Purification of α-Maltotetraose-formingexo-Amylase ofPseudomonas stutzeri—Two-forms of the Amylase and Their Enzymatic Properties

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Abstract
Two active forms 83 (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™ 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 ko) for starch, glycogens and short chain amylose (DP=17), and 5) the type of inhibition and inhibitor constants (Ki) of some microbial α-amylase inhibitors. Both purified F-1 and F-2 attacked starch from the non-reducing end to produce α-anomer oligosaccharides; these results confirmed that both enzymes were exo-α-amylases as reported previously.

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