Biocatalysts for Industry pp 257-283 | Cite as
Protein Engineering of Subtilisin
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.
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 ProteinPreview
Unable to display preview. Download preview PDF.
References
- 1.C: H. Wong, Science 244, 1145 (1989).PubMedCrossRefGoogle Scholar
- 2.H. Yamada and S. Shimizu, Angew. Chem. Int. Ed. Eng 27, 622 (1988).CrossRefGoogle Scholar
- 3.G. M. Whitesides and C.-H. Wong, Angew. Chem. Int. Ed. Eng 24, 617 (1985).CrossRefGoogle Scholar
- 4.J. B Jones, Tetrahedron 42,3351 (1986).Google Scholar
- 5.R. Porter and S. Clark, eds., Enzymes in Organic Synthesis, Pitman, London (1985).Google Scholar
- 6.E. Polastro, A. Walker, and H. Teeuwen, Bio/technology 7, 1238 (1989).Google Scholar
- 7.R. Wetzel, Protein Engineering 1, 3 (1986).PubMedCrossRefGoogle Scholar
- 8.R. J Leatherbarrow and A. R. Fersht, Protein Engineering 1,7 (1986).Google Scholar
- 9.D. L. Oxender and C. F. Fox, Protein Engineering, Alan R. Liss, New York (1987) Google Scholar
- 10.J. R. Knowles, Science 236, 1252 (1987).PubMedCrossRefGoogle Scholar
- 11.J. A. Wells and D. A. Estell, TIBS 13, 291 (1988).PubMedGoogle Scholar
- 12.W. F. Degrado, Z. R. Wasserman, and J. D. Lear, Science 243, 622 (1989).PubMedCrossRefGoogle Scholar
- 13.G. D. Fasman, TIBS 163, 295 (1989).Google Scholar
- 14.A. Warshel and S. T. Russel, Q. Rev. Biophys. 17, 283 (1984).PubMedCrossRefGoogle Scholar
- 15.A. R. Fersht and M. J. E. Sternberg, Protein Engineering 2, 527 (1989).PubMedCrossRefGoogle Scholar
- 16.J. Kraut, Ann. Rev. Biochem. 46, 331 (1977).CrossRefGoogle Scholar
- 17.A. Warshel, G. Naray-Szabo, F. Sussman, and J.-K. Hwang, Biochemistry 28, 3629 (1989).PubMedCrossRefGoogle Scholar
- 18.M. Philipp and M. L. Bender, Molecular and Cellular Biochemistry 51, 5 (1983).PubMedCrossRefGoogle Scholar
- 19.I. B. Svendsen, Carlsberg Res. Commun. 41, 237 (1976).CrossRefGoogle Scholar
- 20.J. D. Robertus, J. Kraut, R. A. Alden, and J. J. Birktoft, Biochemistry 11, 4293 (1972).PubMedCrossRefGoogle Scholar
- 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.S. Hirono, H. Akagawa, Y. Mitsui, and Y. litika, J. Mol. Biol. 178, 389 (1984).PubMedCrossRefGoogle Scholar
- W. Bode, E. Papamokos, D. Musil, U. Seemueller, and H. Fritz, EMBO J. 5 813 (1986).Google Scholar
- 24.C. A. McPhalen, I. Svendsen, I. Jonassen, and M. N. G. James, Proc. Natl. Acad. Sci. USA 82, 7242 (1985).CrossRefGoogle Scholar
- 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.J. A. Wells, E. Ferrari, D. J. Henner, D. A. Estell, and E. Y. Chen, Nucleic Acids Res. 11, 7911 (1983).Google Scholar
- 27.J. Yang, E. Ferrari, and D. J. Henner, J. Bacteriol. 160, 15 (1984).PubMedGoogle Scholar
- 28.D. A. Estell, T. P. Graycar, and J. A. Wells, J. BioL Chem. 260, 6518 (1985).PubMedGoogle Scholar
- 29.M. V. Arbige and W. H. Pitcher, TIBTECH 7, 330 (1989).CrossRefGoogle Scholar
- 30.R. E. Offord, Protein Engineering 1, 151 (1987).PubMedCrossRefGoogle Scholar
- 31.D. Woo, I. Clark-Lewis, B. Chait, and S. Kent, Protein Engineering 3, 29 (1989).PubMedGoogle Scholar
- 32.A. Proudfoot, K. Rose, and C. Wallace, J. Biol. Chem. 264, 8764 (1989).PubMedGoogle Scholar
- 33.L. Polgar and M. L. Bender, J. Am. Chem. Soc 88, 3153 (1966).CrossRefGoogle Scholar
- 34.K. E. Neet and D. E. Koshland, Jr., Proc. NatL Acad. Sci. USA 56, 1606 (1966).CrossRefGoogle Scholar
- 35.E. T. Kaiser and D. S. Lawrence, Science 226, 505 (1984).PubMedCrossRefGoogle Scholar
- 36.L. M. Bech and K. Breddam, Carlsberg Res. Commun. 53, 381 (1988).Google Scholar
- 37.M. J. Zoller and M. Smith, Methods in Enzymology 100, 468 (1983).PubMedCrossRefGoogle Scholar
- 38.P. Carter, Methods in Enzymology 154, 382 (1987).PubMedCrossRefGoogle Scholar
- 39.J. A. Wells, M. Vasser, and D. B. Powers, Gene 34, 315 (1985).PubMedCrossRefGoogle Scholar
