Journal of Molecular Modeling

, 24:229 | Cite as

Investigating the structural properties of the active conformation BTL2 of a lipase from Geobacillus thermocatenulatus in toluene using molecular dynamic simulations and engineering BTL2 via in-silico mutation

  • Aslı YenenlerEmail author
  • Alessandro Venturini
  • Huseyin Cahit Burduroglu
  • Osman Uğur Sezerman
Original Paper


The discovery or development of thermoalkalophilic lipases that show high levels of catalytic activity in organic solvents would have important industrial ramifications. However, this goal is yet to be achieved because organic solvents induce structural changes in lipases that suppress their catalytic abilities. A deep understanding of these structural changes to lipases in the presence of organic solvents is required before strategies can be devised to stop them from occurring. In this work, we investigated the effects of an organic reaction medium, toluene, on the structure of the Bacillus thermocatenulatus lipase BTL2 using MD simulation. The main aims were to identify the regions of the protein that are particularly sensitive to the presence of an organic solvent, and how the presence of a hydrophobic medium affects the overall stability of the enzyme. Upon analyzing how the behavior of the enzyme differed in aqueous and hydrophobic media, it was found that many significant zones of the protein suffer in the presence of an organic solvent, which increases the rigidity of the system. This was readily apparent when we investigated important noncovalent interactions (salt bridges) and probed how distances between the atoms of the catalytic triad Ser114, Asp318, and His359 change in the presence of toluene. Moreover, the high tendency for the system to destabilize in toluene was explained by the results of FoldX calculations. Calculations showed that the addition of a small amount of water to the hydrophobic reaction environment should restore the required flexibility of BTL2. The insights gained from the analysis of our simulations allowed us to propose a modification of BTL2, the G116P mutation, that should result in the structural behavior of BTL2 in organic solvent being closer to that of BTL2 in water.


BTL2 Bacillus thermocatenulatus lipase Geobacillus thermocatenulatus Organic solvent Molecular dynamic simulations (MD) 



We wish to especially thank Associate Prof. Dr. Emel Timuçin for detailed discussions of BTL2 in organic solvents and for helping with advanced MD theory. This work was supported by The Scientific and Technological Research Council of Turkey as a bilateral program with ISOF-CNR with a grant number of 215Z712.

Supplementary material

894_2018_3753_MOESM1_ESM.docx (20.5 mb)
ESM 1 (DOCX 21030 kb)


