Isolation and characterization of novel cellulase and pectinase producing bacteria
Paudel, Yagya Prasad
Master of Science
SubjectCellulases and pectinases (industrial applications)
Microbial cellulases and pectinases
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Cellulases and pectinases are the major cell wall degrading enzymes. The microorganisms producing these enzymes have a wide range of industrial applications. In this research, 17 bacterial isolates from rotting wood samples were screened for their cellulase activity by carboxymethyl cellulose (CMC) plate assay. Similarly, a bacterial strain isolated from the gut of western honey bee (Apis mellifera L.) showed polygalacturonase activity by pectin agar plate assay. The bacterial isolates showing higher region of depolymerisation were further assayed for their activities for producing the enzymes quantitatively and they were identified on the basis of 16S rDNA sequence analysis. The protein gel was run using SDS-PAGE for molecular weight determination of the cellulase and polygalacturonase. The sequences of two isolates producing cellulase (Bacillus sp K1 and Bacillus sp. A0) and one isolate producing polygalacturonase (Bacillus sp. HD2) were successfully uploaded to the NCBI data base. The enzymes produced by isolates K1 and HD2 were characterized. The isolate K1 produced the maximum CMCase at pH 6 and 50 oC in presence of peptone (1%) as a source of nitrogen. The enzyme activity was stimulated by Ca2+ (2 mM) by 20% over the control. Agave biomass was fermented by using two cellulase producing isolates K1 and A0 and ethanol was detected by using micro-dichromate method. Both the strains produced ethanol using untreated Agave biomass. Similarly, the polygalacturonase produced by HD2 strain exhibited enzyme activity in a wide range of pH from pH 5-12. The production was enhanced by using yeast extract (3%) in the production medium and the enzyme activity was stimulated by Ca2+ (2 mM) and SDS (200 mM). In SDS-PAGE gel, the molecular weights of cellulase enzymes produced by K1 and A0 were ~36 kDa and ~40 kDa respectively and the two clear bands of polygalacturonases produced by isolate HD2 were found at ~36 kDa and ~72 kDa.