Purification and characterization of a novel extracellular protease from a halo-alkaliphilic Bacillus sp 17N-1, active in polar organic solvents

 
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Purification and characterization of a novel extracellular protease from a halo-alkaliphilic Bacillus sp 17N-1, active in polar organic solvents (EN)

Papamichael, E. M. (EN)

Πανεπιστήμιο Ιωαννίνων. Σχολή Θετικών Επιστημών. Τμήμα Χημείας (EL)
Papamichael, E. M. (EN)

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 kcat/Km and/or kcat 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 pKas, rate constants, activation energies and other important features were estimated from the profiles of parameters kcat/Km, kcat 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. (EN)

halo-alkaliphiles (EN)

Πανεπιστήμιο Ιωαννίνων (EL)
University of Ioannina (EN)

Environmental Technology (EN)

Αγγλική γλώσσα

2010

<Go to ISI>://000278856500002

Taylor & Francis (EN)



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