Purification and characterization of two extracellular endochitinases from Massilia timonae.
Two extracellular chitinases (designated as Chi-56 and Chi-64) produced by Massilia timonae were purified by ion-exchange chromatography, ammonium sulfate precipitation, and gel-filtration chromatography. The molecular mass of Chi-56 was 56 kDa as determined by both SDS-PAGE and gel-filtration chromatography. On the other hand, Chi-64 showed a molecular mass of 64 kDa by SDS-PAGE and 28 kDa by gel-filtration chromatography suggesting that its properties may be different from those of Chi-56. The optimum temperature, optimum pH, pI, K(m), and V(max) of Chi-56 were 55 degrees C, pH 5.0, pH 8.5, 1.1 mg mL(-1), and 0.59 micromol microg(-1)h(-1), respectively. For Chi-64, these values were 60 degrees C, pH 5.0, pH 8.5, 1.3 mg mL(-1), and 1.36 micromol microg(-1)h(-1), respectively. Both enzymes were stimulated by Mn(2+) and inhibited by Hg(2+), and neither showed exochitinase activity. The N-terminal sequences of Chi-56 and Chi-64 were determined to be Q-T-P-T-Y-T-A-T-L and Q-A-D-F-P-A-P-A-E, respectively.
Adrangi
Sina
S
Department of Pharmaceutical Biotechnology, Biotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, PO Box 14155-6451, Tehran 14174, Iran.
Faramarzi
Mohammad Ali
MA
Shahverdi
Ahmad Reza
AR
Sepehrizadeh
Zargham
Z
D000595:Amino Acid Sequence; D002688:Chitinases / Q000737:chemistry / Q000302:isolation & purification*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D042802:Oxalobacteraceae / Q000201:enzymology*; D013696:Temperature
D002688:Chitinases