Cloning and identification of novel hydrolase genes from a dairy cow rumen metagenomic library and characterization of a cellulase gene.
BACKGROUND
Interest in cellulose degrading enzymes has increased in recent years due to the expansion of the ellulosic biofuel industry. The rumen is a highly adapted environment for the degradation of cellulose and a promising source of enzymes for industrial use. To identify cellulase enzymes that may be of such use we have undertaken a functional metagenomic screen to identify cellulase enzymes from the bacterial community in the rumen of a grass-hay fed dairy cow.


RESULTS
Twenty five clones specifying cellulose activity were identified. Subcloning and sequence analysis of a subset of these hydrolase-positive clones identified 10 endoglucanase genes. Preliminary characterization of the encoded cellulases was carried out using crude extracts of each of the subclones. Zymogram analysis using carboxymethylcellulose as a substrate showed a single positive band for each subclone, confirming that only one functional cellulase gene was present in each. One cellulase gene, designated Cel14b22, was expressed at a high level in Escherichia coli and purified for further characterization. The purified recombinant enzyme showed optimal activity at pH 6.0 and 50°C. It was stable over a broad pH range, from pH 4.0 to 10.0. The activity was significantly enhanced by Mn2+ and dramatically reduced by Fe3+ or Cu2+. The enzyme hydrolyzed a wide range of beta-1,3-, and beta-1,4-linked polysaccharides, with varying activities. Activities toward microcrystalline cellulose and filter paper were relatively high, while the highest activity was toward Oat Gum.


CONCLUSIONS
The present study shows that a functional metagenomic approach can be used to isolate previously uncharacterized cellulases from the rumen environment.
Agriculture and Agri-Food Canada, Lethbridge Research Centre, Lethbridge, Alberta, T1J 4B1, Canada.
Xia
X
Gong
Robert J
RJ
Gruninger
Meng
M
Qi
Lyn
L
Paterson
Robert J
RJ
Forster
Ron M
RM
Teather
Tim A
TA
McAllister
D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002266:Carboxymethylcellulose Sodium / Q000378:metabolism*; D002417:Cattle; D044602:Cellulases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003300:Copper / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D015698:Genomic Library; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008345:Manganese / Q000378:metabolism; D054892:Metagenome; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012417:Rumen / Q000382:microbiology*; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D008345:Manganese; D003300:Copper; D007501:Iron; D044602:Cellulases; D002266:Carboxymethylcellulose Sodium