Purification and characterization of an intracellular cycloalternan-degrading enzyme from Bacillus sp. NRRL B-21195

A novel intracellular cycloalternan-degrading enzyme (CADE) was purified to homogeneity from the cell pellet of Bacillus sp. NRRL B-21195. The enzyme has a molecular mass of 125 kDa on SDS-PAGE. The pH optimum was 7.0, and the enzyme was stable from pH 6.0 to 9.2. The temperature optimum was 35 °C and the enzyme exhibited stability up to 50 °C. The enzyme hydrolyzed cycloalternan [CA; cyclo{→ 6)-α-d-Glcp-(1 → 3)-α-d-Glcp-(1 → 6)-α-d-Glcp-(→ 3)-α-d-Glcp-(1 →}] as the best substrate, to produce only isomaltose via an intermediate, α-isomaltosyl-(1 → 3)-isomaltose. This enzyme also hydrolyzed isomaltosyl substrates, such as panose, α-isomaltosyl-(1 → 4)-maltooligosaccharides, α-isomaltosyl-(1 → 3)-glucose, and α-isomaltosyl-(1 → 3)-isomaltose to liberate isomaltose. Neither maltooligosaccharides nor isomaltooligosaccharides were hydrolyzed by the enzyme, indicating that CADE requires α-isomaltosyl residues connected with (1 → 4)- or (1 → 3)-linkages. The Km value of cycloalternan (1.68 mM) was 20% of that of panose (8.23 mM). The kcat value on panose (14.4 s−1) was not significantly different from that of cycloalternan (10.8 s−1). Judging from its specificity, the systematic name of the enzyme should be cycloalternan isomaltosylhydrolase. This intracellular enzyme is apparently involved in the metabolism of starch via cycloalternan in Bacillus sp. NRRL B-21195, its role being to hydrolyze cycloalternan inside the cells.