Purification and characterization of a raw-starch digesting amylase from a soil bacterium--Cytophaga sp.
A newly isolated bacterium from soil, identified as Cytophaga sp. was found to produce raw-starch digesting amylase. The enzyme was purified from 24-hr cultured medium through ammonium sulfate fractionation, DEAE-Sepharose CL 6B ion exchange chromatography and Sephacryl S-200 gel filtration. The preparation was proved to be homogeneous by SDS-PAGE. The subunit molecular weight determined by SDS-PAGE was 59 KD. The optimum temperature was 50 degrees C on soluble starch and 60 degrees C on raw starch. The optimum pH was in the range of 4.5 to 6.5 on soluble starch and 6.5 to 9.5 on raw starch. In the presence of Mn+2, Cu+2 or Zn+2, the enzyme activity on either substrate was inhibited. Dinitrofluorobenzene, N-bromosuccinimide and trinitrobenzene sulfonic acid all showed inhibitory effect on the enzyme acting on both substrates.
Jeang
C L
CL
Department of Food Science, National Chung Hsing University, Taichung, Taiwan.
Lee
Y H
YH
Chang
L W
LW
D000645:Ammonium Sulfate; D000681:Amylases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D001974:Bromosuccinimide / Q000494:pharmacology; D002413:Cations, Divalent; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017077:Culture Media, Conditioned / Q000737:chemistry; D003589:Cytophaga / Q000201:enzymology*; D004139:Dinitrofluorobenzene / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005590:Fractional Precipitation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013213:Starch / Q000378:metabolism*; D013696:Temperature; D014302:Trinitrobenzenesulfonic Acid / Q000494:pharmacology
D002413:Cations, Divalent; D017077:Culture Media, Conditioned; D014302:Trinitrobenzenesulfonic Acid; D013213:Starch; D004139:Dinitrofluorobenzene; D000681:Amylases; D001974:Bromosuccinimide; D000645:Ammonium Sulfate