Abstract
Adsorption of lipases on hydrophobic supports is a very easy immobilization protocol and it yields very interesting immobilized lipase derivatives. The open and active form of lipase molecules becomes stabilized by strong adsorption on the support surface. By using very rigid hydrophobic supports (e.g., methacrylate), lipase derivatives are very useful to catalyze biotransformations in fully anhydrous organic media (solvents, solvent-free systems, etc.) and design of continuous flow reactors. In addition to that, the design of different lipase derivatives allows the modulation of functional properties of the derivatives. In this chapter, methodology of immobilization into hydrophobic carriers is described using as case study the preparation of immobilized biocatalysts of Thermomyces lanuginosus lipase (TLL), and the following particular features will be discussed:
-
1.
Adsorption on hydrophobic supports yields lipase derivatives that are much more active and stable than other immobilized lipase derivatives.
-
2.
Regioselectivity can be modulated, for example, TLL adsorbed on divinyl benzene hydrophobic supports retains a 1,3 regioselectivity during ethanolysis of oils. On the contrary, the enzyme adsorbed on octadecyl supports loses the regioselectivity and allows the complete ethanolysis of oils (e.g., biodiesel synthesis).
-
3.
TLL adsorbed on octadecyl supports with large pore size (60 nm) is tenfold more active for ethanolysis in solvent-free systems than TLL derivatives adsorbed on supports with small pore size (10 nm).
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Kumar A, Khan A, Malhotra S, Mosurkal R, Dhawan A, Pandey MK, Singh BK, Kumar R, Prasad AK, Sharma SK, Samuelson LA, Cholli AL, Len C, Richards NGJ, Kumar J, Haag R, Watterson AC, Parmar VS (2016) Synthesis of macromolecular systems via lipase catalyzed biocatalytic reactions: applications and future perspectives. Chem Soc Rev 45:6855–6887
Borowiecki P, Justyniak I, Ochal Z (2017) Lipase-catalyzed kinetic resolution approach toward enantiomerically enriched 1-(β-hydroxypropyl)indoles. Tetrahedron Asymmetry 28:1717–1732
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 75: 99–120
Molinari F, de Mattos MC (2017) Chemoenzymatic synthesis of (S)-Pindolol using lipases. Appl Catal A Gen 546:7–14
Merza F, Fawzy A, AlNashef I, Al-Zuhair S, Taher H (2018) Effectiveness of using deep eutectic solvents as an alternative to conventional solvents in enzymatic biodiesel production from waste oils. Energy Rep 4:77–83
Rodrigues RC, Fernandez-Lafuente R (2010) Lipase from Rhizomucor miehei as an industrial biocatalyst in chemical process. J Mol Catal B Enzym 64:1–22
Neta NDAS, dos Santos JCS, Sancho SDO, Rodrigues S, Gonçalves LRB, Rodrigues LR, Teixeira JA (2012) Enzymatic synthesis of sugar esters and their potential as surface-active stabilizers of coconut milk emulsions, Food Hydrocoll 27: 324–331
Verdasco-Martín CM, Villalba M, dos Santos JCS, Tobajas M, FernandezLafuente R, Otero C (2016) Effect of chemical modification of Novozym 435 on its performance in the alcoholysis of camelina oil. Biochem Eng J 111: 75–86
Rios NS, Pinheiro MP, Lima MLB, Freire DMG, da Silva IJ, Rodríguez-Castellón E, de Sant’Ana HB, Macedo AC, Gonçalves LRB (2018) Pore-expanded SBA15 for the immobilization of a recombinant Candida antarctica lipase B: application in esterification and hydrolysis as model reactions. Chem Eng Res Des 129: 12-24
Matuoog N, Li K, Yan Y (2018) Thermomyces lanuginosus lipase immobilized on magnetic nanoparticles and its application in the hydrolysis of fish oil. J Food Biochem 42(5):e12549
Imanparast S, Hamedi J, Faramarzi MA (2018) Enzymatic esterification of acylglycerols rich in omega-3 from flaxseed oil by an immobilized solvent-tolerant lipase from Actinomadura sediminis UTMC 2870 isolated from oil-contaminated soil. Food Chem 245:934–942
Zhang Y, He S, Simpson BK (2018) Enzymes in food bioprocessing — novel food enzymes, applications, and related techniques. Curr Opin Food Sc 19:30–35
