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.
Genomic analysis of the hyperthermophilic archaeon Pyrococcus furiosus revealed the presence of an open reading frame (ORF PF1939) similar to the enzymes in glycoside hydrolase family 13. This amylolytic enzyme, designated PFTA (Pyrococcus furiosus thermostable amylase), was cloned and expressed in Escherichia coli. The recombinant PFTA was extremely thermostable, with an optimum temperature of 90 degrees C. The substrate specificity of PFTA suggests that it possesses characteristics of both alpha-amylase and cyclodextrin-hydrolyzing enzyme. Like typical alpha-amylases, PFTA hydrolyzed maltooligosaccharides and starch to produce mainly maltotriose and maltotetraose. However, it could also attack and degrade pullulan and beta-cyclodextrin, which are resistant to alpha-amylase, to primarily produce panose and maltoheptaose, respectively. Furthermore, acarbose, a potent alpha-amylase inhibitor, was drastically degraded by PFTA, as is typical of cyclodextrin-hydrolyzing enzymes. These results confirm that PFTA possesses novel catalytic properties characteristic of both alpha-amylase and cyclodextrin-hydrolyzing enzyme.
Yang
Sung-Jae
SJ
Department of Food Science and Technology, Seoul National University, Shillim-dong, Kwanak-gu, Seoul 151-742, Korea.
Lee
Hee-Seob
HS
Park
Cheon-Seok
CS
Kim
Yong-Ro
YR
Moon
Tae-Wha
TW
Park
Kwan-Hwa
KH
D000595:Amino Acid Sequence; D001483:Base Sequence; D002240:Carbohydrate Sequence; D003505:Cyclodextrins / Q000378:metabolism*; D019641:DNA, Archaeal / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D019766:Genes, Archaeal; D006358:Hot Temperature; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism*
D003505:Cyclodextrins; D019641:DNA, Archaeal; D009844:Oligosaccharides; D011994:Recombinant Proteins; D000516:alpha-Amylases