Applied Microbiology and Biotechnology

, Volume 97, Issue 9, pp 3965–3978 | Cite as

Functional and structural studies of a novel cold-adapted esterase from an Arctic intertidal metagenomic library

  • Juan FuEmail author
  • Hanna-Kirsti S. Leiros
  • Donatella de Pascale
  • Kenneth A. Johnson
  • Hans-Matti Blencke
  • Bjarne Landfald
Biotechnologically Relevant Enzymes and Proteins


A novel cold-adapted lipolytic enzyme gene, est97, was identified from a high Arctic intertidal zone sediment metagenomic library. The deduced amino acid sequence of Est97 showed low similarity with other lipolytic enzymes, the maximum being 30 % identity with a putative lipase from Vibrio caribbenthicus. Common features of lipolytic enzymes, such as the GXSXG sequence motif, were detected. The gene product was over-expressed in Escherichia coli and purified. The recombinant Est97 (rEst97) hydrolysed various ρ-nitrophenyl esters with the best substrate being ρ-nitrophenyl hexanoate (K m and k cat of 39 μM and 25.8 s−1, respectively). This esterase activity of rEst97 was optimal at 35 °C and pH 7.5 and the enzyme was unstable at temperatures above 25 °C. The apparent melting temperature, as determined by differential scanning calorimetry was 39 °C, substantiating Est97 as a cold-adapted esterase. The crystal structure of rEst97 was determined by the single wavelength anomalous dispersion method to 1.6 Å resolution. The protein was found to have a typical α/β-hydrolase fold with Ser144-His226-Asp197 as the catalytic triad. A suggested, relatively short lid domain of rEst97 is composed of residues 80–114, which form an α-helix and a disordered loop. The cold adaptation features seem primarily related to a high number of methionine and glycine residues and flexible loops in the high-resolution structures.


Esterase Cold adapted Metagenomic Crystal structure Thermolabile 



The work was supported by grant from The University of Tromsø, the regional marine biotechnology program MABIT and the National Functional Genomics Program (FUGE) of the Research Council of Norway (RCN). Provision of beamtime at ID29 at the ESRF is also gratefully acknowledged. We would like to thank Prof. Pompea Del Vecchio (University of Naples, Naples) and Dr. Concetta De Santi (IBP-CNR, Naples) for their kind help in CD and DSC measurements and helpful discussions.


