Skip to main content
SpringerLink
Log in

Structural insights into methanol-stable variants of lipase T6 from Geobacillus stearothermophilus

  • Biotechnologically relevant enzymes and proteins
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Enzymatic production of biodiesel by transesterification of triglycerides and alcohol, catalyzed by lipases, offers an environmentally friendly and efficient alternative to the chemically catalyzed process while using low-grade feedstocks. Methanol is utilized frequently as the alcohol in the reaction due to its reactivity and low cost. However, one of the major drawbacks of the enzymatic system is the presence of high methanol concentrations which leads to methanol-induced unfolding and inactivation of the biocatalyst. Therefore, a methanol-stable lipase is of great interest for the biodiesel industry. In this study, protein engineering was applied to substitute charged surface residues with hydrophobic ones to enhance the stability in methanol of a lipase from Geobacillus stearothermophilus T6. We identified a methanol-stable variant, R374W, and combined it with a variant found previously, H86Y/A269T. The triple mutant, H86Y/A269T/R374W, had a half-life value at 70 % methanol of 324 min which reflects an 87-fold enhanced stability compared to the wild type together with elevated thermostability in buffer and in 50 % methanol. This variant also exhibited an improved biodiesel yield from waste chicken oil compared to commercial Lipolase 100L® and Novozyme® CALB. Crystal structures of the wild type and the methanol-stable variants provided insights regarding structure-stability correlations. The most prominent features were the extensive formation of new hydrogen bonds between surface residues directly or mediated by structural water molecules and the stabilization of Zn and Ca binding sites. Mutation sites were also characterized by lower B-factor values calculated from the X-ray structures indicating improved rigidity.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Adams PD, Afonine PV, Bunkóczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung L-W, Kapral GJ, Grosse-Kunstleve RW, McCoy AJ, Moriarty NW, Oeffner R, Read RJ, Richardson DC, Richardson JS, Terwilliger TC, Zwart PH (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D 66(2):213–221

    Article  CAS  PubMed  Google Scholar 

  • Al-Zuhair S (2007) Production of biodiesel: possibilities and challenges. Biofuels Bioprod Bioref 1(1):57–66

    Article  CAS  Google Scholar 

  • Arnold FH (1990) Engineering enzymes for non-aqueous solvents. Trends Biotechnol 8:244–249

    Article  CAS  PubMed  Google Scholar 

  • Badoei-Dalfard A, Khajeh K, Asghari SM, Ranjbar B, Karbalaei-Heidari HR (2010) Enhanced activity and stability in the presence of organic solvents by increased active site polarity and stabilization of a surface loop in a metalloprotease. J Biochem 148(2):231–238

    Article  CAS  PubMed  Google Scholar 

  • Bajaj A, Lohan P, Jha PN, Mehrotra R (2010) Biodiesel production through lipase catalyzed transesterification: an overview. J Mol Catal B Enzym 62(1):9–14

    Article  CAS  Google Scholar 

  • Bélafi-Bakó K, Kovács F, Gubicza L, Hancsók J (2002) Enzymatic biodiesel production from sunflower oil by Candida antarctica lipase in a solvent-free system. Biocatal Biotransform 20(6):437–439

    Article  Google Scholar 

  • 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(1–2):248–254

    Article  CAS  PubMed  Google Scholar 

  • Chakravorty D, Parameswaran S, Dubey V, Patra S (2012) Unraveling the rationale behind organic solvent stability of lipases. Appl Biochem Biotechnol 167(3):439–461

    Article  CAS  PubMed  Google Scholar 

  • Dizge N, Aydiner C, Imer DY, Bayramoglu M, Tanriseven A, Keskinler B (2009) Biodiesel production from sunflower, soybean, and waste cooking oils by transesterification using lipase immobilized onto a novel microporous polymer. Bioresour Technol 100(6):1983–1991

    Article  CAS  PubMed  Google Scholar 

  • Doukyu N, Ogino H (2010) Organic solvent-tolerant enzymes. Biochem Eng J 48(3):270–282

    Article  CAS  Google Scholar 

  • Dror A, Shemesh E, Dayan N, Fishman A (2014) Protein engineering by random mutagenesis and structure-guided consensus of Geobacillus stearothermophilus lipase T6 for enhanced stability in methanol. Appl Environ Microbiol 80(4):1515–1527

