Skip to main content
SpringerLink
Log in

Recent Advances in Directed Evolution of Stereoselective Enzymes

  • Chapter
  • First Online:
Directed Enzyme Evolution: Advances and Applications

Abstract

Directed evolution of enzymes provides a prolific source of biocatalysts for asymmetric reactions in organic chemistry and biotechnology. Nowadays, the real challenge in this research area is the development of mutagenesis methods and strategies which ensure the formation of small and highest-quality mutant libraries requiring minimal screening effort. This chapter constitutes a critical analysis of recent developments. Saturation mutagenesis at sites lining the binding pocket using highly reduced amino acid alphabets has emerged as the superior approach for evolving stereoselectivity.

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 139.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 179.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 179.99
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

  1. (a) Drauz K, Gröger H, May O (eds) (2012) Enzyme catalysis in organic synthesis, 3rd edn. Wiley-VCH, Weinheim; (b) Faber K (2011) Biotransformations in organic chemistry, 6th edn. Springer, Heidelberg; (c) Liese A, Seelbach K, Wandrey C (eds) (2006) Industrial biotransformations, Wiley-VCH, Weinheim.

    Google Scholar 

  2. (a) Pleiss J (2012) Rational design of enzymes. In: Drauz K, Gröger H, May O (eds) Enzyme catalysis in organic synthesis, 3rd edn. Wiley-VCH, Weinheim, p 89–117; (b) Ema T, Nakano Y, Yoshida D, Kamata S, Sakai T (2012) Redesign of enzyme for improving catalytic activity and enantioselectivity toward poor substrates: manipulation of the transition state. Org Biomol Chem 10:6299–6308; (c) Steiner K, Schwab H (2012) Recent advances in rational approaches for enzyme engineering. Comput Struct Biotechnol J 2:e201209010.

    Google Scholar 

  3. Reviews of directed evolution: (a) Bommarius AS (2015) Biocatalysis, a status report. Annu Rev Chem Biomol Eng 6:319–345; (b) Denard CA, Ren H, Zhao H (2015) Improving and repurposing biocatalysts via directed evolution. Curr Opin Chem Biol 25:55–64; (c) 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; (d) Gillam EMJ, Copp JN, Ackerley DF (eds) (2014) Directed evolution library creation. In: Methods in molecular biology, Humana Press, Totowa; (e) Widersten M (2014) Protein engineering for development of new hydrolytic biocatalysts. Curr Opin Chem Biol 21:42–47; (f) Reetz MT (2012) Directed evolution of enzymes. In Drauz K, Gröger H, May O (eds) Enzyme catalysis in organic synthesis, 3rd edn. Wiley-VCH, Weinheim, p 119–190

    Google Scholar 

  4. Review of directed evolution of protein thermostability: (a) Arnold FH (1998) Design by directed evolution. Acc Chem Res 31:125–131; (b) Petrounia IP, Arnold FH (2000) Designed evolution of enzymatic properties. Curr Opin Biotechnol 11: 325–330; (c) Polizzi KM, Bommarius AS, Broering JM, Chaparro-Riggers JF (2007) Stability of biocatalysts. Curr Opin Chem Biol 11:220–225; (d) Tokuriki N, Tawfik DS (2009) Stability effects of mutations and protein evolvability. Curr Opin Struct Biol 19:596–604

    Google Scholar 

  5. Reviews of directed evolution of stereoselectivity[3f]: (a) Reetz MT (2011) Laboratory evolution of stereoselective enzymes: a prolific source of catalysts for asymmetric reactions. Angew Chem Int Ed 50:138–174; (b) Reetz MT (2013) Biocatalysis in organic chemistry and biotechnology: past, present, and future. J Am Chem Soc 135:12480–12496

    Google Scholar 

  6. Reetz MT, Zonta A, Schimossek K, Liebeton K, Jaeger KE (1997) Creation of enantioselective biocatalysts for organic chemistry by in vitro evolution. Angew Chem Int Ed Eng 36:2830–2832

