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Characterisation of an l-Haloacid Dehalogenase from the Marine Psychrophile Psychromonas ingrahamii with Potential Industrial Application

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Abstract

The recombinant l-haloacid dehalogenase from the marine bacterium Psychromonas ingrahamii has been cloned and over-expressed in Escherichia coli. It shows activity towards monobromoacetic (100 %), monochloroacetic acid (62 %), S-chloropropionic acid (42 %), S-bromopropionic acid (31 %), dichloroacetic acid (28 %) and 2-chlorobutyric acid (10 %), respectively. The l-haloacid dehalogenase has highest activity towards substrates with shorter carbon chain lengths (≤C3), without preference towards a chlorine or bromine at the α-carbon position. Despite being isolated from a psychrophilic bacterium, the enzyme has mesophilic properties with an optimal temperature for activity of 45 °C. It retains above 70 % of its activity after being incubated at 65 °C for 90 min before being assayed at 25 °C. The enzyme is relatively stable in organic solvents as demonstrated by activity and thermal shift analysis. The V max and K m were calculated to be 0.6 μM min−1 mg−1 and 1.36 mM with monobromoacetic acid, respectively. This solvent-resistant and stable l-haloacid dehalogenase from P. ingrahamii has potential to be used as a biocatalyst in industrial processes.

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Abbreviations

l-HAD:

l-Haloacid dehalogenase

PinHAD:

Psychromonas ingrahamii l-haloacid dehalogenase

References

  • Breezee J, Cady N, Staley JT (2004) Subfreezing growth of the sea ice bacterium “Psychromonas ingrahamii” Microb. Ecol 47:300–304

    CAS  Google Scholar 

  • Brinboim H, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523

    Article  Google Scholar 

  • Burroughs AM, Allen KN, Dunaway-Mariano D, Aravind L (2006) Evolutionary genomics of the HAD superfamily: understanding the structural adaptations and catalytic diversity in a superfamily of phosphoesterases and allied enzymes. J Mol Biol 361:1003–1034

    Article  CAS  PubMed  Google Scholar 

  • Cabrita MT, Vale C, Rauter AP (2010) Halogenated compounds from marine algae. Marine Drugs 8:2301–2317

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Cavicchioli R, Siddiqui KS, Andrew D, Sowers KR (2002) Low-temperature extremophiles and their applications. Curr Opin Biotechnol 13:253–261

    Article  CAS  PubMed  Google Scholar 

  • Collins T, Meuwis MA, Gerday C, Feller G (2003) Activity, stability and flexibility in glycosidases adapted to extreme thermal environments. J Mol Biol 328:419–428

    Article  CAS  PubMed  Google Scholar 

  • D'Amico S, Marx JC, Gerday C, Feller G (2003) Activity-stability relationship in extremophilic enzymes. J Biol Chem 278:7891–7896

    Article  PubMed  Google Scholar 

  • Daniel RM, Danson MJ (2010) A new understanding of how temperature affects the catalytic activity of enzymes. Trends Biochem Sci 35:584–591

    Article  CAS  PubMed  Google Scholar 

  • DeLano WL (2002) The PyMOL Molecular Graphics System, DeLano Scientific, Palo Alto, CA.

  • Feller G (2003) Molecular adaptations to cold in psychrophilic enzymes. Cell Mol Life Sci 60:648–662

    Article  CAS  PubMed  Google Scholar 

  • Field JA, Verhagen FJM, De Jong E (1995) Natural organohalogen production by Basidiomycetes. Trends Biotechnol 13:451–456

    Article  CAS  Google Scholar 

  • Georlette D, Damiens B, Blaise V, Depiereux E, Uversky VN, Gerday C, Feller G (2003) Structural and functional adaptions to extreme temperatures in psychrophilic, mesophilic and thermophilic DNA ligases. J Biol Chem 278:37015–37023

    Article  CAS  PubMed  Google Scholar 

  • Georlette D, Blaise V, Collins T, Gratia E, D'Amico S, Hoyoux A, Marx JC, Sonan G, Feller G, Gerday C (2004) Some like it cold: biocatalysis at low temperatures. FEMS Microbiol Rev 28:25–42

    Article  CAS  PubMed  Google Scholar 

  • Gerike U, Danson MJ, Russell NJ, Hough DW (1997) Sequencing and expression of the gene encoding a cold active citrate synthase from an Antarctic bacterium, strain DS2 3R. Eur J Biochem 248:49–57

    Article  CAS  PubMed  Google Scholar 

  • Gerike U, Danson MJ, Hough DW (2001) Cold–active citrate synthase: mutagenesis of active-site residues. Prot Eng 14:655–661

