Skip to main content
SpringerLink
Log in
SpringerLink
Log in

Evolving Enzymes for Biocatalysis

  • Reference work entry
  • First Online:
Consequences of Microbial Interactions with Hydrocarbons, Oils, and Lipids: Production of Fuels and Chemicals

Part of the book series: Handbook of Hydrocarbon and Lipid Microbiology ((HHLM))

Abstract

This chapter covers concepts developed for the directed evolution of enzymes. The principle strategy is given in comparison to rational protein design followed by a description of the most prominent methods for creation of mutant libraries. Screening and selection strategies to identify the best hits in these libraries are presented followed by several assays developed for a range of enzyme classes. Finally, selected examples for the successful application of evolutionary methods to optimize biocatalysts are given.

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

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 359.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 329.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  • Acker MG, Auld DS (2014) Considerations for the design and reporting of enzyme assays in high-throughput screening applications. Perspect Sci 1:56–73

    Article  Google Scholar 

  • Arnold FH, Georgiou G (eds) (2003a) Directed enzyme evolution: screening and selection methods. Humana Press, Totawa

    Google Scholar 

  • Arnold FH, Georgiou G (eds) (2003b) Directed evolution library creation: methods and protocols. Humana Press, Totawa

    Google Scholar 

  • Bartsch S, Kourist R, Bornscheuer UT (2008) Complete inversion of enantioselectivity towards acetylated tertiary alcohols by a double mutant of a Bacillus subtilis esterase. Angew Chem Int Ed 47:1508–1511

    Article  Google Scholar 

  • Baumann M, Stürmer R, Bornscheuer UT (2001) A high-throughput-screening method for the identification of active and enantioselective hydrolases. Angew Chem Int Ed 40:4201–4204

    Article  CAS  Google Scholar 

  • Baxter S, Royer S, Grogan G, Brown F, Holt-Tiffin KE, Taylor IN, Fotheringham IG, Campopiano DJ (2012) An improved racemase/acylase biotransformation for the preparation of enantiomerically pure amino acids. J Am Chem Soc 134:19310–19313

    Article  CAS  PubMed  Google Scholar 

  • Biles BD, Connolly BA (2004) Low-fidelity Pyrococcus furiosus DNA polymerase mutants useful in error-prone PCR. Nucleic Acids Res 32:e176

    Article  PubMed  PubMed Central  Google Scholar 

  • Bornscheuer UT (2016) Protein engineering: beating the odds. Nat Chem Biol 12:54–55

    Article  CAS  PubMed  Google Scholar 

  • Bornscheuer U, Huisman G, Kazlauskas R, Lutz S, Moore J, Robins K (2012) Engineering the third wave of biocatalysis. Nature 485:185–194

    Article  CAS  PubMed  Google Scholar 

  • Brundiek H, Evitt AS, Kourist R, Bornscheuer UT (2012) Creation of a highly trans fatty acid selective lipase by protein engineering. Angew Chem Int Ed 51:412–414

    Article  CAS  Google Scholar 

  • Caldwell RC, Joyce GF (1992) Randomization of genes by PCR mutagenesis. PCR Methods Appl 2:28–33

    Article  Google Scholar 

  • Chen B, Lim S, Kannan A, Alford SC, Sunden F, Herschlag D, Dimov IK, Baer TM, Cochran JR (2016) High-throughput analysis and protein engineering using microcapillary arrays. Nat Chem Biol 12:76–81

    Article  CAS  PubMed  Google Scholar 

  • Colin P-Y, Kintses B, Gielen F, Miton CM, Fischer G, Mohamed MF, Hyvonen M, Morgavi DP, Janssen DB, Hollfelder F (2015) Ultrahigh-throughput discovery of promiscuous enzymes by picodroplet functional metagenomics. Nat Commun 6. https://doi.org/10.1038/ncomms10008

  • Currin A, Swainston N, Day PJ, Kell DB (2015) Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently. Chem Soc Rev 44:1172–1239

    Article  CAS  PubMed  Google Scholar 

  • Dombkowski AA, Sultana KZ, Craig DB (2014) Protein disulfide engineering. FEBS Lett 588:206–212

