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Procedure for the simultaneous large‐scale isolation of pullulanase and 1,4‐α‐glucan phosphorylase from Klebsiella pneumoniae involving liquid–liquid separations
Author(s) -
Hustedt H.,
Kroner K. H.,
Stach W.,
Kula M.R.
Publication year - 1978
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.260201212
Subject(s) - pullulanase , chemistry , chromatography , biochemistry , enzyme
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.