A maltotriose-producing alpha-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 alpha-amylase from T. fusca NTU22 is a monomer. The optimum pH and temperature for the purified enzyme were 7.0 and 60degreesC, respectively. About 70% of the original activity still remained after treatment at 60degreesC for 3 h. The enzyme activity was completely inhibited by 1 mM Hg2+. 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. (C) 2004 Elsevier Inc. All rights reserved.