Molecular cloning and characterization of a glycosyl hydrolase family 9 cellulase distributed throughout the digestive tract of the cricket Teleogryllus emma.
A novel endogenous beta-1,4-endoglucanase (EG) gene belonging to the glycosyl hydrolase family 9 (GHF 9) that is distributed throughout the digestive tract of the cricket Teleogryllus emma was cloned and characterized. This gene, named TeEG-I, consists of eight exons encoding 453 amino acid residues and exists as a single copy in the T. emma genome. TeEG-I possesses all the features, including signature motifs and catalytic domains, of GHF 9 members, sharing high levels of identity with the termite, Mastotermes darwiniensis (64% protein sequence identity), and the cockroach, Panesthia cribrata (62%), GHF 9 cellulases. Recombinant TeEG-I, which is expressed as a 47-kDa polypeptide in baculovirus-infected insect Sf9 cells, showed an optimal pH and temperature of pH 5.0 and 40 degrees C. The K(m) and V(max) values for digestion of carboxymethyl cellulose were 5.4 mg/ml and 3118.4 U/mg, respectively. Northern and Western blot analyses revealed that TeEG-I is present throughout the digestive tract, which correlated with the TeEG-I distribution and cellulase activity in the digestive tract as assayed by immunofluorescence staining and enzyme activity assay, respectively. These results indicate that TeEG-I is distributed throughout the entire digestive tract of T. emma, suggesting a functional role of endogenous TeEG-I in a sequential cellulose digestion process throughout the T. emma digestion tract.
College of Natural Resources and Life Science, Dong-A University, Busan 604-714, South Korea.
Namjung
N
Kim
Young Moo
YM
Choo
Kwang Sik
KS
Lee
Seong Jin
SJ
Hong
Kwang Youl
KY
Seol
Yeon Ho
YH
Je
Hung Dae
HD
Sohn
Byung Rae
BR
Jin
D000595:Amino Acid Sequence; D000818:Animals; D015139:Blotting, Southern; D015153:Blotting, Western; D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D041981:Gastrointestinal Tract / Q000201:enzymology*; D006135:Gryllidae / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
D002480:Cellulase