Spanò Delia, Pospiskova Kristyna, Safarik Ivo, Pisano Maria Barbara, Pintus Francesca, Floris Giovanni, Medda Rosaria
Department of Sciences of Life and Environment, University of Cagliari, I-09042 Monserrato (CA), Italy.
Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 27, 783 71 Olomouc, Czech Republic.
Protein Expr Purif. 2015 Dec;116:152-8. doi: 10.1016/j.pep.2015.08.026. Epub 2015 Aug 28.
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.
本文论述了大戟乳胶中III类内切几丁质酶的纯化。所描述的纯化方法包括使用磁性几丁质颗粒的有效新型分离步骤。磁性亲和吸附剂的应用显著简化并缩短了纯化过程。此步骤及随后的DEAE-纤维素色谱法能够获得均一形式的几丁质酶。在非变性条件下的聚丙烯酰胺凝胶电泳（PAGE）上出现一条蛋白带，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）图谱显示一条独特的36.5±2 kDa蛋白带。在50℃、pH 5.0时观察到最佳几丁质酶活性。大戟乳胶几丁质酶能够水解胶体几丁质，反应产物为N-乙酰-D-葡萄糖胺、壳二糖和壳三糖。此外，我们观察到钙和镁离子可增强几丁质酶活性。最后，我们克隆了编码大戟乳胶几丁质酶的cDNA。部分cDNA核苷酸序列包含762 bp，推导的氨基酸序列（254个氨基酸）与几种植物III类内切几丁质酶的序列同源。
