Preparation and Some Properties of a Novel Maltopentaose-Forming Enzyme of a Pseudomonas species

Abstract

A novel enzyme, of a Pseudomonas species, was purified by ammonium sulfate fractionation and column chromatographies on DEAE-Toyopearl 650M and Toyopearl HW-55s.
The activity recovery was 87 % at the ammonium sulfate fractionation step and 44 % at the final step of the purification, respectively. The purified enzyme was homogeneous electrophoretically and its molecular weight was 72, 000. The optimum pH and temperature were 6.5 and 55°C, respectively. The enzyme was stable up to 45 C in the pH range of 6.5 to 9.0 for 1 hr, and thermostable in the presence of 2 mM CaCl₂ up to 50°C. The isoelectric point of the enzyme was 6.5. The enzyme activity was inhibited by metal ions such as Zn²⁺, Cu²⁺, Ag⁺and Hg²⁺, and enhanced by Rb⁺, Mg²⁺, Co²⁺and Ca²⁺.
The enzyme specifically produced maltopentaose from starch in the initial stage of the reaction, finally producing maltose and maltotriose. The enzyme did not act on glucose, maltose or maltotriose, and maltohexaose was considerably resistant to the enzyme's action. In the initial stage of hydrolysis, G₂ and G₅ were formed from G₇, and thus G₃+G₅ from G₈, G₄+G₅ from G₉, G₅+G₅ from G₁₀, and G₆+G₅ from G₁₁. Through the next action, G₄ and G₅ were degraded to G₂+G₂ and G₂+G₃, respectively. The enzyme action proceeds over the branching points of amylopectin to produce large amounts of G2 and G3, and a residual amount of oligosaccharides having less than 10 glucose units in the final stage of the reaction.