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Efficient biotechnological synthesis of flavor esters using a low-cost biocatalyst with immobilized Rhizomucor miehei lipase

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Abstract

In this work, the synthesis of two fruit flavor esters, namely methyl and ethyl butyrate, by lipase from Rhizomucor miehei immobilized onto chitosan in the presence of the surfactant sodium dodecyl sulfate SDS was investigated. In the optimized conditions, maximum esterification yield for ethyl butyrate and methyl butyrate was (92 ± 1%) and (89 ± 1%), respectively. Esterification yields for both reactions were comparable or even superior to the ones achieved when the synthesis was catalyzed by a commercial enzyme, Lipozyme®, at the same reaction conditions. For ethyl butyrate, the developed biocatalyst was used for seven consecutive cycles of reaction with retention of its catalytic activity. For methyl butyrate synthesis the biocatalyst was used for four consecutive cycles without loss of its catalytic activity. The results show that chitosan may be employed in obtaining biocatalysts with high catalytic efficiency and can successfully replace the currently commercial available biocatalysts.

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References

  1. Singh M, Singh S, Singh RS, Chisti Y, Banerjee UC (2008) Transesterification of primary and secondary alcohols using Pseudomonas aeruginosa lipase. Bioresour Technol 99(7):2116–2120

    Article  CAS  PubMed  Google Scholar 

  2. Alvarez-Macarie E, Baratti J (2000) Short chain flavour ester synthesis by a new esterase from Bacillus licheniformis. J Mol Catal B 10(4):377–383

    Article  CAS  Google Scholar 

  3. de Barros DPC, Fonseca LP, Fernandes P, Cabral JMS, Mojovic L (2009) Biosynthesis of ethyl caproate and other short ethyl esters catalyzed by cutinase in organic solvent. J Mol Catal B 60(3–4):178–185

    Article  CAS  Google Scholar 

  4. Kiss MA, Sefanovits-Bányai É, Tóth Á, Boross L (2004) Extractive synthesis of ethyl-oleate using alginate gel co-entrapped yeast cells and lipase enzyme. Eng Life Sci 4(5):460–464

    Article  CAS  Google Scholar 

  5. Hasan F, Shah AA, Hameed A (2006) Industrial applications of microbial lipases. Enzym Microb Technol 39(2):235–251

    Article  CAS  Google Scholar 

  6. Rueda N, dos Santos CS, Rodriguez MD, Albuquerque TL, Barbosa O, Torres R, Ortiz C, Fernandez-Lafuente R (2016) Reversible immobilization of lipases on octyl-glutamic agarose beads: a mixed adsorption that reinforces enzyme immobilization. J Mol Catal B 128:10–18

    Article  CAS  Google Scholar 

  7. dos Santos JCS, Bonazza HL, de Matos LJBL, Carneiro EA, Barbosa O, Fernandez-Lafuente R, Gonçalves LRB, de Sant’ Ana HB, Santiago-Aguiar RS (2017) Immobilization of CALB on activated chitosan: application to enzymatic synthesis in supercritical and near-critical carbon dioxide. Biotechnol Reports 14:16–26

    Article  Google Scholar 

  8. Orrego CE, Valencia JS, Zapata C (2009) Candida rugosa lipase supported on high crystallinity chitosan as biocatalyst for the synthesis of 1-butyl oleate. Catal Lett 129(3–4):312–322

    Article  CAS  Google Scholar 

  9. Berger RG (2009) Biotechnology of flavours—the next generation. Biotechnol Lett 31(11):1651–1659

    Article  CAS  PubMed  Google Scholar 

  10. Galvão WS, Pinheiro BB, Golçalves LRB et al (2018) Novel nanohybrid biocatalyst: application in the kinetic resolution of secondary alcohols. J Mater Sci. https://doi.org/10.1007/s10853-018-2641-5

