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.