Purification and Characterization of Two Chitinases from the Leaves of Pokeweed (Phytolacca americana)

Abstract

Two chitinases, designated PLC-A and PLC-B, were purified from the leaves of pokeweed (Phytolacca americana) using DEAE-cellulose column chromatography followed by gel filtration on Sephadex G-75, hydrophobic column chromatography, and ion-exchange FPLC. PLC-A and PLC-B are acidic and basic proteins having molecular masses of 25 and 29kDa, and isoelectric points of 3.7 and 9.5, respectively. On the basis of their partial amino acid sequences, it was seen that PLC-A and PLC-B belong to class II and class III chitinases, respectively. The optimal pH of PLC-A toward glycolchitin is pH 4.5 and hydrolyzed (GlcNAc)₄ into 2(GlcNAc)₂, and (GlcNAc)₅ - ₆ into (GlcNAc)₂ and (GlcNAc)₃. On the other hand, PLC-B has two optimal pHs at 3 and 7 toward glycolchitin and hydrolyzed (GlcNAc)₅ into GlcNAc and (GlcNAc)₄, and (GlcNAc)₆ into GlcNAc, (GlcNAc)₂, and (GlcNAc)₄.