    Article  Google Scholar 

  11. Lima GV, da Silva MR, de Sousa Fonseca T et al (2017) Chemoenzymatic synthesis of (S)-pindolol using lipases. Appl Catal A 546:7–14

    Article  CAS  Google Scholar 

  12. Melo A, Silva F, dos Santos J, Fernández-Lafuente R, Lemos T, Dias Filho F (2017) Synthesis of benzyl acetate catalyzed by lipase immobilized in nontoxic chitosan-polyphosphate beads. Molecules 22(12):2165

    Article  PubMed Central  CAS  Google Scholar 

  13. Pinheiro MP, Rios NS, Fonseca T, de S, Bezerra, de A F, Rodríguez-Castellón E, Fernandez-Lafuente R, Carlos de Mattos M, dos Santos JCS, Gonçalves LRB (2018) Kinetic resolution of drug intermediates catalyzed by lipase B from Candida antarctica immobilized on immobead-350. Biotechnol Prog 34(4):878–889

    Article  CAS  PubMed  Google Scholar 

  14. Bornscheuer UT, Kazlauskas RJ (2006) Hydrolases in organic synthesis: regio- and stereoselective biotransformations, 2nd edn. Wiley, Weinheim. https://doi.org/10.1002/3527607544

    Book  Google Scholar 

  15. Fernandez-Lorente G, Godoy CA, Mendes AA, Lopez-Gallego F, Grazu V, de Las Rivas B, Palomo JM, Hermoso J, Fernandez-Lafuente R, Guisan JM (2008) Solid-phase chemical amination of a lipase from Bacillus thermocatenulatus to improve its stabilization via covalent immobilization on highly activated glyoxyl-agarose. Biomacromolecules 9(9):2553–2561

    Article  CAS  PubMed  Google Scholar 

  16. Koeller KM, Wong C-H (2001) Enzymes for chemical synthesis. Nature 409(6817):232–240

    Article  CAS  PubMed  Google Scholar 

  17. Palomo JM, Muñoz G, Fernández-Lorente G, Mateo C, Fuentes M, Guisan JM, Fernández-Lafuente R (2003) Modulation of Mucor miehei lipase properties via directed immobilization on different hetero-functional epoxy resins: hydrolytic resolution of (R,S)-2-butyroyl-2-phenylacetic acid. J Mol Catal B 21(4–6):201–210

    Article  CAS  Google Scholar 

  18. Rios NS, Pinheiro BB, Pinheiro MP, Bezerra RM, dos Santos JCS, Barros Gonçalves LR (2018) Biotechnological potential of lipases from Pseudomonas: sources, properties and applications. Process Biochem. https://doi.org/10.1016/j.procbio.2018.09.003

    Article  Google Scholar 

  19. Raghavendra T, Sayania D, Madamwar D (2010) Synthesis of the ‘green apple ester’ ethyl valerate in organic solvents by Candida rugosa lipase immobilized in MBGs in organic solvents: effects of immobilization and reaction parameters. J Mol Catal B 63(1–2):31–38

    Article  CAS  Google Scholar 

  20. Krajewska B (2004) Application of chitin- and chitosan-based materials for enzyme immobilizations: a review. Enzyme Microb Technol 35(2–3):126–139

    Article  CAS  Google Scholar 

  21. Bonazza HL, Manzo RM, Santos JCS, Mammarella EJ (2017) Operational and thermal stability analysis of thermomyces lanuginosus lipase covalently immobilized onto modified chitosan supports. Appl Biochem Biotechnol. https://doi.org/10.1007/s12010-017-2546-9

    Article  PubMed  Google Scholar 

  22. Rodrigues DS, Mendes AA, Adriano WS, Gonçalves LRB, Giordano RLC (2008) Multipoint covalent immobilization of microbial lipase on chitosan and agarose activated by different methods. J Mol Catal B 51(3–4):100–109

