Identification and Characterization of a Cellulase-Encoding Gene from the Buffalo Rumen Metagenomic Library

• Published in: Bioscience, biotechnology, and biochemistry biotechnology, and biochemistry, 2012, Vol.76 (6), p.1075-1084
• Main Authors: NGUYEN, Nhung Hong, MARUSET, Lalita, UENGWETWANIT, Tanaporn, MHUANTONG, Wuttichai, HARNPICHARNCHAI, Piyanun, CHAMPREDA, Verawat, TANAPONGPIPAT, Sutipa, JIRAJAROENRAT, Kanya, RAKSHIT, Sudip K., EURWILAICHITR, Lily, PONGPATTANAKITSHOTE, Somchai
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animal Feed; Animals; Bacteria; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Biological and medical sciences; buffalo rumen; Buffaloes; Cellulase; Cellulases - genetics; Cellulases - isolation & purification; Cellulases - metabolism; Cellulose; Cloning, Molecular; Encoding; Enzyme Stability; Enzymes; Fundamental and applied biological sciences. Psychology; Gene Library; Genes; Genetic Vectors; Hydrogen-Ion Concentration; Libraries; Lignin - metabolism; Metagenome; Metagenomics; Microbial Consortia - genetics; Molecular Sequence Data; Open Reading Frames; Phylogeny; pyrosequencing; Rumen - enzymology; Rumen - microbiology; Sequence Analysis, DNA; Temperature; xylanase; Xylans - metabolism
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.110786

Microorganisms residing in the rumens of cattle represent a rich source of lignocellulose-degrading enzymes, since their diet consists of plant-based materials that are high in cellulose and hemicellulose. In this study, a metagenomic library was constructed from buffalo rumen contents using pCC1FOS fosmid vector. Ninety-three clones from the pooled library of approximately 10,000 clones showed degrading activity against AZCL-HE-Cellulose, whereas four other clones showed activity against AZCL-Xylan. Contig analysis of pyrosequencing data derived from the selected strongly positive clones revealed 15 ORFs that were closely related to lignocellulose-degrading enzymes belonging to several glycosyl hydrolase families. Glycosyl hydrolase family 5 (GHF5) was the most abundant glycosyl hydrolase found, and a majority of the GHF5s in our metagenomes were closely related to several ruminal bacteria, especially ones from other buffalo rumen metagenomes. Characterization of BT-01, a selected clone with highest cellulase activity from the primary plate screening assay, revealed a cellulase encoding gene with optimal working conditions at pH 5.5 at 50 °C. Along with its stability over acidic pH, the capability efficiently to hydrolyze cellulose in feed for broiler chickens, as exhibited in an in vitro digestibility test, suggests that BT-01 has potential application as a feed supplement.