Applied Microbiology and Biotechnology

, Volume 101, Issue 8, pp 3177–3187 | Cite as

Amino acid substitutions in random mutagenesis libraries: lessons from analyzing 3000 mutations

  • Jing Zhao
  • Victorine Josiane Frauenkron-Machedjou
  • Tsvetan Kardashliev
  • Anna Joëlle Ruff
  • Leilei Zhu
  • Marco Bocola
  • Ulrich Schwaneberg
Biotechnologically relevant enzymes and proteins


The quality of amino acid substitution patterns in random mutagenesis libraries is decisive for the success in directed evolution campaigns. In this manuscript, we provide a detailed analysis of the amino acid substitutions by analyzing 3000 mutations of three random mutagenesis libraries (1000 mutations each; epPCR with a low-mutation and a high-mutation frequency and SeSaM-Tv P/P) employing lipase A from Bacillus subtilis (bsla). A comparison of the obtained numbers of beneficial variants in the mentioned three random mutagenesis libraries with a site saturation mutagenesis (SSM) (covering the natural diversity at each amino acid position of BSLA) concludes the diversity analysis. Seventy-six percent of the SeSaM-Tv P/P-generated substitutions yield chemically different amino acid substitutions compared to 64% (epPCR-low) and 69% (epPCR-high). Unique substitutions from one amino acid to others are termed distinct amino acid substitutions. In the SeSaM-Tv P/P library, 35% of all theoretical distinct amino acid substitutions were found in the 1000 mutation library compared to 25% (epPCR-low) and 26% (epPCR-high). Thirty-six percent of distinct amino acid substitutions found in SeSaM-Tv P/P were unobtainable by epPCR-low. Comparison with the SSM library showed that epPCR-low covers 15%, epPCR-high 18%, and SeSaM-Tv P/P 21% of obtainable beneficial amino acid positions. In essence, this study provides first insights on the quality of epPCR and SeSaM-Tv P/P libraries in terms of amino acid substitutions, their chemical differences, and the number of obtainable beneficial amino acid positions.


Protein engineering Directed evolution Random mutagenesis epPCR SeSaM Lipase 

Supplementary material

253_2016_8035_MOESM1_ESM.pdf (1.5 mb)
ESM 1(PDF 1537 kb)


