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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 한국미생물학회 2013 The journal of microbiology Vol.51 No.3

        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.

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        Proteome‑wide subtractive approach to prioritize a hypothetical protein of XDR‑Mycobacterium tuberculosis as potential drug target

        Reaz Uddin,Quratulain Nehal Siddiqui,Muhammad Sufian,Syed Sikander Azam,Abdul Wadood 한국유전학회 2019 Genes & Genomics Vol.41 No.11

        Background Among the resistant isolates of MTB, multidrug resistant tuberculosis (MDR-TB) and extensively drug resistant tuberculosis (XDR-TB) have been the areas of growing concern. The genomic analysis showed that the respective genomic pool of the XDR-MTB proteome contains more than 30% of the hypothetical proteins for which no functions have been annotated yet. This class of proteins presumably have their own importance to complete genome and proteome information. The bioinformatics advancements have helped to annotate those hypothetical proteins by using various computational tools and have potential to classify them functionally. Objective The objective of this study was to propose a new and unique drug target against the deadly Mycobacterium tuberculosis using Bioinformatics approaches to characterize the hypothetical proteins. Results We stepwise reduced the hypothetical proteins (total number: 1256) out of the complete proteome to only 26 essential hypothetical proteins. Out of those 26 proteins, the protein WP_003401246.1 was computationally characterized as the druggable target. Conclusion The study proposed a hypothetical protein from complete proteome of the XDR-MTB as a new drug target against which new drug candidates can be proposed. Hence, the study opens up the new avenues in the areas of drug discovery against deadly M. tuberculosis.

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