Purification, characterization, cDNA cloning and nucleotide sequencing of a cellulase from the yellow‐spotted longicorn beetle, Psacothea hilaris

A cellulase (endo‐β‐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 µmol·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 µmol·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.