Identification and characterisation of Bacillus subtilis as cellulase-producing bacteria

A.K.R. Emmyrafedziawati

Abstract

Three cellulolytic bacteria were isolated from oil palm empty fruit bunch (EFB) compost under aerobic conditions. The genomic DNA extracted from isolates C4, C12 and EB6 were amplified using specific primers L15 and L73 to determine the presence of genes encoding cellulase CelL15 and CelL73 respectively. The presence of the expected lengths of nucleotide bands at 1,500 bp and 730 bp respectively, indicated the presence of the putative cellulase genes in these isolates. The genes encoding the cellulases CelL15 and CelL73 were cloned into the Bacillus subtilis expression vector Escherichia coli strain JM107 to identify the cellulase gene in the recombinant plasmids. The filter paper assay (FPase) on C4, C12 and EB6 were determined after 48 h of incubation period at 37 °C. C12 isolate showed the highest FPase activity at 1.733 ± 0.023 FPU/ml followed by C4 at 1.718 ± 0.006 FPU/ml. EB6 showed the lowest FPase activity at 1.695 ± 0.006 FPU/ml. However, the ANOVA results showed that all three isolates of B.subtilis had no significant differences (p >0.05) in the FPase activity. The sequence alignment of the 3 isolates C4, C12 and EB6 showed that C4 and EB6 were the same strain while C12 differed slightly and this was confirmed by the presence of 2 cellulase genes in C12 compared to only 1 in C4 and EB6.

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