Overexpression and characterization of a carboxypeptidase from the hyperthermophilic archaeon Thermococcus sp NA1
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Title
- Overexpression and characterization of a carboxypeptidase from the hyperthermophilic archaeon Thermococcus sp NA1
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Author(s)
- Lee, Hyun Sook; Kim, Yun Jae; Bae, Seung Seob; Jeon, g Ho Jeon; Lim, Jae Kyu; Kang, Sung Gyun; Lee, Jung-Hyun
- KIOST Author(s)
- Lee, Hyun Sook(이현숙); Kim, Yun Jae(김윤재); Kang, Sung Gyun(강성균); Lee, Jung Hyun(이정현)
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Alternative Author(s)
- 이현숙; 김윤재; 배승섭; 전정호; 강성균; 이정현
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Publication Year
- 2006-05
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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 I 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 61kDa and 125kDa 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 degrees C (k(cat)/K-m = 8.3mM(-1) s(-1)). In comparison with that of P. furiosus CP (k(cat)/K-m = 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.
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ISSN
- 0916-8451
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URI
- https://sciwatch.kiost.ac.kr/handle/2020.kiost/4890
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DOI
- 10.1271/bbb.70.1140
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Bibliographic Citation
- BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, v.70, no.5, pp.1140 - 1147, 2006
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Publisher
- TAYLOR & FRANCIS LTD
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Subject
- THERMUS-AQUATICUS YT-1; PYROCOCCUS-FURIOSUS; THERMOSTABLE CARBOXYPEPTIDASE; PURIFICATION; SEQUENCE; GENOME; TAQ
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Keywords
- genomic analysis; hyperthermophile; cloning; expression; carboxypeptidase
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Type
- Article
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Language
- English
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Document Type
- Article
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