    Article  CAS  Google Scholar 

  7. 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 

  8. Reetz MT, Puls M, Carballeira JD, Vogel A, Jaeger KE, Eggert T, Thiel W, Bocola M, Otte N (2007) Learning from directed evolution: further lessons from theoretical investigations into cooperative mutations in lipase enantioselectivity. Chembiochem 8:106–112

    Article  CAS  PubMed  Google Scholar 

  9. Zha DX, Wilensek S, Hermes M, Jaeger KE, Reetz MT (2001) Complete reversal of enantioselectivity of an enzyme-catalyzed reaction by directed evolution. Chem Commun 24:2664–2665

    Article  Google Scholar 

  10. Reetz MT (2004) Controlling the enantioselectivity of enzymes by directed evolution: practical and theoretical ramifications. Proc Natl Acad Sci U S A 101:5716–5722

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Reetz MT, Bocola M, Carballeira JD, Zha DX, Vogel A (2005) Expanding the range of substrate acceptance of enzymes: combinatorial active-site saturation test. Angew Chem Int Ed 44:4192–4196

    Article  CAS  Google Scholar 

  12. (a) Reetz MT, Wang LW, Bocola M (2006) Directed evolution of enantioselective enzymes: iterative cycles of CASTing for probing protein-sequence space. Angew Chem Int Ed 45:1236–1241; (b) Reetz MT (2005) Evolution im Reagenzglas: Neue Perspektiven für die Weiße Biotechnologie. Tätigkeitsberichte der Max-Planck-Gesellschaft 327–331

    Google Scholar 

  13. (a) Reetz MT, Kahakeaw D, Lohmer R (2008) Addressing the numbers problem in directed evolution. Chembiochem 9:1797–1804; (b) Clouthier CM, Kayser MM, Reetz MT (2006) Designing new Baeyer-Villiger monooxygenases using restricted CASTing. J Org Chem 71:8431–8437

    Google Scholar 

  14. (a) Firth AE, Patrick WM (2008) GLUE-IT and PEDEL-AA: new programmes for analyzing protein diversity in randomized libraries. Nucleic Acids Res 36 (Web Server issue):W281–285; (b) Denault M, Pelletier JN (2007) Protein library design and screening: working out the probabilities. In: Arndt KM, Müller KM (eds) Protein engineering protocols, Humana Press, Totowa, p 127–154

    Google Scholar 

  15. Nov Y (2012) When second best is good enough: another probabilistic look at saturation mutagenesis. Appl Environ Microbiol 78:258–262

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Reetz MT, Prasad S, Carballeira JD, Gumulya Y, Bocola M (2010) Iterative saturation mutagenesis accelerates laboratory evolution of enzyme stereoselectivity: rigorous comparison with traditional methods. J Am Chem Soc 132:9144–9152

    Article  CAS  PubMed  Google Scholar 

  17. Reetz MT (2013) The importance of additive and non-additive mutational effects in protein engineering. Angew Chem Int Ed 52:2658–2666

    Article  CAS  Google Scholar 

  18. Parikh MR, Matsumura I (2005) Site-saturation mutagenesis is more efficient than DNA shuffling for the directed evolution of beta-fucosidase from beta-galactosidase. J Mol Biol 352:621–628

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Acevedo-Rocha CG, Hoebenreich S, Reetz MT (2014) Iterative saturation mutagenesis: a powerful approach to engineer proteins by systematically simulating Darwinian evolution. Methods Mol Biol 1179:103–128

    Article  PubMed  Google Scholar 

  20. Sun Z, Wikmark Y, Bäckvall J-E, Reetz MT (2016) New concepts for increasing the efficiency in directed evolution of stereoselective enzymes. Chem Eur J 22:5046–5054

    Article  CAS  PubMed  Google Scholar 

  21. (a) Sun Z, Lonsdale R, Kong XD, Xu JH, Zhou J, Reetz MT (2015) Reshaping an enzyme binding pocket for enhanced and inverted stereoselectivity: use of smallest amino acid alphabets in directed evolution. Angew Chem Int Ed 54:12410–12415; (b) Sandström AG, Wikmark Y, Engström K, Nyhlen J, Bäckvall JE (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

