Cloning and Expression of the α-Amylase Gene from the Hyperthermophilic Archaeon Pyrococcus sp. KOD1,and Characterization of the Enzyme
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概要
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A hyperthermophilic archaeon, Pyrococcus sp. KOD1,produces extracellular thermostable α-amylase (ApkA). The apkA gene was cloned and expressed in Escherichia coli. Nucleotide sequence analysis showed that the apkA gene is composed of 1,383 hp (461 amino acid residues) and contains a signal sequence (26 N-terminal amino acid residues). The molecular weight of the mature enzyme was estimated to be 49,456 Da with 435 amino acid residues. The deduced amino acid sequence of the mature enzyme was compared with those of α-amylase and other starch-degrading enzymes from a variety of organisms. The overall homology to other amylases was not high (below 40%); however, high homology was found in the four conserved regions of the α-amylase family. The cloned apkA was overexpressed using the T7 RNA polymerase expression system.The enzyme was purified by ammonium sulfate fractionation, heat treatment, anion-exchange chromatography, and hydrophobic interaction chromatography. The optimum temperature and pH for the enzyme activity were found to be 90°C and pH 6.5,and the enzyme was considerably stable even after heating at 90°C for 60 min. The thermostability of the enzyme was enhanced in the presence of 2 mM Ca^<2+>. ApkA hydrolyzed soluble starch and glycogen to form maltose and maltotriose as the major products, and very weakly hydrolyzed pullulan to form panose or isopanose, suggesting cleavage at the α-1,4-linkage of pullulan. The steady-state kinetic constants were determined for various substrates, and ApkA was found to show high affinity to highly branched polysaccharides such as glycogen.
- 社団法人日本生物工学会の論文
- 1996-09-25
著者
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Takagi Masahiro
School Of Materials Science Japan Advanced Institute Of Science And Technology
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Takagi M
School Of Materials Science Japan Advanced Institute Of Science And Technology
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FUJIWARA SHINSUKE
Department of Bioscience, School of Science and Technology, Kwansei Gakuin University
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IMANAKA Tadayuki
Department of Synthetic and Biological Chemistry, Graduate School of Engineering, Kyoto University
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TAKAGI MASAHIRO
Department of Biotechnology, Graduate School of Engineering, Osaka University
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TACHIBANA YOSHIHISA
Research and Development Center, Nagase Co.Ltd.
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Leclere Martha
Department Of Biotechnology Faculty Of Engineering Osaka University
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Fujiwara S
School Of Science And Engineering Department Of Life Science Kwansei-gakuin University
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Fujiwara Shinsuke
Department Of Biotechnology Graduate School Of Engineering Osaka University
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Fujiwara Shinsuke
Department Of Bioscience School Of Science And Technology Kwansei Gakuin University
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TACHIBANA YOSHIHISA
Nagase Biochemicals Ltd.
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Takagi M
Nagasaki Univ. Nagasaki Jpn
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Imanaka Tadayuki
Department Of Applied Biotechnology Faculty Of Engineering Osaka University
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Fujiwara Shinsuke
School Of Science And Engineering Department Of Life Science Kwansei-gakuin University
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Tachibana Yoshihisa
Research And Development Center Nagase Co.ltd.
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Takagi Masahiro
Department Of Biotechnology Faculty Of Engineering Osaka University
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Fujiwara Shinsuke
Department Of Bioscience Graduate School Of Science And Technology Kwansei-gakuin University:researc
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