Dubiumin, a chymotrypsin-like serine protease from the seeds of Solanum dubium Fresen.
A serine protease was purified 6.7-fold and with 35% recovery from the seeds Solanum dubium Fresen by a simple purification procedure that combined ammonium sulfate fractionation, cation exchange and gel filtration chromatographies. The enzyme, named dubiumin, has a molecular mass of 66kDa as estimated by gel filtration and SDS-PAGE. Carbohydrate staining established the existence of a carbohydrate moiety attached to the enzyme. Inhibition of enzyme activity by serine protease inhibitors such as PMSF and chymostatin indicated that the enzyme belongs to the chymotrypsin-like serine protease class. Dubiumin is a basic protein with pI value of 9.3, acts optimally at pH 11.0, and is stable over a wide range of pH (3.0-12.0). The enzyme is also thermostable retaining complete activity at 60 degrees C after 1h and acts optimally at 70 degrees C for 30 min. Furthermore, it is highly stable in the presence of various denaturants (2.0% SDS, 7.0M urea and 3.0M guanidine hydrochloride) and organic solvents [CH(3)CN-H(2)O (1:1, v/v) and MeOH-H(2)O (1:1, v/v)] when incubated for 1h. The enzyme showed a high resistance to autodigestion even at low concentrations.
Mohamed Ahmed
Isam A
IA
United Graduate School of Agricultural Science, Tottori University, Koyama, Tottori 680-8553, Japan.
Morishima
Isao
I
Babiker
Elfadil E
EE
Mori
Nobuhiro
N
D000818:Animals; D005591:Chemical Fractionation; D002850:Chromatography, Gel; D053818:Chymases / Q000737:chemistry* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008892:Milk / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification; D011480:Protease Inhibitors / Q000737:chemistry; D011489:Protein Denaturation; D012639:Seeds / Q000201:enzymology*; D020539:Sequence Analysis, Protein; D032322:Solanum / Q000201:enzymology*; D012997:Solvents / Q000737:chemistry; D013696:Temperature
D010940:Plant Proteins; D011480:Protease Inhibitors; D012997:Solvents; D053818:Chymases