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.
Kim
Namjung
N
College of Natural Resources and Life Science, Dong-A University, Busan 604-714, South Korea.
Choo
Young Moo
YM
Lee
Kwang Sik
KS
Hong
Seong Jin
SJ
Seol
Kwang Youl
KY
Je
Yeon Ho
YH
Sohn
Hung Dae
HD
Jin
Byung Rae
BR
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