
TGGTACTGTATGAGACCACGCCTACCTCGACTTCATGTTTGAAACCAGTCAGTAATTCTC360 
TATGAACATGAAACAACACATTGATCTCTGTAACATCTCATTGCATAGTAAACCTTCTTA420 
CATTGATTACTGGCTATGAACAAAGGTTGTAGGGTAGGTAACGAAAAAAAAAAAAAAAAA480 
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACTCGAG536 
(2) INFORMATION FOR SEQ ID NO: 2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 243 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Trichoderma reesei 
(B) STRAIN: QM9414 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: 
GAATTCGGCACGAGCTCTTAACCAACCACCAAACTACAGCCACCCACCATGTCCGCCCAA60 
GACTACTACGTCCAGCGCCTCCGGAGGCAGCAGCAACGGTTATCCTCCTCAGCAGTACCA120 
CCAGCAGCAGCAACAGCCATACGGCCAGCAGCAGCCAGGGTATGACCAGCAGTACCCCGG180 
CGCCGGAGTCGATGGAGGCCCCGACGGAGAGCGCGGCCTCGGCGCGACCCTCGTCGGCGG240 
CGG243 
(2) INFORMATION FOR SEQ ID NO: 3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 289 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Trichoderma reesei 
(B) STRAIN: QM9414 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: 
CTGCAGCCGCCGGAGCCATTGGGGCGAATCTCATTGAGAACGCATACAAGAAGCACAAGG60 
AGGAAAAGATGTACAATGATGGCGGACACCATGGCCATCACCACCATTCGCATCATCATA120 
AGCACTAGAATGATTTGATGAAGAATATGACATTGCATGCCTGCTACATACGTAGATTAT180 
GATTGGGGGAGGGGCGTTTTTGATGAATATATATATATATCAATAAAAAAAAAAAAAAAA240 
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACTCGAG289 
(2) INFORMATION FOR SEQ ID NO: 4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 865 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Trichoderma reesei 
(B) STRAIN: QM9414 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: 
GAATTCGGCACGAGTTTTTTTTTTTTTTTTTACAGTATACGTTAGGTGTATTCGAACGAA60 
AGCCTGAAATAGGTACAAATAGCCACAACAGAAACGATTGTCTGACCTCCACTGAGAAAA120 
TCCCCCTCGTCCCCCAGACAGGTCTGATCAAACACCAGCTATCTACCTGCAAATGCCCCA180 
TATGTAGGTCACACAGTAAGGAGTGGATTCGCTATATACATCGGGTACGTTCGCATCACC240 
CCATGGCAAGGGAGGTGACTTAAGCAAAACCGCCACTAACCACAAAGCTCAACTGCATAG300 
TATCGACTTCAAGGAAAACACGGACAAATAATCATCATGGTTGCCTTTTGCAGCCTCATC360 
TGCGCTCTCACGAGGCATCGCCAGTACTCTGGCGATGCCCACAGGCTCGAGCCTGAGAGC420 
AGTGTCAACGTCACAGAGCGTGGCATGTACGACTTTGTTCTTGGAGCTCACAATGATCAT480 
CGCCGTCGTGCTAGCATCAACTACGACCAAAACTACCAAACTGGCGGACAAGTCAGCTAT540 
TCGCCTTCCAACACTGGCTTCTCAGTGAACTGGAACACTCAAGATGACTTTGTTGTGGGC600 
GTTGGTTGGACGACTGGATCTTCTGCTCCCATCAACTTTGGCGGCTCTTTTAGTGTCAAC660 
AGCGGAACTGGCCTGCTTTCCGTCTATGGCTGGAGCACCAACCCACTGGTTGAGTACTAC720 
ATCATGGAGGACAACCACAACTACCCAGCACAGGGCACCGTCAAGGGAACCGTCAACAGC780 
GACGGGGCACTTACACCATCTTGGGGGATTACCGTGTAACGAGCCTTCCATCCAGGGTAC840 
AGCGCCTTCAACCGTACATTTCCGT865 
(2) INFORMATION FOR SEQ ID NO: 5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 319 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Trichoderma reesei 
(B) STRAIN: QM9414 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: 
CAGATGAACTACCCAGGTTGTCGCTGTCGAAAGGCTGGGGTGGTAGTGGTTCTGCCTCAC60 
AGAGTGTCAGCAACTAGGTTCTGTTGATGTTGACTTGGAGTGGATGAGGGGTTTGAGCTG120 
GTATGTAGTATTGGGGTGGTTAGTGAGTTAACTTGACAGACTGCACTTTGGCAACAGAGC180 
CGACGATTAAGAGATTGCTGTCATGTAACTAAAGTAGCCTGCCTTTGACGCTGTATGCTC240 
ATGATACATGCGTGACATCGAAATATATCAGCCAAAGTATCCGTCCGGCGAAAAAAAAAA300 
AAAAAAAAAAAAACTCGAG319 
(2) INFORMATION FOR SEQ ID NO: 6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(iv) FRAGMENT TYPE: N-terminal 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: 
XaaXaaXaaPheValThrIleSerXaaThrGlnXaaXaaIleAsp 
151015 
(2) INFORMATION FOR SEQ ID NO: 7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(iv) FRAGMENT TYPE: N-terminal 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: 
XaaXaaXaaXaaXaaThrIleSerGlyThrGlnXaaXaaIleAsp 
151015 
__________________________________________________________________________