  1. Ahmad S, Kumar V, Ramanand KB, Rao NM (2012) Probing protein stability and proteolytic resistance by loop scanning: a comprehensive mutational analysis. Protein Sci 21(3):433–446. doi:10.1002/pro.2029 CrossRefPubMedPubMedCentralGoogle Scholar
  2. Arango Gutierrez E, Mundhada H, Meier T, Duefel H, Bocola M, Schwaneberg U (2013) Reengineered glucose oxidase for amperometric glucose determination in diabetes analytics. Biosens Bioelectron 50:84–90. doi:10.1016/j.bios.2013.06.029 CrossRefPubMedGoogle Scholar
  3. Cheng F, Zhu L, Lue H, Bernhagen J, Schwaneberg U (2014) Directed arginine deiminase evolution for efficient inhibition of arginine-auxotrophic melanomas. Appl Microbiol Biot 99(3):1237–1247. doi:10.1007/s00253-014-5985-z CrossRefGoogle Scholar
  4. Chronopoulou EG, Labrou NE (2011) Site-saturation mutagenesis: a powerful tool for structure-based design of combinatorial mutation libraries. Curr Protoc Protein Sci 26(6):26.6.1–26.6.10. doi:10.1002/0471140864.ps2606s63 CrossRefGoogle Scholar
  5. Cobb RE, Chao R, Zhao HM (2013) Directed evolution: past, present, and future. AICHE J 59(5):1432–1440. doi:10.1002/aic.13995 CrossRefPubMedPubMedCentralGoogle Scholar
  6. Crooks GE, Hon G, Chandonia JM, Brenner SE (2004) WebLogo: a sequence logo generator. Genome Res 14(6):1188–1190. doi:10.1101/gr.849004 CrossRefPubMedPubMedCentralGoogle Scholar
  7. Dennig A, Shivange AV, Marienhagen J, Schwaneberg U (2011) OmniChange: the sequence independent method for simultaneous site-saturation of five codons. PLoS One 6(10):e26222. doi:10.1371/journal.pone.0026222 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 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 Microb 80(4):1515–1527. doi:10.1128/AEM.03371-13 CrossRefGoogle Scholar
  9. Firnberg E, Ostermeier M (2012) PFunkel: efficient, expansive, user-defined mutagenesis. PLoS One 7(12):e52031. doi:10.1371/journal.pone.0052031 CrossRefPubMedPubMedCentralGoogle Scholar
  10. Firth AE, Patrick WM (2005) Statistics of protein library construction. Bioinformatics 21(15):3314–3315. doi:10.1093/bioinformatics/bti516 CrossRefPubMedGoogle Scholar
  11. Firth AE, Patrick WM (2008) GLUE-IT and PEDEL-AA: new programmes for analyzing protein diversity in randomized libraries. Nucleic Acids Res 36:W281–W285. doi:10.1093/nar/gkn226 CrossRefPubMedPubMedCentralGoogle Scholar
  12. Frauenkron-Machedjou VJ, Fulton A, Zhu L, Anker C, Bocola M, Jaeger KE, Schwaneberg U (2015) Towards understanding directed evolution: more than half of all amino acid positions contribute to ionic liquid resistance of Bacillus subtilis lipase A. Chembiochem 16(6):937–945. doi:10.1002/cbic.201402682 CrossRefPubMedGoogle Scholar
  13. Jain PC, Varadarajan R (2014) A rapid, efficient, and economical inverse polymerase chain reaction-based method for generating a site saturation mutant library. Anal Biochem 449:90–98. doi:10.1016/j.ab.2013.12.002 CrossRefPubMedGoogle Scholar
  14. Jimenez-Morales D, Liang J, Eisenberg B (2012) Ionizable side chains at catalytic active sites of enzymes. Eur Biophys J 41(5):449–460. doi:10.1007/s00249-012-0798-4 CrossRefPubMedPubMedCentralGoogle Scholar
  15. Kumar A, Singh S (2013) Directed evolution: tailoring biocatalysts for industrial applications. Crit Rev Biotechnol 33(4):365–378. doi:10.3109/07388551.2012.716810 CrossRefPubMedGoogle Scholar
  16. Lauchli R, Rabe KS, Kalbarczyk KZ, Tata A, Heel T, Kitto RZ, Arnold FH (2013) High-throughput screening for terpene-synthase-cyclization activity and directed evolution of a terpene synthase. Angew Chem Int Ed 52(21):5571–5574. doi:10.1002/anie.201301362 CrossRefGoogle Scholar
  17. Lehmann C, Bocola M, Streit WR, Martinez R, Schwaneberg U (2014) Ionic liquid and deep eutectic solvent-activated CelA2 variants generated by directed evolution. Appl Microbiol Biot 98(12):5775–5785. doi:10.1007/s00253-014-5771-y CrossRefGoogle Scholar
  18. Li ZW, Roccatano D, Lorenz M, Schwaneberg U (2012) Directed evolution of subtilisin E into a highly active and guanidinium chloride- and sodium dodecylsulfate-tolerant protease. Chembiochem 13(5):691–699. doi:10.1002/cbic.201100714 CrossRefPubMedGoogle Scholar
  19. LinGoerke JL, Robbins DJ, Burczak JD (1997) PCR-based random mutagenesis using manganese and reduced dNTP concentration. BioTechniques 23(3):409–412Google Scholar
  20. Liu HF, Zhu LL, Bocola M, Chen N, Spiess AC, Schwaneberg U (2013) Directed laccase evolution for improved ionic liquid resistance. Green Chem 15(5):1348–1355. doi:10.1039/C3GC36899H CrossRefGoogle Scholar
  21. Martinez R, Jakob F, Tu R, Siegert P, Maurer KH, Schwaneberg U (2013) Increasing activity and thermal resistance of Bacillus gibsonii alkaline protease (BgAP) by directed evolution. Biotechnol Bioeng 110(3):711–720. doi:10.1002/bit.24766 CrossRefPubMedGoogle Scholar
