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Critical Factors to High Thermostability of an ${\alpha}$-Amylase from Hyperthermophilic Archaeon Thermococcus onnurineus NA1  

Lim, Jae-Kyu (Korean Ocean Research & Development Institute)
Lee, Hyun-Sook (Korean Ocean Research & Development Institute)
Kim, Yun-Jae (Korean Ocean Research & Development Institute)
Bae, Seung-Seob (Korean Ocean Research & Development Institute)
Jeon, Jeong-Ho (Korean Ocean Research & Development Institute)
Kang, Sung-Gyun (Korean Ocean Research & Development Institute)
Lee, Jung-Hyun (Korean Ocean Research & Development Institute)
Publication Information
Journal of Microbiology and Biotechnology / v.17, no.8, 2007 , pp. 1242-1248 More about this Journal
Abstract
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 $80^{\circ}C$ 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 $90^{\circ}C$, despite the high similarity to ${\alpha}$-amylases from Pyrococcus. Factors affecting the thermostability were considered to enhance the thermo stability. The presence of $Ca^{2+}$ seemed to be critical, significantly changing $t_{1/2}$ at $90^{\circ}C$ to 153 min by the addition of 0.5 mM $Ca^{2+}$. On the other hand, the thermostability was not enhanced by the addition of $Zn^{2+}$ or other divalent metals, irrespective of the concentration. The mutagenetic study showed that the recovery of zinc-binding residues (His175 and Cys189) enhanced the thermo stability, indicating that the residues involved in metal binding is very critical for the thermostability.
Keywords
Genomic analysis; hyperthermophile; cloning and expression; ${\alpha}$-amylase;
Citations & Related Records
Times Cited By KSCI : 5  (Citation Analysis)
Times Cited By Web Of Science : 9  (Related Records In Web of Science)
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