Abstract:
The use of lignocellulosic biomass for bioethanol production is largely dependent on
cost effective production of cellulase enzymes and most importantly, the availability
of cellulases with sufficient β-glucosidase activity for complete hydrolysis of cellulose
to glucose. Commercial cellulase preparations are often inefficient in the complete
hydrolysis of cellulose to glucose. The addition of β-glucosidases to commercial
cellulase preparations may enhance cellulolytic activity in the saccharification of
cellulose to fermentable sugars.
A β-glucosidase producing thermophilic bacterium, Anoxybacillus sp. KTC2 was
isolated from a hot geyser in the Zambezi Valley, Zimbabwe. The bacterium
identified through biochemical tests and 16S rDNA sequencing, had an optimal
growth temperature and pH of 60˚C and pH 8, respectively. The β-glucosidase
enzyme had an optimal temperature of 60˚C and a broad pH range for activity,
between 4.5 and 7.5 with an optimum at pH 7. The β-glucosidase enzyme retained
almost 100% activity after 24 hours’ incubation at 50˚C.
The Anoxybacillus sp. KTC2 β-glucosidase was partially purified and a partial amino
acid sequence obtained through MALDI-TOF analysis. The whole genome of
Anoxybacillus sp KTC2 β-glucosidase was sequenced and a β-glucosidase gene
identified. The deduced amino acid sequence corresponded to the peptide
sequences obtained through MALDI-TOF, confirming the presence of the a β glucosidase on the genome of Anoxybacillus sp KTC2. Analysis of the deduced
amino acid sequence revealed that the β-glucosidase enzyme belongs to the GH
family 1. The β-glucosidase gene was isolated by PCR and successfully cloned into
an E. coli expression system.
The saccharification efficiency of the β-glucosidase enzyme was evaluated through
the creation of enzyme cocktails with the commercial cellulase preparation,
CelluclastTM. CelluclastTM with the Anoxybacillus sp KTC2 β-glucosidase were used
to hydrolyse pure Avicel cellulose, at 50˚C over a 96 hour reaction time. The
Anoxybacillus sp KTC2 β-glucosidase enabled a 25% decrease in the total cellulose
loading without a decrease in the amount of glucose released.
