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Synthesis of fatty acid esters and diacylglycerols at elevated temperatures by alkalithermophilic lipases from Thermosyntropha lipolytica

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Journal of Industrial Microbiology & Biotechnology

Abstract

LipA and LipB of Thermosyntropha lipolytica DSM 11003 as previously published are the most alkalithermophilic (pH 25°Copt  = 9.4–9.6, T opt = 96°C) and thermostable (T 24 h1/2  = 74–76°C) lipases currently known. The purified enzymes were analyzed in organic solvents for their ability to catalyze synthesis of diacylglycerols and various alcohol fatty acids. To obtain 100% recovery and avoid a 40% and 50% loss of catalytic activity during lyophilization of purified LipA and LipB, respectively, addition of 1 mg/ml bovine serum albumin (BSA) and 25% polyethylene glycol (PEG400) was required. LipA and LipB catalyzed esterification of fatty acids and alcohols with the highest yields for octyl oleate (LipA) and lauryl oleate (LipB) and also catalyzed synthesis of 1,3-dioleoyl glycerol, 1-oleoyl-3-lauroyl glycerol, and 1-oleoyl-3-octoyl glycerol. Isooctane was the most efficient solvent for esterification reactions at 85°C. Similar to the positional specificity for the hydrolytic reaction in aqueous solutions, LipA and LipB catalyzed in organic solvents the synthesis of diacylglycerol with esterification of position 1 and 3 with a yield of 62% for di-oleoyl glycerol. The reported conversion rates do not represent the full potential of these enzymes, since only 1/100th–1/1,000th of the protein concentrations usually used in commercial processes were available. However, use of slightly increased protein concentrations confirmed the trend to higher yields with higher protein concentrations. The obtained specificity and variety of the reactions catalyzed by LipA and LipB, and their high thermostability allowing synthesis to occur at 90°C, demonstrate their great potentials for industrial applications, particularly in structured lipid biosynthesis for substrates that are less soluble at mesobiotic temperatures.

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References

  1. Adachi S, Kobayashi T (2005) Synthesis of esters by immobilized-lipase-catalyzed condensation reaction of sugars and fatty acids in water-miscible organic solvent. J Biosci Bioeng 99:87–94

    Article  PubMed  CAS  Google Scholar 

  2. Afach G, Kawanami Y, Izumori K (2005) Synthesis of d-allose fatty acid esters via lipase-catalyzed regioselective transesterification. Biosci Biotechnol Biochem 69:833–835

    Article  PubMed  CAS  Google Scholar 

  3. Affleck R, Haynes CA, Clark DS (1992) Solvent dielectric effects on protein dynamics. Proc Natl Acad Sci USA 89:5167–5170

    Article  PubMed  CAS  Google Scholar 

  4. Akimoto T, Nagase Y (2003) Novel transdermal drug penetration enhancer: synthesis and enhancing effect of alkyldisiloxane compounds containing glucopyranosyl group. J Control Release 88:243–252

    Article  PubMed  CAS  Google Scholar 

  5. Ausubel FM (2001) Current protocols in molecular biology. Wiley, New York

  6. Balcao VM, Paiva AL, Malcata FX (1996) Bioreactors with immobilized lipases: state of the art. Enzyme Microb Technol 18:392–416

    Article  PubMed  CAS  Google Scholar 

  7. Bornscheuer UT, Bessler C, Srinivas R, Krishna SH (2002) Optimizing lipases and related enzymes for efficient application. Trends Biotechnol 20:433–437

    Article  PubMed  CAS  Google Scholar 

  8. Burke PA, Griffin RG, Klibanov AM (1993) Solid-State nuclear magnetic resonance investigation of solvent dependence of tyrosyl ring motion in an enzyme. Biotechnol Bioeng 42:87–94

    Article  PubMed  CAS  Google Scholar 

  9. Carrea G, Ottolina G, Riva S (1995) Role of solvents in the control of enzyme selectivity in organic media. Trends Biotechnol 13:63–70

    Article  CAS  Google Scholar 

  10. Carrea G, Riva S (2008) Organic synthesis with enzymes in non-aqueous media. Wiley, Weinheim, pp 8–21, 53–58

  11. Castillo E, Pezzotti F, Navarro A, Lopez-Munguia A (2003) Lipase-catalyzed synthesis of xylitol monoesters: solvent engineering approach. J Biotechnol 102:251–259

    Article  PubMed  CAS  Google Scholar 

  12. Cowan DA (1997) Thermophilic proteins: stability and function in aqueous and organic solvents. Comp Biochem Physiol A Physiol 118:429–438

    Article  PubMed  CAS  Google Scholar 

  13. Debulis K, Klibanov AM (1993) Dramatic enhancement of enzymatic activity in organic solvents by lyoprotectants. Biotechnol Bioeng 41:566–571

    Article  PubMed  CAS  Google Scholar 

  14. Estrada-Mondaca S, Fournier D (1998) Stabilization of recombinant Drosophila acetylcholinesterase. Protein Expr Purif 12:166–172