- 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.D. Botstein and D. Shortle, Science 229, 1193 (1985).PubMedCrossRefGoogle Scholar
- 42.M. Smith, Ann. Rev. Genet. 19, 423 (1985).PubMedCrossRefGoogle Scholar
- 43.S. S. Ner, D. B. Goodin, and M. Smith, DNA 7, 127 (1988).PubMedCrossRefGoogle Scholar
- 44.D. W. Leung, E. Chen, and D. V. Goeddel, Techniques 1, 11 (1989).Google Scholar
- 45.R. Menzel, Anal. Biochem. 181, 40 (1989).PubMedCrossRefGoogle Scholar
- 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.A. R. Fersht, Enzyme Structure and Mechanism, 2nd ed., Freeman, San Francisco (1985).Google Scholar
- 48.I. Schechter and A. Berger, Biochem. Biophys. Res. Commun 27, 157 (1967).CrossRefGoogle Scholar
- 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.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.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.R. Bott, personal communication.Google Scholar
- 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.J. A. Wells, B. C. Cunningham, T. P. Graycar, and D. A. Este11, Proc. Natl. Acad. Sci. USA 84, 5167 (1987).PubMedCrossRefGoogle Scholar
- 55.C. McPhalen, H. Schnebli, and M. James, FEBS Lett. 188, 55 (1985).PubMedCrossRefGoogle Scholar
- 56.P. Carter and J. A. Wells, Science 237, 394 (1987).PubMedCrossRefGoogle Scholar
- 57.P. Carter, B. Nilsson, J. P. Bumier, D. Burdick, and J. Wells, Proteins: Structure, Function, and Genetics 6, 240 (1989).Google Scholar
- 58.F. Graham, G. Gray, C. Carmona, E. Ferrari, and D. Estell, manuscript in preparation.Google Scholar
- 59.S. D. Power, R. M. Adams, and J. A. Wells, Proc. Natl. Acad. Sci. USA 83, 3096 (1986).PubMedCrossRefGoogle Scholar
- 60.P. Valenzuela and M. L. Bender, Biochim. Biophys. Acta 250, 538 (1971).PubMedCrossRefGoogle Scholar
- 61.A. J. Russell and A. R. Fersht, Nature 328, 496 (1987).PubMedCrossRefGoogle Scholar
- 62.T. P. Graycar and D. A. Estell, unpublished results (1986).Google Scholar
- 63.C. E. Stauffer and D. Etson, J. Biol. Chem 244, 5333 (1969).PubMedGoogle Scholar
- 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.J. Wells, B. Cunningham, R. Bott, R. Adams, S. Power, T. Graycar, and D. Estell, unpublished results (1987).Google Scholar
- 66.J. A. Wells and P. B. Powers, J. Biol. Chem 261, 6564 (1986).PubMedGoogle Scholar
- 67.B. A. Katz and A. Kossiakofl; J. Biol. Chem 261, 15480 (1986).PubMedGoogle Scholar
- 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.C. Mitchinson and J. A. Wells, Biochemistry 28, 4807 (1989).PubMedCrossRefGoogle Scholar
- 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.L. J. Perry and R. Wetzel, Science 226, 555 (1984).PubMedCrossRefGoogle Scholar
- 72.R. Wetzel, Trends Biochem. Sci 12, 478 (1987).CrossRefGoogle Scholar
- 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.J. Drenth, W. G. Hol, and J. Jansonius, Eur. J. Biochem 26, 177 (1972).PubMedCrossRefGoogle Scholar
- 75.G. Voordouw, C. Milo, and R. S. Roche, Biochemistry 15, 3716 (1976).PubMedCrossRefGoogle Scholar
- 76.K. Morihara, Trends Biotechnol. 5, 164 (1987).CrossRefGoogle Scholar
- 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.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.T. Nakatsuka, T. Sasaki, and E. T. Kaiser, J. Am. Chem. Soc 109, 3808 (1987).CrossRefGoogle Scholar
- 80.Z.-P. Wu and D. Hilvert, J. Am. Chem. Soc 111, 4513 (1989).CrossRefGoogle Scholar
- 81.B. Cambou and A. M. Klibanov, J. Am. Chem. Soc 106, 2687 (1984).CrossRefGoogle Scholar
- 82.C.-S. Chen, S.-H. Wu, G. Girdaukus, and C. J. Sih, J. Am. Chem. Soc 109, 2812 (1987).CrossRefGoogle Scholar
- 83.S. Riva, J. Chopineau, A. P. G. Kieboom, and A. M. Klibanov, J. Am. Chem. Soc 110, 584 (1988).CrossRefGoogle Scholar
- 84.S.-H. Wu, L.-C. Lo, S.-T. Chen, and K.-T. Wang, J. Org . Chem. 54, 4220 (1989).CrossRefGoogle Scholar
- 85.M. Arbige, D. Estell, M. Pepsin, and A. J. Poulose, European Patent Office Application No. 0260105.Google Scholar