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

Abstract

A cellulase (endo-b-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 lmolAE min. TLC analysis showed that the cellulase produces cellotriose and cellobiose from insoluble cellooligosaccharides. However, a glucose assay linked with glucose oxidase detected a small amount of glucose, with a productivity of 0.072 lmolAEmin. 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 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; insect; purification.Cellulase (endo-b-1,4-glucanase) is a widespread enzyme in micro-organisms such as bacteria and fungi [1,2]. Until recently it was believed that cellulose digestion in animals was mediated by microbial cellulase activity in their intestine, and that no animals possessed endogenous cellulase. This traditional view of cellulase activity in animals was challenged by two reports of endogenous animal cellulase genes from plant-parasitic nematodes and a termite [3,4]. Since these discoveries, a number of other animal cellulase genes have been reported (summarized in [5]).Glycosyl hydrolases are categorized into 90 families according to amino-acid sequence similarity and hydrophobic cluster analysis, and among them, cellulases are found in 14 families [6,7] (refer also to: http://afmb.cnrs-mrs.fr/ CAZY/index.html). Known animal cellulases belong to three glycosyl hydrolase families (GHFs): GHF 5 (plantparasitic nematodes), GHF 9 (termites, cockroaches and crayfish) and GHF 45 (mussel and beetle). These three families are structurally unrelated and their evolutionary origins are likely to be independent.Larvae of the yellow-spotted longicorn beetle, Psacothea hilaris, feed on mulberry and fig trees, tunneling inside the stems and ingesting the living wood. The major constituent is cellulose (44.6%), followed by hemicellulose (28.5%); soluble sugars constitute only 4.7% of the dry weight of the wood [8]. The habitat of P. hilaris larvae suggests that they possess the ability to digest cellulo...

Plant cell wall degrading enzymes, pectinase and cellulase, in the digestive system of the red palm weevil, Rhynchophorus ferrugineus (Coleoptera: Curculionidae)

“…Cellulase (endo-β-1,4-glucanase) activity in Apriona germari (Coleoptera: Cerambycidae) and Anoplophora glabripennis (Coleoptera: Cerambycidae) was optimum at pH 4.4 and 5.2, respectively (Yin et al 2000;Li et al 2008). The studies on the isolation of cellulase from beetles demonstrated that optimum pH for the highest activity of cellulase from the larval gut of Psacothea hilaris against CMC was 5.5 (Sugimura et al 2003). The results showed that CaCl 2 and MgCl 2 increased significantly endo-β-1,4-glucanase activity in R. ferrugineus.…”

Microbial diversity in the larval gut of field and laboratory populations of the sugarcane weevil Sphenophorus levis (Coleoptera, Curculionidae)

“…Cellulolytic activities have been detected in the gut of beetle larvae, thus, the gut is considered to be a site for lignocellulose digestion (Watanabe and Tokuda, 2001). The expression of endogenous cellulases has been confirmed in the gut of mustard beetles, Phaedon cochlearine (Chrysomelidae; Sugimura et al, 2003). Thus, studies describing the microbiota of S. levis larvae may contribute to find novel cellulose-degrading genes within the gut of insect.…”

Current knowledge on genes and genomes of phytophagous beetles (Coleoptera: Chrysomeloidea, Curculionoidea): a review

“…There are genes that show higher levels of expression in certain tissues than in others or in specific stages of the beetle life cycle, so that studies focused on these genes benefit from a tissue or stage specific approach. This is the case of many studies interested in the characterisation of digestive enzymes, which use gut tissue as starting point (Girard & Jouanin, 1999a,b;Pedra et al, 2003;Sugimura et al, 2003;Zhu-Salzman et al, 2003;Bown et al, 2004;Gruden et al, 2004), or those centred in metabolic enzymes, focusing on the insect fat body, an organ involved in many homeostatic mechanisms and active centre of metabolic activity, where food reserves are stored in the insect and the maintenance of the balance between resources and requirements takes place (Smith et al, 1994;Vermunt et al, 1997Vermunt et al, , 1998Saito et al, 2004). In the case of genes specific of life cycle stages, those involved in the diapause or dormancy are a good example (Yocum, 2001(Yocum, , 2003.…”