  1. Amann RI, Ludwig W, Schleifer KH (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59:143–169Google Scholar
  2. Arpigny JL, Jaeger KE (1999) Bacterial lipolytic enzymes: classification and properties. Biochem J 343:177–183CrossRefGoogle Scholar
  3. Bateman A, Birney E, Cerruti L, Durbin R, Etwiller L, Eddy SR, Griffiths-Jones S, Howe KL, Marshall M, Sonnhammer EL (2002) The Pfam protein families database. Nucleic Acids Res 30:276–280CrossRefGoogle Scholar
  4. Bornscheuer UT (2002) Microbial carboxyl esterases: classification, properties and application in biocatalysis. FEMS Microbiol Rev 26:73–81CrossRefGoogle Scholar
  5. Byun JS, Rhee JK, Kim ND, Yoon J, Kim DU, Koh E, Oh JW, Cho HS (2007) Crystal structure of hyperthermophilic esterase EstE1 and the relationship between its dimerization and thermostability properties. BMC Struct Biol 7:47–57CrossRefGoogle Scholar
  6. Choo D-W, Kurihara T, Suzuki T, Soda K, 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 64:486–491Google Scholar
  7. Chu X, He H, Guo C, Sun B (2008) Identification of two novel esterases from a marine metagenomic library derived from South China Sea. Appl Environ Microbiol 80:615–625Google Scholar
  8. Cieśliński H, Białkowska A, Długołęcka A, Daroch M, Tkaczuk KL, Kalinowska H, Kur J, Turkiewicz M (2007) A cold-adapted esterase from psychrotrophic Pseudoalteromas sp. strain 643A. Arch Microbiol 188:27–36CrossRefGoogle Scholar
  9. Cohen SX, Ben Jelloul M, Long F, Vagin A, Knipscheer P, Lebbink J, Sixma TK, Lamzin VS, Murshudov GN, Perrakis A (2008) ARP/wARP and molecular replacement: the next generation. Acta Crystallogr D: Biol Crystallogr 64:49–60CrossRefGoogle Scholar
  10. Collaborative Computational Project-4 (1994) The CCP4 suite: Programs for protein crystallography. Acta Crystallogr D: Biol Crystallogr 50:760–763CrossRefGoogle Scholar
  11. Cowtan K (2008) Fitting molecular fragments into electron density. Acta Crystallogr D: Biol Crystallogr 64:83–89CrossRefGoogle Scholar
  12. de Pascale D, Cusano AM, Autore F, Parrilli E, di Prisco G, Marino G, Tutino ML (2008) The cold-active Lip1 lipase from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 is a member of a new bacterial lipolytic enzyme family. Extremophiles 12:311–323CrossRefGoogle Scholar
  13. De Santi C, Tutino ML, Mandrich L, Giuliani M, Parrilli E, Del Vecchio P, de Pascale D (2010) The hormone-sensitive lipase from Psychrobacter sp. TA144: new insight in the structural/functional characterization. Biochimie 92:949–957CrossRefGoogle Scholar
  14. Di Tommaso P, Moretti S, Xenarios I, Orobitg M, Montanyola A, Chang J-M, Taly J-F, Notredame C (2011) T-coffee: a webserver for the multiple sequence alignment of protein and RNA sequences using structural information and homology extension. Nucl Acids Res 39(suppl 2):W13–W17CrossRefGoogle Scholar
  15. Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D: Biol Crystallogr 60:2126–2132CrossRefGoogle Scholar
  16. Ericsson DJ, Kasrayan A, Johansson P, Bergfors T, Sandström AG, Bäckvall JE, Mowbray SL (2008) X-ray structure of Candida antarctica lipase A shows a novel lid structure and a likely mode of interfacial activation. J Mol Biol 376:109–119CrossRefGoogle Scholar
  17. Fedøy AE, Yang N, Martinez A, Leiros H-KS, Steen IH (2007) Structural and functional properties of isocitrate dehydrogenase from the psychrophilic bacterium Desulfotalea psychrophila reveal a cold-active enzyme with an unusual high thermal stability. J Mol Biol 372:130–149CrossRefGoogle Scholar
  18. Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791CrossRefGoogle Scholar
  19. Fu C, Hu Y, Xie F, Guo H, Ashforth EJ, Polyak SW, Zhu B, Zhang L (2011) Molecular cloning and characterization of a new cold-active esterase from a deep-sea metagenomic library. Appl Microbiol Biotechnol 90:961–970CrossRefGoogle Scholar
  20. Georlette D, Blaise V, Collins T, D'Amico S, Gratia E, Hoyoux A, Marx JC, Sonan G, Feller G, Gerday C (2004) Some like it cold: biocatalysis at low temperatures. FEMS Microbiol Rev 28:25–42CrossRefGoogle Scholar