    Article  CAS  Google Scholar 

  • Gomes J, Steiner W (2004) The biocatalytic potential of extremophiles and extremozymes. Food Technol Biotechnol 42:223–235

    CAS  Google Scholar 

  • Gouet P, Courcelle E, Stuart DI, Metoz F (1999) ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 15:305–308

    Article  CAS  PubMed  Google Scholar 

  • Gribble GW (1996) The diversity of natural organochlorines in living organisms. Pure Appl Chem 68:1699–1712

    Article  CAS  Google Scholar 

  • Gribble GW (1999) The diversity of naturally occurring organobromine compounds. Chem Soc Rev 28:335–346

    Article  CAS  Google Scholar 

  • Haki GD, Rakshit SK (2003) Developments in industrially important thermostable enzymes: a review. Bioresour Technol 89:17–34

    Article  CAS  PubMed  Google Scholar 

  • Hasan AK, Takada H, Esaki N, Soda K (1991) Catalytic action of l-2-haloacid dehalogenase on long chain l-2-haloalkanoic acids in organic solvents. Biotechnol Bioeng 38:1114–1117

    Article  CAS  PubMed  Google Scholar 

  • Holloway P, Trevors JT, Lee H (1998) A colorimetric assay for detecting haloalkane dehalogenase activity. J Microbiol Methods 32:31–36

    Article  CAS  Google Scholar 

  • Inatomi K, Ohba M, Oshima T (1983) Chemical properties of proteinaceous cell wall from an acido-thermophile, Sulfolobus acidocaldarius. Chemistry Letters:1191–1194

  • Kurata K, Taniguchi K, Agatsuma Y, Suzuki M (1998) Diterpenoid feeding-deterrents from Laurencia saitoi. Phytochemistry 47:363–369

    Article  CAS  Google Scholar 

  • Kurihara T, Liu JQ, Nardi-Dei V, Koshikawa H, Esaki N, Soda K (1995) Comprehensive site-directed mutagenesis of L-2-haloacid dehalogenase to probe catalytic amino acid residues. J Biochem 117:1317–1322

    CAS  PubMed  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  PubMed  Google Scholar 

  • Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948

    Article  CAS  PubMed  Google Scholar 

  • Leatherbarrow R J, Horley U K (2007) GraFit Version 6, Erithacus Software Ltd.

  • Lee CK, Daniel RM, Shepherd C, Saul D, Cary SC, Danson MJ, Eisenthal R, Peterson ME (2007) Eurythermalism and temperature dependence of enzyme activity. FASEB J 8:1934–1941

    Article  Google Scholar 

  • Li YF (1999) Global technical hexachlorocyclohexane usage and its contamination consequences in the environment: from 1948 to 1997. Sci Total Environ 232:121–158

    Article  CAS  Google Scholar 

  • Liu JQ, Kurihara T, Hasan AK, Nardi-Dei V, Koshikawa H, Esaki N, Soda K (1994) Purification and characterization of thermostable and nonthermostable 2-haloacid dehalogenases with different stereospecificities from Pseudomonas sp. strain YL. Appl Environ Microbiol 60:2389–2393

    PubMed Central  CAS  PubMed  Google Scholar 

  • Liu JQ, Kurihara T, Ichiyama S, Tsunasawa S, Miyagi, Kawasaki H, Soda K, Esaki (1998) Reaction mechanism of fluoroacetate dehalogenase from Moraxella sp. B J Biol Chem 273:30897–30902

    Article  CAS  PubMed  Google Scholar 

  • MOE 2011.10, Chemical Computing Group Inc., www.chemcomp.com

  • Nagata Y, Endo R, Ito M, Ohtsubo Y, Tsuda M (2007) Aerobic degradation of lindane (hexachlorocyclohexane) in bacteria and its biochemical and molecular basis. Appl Microbiol Biotechnol 76:741–752

    Article  CAS  PubMed  Google Scholar 

  • Nightingale PD, Malin G, Liss PS (1995) Production of chloroform and other low-molecular-weight halocarbons by some species of macroalgae. Limnol Oceanogr 40:680–689

    Article  CAS  Google Scholar 

  • Oikawa T, Kazuoka T, Soda K (2003) Paradoxical thermostable enzymes from psychrophile: molecular characterization and potentiality for biotechnological application. J Mol Catal B: Enzymatic 23:65–70

    Article  CAS  Google Scholar 

  • Paul NA, De Nys R, Steinberg PD (2006) Chemical defence against bacteria in the red alga Asparagopsis armata: linking structure with function. Mar Ecol Prog Ser 306:87–101

