Cloning, sequencing, and expression of the gene encoding amylopullulanase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme.
The gene encoding the Pyrococcus furiosus hyperthermophilic amylopullulanase (APU) was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a single 827-residue polypeptide with a 26-residue signal peptide. The protein sequence had very low homology (17 to 21% identity) with other APUs and enzymes of the alpha-amylase family. In particular, none of the consensus regions present in the alpha-amylase family could be identified. P. furiosus APU showed similarity to three proteins, including the P. furiosus intracellular alpha-amylase and Dictyoglomus thermophilum alpha-amylase A. The mature protein had a molecular weight of 89,000. The recombinant P. furiosus APU remained folded after denaturation at temperatures of < or = 70 degrees C and showed an apparent molecular weight of 50,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Denaturating temperatures of above 100 degrees C were required for complete unfolding. The enzyme was extremely thermostable, with an optimal activity at 105 degrees C and pH 5.5. Ca2+ increased the enzyme activity, thermostability, and substrate affinity. The enzyme was highly resistant to chemical denaturing reagents, and its activity increased up to twofold in the presence of surfactants.
Dong
G
G
Department of Biochemistry, Michigan State University, East Lansing 48824, USA.
Vieille
C
C
Zeikus
J G
JG
D000595:Amino Acid Sequence; D001105:Archaea / Q000201:enzymology* / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004795:Enzyme Stability; D015870:Gene Expression; D005798:Genes, Bacterial; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013379:Substrate Specificity
D001426:Bacterial Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C082507:amylopullulanase