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Production optimization and biochemical characterization of cellulase from Geobacillus species isolated from hot spring water
Subash Khadka, Purnima Baidya, Jyoti Maharjan

Last modified: 2017-08-24

Abstract


ABSTRACT

This study aims to produce and characterize thermostable cellulase from a thermophilic bacterium isolated from hot spring water sample and identify the isolate using phenotypic and molecular methods. Enrichment and culture of water sample was used for isolation of bacterial strains and an isolate with highest cellulase activity was chosen for enzyme production, partial purification and biochemical characterization. Different staining techniques, enzymatic tests and 16S rRNA sequencing were used for identification of the isolate. The organism was Gram positive rod with terminal endospores and growing optimally between 55°C to 65°C and pH 5.5 to 6.5. Based on partial 16S rDNA sequence analysis, the isolate was designated as Geobacillus sp. KP43. Optimum production of cellulase was observed on incubation at 55°C for 18 hours in a medium containing 1% Carboxymethyl cellulose (CMC), 0.04% yeast extract and having pH 6.5. The molecular weight of the enzyme was found to be approximately 66kDa on 12% SDS-PAGE analysis. The partially purified enzyme showed temperature optima of 70°C and demonstrated its activity over a wide pH range from 5 to 11. Further, enzyme activity was markedly stimulated by metal ion Fe++. Apart from cellulases, the isolate also depicted good xylanase (EC 3.2.1.8), cellobiase (EC 3.2.1.21) and amylase (EC 3.2.1.1) activities. Thermophilic growth with a variety of extracellular enzyme activities at elevated temperature as well as in a wide pH range make this organism suitable for biotechnological and industrial applications.

Keywords: Geobacillus, cellulases, thermophilic, hot spring, 16S rDNA

 


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