    Article  CAS  Google Scholar 

  • Riley M, Staley JT, Danchin A, Wang TZ, Brettin TS, Hauser LJ, Land ML, Thompson LS (2008) Genomics of an extreme psychrophile, Psychromonas ingrahamii. BMC Genomics 9:210–239

    Article  PubMed Central  PubMed  Google Scholar 

  • Russell RJ, Gerike U, Danson MJ, Hough DW, Taylor GL (1998) Structural adaptations of the cold-active citrate synthase from an Antarctic bacterium. Structure 6:351–361

    Article  CAS  PubMed  Google Scholar 

  • Rye CA, Isupov MN, Lebedev AA, Littlechild JA (2009) Biochemical and structural studies of a l-haloacid dehalogenase from the thermophilic archaeon Sulfolobus tokodaii. Extremophiles 13:179–190

    Article  CAS  PubMed  Google Scholar 

  • Schmidberger JW, Wilce JA, Tsang JS, Wilce MC (2007) Crystal structures of the substrate free-enzyme, and reaction intermediate of the HAD superfamily member, haloacid dehalogenase DehIVa from Burkholderia cepacia MBA4. J Mol Biol 368:706–717

    Article  CAS  PubMed  Google Scholar 

  • Schoemaker HE, Mink D, Wubbolts MG (2003) Dispelling the myths-biocatalysis in industrial synthesis. Science 299:1694–1697

    Article  CAS  PubMed  Google Scholar 

  • Scott C, Pandey G, Hartley CJ, Jackson CJ, Cheesman MJ, Taylor MC, Pandey R, Khurana JL, Teese M, Coppin CW, Weir K, Jain RK, Lal R, Russell RJ, Oakeshott JG (2008) The enzymatic basis for pesticide bioremediation. Ind J Microbiol 48:65–79

    Article  CAS  Google Scholar 

  • Siddiqui KS, Feller G, D'Amico S, Gerday C, Giaquinto L, Cavicchioli R (2005) The active site is the least stable structure in the unfolding pathway of a multidomain cold-adapted alpha-amylase. J Bacteriol 187:6197–6205

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Siddiqui KS, Cavicchioli R (2006) Cold-adapted enzymes. Annu Rev Biochem 75:403–433

    Article  CAS  PubMed  Google Scholar 

  • Staley JT, Gosink JJ (1999) Poles apart: biodiversity and biogeography of sea ice bacteria. Annu Rev Microbiol 53:189–215

    Article  CAS  PubMed  Google Scholar 

  • Struvay C, Feller G (2012) Optimization to low temperature activity in psychrophilic enzyme. Int J Mol Sci 13:11643–11665

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Trincone A (2011) Marine Biocatalysts: Enzymatic Features and Applications. Marine Drugs 9:478–499

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Tsang JSH, Sam L (1999) Cloning and characterization of a cryptic haloacid dehalogenase from Burkholderia cepacia MBA4. J Bacteriol 181:6003–6009

    PubMed Central  CAS  PubMed  Google Scholar 

  • Valverde C, Orozco A, Becerra A, Jeziorszi MC, Villalobos P, Solis JC (2004) Halometabolites and cellular dehalogenase systems: an evolutionary perspective. Int Rev Cytol 234:143–199

    Article  PubMed  Google Scholar 

  • Van der Ploeg J, Van Hall G, Janssen DB (1991) Characterization of the haloacid dehalogenase from Xanthobacter autotrophicus GJ10 and sequencing of the dhlB gene. J Bacteriol 173:7925–7933

    PubMed Central  PubMed  Google Scholar 

Download references

Acknowledgments

The Biotechnology and Biological Science Research Council, UK and Aquapharm Biodiscovery, Oban are acknowledged for PhD studentship funding (HRN). We thank Dr. Dorothee Gotz and Dr. Andrew Mearns Spragg for useful discussion.

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  1. The Henry Wellcome Building for Biocatalysis, Biosciences, College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK

    Halina R. Novak, Christopher Sayer, Jana Panning & Jennifer A. Littlechild

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  1. Halina R. Novak
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  2. Christopher Sayer
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  3. Jana Panning
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  4. Jennifer A. Littlechild
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Correspondence to Jennifer A. Littlechild.

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Novak, H.R., Sayer, C., Panning, J. et al. Characterisation of an l-Haloacid Dehalogenase from the Marine Psychrophile Psychromonas ingrahamii with Potential Industrial Application. Mar Biotechnol 15, 695–705 (2013). https://doi.org/10.1007/s10126-013-9522-3

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