Purification of .ALPHA.-maltotetraose-forming exo-amylase of Pseudomonas stutzeri - Two-forms of the amylase and their enzymatic properties.

Sakano Yoshiyuki, KASHIYAMA Eiji, KOBAYASHI Tsuneo

doi:10.1271/bbb1961.47.1761

Two active forms (F-1 and F-2) of a maltotetraose-forming amylase [EC 3.2. 1.60 exomaltotetraohydrolase] from Pseudomonas stutzeri were completely separated and highly purified by using Polybuffer exchanger PBE^TM 94, and their enzymatic properties were compared. The purified F-1 and F-2 both showed a single band in polyacrylamide gel electrophoresis with or without sodium dodecyl sulfate. The optimum pH for the action of F-1 and F-2 on starch was exactly the same at 8.0. The isoelectric points of F-1 and F-2 were estimated to be 5.6 and 5.3 by polyacrylamide gel electrofocusing. Except the properties above, other enzymatic properties were very similar for F-1 and F-2: 1) the optimum temperature (45°C at pH 8.0) and the molecular weight (5.5×10⁴), 2) the stabilization by Ca²⁺, and the inhibition by some metal ions and N-bromosuccinimide, 3) the inhibition by cyclomaltodextrins and the inability to hydrolyze cyclomaltodextrins, 4) the kinetic constants (Km and k⁰) for starch, glycogens and short chain amylose (DP=17), and 5) the type of inhibition and inhibitor constants (Ki) of some microbial a-amylase inhibitors. Both purified F-1 and F-2 attacked starch from the non-reducing end to produce a-anomer oligosaccharides; these results confirmed that both enzymeswere exo-a-amylases as reported previously.

Purification of .ALPHA.-maltotetraose-forming exo-amylase of Pseudomonas stutzeri - Two-forms of the amylase and their enzymatic properties.

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