Identification and characterization of a protease (EuRP-61) from Euphorbia resinifera latex

Abstract

A serine protease designated as EuRP-61 was purified from Euphorbia resinifera latex. The N-terminal sequence of 15 amino acids of EuRP-61 supported the conclusion that the enzyme was a serine protease because its amino acid sequence had homology (between 50 and 70% identities) with the subtilisin-like proteases of other plants. EuRP-61 had a molecular weight estimated at 61 kDa analyzed by MALDI-TOF MS. The enzyme could cleave human fibrinogen with optimal conditions at pH 5.0 and 45 °C. The enzyme had a broad range of pH stability from 1 to 14 and tolerance to denaturation up to a temperature of approximately 65–66 °C. EuRP-61 hydrolyzed fibrinogen with a Michaelis constant (Km) of 4.95 ± 0.1 μM; a maximal velocity (Vmax) of 578.1 ± 11.81 ng min−1; and a catalytic efficiency (Vmax/Km) of 116.8 ± 1 ng μM−1 min−1. EuRP-61was crystallized under the condition of sodium iodide (0.2 M), Bis-Tris propane (0.1 M, pH 8.5) and PEG3350 (20%) by the sitting-drop method. The crystal belonged to space group P212121, with unit cell dimension a = 109.91, b = 67.38 and c = 199.45 Å and diffracted X-ray to 2.53 Å resolution. The crystal structure of EuRP-61 will be explored further by special phase solving techniques.