Pullulanase,an enzyme of starch catabolism,is associated with the outer membrane of Klebsiella

Late-log phase cells of Klebsiella sp. 5246 could be converted into spheroplasts with a yield of better than 90% by ethylenediamine tetraacetate/lysozyme treatment in osmotically stabilizing media. Membrane fragments obtained after ultrasonication of spheroplasts were separated by centrifugation to sedimentation equilibrium on a sucrose density gradient. A light membrane fraction with a buoyant density of 1.17±0.02g/cm³ was sought and found to contain the enzymes NADH oxidase, succinate dehydrogenase and D-lactate dehydrogenase. A heavy membrane fraction having a buoyant density of 1.23 ±0.01g/cm³ was characterized by phospholipase A₁ activity and lipopolysaccharide content. By analogy to other gram-negative bacteria, the light and the heavy fraction were assigned, respectively, to the cytoplasmic and the outer membrane of Klebsiella sp. 5246.The organism produced pullulanase in a cellbound form during the exponential phase of growth on soluble starch. Pullulanase was localized exclusively on the outer membrane. Pullulanase is the second protein of the outer membrane with defined enzyme function to become known among gram-negative bacteria, the other one being phospholipase A₁.What had been inferred from physiological studies of growth characteristics on various carbon sources can now be proven directly: Pullulanase implicated in the utilization of branched -glucans in Klebsiella is capable of acting on macromolecular substrates in the environment of the cell by virtue of its association with the outer membrane.Non-Standard Abbreviations EDTA ethylenediamine tetraacetate - SDS sodium dodecyl sulphate - OD optical density List of Enzymes EC 3.2.1. 23 -galactosidase or -D-galactoside galactohydrolase - EC 1.1.1.28 D-lactate dehydrogenase or D-lactate: NAD⁺ oxidoreductase - EC 3.2.1.17 lysozyme or mucopeptide N-acetylmuramoylhydrolase - EC 2.4.1.1 maltodextrin phosphorylase or 1,4--D-glucan: orthophosphate -glucosyltransferase - EC 1.6.99.3 NADH oxidase or NADH: (acceptor) oxidoreductase - EC 3.1.1.32 phospholipase A₁ or phosphatide 1-acylhydrolase - EC 3.2.1.41 pullulanase or pullulan 6-glucanohydrolase - EC 1.3.99.1 succinate dehydrogenase or succinate: (acceptor) oxidoreductase