    Article  PubMed  Google Scholar 

  • Du W, Li W, Sun T, Chen X, Liu D (2008) Perspectives for biotechnological production of biodiesel and impacts. Appl Microbiol Biotechnol 79(3):331–337

    Article  CAS  PubMed  Google Scholar 

  • Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D 60(12 Part 1):2126–2132

    Article  PubMed  Google Scholar 

  • Fernandez-Lafuente R (2010) Lipase from Thermomyces lanuginosus: uses and prospects as an industrial biocatalyst. J Mol Catal B Enzym 62(3–4):197–212

    Article  CAS  Google Scholar 

  • Fischer K (1935) Neues Verfahren zur maßanalytischen Bestimmung des Wassergehaltes von Flüssigkeiten und festen Körpern. Angew Chem 48(26):394–396

    Article  CAS  Google Scholar 

  • Fishman A, Levy I, Cogan U, Shoseyov O (2002) Stabilization of horseradish peroxidase in aqueous-organic media by immobilization onto cellulose using a cellulose-binding-domain. J Mol Catal B Enzym 18(1–3):121–131

    Article  CAS  Google Scholar 

  • Guncheva M, Zhiryakova D (2011) Catalytic properties and potential applications of Bacillus lipases. J Mol Catal B Enzym 68(1):1–21

    Article  CAS  Google Scholar 

  • Gupta R, Gupta N, Rathi P (2004) Bacterial lipases: an overview of production, purification and biochemical properties. Appl Microbiol Biotechnol 64(6):763–781

    Article  CAS  PubMed  Google Scholar 

  • Herman A, Tawfik DS (2007) Incorporating synthetic oligonucleotides via gene reassembly (ISOR): a versatile tool for generating targeted libraries. Protein Eng Des Sel 20(5):219–226

    Article  CAS  PubMed  Google Scholar 

  • Hernández-Martín E, Otero C (2008) Different enzyme requirements for the synthesis of biodiesel: Novozym® 435 and Lipozyme® TL IM. Bioresour Technol 99(2):277–286

    Article  PubMed  Google Scholar 

  • Hsu A-F, Jones K, Foglia TA, Marmer WN (2002) Immobilized lipase-catalysed production of alkyl esters of restaurant grease as biodiesel. Biotechnol Appl Biochem 36(3):181–186

    Article  CAS  PubMed  Google Scholar 

  • Jaeger KE, Dijkstra BW, Reetz MT (1999) Bacterial biocatalysts: molecular biology, three-dimensional structures, and biotechnological applications of lipases. Annu Rev Microbiol 53(1):315–351

    Article  CAS  PubMed  Google Scholar 

  • Jaeger KE, Ransac S, Dijkstra BW, Colson C, van Heuvel M, Misset O (1994) Bacterial lipases. FEMS Microbiol Rev 15(v15i0001):29–63

    Article  CAS  PubMed  Google Scholar 

  • Kaewmeesri R, Srifa A, Itthibenchapong V, Faungnawakij K (2015) Deoxygenation of waste chicken fats to green diesel over Ni/Al2O3: effect of water and free fatty acid content. Energy Fuel 29(2):833–840

    CAS  Google Scholar 

  • Kamal MZ, Yedavalli P, Deshmukh MV, Rao NM (2013) Lipase in aqueous-polar organic solvents: activity, structure, and stability. Protein Sci 22(7):904–915

    Article  CAS  PubMed  Google Scholar 

  • Karmakar A, Karmakar S, Mukherjee S (2010) Properties of various plants and animals feedstocks for biodiesel production. Bioresour Technol 101(19):7201–7210

    Article  CAS  PubMed  Google Scholar 

  • Kawata T, Ogino H (2009) Enhancement of the organic solvent-stability of the LST-03 lipase by directed evolution. Biotechnol Prog 25(6):1605–1611

    CAS  PubMed  Google Scholar 

  • Kim M-H, Kim H-K, Lee J-K, Park S-Y, Oh T-K (2000) Thermostable lipase of Bacillus stearothermophilus: high-level production, purification, and calcium-dependent thermostability. Biosci Biotechnol Biochem 64(2):280–286

    Article  CAS  PubMed  Google Scholar 

  • Klibanov AM (1997) Why are enzymes less active in organic solvents than in water? Trends Biotechnol 15(3):97–101

    Article  CAS  PubMed  Google Scholar 

  • Korman T, Sahachartsiri B, Charbonneau D, Huang G, Beauregard M, Bowie J (2013) Dieselzymes: development of a stable and methanol tolerant lipase for biodiesel production by directed evolution. Biotechnol Biofuels 6(1):70

