Identification, cloning, and expression of a GHF9 cellulase from Tribolium castaneum (Coleoptera: Tenebrionidae).
The availability of sequenced insect genomes has allowed for discovery and functional characterization of novel genes and proteins. We report use of the Tribolium castaneum (Herbst) (red flour beetle) genome to identify, clone, express, and characterize a novel endo-β-1,4-glucanase we named TcEG1 (T. castaneum endoglucanase 1). Sequence analysis of a full-length TcEG1 cDNA clone (1356bp) revealed sequence homology to enzymes in glycosyl hydrolase family 9 (GHF9), and verified presence of a change (Gly for Ser) in the conserved catalytic domain for GHF9 cellulases. This TcEG1 cDNA clone was predicted to encode a 49.5kDa protein with a calculated pI of 5.39. Heterologous expression of TcEG1 in Drosophila S2 cell cultures resulted in secretion of a 51-kDa protein, as determined by Western blotting. The expressed protein was used to characterize TcEG1 enzymatic activity against two cellulose substrates to determine its specificity and stability. Our data support that TcEG1 as a novel endo-β-1,4-glucanase, the first functional characterization of a cellulase enzyme derived from an insect genome with potential applications in the biofuel industry due to its high relative activity at alkaline pH.
Department of Entomology and Plant Pathology, University of Tennessee, 2431 Joe Johnson Drive, Knoxville, TN 37996-4560, USA.
Jonathan D
JD
Willis
Brenda
B
Oppert
Cris
C
Oppert
William E
WE
Klingeman
Juan L
JL
Jurat-Fuentes
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018929:Cell Culture Techniques; D002480:Cellulase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004330:Drosophila / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020049:Isoptera / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014231:Tribolium / Q000145:classification / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D019476:Insect Proteins; D002480:Cellulase