Isolation and characterization of a heat-stable pullulanase from the hyperthermophilic archaeon Pyrococcus woesei after cloning and expression of its gene in Escherichia coli.
The gene encoding an extremely heat-stable pullulanase from the hyperthermophilic archaeon Pyrococcus woesei was cloned and expressed in Escherichia coli. Purification of the enzyme to homogeneity was achieved after heat treatment of the recombinant E. coli cells, affinity chromatography on a maltotriose-coupled Sepharose 6B column, and anion-exchange chromatography on Mono Q. The pullulanase, which was purified 90-fold with a final yield of 15%, is composed of a single polypeptide chain with a molecular mass of 90 kDa. The enzyme is optimally active at 100 degrees C and pH 6.0 and shows 40% activity at 120 degrees C. Enzyme activation up to 370% is achieved in the presence of calcium ions and reducing agents such as beta-mercaptoethanol and dithiothreitol, whereas N-bromosuccinimide and alpha-cyclodextrin are inhibitory. The high rigidity of the heat-stable enzyme is demonstrated by fluorescence spectroscopic studies in the presence of denaturing agents such as sodium dodecyl sulfate. At temperatures above 80 degrees C, the enzyme seems to switch from the compact to the unfolded form, which is accompanied by an apparent shift in the molecular mass from 45 to 90 kDa.
Rüdiger
A
A
Department of Technical Microbiology, Technical University Hamburg-Harburg, Germany.
Jorgensen
P L
PL
Antranikian
G
G
D001105:Archaea / Q000201:enzymology* / Q000235:genetics*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D008670:Metals; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D007202:Indicators and Reagents; D008670:Metals; D006026:Glycoside Hydrolases; C100181:pullulanase