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Shewanella sp. Ke75 esterase with specificity for p-nitorphenyl butyrate: Gene cloning and characterization

  • Young-Ok KimEmail author
  • In-Suk Park
  • Hyung-Kwoun Kim
  • Bo-Hye Nam
  • Hee Jeong Kong
  • Woo-Jin Kim
  • Dong-Gyun Kim
  • Bong-Seok Kim
  • Young-Ju Jee
  • Jung-Hun Song
  • Sang-Jun Lee
Original Article Food Science/Microbiology

Abstract

A bacterial strain that produces a cold-adapted esterase was isolated from tidal flats and identified as Shewanella sp. Ke75. In the present study, the corresponding gene was cloned using the shotgun method. The amino acid sequence deduced from the nucleotide sequence (957 bp) corresponded to a protein of 318 amino acid residues with a calculated molecular weight of 34875 Da. The esterase showed 68 and 57% identities with the putative esterases of Shewanella amazonensis SB2B and Colwellia psychrerythraea 34H, respectively. The esterase contained a putative leader sequence, as well as the conserved catalytic triad (Ser, His, Asp), consensus pentapeptide GXSXG, and oxyanion hole sequence (HG). The protein Ke75 was produced in both soluble and insoluble forms when Escherichia coli cells harboring the gene were cultured at 30°C. The enzyme showed specificity for C4 (butyrate) as a substrate, with little activity toward the other p-nitrophenyl esters tested. The optimum pH and temperature for enzyme activity were pH 9.0 and 30°C, respectively. Relative activity remained up to 60% even at 5°C with an activation energy of 6.29 kcal/mol, which indicated that it was a cold-adapted enzyme. Enzyme activity was enhanced in the presence of Mn2+ ions, but inhibited by Cd2+, Cu2+, Hg2+, and Zn2+ ions.

