Abstract
A key issue in the realization of fuel ethanol production from
lignocellulosics is the utilization of hydrolytic enzymes with special
characteristics (e.g. high cellulase activity at elevated operation
temperatures) for efficient enzymatic hydrolysis of the technical substrate.
Novel thermostable cellulases of the fungal ascomycete Melanocarpus albomyces
are seen as promising candidates for this task. Prior to their introduction to
the actual industrial process, hydrolytic properties of novel enzymes have to
be characterised, preferably using model substrates as the first approach of
investigation.
In our present study, two of the previously identified M. albomyces
cellulases, a 50-kDa endoglucanase (EG) Cel7A, and a 50-kDa cellobiohydrolase
(CBH) Cel7B, produced in recombinant strains of the filamentous fungus
Trichoderma reesei, were purified (DEAE-IEX) and tested in hydrolysis (60°C,
pH 6.0) of crystalline and amorphous cellulose. Enzymes were studied both in
their native forms, in which they do not bear a cellulose binding domain
(CBD), and also with a modified structure, in which they harbour the CBD of T.
reesei CBHI. Time courses of hydrolysis experiments were evaluated via
monitoring the release of reducing sugars by the DNS method. Product profiles
were analysed by high performance anion exchange chromatography (HPAEC).
Both enzymes were active against tested substrates: Cel7B had greater activity
than Cel7A against crystalline cellulose, whereas in the case of amorphous
substrate the order was reversed. Evidence for synergism was obtained when
mixtures of the two enzymes were used with a constant total protein dosage.
The presence of CBD enhanced hydrolysis rate in all experimental
configurations. When individual enzymes were tested, CBD had a greater effect
on the performance of CBH than that of EG, especially on crystalline
substrate. In synergism, the positive role of CBD was even more evident.
Comparison of Cel7B (+/-CBD) to corresponding enzymes of T. reesei (CBHI and
CBHI core, respectively) indicated, that novel CBH of M. albomyces is more
active on amorphous cellulose than the industrially relevant enzyme
preparation from T. reesei under the circumstances applied hereby. Although
using crystalline substrate a reverse order was observed, the higher T optima
(approx. 70°C, at pH 7.0 with up to 60 min reaction time) of novel cellulases
gives evidence for their potential to serve as attractive tools in
high-temperature industrial hydrolysis processes.
Original language | English |
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Publication status | Published - 2005 |
MoE publication type | Not Eligible |
Event | Renewable Resources and Biorefineries - Ghent, Belgium Duration: 19 Sept 2005 → 21 Sept 2005 |
Conference
Conference | Renewable Resources and Biorefineries |
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Country/Territory | Belgium |
City | Ghent |
Period | 19/09/05 → 21/09/05 |