    Article  CAS  Google Scholar 

  23. Silva JA, Macedo GP, Rodrigues DS, Giordano RLC, Gonçalves LRB (2012) Immobilization of Candida antarctica lipase B by covalent attachment on chitosan-based hydrogels using different support activation strategies. Biochem Eng J 60:16–24

    Article  CAS  Google Scholar 

  24. Tufvesson P, Törnvall U, Carvalho J, Karlsson AJ, Hatti-Kaul R (2011) Towards a cost-effective immobilized lipase for the synthesis of specialty chemicals. J Mol Catal B 68(2):200–205

    Article  CAS  Google Scholar 

  25. Villalba M, Verdasco-Martín CM, dos Santos JCS, Fernandez-Lafuente R, Otero C (2016) Operational stabilities of different chemical derivatives of Novozym 435 in an alcoholysis reaction. Enzyme Microb Technol 90:35–44

    Article  CAS  PubMed  Google Scholar 

  26. dos Santos JCS, Garcia-Galan C, Rodrigues RC, de Sant’Ana HB, Gonçalves LRB, Fernandez-Lafuente R (2014) Stabilizing hyperactivated lecitase structures through physical treatment with ionic polymers. Process Biochem 49(9):1511–1515

    Article  CAS  Google Scholar 

  27. de Oliveira UMF, Lima de Matos LJB, de Souza MCM, Pinheiro BB, dos Santos JCS, Gonçalves LRB (2018) Effect of the presence of surfactants and immobilization conditions on catalysts’ properties of Rhizomucor miehei lipase onto chitosan. Appl Biochem Biotechnol 184(4):1263–1285

    Article  PubMed  CAS  Google Scholar 

  28. Rodrigues RC, Godoy CA, Volpato G, Ayub MAZ, Fernandez-Lafuente R, Guisan JM (2009) Immobilization-stabilization of the lipase from Thermomyces lanuginosus: critical role of chemical amination. Process Biochem 44:963–968

    Article  CAS  Google Scholar 

  29. Li X-L, Zhang W-H, Wang Y-D, Dai Y-J, Zhang H-T, Wang Y, Wang H-K, Lu F-P (2014) A high-detergent-performance, cold-adapted lipase from Pseudomonas stutzeri PS59 suitable for detergent formulation. J Mol Catal B 102:16–24

    Article  CAS  Google Scholar 

  30. Helistö P, Korpela T (1998) Effects of detergents on activity of microbial lipases as measured by the nitrophenyl alkanoate esters method. Enzyme Microb Technol 23(1–2):113–117

    Article  Google Scholar 

  31. Holmberg K (2018) Interactions between surfactants and hydrolytic enzymes. Colloids Surf B 168:169–177

    Article  CAS  Google Scholar 

  32. Fernández-Lorente G, Palomo JM, Fuentes M, Mateo C, Guisán JM, Fernández-Lafuente R (2003) Self-assembly of Pseudomonas fluorescens lipase into bimolecular aggregates dramatically affects functional properties. Biotechnol Bioeng 82(2):232–237

    Article  PubMed  CAS  Google Scholar 

  33. Mogensen JE, Sehgal P, Otzen DE (2005) Activation, inhibition, and destabilization of Thermomyces lanuginosus lipase by detergents †. Biochemistry 44(5):1719–1730

    Article  CAS  PubMed  Google Scholar 

  34. Jutila A, Zhu K, Patkar SA, Vind J, Svendsen A, Kinnunen PK (2000) Detergent-induced conformational changes of Humicola lanuginosa lipase studied by fluorescence spectroscopy. Biophys J 78(3):1634–1642

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  36. Aragão VC, Rita M, Porto A, André C, Burkert V, Kalil SJ, Fernandes J, Burkert DM (2011) Response surface methodology approach for the synthesis of ethyl butyrate. Food Technol Biotechnol 49(1):103–110

