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Improving the quality of industrially important enzymes by directed evolution

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Abstract

Directed evolution is a new process for developing industrially viable biocatalysts. This technique does not require a comprehensive knowledge of the relationships between sequence structure and function of proteins as required by protein engineering. It mimics the process of Darwinian evolution in a test tube combining random mutagenesis and recombination with screening or selection for enzyme variants that have the desired properties. Directed evolution helps in enhancing the enzyme performance both in natural and synthetic environments. This article reviews the process of directed evolution and its application to improve substrate specificity, activity, enantioselectivity and thermal stability.

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References

  1. Stemmer WPC: Rapid evolution of a protein in vitro by DNA shuffling. Nature 370: 389–391, 1994

    Google Scholar 

  2. Arnold FH, Volkov AA: Design by directed evolution. Acc Chem Res 31: 125–131, 1998

    Google Scholar 

  3. Kuchner O, Arnold FH: Strategies for the in vitro evolution of protein function: Enzyme evolution by random recombination of improved sequences. J Mol Biol 272: 36–347, 1997

    Google Scholar 

  4. Arnold FH, Volkov AA: Design by directed evolution. Acc Chem Res 31: 125–131, 1998

    Google Scholar 

  5. Arnold FH, Volkov AA: Directed evolution of biocatalysts. Curr Opin Chem Biol 3: 54–59, 1999

    Google Scholar 

  6. Hayashi N, Welschof M, Zewe M, Braunagel M: Simultaneous mutagenesis of antibody CDR region by overlap extension and PCR. Biotechniques 17: 310–315, 1994

    Google Scholar 

  7. Reidhaar-Olson JF, Sauer RT: Combinatorial cassette mutagenesis as a probe of the informational content of protein sequences. Science 241: 53–57, 1988

    Google Scholar 

  8. Stemmer WPC, Morris SK, Wilson BS: Enzymatic inverse PCR: A restriction site independent, single-fragment method for high-efficiency, site-directed mutagenesis. Biotechniques 14: 256–265, 1992

    Google Scholar 

  9. Oliphant AR, Nussbaum AL, Struhl K: Cloning of random-sequence oligodeoxynucleotides. Gene 44: 177–183, 1986 167

    Google Scholar 

  10. Hermes JD, Blacklow SC, Knowles JR: Searching sequence space by definably random mutagenesis: improving the catalytic potency of an enzyme. Proc Natl Acad Sci USA 87: 696–700, 1990

    Google Scholar 

  11. Reidhar-Olson J, Bowie J, Breyer RM, Hug C, Knight KL, Lim WA, Mossing MC, Parsell DA, Shoemaker KR, Sauer RT: Random mutagenesis of protein sequences using oligonucleotide cassettes. Meth Enzymol 208: 564–586, 1991

    Google Scholar 

  12. Arkin A, Youvan DG: An algorithm for protein engineering: Simulations of recursive ensemble mutagenesis. Proc Natl Acad Sci USA 89: 7811–7815, 1992

    Google Scholar 

  13. Delagrave S, Youvan DC: Searching sequence space to engineer proteins: Exponential ensemble mutagenesis. Biotechnology 11: 1548–1552, 1993

    Google Scholar 

  14. Leung DW, Chen E, Cachianes G, Goeddel DV: Nucleotide sequence of the partition function of Escherchia coli plasmid ColE1. DNA 4: 351–355, 1985

    Google Scholar 

  15. Cadwell RC, Joyce GF: Mutagenic PCR. PCR Meth Appl 3: 136–140, 1994

    Google Scholar 

  16. Crameri A, Whitehorn EA, Tate E, Stemmer WPC: Improved green fluorescent protein by molecular evolution using DNA shuffling. Nature Biotech 14: 315–319, 1995

    Google Scholar 

  17. Patten P, Howard RJ, Stemmer WPC: Applications of DNA shuffling to pharmaceuticals and vaccines. Curr Opin Biotechnol 15: 523–530, 1997

    Google Scholar 

  18. Crameri A, Raillard SA, Bermudez E, Stemmer WP: DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature 391: 288–291, 1998

    Google Scholar 

  19. Smith GP: Applied evolution. The progeny of sexual PCR. Nature 370: 324–325, 1994

    Google Scholar 

  20. Stemmer WPC: DNA shuffling by random fragmentation and reassembly in vitro recombination for molecular evolution. Proc Natl Acad Sci USA 91: 10747–10751, 1994

    Google Scholar 

  21. Stemmer WPC: Searching sequence space. Biotechnology 13: 549–553, 1995

    Google Scholar 

  22. Zhao H, Arnold FH: Optimization of DNA shuffling for high fidelity recombination. Nucleic Acid Res 25: 1307–1308, 1997

    Google Scholar 

  23. Lorimer IAJ, Pastan I: Random recombination of antibody single chain Fv sequences after fragmentation with Dnase I in the presence of Mn2+. Nucleic Acid Res 23: 3067–3068, 1995

