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 Pyr ococcus woesei has been cloned and sequenced. The 1.4-kb protein-coding seq uence is identical to that of the corresponding alpha-amylase gene of the c losely related species P. furiosus. By using a shuttle cloning vector for h alophilic bacteria, the P. woesei alpha-amylase was expressed in the modera te halophile Halomonas elongata, under the control of a native H. elongata promoter. The hyperthermophilic amylase activity expressed in the halophili c host was recovered completely in the crude membrane fraction of cell homo genates, 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, meta l ion interactions, optimal temperature and pH values for the crude and pur ified recombinant alpha-amylase were comparable with those of the native py rococcal enzyme. The P. woesei amylase activity expressed in H. elongata wa s consistently detected in the cells upon growth on a wide range of NaCl co ncentrations (0.7-2.5 mol l(-1)). To our knowledge, this is the first repor t on the expression of an archaeal gene (P. woesei alpha-amylase) in a mode rate halophilic host which serves as a cell factory able to grow under extr eme salt conditions and with very simple nutritional requirements.