Your browser doesn't support javascript.
loading
Purification, characterization and molecular cloning of the major chitinase from Tenebrio molitor larval midgut.
Genta, Fernando A; Blanes, Lucas; Cristofoletti, Plínio T; do Lago, Claudimir L; Terra, Walter R; Ferreira, Clélia.
Afiliação
  • Genta FA; Instituto de Química, Departamento de Bioquímica, Universidade de São Paulo, C.P. 26077, 05513-970 São Paulo, Brazil.
Insect Biochem Mol Biol ; 36(10): 789-800, 2006 Oct.
Article em En | MEDLINE | ID: mdl-17027845
Insect chitinases are involved in degradation of chitin from the exoskeleton cuticle or from midgut peritrophic membrane during molts. cDNAs coding for insect cuticular and gut chitinases were cloned, but only chitinases from moulting fluid were purified and characterized. In this study the major digestive chitinase from T. molitor midgut (TmChi) was purified to homogeneity, characterized and sequenced after cDNA cloning. TmChi is secreted by midgut epithelial cells, has a molecular weight of 44 kDa and is unstable in the presence of midgut proteinases. TmChi shows strong substrate inhibition when acting on umbelliferyl-derivatives of chitobio- and chitotriosaccharides, but has normal Michaelis kinetics with the N-acetylglucosamine derivative as substrate. TmChi has very low activity against colloidal chitin, but effectively converts oligosaccharides to shorter fragments. The best substrate for TmChi is chitopentaose, with highest k(cat)/K(M) value. Sequence analysis and chemical modification experiments showed that the TmChi active site contains carboxylic groups and a tryptophane, which are known to be important for catalysis in family 18 chitinases. Modification with p-hidroximercuribenzoate of a cysteine residue, which is exposed after substrate binding, leads to complete inactivation of the enzyme. TmChi mRNA encodes a signal peptide plus a protein with 37 kDa and high similarity with other insect chitinases from family 18. Surprisingly, this gene does not encode the C-terminal Ser-Thr-rich connector and chitin-binding domain normally present in chitinases. The special features of TmChi probably result from its adaptation to digest chitin-rich food without damaging the peritrophic membrane.
Assuntos
Buscar no Google
Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Tenebrio / Quitinases / Proteínas de Insetos Limite: Animals Idioma: En Revista: Insect Biochem Mol Biol Assunto da revista: BIOLOGIA MOLECULAR / BIOQUIMICA Ano de publicação: 2006 Tipo de documento: Article País de afiliação: Brasil País de publicação: Reino Unido
Buscar no Google
Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Tenebrio / Quitinases / Proteínas de Insetos Limite: Animals Idioma: En Revista: Insect Biochem Mol Biol Assunto da revista: BIOLOGIA MOLECULAR / BIOQUIMICA Ano de publicação: 2006 Tipo de documento: Article País de afiliação: Brasil País de publicação: Reino Unido