  21. Goldstone DC, Villas-Boas SG, Till M, Kelly WJ, Attwood GT, Arcus VL (2010) Structural and functional characterization of a promiscuous feruloyl esterase (Est1E) from the rumen bacterium Butyrivibrio proteoclasticus. Proteins 78:1457–1469Google Scholar
  22. Gupta R, Gupta N, Rathi P (2004) Bacterial lipases: an overview of production, purification and biochemical properties. App Microbiol Biotechnol 64:763–781CrossRefGoogle Scholar
  23. Hårdeman F, Sjöling S (2007) Metagenomic approach for the isolation of a novel low-temperature-active lipase from uncultured bacteria of marine sediment. FEMS Microbiol Ecol 59:524–534CrossRefGoogle Scholar
  24. Hasan F, Shah AA, Hameed A (2006) Industrial applications of microbial lipases. Enzyme Microb Technol 39:235–251CrossRefGoogle Scholar
  25. Hausmann S, Jaeger KE (2010) Lipolytic enzymes from bacteria. In: Timmis KN (ed) Handbook of hydrocarbon and lipid microbiology. Springer, Berlin, pp 1099–1126CrossRefGoogle Scholar
  26. Heath C, Xiao PH, Cary SC, Cowan D (2009) Identification of a novel alkaliphilic esterase active at low temperatures by screening a metagenomic library from Antarctic desert soil. Appl Environ Microbiol 75:4657–4659CrossRefGoogle Scholar
  27. Holm L, Rosenström P (2010) Dali server: conservation mapping in 3D. Nucleic Acids Res 38:W545–W549CrossRefGoogle Scholar
  28. Houde A, Kademi A, Leblanc D (2004) Lipases and their industrial applications: an overview. Appl Biochem Biotechnol 118:155–170CrossRefGoogle Scholar
  29. Hu Y, Fu C, Huang Y, Yin Y, Cheng G, Lei F, Lu N, Li J, Ashforth EJ, Zhang L, Zhu B (2010) Novel lipolytic genes from the microbial metagenomic library of the South China Sea marine sediment. FEMS Microbiol Ecol 72:228–237CrossRefGoogle Scholar
  30. Hu XP, Heath C, Taylor MP, Tuffin M, Cowan D (2012) A novel, extremely alkaliphilic and cold-active esterase from Antarctic desert soil. Extremophiles 16:79–86CrossRefGoogle Scholar
  31. Jeon JH, Kim JT, Kang SG, Lee JH, Kim SJ (2009a) Characterization and its potential application of two esterases derived from the arctic sediment metagenome. Mar Biotechnol (NY) 11:307–316CrossRefGoogle Scholar
  32. Jeon JH, Kim JT, Kim YJ, Kim HK, Lee HS, Kang SG, Kim SJ, Lee JH (2009b) Cloning and characterization of a new cold-active lipase from a deep-sea sediment metagenome. Appl Microbiol Biotechnol 81:865–874CrossRefGoogle Scholar
  33. Jeon JH, Lee HS, Kim JT, Kim SJ, Choi SH, Kang SG, Lee JH (2011) Identification of a new subfamily of salt-tolerant esterases from a metagenomic library of tidal flat sediment. Appl Microbiol Biotechnol 93:623–631CrossRefGoogle Scholar
  34. Joseph B, Ramteke PW, Thomas G (2008) Cold active microbial lipases: some hot issues and recent developments. Biotechnol Adv 26:457–470CrossRefGoogle Scholar
  35. Kabsch W (1993) Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J Appl Cryst 26:795–800CrossRefGoogle Scholar
  36. Kim BS, Oh HM, Kang H, Park SS, Chun J (2004) Remarkable bacterial diversity in the tidal flat sediment as revealed by 16S rDNA analysis. J Microbiol Biotechnol 14:205–211Google Scholar
  37. Kim BS, Oh HM, Kang H, Chun J (2005) Archaeal diversity in tidal flat sediment as revealed by 16S rDNA analyis. J Microbiol 43:144–151Google Scholar
  38. Kim EY, Oh KH, Lee MH, Kang CH, Oh TK, Yoon JH (2009) Novel cold-adapted alkaline lipase from an intertidal flat metagenome and proposal for a new family of bacterial lipases. Appl Environ Microbiol 75:257–260CrossRefGoogle Scholar
  39. Lee MH, Lee CH, Oh TK, Song JK, 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 Microbiol 72:7406–7409CrossRefGoogle Scholar
  40. Leiros H-KS, Brandsdal BO, McSweeney SM (2010) Biophysical characterization and mutational analysis of the antibiotic resistance protein NimA from Deinococcus radiodurans. Biochim Biophys Acta 1804:967–76CrossRefGoogle Scholar
