Prospecting for Cellulase Enzymes Based on Sequences and Functional Screening from Metagenomic LibrariesC. Wiwat* and J. Sillapee
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Microorganisms dominate the biosphere, yet current methods of culture reveal less than 1% of the microbial diversity. There is a vast amount of information held within the genomes of uncultured microorganisms, and metagenomics is one of the key technologies used to access and investigate this potential. Cellulose is an abundant biopolymer and sustainable bioconversion from agricultural waste to value products such as biofuel. Thus cellulase is attractive for their potential in the biotechnological application. Mangrove soils are in general acidic in nature and extremely salt condition can appear some bacterial strains that can tolerant to salinity condition and also can produce extracellular cellulase enzyme. In order to exploit the most of microbial diversity resources to find bioactive compounds or genes, mangrove soil metagenomic libraries were constructed. DNA was directly extracted from the selected mangrove soil, Satul province, fragments of the soil DNA were ligated to an expression plasmid pBluescriptII SK(+), transformed to surrogate host Escherichia coli DH5?, and the white clones out on LB medium were selected to accumulate libraries. More than 100 positive clones with average insert size of about 1 kb were collected. The libraries were screened for cellulase enzyme producer. Over 20 clones with cellulase producing were found out from these libraries by cellulase screening method using carboxymethyl cellulose as a substrate. The two cellulolytic clones, celP8 and celP24, were shown the best activity after screening. There were chosen for sequencing and the size of the inserted gene of celP8 and celP24 are 421 and 127 bp, respectively. Theoretical predicted size of protein of celP8 and celP24 are 15,436 dal and 4,656 dal, respectively. Addition with blasting in GenBank, there are no similar sequences found from all known cellulase genes. Further characterization, the optimal pH of celP8 and celP24 were presented at pH 10.0 and 11.0, respectively. While the optimal temperature of celP8 and celP24 were 35?C and 25?C, respectively. SDS-PAGE and zymogram analysis demonstrated that celP8 and celP24 had CMCase activity indicating those are endoglucanases. Even their homology searches were no cellulose-binding domain (CBD) or conserved domain of any classes of cellulases. It is suggested that they may be new or undiscovered subclass of cellulolytic enzyme from mangrove soils from the south of Thailand.
Keyword:
Metagenome, Mangrove soil, Cellulase.
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