    Google Scholar 

  22. (a) Zhang ZG, Lonsdale R, Sanchis J, Reetz MT (2014) Extreme synergistic mutational effects in the directed evolution of a baeyer-villiger monooxygenase as catalyst for asymmetric sulfoxidation. J Am Chem Soc 136:17262–17272; (b) Sun Z, Lonsdale R, Wu L, Li G, Li A, Wang J, Zhou J, Reetz MT (2016) Structure-guided triple code saturation mutagenesis: efficient tuning of the stereoselectivity of an epoxide hydrolase. ACS Catal 6:1590–1597; (c) Sun Z, Lonsdale R, Ilie A, Li G, Zhou J, Reetz MT (2016) Catalytic asymmetric reduction of difficult-to-reduce ketones: triple code saturation mutagenesis of an alcohol dehydrogenase. ACS Catal 6:1598–1605

    Google Scholar 

  23. (a) Bougioukou DJ, Kille S, Taglieber A, Reetz MT (2009) Directed Evolution of an enantioselective enoate-reductase: testing the utility of iterative saturation mutagenesis. Adv Synth Catal 351:3287–3305; (b) Sullivan B, Walton AZ, Stewart JD (2013) Library construction and evaluation for site saturation mutagenesis. Enzym Microb Technol 53:70–77

    Google Scholar 

  24. Polizzi KM, Parikh M, Spencer CU, Matsumura I, Lee JH, Realff MJ, Bommarius AS (2006) Pooling for improved screening of combinatorial libraries for directed evolution. Biotechnol Prog 22:961–967

    Article  CAS  PubMed  Google Scholar 

  25. Acevedo-Rocha CG, Reetz MT, Nov Y (2015) Economical analysis of saturation mutagenesis experiments. Sci Rep 5:10654

    Article  PubMed  PubMed Central  Google Scholar 

  26. Kwan DH, Constantinescu I, Chapanian R, Higgins MA, Kotzler MP, Samain E, Boraston AB, Kizhakkedathu JN, Withers SG (2015) Toward efficient enzymes for the generation of universal blood through structure-guided directed evolution. J Am Chem Soc 137:5695–5705

    Article  CAS  PubMed  Google Scholar 

  27. (a) Whitehouse CJ, Bell SG, Wong LL (2012) P450(BM3) (CYP102A1): connecting the dots. Chem Soc Rev 41:1218–1260; (b) Fasan R (2012) Tuning P450 enzymes as oxidation catalysts. ACS Catal 2:647–666; (c) Bernhardt R, Urlacher VB (2014) Cytochromes P450 as promising catalysts for biotechnological application: chances and limitations. Appl. Microbiol Biotechnol 98:6185–6203; (d) Holtmann D, Fraaije MW, Arends IW, Opperman DJ, Hollmann F (2014) The taming of oxygen: biocatalytic oxyfunctionalisations. Chem Commun 50:13180–13200; (e) Roiban GD, Reetz MT (2015) Expanding the toolbox of organic chemists: directed evolution of P450 monooxygenases as catalysts in regio- and stereoselective oxidative hydroxylation. Chem Commun 51:2208–2224; (f) Girvan HM, Munro AW (2016) Applications of microbiol cytochrome P450 enzymes in biotechnology and synthetic biology. Curr Opin Chem Biol 31:136–145

    Google Scholar 

  28. (a) Tang WL, Li Z, Zhao H (2010) Inverting the enantioselectivity of P450pyr monooxygenase by directed evolution. Chem Commun 46:5461–5463; (b) Pham SQ, Pompidor G, Liu J, Li XD, Li Z (2012) Evolving P450pyr hydroxylase for highly enantioselective hydroxylation at non-activated carbon atom. Chem Commun 48:4618–4620

    Google Scholar 

  29. Lonsdale R, Harvey JN, Mulholland AJ (2010) Inclusion of dispersion effects significantly improves accuracy of calculated reaction barriers for cytochrome P450 catalyzed reactions. J Phys Chem Lett 1:3232–3237

    Article  CAS  Google Scholar 

  30. Agudo R, Roiban GD, Reetz MT (2012) Achieving regio- and enantioselectivity of P450-catalyzed oxidative CH activation of small functionalized molecules by structure-guided directed evolution. Chembiochem 13:1465–1473

