Pullulanase from the hyperthermophilic bacterium Thermotoga maritima: purification by beta-cyclodextrin affinity chromatography

This is the first report about the isolation of a type I pullulanase from a hyperthermophilic bacterium, Thermotoga maritima strain MSB8. Purification of the enzyme from a cleared cell-free extract was achieved by anion-excha nge chromatography and beta-cyclodextrin affinity chromatography. Using thi s convenient two-step method we have purified the pullulanase 406-fold with a 26% yield. The purified enzyme displayed maximum pullulan hydrolysis at pH 5.9 and 90 degrees C (15-min assay) and was remarkably resistant against thermoinactivation, having a half-life at 90 degrees C of about 3.5 h. To our knowledge, the T, maritima pullulanase is the most thermostat,le type I pullulanase known to date. The affinity-based purification protocol descri bed here may be useful for the efficient isolation of other pullulanases. ( C) 2000 Elsevier Science B.V. All rights reserved.