Procedure for the simultaneous large‐scale isolation of pullulanase and 1,4‐α‐glucan phosphorylase from <i>Klebsiella pneumoniae</i> involving liquid–liquid separations

Abstract

A procedure for the simultaneous large-scale isolation of pullulanase and 1,4-alpha-glucan phosphorylase from Klebsiella pneumoniae is described. The pullulanase is solubilized from the cell wall by cholate treatment; cells and cell debris are removed by partition in a poly(ethylene glycol) (PEG)-dextran two-phase system and from the upper (PEG) phase of this system the pullulanase is isolated by ultrafiltration and precipitation with N-cetyl,N-,N-,N-trimethyl ammonium bromide to a purity of about 80% with a yield of 70%. The preparations are free of alpha-amylase activity. The cell containing dextran-rich phase is passed through a Manton-Gaulin homogenizer. Then the phosphorylase is separated from the cell debris by partition in a second PEG-dextran system. From the top phase of this system the phosphorylase is isolated by distribution in a PEG-salt two-phase system followed by batch adsorption on carboxymethyl-Sephadex in a yield of 55%, a purity of around 90%, and nearly free of glycosyltransferase activity. All steps in the isolation of the two enzymes can be performed easily in a large scale.