An unusual thermostable aspartic protease from the latex of Ficus racemosa (L.).
The most extensively studied ficins have been isolated from the latex of Ficus glabrata and Ficus carica. However the proteases (ficins) from other species are less known. The purification and characterization of a protease from the latex of Ficus racemosa is reported. The enzyme purified to homogeneity is a single polypeptide chain of molecular weight of 44,500+/-500Da as determined by MALDI-TOF. The enzyme exhibited a broad spectrum of pH optima between pH 4.5-6.5 and showed maximum activity at 60+/-0.5 degrees C. The enzyme activity was completely inhibited by pepstatin-A indicating that the purified enzyme is an aspartic protease. Far-UV circular dichroic spectra revealed that the purified enzyme contains predominantly beta-structures. The purified protease is thermostable. The apparent T(m), (mid point of thermal inactivation) was found to be 70+/-0.5 degrees C. Thermal inactivation was found to follow first order kinetics at pH 5.5. Activation energy (E(a)) was found to be 44.0+/-0.3kcal mol(-1). The activation enthalpy (DeltaH *), free energy change (DeltaG *) and entropy (DeltaS *) were estimated to be 43+/-4kcal mol(-1), -26+/-3kcal mol(-1) and 204+/-10cal mol(-1)K(-1), respectively. Its enzymatic specificity studied using oxidized B chain of insulin indicates that the protease preferably hydrolyzed peptide bonds C-terminal to glutamate, leucine and phenylalanine (at P1 position). The broad specificity, pH optima and elevated thermal stability indicate the protease is distinct from other known ficins and would find applications in many sectors for its unique properties.
Devaraj
K B
KB
Department of Protein Chemistry and Technology, Central Food Technological Research Institute, Mysore 570 020, India.
Gowda
Lalitha R
LR
Prakash
V
V
D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D002384:Catalysis; D004063:Digestion; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D030681:Ficus / Q000201:enzymology*; D005502:Food; D006863:Hydrogen-Ion Concentration; D007840:Latex / Q000737:chemistry*; D011480:Protease Inhibitors / Q000494:pharmacology; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
D007840:Latex; D011480:Protease Inhibitors; D011506:Proteins; D016282:Aspartic Acid Endopeptidases