    Article  PubMed  CAS  Google Scholar 

  15. Ferrato F, Carriere F, Sarda L, Verger R (1997) A critical reevaluation of the phenomenon of interfacial activation. Methods Enzymol 286:327–347

    Article  PubMed  CAS  Google Scholar 

  16. Gerday C, Aittaleb M, Bentahir M, Chessa JP, Claverie P, Collins T, D’Amico S, Dumont J, Garsoux G, Georlette D, Hoyoux A, Lonhienne T, Meuwis MA, Feller G (2000) Cold-adapted enzymes: from fundamentals to biotechnology. Trends Biotechnol 18:103–107

    Article  PubMed  CAS  Google Scholar 

  17. Ghanem A, Schurig V (2000) Lipase-catalyzed irreversible transesterification of 1-(2-furyl)ethanol using isopropenyl acetate. Chirality 13:118–123

    Article  Google Scholar 

  18. Griebenow K, Klibanov AM (1995) Lyophilization-induced reversible changes in the secondary structure of proteins. Proc Natl Acad Sci USA 92:10969–10976

    Article  PubMed  CAS  Google Scholar 

  19. Griebenow K, Klibanov AM (1996) On protein denaturation in aqueous-organic mixtures but not in pure organic solvents. J Am Chem Soc 118:11695–11700

    Article  CAS  Google Scholar 

  20. Jaeger KE, Dijkstra BW, Reetz MT (1999) Bacterial biocatalysts: molecular biology, three-dimensional structures, and biotechnological applications of lipases. Annu Rev Microbiol 53:315–351

    Article  PubMed  CAS  Google Scholar 

  21. Jaeger KE, Eggert T (2002) Lipases for biotechnology. Curr Opin Biotechnol 13:390–397

    Article  PubMed  CAS  Google Scholar 

  22. Jaeger KE, Ransac S, Dijkstra BW, Colson C, van Heuvel M, Misset O (1994) Bacterial lipases. FEMS Microbiol Rev 15:29–63

    Article  PubMed  CAS  Google Scholar 

  23. Jaeger KE, Reetz MT (1998) Microbial lipases form versatile tools for biotechnology. Trends Biotechnol 16:396–403

    Article  PubMed  CAS  Google Scholar 

  24. Jeffrey GA, Saenger W (1991) Hydrogen bonding in biological structures. Springer, Berlin

    Google Scholar 

  25. Joseph B, Ramteke PW, Thomas G (2008) Cold active microbial lipases: some hot issues and recent developments. Biotechnol Adv 26:457–470

    Article  PubMed  CAS  Google Scholar 

  26. Kumar A, Gross RA (2000) Candida antartica lipase B catalyzed polycaprolactone synthesis: effects of organic media and temperature. Biomacromolecules 1:133–138

    Article  PubMed  CAS  Google Scholar 

  27. Liao HF, Tsai WC, Chang SW, Shieh CJ (2003) Application of solvent engineering to optimize lipase-catalyzed 1, 3-diglyacylcerols by mixture response surface methodology. Biotechnol Lett 25:1857–1861

    Article  PubMed  CAS  Google Scholar 

  28. Maki KC, Davidson MH, Tsushima R, Matsuo N, Tokimitsu I, Umporowicz DM, Dicklin MR, Foster GS, Ingram KA, Anderson BD, Frost SD, Bell M (2002) Consumption of diacylglycerol oil as part of a reduced-energy diet enhances loss of body weight and fat in comparison with consumption of a triacylglycerol control oil. Am J Clin Nutr 76:1230–1236

    PubMed  CAS  Google Scholar 

  29. Miyazawa T, Kurita S, Shimaoka M, Ueji S, Yamada T (1999) Resolution of racemic carboxylic acids via the lipase-catalyzed irreversible transesterification of vinyl esters. Chirality 11:554–560

    Article  PubMed  CAS  Google Scholar 

  30. Murase T, Mizuno T, Omachi T, Onizawa K, Komine Y, Kondo H, Hase T, Tokimitsu I (2001) Dietary diacylglycerol suppresses high fat and high sucrose diet-induced body fat accumulation in C57BL/6 J mice. J Lipid Res 42:372–378

    PubMed  CAS  Google Scholar 

  31. Nagao T, Watanabe H, Goto N, Onizawa K, Taguchi H, Matsuo N, Yasukawa T, Tsushima R, Shimasaki H, Itakura H (2000) Dietary diacylglycerol suppresses accumulation of body fat compared to triacylglycerol in men in a double-blind controlled trial. J Nutr 130:792–797

    PubMed  CAS  Google Scholar 

  32. Park HJ, Choi WJ, Huh EC, Lee EY, Choi CY (1999) Production of optically active ketoprofen by direct enzymatic esterification. J Biosci Bioeng 87:545–547

    Article  PubMed  CAS  Google Scholar 

  33. Patel RN (2000) Microbial/enzymatic synthesis of chiral drug intermediates. Adv Appl Microbiol 47:33–78

    Article  PubMed  CAS  Google Scholar 

  34. Pirozzi D, Greco G Jr (2006) Lipase-catalyzed transformations for the synthesis of butyl lactate: a comparison between esterification and transesterification. Biotechnol Prog 22:444–448

