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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

Abstract

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.

Keywords

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.

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Copyright information

© Springer-Verlag Vienna 2000

Authors and Affiliations

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

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