    Article  CAS  PubMed  Google Scholar 

  31. Kille S, Zilly FE, Acevedo JP, Reetz MT (2011) Regio- and stereoselectivity of P450-catalysed hydroxylation of steroids controlled by laboratory evolution. Nat Chem 3:738–743

    Article  CAS  PubMed  Google Scholar 

  32. Le-Huu P, Heidt T, Claasen B, Laschat S, Urlacher VB (2015) Chemo-, regio-, and stereoselective oxidation of the monocyclic diterpenoid beta-cembrenediol by P450 BM3. ACS Catal 5:1772–1780

    Article  CAS  Google Scholar 

  33. Zhang K, Shafer BM, Demars MD 2nd, Stern HA, Fasan R (2012) Controlled oxidation of remote sp3 C-H bonds in artemisinin via P450 catalysts with fine-tuned regio- and stereoselectivity. J Am Chem Soc 134:18695–18704

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. (a) Chen MM, Snow CD, Vizcarra CL, Mayo SL, Arnold FH (2012) Comparison of random mutagenesis and semi-rational designed libraries for improved cytochrome P450 BM3-catalyzed hydroxylation of small alkanes. Prot Eng Des Sel 25:171–178; (b) Ritter C, Nett N, Acevedo-Rocha CG, Lonsdale R, Kraling K, Dempwolff F, Hoebenreich S, Graumann PL, Reetz MT, Meggers E (2015) Bioorthogonal enzymatic activation of caged compounds. Angew Chem Int Ed 54:13440–13443; (c) Dennig A, Lulsdorf N, Liu H, Schwaneberg U (2013) Regioselective o-hydroxylation of monosubstituted benzenes by P450 BM3. Angew Chem Int Ed 52:8459–8462; (d) Agudo R, Roiban GD, Lonsdale R, Ilie A, Reetz MT (2015) Biocatalytic route to chiral acyloins: P450-catalyzed regio- and enantioselective alpha-hydroxylation of ketones. J Org Chem 80:950–956; (e) Hoebenreich S, Zilly FE, Acevedo-Rocha CG, Zilly M, Reetz MT (2015) Speeding up directed evolution: combining the advantages of solid-phase combinatorial gene synthesis with statistically guided reduction of screening effort. ACS Synth Biol 4:317–331; (f) Roiban GD, Agudo R, Reetz MT (2014) Cytochrome P450 catalyzed oxidative hydroxylation of achiral organic compounds with simultaneous creation of two chirality centers in a single C-H activation step. Angew Chem Int Ed 53:8659–8663; (g) Hu S, Huang J, Mei LH, Yu Q, Yao SJ, Jin ZH (2010) Altering the regioselectivity of cytochrome P450 BM-3 by saturation mutagenesis for the biosynthesis of indirubin. J Mol Catal B-Enzym 67:29–35; (h) Nguyen KT, Virus C, Gunnewich N, Hannemann F, Bernhardt R (2012) Changing the regioselectivity of a P450 from C15 to C11 hydroxylation of progesterone. Chembiochem 13:1161–1166; (i) Yang Y, Liu J, Li Z (2014) Engineering of P450pyr hydroxylase for the highly regio- and enantioselective subterminal hydroxylation of alkanes. Angew Chem Int Ed 53:3120–3124

    Google Scholar 

  35. (a) Reinen J, Vredenburg G, Klaering K, Vermeulen NPE, Commandeur JNM, Honing M, Vos JC (2015) Selective whole-cell biosynthesis of the designer drug metabolites 15-or 16-ß-hydroxynorethisterone by engineered Cytochrome P450 BM3 mutants. J Mol Catal B-Enzym 121:64–74; (b) Bruhlmann F, Bosijokovic B, Ullmann C, Auffray P, Fourage L, Wahler D (2013) Directed evolution of a 13-hydroperoxide lyase (CYP74B) for improved process performance. J Biotechnol 163:339–345

    Google Scholar 

  36. Reetz MT, Brunner B, Schneider T, Schulz F, Clouthier CM, Kayser MM (2004) Directed evolution as a method to create enantioselective cyclohexanone monooxygenases for catalysis in Baeyer-Villiger reactions. Angew Chem Int Ed 43:4075–4078

