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Protein Engineering of Subtilisin

  • Thomas P. Graycar
Chapter
Part of the Topics in Applied Chemistry book series (TAPP)

Abstract

The use of enzymes as catalysts in organic synthesis reactions has expanded rapidly in recent years.1–5 In contrast to conventional organic reactions enzyme-catalyzed reactions offer the potential of highly stereo-selective or regioselective transformations. The value of enzymes in organic synthesis can be further attributed to the high reactivity and mild conditions characteristic of enzyme-catalyzed reactions. The increased availability and lower cost of enzymes resulting from the development of recombinant DNA technology has also stimulated interest in the use of enzymes for the production of fine organic chemicals. An apparent impediment to the widespread utilization of enzymes as commercial biocatalysts, however, is the difficulty encountered in optimizing their use in industrial processes. Although enzymes are capable of reacting with compounds having structures similar to that of their natural substrates, the catalytic efficiency of reactions involving nonnatural substrates is often suboptimal. In addition, the inherent instability of proteins and the sensitivity of enzyme activity to alterations in pH further limit their utility in industry. Hence, despite the known advantages of enzymes their use as biocatalysts is currently limited to less than 5% of the total industrial enzyme market.6 In the past, the only methods available to optimize the performance of an enzyme involved chemical modification of residues on the surface of the enzyme or else random mutagenesis of the corresponding gene. Now, however, the recent development of protein engineering has made it possible to redesign the structure of an enzyme and tailor its functional properties for a particular application, thereby greatly enhancing the potential to create novel industrial biocatalysts.

Table 1

Functional Properties of Enzymes which Can Be Modified by Protein Engineering

Stability

Specificity

Temperature

Substrate

Denaturants

Nucleophile

pH

Catalytic rate

Organic solvents

pH Activity profile

Oxidation

Allosteric regulation

Proteolysis

Antigenicity

Keywords

Catalytic Efficiency Protein Engineering Mutant Enzyme Random Mutagenesis Hybrid Protein 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
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References

