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- Cloning and Functional Characterization of Endo-β-1,4-Glucanase Gene from Metagenomic Library of Vermicompost
- Muhammad Yasir , Haji Khan , Syed Sikander Azam , Amar Telke , Seon Won Kim , Young Ryun Chung
- J. Microbiol. 2013;51(3):329-335. Published online June 28, 2013
- DOI: https://doi.org/10.1007/s12275-013-2697-5
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- 14 Scopus
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Abstract
- In the vermicomposting of paper mill sludge, the activity of earthworms is very dependent on dietetic polysaccharides including cellulose as energy sources. Most of these polymers are degraded by the host microbiota and considered potentially important source for cellulolytic enzymes. In the present study, a metagenomic library was constructed from vermicompost (VC) prepared with paper mill sludge and dairy sludge (fresh sludge, FS) and functionally screened for cellulolytic activities. Eighteen cellulase expressing clones were isolated from about 89,000 fosmid clones libraries. A short fragment library was constructed from the most active positive clone (cMGL504) and one open reading frame (ORF) of 1,092 bp encoding an endo-β-1,4-glucanase was indentified which showed 88% similarity with Cellvibrio mixtus cellulase A gene. The endo-β-1,4-glucanase cmgl504 gene was overexpressed in Escherichia coli. The purified recombinant cmgl504 cellulase displayed activities at a broad range of temperature (25–55°C) and pH (5.5–8.5). The enzyme degraded carboxymethyl cellulose (CMC) with 15.4 U, while having low activity against avicel. No detectable activity was found for xylan and laminarin. The enzyme activity was stimulated by potassium chloride. The deduced protein and three-dimensional structure of metagenomederived cellulase cmgl504 possessed all features, including general architecture, signature motifs, and N-terminal signal peptide, followed by the catalytic domain of cellulase belonging to glycosyl hydrolase family 5 (GHF5). The cellulases cloned in this work may play important roles in the degradation of celluloses in vermicomposting process and could be exploited for industrial application in future.
- Identification and Functional Analysis of a Gene Encoding β-Glucosidase from the Brown-Rot Basidiomycete Fomitopsis palustris
- Hwang-Woo Ji , Chang-Jun Cha
- J. Microbiol. 2010;48(6):808-813. Published online January 9, 2011
- DOI: https://doi.org/10.1007/s12275-010-0482-2
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- 7 Scopus
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Abstract
- The brown-rot basidiomycete Fomitopsis palustris is known to degrade crystalline cellulose (Avicel) and produce three major cellulases, exoglucanases, endoglucanases, and β-glucosidases. A novel β-glucosidase designated as Cel3A was identified from F. palustris grown at the expense of Avicel. The deduced amino acid sequence of Cel3A showed high homology with those of other fungal β-glucosidases that belong to glycosyl hydrolase (GH) family 3. The sequence analysis also indicated that Cel3A contains the N- and C-terminal domains of GH family 3 and Asp-209 was conserved as a catalytic nucleophile. The cloned gene was successfully expressed in the yeast Pichia pastoris and the recombinant protein exhibited β-glucosidase activity with cellobiose and some degree of thermostability. Considering the size and sequence of the protein, the β-glucosidase identified in this study is different from the protein purified directly from F. palustris in the previous study. Our results suggest that the fungus possesses at least two β-glucosidase genes.
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