>Magister Scientiae - MSc / Biofuels are currently recognised as the most viable source of energy to replace
depleting fossil fuel reserves, with bioethanol the most popular alternative alcohol fuel.
Producing bioethanol from agricultural waste residues is a feasible socio-economic
industrial process. Lignocellulose, from which plant material is composed, is highly
recalcitrant to enzymatic degradation and therefore requires a suite of enzymes for
complete hydrolysis of the biomass. Metagenomes, particularly from extreme
environments, represent an unlimited resource for the discovery of novel biocatalysts for
inclusion in industrial processes. Here we report on the cloning and functional
characterisation of a novel thermophilic cellulase identified by the functional screening
of a Malawian, hotspring sediment metagenomic library. The gene encoding the
cellulase, celMHS, composed of 2,705 nucleotides and encoded a polypeptide of 905
amino acids with a predicted molecular mass of about 98 kDa. The in silico translated
protein, CelMHS, contained a putative transmembrane domain, a family 4 carbohydrate
binding motive (CBM 4), a truncated glycoside hydrolase family 42 (GH42) domain and
a N-terminal region that does not have sequence similarity to any previously described
domains. Functional characterisation of the recombinant CelMHS demonstrated that the
protein displayed an optimal pH of 6.0 and temperature of 100°C. CelMHS had high
specific activity toward substrates comprising of β-1,4 linked glucose subunits such as
carboxymethyl cellulose, β-D-glucan from barley and lichenan, however, some activity
was also observed against avicel, a crystalline cellulose substrate. HPLC analysis of the
hydrolysis products produced by CelMHS indicates that this particular enzyme prefers
longer chain oligosaccharides. This is, to the best of our knowledge, the first
investigation describing the cloning and characterization of a carbohydrate hydrolysing
enzyme comprised of the unique sequence architecture: a partial GH42 catalytic
domain, a CBM 4 and a unique N-domain sequence.
Key words: cellulose, cellulases, lignocellulosic biomass, bioethanol, saccharification,
hydrolysis, metagenomic library, thermophilic
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:uwc/oai:etd.uwc.ac.za:11394/4254 |
| Date | January 2013 |
| Creators | January, Timna |
| Contributors | Tuffin, Marla |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Rights | University of the Western Cape |
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