Tan T, Lu J, Nie K, Deng L, Wang F (2010) Biodiesel production with immobilized lipase: a review. Biotechnol Adv 28:628–634
Zhang B, Weng Y, Xu H, Mao Z (2012) Enzyme immobilization for biodiesel production. Appl Microbiol Biotechnol 93:61–70
Bezerra RM, Neto DMA, Galvão WS, Rios NS, Carvalho ACLM, Correa MA, Bohn F, Fernandez-Lafuente R, Fechine PBA, de Mattos MC, dos Santos JCS, Gonçalves LRB (2017) Design of a lipase-nano particle biocatalysts and its use in the kinetic resolution of medicament precursors. Biochem Eng J 125:104–115
Cubides-Roman DC, Pérez VH, de Castro HF, Orrego CE, Giraldo OH, Silveira GS, David GF (2017) Ethyl esters (biodiesel) production by Pseudomonas fluorescens lipase immobilized on chitosan with magnetic properties in a bioreactor assisted by electromagnetic field. Fuel 196:481–487
Brzozowski AM, Derewenda U, Derewenda ZS, Dodson GG, Lawson DM, Turkenburg JP, Bjorkling F, Huge-Jensen B, Patkar SA, Thim L (1991) A model for interfacial activation in lipases from the structure of a fungal lipase-inhibitor complex. Nature 351:491–494
Brady L, Brzozowski AM, Derewenda ZS, Dodson E, Dodson G, Tolley S, Turkenburg JP, Christiansen L, Huge-Jensen B, Norskov L, Thim L, Menge U (1990) A serine protease triad forms the catalytic center of a triacylglycerol lipase. Nature 343:767–770
Sarda L, Desnuelle P (1958) Action de la lipase pancreatique sur les esteres en emulsion. Biochim Biphys Acta 30:513–521
Miled N, Beisson F, de Caro J, de Caro A, Arondel V, Verger R (2001) Interfacial catalysis by lipases. J Mol Catal B Enzym 11:65–171
Bastida A, Sabuquillo P, Armisen P, Fernández-Lafuente R, Huguet J, Guisán JM (1998) A single step purification, immobilization and hyperactivation of lipases via interfacial adsorption on strongly hydrophobic supports. Biotechnol Bioeng 58:486–493
Fernandez-Lorente G, Palomo JM, Guisan JM, Fernandez-Lafuente R (2007) Effect of the immobilization protocol in the activity, stability, and enantioslectivity of Lecitase® ultra. J Mol Catal B Enzym 47:99–104
Palomo JM, Fernandez-Lorente G, Mateo C, Fuentes M, Fernandez-Lafuente R, Guisan JM (2002) Modulation of the enantioselectivity of Candida antarctica B lipase via conformational engineering: kinetic resolution of (±)--hydroxy-phenylacetic acid derivatives. Tetrahedron Asymmetr 13:1337–1345
Palomo JM, Fernandez-Lorente G, Mateo C, Ortiz C, Fernandez-Lafuente R, Guisan JM (2002) Modulation of the enantioselectivity of lipases via controlled immobilization and medium engineering: hydrolytic resolution of mandelic acid esters. Enzym Microb Technol 31:775–783
Palomo JM, Fernandez-Lorente G, Mateo C, Fernandez-Lafuente R, Guisan JM (2002) Enzymatic resolution of (±)-trans-4-(4-fluorophenyl)-6-oxo-piperidin-3-ethyl carboxylate, an intermediate in the synthesis of (−)-paroxetine. Tetrahedron Asymmetry 13:2375–2381
Fernandez-Lorente G, Fernandez-Lafuente R, Palomo JM, Mateo C, Bastida A, Coca J et al (2001) Biocatalyst engineering exerts a dramatic effect on selectivity of hydrolysis catalyzed by immobilized lipases in aqueous medium. J Mol Catal B Enzym 11:649–656
Palomo JM, Fernandez-Lorente G, Mateo C, Fuentes M, Guisan JM, Fernandez-Lafuente R (2002) Enzymatic production of (3S, 4R)-(−)-4-(4-fluorophenyl)-6-oxo-piperidin-3-carboxylic acid using a commercial preparation from Candida antarctica a: the role of a contaminant esterase. Tetrahedron Asymmetry 13:2653–2659
Palomo JM, Munoz G, Fernandez-Lorente G, Mateo C, Fuentes M, Guisan JM (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 Enzym 21:201–210
Palomo JM, Fernandez-Lorente G, Rua ML, Guisan JM (2003) Fernandez-Lafuente R. evaluation of the lipase from Bacillus thermocatenulatus as an enantioselective biocatalyst. Tetrahedron Asymmetry 14:3679–3687
Wilson L, Palomo JM, Fernández-Lorente G, Illanes A, Guisán JM, Fernández-Lafuente R (2006) Improvement of the functional properties of a thermostable lipase from alcaligenes sp. via strong adsorption on hydrophobic supports. Enzym Microb Technol 38:975–980