    Article  CAS  PubMed  Google Scholar 

  • Dörr M, Fibinger MP, Last D, Schmidt S, Santos‐Aberturas J, Böttcher D, Hummel A, Vickers C, Voss M, Bornscheuer UT (2016) Fully automatized high throughput enzyme library screening using a robotic platform. Biotechnol Bioeng 113:1421–1432. https://doi.org/10.1002/bit.25925

    Article  PubMed  Google Scholar 

  • Engström K, Nyhlen J, Sandström AG, Backväll JE (2010) Directed evolution of an enantioselective lipase with broad substrate scope for hydrolysis of alpha-substituted esters. J Am Chem Soc 132(20):7038–7042

    Article  PubMed  Google Scholar 

  • Enoki J, Meisborn J, Müller A, Kourist R (2016) A multi-enzymatic cascade reaction for the stereoselective production of γ-oxyfunctionalyzed amino acids. Front Microbiol. https://doi.org/10.3389/fmicb.2016.00425

  • Fernández‐Álvaro E, Snajdrova R, Jochens H, Davids T, Böttcher D, Bornscheuer UT (2011) A combination of in vivo selection and cell sorting for the identification of enantioselective biocatalysts. Angew Chem Int Ed 50:8584–8587

    Article  Google Scholar 

  • Fibla J, Gonzalezduarte R (1993) Colorimetric assay to determine alcohol-dehydrogenase activity. J Biochem Biophys Methods 26:87–93

    Article  CAS  PubMed  Google Scholar 

  • Fox RJ, Davis SC, Mundorff EC, Newman LM, Gavrilovic V, Ma SK, Chung LM, Ching C, Tam S, Muley S, Grate J, Gruber J, Whitman JC, Sheldon RA, Huisman GW (2007) Improving catalytic function by ProSAR-driven enzyme evolution. Nat Biotechnol 25:338–344

    Article  CAS  PubMed  Google Scholar 

  • Gassmeyer SK, Yoshikawa H, Enoki J, Hülsemann N, Stoll R, Miyamoto K, Kourist R (2015) STD NMR based protein engineering of the unique arylpropionate racemase AMDase G74C. ChemBioChem 16:1943–1949

    Article  CAS  Google Scholar 

  • Gassmeyer S, Wetzig J, Mügge C, Assmann M, Enoki J, Hilterhaus L, Zuhse R, Miyamoto K, Liese A, Kourist R (2016) Arylmalonate decarboxylase-catalyzed asymmetric synthesis of both enantiomers of optically pure flurbiprofen. ChemCatChem 8:916–921

    Article  CAS  Google Scholar 

  • Grognux J, Reymond JL (2004) Classifying enzymes from selectivity fingerprints. ChemBioChem 5:826–831

    Article  CAS  PubMed  Google Scholar 

  • Heinze B, Kourist R, Fransson L, Hult K, Bornscheuer UT (2007) Highly enantioselective kinetic resolution of two tertiary alcohols using mutants of an esterase from Bacillus subtilis. Protein Eng Des Sel 20:125–131

    Article  CAS  PubMed  Google Scholar 

  • Heitman J, Sun S, James TY (2013) Evolution of fungal sexual reproduction. Mycologia 105:1–27

    Article  CAS  PubMed  Google Scholar 

  • Henke E, Bornscheuer UT, Schmid RD, Pleiss J (2003) A molecular mechanism of enantiorecognition of tertiary alcohols by carboxylesterases. ChemBioChem 4:485–493

    Article  CAS  PubMed  Google Scholar 

  • Horsman GP, Liu AMF, Henke E, Bornscheuer UT, Kazlauskas RJ (2003) Mutations in distant residues moderately increase the enantioselectivity of Pseudomonas fluorescens esterase towards methyl 3-bromo-2-methyl propanoate and ethyl 3-phenylbutyrate. Chem Eur J 9:1933–1939

    Article  CAS  PubMed  Google Scholar 

  • Ljima Y, Matoishi K, Terao Y, Doi N, Yanagawa H, Ohta H (2005) Inversion of enantioselectivity of asymmetric biocatalytic decarboxylation by site-directed mutagenesis based on the reaction mechanism. Chem Commun 21:877–879

