Identification and characterization of a novel raw-starch-degrading α-amylase (AmyASS) from the marine fish pathogen Aeromonas salmonicida ssp. salmonicida

Abstract

A glycoside hydrolase family protein, designated AmyASS, from the marine fish pathogen Aeromonas salmonicida ssp. salmonicida A449 was expressed in Escherichia coli as inclusion bodies, which was renatured by a simple stepwise dialysis. AmyASS has more than 50% identity with many putative glycosidases. Based on the mode of hydrolytic action and the type of substrates, AmyASS was clearly defined as a saccharifying-type α-amylase, which is the third extracellular α-amylase of A. salmonicida ssp. salmonicida. AmyASS showed a raw starch digesting ability, and the specific activity toward raw rice starch (23.5±1.6Umg−1) was much higher than that toward raw wheat starch and raw mung starch. The catalytic region of AmyASS has 50% identity with the raw starch-degrading α-amylase AmyP, whereas AmyASS does not possess a starch-binding domain (SBD). AmyASS was optimally active at pH 6.0 and 40°C toward soluble starch and at pH 7.0 and 40°C toward raw starch. The enzyme exhibited stability under acidic (pH 6.0) conditions, while the activity decreased rapidly under neutral (pH 7.0) conditions. The AmyASS activity was stimulated by Ca2+, Co2+ and Cu2+.