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Cloning and Characterization of an Alpha-amylase Gene from the Hyperthermophilic Archaeon Thermococcus ThioreducensThe gene encoding an extracellular a-amylase, TTA, from the hyperthermophilic archaeon Thermococcus thioreducens was cloned and expressed in Escherichia coli. Primary structural analysis revealed high similarity with other a-amylases from the Thermococcus and Pyrococcus genera, as well as the four highly conserved regions typical for a-amylases. The 1374 bp gene encodes a protein of 457 amino acids, of which 435 constitute the mature protein preceded by a 22 amino acid signal peptide. The molecular weight of the purified recombinant enzyme was estimated to be 43 kDa by denaturing gel electrophoresis. Maximal enzymatic activity of recombinant TTA was observed at 90 C and pH 5.5 in the absence of exogenous Ca(2+), and the enzyme was considerably stable even after incubation at 90 C for 2 hours. The thermostability at 90 and 102 C was enhanced in the presence of 5 mM Ca(2+). The extraordinarily high specific activity (about 7.4 x 10(exp 3) U/mg protein at 90 C, pH 5.5 with soluble starch as substrate) together with its low pH optimum makes this enzyme an interesting candidate for starch processing applications.
Document ID
Document Type
Preprint (Draft being sent to journal)
Bernhardsdotter, Eva C. M. J.
(Alabama Univ. Huntsville, AL, United States)
Pusey, Marc L.
(NASA Marshall Space Flight Center Huntsville, AL, United States)
Ng, Joseph D.
(Alabama Univ. Huntsville, AL, United States)
Garriott, Owen K.
(Alabama Univ. Huntsville, AL, United States)
Date Acquired
August 21, 2013
Publication Date
January 1, 2004
Subject Category
Life Sciences (General)
Distribution Limits
Work of the US Gov. Public Use Permitted.
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