title: Molecular cloning and functional expression of chitinase-encoding cDNA from the cabbage moth, Mamestra brassicae.
abstract: Chitinase is a rate-limiting and endo-splitting enzyme involved in the bio-degradation of chitin, an important component of the cuticular exoskeleton and peritrophic matrix in insects. We isolated a cDNA-encoding chitinase from the last larval integument of the cabbage moth, Mamestra brassicae (Lepidoptera; Noctuidae), cloned the ORF cDNA into E. coli to confirm its functionality, and analyzed the deduced amino acid sequence in comparison with previously described lepidopteran chitinases. M. brassicae chitinase expressed in the transformed E. coli cells with the chitinase-encoding cDNA enhanced cell proliferation to about 1.6 times of the untransformed wild type strain in a colloidal chitin-including medium with only a very limited amount of other nutrients. Compared with the wild type strain, the intracellular levels of chitin degradation derivatives, glucosamine and N-acetylglucosamine were about 7.2 and 2.3 times higher, respectively, while the extracellular chitinase activity was about 2.2 times higher in the transformed strain. The ORF of M. brassicae chitinaseencoding cDNA consisted of 1686 nucleotides (562 amino acid residues) except for the stop codon, and its deduced amino acid composition revealed a calculated molecular weight of 62.7 and theoretical pI of 5.3. The ORF was composed of N-terminal leading signal peptide (AA 1-20), catalytic domain (AA 21-392), linker region (AA 393-498), and C-terminal chitin-binding domain (AA 499-562) showing its characteristic structure as a molting fluid chitinase. In phylogenetic analysis, the enzymes from 6 noctuid species were grouped together, separately from a group of 3 bombycid and 1 tortricid enzymes, corresponding to their taxonomic relationships at both the family and genus levels.
authors: Department of Biological Sciences, Hannam University, Daejeon 306-791, Korea.
authors: Aron
authors: A
authors: Paek
authors: Hee Yun
authors: HY
authors: Park
authors: Seong Eun
authors: SE
authors: Jeong
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D048708:Cell Growth Processes / Q000502:physiology; D002686:Chitin / Q000235:genetics / Q000378:metabolism; D002688:Chitinases / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005944:Glucosamine / Q000235:genetics / Q000378:metabolism; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism; D007814:Larva; D008969:Molecular Sequence Data; D009036:Moths / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D016366:Open Reading Frames; D011485:Protein Binding; D021382:Protein Sorting Signals; D016415:Sequence Alignment
chemical_list: D018076:DNA, Complementary; D019476:Insect Proteins; D021382:Protein Sorting Signals; D002686:Chitin; D002688:Chitinases; D005944:Glucosamine