    Google Scholar 

  37. Romero MD, Calvo L, Alba C, Daneshfar A (2007) A kinetic study of isoamyl acetate synthesis by immobilized lipase-catalyzed acetylation in n-hexane. J Biotechnol 127(2):269–277

    Article  CAS  PubMed  Google Scholar 

  38. Barbosa O, Ortiz C, Berenguer-Murcia Á, Torres R, Rodrigues RC, Fernandez-Lafuente R (2014) Glutaraldehyde in bio-catalysts design: a useful crosslinker and a versatile tool in enzyme immobilization. RSC Adv 4(4):1583

    Article  CAS  Google Scholar 

  39. Ozyilmaz G, Gezer E (2010) Production of aroma esters by immobilized Candida rugosa and porcine pancreatic lipase into calcium alginate gel. J Mol Catal B 64(3–4):140–145

    Article  CAS  Google Scholar 

  40. Krishna SH, Manohar B, Divakar S, Karanth NG (1999) Lipase-catalyzed synthesis of isoamyl butyrate: optimization by response surface methodology. J Am Oil Chem Soc 76(12):1483–1488

    Article  CAS  Google Scholar 

  41. Foresti ML, Ferreira ML (2007) Chitosan-immobilized lipases for the catalysis of fatty acid esterifications. Enzyme Microb Technol 40(4):769–777

    Article  CAS  Google Scholar 

  42. Romero MD, Calvo L, Alba C, Daneshfar A, Ghaziaskar HS (2005) Enzymatic synthesis of isoamyl acetate with immobilized Candida antarctica lipase in n-hexane. Enzyme Microb Technol 37:42–48

    Article  CAS  Google Scholar 

  43. Dave R, Madamwar D (2008) Candida rugosa lipase immobilized in Triton-X100 microemulsion based organogels (MBGs) for ester synthesis. Process Biochem 43(1):70–75

    Article  CAS  Google Scholar 

  44. Marty A, Chulalaksananukul W, Willemot RM, Condoret JS (1992) Kinetics of lipase-catalyzed esterification in supercritical CO2. Biotechnol Bioeng 39(3):273–280

    Article  CAS  PubMed  Google Scholar 

  45. Ceni G, Lerin LA, de Conto JF et al (2010) Optimization of 1-glyceryl benzoate production by enzymatic transesterification in organic solvents. Enzyme Microb Technol 46(2):107–112

    Article  CAS  Google Scholar 

  46. Belafi-Bako K, Badr AK, Nemestothy N, Ehrenstein U, Gubicza L (2003) Kinetics of ethyl acetate formation by lipase in organic solvent and solvent-free system. Chem Pap 57(4):278–281

    CAS  Google Scholar 

  47. Langrand G, Rondot N, Triantaphylides C, Baratti J (1990) Short chain flavour esters synthesis by microbial lipases. Biotechnol Lett 12(8):581–586

    Article  CAS  Google Scholar 

  48. Pires-Cabral P, da Fonseca MMR, Ferreira-Dias S (2009) Synthesis of ethyl butyrate in organic media catalyzed by Candida rugosa lipase immobilized in polyurethane foams: a kinetic study. Biochem Eng J 43(3):327–332

    Article  CAS  Google Scholar 

  49. Zaidi A, Gainer J, Carta G, Mrani A, Kadiri T, Belarbi Y, Mir A (2002) Esterification of fatty acids using nylon-immobilized lipase in n-hexane: kinetic parameters and chain-length effects. J Biotechnol 93(3):209–216

    Article  CAS  PubMed  Google Scholar 

  50. Paiva AL, Balcão VM, Malcata FX (2000) Kinetics and mechanisms of reactions catalyzed by immobilized lipases. Enzyme Microb Technol 27(3–5):187–204

    Article  CAS  PubMed  Google Scholar 

  51. Gonçalves LRB, Ferreira ALO, Fernandez-Lafuente R, Guisan JM, Giordano RC, Giordano RLC (2008) Influence of mass transfer limitations on the enzymatic synthesis of β-lactam antibiotics catalyzed by penicillin G acylase immobilized on glioxil-agarose. Bioprocess Biosyst Eng 31(5):411–418