  1. 1.
    Gurung N, Ray S, Bose S, Rai V (2013) A broader view: microbial enzymes and their relevance in industries, medicine, and beyond. Biomed Res Int 2013:329121. CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Park H, Lee K, Chi Y, Jeong S (2005) Effects of methanol on the catalytic properties of porcine pancreatic lipase. J Microbiol Biotechnol 15(2):296–301Google Scholar
  3. 3.
    Gupta R, Gupta N, Rathi P (2004) Bacterial lipases: an overview of production, purification and biochemical properties. Appl Microbiol Biotechnol 64(6):763–781. CrossRefPubMedGoogle Scholar
  4. 4.
    Grbavcic SZ, Dimitrijevic-Brankovic SI, Bezbradica DI, Siler-Marinkovic SS, Knezevic ZD (2007) Effect of fermentation condition on lipase production by Candida utilis. J Serb Chem Soc 72(8-9):757–765Google Scholar
  5. 5.
    Klibanov AM (1997) Why are enzymes less active in organic solvents than in water? Trends Biotechnol 15(3):97–101. CrossRefPubMedGoogle Scholar
  6. 6.
    Gutman AL, Meyer E, Kalerin E, Polyak F, Sterling J (1992) Enzymatic resolution of racemic amines in a continuous reactor in organic solvents. Biotechnol Bioeng 40(7):760–767. CrossRefPubMedGoogle Scholar
  7. 7.
    Schmidt-Dannert C, Rua ML, Atomi H, Schmid RD (1996) Thermoalkalophilic lipase of Bacillus thermocatenulatus. I. Molecular cloning, nucleotide sequence, purification and some properties. Biochim Biophys Acta 1301(1-2):105–114Google Scholar
  8. 8.
    Dodson G, Verma CS (2006) Protein flexibility: its role in structure and mechanism revealed by molecular simulations. Cell Mol Life Sci 63(2):207–219. CrossRefPubMedGoogle Scholar
  9. 9.
    Kumar A, Dhar K, Kanwar SS, Arora PK (2016) Lipase catalysis in organic solvents: advantages and applications. Biol Proced Online 18:2. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Herbert RA (1992) A perspective on the biotechnological potential of extremophiles. Trends Biotechnol 10(11):395–402CrossRefPubMedGoogle Scholar
  11. 11.
    Jaeger KE, Ransac S, Dijkstra BW, Colson C, van Heuvel M, Misset O (1994) Bacterial lipases. FEMS Microbiol Rev 15(1):29–63CrossRefPubMedGoogle Scholar
  12. 12.
    Riberio DB, de Castro AM, Coelho MAZ, Freiere DMG (2011) Production and use of lipases in bioenergy: a review from the feedstocks to the biodiesel production. Enzym Res 615803.
  13. 13.
    Jeong ST, Kim HK, Kim SJ, Chi SW, Pan JG, Oh TK, Ryu SE (2002) Novel zinc-binding center and a temperature switch in the Bacillus stearothermophilus L1 lipase. J Biol Chem 277(19):17041–17047.
  14. 14.
    Carrasco-Lopez C, Godoy C, de Las Rivas B, Fernandez-Lorente G, Palomo JM, Guisan JM, Fernandez-Lafuente R, Martinez-Ripoll M, Hermoso JA (2009) Activation of bacterial thermoalkalophilic lipases is spurred by dramatic structural rearrangements. J Biol Chem 284(7):4365–4372. CrossRefPubMedGoogle Scholar
  15. 15.
    Trodler P, Pleiss J (2008) Modeling structure and flexibility of Candida antarctica lipase B in organic solvents. BMC Struct Biol 8:9.
  16. 16.
    Tejo BA, Salleh AB, Pleiss J (2004) Structure and dynamics of Candida rugosa lipase: the role of organic solvent. J Mol Model 10(5-6):358–366.
  17. 17.
    Bayram Akcapinar G, Venturini A, Martelli PL, Casadio R, Sezerman UO (2015) Modulating the thermostability of endoglucanase I from Trichoderma reesei using computational approaches. Protein Eng Des Sel 28(5):127–135.
  18. 18.
    Phillips JC, Braun R, Wang W, Gumbart J, Tajkhorshid E, Villa E, Chipot C, Skeel RD, Kale L, Schulten K (2005) Scalable molecular dynamics with NAMD. J Comput Chem 26(16):1781–1802. CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Brooks BR, Brooks 3rd CL, Mackerell Jr AD, Nilsson L, Petrella RJ, Roux B, Won Y, Archontis G, Bartels C, Boresch S, Caflisch A, Caves L, Cui Q, Dinner AR, Feig M, Fischer S, Gao J, Hodoscek M, Im W, Kuczera K, Lazaridis T, Ma J, Ovchinnikov V, Paci E, Pastor RW, Post CB, Pu JZ, Schaefer M, Tidor B, Venable RM, Woodcock HL, Wu X, Yang W, York DM, Karplus M (2009) CHARMM: the biomolecular simulation program. J Comput Chem 30(10):1545–1614. CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    MacKerell AD, Bashford D, Bellott M, Dunbrack RL, Evanseck JD, Field MJ, Fischer S, Gao J, Guo H, Ha S, Joseph-McCarthy D, Kuchnir L, Kuczera K, Lau FT, Mattos C, Michnick S, Ngo T, Nguyen DT, Prodhom B, Reiher WE, Roux B, Schlenkrich M, Smith JC, Stote R, Straub J, Watanabe M, Wiorkiewicz-Kuczera J, Yin D, Karplus M (1998) All-atom empirical potential for molecular modeling and dynamics studies of proteins. J Phys Chem B 102(18):3586–3616. CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    MacKerell Jr AD, Feig M, Brooks 3rd CL (2004) Improved treatment of the protein backbone in empirical force fields. J Am Chem Soc 126(3):698–699. CrossRefPubMedGoogle Scholar
