Overexpression and Characterization of a Carboxypeptidase from the Hyperthermophilic Archaeon <I>Thermococcus</I> sp. NA1
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- LEE Hyun Sook
- Korea Ocean Research & Development Institute
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- KIM Yun Jae
- Korea Ocean Research & Development Institute
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- BAE Seung Seob
- Korea Ocean Research & Development Institute
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- JEON Jeong Ho
- Korea Ocean Research & Development Institute
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- LIM Jae Kyu
- Korea Ocean Research & Development Institute
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- KANG Sung Gyun
- Korea Ocean Research & Development Institute
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- LEE Jung-Hyun
- Korea Ocean Research & Development Institute
Bibliographic Information
- Other Title
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- Overexpression and Characterization of a Carboxypeptidase from the Hyperthermophilic Archaeon Thermococcus sp. NA1
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Abstract
Genomic analysis of a hyperthermophilic archaeon, Thermococcus sp. NA1, revealed the presence of an 1,497 bp open reading frame, encoding a protein of 499 amino acids. The deduced amino acid sequence was similar to thermostable carboxypeptidase 1 from Pyrococcus furiosus, a member of peptidase family M32. Five motifs, including the HEXXH motif with two histidines coordinated with the active site metal, were conserved. The carboxypeptidase gene was cloned and overexpressed in Escherichia coli. Molecular masses assessed by SDS–PAGE and gel filtration were 61 kDa and 125 kDa respectively, which points to a dimeric structure for the recombinant enzyme, designated TNA1_CP. The enzyme showed optimum activity toward Z-Ala-Arg at pH 6.5 and 70–80 °C (kcat⁄Km=8.3 mM−1 s−1). In comparison with that of P. furiosus CP (kcat⁄Km=667 mM−1 s−1), TNA1_CP exhibited 80-fold lower catalytic efficiency. The enzyme showed broad substrate specificity with a preference for basic, aliphatic, and aromatic C-terminal amino acids. This broad specificity was confirmed by C-terminal ladder sequencing of porcine N-acetyl-renin substrate by TNA1_CP.
Journal
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- Bioscience, Biotechnology, and Biochemistry
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Bioscience, Biotechnology, and Biochemistry 70 (5), 1140-1147, 2006
Japan Society for Bioscience, Biotechnology, and Agrochemistry
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Details 詳細情報について
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- CRID
- 1390282681449508480
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- NII Article ID
- 10018531161
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- NII Book ID
- AA10824164
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- ISSN
- 13476947
- 09168451
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- NDL BIB ID
- 7926092
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- Text Lang
- en
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- Data Source
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- JaLC
- NDL
- Crossref
- CiNii Articles
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- Abstract License Flag
- Disallowed