    Article  PubMed  CAS  Google Scholar 

  35. Sabally K, Karboune S, Yeboah FK, Kermasha S (2005) Lipase-catalyzed esterification of selected phenolic acids with linolenyl alcohols in organic solvent media. Appl Biochem Biotechnol 127:17–27

    Article  PubMed  CAS  Google Scholar 

  36. Salameh M, Wiegel J (2007) Lipases from extremophiles and potential for industrial applications. Adv Appl Microbiol 61:253–283

    Article  PubMed  CAS  Google Scholar 

  37. Salameh MA, Wiegel J (2007) Purification and characterization of two highly thermophilic alkaline lipases from Thermosyntropha lipolytica. Appl Environ Microbiol 73:7725–7731

    Article  PubMed  CAS  Google Scholar 

  38. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor

  39. Sarda L, Desnuelle P (1958) Actions of pancreatic lipase on esters in emulsions. Biochim Biophys Acta 30:513–521

    Article  PubMed  CAS  Google Scholar 

  40. Schmid A, Dordick JS, Hauer B, Kiener A, Wubbolts M, Witholt B (2001) Industrial biocatalysis today and tomorrow. Nature 409:258–268

    Article  PubMed  CAS  Google Scholar 

  41. Schmidt-Dannert C (1999) Recombinant microbial lipases for biotechnological applications. Bioorg Med Chem 7:2123–2130

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  43. Serri NA, Kamaruddin AH, Long WS (2006) Studies of reaction parameters on synthesis of citronellyl laurate ester via immobilized Candida rugosa lipase in organic media. Bioprocess Biosyst Eng 29:253–260

    Article  PubMed  CAS  Google Scholar 

  44. Svetlitshnyi V, Rainey F, Wiegel J (1996) Thermosyntropha lipolytica gen nov., sp. nov., a lipolytic, anaerobic, alkalitolerant, thermophilic bacterium utilizing short- and long-chain fatty acids in syntrophic coculture with a methanogenic archaeum. Int J Syst Bacteriol 46:1131–1137

    Article  PubMed  CAS  Google Scholar 

  45. Volkin DB, Staubli A, Langer R, Klibanov AM (1991) Enzyme thermoinactivation in anhydrous organic solvents. Biotechnol Bioeng 37:843–853

    Article  PubMed  CAS  Google Scholar 

  46. Wang N, Liu BK, Wu Q, Wang JL, Lin XF (2005) Regioselective enzymatic synthesis of non-steroidal anti-inflammatory drugs containing glucose in organic media. Biotechnol Lett 27:789–792

    Article  PubMed  CAS  Google Scholar 

  47. Watanabe Y, Miyawaki Y, Adachi S, Nakanishi K, Matsuno R (2001) Equilibrium constant for lipase-catalyzed condensation of mannose and lauric acid in water-miscible organic solvents. Enzyme Microb Technol 29:494–498

    Article  CAS  Google Scholar 

  48. Watanabe Y, Miyawaki Y, Adachi S, Nakanishi K, Matsuno R (2000) Synthesis of lauroyl saccharides through lipase-catalyzed condensation in microaqueous water-miscible solvents. J Mol Catal B Enzym 10:241–247

    Article  CAS  Google Scholar 

  49. Weber N, Mukherjee KD (2004) Solvent-free lipase-catalyzed preparation of diacylglycerols. J Agric Food Chem 52:5347–5353

    Article  PubMed  CAS  Google Scholar 

  50. Wescott CR, Klibanov AM (1994) The solvent dependence of enzyme specificity. Biochim Biophys Acta 1206:1–9

    PubMed  CAS  Google Scholar 

  51. Xu YL, Liu ZS, Wang HF, Yan C, Gao RY (2005) Chiral recognition ability of an (S)-naproxen-imprinted monolith by capillary electrochromatography. Electrophoresis 26:804–811

    Article  PubMed  CAS  Google Scholar 

  52. Zaks A, Klibanov AM (1988) Enzymatic catalysis in nonaqueous solvents. J Biol Chem 263:3194–3201

    PubMed  CAS  Google Scholar 

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  1. Moh’d A. Salameh

    Present address: Mayo Clinic, Cancer Research Center, Jacksonville, FL, 32224, USA

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  1. Department of Microbiology, University of Georgia, 1000 Cedar Street, Biological Sciences Bldg Rm 211-212C, Athens, GA, 30602-2605, USA

    Juergen Wiegel

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Correspondence to Juergen Wiegel.

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Salameh, M.A., Wiegel, J. Synthesis of fatty acid esters and diacylglycerols at elevated temperatures by alkalithermophilic lipases from Thermosyntropha lipolytica . J Ind Microbiol Biotechnol 36, 1281–1287 (2009). https://doi.org/10.1007/s10295-009-0610-3

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  • DOI: https://doi.org/10.1007/s10295-009-0610-3

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