    Article  CAS  PubMed  Google Scholar 

  • Kulschewski T, Sasso F, Secundo F, Lotti M, Pleiss J (2013) Molecular mechanism of deactivation of C. antarctica lipase B by methanol. J Biotechnol 168(4):462–469

    Article  CAS  PubMed  Google Scholar 

  • Leslie A, Joint C (1992) ESF-EACMB newsletter on protein crystallography No. 26. Daresbury Laboratory, Warrington

    Google Scholar 

  • Li Y, Du W, Liu D (2014) Exploration on the effect of phospholipids on free lipase-mediated biodiesel production. J Mol Catal B Enzym 102:88–93

    Article  CAS  Google Scholar 

  • Liu K-S (1994) Preparation of fatty acid methyl esters for gas-chromatographic analysis of lipids in biological materials. J Am Oil Chem Soc 71(11):1179–1187

    Article  CAS  Google Scholar 

  • Lotti M, Pleiss J, Valero F, Ferrer P (2014) Effects of methanol on lipases: molecular, kinetic and process issues in the production of biodiesel. Biotechnol J:1–9

  • Maceiras R, Vega M, Costa C, Ramos P, Márquez MC (2011) Enzyme deactivation during biodiesel production. Chem Eng J 166(1):358–361

    Article  CAS  Google Scholar 

  • Martinez P, Arnold FH (1991) Surface charge substitutions increase the stability of α-lytic protease in organic solvents. J Am Chem Soc 113(16):6336–6337

    Article  CAS  Google Scholar 

  • McCoy A (2007) Solving structures of protein complexes by molecular replacement with Phaser. Acta Crystallogr D 63(1):32–41

    Article  CAS  PubMed  Google Scholar 

  • Meher LC, Vidya Sagar D, Naik SN (2006) Technical aspects of biodiesel production by transesterification—a review. Renew Sust Energ Rev 10(3):248–268

    Article  CAS  Google Scholar 

  • Meshulam-Simon G (2001) Isolation and characterization of lipases from thermophilic bacteria for the preparation of optically active compounds. Ph.D. thesis, Technion—Israel Institute of technology. Haifa. Israel

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

    Article  CAS  Google Scholar 

  • Nie K, Xie F, Wang F, Tan T (2006) Lipase catalyzed methanolysis to produce biodiesel: optimization of the biodiesel production. J Mol Catal B Enzym 43(1–4):142–147

    Article  CAS  Google Scholar 

  • Nizar NNA, NMJ M, Hashim DM (2013) Differentiation of lard, chicken fat, beef fat and mutton fat by GCMS and EA-IRMS techniques. J Oleo Sci 62(7):459–464

    Article  CAS  Google Scholar 

  • Noureddini H, Gao X, Philkana RS (2005) Immobilized Pseudomonas cepacia lipase for biodiesel fuel production from soybean oil. Bioresour Technol 96(7):769–777

    Article  CAS  PubMed  Google Scholar 

  • Otwinowski Z, Minor W (2001) DENZO and SCALEPACK. In: Rossmann MG, Arnold E (eds) International tables for crystallography volume F: crystallography of biological macromolecules. International tables for crystallography, vol F. Springer, Netherlands, pp 226–235

    Chapter  Google Scholar 

  • Park H, Joo J, Park K, Yoo Y (2012) Stabilization of Candida antarctica lipase B in hydrophilic organic solvent by rational design of hydrogen bond. Biotechnol Bioprocess Eng 17(4):722–728

    Article  CAS  Google Scholar 

  • Raita M, Laothanachareon T, Champreda V, Laosiripojana N (2011) Biocatalytic esterification of palm oil fatty acids for biodiesel production using glycine-based cross-linked protein coated microcrystalline lipase. J Mol Catal B Enzym 73(1–4):74–79

    Article  CAS  Google Scholar 

  • Reetz MT, Carballeira JD (2007) Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes. Nat Protoc 2(4):891–903

    Article  CAS  PubMed  Google Scholar 

  • Reetz MT, Soni P, Fernandez L, Gumulya Y, Carballeira JD (2010) Increasing the stability of an enzyme toward hostile organic solvents by directed evolution based on iterative saturation mutagenesis using the B-FIT method. Chem Commun 46(45):8657–8658

