Screening of solvents for favoring hydrolytic activity of Candida antarctica Lipase B

  • Bartłomiej ZieniukEmail author
  • Agata Fabiszewska
  • Ewa Białecka-Florjańczyk
Research Paper


Lipases are a group of enzymes of considerable significance in organic synthesis, among which Candida antarctica lipase B (CALB) is one of the most widely studied enzymes. The activity of the biocatalyst has been intensively characterized in many organic media, but this paper aimed to compare the effect of 20 different solvents on the activity of CALB in the hydrolysis of p-nitrophenyl laurate. Nonpolar, polar aprotic, and polar protic solvents were used for enzyme pretreatment and then entered the composition of mixed solvents reaction medium. An impact of solvents on solvation processes affecting the catalysis steps, protein denaturation, and changes of its conformation was discussed. Moreover the hydrolytic activity of CALB with partition coefficient (logP) of the solvent used was correlated. It was emphasized that the substrate solubility plays an important role in solvent selection. In the presence of hydrophobic solvents, hydration layer becomes more hydrophobic facilitating the substrate access to the enzyme surface. In turn, polar compounds are good solvents for organic substrates facilitating the penetration of the aqueous layer that surrounds the surface of the enzyme. Two variants proved to be favorable for ester hydrolysis reaction: isooctane or polar solvent such as acetone, tert -butyl methyl ether, tert-butanol or acetonitrile.


Candida antarctica lipase B Organic solvent Lipase activity logP 


Author contributions

BZ and EB-F: conceptualization; BZ: investigation; BZ and AF: methodology; BZ, AF, and EB-F: writing—original draft preparation; EB-F: supervision.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.


