Purification and some properties of an extracellular exo-maltohexaohydrolase from an Aerobacter aerogenes mutant.

Abstract

An extracellular exo-maltohexaohydrolase [EC 3.2.1.98] was obtained from the culture medium of a mutant from Aerobacter aerogenes (Klebsiella pneumoniae). This enzyme was purified by ammonium sulfate precipitation, DEAE-cellulose column chromatography and Sephadex G-100 gel filtration to 1, 100 fold of the activity of the original cuture liquor. It gave a single band on disc gel electrophoresis, and the molecular weight by SDS disc gel electrophoresis and Sephadex G-100 gel filtration were 65, 000 and 48, 000, respectively. In isoelectric focusing, the extracellular enzyme showed three bands, at isoelectric points (p1) of 7. 20, 7.60 and 7.80. This amylase showed maximum activity at 52°C and pH 7.0. The pH-stability range was relatively wide, the enzyme retaining more than 80% of its initial activity in the range of pH 5.0 to 10.0. It was stable below 50°C, and thermostability was improved by the addition of calcium ion. The relative reaction rates of the enzyme on various substrates were also determined. The enzyme produced maltohexaose from starch, amylose and amylopectin by exo-attack, but did not act on a-, B- or r-cyclodextrin, pullulan and maltose. The enzyme acted on Q-limit dextrins of amylopectin and glycogen to form branched oligosaccharides. The characteristics of the enzyme were similar to those of the cell-bound enzyme reported in the previous paper.