Biocatalysis pp 97-106 | Cite as

Comparative Study of Substrate-and Stereospecificity of Penicillin G amidases from Different Sources and Hybrid Isoenzymes

  • Boris Galunsky
  • Karsten Lummer
  • Volker Kasche


Four natural pencillin G amidase variants from different sources and two genetically constructed hybrid enzymes were produced and purified to homogeneity. The specificity constants of one enzyme (E. coli) were found to differ six orders of magnitude for hydrolytic transformations within a wide range of substrates. The substrate specificity of the homologous penicillin amidases was found to differ less than one order of magnitude for hydrolysis of the most specific and up to two orders of magnitude for the less specific substrates. The Si-substrate specificity in hydrolytic and transfer reactions (studied mainly with the E. coli enzyme) varied more than three orders of magnitude for the different substrates. The penicillin amidases were found to be R-specific in the S1-binding site and S-specific in the Si-binding site. The S1-stereoselectivity differs less than one order of magnitude for the different variants. The Si-stereoselectivity is more pronounced, increases with nucleophile specificity, and was found to differ up to three orders of magnitude in transfer reactions for the enzyme from E. coli. The observed variation of enatioselectivity for different penicillin amidases and one substrate can also be achieved by changes in temperature. Comparison of substrate-and stereospecificity of penicillin amidases from different sources and hybrid isoenzymes suggests that similar changes can be expected for enzyme variants derived by rational protein design or directed evolution.