    Google Scholar 

  • Janes LE, Kazlauskas RJ (1997) Quick E. A fast spectroscopic method to measure the enantioselectivity of hydrolases. J Org Chem 62:4560–4561

    Article  CAS  Google Scholar 

  • Kan SJ, Lewis RD, Chen K, Arnold FH (2016) Directed evolution of cytochrome c for carbon–silicon bond formation: bringing silicon to life. Science 354(6315):1048–1051

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Köninger K, Gomez-Baraibar A, Mügge C, Paul C, Hollmann F, Nowaczyk M, Kourist R (2016) Recombinant cyanobacteria as tools for asymmetric C = C bond reduction fueled by biocatalytic water oxidation. Angew Chem Int Ed 55:5582–5585. https://doi.org/10.1002/anie.201601200

  • Koudelakova T, Chaloupkova R, Brezovsky J, Prokop Z, Sebestova E, Hesseler M, Khabiri M, Plevaka M, Kulik D, Kuta Smatanova I (2013) Engineering enzyme stability and resistance to an organic cosolvent by modification of residues in the access tunnel. Angew Chem Int Ed 52:1959–1963

    Article  CAS  Google Scholar 

  • Kourist R, Bartsch S, Bornscheuer UT (2007) Highly enantioselective synthesis of arylaliphatic tertiary alcohols using mutants of an esterase from Bacillus subtilis. Adv Synth Catal 349:1393–1398

    Article  CAS  Google Scholar 

  • Kourist R, Jochens H, Bartsch S, Kuipers R, Padhi SK, Gall M, Böttcher D, Joosten HJ, Bornscheuer UT (2010) The alpha/beta-hydrolase fold 3DM database (ABHDB) as a tool for protein engineering. ChemBioChem 11:1635–1643. https://doi.org/10.1002/cbic.201000213

  • Leroy E, Bensel N, Reymond JL (2003) A low background high-throughput screening (HTS) fluorescence assay for lipases and esterases using acyloxymethylethers of umbelliferone. Bioorg Med Chem Lett 13:2105–2108

    Article  CAS  PubMed  Google Scholar 

  • Leung DW, Chen E, Goeddel DV (1989) A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique 1:11–15

    Google Scholar 

  • Liebeton K, Zonta A, Schimossek K, Nardini M, Lang D, Dijkstra BW, Reetz MT, Jaeger KE (2000) Directed evolution of an enantioselective lipase. Chem Biol 7:709–718

    Article  CAS  PubMed  Google Scholar 

  • Liu AMF, Somers NA, Kazlauskas RJ, Brush TS, Zocher F, Enzelberger MM, Bornscheuer UT, Horsman GP, Mezzetti A, Schmidt-Dannert C, Schmid RD (2001) Mapping the substrate selectivity of new hydrolases using colorimetric screening: lipases from Bacillus thermocatenulatus and Phiostoma piliferum, esterases from Pseudomonas fluorescens and Streptomyces diastatochromogenes. Tetrahedron: Asymmetry 12:545–556

    Article  Google Scholar 

  • Lutz S, Bornscheuer UT (eds) (2008) Protein engineering handbook. Wiley VCH, Weinheim

    Google Scholar 

  • Lutz S, Ostermeier M, Benkovic SJ (2001) Rapid generation of incremental truncation libraries for protein engineering using alpha phosphorothioate nucleotides. Nucleic Acids Res 29:e16

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mate DM, Alcalde M (2015) Laccase engineering: from rational design to directed evolution. Biotechnol Adv 33:25–40

    Article  CAS  PubMed  Google Scholar 

  • Mayer KM, Arnold FH (2002) A colorimetric assay to quantify dehydrogenase activity in crude cell lysates. J Biomol Screen 7:135–140

    Article  CAS  PubMed  Google Scholar 

  • Meyer MM, Hochrein L, Arnold FH (2006) Structure-guided SCHEMA recombination of distantly related β-lactamases. Prot Eng Des Sel 19:563–570