  1. 1.
    C: H. Wong, Science 244, 1145 (1989).PubMedCrossRefGoogle Scholar
  2. 2.
    H. Yamada and S. Shimizu, Angew. Chem. Int. Ed. Eng 27, 622 (1988).CrossRefGoogle Scholar
  3. 3.
    G. M. Whitesides and C.-H. Wong, Angew. Chem. Int. Ed. Eng 24, 617 (1985).CrossRefGoogle Scholar
  4. 4.
    J. B Jones, Tetrahedron 42,3351 (1986).Google Scholar
  5. 5.
    R. Porter and S. Clark, eds., Enzymes in Organic Synthesis, Pitman, London (1985).Google Scholar
  6. 6.
    E. Polastro, A. Walker, and H. Teeuwen, Bio/technology 7, 1238 (1989).Google Scholar
  7. 7.
    R. Wetzel, Protein Engineering 1, 3 (1986).PubMedCrossRefGoogle Scholar
  8. 8.
    R. J Leatherbarrow and A. R. Fersht, Protein Engineering 1,7 (1986).Google Scholar
  9. 9.
    D. L. Oxender and C. F. Fox, Protein Engineering, Alan R. Liss, New York (1987) Google Scholar
  10. 10.
    J. R. Knowles, Science 236, 1252 (1987).PubMedCrossRefGoogle Scholar
  11. 11.
    J. A. Wells and D. A. Estell, TIBS 13, 291 (1988).PubMedGoogle Scholar
  12. 12.
    W. F. Degrado, Z. R. Wasserman, and J. D. Lear, Science 243, 622 (1989).PubMedCrossRefGoogle Scholar
  13. 13.
    G. D. Fasman, TIBS 163, 295 (1989).Google Scholar
  14. 14.
    A. Warshel and S. T. Russel, Q. Rev. Biophys. 17, 283 (1984).PubMedCrossRefGoogle Scholar
  15. 15.
    A. R. Fersht and M. J. E. Sternberg, Protein Engineering 2, 527 (1989).PubMedCrossRefGoogle Scholar
  16. 16.
    J. Kraut, Ann. Rev. Biochem. 46, 331 (1977).CrossRefGoogle Scholar
  17. 17.
    A. Warshel, G. Naray-Szabo, F. Sussman, and J.-K. Hwang, Biochemistry 28, 3629 (1989).PubMedCrossRefGoogle Scholar
  18. 18.
    M. Philipp and M. L. Bender, Molecular and Cellular Biochemistry 51, 5 (1983).PubMedCrossRefGoogle Scholar
  19. 19.
    I. B. Svendsen, Carlsberg Res. Commun. 41, 237 (1976).CrossRefGoogle Scholar
  20. 20.
    J. D. Robertus, J. Kraut, R. A. Alden, and J. J. Birktoft, Biochemistry 11, 4293 (1972).PubMedCrossRefGoogle Scholar
  21. 21.
    T. L. Poulos, R. A. Alden, J. J. Birktoft, S. T. Freer, and J. Kraut, J. Biol. Chem. 251, 1097 (1976).PubMedGoogle Scholar
  22. 22.
    S. Hirono, H. Akagawa, Y. Mitsui, and Y. litika, J. Mol. Biol. 178, 389 (1984).PubMedCrossRefGoogle Scholar
  23. W. Bode, E. Papamokos, D. Musil, U. Seemueller, and H. Fritz, EMBO J. 5 813 (1986).Google Scholar
  24. 24.
    C. A. McPhalen, I. Svendsen, I. Jonassen, and M. N. G. James, Proc. Natl. Acad. Sci. USA 82, 7242 (1985).CrossRefGoogle Scholar
  25. 25.
    R. R. Bott, M. Ultsch, A. Kossiakoff, T. P. Graycar, B. Katz, and S. Power, J. Biol. Chem. 263, 7895 (1988).PubMedGoogle Scholar
  26. 26.
    J. A. Wells, E. Ferrari, D. J. Henner, D. A. Estell, and E. Y. Chen, Nucleic Acids Res. 11, 7911 (1983).Google Scholar
  27. 27.
    J. Yang, E. Ferrari, and D. J. Henner, J. Bacteriol. 160, 15 (1984).PubMedGoogle Scholar
  28. 28.
    D. A. Estell, T. P. Graycar, and J. A. Wells, J. BioL Chem. 260, 6518 (1985).PubMedGoogle Scholar
  29. 29.
    M. V. Arbige and W. H. Pitcher, TIBTECH 7, 330 (1989).CrossRefGoogle Scholar
  30. 30.
    R. E. Offord, Protein Engineering 1, 151 (1987).PubMedCrossRefGoogle Scholar
  31. 31.
    D. Woo, I. Clark-Lewis, B. Chait, and S. Kent, Protein Engineering 3, 29 (1989).PubMedGoogle Scholar
  32. 32.
    A. Proudfoot, K. Rose, and C. Wallace, J. Biol. Chem. 264, 8764 (1989).PubMedGoogle Scholar
  33. 33.
