Critical factors to high thermostability of an alpha-amylase from hyperthermophilic archaeon Thermococcus onnurineus NA1.
Genomic analysis of a hyperthermophilic archaeon, Thermococcus onnurineus NA1 [1], revealed the presence of an open reading frame consisting of 1,377 bp similar to alpha-amylases from Thermococcales, encoding a 458-residue polypeptide containing a putative 25-residue signal peptide. The mature form of the alpha-amylase was cloned and the recombinant enzyme was characterized. The optimum activity of the enzyme occurred at 80 degrees C and pH 5.5. The enzyme showed a liquefying activity, hydrolyzing maltooligosaccharides, amylopectin, and starch to produce mainly maltose (G2) to maltoheptaose (G7), but not pullulan and cyclodextrin. Surprisingly, the enzyme was not highly thermostable, with half-life (t(1/2)) values of 10 min at 90 degrees C, despite the high similarity to alpha-amylases from Pyrococcus. Factors affecting the thermostability were considered to enhance the thermostability. The presence of Ca2+ seemed to be critical, significantly changing t(1/2) at 90 degrees C to 153 min by the addition of 0.5 mM Ca2+. On the other hand, the thermostability was not enhanced by the addition of Zn2+ or other divalent metals, irrespective of the concentration. The mutagenetic study showed that the recovery of zinc-binding residues (His175 and Cys189) enhanced the thermostability, indicating that the residues involved in metal binding is very critical for the thermostability.
Korean Ocean Research & Development Institute, Ansan P O. Box 29, Seoul 425-600, Korea.
Jae Kyu
JK
Lim
Hyun Sook
HS
Lee
Yun Jae
YJ
Kim
Seung Seob
SS
Bae
Jeong Ho
JH
Jeon
Sung Gyun
SG
Kang
Jung-Hyun
JH
Lee
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D003001:Cloning, Molecular; D003067:Coenzymes / Q000494:pharmacology; D019641:DNA, Archaeal / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics*; D015870:Gene Expression; D005936:Glucans / Q000378:metabolism; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013696:Temperature; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D019843:Archaeal Proteins; D002413:Cations, Divalent; D003067:Coenzymes; D019641:DNA, Archaeal; D005936:Glucans; D009844:Oligosaccharides; D011134:Polysaccharides; D011994:Recombinant Proteins; C021705:maltooligosaccharides; C031146:maltoheptaose; D000516:alpha-Amylases; D002118:Calcium