  1. 1.
    Johannes T, Simurdiak MR, Zhao H (2006) Biocatalysis. In: Lee S (ed) Encyclopedia of chemical processing. Taylor & Francis Group, New York.Google Scholar
  2. 2.
    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(4):293–308. CrossRefPubMedGoogle Scholar
  3. 3.
    Höck H, Engel S, Weingarten S, Keul H, Schwaneberg U, Möller M, Bocola M (2018) Comparison of Candida antarctica lipase B variants for conversion of ε-caprolactone in aqueous medium—Part 2. Polymers 10(5):524. CrossRefPubMedCentralGoogle Scholar
  4. 4.
    Kundys A, Białecka-Florjańczyk E, Fabiszewska A, Małajowicz J (2018) Candida antarctica lipase B as catalyst for cyclic esters synthesis, their polymerization and degradation of aliphatic polyesters. J Polym Environ 26:396–407. CrossRefGoogle Scholar
  5. 5.
    Rosset IG, Cavalheiro MCHT, Assaf EM, Porto ALM (2013) Enzymatic esterification of oleic acid with aliphatic alcohols for the biodiesel production by Candida antarctica lipase. Catal Lett 143:863–872. CrossRefGoogle Scholar
  6. 6.
    Wang B, Zhang C, He Q, Qin H, Liang G, Liu W (2018) Efficient resolution of (R, S)-1-(1-naphthyl)ethylamine by Candida antarctica lipase B in ionic liquids. Mol Catal 448:116–121. CrossRefGoogle Scholar
  7. 7.
    Saik AYH, Lim YY, Stanslas J, Choo WS (2017) Enzymatic synthesis of quercetin oleate esters using Candida antarctica lipase B. Biotechnol Lett 39:297–304. CrossRefPubMedGoogle Scholar
  8. 8.
    Stevenson DE, Wibisono R, Jensen DJ, Stanley RA, Cooney JM (2006) Direct acylation of flavonoid glycosides with phenolic acids catalysed by Candida antarctica lipase B (Novozym 435®). Enzyme Microb Technol 39(6):1236–1241. CrossRefGoogle Scholar
  9. 9.
    Kapturowska AU, Stolarzewicz IA, Krzyczkowska J, Białecka-Florjańczyk E (2012) Studies on the lipolytic activity of sonicated enzymes from Yarrowia lipolytica. Ultrason Sonochem 19(1):186–191. CrossRefPubMedGoogle Scholar
  10. 10.
    Sangster J (1989) Octanol-water partition coefficients of simple organic compounds. J Phys Chem Ref Data 18(3):1111–1227. CrossRefGoogle Scholar
  11. 11.
    Murov SL (2018) Properties of solvents used in organic chemistry. Accessed 26 June 2019
  12. 12.
    Reichardt C, Welton T (2010) Appendix A. properties, purification, and use of organic solvents. In: Reichardt C, Welton T (eds) Solvents and solvent effects in organic chemistry. Wiley, WeinheimGoogle Scholar
  13. 13.
    Jaeger KE, Ransac S, Dijkstra BW, Colson C, van Heuvel M, Misset O (1994) Bacterial lipases. FEMS Microbiol Lett 15:29–63. CrossRefGoogle Scholar
  14. 14.
    Matsumoto M, Kida K, Kondo K (2001) Enhanced activities of lipase pretreated with organic solvents. J Chem Technol Biotechnol 76(10):1070–1073. CrossRefGoogle Scholar
  15. 15.
    Aguieiras ECG, Ribeiro DS, Couteiro PP, Bastos CMB, de Queiroz DS, Parreira JM, Langone MAP (2016) Investigation of the reuse of immobilized lipases in biodiesel synthesis: influence of different solvents in lipase activity. Appl Biochem Biotechnol 179:485–496. CrossRefPubMedGoogle Scholar
  16. 16.
    Liu Y, Zhang X, Tan H, Yan Y, Hameed BH (2010) Effect of pretreatment by different organic solvents on esterification activity and conformation of immobilized Pseudomonas cepacia lipase. Process Biochem 45:1176–1180. CrossRefGoogle Scholar
  17. 17.
    Lotti M, Pleiss J, Valero F, Ferrer P (2015) Effects of methanol on lipases: molecular, kinetic and process issues in the production of biodiesel. Biotechnol. J. 10:22–30. CrossRefPubMedGoogle Scholar
  18. 18.
    Yang Z, Russel AJ (1996) Fundamentals of non-aqueous enzymology. In: Koskinen A, Klibanov A (eds) Enzymatic reactions in organic media. Springer, NetherlandsGoogle Scholar
  19. 19.
    Du W, Xu Y, Zeng J, Liu D (2004) Novozym 435-catalysed transesterification of crude soya bean oils for biodiesel production in a solvent-free medium. Biotechnol Appl Biochem 40:187–190. CrossRefPubMedGoogle Scholar
  20. 20.
    Samukawa T, Kaieda M, Matsumoto T, Ban K, Kondo A, Shimada Y, Noda H, Fukuda H (2000) Pretreatment of immobilized Candida antarctica lipase for biodiesel fuel production from plant oil. J Biosci Bioeng 90(2):180–183. CrossRefPubMedGoogle Scholar
  21. 21.
    Graber M, Irague R, Rosenfeld E, Lamare S, Franson L, Hult K (2007) Solvent as a competitive inhibitor for Candida antarctica lipase B. Biochim. Biophys. Acta 1774:1052–1057. CrossRefPubMedGoogle Scholar
  22. 22.