    Article  PubMed  CAS  Google Scholar 

  52. Gandhi NN, Sawant SB, Joshi JB (1995) Studies on the lipozyme-catalyzed synthesis of butyl laurate. Biotechnol Bioeng 46(1):1–12

    Article  CAS  PubMed  Google Scholar 

  53. Shu C, Cai J, Huang L, Zhu X, Xu Z (2011) Biocatalytic production of ethyl butyrate from butyric acid with immobilized Candida rugosa lipase on cotton cloth. J Mol Catal B 72(3–4):139–144

    Article  CAS  Google Scholar 

  54. Klibanov AM (2001) Improving enzymes by using them in organic solvents. Nature 409(6817):241–246

    Article  CAS  PubMed  Google Scholar 

  55. Dordick JS (1989) Enzymatic catalysis in monophasic organic solvents. Enzyme Microb Technol 11(4):194–211

    Article  CAS  Google Scholar 

  56. Ghamgui H, Karra-Chaâbouni M, Gargouri Y (2004) 1-Butyl oleate synthesis by immobilized lipase from Rhizopus oryzae: a comparative study between n-hexane and solvent-free system. Enzyme Microb Technol 35(4):355–363

    Article  CAS  Google Scholar 

  57. Martins AB, Graebin NG, Lorenzoni ASG, Fernandez-Lafuente R, Ayub MAZ, Rodrigues RC (2011) Rapid and high yields of synthesis of butyl acetate catalyzed by Novozym 435: reaction optimization by response surface methodology. Process Biochem 46(12):2311–2316

    Article  CAS  Google Scholar 

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Acknowledgements

The authors would like to thank the Brazilian research-funding agencies FUNCAP (BP3-0139-00005.01.00/18), CNPq and CAPES. The data used in this article originated from the Ph.D. Thesis of Ulisses Marcondes Freire de Oliveira (ORCID ID: orcid.org/0000-0002-5479-4905), available in: (http://repositorio.ufc.br/bitstream/riufc/17222/1/2012_tese_%20umfoliveira.pdf?). This Doctoral thesis has been submitted (2012) to the Postgraduate Program in Biotechnology of the Northeast Biotechnology at Federal University of Ceará—UFC as part of the requirements for obtaining title Ph.D. in Industrial Biotechnology. Thesis Advisor: Prof. Dr. Luciana Rocha Barros Gonçalves.

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Authors and Affiliations

  1. Departamento de Engenharia Química, Universidade Federal do Ceará, Campus do Pici, Bloco 709, CEP 60455-760, Fortaleza, CE, Brazil

    Ulisses M. F. de Oliveira, Leonardo J. B. Lima de Matos, Bruna B. Pinheiro & Luciana R. B. Gonçalves

  2. Instituto de Engenharias e Desenvolvimento Sustentável, Universidade da Integração Internacional da Lusofonia Afro-Brasileira, CEP 62785-000, Acarape, CE, Brazil

    Maria Cristiane M. de Souza & José C. S. dos Santos

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  1. Ulisses M. F. de Oliveira
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  2. Leonardo J. B. Lima de Matos
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  3. Maria Cristiane M. de Souza
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  4. Bruna B. Pinheiro
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  5. José C. S. dos Santos
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  6. Luciana R. B. Gonçalves
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Correspondence to José C. S. dos Santos or Luciana R. B. Gonçalves.

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de Oliveira, U.M.F., Lima de Matos, L.J.B., de Souza, M.C.M. et al. Efficient biotechnological synthesis of flavor esters using a low-cost biocatalyst with immobilized Rhizomucor miehei lipase. Mol Biol Rep 46, 597–608 (2019). https://doi.org/10.1007/s11033-018-4514-z

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