title: Chitinase III in Euphorbia characias latex: Purification and characterization.
abstract: This paper deals with the purification of a class III endochitinase from Euphorbia characias latex. Described purification method includes an effective novel separation step using magnetic chitin particles. Application of magnetic affinity adsorbent noticeably simplifies and shortens the purification procedure. This step and the subsequently DEAE-cellulose chromatography enable to obtain the chitinase in homogeneous form. One protein band is present on PAGE in non-denaturing conditions and SDS-PAGE profile reveals a unique protein band of 36.5 ± 2 kDa. The optimal chitinase activity is observed at 50 °C, pH 5.0. E. characias latex chitinase is able to hydrolyze colloidal chitin giving, as reaction products, N-acetyl-D-glucosamine, chitobiose and chitotriose. Moreover, we observed that calcium and magnesium ions enhance chitinase activity. Finally, we cloned the cDNA encoding the E. characias latex chitinase. The partial cDNA nucleotide sequence contains 762 bp, and the deduced amino acid sequence (254 amino acids) is homologous to the sequence of several plant class III endochitinases.
authors: Department of Sciences of Life and Environment, University of Cagliari, I-09042 Monserrato (CA), Italy.
authors: Delia
authors: D
authors: Spanò
authors: Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 27, 783 71 Olomouc, Czech Republic.
authors: Kristyna
authors: K
authors: Pospiskova
authors: Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 27, 783 71 Olomouc, Czech Republic; Department of Nanobiotechnology, Institute of Nanobiology and Structural Biology of GCRC, Na Sadkach 7, 370 05 Ceske Budejovice, Czech Republic.
authors: Ivo
authors: I
authors: Safarik
authors: Department of Public Health, Clinical and Molecular Medicine, University of Cagliari, I-09042 Monserrato (CA), Italy.
authors: Maria Barbara
authors: MB
authors: Pisano
authors: Department of Sciences of Life and Environment, University of Cagliari, I-09042 Monserrato (CA), Italy.
authors: Francesca
authors: F
authors: Pintus
authors: Department of Sciences of Life and Environment, University of Cagliari, I-09042 Monserrato (CA), Italy.
authors: Giovanni
authors: G
authors: Floris
authors: Department of Sciences of Life and Environment, University of Cagliari, I-09042 Monserrato (CA), Italy. Electronic address: rmedda@unica.it.
authors: Rosaria
authors: R
authors: Medda
mesh_terms: D000595:Amino Acid Sequence; D002686:Chitin / Q000378:metabolism*; D002688:Chitinases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D028481:Euphorbia / Q000737:chemistry / Q000201:enzymology*; D006868:Hydrolysis; D008969:Molecular Sequence Data
chemical_list: D002686:Chitin; D002688:Chitinases
keywords: Chitin; Chitinase; Euphorbia characias; Latex; Magnetic particles