    L. Polgar and M. L. Bender, J. Am. Chem. Soc 88, 3153 (1966).CrossRefGoogle Scholar
  34. 34.
    K. E. Neet and D. E. Koshland, Jr., Proc. NatL Acad. Sci. USA 56, 1606 (1966).CrossRefGoogle Scholar
  35. 35.
    E. T. Kaiser and D. S. Lawrence, Science 226, 505 (1984).PubMedCrossRefGoogle Scholar
  36. 36.
    L. M. Bech and K. Breddam, Carlsberg Res. Commun. 53, 381 (1988).Google Scholar
  37. 37.
    M. J. Zoller and M. Smith, Methods in Enzymology 100, 468 (1983).PubMedCrossRefGoogle Scholar
  38. 38.
    P. Carter, Methods in Enzymology 154, 382 (1987).PubMedCrossRefGoogle Scholar
  39. 39.
    J. A. Wells, M. Vasser, and D. B. Powers, Gene 34, 315 (1985).PubMedCrossRefGoogle Scholar
  40. 40.
    P. N. Bryan, M. L. Rollence, M. W. Pantoliano, J. Wood, B. C. Finzel, G. L. Gilliland, A. J. Howard, and T. L. Poulos, Proteins: Structure, Function, and Genetics 1, 326 (1986).Google Scholar
  41. 41.
    D. Botstein and D. Shortle, Science 229, 1193 (1985).PubMedCrossRefGoogle Scholar
  42. 42.
    M. Smith, Ann. Rev. Genet. 19, 423 (1985).PubMedCrossRefGoogle Scholar
  43. 43.
    S. S. Ner, D. B. Goodin, and M. Smith, DNA 7, 127 (1988).PubMedCrossRefGoogle Scholar
  44. 44.
    D. W. Leung, E. Chen, and D. V. Goeddel, Techniques 1, 11 (1989).Google Scholar
  45. 45.
    R. Menzel, Anal. Biochem. 181, 40 (1989).PubMedCrossRefGoogle Scholar
  46. 46.
    G. L. Gray, S. E. Mainzer, M. W. Rey, M. H. Lamsa, K. L. Kindle, C. Carmona, and C. Requadt, J. Bacteriol. 166, 635 (1986).PubMedGoogle Scholar
  47. 47.
    A. R. Fersht, Enzyme Structure and Mechanism, 2nd ed., Freeman, San Francisco (1985).Google Scholar
  48. 48.
    I. Schechter and A. Berger, Biochem. Biophys. Res. Commun 27, 157 (1967).CrossRefGoogle Scholar
  49. 49.
    D. A. Estell, T. P. Graycar, J. V. Miller, D. B. Powers, J. P. Bumier, P. G. Ng, and J. A. Wells, Science 233, 659 (1986).PubMedCrossRefGoogle Scholar
  50. 50.
    R. Bott and M. Ultsch, in: Fifth International Symposium on the Genetics of Industrial Microorganisms ( M. Alacevic, D. Hranueli, and Z. Toman, eds.), Pliva, Zagreb (1986).Google Scholar
  51. 51.
    J. A. Wells, D. B. Powers, R. R. Bott, T. P. Graycar, and D. A. Estell, Proc. Natl. Acad. Sci. USA 84, 1219 (1987).CrossRefGoogle Scholar
  52. 52.
    R. Bott, personal communication.Google Scholar
  53. 53.
    R. Bott, M. Ultsch, J. Wells, D. Powers, D. Burdick, M. Struble, J. Burnier, D. Estell, J. Miller, T. Graycar, R. Adams, and S. Power, in: Biotechnology in Agricultural Chemistry (ACS Symposium Series No. 334, H. LeBaron, R. Mumma, R. Honeycutt, and J. Duesing), 139 (1987).Google Scholar
  54. 54.
    J. A. Wells, B. C. Cunningham, T. P. Graycar, and D. A. Este11, Proc. Natl. Acad. Sci. USA 84, 5167 (1987).PubMedCrossRefGoogle Scholar
  55. 55.
    C. McPhalen, H. Schnebli, and M. James, FEBS Lett. 188, 55 (1985).PubMedCrossRefGoogle Scholar
  56. 56.
    P. Carter and J. A. Wells, Science 237, 394 (1987).PubMedCrossRefGoogle Scholar
  57. 57.
    P. Carter, B. Nilsson, J. P. Bumier, D. Burdick, and J. Wells, Proteins: Structure, Function, and Genetics 6, 240 (1989).Google Scholar
  58. 58.
    F. Graham, G. Gray, C. Carmona, E. Ferrari, and D. Estell, manuscript in preparation.Google Scholar
  59. 59.
    S. D. Power, R. M. Adams, and J. A. Wells, Proc. Natl. Acad. Sci. USA 83, 3096 (1986).PubMedCrossRefGoogle Scholar
  60. 60.
    P. Valenzuela and M. L. Bender, Biochim. Biophys. Acta 250, 538 (1971).PubMedCrossRefGoogle Scholar
