Purification, Characterization, and Solvent-Induced Thermal Stabilization of Ficin from Ficus carica

Ficin (EC 3.4.22.3), a cysteine proteinase isolated from the latex of a Ficus tree, is known to occur
in multiple forms. Although crude ficin is of considerable commercial importance, ficin as such has
not been fully characterized. A major ficin from the commercial crude proteinase mixture preparation
of Ficus carica was purified and characterized. The purified enzyme was homogeneous in both sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel-filtration chromatography
and is a single polypeptide chain protein with a molecular mass of 23 100 ( 300 Da as determined
by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF). The enzyme was active
in the pH range of 6.5-8.5, and maximum activity was observed at pH 7.0. The N-terminal core
sequence of ficin has homology with N-terminal sequences of plant cysteine proteinases. The enzyme
contains three disulfide bonds and a single free cysteine residue at the active site. The effect of
co-solvents, such as sorbitol, trehalose, sucrose, and xylitol, on the thermal stability of ficin was
determined by activity measurements, fluorescence, and thermal denaturation studies. The apparent
thermal denaturation temperature (Tm) of ficin was significantly increased from the control value of
72 ( 1 °C in the presence of all co-solvents. However, the maximum stabilization effect was observed
in terms of thermal stabilization by the co-solvent trehalose.