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-exchange chromatography and beta-cyclodextrin affinity chromatography. Using this 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 thermostable type I pullulanase known to date. The affinity-based purification protocol described here may be useful for the efficient isolation of other pullulanases.
Kriegshäuser
G
G
Institut für Mikrobiologie und Genetik, Georg-August-Universität, Göttingen, Germany.
Liebl
W
W
D002846:Chromatography, Affinity / Q000379:methods*; D002852:Chromatography, Ion Exchange; D003505:Cyclodextrins / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000302:isolation & purification*; D020124:Thermotoga maritima / Q000201:enzymology*; D047392:beta-Cyclodextrins
D003505:Cyclodextrins; D047392:beta-Cyclodextrins; D006026:Glycoside Hydrolases; C100181:pullulanase; C031215:betadex