    Article  CAS  Google Scholar 

  37. Mihovilovic MD, Rudroff F, Winninger A, Schneider T, Schulz F, Reetz MT (2006) Microbial Baeyer-Villiger oxidation: stereopreference and substrate acceptance of cyclohexanone monooxygenase mutants prepared by directed evolution. Org Lett 8:1221–1224

    Article  CAS  PubMed  Google Scholar 

  38. (a) Fraaije MW, Wu J, Heuts DP, van Hellemond EW, Spelberg JH, Janssen DB (2005) Discovery of a thermostable Baeyer-Villiger monooxygenase by genome mining. Appl Microbiol Biotechnol 66:393–400; (b) Malito E, Alfieri A, Fraaije MW, Mattevi A (2004) Crystal structure of a Baeyer-Villiger monooxygenase. Prod Natl Acad Sci 101:13157–13162

    Google Scholar 

  39. Reviews of directed evolution of Baeyer-Villiger monooxygenases: (a) Zhang ZG, Parra LP, Reetz MT (2012) Protein engineering of stereoselective Baeyer-Villiger monooxygenases. Chem Eur J 18:10160–10172; (b) Balke K, Kadow M, Mallin H, Sass S, Bornscheuer UT (2012) Discovery, application and protein engineering of Baeyer-Villiger monooxygenases for organic synthesis. Org Biomol Chem 10:6249–6265

    Google Scholar 

  40. Reetz MT, Wu S (2008) Greatly reduced amino acid alphabets in directed evolution: making the right choice for saturation mutagenesis at homologous enzyme positions. Chem Commun 43:5499–5501

    Article  Google Scholar 

  41. Kirschner A, Bornscheuer UT (2006) Kinetic resolution of 4-hydroxy-2-ketones catalyzed by a Baeyer-Villiger monooxygenase. Angew Chem 45:7004–7006

    Article  CAS  Google Scholar 

  42. Wu S, Acevedo JP, Reetz MT (2010) Induced allostery in the directed evolution of an enantioselective Baeyer-Villiger monooxygenase. Prod Natl Acad Sci USA 107:2775–2780

    Article  CAS  Google Scholar 

  43. Ichiye T, Karplus M (1991) Collective motions in proteins – a covariance analysis of atomic fluctuations in molecular-dynamics and normal mode simulations. Proteins 11:205–217

    Article  CAS  PubMed  Google Scholar 

  44. Tang L, Gao H, Zhu X, Wang X, Zhou M, Jiang R (2012) Construction of “small-intelligent” focused mutagenesis libraries using well-designed combinatorial degenerate primers. BioTechniques 52:149–158

    CAS  PubMed  Google Scholar 

  45. Reetz MT, Sanchis J (2008) Constructing and analyzing the fitness landscape of an experimental evolutionary process. Chembiochem 9:2260–2267

    Article  CAS  PubMed  Google Scholar 

  46. Gumulya Y, Sanchis J, Reetz MT (2012) Many pathways in laboratory evolution can lead to improved enzymes: how to escape from local minima. Chembiochem 13:1060–1066

    Article  CAS  PubMed  Google Scholar 

  47. Wu Q, Soni P, Reetz MT (2013) Laboratory evolution of enantiocomplementary Candida antarctica lipase B mutants with broad substrate scope. J Am Chem Soc 135:1872–1881

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  Google Scholar 

  49. Wikmark Y, Svedendahl Humble M, Bäckvall JE (2015) Combinatorial library based engineering of Candida antarctica lipase A for enantioselective transacylation of sec-alcohols in organic solvent. Angew Chem Int Ed 54:4284–4288

