This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Dong, G. Articles by Zeikus, J. G. Search for Related Content PubMed PubMed Citation Articles by Dong, G. Articles by Zeikus, J. G. Agricola Articles by Dong, G. Articles by Zeikus, J. G.

 Previous Article  |  Next Article 

Appl. Environ. Microbiol., 09 1997, 3569-3576, Vol 63, No. 9
Copyright © 1997, American Society for Microbiology

Cloning, sequencing, and expression of the gene encoding extracellular alpha-amylase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme

G Dong, C Vieille, A Savchenko and JG Zeikus
Department of Biochemistry, Michigan State University, East Lansing 48824, USA.

The gene encoding the hyperthermophilic extracellular alpha-amylase from Pyrococcus furiosus was cloned by activity screening in Escherichia coli. The gene encoded a single 460-residue polypeptide chain. The polypeptide contained a 26-residue signal peptide, indicating that this Pyrococcus alpha-amylase was an extracellular enzyme. Unlike the P. furiosus intracellular alpha-amylase, this extracellular enzyme showed 45 to 56% similarity and 20 to 35% identity to other amylolytic enzymes of the alpha-amylase family and contained the four consensus regions characteristic of that enzyme family. The recombinant protein was a homodimer with a molecular weight of 100,000, as estimated by gel filtration. Both the dimer and monomer retained starch-degrading activity after extensive denaturation and migration on sodium dodecyl sulfate-polyacrylamide gels. The P. furiosus alpha- amylase was a liquefying enzyme with a specific activity of 3,900 U mg- 1 at 98 degrees C. It was optimally active at 100 degrees C and pH 5.5 to 6.0 and did not require Ca2+ for activity or thermostability. With a half-life of 13 h at 98 degrees C, the P. furiosus enzyme was significantly more thermostable than the commercially available Bacillus licheniformis alpha-amylase (Taka-therm).

This article has been cited by other articles:

• Lee, H.-S., Shockley, K. R., Schut, G. J., Conners, S. B., Montero, C. I., Johnson, M. R., Chou, C.-J., Bridger, S. L., Wigner, N., Brehm, S. D., Jenney, F. E. Jr., Comfort, D. A., Kelly, R. M., Adams, M. W. W. (2006). Transcriptional and Biochemical Analysis of Starch Metabolism in the Hyperthermophilic Archaeon Pyrococcus furiosus. J. Bacteriol. 188: 2115-2125 [Abstract] [Full Text]  
• Yang, S.-J., Lee, H.-S., Park, C.-S., Kim, Y.-R., Moon, T.-W., Park, K.-H. (2004). Enzymatic Analysis of an Amylolytic Enzyme from the Hyperthermophilic Archaeon Pyrococcus furiosus Reveals Its Novel Catalytic Properties as both an {alpha}-Amylase and a Cyclodextrin-Hydrolyzing Enzyme. Appl. Environ. Microbiol. 70: 5988-5995 [Abstract] [Full Text]  
• Nonaka, T., Fujihashi, M., Kita, A., Hagihara, H., Ozaki, K., Ito, S., Miki, K. (2003). Crystal Structure of Calcium-free {alpha}-Amylase from Bacillus sp. Strain KSM-K38 (AmyK38) and Its Sodium Ion Binding Sites. J. Biol. Chem. 278: 24818-24824 [Abstract] [Full Text]  
• Linden, A., Mayans, O., Meyer-Klaucke, W., Antranikian, G., Wilmanns, M. (2003). Differential Regulation of a Hyperthermophilic alpha -Amylase with a Novel (Ca,Zn) Two-metal Center by Zinc. J. Biol. Chem. 278: 9875-9884 [Abstract] [Full Text]  
• Worthington, P., Hoang, V., Perez-Pomares, F., Blum, P. (2003). Targeted Disruption of the {alpha}-Amylase Gene in the Hyperthermophilic Archaeon Sulfolobus solfataricus. J. Bacteriol. 185: 482-488 [Abstract] [Full Text]  
• Richardson, T. H., Tan, X., Frey, G., Callen, W., Cabell, M., Lam, D., Macomber, J., Short, J. M., Robertson, D. E., Miller, C. (2002). A Novel, High Performance Enzyme for Starch Liquefaction. DISCOVERY AND OPTIMIZATION OF A LOW pH, THERMOSTABLE alpha -AMYLASE. J. Biol. Chem. 277: 26501-26507 [Abstract] [Full Text]  
• Vieille, C., Zeikus, G. J. (2001). Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability. Microbiol. Mol. Biol. Rev. 65: 1-43 [Abstract] [Full Text]  
• Tanaka, T., Fujiwara, S., Nishikori, S., Fukui, T., Takagi, M., Imanaka, T. (1999). A Unique Chitinase with Dual Active Sites and Triple Substrate Binding Sites from the Hyperthermophilic Archaeon Pyrococcus kodakaraensis KOD1. Appl. Environ. Microbiol. 65: 5338-5344 [Abstract] [Full Text]  
• Tachibana, Y., Kuramura, A., Shirasaka, N., Suzuki, Y., Yamamoto, T., Fujiwara, S., Takagi, M., Imanaka, T. (1999). Purification and Characterization of an Extremely Thermostable Cyclomaltodextrin Glucanotransferase from a Newly Isolated Hyperthermophilic Archaeon, a Thermococcus sp.. Appl. Environ. Microbiol. 65: 1991-1997 [Abstract] [Full Text]  
• Driskill, L. E., Kusy, K., Bauer, M. W., Kelly, R. M. (1999). Relationship between Glycosyl Hydrolase Inventory and Growth Physiology of the Hyperthermophile Pyrococcus furiosus on Carbohydrate-Based Media. Appl. Environ. Microbiol. 65: 893-897 [Abstract] [Full Text]