tailieunhanh - Báo cáo khoa học: Purification, characterization, cDNA cloning and nucleotide sequencing of a cellulase from the yellow-spotted longicorn beetle, Psacothea hilaris

A cellulase (endo-b-1,4-glucanase, EC ) was purified from the gut of larvae of the yellow-spotted longicorn beetle Psacothea hilarisby 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 cellulasedegradedcarboxymethylcellulose (CMC), insoluble cello-oligosaccharide (average degree of polymerization 34) and soluble cello-oligosaccharides longer than cellotriose, but not crystalline cellulose or cellobiose. . | Eur. J. Biochem. 270 3455-3460 2003 FEBS 2003 doi Purification characterization cDNA cloning and nucleotide sequencing of a cellulase from the yellow-spotted longicorn beetle Psacothea hilaris Masahiro Sugimura1 Hirofumi Watanabe1 Nathan Lo1 and Hitoshi Saito2 National Institute of Agrobiological Sciences Ibaraki Japan department of Applied Biology Faculty of Textile Science Kyoto Institute of Technology Japan A cellulase endo-b-1 4-glucanase EC was purified from the gut oflarvae 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 Ltmol-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 pmol-min-1- mg protein -1. The optimal pH of P. hilaris cellulase was 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 aminoacid 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. Keywords cDNA cloning cellulase endoglucanase .