Decolorization of crude latex by activated charcoal, purification and physico-chemical characterization of religiosin, a milk-clotting serine protease from the latex of Ficus religiosa.
The crude latex of Ficus religiosa is decolorized by activated charcoal. Decolorization follows the Freundlich and Langmuir equations. A serine protease, named religiosin, has been purified to homogeneity from the decolorized latex using anion exchange chromatography. Religiosin is a glycoprotein with a molecular mass of 43.4 kDa by MALDI-TOF. Religiosin is an acidic protein with a pI value of 3.8 and acts optimally at pH 8.0-8.5 and temperature 50 degrees C. The proteolytic activity of religiosin is strongly inhibited by PMSF and chymostatin indicating that the enzyme is a serine protease. The extinction coefficient (epsilon(1%)(280)) of religiosin is 29.47 M(-1) cm(-1)with 16 tryptophan, 26 tyrosine, and 11 cysteine residues per molecule. The enzyme shows broad substrate specificity against natural as well as synthetic substrates with an apparent K(m) of 0.066 mM and 6.25 mM using casein and Leu-pNA, respectively. MS/MS analysis confirms the novelty of the enzyme. Religiosin is highly stable against denaturants, metal ions, and detergents as well as over a wide range of pH and temperature. In addition, the enzyme exhibits milk-clotting as well as detergent activity.
Kumari
Moni
M
Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India.
Sharma
Anurag
A
Jagannadham
M V
MV
D000818:Animals; D002417:Cattle; D002606:Charcoal / Q000737:chemistry*; D002852:Chromatography, Ion Exchange; D004795:Enzyme Stability; D030681:Ficus / Q000737:chemistry / Q000201:enzymology*; D006868:Hydrolysis; D007526:Isoelectric Point; D007840:Latex / Q000737:chemistry*; D008892:Milk / Q000737:chemistry; D008970:Molecular Weight; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D057057:Serine Proteases / Q000737:chemistry* / Q000302:isolation & purification*; D013379:Substrate Specificity
D007840:Latex; D010940:Plant Proteins; D002606:Charcoal; D057057:Serine Proteases