Purification, characterization, cDNA cloning and nucleotide sequencing of a cellulase from the yellow-spotted longicorn beetle, Psacothea hilaris.
A cellulase (endo-beta-1,4-glucanase, EC 3.2.1.4) was purified from the gut of larvae of the yellow-spotted longicorn beetle Psacothea hilaris by acetone precipitation and elution from gels after native PAGE and SDS/PAGE with activity staining. The purified protein formed a single band, and the molecular mass was estimated to be 47 kDa. The purified cellulase degraded carboxymethylcellulose (CMC), insoluble cello-oligosaccharide (average degree of polymerization 34) and soluble cello-oligosaccharides longer than cellotriose, but not crystalline cellulose or cellobiose. The specific activity of the cellulase against CMC was 150 micro mol.min-1.(mg protein)-1. TLC analysis showed that the cellulase produces cellotriose and cellobiose from insoluble cello-oligosaccharides. However, a glucose assay linked with glucose oxidase detected a small amount of glucose, with a productivity of 0.072 micro mol.min-1.(mg protein)-1. The optimal pH of P. hilaris cellulase was 5.5, close to the pH in the midgut of P. hilaris larvae. The N-terminal amino-acid sequence of the purified P. hilaris cellulase was determined and a degenerate primer designed, which enabled a 975-bp cDNA clone containing a typical polyadenylation signal to be obtained by PCR and sequencing. The deduced amino-acid sequence of P. hilaris cellulase showed high homology to members of glycosyl hydrolase family 5 subfamily 2, and, in addition, a signature sequence for family 5 was found. Thus, this is the first report of a family 5 cellulase from arthropods.
Sugimura
Masahiro
M
National Institute of Agrobiological Sciences, Ibaraki, Japan.
Watanabe
Hirofumi
H
Lo
Nathan
N
Saito
Hitoshi
H
D000818:Animals; D002480:Cellulase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism; D003001:Cloning, Molecular; D001517:Coleoptera / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D005766:Gastrointestinal Contents / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007814:Larva / Q000201:enzymology / Q000235:genetics / Q000378:metabolism
D002482:Cellulose; D002480:Cellulase