    Google Scholar 

  24. Takato Y, Shinya O, Hiroyoki K: Directed evolution of an aspartate aminotransferase with new substrate specificities. Proc Natl Acad Sci USA 95: 5511–5515, 1998

    Google Scholar 

  25. Shiya O, Akihiro O, Takato Y, Hiroyuki K: Redesigning the substrate specificity of an enzyme by cumulative effects of the mutations of nonactive site residues. J Biol Chem 274: 2344–2349

  26. Brannon DR, Mabe JA, Fukuda DS: De esterification of cephalosporin para-ntrobenzyl ester by microbial enzymes. J Antibiotics 29: 121–124, 1976

    Google Scholar 

  27. Jeffrey C, Moore, Frances H, Arnold: Directed evolution of para-nitrobenzyl esterase for aqueous-organic solvents. Nature Biotechnol 14: 458–467, 1996

    Google Scholar 

  28. Abelskov AK, Smith AT, Rasmussen CB, Dunford HB, Welinder KG: Ph dependence and structural interpretation of the reactions of Coprinus cinereus peroxidase with hydrogen peroxide, ferulic acid. Biochem 36: 9453–9463, 1997

    Google Scholar 

  29. Poulos TL, Kraut J: The stereochemistry of peroxidase catalysts. J Biol Chem 255: 8199–8205, 1980

    Google Scholar 

  30. Joel R, Cherry MH, Lamsa P, Jesper V, Allan S, Aubrey J, Anders H: Directed evolution of fungal peroxidase. Nature Biotechnol 17: 379–384, 1999

    Google Scholar 

  31. Roth NJ, Huber RE: The β-galactosidase (Escherichia coli) reaction is partly facilitated by interactions of His-540 with the C6 hydroxyl of galactose. J Biol Chem 271: 14296–14301, 1996

    Google Scholar 

  32. Zhang J-H, Dawes G, Williem PC, Stemmer: Directed evolution of a fucosidase from a galactosidase by DNA shuffling and screening. Proc Natl Acad Sci USA 94: 4504–4509, 1997

    Google Scholar 

  33. Hall BG: Changes in the substrate specificities of an enzyme during directed evolution of new functions. Biochemistry 20: 4042–4049, 1981

    Google Scholar 

  34. Juers DH, Huber RE, Matthews BW: Structural comparisons of TIM barrel proteins suggest functional and evolutionary relationships between β-galactosidase and other glycohydrolases. Prot Sci 8: 122–136, 1999

    Google Scholar 

  35. Allen SJ, Holbrook JJ: Production of an activated form of Bacillus stearothermophilus L-2-hydroxyacid dehydrogenase by directed evolution. Prot Eng 13: 5–7, 2000

    Google Scholar 

  36. Kumamaru T, Suenaga H, Mitsuoka M, Watanabe T, Furukawa K: Enhanced degradation of polychlorinated biphenyls by directed evolution of biphenyl dioxygenase. Nature Biotechnol 16: 663–666, 1998

    Google Scholar 

  37. Szostak JW: In vitro selection and directed evolution. Harvey Lect 98: 95–118, 1997

    Google Scholar 

  38. Wills C, Kratofil P, Londo D, Martin ?: Characterisation of the two alcohol dehydrogenases of Zymomonas mobilis. Arch Biochem Biophys 210: 775–785, 1981

    Google Scholar 

  39. Jun H, Takato Y, Yoshinaro K, Seiki K: Directed evolution of thermostable kanamycin-resistance gene: A convenient selection marker for Thermus thermophiles. J Biochem 126: 951–956, 1999

    Google Scholar 

  40. Giver L, Gershenson A, Freskgard PO, Arnold FH: Directed evolution of a thermostable esterase. Proc Natl Acad Sci USA 95: 12809–12813, 1998

    Google Scholar 

  41. Miyazaki K, Wintrode PL, Grayling RA, Rubingh DN, Arnold FH: Directed evolution study of temperature adaptation in a psychrophilic enzyme. Mol Biol 297: 1015–1026, 2000

    Google Scholar 

  42. Song JK, Rhee H: Simultaneous enhancement of thermostability and catalytic activity of phospholipase A(1) by evolutionary molecular engineering. Appl Environ Microbiol 66: 890–894, 2000

    Google Scholar 

  43. Lebbink JH, Kaper T, Bron P, van Der Oost J, de Vos WM: Improving low-temperature catalysis in the hyperthermostable Pyrococcus furiosus β-glucosidase by directed evolution. Biochemistry 39: 3656–3665, 2000

    Google Scholar 

  44. Zhao H, Arnold FH: Directed evolution of β-glucosidase A from Paenibacillus polymyxa to thermal resistance. J Biol Chem 275: 13708–13712, 2000

    Google Scholar 

  45. Akanuma S, Yamagishi A, Tanaka N, Oshima T: Directed evolution converts subtilisin E into a functional equivalent of thermitase. Prot Eng 12: 47–53, 1999

