Biochemical aspects of a serine protease from Caesalpinia echinata Lam. (Brazilwood) seeds: a potential tool to access the mobilization of seed storage proteins

Abstract

Several proteins have been isolated from seeds of leguminous, but this is the first report that a protease was obtained from seeds of Caesalpinia echinata Lam., a tree belonging to the Fabaceae family. This enzyme was purified to homogeneity by hydrophobic interaction and anion exchange chromatographies and gel filtration. This 61-kDa serine protease (CeSP) hydrolyses H-D-prolyl-L-phenylalanyl-L-arginine-p-nitroanilide (K(m) 55.7 μM) in an optimum pH of 7.1, and this activity is effectively retained until 50 °C. CeSP remained stable in the presence of kosmotropic anions (PO(4) (3-), SO(4) (2-), and CH(3)COO(-)) or chaotropic cations (K(+) and Na(+)). It is strongly inhibited by TLCK, a serine protease inhibitor, but not by E-64, EDTA or pepstatin A. The characteristics of the purified enzyme allowed us to classify it as a serine protease. The role of CeSP in the seeds cannot be assigned yet but is possible to infer that it is involved in the mobilization of seed storage proteins.

Figure 1

Elution profile of CeSP purification. (a) Hydrophobic interaction chromatography, where a Hitrap Phenyl column was equilibrated with 50 mM phosphate buffer, pH 7.0, 1.0 M (NH₄)₂SO₄, and proteins were eluted with (NH₄)₂SO₄ (0.95, 0.25, and 0.13 M). (b) Anion exchange chromatography, where a Resource Q column was equilibrated with 50 mM Tris buffer, pH 7.5, and proteins were eluted with a linear NaCl (0 to 0.50 M) gradient. (c) Gel filtration, where a Superdex 75 column was equilibrated with 50 mM phosphate buffer, pH 7.0, 0.15 M NaCl, and proteins were eluted in the same buffer. Inset: molecular masses as function of protein elution. Standard proteins: BSA (67.0 kDa), ovalbumin (43.0 kDa), chymotrypsinogen A (25.0 kDa) and ribonuclease A (13.7 kDa). All fractions were monitored by the absorbance at 280 nm (●) and enzymatic activity (°).

Figure 2

SDS-PAGE of CeSP. Gel 12%. Lane 1: molecular mass markers: myosin (200 kDa), β-galactosidase (126 kDa), bovine albumin (82.0 kDa), carbonic anhydrase (38.7 kDa), soybean trypsin inhibitor (32.0 kDa), and lysozyme (17.3 kDa). Lane 2: purified CeSP (50 μg), under reducing conditions.

Figure 3

Effect of pH and temperature on the CeSP activity. (a) Optimum pH for the protease activity was determined in the H-D-Pro-Phe-Arg-pNA hydrolysis in 50 mM acetate/borate/phosphate buffer in the pH range of 2.0 to 11.5 for 20 min at 37°C. (b) For thermal stability studies, CeSP was kept in Tris buffer pH 7.1 for 30 min at different temperatures (20–60°C). Then, enzyme activity in the H-D-Pro-Phe-Arg-pNA hydrolysis was determined at 37°C in the same buffer.

Figure 4

Salt influence on CeSP activity. Hydrolysis of H-D-Pro-Phe-Arg-pNA by CeSP was determined in absence or presence of (a) NaCl (●), NaBr (■), NaI (▲), Na₂SO₄ (◊), NaH₂PO₄ (°), and NaC₂H₃O₂ (♦); (b) KCl (●), NaCl (♦), NH₄Cl (°), MgCl₂ (□), and CaCl₂ (■).