    Article  CAS  Google Scholar 

  50. (a) Ma J, Wu L, Guo F, Gu J, Tang X, Jiang L, Liu J, Zhou J, Yu H (2013) Enhanced enantioselectivity of a carboxyl esterase from Rhodobacter sphaeroides by directed evolution. Appl Microbiol Biotechnol 97:4897–4906; (b) Luan ZJ, Li FL, Dou S, Chen Q, Kong XD, Zhou JH, Yu HL, Xu JH (2015) Substrate channel evolution of an esterase for the synthesis of cilastatin. Catal Sci Technol 5:2622–2629; (c) Godinho LF, Reis CR, van Merkerk R, Poelarends GJ, Quax WJ (2012) An esterase with superior activity and enantioselectivity towards 1,2-o-isopropylideneglycerol esters obtained by protein design. Adv Synth Catal 354:3009–3015; (d) Nobili A, Tao Y, Pavlidis IV, van den Bergh T, Joosten HJ, Tan T, Bornscheuer UT (2015) Simultaneous use of in silico design and a correlated mutation network as a tool to efficiently guide enzyme engineering. Chembiochem 16:805–810

    Google Scholar 

  51. (a) Gupta RD, Tawfik DS (2008) Directed enzyme evolution via small and effective neutral drift libraries. Nat Methods 5:939–942; (b) Kaltenbach M, Tokuriki, N (2014) Generation of effective libraries by neutral drift. Methods Molec Biol 1179:69–81; (c) Smith WS, Hale JR, Neylon C (2011) Applying neutral drift to the directed molecular evolution of a ß-glucuronidase into a ß-galactosidase: two different evolutionary pathways lead to the same variant. BMC Res Notes 4:138; (d) Bernath-Levin K, Shainsky J, Sigawi L, Fishman A (2014) Directed evolution of nitrobenzene dioxygenase for the synthesis of the antioxidant hydroxytyrosol. Appl Microbiol Biotechnol 98:4975–4985

    Google Scholar 

  52. (a) Eigen M, McCaskill J, Schuster P (1988) Molecular quasi-species. J Phys Chem 92:6881–6891; (b) Kurtovic S, Mannervik B (2009) Identification of emerging quasi-species in directed enzyme evolution. Biochemistry 48:9330–9339

    Google Scholar 

  53. Savile CK, Janey JM, Mundorff EC, Moore JC, Tam S, Jarvis WR, Colbeck JC, Krebber A, Fleitz FJ, Brands J, Devine PN, Huisman GW, Hughes GJ (2010) Biocatalytic asymmetric synthesis of chiral amines from ketones applied to sitagliptin manufacture. Science 329:305–309

    Article  CAS  PubMed  Google Scholar 

  54. Chen Z, Zhao H (2005) Rapid creation of a novel protein function by in vitro coevolution. J Mol Biol 348:1273–1282

    Article  CAS  PubMed  Google Scholar 

  55. (a) Ostermeier M, Benkovic SJ (2000) Evolution of protein function by domain swapping. Adv Protein Chem 55:29–77; (b) Park AH, Park HY, Sohng JK, Lee HC, Liou K, Yoon YJ, Km BG (2009) Expanding substrate specificity of GT-B fold glycosyltransferase via domain swapping and high-throughput screening. Biotechnol Bioeng 102:988–994

    Google Scholar 

  56. (a) Yu Y, Lutz S (2011) Circular permutation: a different way to engineer enzyme structure and function. Trends Biotechnol 29:18–25; (b) Qian Z, Lutz S (2005) Improving the catalytic activity of Candida antarctica lipase B by circular permutation. J Am Chem Soc 127:13466–13467; (c) Yu Y, Lutz S (2010) Improved triglyceride transesterification by circular permutated Candida antarctica lipase B. Biotechnol Bioeng 105:44–50.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Chemistry, Philipps-Universität, Marburg, Germany

    Manfred T. Reetz

Authors
  1. Manfred T. Reetz
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Manfred T. Reetz .

Editor information

Editors and Affiliations

  1. Department of Biocatalysis, Institute of Catalysis and Petrochemistry, CSIC, Madrid, Spain

    Miguel Alcalde

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer International Publishing AG

About this chapter

Cite this chapter

Reetz, M.T. (2017). Recent Advances in Directed Evolution of Stereoselective Enzymes. In: Alcalde, M. (eds) Directed Enzyme Evolution: Advances and Applications. Springer, Cham. https://doi.org/10.1007/978-3-319-50413-1_3

Download citation

Publish with us

Policies and ethics

Search

Navigation