Comparative Study of Substrate- and Stereospecificity of Penicillin G Amidases from Different Sources and Hybrid Isoenzymes |
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Authors: | Boris Galunsky Karsten Lummer Volker Kasche |
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Institution: | Arbeitsbereich Biotechnologie II, Technische Universit?t Hamburg-Harburg, D-21071 Hamburg, Germany, DE
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Abstract: | Summary. 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 -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 S
1-binding site and S-specific in the -binding site. The S
1-stereoselectivity differs less than one order of magnitude for the different variants. The -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.
Received December 20, 1999. Accepted (revised) February 4, 2000 |
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Keywords: | , ,Homologous enzymes, Penicillin amidase (acylase), Stereospecificity, Substrate specificity, |
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