Identification and characterization of a cellulase-encoding gene from the buffalo rumen metagenomic library.
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.
Food Engineering and Bioprocess Technology, Asian Institute of Technology, Pathumthani, Thailand.
Nhung Hong
NH
Nguyen
Lalita
L
Maruset
Tanaporn
T
Uengwetwanit
Wuttichai
W
Mhuantong
Piyanun
P
Harnpicharnchai
Verawat
V
Champreda
Sutipa
S
Tanapongpipat
Kanya
K
Jirajaroenrat
Sudip K
SK
Rakshit
Lily
L
Eurwilaichitr
Somchai
S
Pongpattanakitshote
D000595:Amino Acid Sequence; D000821:Animal Feed; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002020:Buffaloes; D044602:Cellulases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D015723:Gene Library; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D008031:Lignin / Q000378:metabolism*; D054892:Metagenome; D056186:Metagenomics; D059013:Microbial Consortia / Q000235:genetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010802:Phylogeny; D012417:Rumen / Q000201:enzymology / Q000382:microbiology; D017422:Sequence Analysis, DNA; D013696:Temperature; D014990:Xylans / Q000378:metabolism
D001426:Bacterial Proteins; D014990:Xylans; C036909:lignocellulose; D008031:Lignin; D044602:Cellulases