Abstract
Trichoderma reesei is a biotechnically important
filamentous fungus used commercially in enzyme
production. T. reesei is also one of the best known
cellulolytic organisms, producing readily and in large
quantities a complete set of extracellular cellulases for
the degradation of crystalline cellulose. In addition to
T. reesei, a wide variety of other bacteria and fungi
also produce cellulolytic enzymes. Cellulases originating
from various organisms and having different
characteristics are used industrially in many
applications, such as in the textile industry in
finishing of denim fabric to impart a stonewashed
appearance (biostoning) and in biofinishing of cotton.
In this work T. reesei strains producing significant
amounts of homologous and heterologous cellulases and
having defined cellulase profiles were constructed for
specific industrial applications, i.e. biostoning and
biofinishing of cotton. The production of T. reesei
endoglucanase II (EGII), cellobiohydrolases I and II
(CBHI and CBHII) was improved in separate strains.
Strains producing high amounts of EGI and EGII without
CBHs or CBHI and CBHII without the main EGs were also
constructed. The cellulase genes were expressed under the
powerful T. reesei cbh1 promoter; in a transformant
overproducing both CBHI and CBHII, the cbh2 promoter was
also used for cbh2 expression. The level of endoglucanase
activity produced by the EGII-overproducing transformants
correlated with the copy number of the egl2 expression
cassette. Production of the major secreted cellulase CBHI
was increased up to 1.5-fold and production of CBHII
fourfold compared with the parent strain. In
transformants overproducing both CBHI and CBHII,
production of CBHI was increased up to 1.6-fold and
production of CBHII up to 3.4-fold as compared with the
host strain and approximately similar amounts of CBHII
protein were produced by using the cbh1 or cbh2
promoters.
The enzyme preparation with elevated EGII content most
clearly improved the biostoning of denim fabric and the
biofinishing of cotton fabric. Better depilling and
visual appearance were achieved with the enzyme
preparation having an elevated CBHII content compared to
the wild type preparation in biofinishing of cotton, but
the improvement was not as pronounced as in the case of
the EGII preparation.
Novel neutral cellulases were demonstrated to have
potential in biostoning. The cellulase preparation of the
thermophilic fungus Melanocarpus albomyces was found to
be effective in releasing dye from indigo-dyed denim and
to cause low backstaining at neutral pH. M. albomyces
produces at least three cellulases and these cellulases
with an effect on biostoning were purified and the genes
encoding them were cloned and sequenced. Ma 20 kDa EGV
(Ma Cel45A) belongs to the glycosyl hydrolase family 45
and the 50 kDa EGI (Ma Cel7A) and CBHI (Ma Cel7B) to
family 7. None of the cellulases harbours a cellulose
binding domain. Especially purified Ma Cel45A performed
well in biostoning. The Ma cellulases were produced in T.
reesei under the T. reesei cbh1 promoter for biostoning
applications. The endoglucanase production levels of Ma
cel45A- and cel7A-transformants were several times higher
than those of the parental M. albomyces strain. The
cellulase preparation produced by the recombinant Ma
cel45A transformant performed well at neutral pH in the
finishing of denim fabric and caused considerably less
backstaining than the acid cellulase product of T.
reesei.
Original language | English |
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Qualification | Doctor Degree |
Awarding Institution |
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Supervisors/Advisors |
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Award date | 5 Nov 2004 |
Place of Publication | Espoo |
Publisher | |
Print ISBNs | 951-38-6416-2 |
Electronic ISBNs | 951-38-6417-0 |
Publication status | Published - 2004 |
MoE publication type | G5 Doctoral dissertation (article) |
Keywords
- cellulase
- cloning
- Trichoderma reesei
- Melanocarpus albomyces
- homologous and heterologous gene expression
- biostoning
- biofinishing
- textile industry