  22. Patrick WM, Firth AE, Blackburn JM (2003) User-friendly algorithms for estimating completeness and diversity in randomized protein-encoding libraries. Protein Eng 16(6):451–457. doi:10.1093/protein/gzg057 CrossRefPubMedGoogle Scholar
  23. Petritis K, Kangas LJ, Yan B, Monroe ME, Strittmatter EF, Qian WJ, Adkins JN, Moore RJ, Xu Y, Lipton MS, Ii DGC, Smith RD (2006) Improved peptide elution time prediction for reversed-phase liquid chromatography-MS by incorporating peptide sequence information. Anal Chem 78(14):5026–5039. doi:10.1021/ac060143p CrossRefPubMedPubMedCentralGoogle Scholar
  24. Quin MB, Schmidt-Dannert C (2011) Engineering of biocatalysts: from evolution to creation. ACS Catal 1:1017–1021. doi:10.1021/cs200217t CrossRefPubMedPubMedCentralGoogle Scholar
  25. Rasila TS, Pajunen MI, Savilahti H (2009) Critical evaluation of random mutagenesis by error-prone polymerase chain reaction protocols, Escherichia coli Mutator strain, and hydroxylamine treatment. Anal Biochem 388:71–80. doi:10.1016/j.ab.2009.02.008 CrossRefPubMedGoogle Scholar
  26. Ruff AJ, Dennig A, Schwaneberg U (2013) To get what we aim for-progress in diversity generation methods. FEBS J 280(13):2961–2978. doi:10.1111/febs.12325 CrossRefPubMedGoogle Scholar
  27. Shivange AV, Marienhagen J, Mundhada H, Schenk A, Schwaneberg U (2009) Advances in generating functional diversity for directed protein evolution. Curr Opin Chem Biol 13(1):19–25. doi:10.1016/j.cbpa.2009.01.019 CrossRefPubMedGoogle Scholar
  28. Shivange AV, Serwe A, Dennig A, Roccatano D, Haefner S, Schwaneberg U (2012) Directed evolution of a highly active Yersinia mollaretii phytase. Appl Microbiol Biot 95(2):405–418. doi:10.1007/s00253-011-3756-7 CrossRefGoogle Scholar
  29. Tee KL, Schwaneberg U (2007) Directed evolution of oxygenases: screening systems, success stories and challenges. Comb Chem High Throughput Screen 10(3):197–217. doi:10.2174/138620707780126723 CrossRefPubMedGoogle Scholar
  30. Tee KL, Wong TS (2013) Polishing the craft of genetic diversity creation in directed evolution. Biotechnol Adv 31(8):1707–1721. doi:10.1016/j.biotechadv.2013.08.021 CrossRefPubMedGoogle Scholar
  31. Torres-Salas P, Mate DM, Ghazi I, Plou FJ, Ballesteros AO, Alcalde M (2013) Widening the pH activity profile of a fungal laccase by directed evolution. Chembiochem 14(8):934–937. doi:10.1002/cbic.201300102 CrossRefPubMedGoogle Scholar
  32. Tracewell CA, Arnold FH (2009) Directed enzyme evolution: climbing fitness peaks one amino acid at a time. Curr Opin Chem Biol 13(1):3–9. doi:10.1016/j.cbpa.2009.01.017 CrossRefPubMedPubMedCentralGoogle Scholar
  33. van Pouderoyen G, Eggert T, Jaeger KE, Dijkstra BW (2001) The crystal structure of Bacillus subtilis lipase: a minimal alpha/beta hydrolase fold enzyme. J Mol Biol 309(1):215–226. doi:10.1006/jmbi.2001.4659 CrossRefPubMedGoogle Scholar
  34. Verma R, Schwaneberg U, Roccatano D (2012) MAP(2.0)3D: a sequence/structure based server for protein engineering. ACS Synth Biol 1(4):139–150. doi:10.1021/sb200019x CrossRefPubMedGoogle Scholar
  35. Wong TS, Roccatano D, Schwaneberg U (2007a) Are transversion mutations better? A mutagenesis assistant program analysis on P450 BM-3 heme domain. Biotechnol J 2(1):133–142. doi:10.1002/biot.200600201 CrossRefPubMedGoogle Scholar
  36. Wong TS, Roccatano D, Schwaneberg U (2007b) Challenges of the genetic code for exploring sequence space in directed protein evolution. Biocatal Biotransformation 25(2–4):229–241. doi:10.1080/10242420701444280 CrossRefGoogle Scholar
  37. Wong TS, Roccatano D, Zacharias M, Schwaneberg U (2006) A statistical analysis of random mutagenesis methods used for directed protein evolution. J Mol Biol 355(4):858–871. doi:10.1016/j.jmb.2005.10.082 CrossRefPubMedGoogle Scholar
  38. Yedavalli P, Rao NM (2013) Engineering the loops in a lipase for stability in DMSO. Protein Eng Des Sel 26(4):317–324. doi:10.1093/protein/gzt002 CrossRefPubMedGoogle Scholar
  39. Zhao J, Jia N, Jaeger KE, Bocola M, Schwaneberg U (2015) Ionic liquid activated Bacillus subtilis lipase A variants through cooperative surface substitutions. Biotechnol Bioeng 112(10):1997–2004. doi:10.1002/bit.25617 CrossRefPubMedGoogle Scholar
  40. Zhao J, Kardashliev T, Ruff AJ, Bocola M, Schwaneberg U (2014) Lessons from diversity of directed evolution experiments by an analysis of 3,000 mutations. Biotechnol Bioeng 111(12):2380–2389. doi:10.1002/bit.25302 CrossRefPubMedGoogle Scholar
  41. Zheng L, Baumann U, Reymond JL (2004) An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucleic Acids Res 32(14):e115. doi:10.1093/nar/gnh110 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  1. 1.Lehrstuhl für BiotechnologieRWTH Aachen UniversityAachenGermany
  2. 2.Tianjin Institute of Industrial BiotechnologyChinese Academy of SciencesTianjinChina

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