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 (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.
Devaraj
Kamsagara Basavarajappa
KB
Department of Protein Chemistry and Technology, Central Food Technological Research Institute, Mysore, India.
Kumar
Parigi Ramesh
PR
Prakash
Vishweshwaraiah
V
D000595:Amino Acid Sequence; D004795:Enzyme Stability; D005361:Ficain / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D030681:Ficus / Q000737:chemistry / Q000201:enzymology*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D016415:Sequence Alignment; D012997:Solvents / Q000737:chemistry
D010940:Plant Proteins; D012997:Solvents; D005361:Ficain