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

Abstract

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 (T(m)) of ficin was significantly increased from the control value of 72 +/- 1 degrees 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.

Characterization of acid-induced molten globule like state of ficin

“…It is a single polypeptide chain protein molecule of molecular mass 23,100 Da. The enzyme is active at neutral pH and looses its activity completely below pH 3.0 [22]. In the present study, the structural characterization of molten globule state of ficin at acidic pH has been examined.…”

“…Ficin was purified from the commercial crude preparation (Sigma) according to the method described in our earlier report [22]. The homogeneity of the purified ficin was evaluated by SDS-PAGE to confirm the purity.…”

Nutritional and pharmacological importance of Ficus carica - A review

“…C3R (cyanidin-3-O-rutinoside) contributed 92% of the total antioxidant capacity of the anthocyanin fraction. Fruits of the Mission variety contained the highest levels of polyphenols, flavonoids, and anthocyanins and exhibited the highest antioxidant capacity [70].Six organic acids were identified in the fig leaves including: oxalic, citric, malic, quinic, shikimic, and fumaric acids [69].Ficins, cysteine endoproteolytic proteases were isolated from Ficus carica latex, these included ficins A, B, C, D1, D2 and E [71][72].Many volatile compounds were isolated from Ficus carica fructus included 2,3-butane-diol, tetramethyl-decane, trimethylundecane, octadecane, carvacrol, ß-Caryophyllene, caryophyllene-oxid and apiol [68].The volatile profile of fresh fruits (pulp and peel) and leaves of Portuguese Ficus carica white (Pingo de Mel and Branca Tradicional) and dark (Borrasota Tradicional, Verbera Preta and Preta Tradicional) varieties revealed the presence of fifty-nine compounds including (aldehydes, alcohols, ketones, esters, monoterpenes, sesquiterpenes, norisoprenoids). The highest diversity of compounds was found in leaves (40), followed by pulps (30) and peels (27), Pulps and peels were distinguished from leaves by their abundance of monoterpenes and aldehydes.…”

Native and Biotechnologically Engineered Plant Proteases with Industrial Applications

“…As of today, only three fragments of ficin have been studied (a fragment around the catalytic Cys, a fragment around the catalytic His, and Nterminal fragment). The amino acid sequence determined for neighboring residues of the active-site Cys was found to closely resemble the corresponding sequence in papain (Devaraj et al 2008). …”