    Kumar A, Dhar K, Kanwar SS, Arora PK (2016) Lipase catalysis in organic solvents: advantages and applications. Biol Proced Online 18:2. CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Garcia-Alles L, Gotor V (1998) Alcohol inhibition and specificity studies of Lipase B from Candida antarctica in organic solvents. Biotechnol Bioeng 59(2):163–170.;2-F CrossRefPubMedGoogle Scholar
  24. 24.
    Ingenbosch KN, Rousek A, Wunschik DS, Hoffmann-Jacobsen K (2019) A fluorescence-based activity assay for immobilized lipases in non-native media. Anal Biochem 569:22–27. CrossRefPubMedGoogle Scholar
  25. 25.
    Roy S, Jana B, Bagchi B (2012) Dimethyl sulfoxide induced structural transformations and non-monotonic concentration dependence of conformational fluctuation around active site of lysozyme. J Chem Phys 136(11):115103. CrossRefPubMedGoogle Scholar
  26. 26.
    Pleiss J, Fischer M, Schmid RD (1998) Anatomy of lipase binding sites: the scissile fatty acid binding site. Chem Phys Lipids 93:67–80. CrossRefPubMedGoogle Scholar
  27. 27.
    Kim HS, Ha SH, Sethaphong L, Koo Y, Yingling YG (2014) The relationship between enhanced enzyme activity and structural dynamics in ionic liquids: a combined computational and experimental study. Phys Chem Chem Phys 16:2944–2953. CrossRefPubMedGoogle Scholar
  28. 28.
    Stauch B, Fisher SJ, Cianci M (2015) Open and closed states of Candida antarctica lipase B: protonation and the mechanism of interfacial activation. J Lipid Res 56(12):2348–2358. CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Li C, Tan T, Zhang H, Feng W (2010) Analysis of the conformational stability and activity of Candida antarctica lipase B in organic solvents: insight from molecular dynamics and quantum mechanics/simulations. J Biol Chem 285(37):28434–28441. CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Faber K (2011) Biotransformations in organic chemistry, 6th edn. Springer, BerlinCrossRefGoogle Scholar
  31. 31.
    Reis P, Holmnerg K, Debeche T, Folmer B, Fauconnot L, Watzke H (2006) Lipase-catalyzed reactions at different surfaces. Langmuir 22:8169–8177. CrossRefPubMedGoogle Scholar
  32. 32.
    Reis P, Watzke H, Leser M, Holmberg K, Miller R (2010) Interfacial mechanism of lipolysis as self-regulated process. Biophys Chem 147:93–103. CrossRefPubMedGoogle Scholar
  33. 33.
    Zisis T, Freddolino PL, Turunen P, van Teeseling MCF, Rowan AE, Blank KG (2015) Interfacial activation of Candida antarctica lipase B: combined evidence from experiment and simulation. Biochemistry 54(38):5969–5979. CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Su E, Wei D (2008) Improvement in lipase-catalyzed methanolysis of triacylglycerols for biodiesel production using a solvent engineering method. J Mol Catal B: Enzym 55:118–125. CrossRefGoogle Scholar
  35. 35.
    Castillo E, Casas-Godoy L, Sandoval G (2015) Medium-engineering: a useful tool for modulating lipase activity and selectivity. Biocatalysis 1:178–188. CrossRefGoogle Scholar
  36. 36.
    Priego J, Ortiz-Nava C, Carrillo-Morales M, Lopez-Munguia A, Escalante J, Castillo E (2009) Solvent engineering: an effective tool to direct chemoselectivity in a lipase-catalyzed Michael addition. Tetrahedron 65:536–539. CrossRefGoogle Scholar
  37. 37.
    Cui C, Xie R, Tao Y, Zeng Q, Chen B (2015) Improving performance of Yarrowia lipolytica lipase lip2-catalyzed kinetic resolution of (R, S)-1-phenylethanol by solvent engineering. Biocatal Biotransformation 33(1):38–43. CrossRefGoogle Scholar
  38. 38.
    Castillo E, Pezzotti F, Navarro A, Lopez-Munguia A (2003) Lipase-catalyzed synthesis of xylitol monoesters: solvent engineering approach. J Biotechnol 102(3):251–259. CrossRefPubMedGoogle Scholar
  39. 39.
    Bellot JC, Choisnard L, Castillo E, Marty A (2001) Combining solvent engineering and thermodynamic modeling to enhance selectivity during monoglyceride synthesis by lipase-catalyzed esterification. Enzyme Microb Technol 28:362–369. CrossRefPubMedGoogle Scholar
  40. 40.
    Sheldon RA, Pereira PC (2017) Biocatalysis engineering: the big picture. Chem Soc Rev 46(10):2678–2691. CrossRefPubMedGoogle Scholar
  41. 41.
    Trodler P, Pleiss J (2008) Modeling structure and flexibility of Candida antarctica lipase B in organic solvents. BMC Struct Biol 8:9. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Chemistry, Institute of Food SciencesWarsaw University of Life SciencesWarsawPoland

Personalised recommendations