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Functional expression and structural characterization of ORF cDNA encoding chitinase of the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae)

Author:
Park, Hee Yun, Paek, Aron, Jeong, Seong Eun
Source:
Journal of Asia-Pacific entomology 2012 v.15 no.1 pp. 45-50
ISSN:
1226-8615
Subject:
Bombycidae, Escherichia coli, N-acetylglucosamine, Spodoptera exigua, active sites, amino acids, cell growth, chitin, chitinase, complementary DNA, exoskeleton, glucosamine, insects, instars, molecular weight, molting, nucleotides, nutrients, open reading frames, peritrophic membrane, phylogeny, signal peptide, stop codon
Abstract:
Chitin is an important component of the exoskeleton and peritrophic matrix in insects. Its bio-degradation is initiated by the endo-splitting chitinase. We cloned an ORF cDNA encoding chitinase from the last instar larva of the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae), into E. coli to confirm its functionality. Its amino acid sequence was compared with previously described lepidopteran chitinases. S. exigua chitinase expression enhanced cell growth approx. 1.5 fold in transformed E. coli than in the wild strain in a 1% colloidal chitin-containing medium with insufficient regular nutrients. Compared with the wild strain, the two intracellular chitin degradation derivatives, glucosamine and N-acetylglucosamine, increased approx. 5.8 and 1.5 fold, respectively, and extracellular chitinase activity in the transformed strain was about 1.6 fold higher. The ORF of S. exigua chitinase-encoding cDNA including stop codon was composed of 1674bp nucleotides and the calculated molecular weight of the deduced 557 amino acid residues was about 62.6kDa. The ORF consisted of an N-terminal leading signal peptide (AA 1–20), a catalytic domain (AA 21–392), a linker region (AA 393–493), and a C-terminal chitin-binding domain (AA 494–557) showing a typical molting fluid chitinase structure. Phylogenetic analysis determined that all 5 noctuid chitinases were grouped together, while two bombycid enzymes and one tortricid enzyme mapped together in one lineage. In the noctuid group, the sub-lineages reflected their taxonomic relationships at the Genus level.
Agid:
870381