Cloning and expression of alpha-amylase from the hyperthermophilic archaeon Pyrococcus woesei in the moderately halophilic bacterium Halomonas elongata.
An extracellular alpha-amylase gene from the hyperthermophilic archaeon Pyrococcus woesei has been cloned and sequenced. The 1.4-kb protein-coding sequence is identical to that of the corresponding alpha-amylase gene of the closely related species P. furiosus. By using a shuttle cloning vector for halophilic bacteria, the P. woesei alpha-amylase was expressed in the moderate halophile Halomonas elongata, under the control of a native H. elongata promoter. The hyperthermophilic amylase activity expressed in the halophilic host was recovered completely in the crude membrane fraction of cell homogenates, suggesting the formation of inclusion bodies or that the secretion machinery of H. elongata may fail to recognize and release the pyrococcal alpha-amylase to the extracellular medium. However, thermal stability, metal ion interactions, optimal temperature and pH values for the crude and purified recombinant alpha-amylase were comparable with those of the native pyrococcal enzyme. The P. woesei amylase activity expressed in H. elongata was consistently detected in the cells upon growth on a wide range of NaCl concentrations (0.7-2.5 mol l-1). To our knowledge, this is the first report on the expression of an archaeal gene (P. woesei alpha-amylase) in a moderate halophilic host which serves as a cell factory able to grow under extreme salt conditions and with very simple nutritional requirements.
Frillingos
S
S
Sector of Organic Chemistry and Biochemistry, Department of Chemistry, University of Ioannina, Ioannina, Greece.
Linden
A
A
Niehaus
F
F
Vargas
C
C
Nieto
J J
JJ
Ventosa
A
A
Antranikian
G
G
Drainas
C
C
D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D019766:Genes, Archaeal; D020632:Halomonas / Q000201:enzymology / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D019711:Pyrococcus / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism*
D011994:Recombinant Proteins; D000516:alpha-Amylases