Maltooligosaccharides (MOS), oligosaccharides composed of 2 to 10 glucopyranose subunits linked by alpha-1,4 bonds, are promising ingredients possessing low sweetness, low osmolality, high water-holding capacity, and crystallization-prevention ability. For improved the cost effectiveness, a novel process for MOS preparation was developed by utilizing sucrose as a raw material instead of starch. In this work, a novel alpha-amylase from Pseudomonas mendocina (PM-Amy) was characterized and applied to produce the MOS from alpha-1,4-glucans obtained by the amylosucrase treatment of sucrose. PM-Amy showed a maximum activity at a pH of 6.0 and 55 degrees C. In the one-pot enzymatic reaction, a maximum MOS yield of 22.8% (w/w) was achieved, while the highest MOS yield (34.1%, w/w) was achieved from the two-step dual enzyme reaction.