Purification and properties of a maltotriose-producing α-amylase from Thermobifida fusca

Abstract

A maltotriose-producing α-amylase from Thermobifida fusca NTU22 was purified 7.2-fold as measured by specific activity from crude culture filtrate by ammonium sulfate fractionation, Sepharose CL-6B and DEAE-Sepharose CL-6B column chromatography. The overall yield of the purified enzyme was 22%. The purified enzyme gave an apparent single protein band on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular mass of purified enzyme as estimated by SDS-PAGE and by gel filtration on Sepharose CL-6B to be 64 and 60 kDa, respectively, indicated that the maltotriose-producing α-amylase from T. fusca NTU22 is a monomer. The optimum pH and temperature for the purified enzyme were 7.0 and 60 °C, respectively. About 70% of the original activity still remained after treatment at 60 °C for 3 h. The enzyme activity was completely inhibited by 1 mM Hg 2+. The K m value for soluble starch was 0.88 mg/ml. The purified enzyme exhibited a high level of activity with raw sago starch. Maltotriose was formed as the major enzymatic product from both soluble starch and raw sago starch.