Purification and characterization of a thermostable neutral metalloprotease I from Chloroflexus aurantiacus J-10-fl.
Chloroflexus aurantiacus J-10-fl was found to contain two types (protease I and protease II) of thermostable proteases which were separated by Butyl-Toyopearl 650 M chromatography. Protease I was purified to electrophoretic homogeneity from the culture broth of C. aurantiacus J-10-fl. The molecular mass of protease I was estimated to be approximately 66 kDa by SDS-PAGE, and the value of approximately 66 kDa was also obtained by the Hedrick-Smith method, indicating that protease I was a monomer. The isoelectric point was 6.2. Protease I activity was inhibited by metalloprotease inhibitors such as EDTA, EGTA, and o-phenanthroline. The optimum pH for the activity of protease I was around 8.0. Addition of Ca2+ increased the pH and heat stabilities of protease I. The activity was stable between pH 4.0-11.0 and up to 75 degrees C, and the maximum activity was observed at 70 degrees C in the presence of 2 mM CaCl2. Protease I was resistant to the treatment by denaturing reagents (8 M urea or 1% SDS) at pH 8.0 and 20 degrees C for 24 h. The sites of cleavage in oxidized insulin B chain by protease I were similar to those by other microbial neutral metalloproteases. Elastase activity of protease I was not detected.
Watanabe
A
A
Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, Sendai, Japan.
Kamio
Y
Y
Kimura
W
W
Izaki
K
K
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology*; D002122:Calcium Chloride / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism; D007526:Isoelectric Point; D008666:Metalloendopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010196:Pancreatic Elastase / Q000378:metabolism; D013696:Temperature
D003470:Culture Media; D007328:Insulin; D010196:Pancreatic Elastase; D008666:Metalloendopeptidases; C044194:microbial metalloproteinases; D002122:Calcium Chloride