Identification and partial characterization of a chitinase and a -1,3- glucanase from Copernicia cerifera wax Marco Antonio L. Cruz a , Valdirene M. Gomes b , Kátia V.S. Fernandes a , Olga L.T. Machado a , José Xavier-Filho a, * a Laboratório de Química e Função de Proteínas e Peptídeos, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense, Av. Alberto Lamego 2000, 28015-620 Campos dos Goytacazes, RJ, Brazil b Laboratório de Fisiologia e Bioquímica de Microrganismos, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense, Av. Alberto Lamego 2000, 28015-620 Campos dos Goytacazes, RJ, Brazil Received 12 June 2001 Abstract The presence of proteins in plant cuticular waxes has been known for a long time. The aim of the present work is to investigate the presence of defense-related proteins in the wax of the “Carnauba” (Copernicia cerifera) tree, an economically important tree of the palm family in the northeastern region of Brazil. Results from our laboratory have shown the presence of protein in different fractions isolated from this wax. A DEAE-Sepharose anion exchange column was employed for further discrimination of proteins from different fractions. The samples were visualized by SDS-Tricine-gel electrophoresis. The acidic and basic fractions were recovered by freeze-drying and fractions with antifungic activity identified by assaying for inhibition of fungi growth, -1,3-glucanase and chitinase activities. Our results showed the presence of enzymatic activities in different wax fractions. Isolated proteins were submitted to automated N-terminal amino acid sequencing and obtained sequences showed the presence of proteins with a high degree of homology to class III chitinases and to -1,3 glucanase. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Plant wax; Chitinase; -1,3-glucanase; Copernicia cerifera; “Carnauba” tree 1. Introduction Insects and pathogens that attack plants are normally faced with a combination of both physical and chemical defenses provided by the plant. The first line of defense of a plant against pathogens is its surface, which it must penetrate if it is to cause infection. Waxes on leaf and fruit surfaces are water-repellent and thereby prevent the forma- tion of a film of water on which pathogens might be deposited and germinate [1]. The presence of proteins in plant cuticular waxes has been known for a long time [12] but only few of these have been identified. Recently, a 9 kDa protein was purified as the major protein from the surface wax of broccoli leaves and its amino acid sequence showed 40–50% identity with non-specific lipid transfer proteins (nsLTPs) isolated from various other plants [16]. Recent work has also shown evidence that nsLTPs present in leaf surfaces may function in the transfer of cutin or wax monomers from the sites of synthesis in the cell to the cuticle [11]. Different types of antimicrobial proteins have been purified from plants, including chitinases, -1,3-glucanases, thionins, ribosome-inactivating proteins, defensins and nsLTPs [6,20]. These proteins have been implicated in the resistance mechanisms of plants against pathogens and insects. For example, chitinases and -1,3-glucanases are lytic enzymes capable of inhibiting fungi development by degrading the pathogen’s cell wall, which is mostly com- posed of chitin and -1,3-glucans [3,15]. The objective of this work was to investigate the pres- ence of defense-related proteins in “Carnauba” tree (Coper- nicia cerifera) leaf wax, an economically important tree native to the northeastern region of Brazil, and relate these proteins to other plant defense proteins. Abbreviations: BSA, bovine serum albumine; PBS, phosphate-buffered saline; RP HPLC, reverse phase high protein liquid chromatography; PVP, polyvinylpyrrolidone * Corresponding author.. E-mail address: xavier@uenf.br (J. Xavier-Filho). Plant Physiol. Biochem. 40 (2002) 11–16 www.elsevier.com/locate/plaphy © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 0 9 8 1 - 9 4 2 8 ( 0 1 ) 0 1 3 4 0 - 7