    Article  CAS  Google Scholar 

  • Miyauchi Y, Kourist R, Uemura D, Miyamoto K (2011) Dramatically improved catalytic activity of an artificial (S)-selective arylmalonate decarboxylase by structure-guided directed evolution. Chem Commun 47:7503–7505. https://doi.org/10.1039/c1cc11953b

  • Molina-Espeja P, Garcia-Ruiz E, Gonzalez-Perez D, Ullrich R, Hofrichter M, Alcalde M (2014) Directed evolution of unspecific peroxygenase from Agrocybe aegerita. Appl Environ Microbiol 80(11):3496–3507

    Article  PubMed  PubMed Central  Google Scholar 

  • Molina Espeja P, Cañellas M, Plou FJ, Hofrichter M, Lucas F, Guallar V, Alcalde M (2016) Synthesis of 1 naphthol by a natural peroxygenase engineered by directed evolution. ChemBioChem 17:341–349

    Article  CAS  PubMed  Google Scholar 

  • Moore JC, Arnold FH (1996) Directed evolution of a para-nitrobenzyl esterase for aqueous-organic solvents. Nat Biotechnol 14:458–467

    Article  CAS  PubMed  Google Scholar 

  • Neylon C (2004) Chemical and biochemical strategies for the randomization of protein encoding DNA sequences: library construction methods for directed evolution. Nucleic Acids Res 32:1448–1459

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Obata R, Nakasako M (2010) Structural basis for inverting the enantioselectivity of arylmalonate decarboxylase revealed by the structural analysis of the Gly74Cys/Cys188Ser mutant in the liganded form. Biochemistry 49:1963–1969. https://doi.org/10.1021/bi9015605

  • Ostermeier M, Lutz S (2003) The creation of ITCHY hybrid protein libraries. In: Arnold FH, Georgiou G (eds) Directed evolution library creation: methods and protocols, Methods in molecular biology. Humana Press, Totowa, pp 129–141

    Chapter  Google Scholar 

  • Ostermeier M, Shim JH, Benkovic SJ (1999) A combinatorial approach to hybrid enzymes independent of DNA homology. Nat Biotechnol 17:1205–1209

    Article  CAS  PubMed  Google Scholar 

  • Packer MS, Liu DR (2015) Methods for the directed evolution of proteins. Nat Rev Gen 16:379–394

    Article  CAS  Google Scholar 

  • Patel PH, Kawate H, Adman E, Ashbach M, Loeb LA (2001) A single highly mutable catalytic site amino acid is critical for DNA polymerase fidelity. J Biol Chem 276:5044–5051

    Article  CAS  PubMed  Google Scholar 

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

    CAS  Google Scholar 

  • Reetz MT, Wilensek S, Zha D, Jaeger KE (2001) Directed evolution of an enantioselective enzyme through combinatorial multiple-cassette mutagenesis. Angew Chem Int Ed 40:3589–3591

    Article  CAS  Google Scholar 

  • Reetz MT, Carballeira JD, Vogel A (2006) Iterative saturation mutagenesis on the basis of B factors as a strategy for increasing protein thermostability. Angew Chem Int Ed 45:7745–7751

    Article  CAS  Google Scholar 

  • Reetz MT, Soni P, Fernandez L (2009) Knowledge-guided laboratory evolution of protein thermolability. Biotechnol Bioeng 102:1712–1717

    Article  CAS  PubMed  Google Scholar 

  • Reymond JL (ed) (2005) Enzyme assays. Wiley-VCH, Weinheim

    Google Scholar 

  • Sandström AG, Engström K, Jyhlén J, Kasrayan A, Bäckvall J-E (2009) Directed evolution of Candida antarctica lipase A using an episomally replicating yeast plasmid. Protein Eng Des Sel 22(7):413–420

    Article  PubMed  Google Scholar 

  • Sandström AG, Wikmark Y, Engström K, Nyhlén J, Bäckvall J-e (2012) Combinatorial reshaping of the Candida antarctica lipase A substrate pocket for enantioselectivity using an extremely condensed library. Proc Natl Acad Sci U S A 109:78–83. https://doi.org/10.1073/pnas.1111537108

