Procedure for the simultaneous large-scale isolation of pullulanase and 1,4-α-glucan phosphorylase from Klebsiella pneumoniae involving liquid-liquid separations

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 α‐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.