Keywords

cold-adapted esterase gene expression Shewanella sp. substrate specificity 

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References

  1. Arpigny JL and Jaeger KE (1999) Bacterial lipolytic enzymes: classification and properties. Biochem J 343, 177–183.CrossRefGoogle Scholar
  2. Bornscheuer UT (2002) Microbial carboxylesterases: classification, properties and applications in biocatalysis. FEMS Microbiol Rev 26, 73–81.CrossRefGoogle Scholar
  3. Bornscheuer UT and Kazlauskas RJ (1999) Hydrolases in organic synthesis — regio- and stereoselective biotransformations. Wiley-VCH, Weinheim, Germany.Google Scholar
  4. Choo DW, Kurihara T, Suzuki T, Soda K, and Esaki N (1998) A cold-adapted lipase of an alaskan psychrotroph, Pseudomonas sp. strain B11-1: gene cloning and enzyme purification and characterization. Appl Environ Microbiol 64, 486–491.Google Scholar
  5. Feller G, Thiry M, Arpigny JL, and Gerday C (1991) Nucleotide sequence of the lipase gene Lip2 from the antarctic psychrotrophic Moraxella TA144 and site-specific mutagenesis of the conserved serine and histidine resides. DNA Cell Biol 10, 381–388.CrossRefGoogle Scholar
  6. Grochulski P, Li Y, Schrag JD, Bouthillier F, Smith P, Harrison D et al. (1993) Insights into interfacial activation from an open structure of Candida rugosa lipase. J Biol Chem 268, 12843–12847.Google Scholar
  7. Gupta R, Gupta N, and Rathi P (2004) Bacterial lipases: An overview of production, purification and biochemical properties. Appl Microbiol Biotechnol 64, 763–781.CrossRefGoogle Scholar
  8. Hasan F, Shah AA, and Hameed A (2006) Industrial applications of microbial lipases. Enzyme Microb Technol 39, 234–251.CrossRefGoogle Scholar
  9. Jaeger KE and Eggert T (2002) Lipases for biotechnology. Currt Opin Biotechnol 13, 390–397.CrossRefGoogle Scholar
  10. Jaeger KE, Dijkstra B, and Reetz MT (1999) Bacterial biocatalysis: Molecular biology, three-dimensional structures, and biotechnological applications of lipases. Ann Rev Microbiol 53, 315–351.CrossRefGoogle Scholar
  11. Joseph B, Ramteke PW, and Thomas G (2008) Cold active microbial lipases: some hot issues and recent developments. Biotechnol Adv 26, 457–470.CrossRefGoogle Scholar
  12. Kim BS, Oh HM, Kang HJ, Park SS, and Chun J (2004) Remarkable bacterial diversity in the tidal flat sediments as revealed by 16S rDNA analysis. J Microbial Biotechnol 24, 205–211.Google Scholar
  13. Kim HE and Kim KR (2002) Purification and characterization of an esterase from Acinetobacter lwoffii 16C-1. Curr Microbiol 44, 401–405.CrossRefGoogle Scholar
  14. Kim YO, Park IS, Kim HK, Nam BH, Kong HJ, Kim WJ et al. (2011) A novel cold-adapted esterase from Salinisphaera sp. P7-4: Gene cloning, overexpression, and characterization. J Gen Appl Microbiol 57, 357–364.CrossRefGoogle Scholar
  15. Kulakova L, Galkin A, Nakayama T, Nishino T, and Esaki N (2004) Coldactive esterase form Psychrobacter sp. Ant300: Gene cloning, characterization, and the effects of GlyPro substitution near the active site on its catalytic activity and stability. Biochim Biophys Acta 1696, 59–65.CrossRefGoogle Scholar
  16. Lee HK, Min JA, Sung HK, Won HS, and Byeong CJ (2003) Purification and characterization of cold active lipase from psychrotrophic Aeromonas sp. LPB4. J Microbiol 41, 22–27.Google Scholar
  17. Lee MH, Lee CH, Oh TK, Song JK, and Yoon JH (2006) Isolation and characterization of a novel lipase from a metagenomic library of tidal flat sediments: evidence for a new family of bacterial lipases. Appl Environ Microbial 72, 7406–7409.CrossRefGoogle Scholar
  18. Martinez C, Nicolas A, van Tilbeurgh H, Egloff MP, Cudrey C, Verger R et al. (1994) Cutinase, a lipolytic enzyme with a preformed oxyanion hole. Biochem 33, 83–89.CrossRefGoogle Scholar
  19. Park HY, Jeon JH, Kang SG, Lee JH, Lee SA, and Kim HK (2007) Functional expression and refolding of new alkaline esterase, EM2L8 from deep-sea sediment metagenome. Protein Expres Purif 52, 340–347.CrossRefGoogle Scholar
  20. Ryu HS, Kim HK, Choi WC, Kim MH, Park SY, Han NS et al. (2006) New cold-adapted lipase from Photobacterium lipolyticum sp. nov. that is closely relayed to filamentous fungal lipases. Appl Microbiol Biotechnol 70, 321–326.CrossRefGoogle Scholar
  21. Saito H and Miura KI (1963) Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim Biophys Acta 72, 619–629.CrossRefGoogle Scholar
  22. Suzuki T, Nakayama T, Choo DW, Hirano Y, Kurihara T, Nishino T et al. (2003) Cloning, heterologous expression, renaturation, and characterization of a cold-adapted esterase with unique primary structure from a psychrotroph Pseudomonas sp. strain B11-1. Protein Expr Purif 30, 171–178.CrossRefGoogle Scholar
  23. Suzuki T, Nakayama T, Kurihara T, Nishino T, and Esaki N (2001) Cold active lipolytic activity of psychrotrophic Acinetobacter sp. strain no. 6. J Biosci Bioeng 92, 144–148.Google Scholar
  24. Suzuki T, Nakayama T, Kurihara T, Nishino T, and Esaki N (2002a) A cold active esterase with a substrate preference for vinyl esters from a psychrotroph, Acinetobacter sp. strain no. 6: Gene cloning, purification, and characterization, J Mol Catal B Enzym 16, 255–263.CrossRefGoogle Scholar
  25. Suzuki T, Nakayama T, Kurihara T, Nishino T, and Esaki N (2002b) Primary structure and catalytic properties of a cold-active esterase from a psychrotroph, Acinetobacter sp. strain no. 6 isolated from Siberian soil. Biosci Biotechnol Biochem 66, 1682–1690.CrossRefGoogle Scholar
  26. Thompson JD, Higgins DG, and Gibson TJ (1994) CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.CrossRefGoogle Scholar
  27. Wei P, Bai L, Song W, and Hao G (2009) Characterization of two soil metagenome-derived lipases with high specificity for p-nitrophenyl palmitate Arch Microbiol 191, 233–240.CrossRefGoogle Scholar

Copyright information

© The Korean Society for Applied Biological Chemistry 2013

Authors and Affiliations

  • Young-Ok Kim
    • 1
    Email author
  • In-Suk Park
    • 1
  • Hyung-Kwoun Kim
    • 2
  • Bo-Hye Nam
    • 1
  • Hee Jeong Kong
    • 1
  • Woo-Jin Kim
    • 1
  • Dong-Gyun Kim
    • 1
  • Bong-Seok Kim
    • 1
  • Young-Ju Jee
    • 1
  • Jung-Hun Song
    • 3
  • Sang-Jun Lee
    • 1
  1. 1.Biotechnology Research DivisionNational Fisheries Research and Development InstituteBusanRepublic of Korea
  2. 2.Division of BiotechnologyThe Catholic University of KoreaGyeonggidoRepublic of Korea
  3. 3.Division of Marine Business & Economics, College of Fisheries SciencePukyong National UniversityBusanRepublic of Korea

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