    Article  PubMed  Google Scholar 

  • Schmidt M, Hasenpusch D, Kahler M, Kirchner U, Wiggenhorn K, Lange W, Bornscheuer UT (2006) Directed evolution of an esterase from Pseudomonas fluorescens yields a mutant with excellent enantioselectivity and activity for the kinetic resolution of a chiral building block. ChemBioChem 7:805–809

    Article  CAS  PubMed  Google Scholar 

  • Schmidt S, Scherkus C, Muschiol J, Menyes U, Winkler T, Hummel W, Gröger H, Liese A, Herz HG, Bornscheuer UT (2015) An enzyme cascade synthesis of ε-caprolactone and its oligomers. Angew Chem Int Ed 54:2784–2787

    Article  CAS  Google Scholar 

  • Schrewe M, Ladkau N, Bühler B, Schmid A (2013) Direct terminal alkylamino-functionalization via multistep biocatalysis in one recombinant whole-cell catalyst. Adv Synth Catal 355:1693–1697

    Article  CAS  Google Scholar 

  • Stemmer WPC (1994) Rapid evolution of a protein by in vitro DNA shuffling. Nat Biotechnol 370:389–391

    CAS  Google Scholar 

  • Tee KL, Wong TS (2013) Polishing the craft of genetic diversity creation in directed evolution. Biotechnol Adv 31:1707–1721

    Article  CAS  PubMed  Google Scholar 

  • Terao Y, Miyamoto K, Ohta H (2006) Improvement of the activity of arylmalonate decarboxylase by random mutagenesis. Appl Microbiol Biotechnol 73:647–653

    Article  CAS  PubMed  Google Scholar 

  • Udit AK, Silberg JJ, Sieber V (2003) Sequence homology-independent protein recombination, SHIPREC. In: Arnold FH, Georgiou G (eds) Directed evolution library creation: methods and protocols, Methods in molecular biology. Humana Press, Totowa, pp 153–164

    Chapter  Google Scholar 

  • Wahler D, Reymond JL (2002) The adrenaline test for enzymes. Angew Chem Int Ed 41:1229–1232

    Article  CAS  Google Scholar 

  • Wahler D, Boujard O, Lefevre F, Reymond JL (2004) Adrenaline profiling of lipases and esterases with 1,2-diol and carbohydrate acetates. Tetrahedron 60:703–710

    Article  CAS  Google Scholar 

  • Yang GY, Shamsuddin AM (1996) Gal-GalNAc: a biomarker of colon carcinogenesis. Histol Histopathol 11:801–806

    CAS  PubMed  Google Scholar 

  • Yoshida S, Enoki J, Kourist R, Miyamoto K (2015) Engineered hydrophobic pocket of (S)-selective arylmalonate decarboxylase variant by simultaneous saturation mutagenesis to improve catalytic performance. Biosci Biotechnol Biochem 79:1965–1971

    Article  CAS  PubMed  Google Scholar 

  • Zhao H (1998) Molecular evolution by staggered extension process (StEP) in vitro recombination. Nat Biotechnol 16:258–261

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Greifswald University, Felix-Hausdorff-Str. 4, 17487, Greifswald, Germany

    Uwe T. Bornscheuer

  2. Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, Graz, 8010, Austria

    Robert Kourist

Authors
  1. Uwe T. Bornscheuer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Robert Kourist
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Robert Kourist .

Editor information

Editors and Affiliations

  1. Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea (Republic of)

    Sang Yup Lee

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer International Publishing AG, part of Springer Nature

About this entry

Check for updates. Verify currency and authenticity via CrossMark

Cite this entry

Bornscheuer, U.T., Kourist, R. (2017). Evolving Enzymes for Biocatalysis. In: Lee, S. (eds) Consequences of Microbial Interactions with Hydrocarbons, Oils, and Lipids: Production of Fuels and Chemicals. Handbook of Hydrocarbon and Lipid Microbiology . Springer, Cham. https://doi.org/10.1007/978-3-319-50436-0_217

Download citation

Publish with us

Policies and ethics

Search

Navigation