Diversity in Size and Alkaliphily of Thermostable α-Amylase-Pullulanases (AapT) Produced by Recombinant Escherichia coli, Bacillus subtilis and the Wild-Type Bacillus sp.
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概要
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Alkaliphilic Bacillus sp. XAL601 produces a thermostable and alkaliphilic α-amylase-pullulanase of high molecular weight (AapT; M.W. 220 kDa). The aapT gene, which had been cloned and expressed in Escherichia coli, was subcloned in Bacillus subtilis ANA-1. The enzyme expressed by recombinant B. subtilis ANA-1 was detected as two truncated forms (AapT-1,85 kDa; AapT-2,135 kDa) on an amylase activity staining gel, while recombinant E. coli expressed a single amylase with a molecular weight of 220 kDa (AapT), which corresponded to the molecular weight calculated from the open reading frame of aapT. The optimum temperature for the activities of AapT-1 (85 kDa) and AapT-2 (135 kDa) was 70℃, which is the same as that of the full-sized AapT (220 kDa) from E. coli. In contrast, the optimum pH for the activities of AapT-1 and AapT-2 were pH 7.0 and pH 8.0,respectively, whereas that of the AapT (220 kDa) was pH 9.0. These observations indicated that the optimum pH for the activity of AapT was changed from the alkaline to the neutral region when the enzyme was expressed in a lower molecular weight truncated form. Furthermore, amino acid sequence alignment suggested that AapT was truncated in its C terminal region. Therefore, the noncatalytic C-terminal region may be responsible for the high optimum pH of the enzyme activity. In addition, activity staining and further analysis of AapT from the original strain, Bacillus sp. XAL601,showed glycosylation of the enzyme.
- 社団法人日本生物工学会の論文
- 1996-06-25
著者
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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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Imanaka Tadayuki
Department Of Applied Biotechnology Faculty Of Engineering Osaka University
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Lee Sang-pil
Department Of Biotechnology Faculty Of Engineering Osaka University
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Takagi Masahiro
Department Of Biotechnology Faculty Of Engineering Osaka University
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