    Article  CAS  Google Scholar 

  • Rodrigues RC, Volpato G, Ayub MAZ, Wada K (2008) Lipase-catalyzed ethanolysis of soybean oil in a solvent-free system using central composite design and response surface methodology. J Chem Technol Biotechnol 83(6):849–854

    Article  CAS  Google Scholar 

  • Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  • Shimada Y, Watanabe Y, Samukawa T, Sugihara A, Noda H, Fukuda H, Tominaga Y (1999) Conversion of vegetable oil to biodiesel using immobilized Candida antarctica lipase. J Am Oil Chem Soc 76(7):789–793

    Article  CAS  Google Scholar 

  • Smith RR, Canady WJ (1992) Solvation effects upon the thermodynamic substrate activity; correlation with the kinetics of enzyme catalyzed reactions. I. Effects of added reagents such as methanol upon alpha-chymotrypsin. Biophys Chem 43(2):173–187

    Article  CAS  PubMed  Google Scholar 

  • Soumanou MM, Bornscheuer UT (2003) Improvement in lipase-catalyzed synthesis of fatty acid methyl esters from sunflower oil. Enzym Microb Technol 33(1):97–103

    Article  CAS  Google Scholar 

  • Tan T, Lu J, Nie K, Deng L, Wang F (2010) Biodiesel production with immobilized lipase: a review. Biotechnol Adv 28(5):628–634

    Article  CAS  PubMed  Google Scholar 

  • Tanaka A (1998) Differential scanning calorimetric studies on the thermal unfolding of Pseudomonas cepacia lipase in the absence and presence of alcohols. J Biochem 123(2):289–293

    Article  CAS  PubMed  Google Scholar 

  • Tyndall JDA, Sinchaikul S, Fothergill-Gilmore LA, Taylor P, Walkinshaw MD (2002) Crystal structure of a thermostable lipase from Bacillus stearothermophilus P1. J Mol Biol 323(5):859–869

    Article  CAS  PubMed  Google Scholar 

  • Vazquez-Figueroa E, Yeh V, Broering JM, Chaparro-Riggers JF, Bommarius AS (2008) Thermostable variants constructed via the structure-guided consensus method also show increased stability in salts solutions and homogeneous aqueous-organic media. Protein Eng Des Sel 21(11):673–680

    Article  CAS  PubMed  Google Scholar 

  • Watanabe Y, Shimada Y, Sugihara A, Tominaga Y (2002) Conversion of degummed soybean oil to biodiesel fuel with immobilized Candida antarctica lipase. J Mol Catal B Enzym 17(3–5):151–155

  • Xu Y, Du W, Zeng J, Liu D (2004) Conversion of soybean oil to biodiesel fuel using lipozyme TL IM in a solvent-free medium. Biocatal Biotransform 22(1):45–48

    Article  CAS  Google Scholar 

  • Yagiz F, Kazan D, Akin AN (2007) Biodiesel production from waste oils by using lipase immobilized on hydrotalcite and zeolites. Chem Eng J 134(1–3):262–267

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This project was funded in part by the Israel Ministry of Environmental Protection, grant number 132-2-2. This research benefited from use of the Technion Center of Structural Biology facility of the Lorry I. Lokey Center for Life Sciences and Engineering and the Russell Berrie Nanotechnology Institute. We thank Dr. Hay Dvir from the Technion Center of Structural Biology facility for assistance in diffraction data collection. We also thank the staff of the European Synchrotron Radiation Facility in France, beamline BM14, for the provision of synchrotron radiation facilities and assistance.

Author information

Authors and Affiliations

  1. Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa, 32000, Israel

    Adi Dror, Margarita Kanteev, Irit Kagan, Shalev Gihaz & Ayelet Fishman

  2. Macromolecular Crystallography Research Center (MCRC), Department of Life Sciences & NIBN, Ben-Gurion University of the Negev, Beer-Sheva, Israel

    Anat Shahar

Authors
  1. Adi Dror
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Margarita Kanteev
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Irit Kagan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shalev Gihaz
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Anat Shahar
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ayelet Fishman
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ayelet Fishman.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 397 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Dror, A., Kanteev, M., Kagan, I. et al. Structural insights into methanol-stable variants of lipase T6 from Geobacillus stearothermophilus . Appl Microbiol Biotechnol 99, 9449–9461 (2015). https://doi.org/10.1007/s00253-015-6700-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-015-6700-4

Keywords

Search

Navigation