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±2kDa. 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 762bp, and the deduced amino acid sequence (254 amino acids) is homologous to the sequence of several plant class III endochitinases.

Chitinase III in Euphorbia characias latex: Purification and characterization

SPANO', DELIA;PISANO, MARIA BARBARA;PINTUS, FRANCESCA;FLORIS, GIOVANNI;MEDDA, ROSARIA
Ultimo
2015

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±2kDa. 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 762bp, and the deduced amino acid sequence (254 amino acids) is homologous to the sequence of several plant class III endochitinases.
Chitin; Chitinase; Euphorbia characias; Latex; Magnetic particles
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11584/117349
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