Phenylacetic Acid Acyl Moiety Enzyme Variant Specificity Constant Acyl Transfer 
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.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Rubingh DN (1997)Curr Opin Biotechnol 8: 417CrossRefGoogle Scholar
  2. 2.
    Takagi H (1993)Int J Biochem 25: 307CrossRefGoogle Scholar
  3. 3.
    Joyet P, Decklerk N, Gaillardin C (1992) Bio-Technology 10: 1579Google Scholar
  4. 4.
    You L, Arnold FH (1994)Protein Eng 9: 77CrossRefGoogle Scholar
  5. 5.
    Rubingh DN (1996) Engineering Proteases with Improved Properties for Detergents. In: Savage L, Southborough MA (eds) Enzyme Technology for Industrial Applications. IBC, p 98Google Scholar
  6. 6.
    Stemmer WPC (1994)Nature 370: 389CrossRefGoogle Scholar
  7. 7.
    Stemmer WPC (1994)Proc Natl Acad Sci 91: 10747CrossRefGoogle Scholar
  8. 8.
    Arnold FH (1996) Chem Eng Sci 51: 5091CrossRefGoogle Scholar
  9. 9.
    Arnold FH, Moor JC (1997) Adv Biochem Eng 58: 1Google Scholar
  10. 10.
    Moore JC, Arnold FH (1996)Nat Biotechnol 14: 458CrossRefGoogle Scholar
  11. 11.
    Virden R (1990) Biotechnol Gen Eng Rev 8: 189Google Scholar
  12. 12.
    Kasche V (1986) Enzyme Microb Technol 8: 4CrossRefGoogle Scholar
  13. 13.
    Brugging A, Roos EC, de Vroom E (1998) Organic Process Research and Development 2: 128CrossRefGoogle Scholar
  14. 14.
    Schumacher G, Sizmann D, Haug H, Buckel P, Bock A (1986)Nucleic Acids Res 14: 5713CrossRefGoogle Scholar
  15. 15.
    Barbero JL, Buesa JM, Gonzales de Buitrago G, Mendez E, Perez Aranda A, Garcia JL (1986)Gene 49: 69CrossRefGoogle Scholar
  16. 16.
    Ljubijankic C, Konstantinovic M, Glisin V (1992)DANN Seq 3: 195Google Scholar
  17. 17.
    Verhaert RMD, Riemens AM, van der Laan J-M, van Duin J, Quax WJ (1997)Appl Environ Microbiol 63: 3412Google Scholar
  18. 18.
    Piotraschke E, Nurk A, Galunsky B, Kasche V (1994)Biotechnol Lett 16: 119CrossRefGoogle Scholar
  19. 19.
    Martin L, Prieto MA, Cortes E, Garcia JL (1995) FEMS Microbiol Lett 125: 287CrossRefGoogle Scholar
  20. 20.
    Ohashi H, Katsuna Y, Hashizume T, Abe SN, Kajiura H, Hattori H, Kamei T, Yano M (1988) Appl Environ Microbiol 54: 2603Google Scholar
  21. 21.
    Olsen GJ, Woese CR and Overbeek R (1994)J Bacteriol 176: 1Google Scholar
  22. 22.
    Shewale JG, Sivaraman H (1989)Process Biochem 24: 146Google Scholar
  23. 23.
    Shewale JG, Deshpande BS, Sudhakaran VK, Ambedkar SS (1990)Process Biochem 25: 97Google Scholar
  24. 24.
    Duggleby H, Tolley SP, Hill CP, Dodson EJ, Dodson G, Moody PCE (1995) Nature 373: 264CrossRefGoogle Scholar
  25. 25.
    Brannigan JA, Dodson G, Duggleby H, Moody PCE, Smith JL, Tomchik DR, Murzin AG (1995)Nature 378: 416CrossRefGoogle Scholar
  26. 26.
    Konecny J (1981) Biotechnol Lett3: 112CrossRefGoogle Scholar
  27. 27.
    Haufler U (1987) PhD Thesis, University of Bremen, GermanyGoogle Scholar
  28. 28.
    Svedas VK, Margolin AL, Sherestyuk CF, Klesov AA, Berezin IV (1977)Bioorgan Khim (Russ) 3: 546Google Scholar
  29. 29.
    Schechter I, Berger A (1967) Biochem Biophys Res Commun 27: 157CrossRefGoogle Scholar
  30. 30.
    Kasche V, Galunsky B, Nurk A, Piotraschke E, Rieks A (1996) Biotechnol Lett 18: 455CrossRefGoogle Scholar
  31. 31.
    Niersbach H, Kühne A, Tischer W, Weber M, Wedekind F, Plapp R (1995)Appl Microbiol Biotechnol 43: 679CrossRefGoogle Scholar
  32. 32.
    Stambolieva N, Mincheva Z, Galunsky B, Kalcheva V (1992)Enzyme Microb Technol 14: 496CrossRefGoogle Scholar
  33. 33.
    Vojtisek V, Slezak J (1975) Folia Microbiol (Praha) 20: 224CrossRefGoogle Scholar
  34. 34.
    Svedas, V, Guranda D, van Langen L, van Rantwijk F, Sheldon R (1997) FEBS Lett 417: 414CrossRefGoogle Scholar
  35. 35.
    Kasche V, Haufler U, Markowsky D, Ziech A, Galunsky B (1987) Ann NY Acad Sci 501: 97CrossRefGoogle Scholar
  36. 36.
    Ignatova Z, Stoeva S, Galunsky B, Hörnle C, Nurk A, Piotraschke E, Voelter W, Kasche V (1998) Biotechnol Lett 20: 997CrossRefGoogle Scholar
  37. 37.
    Prieto I, Martin J, Arche R, Fernandez P, Perez-Aranda A, Barbero JL (1990)Appl Microbiol Biotechnol 33: 553CrossRefGoogle Scholar
  38. 38.
    Williams JA, Zuzel TJ (1985)J Cell Biochem 9B: 99Google Scholar
  39. 39.
    Martin J, Prieto R, Barbero JL, Perez-Gil J, Mencheno JM, Arche R (1990) Biochim Biophys Acta 1037: 133CrossRefGoogle Scholar
  40. 40.
    Forney LJ, Wong DCL, Ferber DM (1989) Appl Environ Microbiol 55: 2550Google Scholar
  41. 41.
    Waldmann H, Sebastian D (1994)Chem Rev 94: 911CrossRefGoogle Scholar
  42. 42.
    Svedas VK, Savchenko MV, Beltser AI, Guranda DF (1996)Ann NY Acad Sci 799: 659CrossRefGoogle Scholar
  43. 43.
    Waldmann H (1989)Tetrahedron Lett 30: 3057CrossRefGoogle Scholar
  44. 44.
    Dineva M, Galunsky B, Kasche V, Petkov D (1993) Bioorg Med Chem Lett 3: 2781CrossRefGoogle Scholar
  45. 45.
    Stoineva I, Galunsky B, Lozanov VS, Ivanov IP, Petkov DD (1992)Tetrahedron 48: 1115CrossRefGoogle Scholar
  46. 46.
    Vrudhula VM, Senter PD, Fischer KJ, Wallace PM (1993)J Med Chem 36: 919CrossRefGoogle Scholar
  47. 47.
    Alkema WBL, Prins AK, Janssen DB (1999) 4th International Symposium on Biocatalysis and Biotransformations, Giardini Naxos-Taormina, Italy, Abstract P112Google Scholar
  48. 48.
    Stambolieva N, Mincheva Z, Galunsky B (1998)Biocat Biotrans 16: 225CrossRefGoogle Scholar
  49. 49.
    Collins AN, Sheldrake GN, Crosby J (eds) (1992) Chyrality in Industry. Wiley, ChichesterGoogle Scholar
  50. 50.
    Rakels JLL, Paffen MT, Straathof AJJ, Heijnen JJ (1994) Enzyme Microb Technol 16: 791CrossRefGoogle Scholar
  51. 51.
    Zmiewski MJ, Briggs, BS Tompson AR, Wright IG (1991)Tetrahedron Lett 32: 1621CrossRefGoogle Scholar
  52. 52.
    Bornscheuer UT, Altenbuchner J, Meyer HH (1998)Biotechnol Bioeng 58: 554CrossRefGoogle Scholar
  53. 53.
    Galunsky B, Ignatova S, Kasche V (1997)Biochim Biophys Acta 1343: 130CrossRefGoogle Scholar
  54. 54.
    Suyama TJ (1965) Chem Abstr 63: 7095Google Scholar
  55. 55.
    Daumy GO, McColl AS, Apostolakos D (1982)J Bacteriol 152: 104Google Scholar
  56. 56.
    Kasche V, Gottschlich N, Lindberg A, Niebuhr-Redder C, Schmieding J (1994)J Chromatog 660: 137CrossRefGoogle Scholar
  57. 57.
    Kutzbach C, Rauenbusch E (1974)Hoppe-Seyler’s Z Physiol Chem 354: 45CrossRefGoogle Scholar
  58. 58.
    Kasche V (1985)Biotechnol Lett 7: 877CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Vienna 2000

Authors and Affiliations

  • Boris Galunsky
  • Karsten Lummer
  • Volker Kasche
    • 1
  1. 1.Arbeitsbereich Biotechnologie IITechnische Universität Hamburg-HarburgHamburgGermany

Personalised recommendations