Cabrera Z, Fernandez-Lorente G, Fernandez-Lafuente R, Palomo JM, Guisan JM (2009) Novozym 435 displays very different selectivity compared to lipase from Candida antarctica B adsorbed on other hydrophobic supports. J Mol Catal B Enzym 57:171–176
Wong CH, Whitesides GM (1994) Enzymes in synthetic organic chemistry. Pergamon, New York
T Forutani T, Masakatsu F, Ooshima H (1996) Enzym Microb Technol 19:578–584
Santinello E, Ferrabochi P, Grisenti P (1993) Enzym Microb Technol 15:367–382
Guisan JM, Sabuquillo P, Fernández-Lafuente R, Fernández-Lorente G, Mateo C, Halling PJ, Kennedy D, Miyata E, Re D (2001) Preparation of new lipases derivatives with high activity-stability in anhydrous media: adsorption on hydrophobic supports plus hydrophilization with polyethylenimine. J Mol Catal B Enzym 11:817–824
Moreno-Perez S, Turati DFM, Borges JP, Luna P, Señorans FJ, Guisan JM, Fernandez-Lorente G (2017) Critical role of different immobilized biocatalysts of a given lipase in the selective ethanolysis of sardine oil. J Agric Food Chem 65:117–122
Abreu Silveira E, Moreno-Perez S, Basso A, Serban S, Pestana Mamede R, Tardioli PW, Sanchez Farinas C, Rocha-Martin J, Fernandez-Lorente G, Guisan JM (2017) Modulation of the regioselectivity of Thermomyces lanuginosus lipase via biocatalyst engineering for the Ethanolysis of oil in fully anhydrous medium. BMC Biotechnol 17:88
Abreu Silveira E, Moreno-Perez S, Basso A, Serban S, Pestana-Mamede R, Tardioli PW, Sanchez-Farinas C, Castejon N, Fernandez-Lorente G, Rocha-Martin J, Guisan JM (2019) Biocatalyst engineering of Thermomyces Lanuginosus lipase adsorbed on hydrophobic supports: modulation of enzyme properties for ethanolysis of oil in solvent-free systems. J Biotechnol 289:126–134
Martínez-Force E, Muñoz-Ruz J, Fernández-Martínez JM, Garces R (2000) WO 2000/074471 A1
Woods BL, Walker RA (2013) pH effects on molecular adsorption and solvation of p-nitrophenol at silica/aqueous interfaces. J Phys Chem A 117:6224–6233
Basso A, Hesseler M, Serban S (2016) Hydrophobic microenvironment optimization for efficient immobilization of lipases on octadecyl functionalised resins. Tetrahedron 72:7323–7328
Fernández-Lafuente R (2010) Lipasa de Thermomyces lanuginosus : usos y perspectivas como biocatalizador industrial. J Mol Catal B Enzym 62:197–212
Ison AP, Macrae AR, Smith CG, Bosley J (1994) Mass transfer effects in solvent-free fat interesterification reactions: influences on catalyst design. Biotechnol Bioeng 43:122–130
Trubiano G, Borio D, Errazu A (2007) Influence of the operating conditions and the external mass transfer limitations on the synthesis of fatty acid esters using a Candida antarctica lipase. Enzym Microb Technol 40:716–722
Lei Z, Jiang Q (2011) Synthesis and properties of immobilized pectinase onto the macroporous polyacrylamide microspheres. J Agric Food Chem 59:2592–2599
Zaak H, Siar E-H, Kornecki JF, Fernandez-Lopez L, Pedrero SG, Virgen-Ortíz JJ, Fernandez-Lafuente R (2017) Effect of immobilization rate and enzyme crowding on enzyme stability under different conditions. The case of lipase from Thermomyces lanuginosus immobilized on octyl agarose beads. Process Biochem 56:117–123
Zhang DH, Yuwen LX, Li C, Li YQ (2012) Effect of poly(vinyl acetate–acrylamide) microspheres properties and steric hindrance on the immobilization of Candida rugosa lipase. Bioresour Technol 124:233–236
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Fernandez-Lorente, G., Rocha-Martín, J., Guisan, J.M. (2020). Immobilization of Lipases by Adsorption on Hydrophobic Supports: Modulation of Enzyme Properties in Biotransformations in Anhydrous Media. In: Guisan, J., Bolivar, J., López-Gallego, F., Rocha-Martín, J. (eds) Immobilization of Enzymes and Cells. Methods in Molecular Biology, vol 2100. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0215-7_9
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0215-7_9
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0214-0
Online ISBN: 978-1-0716-0215-7
eBook Packages: Springer Protocols