Purification and characterization of a highly stable cysteine protease from the latex of Ervatamia coronaria.
A highly stable cysteine protease was purified to homogeneity from the latex of Ervatamia coronaria by a simple purification procedure involving ammonium sulfate precipitation and ion-exchange chromatography. The molecular mass was estimated to be approximately 25,000 Da by SDS-PAGE and gel filtration. The extinction coefficient (epsilon 280 nm 1%) of the enzyme was 24.6. The enzyme hydrolyzed denatured natural substrates like casein, hemoglobin, azoalbumin, and azocasein with a high specific activity but showed low specific activity towards synthetic substrates. The pH and temperature optima were 7.5-8.0 and 50 degrees C respectively. The activity of the enzyme was strongly inhibited by thiol-specific inhibitors like leupeptin, iodoacetamide, PCMB, NEM, and mercuric chloride. The striking property of this enzyme was its stability over a wide pH range (2-12) and other extreme conditions of temperature, denaturants, and organic solvents. The N-terminal sequence showed marked similarity to known cysteine proteases.
Sundd
M
M
Molecular Biology Unit, Banaras Hindu University, Varanasi, India.
Kundu
S
S
Pal
G P
GP
Medicherla
J V
JV
D000595:Amino Acid Sequence; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D007840:Latex / Q000737:chemistry; D008969:Molecular Sequence Data; D010946:Plants, Medicinal / Q000201:enzymology*
D007840:Latex; D003546:Cysteine Endopeptidases; C116225:ervatamin