  61. 61.
    A. J. Russell and A. R. Fersht, Nature 328, 496 (1987).PubMedCrossRefGoogle Scholar
  62. 62.
    T. P. Graycar and D. A. Estell, unpublished results (1986).Google Scholar
  63. 63.
    C. E. Stauffer and D. Etson, J. Biol. Chem 244, 5333 (1969).PubMedGoogle Scholar
  64. 64.
    M. O. Dayhoff, R. M. Schwartz, and B. C. Orcutt, in: Atlas of Protein Sequence and Structure (M. O. Dayhoff, ed.), Vol. 5, Supplement 3, 345 (1978), National Biomedical Research Foundation, Georgetown University Medical Center, Washington, D.C.Google Scholar
  65. 65.
    J. Wells, B. Cunningham, R. Bott, R. Adams, S. Power, T. Graycar, and D. Estell, unpublished results (1987).Google Scholar
  66. 66.
    J. A. Wells and P. B. Powers, J. Biol. Chem 261, 6564 (1986).PubMedGoogle Scholar
  67. 67.
    B. A. Katz and A. Kossiakofl; J. Biol. Chem 261, 15480 (1986).PubMedGoogle Scholar
  68. 68.
    M. W. Pantoliano, R. C. Ladner, P. N. Bryan, M. L. Rollence, J. F. Wood, and T. L. Poulos, Biochemistry 26, 2077 (1987).PubMedCrossRefGoogle Scholar
  69. 69.
    C. Mitchinson and J. A. Wells, Biochemistry 28, 4807 (1989).PubMedCrossRefGoogle Scholar
  70. 70.
    J. E. Villafranca, E. E. Howell, D. H. Voet, M. S. Strobel, R. C. Ogden, J. N. Ableson, and J. Kraut, Science 222, 782 (1983).PubMedCrossRefGoogle Scholar
  71. 71.
    L. J. Perry and R. Wetzel, Science 226, 555 (1984).PubMedCrossRefGoogle Scholar
  72. 72.
    R. Wetzel, Trends Biochem. Sci 12, 478 (1987).CrossRefGoogle Scholar
  73. 73.
    A. Pahler, A. Banarjee, J. K. Dattagapta, T. Fujiwara, K. Linder, G. P. Pal, D. Snick, G. Weber, and W. Saenger, EMBO J. 3, 1311 (1984).PubMedGoogle Scholar
  74. 74.
    J. Drenth, W. G. Hol, and J. Jansonius, Eur. J. Biochem 26, 177 (1972).PubMedCrossRefGoogle Scholar
  75. 75.
    G. Voordouw, C. Milo, and R. S. Roche, Biochemistry 15, 3716 (1976).PubMedCrossRefGoogle Scholar
  76. 76.
    K. Morihara, Trends Biotechnol. 5, 164 (1987).CrossRefGoogle Scholar
  77. 77.
    C. F. Barbas, III, J. R. Matos, J. B. West, and C: H. Wong, J. Am. Chem. Soc 110, 5162 (1988).CrossRefGoogle Scholar
  78. 78.
    J. B. West, J. Scholten, N. J. Stolowich, J. L. Hogg, A. I. Scott, and C.-H. Wong, J. Am. Chem. Soc 110, 3709 (1988).CrossRefGoogle Scholar
  79. 79.
    T. Nakatsuka, T. Sasaki, and E. T. Kaiser, J. Am. Chem. Soc 109, 3808 (1987).CrossRefGoogle Scholar
  80. 80.
    Z.-P. Wu and D. Hilvert, J. Am. Chem. Soc 111, 4513 (1989).CrossRefGoogle Scholar
  81. 81.
    B. Cambou and A. M. Klibanov, J. Am. Chem. Soc 106, 2687 (1984).CrossRefGoogle Scholar
  82. 82.
    C.-S. Chen, S.-H. Wu, G. Girdaukus, and C. J. Sih, J. Am. Chem. Soc 109, 2812 (1987).CrossRefGoogle Scholar
  83. 83.
    S. Riva, J. Chopineau, A. P. G. Kieboom, and A. M. Klibanov, J. Am. Chem. Soc 110, 584 (1988).CrossRefGoogle Scholar
  84. 84.
    S.-H. Wu, L.-C. Lo, S.-T. Chen, and K.-T. Wang, J. Org . Chem. 54, 4220 (1989).CrossRefGoogle Scholar
  85. 85.
    M. Arbige, D. Estell, M. Pepsin, and A. J. Poulose, European Patent Office Application No. 0260105.Google Scholar

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© Springer Science+Business Media New York 1991

Authors and Affiliations

  • Thomas P. Graycar
    • 1
  1. 1.Department of EnzymologyGenencor International, IncSouth San FranciscoUSA

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