  22. 22.
    Jorgensen WL, Chandrasekar J, Madura JD, Impey RW, Klein ML (1983) Comparison of simple potential functions for simulating liquid water. J Chem Phys 76:926CrossRefGoogle Scholar
  23. 23.
    Darden T, York D, Pedersen L (1993) Particle mesh Ewald: an Nlog(N) method for Ewald sums in large systems. J Chem Phys B 98:12Google Scholar
  24. 24.
    Essman U, Perera L, Berkowitz M, Darden T, Lee H, Pedersen GG (1995) A smooth particle mesh Ewald method. J Chem Phys 19:8577–8593CrossRefGoogle Scholar
  25. 25.
    Humphrey W, Dalke A, Schulten K (1996) VMD: visual molecular dynamics. J Mol Graph 14(1):33–38 27-38CrossRefPubMedGoogle Scholar
  26. 26.
    Knapp B, Lederer N, Omasits U, Schreiner W (2010) vmdICE: a plug-in for rapid evaluation of molecular dynamics simulations using VMD. J Comput Chem 31(16):2868–2873. CrossRefPubMedGoogle Scholar
  27. 27.
    Schymkowitz J, Borg J, Stricher F, Nys R, Rousseau F, Serrano L (2005) The FoldX web server: an online force field. Nucleic Acids Res 33(web server issue):W382–W388.
  28. 28.
    Guerois R, Nielsen JE, Serrano L (2002) Predicting changes in the stability of proteins and protein complexes: a study of more than 1000 mutations. J Mol Biol 320(2):369–387. CrossRefPubMedGoogle Scholar
  29. 29.
    Krieger E, Koraimann G, Vriend G (2002) Increasing the precision of comparative models with YASARA NOVA—a self-parameterizing force field. Proteins Struct Funct Genet 47(3):393–402.
  30. 30.
    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(6326):491–494. CrossRefPubMedGoogle Scholar
  31. 31.
    Derewenda ZS, Sharp AM (1993) News from the interface: the molecular structures of triacylglyceride lipases. Trends Biochem Sci 18(1):20–25CrossRefPubMedGoogle Scholar
  32. 32.
    Choi WC, Kim MH, Ro HS, Ryu SR, Oh TK, Lee JK (2005) Zinc in lipase L1 from Geobacillus stearothermophilus L1 and structural implications on thermal stability. FEBS Lett 579(16):3461–3466.
  33. 33.
    Kim MH, Kim HK, Lee JK, Park SY, Oh TK (2000) Thermostable lipase of Bacillus stearothermophilus: high-level production, purification, and calcium-dependent thermostability. Biosci Biotechnol Biochem 64(2):280–286.
  34. 34.
    Rua ML, Schmidt-Dannert C, Wahl S, Sprauer A, Schmid RD (1997) Thermoalkalophilic lipase of Bacillus thermocatenulatus large-scale production, purification and properties: aggregation behaviour and its effect on activity. J Biotechnol 56(2):89–102Google Scholar
  35. 35.
    Marti DN, Bosshard HR (2003) Electrostatic interactions in leucine zippers: thermodynamic analysis of the contributions of Glu and his residues and the effect of mutating salt bridges. J Mol Biol 330(3):621–637CrossRefPubMedGoogle Scholar
  36. 36.
    Anderson DE, Becktel WJ, Dahlquist FW (1990) pH-induced denaturation of proteins: a single salt bridge contributes 3–5 kcal/mol to the free energy of folding of T4 lysozyme. Biochemistry 29(9):2403–2408Google Scholar
  37. 37.
    Sanfelice D, Temussi PA (2016) Cold denaturation as a tool to measure protein stability. Biophys Chem 208:4–8. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Faculty of Engineering and Natural Sciences, Molecular Biology, Genetics and BioengineeringSabanci UniversityIstanbulTurkey
  2. 2.Department of Biostatistics and Medical Informatics, School of MedicineAcibadem Mehmet Ali Aydınlar UniversityIstanbulTurkey
  3. 3.Institute of Organic Synthesis and Photoreactivity, National Research Council of ItalyBolognaItaly
  4. 4.Institute of Health Science, Department of Medical BiotechnologyAcibadem Mehmet Ali Aydınlar UniversityIstanbulTurkey

Personalised recommendations