    Google Scholar 

  46. Buchholz F, Angrand PO, Stewart AF: Serial increase in the thermal stability of 3-isopropylmalate dehydrogenase from Bacillus subtilis by experimental evolution. Prot Sci 7: 698–705, 1998

    Google Scholar 

  47. Shaikh AC, Sadowski PD: Chimeras of the flp and cre recombinases: Tests of the mode of cleavage by flp and cre. J Mol Biol 302: 27–48, 2000

    Google Scholar 

  48. Strausberg SL, Alexander PA, Gallagher DT, Gilliland GL, Barnett BL, Bryan PN: Directed evolution of a subtilisin with calcium-independent stability. Biotechnology 13: 669–673, 1995

    Google Scholar 

  49. Joo H, Arisawa A, Lin Z, Arnold FH: A high-throughput digital imaging screen for the discovery and directed evolution of oxygenases. Chem Biol 6: 699–706, 1999

    Google Scholar 

  50. Reetz MT: Evolution in the test tube as a means to create enantioselective enzymes for use in organic synthesis. Sci Prog 83: 157–172, 2000

    Google Scholar 

  51. Reetz MT, Jaeger KE: Enantioselective enzymes for organic synthesis created by directed evolution. Chemistry 6: 407–412, 2000

    Google Scholar 

  52. Henke E, Bornscheuer UT: Directed evolution of an esterase from Pseudomonas fluorescens. Random mutagenesis by error-prone PCR or a mutator strain and identification of mutants showing enhanced enantioselectivity by a resorufin-based fluorescence assay. J Biol Chem 380: 1029–33, 1999

    Google Scholar 

  53. May O, Nguyen PT, Arnold FH: Inverting enantioselectivity by directed evolution of hydantoinase for improved production of L-methionine. Nature Biotechnol 18: 317–320, 2000

    Google Scholar 

  54. Bornscheuer UT, Altenbuchner J, Meyer HH: Directed evolution of an esterase for the stereoselective resolution of a key intermediate in the synthesis of epothilones. Biotechnol Bioeng 58: 554–559, 1998

    Google Scholar 

  55. You L, Arnold FH: Optimizing industrial enzymes by directed evolution. Adv Biochem Eng Biotechnol 58: 1–14, 1997

    Google Scholar 

  56. Chen KQ, Arnold FH: Directed evolution of subtilisin E in Bacillus subtilis to enhance total activity in aqueous dimethylformamide. Prot Eng 9: 77–83, 1996

    Google Scholar 

  57. Matsuura T, Yomo T, Trakulnaleamsai S, Ohashi Y, Yamamoto K, Urabe I: Enzyme engineering for nonaqueous solvents: Random mutagenesis to enhance activity of subtilisin E in polar organic media. Biotechnology 9: 1073–1077, 1991

    Google Scholar 

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

    Google Scholar 

  59. Altamirano MM, Blackburn JM, Aguayo C, Fersht AR: Nonadditivity of mutational effects on the properties of catalase I and its application to efficient directed evolution. Prot Eng 11: 789–795, 1998

    Google Scholar 

  60. Kim GJ, Cheon YH, Kim HS: Directed evolution of new catalytic activity using the α/β-barrel scaffold. Nature 403: 617–622, 2000

    Google Scholar 

  61. Matsumura I, Wallingford JB, Surana NK, Vize PD, Ellington AD: Directed evolution of a novel N-carbamylase/D-hydantoinase fusion enzyme for functional expression with enhanced stability. Biotechnol Bioeng 68: 211–217, 2000

    Google Scholar 

  62. Joo H, Lin Z, Arnold FH: Directed evolution of the surface chemistry of the reporter enzyme β-glucuronidase. Nature Biotechnol 17: 696–701, 1999

    Google Scholar 

  63. Lin Z, Thorsen T, Arnold FH: Laboratory evolution of peroxide-mediated cytochrome P450 hydroxylation. Nature 399: 670–673, 1999

    Google Scholar 

  64. Christians FC, Scapozza L, Crameri A, Folkers G, Stemmer WP: Functional expression of horseradish peroxidase in E. coli by directed evolution. Biotechnol Prog 15: 467–471, 1999

    Google Scholar 

  65. Zhang JH, Dawes G, Stemmer WP: Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling. Nature Biotechnol 17: 259–264, 1999

    Google Scholar 

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Authors and Affiliations

  1. Biotechnology Research Group, University of Ulster at Coleraine, N, Ireland, BT52 1SA, UK

    Rajendra Rani Chirumamilla, Reddivari Muralidhar, Roger Marchant & Poonam Nigam

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  1. Rajendra Rani Chirumamilla
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  2. Reddivari Muralidhar
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  3. Roger Marchant
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  4. Poonam Nigam
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Correspondence to Poonam Nigam.

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Chirumamilla, R.R., Muralidhar, R., Marchant, R. et al. Improving the quality of industrially important enzymes by directed evolution. Mol Cell Biochem 224, 159–168 (2001). https://doi.org/10.1023/A:1011904405002

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