Abstract
Cellulose is an important industrial raw material and a source of
renewable energy. Cellulases catalyse the hydrolysis of cellulose to smaller
oligosaccharides. This catalysis is exploited in many biotechnological
processes. In the present study, a one-domain cellobiohydrolase (Cel7B) from
the thermophilic fungus Melanocarpus albomyces was expressed in Saccharomyces
cerevisiae. The expression plasmid pSV7 contains Cel7B cDNA under a
constitutive triose phosphate isomerase (TPI) promoter, and URA3 was used as
the selection marker. This yeast expression system makes the Cel7B available
for directed evolution methods to improve its characteristics, i.e.
thermostability. The cDNA sequence coding for the M. albomyces Cel7B was
subjected to random mutagenesis by an error-prone PCR method resulting into a
mutation rate on average of 1.7 amino acid changes per gene. After one round
of random mutagenesis, a robotic screen of 15,000 clones was performed. Mutant
libraries were screened in a 96-well microtiter plate format for higher
temperature stability in an enzymatic activity assay based on a soluble
chromophoric substrate, methylumbelliferyl lactoside (MUL). The yeast
supernatants were pre-incubated at 76°C, and the activity after the heat
treatment was compared to the activity of the untreated control samples.
Mutants with improved thermostability were chosen for replica assays, after
which a manual secondary screen was performed. The most interesting mutant
cellulases from the secondary screen are now being purified for more detailed
enzymatic characterisation.
Original language | English |
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Publication status | Published - 2004 |
MoE publication type | Not Eligible |
Event | Summer Course Glycosciences 2004 - Wageningen, Netherlands Duration: 28 Jun 2004 → 1 Jul 2004 |
Course
Course | Summer Course Glycosciences 2004 |
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Country/Territory | Netherlands |
City | Wageningen |
Period | 28/06/04 → 1/07/04 |