Purification and characterization of a novel extracellular protease from a halo-alkaliphilic Bacillus sp. 17N-1, active in polar organic solvents.
A novel enzyme of molecular mass about 29 kDa was purified from the strain halo-alkaliphilic Bacillus sp. 17N-1 and designated protease-B-17N-1. This enzyme is likely to be a cysteine protease; it was found active in media containing EDTAK2 and dithiothreitol, it maintained considerable activity at temperatures 14 degrees C to 33 degrees C and pH 6.50 to 8.50 with optimum k(cat)/Km and/or k(cat) values at pH 7.00 and 25 degrees C. The activity of protease-B-17N-1 was strongly affected by the specific irreversible inhibitor of cysteine proteases E-64, while it remained unaffected by the 3,4-dichloro-isocoumarine, an irreversible inhibitor specific for serine proteases. Protease-B-17N-1 retained full activity at 25 degrees C after 30 min incubation at 8 degrees C or at 33 degrees C; moreover, it was found to be stable and active in the polar organic solvents DMSO and acetonitrile. The enzyme hydrolyzed the substrate Cbz-FR-pNA via Michaelis-Menten kinetics, while it showed insignificant activity for the substrate Suc-AAA-pNA. Valuable pK(a)s, rate constants, activation energies and other important features were estimated from the profiles of parameters k(cat)/Km, k(cat) and Km, versus pH, temperature, and [NaCl]. In addition, interesting results were obtained from the effect of different metallic ions and polar organic solvents on the Michaelis-Menten parameters of protease-B-17N-1, showing that it performs catalysis via a (Cys)-S(-)/(His)-Im(+)H ion-pair, as well as its industrial and biotechnological potential, respectively.
Papamichael
E M
EM
Department of Chemistry, University of Ioannina, Ioannina 45110, Greece. epapamic@cc.uoi.gr
Theodorou
L G
LG
Perisynakis
A
A
Drainas
C
C
D000097:Acetonitriles / Q000737:chemistry; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D057056:Cysteine Proteases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D019216:Metals, Heavy / Q000737:chemistry; D009930:Organic Chemicals / Q000737:chemistry*; D012997:Solvents / Q000737:chemistry*; D013696:Temperature
D000097:Acetonitriles; D001426:Bacterial Proteins; D019216:Metals, Heavy; D009930:Organic Chemicals; D012997:Solvents; D004492:Edetic Acid; D057056:Cysteine Proteases; D004229:Dithiothreitol; D004121:Dimethyl Sulfoxide; C032159:acetonitrile