Cloning, molecular modeling and characterization of acidic cellulase from buffalo rumen and its applicability in saccharification of lignocellulosic biomass
2018
Dadheech, Tripti | Shah, Ravi | Pandit, Ramesh | Hinsu, Ankit | Chauhan, Prakram Singh | Jakhesara, Subhash | Kunjadiya, Anju | Rank, Dharamshibhai | Joshi, Chaitanya
Cellulase hydrolyses the cellulose by cleaving the β-1,4-linkages to produce mono-, oligo- and shorter polysaccharide units. These enzymes have applications in various industries such as pulp and paper, laundry, food and feed, textile, brewing industry and in biofuel production. In the present study we have cloned acid-cellulase gene (Cel-1) from the fosmid library of buffalo rumen metagenomic DNA and functionally expressed it in Escherichia coli. The ORF encoding cellulase consisted of 1176-bp, corresponding to protein of 391 amino acid and has catalytic domain belonging to glycosyl hydrolase family 5. The purified protein has a molecular weight of 43-kDa on SDS-PAGE and its expression was confirmed by western blotting. The tertiary structure of the cellulase (Cel-1) showed a classical (α/β) TIM-like barrel motif. Model surface charge of Cel-1 predicted that surface near active site was mostly negative which might be responsible for the stability of enzyme at lower pH. The pH and temperature for maximum enzyme activity were 4.5 and 45°C respectively. Various metal ions enhanced the enzyme activity and in presence of Mn+2 activity was significantly increased. Cel-1 hydrolyzed pre-treated wheat straw and released reducing sugars (62.60%). These desirable properties of Cel-1 make it attractive for the bioconversion of biomass.
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