Cold-active serine alkaline protease from the psychrophilic bacterium Pseudomonas strain DY-A: enzyme purification and characterization.
An extracellular protease was purified from a deep-sea psychrophilic bacterium strain DY-A which was identified as a Pseudomonas species. The optimal growth and protease-producing temperatures of the strain were all 10 degrees C, and the protease was secreted only at temperatures under 20 degrees C. The enzyme was most active at 40 degrees C and at pH 10.0. It was inhibited by phenylmethyl sulfonylfluoride and diisopropyl fluorophosphate, indicating that it is a serine protease. Chelators such as EDTA, EGTA, 1,10-phenanthroline and 2,2'-bipyridyl produced a decrease of activity. The enzyme was sensitive to denaturing agents such as SDS, urea, and guanidine HCl and resistant to thiol-containing reducing agents such as dithiotreitol. The enzyme was active towards N-succinyl-Ala-Ala-Pro-Phe- p-nitroanilide and N-succinyl-Ala-Ala-Pro-Leu- p-nitroanilide. The native molecular mass of the enzyme determined by native PAGE and SDS-PAGE was 25 kDa.
Zeng
Runying
R
Third Institute of Oceanography, State Oceanic Administration, Daxue Road 178, Xiamen, 361005, Fujian, China. rainz@public.xm.fj.cn
Zhang
Rui
R
Zhao
Jing
J
Lin
Nianwei
N
D002848:Chromatography, DEAE-Cellulose; D003080:Cold Temperature; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D011489:Protein Denaturation; D011549:Pseudomonas / Q000201:enzymology*; D012697:Serine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature
D015842:Serine Proteinase Inhibitors; D012697:Serine Endopeptidases