  41. Liaw RB, Cheng MP, Wu MC, Lee CY (2010) Use of metagenomic approaches to isolate lipolytic genes from activated sludge. J Bioresour Technol 101:8323–8329CrossRefGoogle Scholar
  42. McDonald IK, Thornton JM (1994) Satisfying hydrogen bonding potential in proteins. J Mol Biol 238:777–793CrossRefGoogle Scholar
  43. Nacke H, Will C, Herzog S, Nowka B, Engelhaupt M, Daniel R (2011) Identification of novel lipolytic genes and gene families by screening of metagenomic libraries derived from soil samples of the German Biodiversity Exploratories. FEMS Microbiol Ecol 78:188–201CrossRefGoogle Scholar
  44. Nam KH, Kim M-Y, Kim S-J, Priyadarshi A, Lee WH, Hwang KY (2009) Structural and functional analysis of a novel EstE5 belonging to the subfamily of hormone-sensitive lipase. Biochem Biophys Res Commun 379:553–556CrossRefGoogle Scholar
  45. Rashid N, Shimada Y, Ezaki S, Atomi H, Imanaka T (2001) Low-temperature lipase from psychrotrophic Pseudomonas sp. strain KB700A. Appl Environ Microbiol 67:4064–4069CrossRefGoogle Scholar
  46. Roh C, Villatte F (2008) Isolation of a low-temperature adapted lipolytic enzyme from uncultivated micro-organism. J Appl Microbiol 105:116–123CrossRefGoogle Scholar
  47. Ronquist F, Huelsenbeck JP (2003) MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574CrossRefGoogle Scholar
  48. Ryu HS, Kim HK, Choi WC, Kim MH, Park SY, Han NS, Oh TK, Lee JK (2006) New cold-adapted lipase from Photobacterium lipolyticum sp. nov. that is closely related to filamentous fungal lipases. Appl Microbiol Biotechnol 70:321–326CrossRefGoogle Scholar
  49. Santarossa G, Lafranconi PG, Alquati C, DeGioia L, Alberghina L, Fantucci P, Lotti M (2005) Mutations in the "lid" region affect chain length specificity and thermostability of a Pseudomonas fragi lipase. FEBS Lett 579:2383–2386CrossRefGoogle Scholar
  50. Schneider TR, Pape T (2004) HKL2MAP: a graphical user interface for macromolecular phasing with SHELX programs. J Appl Crystallogr 37:843–844CrossRefGoogle Scholar
  51. Sheldrick GM (2008) A short history of SHELX. Acta Crystallogr Sect A 64:112–122CrossRefGoogle Scholar
  52. Siddiqui KS, Cavicchioli R (2006) Cold-adapted enzymes. Annu Rev Biochem 75:403–433CrossRefGoogle Scholar
  53. Smalås AO, Leiros HK, Os V, Willassen NP (2000) Cold adapted enzymes. Biotechnol Annu Rev 6:1–57CrossRefGoogle Scholar
  54. Suzuki T, Nakayama T, Kurihara T, Nishino T, Esaki N (2002) 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 66:1682–1690CrossRefGoogle Scholar
  55. Suzuki T, Nakayama T, Choo DW, Hirano Y, Kurihara T, Nishino T, Esaki N (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 30:171–178CrossRefGoogle Scholar
  56. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599CrossRefGoogle Scholar
  57. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882CrossRefGoogle Scholar
  58. Uppenberg J, Hansen MT, Patkar S, Jones TA (1994) The sequence, crystal structure determination and refinement of two crystal forms of lipase B from Candida antarctica. Structure 2:293–308CrossRefGoogle Scholar
  59. Vriend G (1990) What if: a molecular modeling and drug design program. J Mol Graph 8:52–56CrossRefGoogle Scholar
  60. Whitmore L, Wallace BA (2008) Protein secondary structure analyses from circular dichroism spectroscopy: methods and reference databases. Biopolymers 89:392–400CrossRefGoogle Scholar
  61. Zhou J, Bruns MA, Tiedje JM (1996) DNA recovery from soils of diverse composition. Appl Environ Microbiol 62:316–322Google Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Juan Fu
    • 1
    Email author
  • Hanna-Kirsti S. Leiros
    • 2
  • Donatella de Pascale
    • 3
  • Kenneth A. Johnson
    • 2
  • Hans-Matti Blencke
    • 1
  • Bjarne Landfald
    • 1
  1. 1.Norwegian College of Fishery ScienceUniversity of TromsøTromsøNorway
  2. 2.The Norwegian Structural Biology Centre (NorStruct), Department of ChemistryUniversity of TromsøTromsøNorway
  3. 3.Institute of Protein BiochemistryNational Research CouncilNaplesItaly

Personalised recommendations