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A. S. Jasnaya, O. V. Jamskova, D. T. Guranda, T. A. Shcherbakova, V. I. Tishkov, V. K. Shvedas
Cloning of penicillin acylase from Escherichia coli.
Catalytic properties of recombinant enzymes
Abstract
Gene of penicillin acylase (PA) from Escherichia coli
has been cloned from a producing strain analogous to strain ATCC 11105.
Optimization of cultivation conditions allowed to obtain up to 130 mg of active
enzyme from per litre of culture broth. A number of single, double and triple
mutants has been produced by site specific mutagenesis. Homogeneous
preparations of wild type enzyme and its mutants have been received due to
isolation and purification. It was shown that 1) isolated enzymes have
correctly folded structure; 2) complexing agents and metal ions do not inhibit
catalytic activity; 3) penicillin acylase mutants are inactivated by
phenylmethylsulphonyl fluoride as effective as a wild type enzyme what allows
to use this reagent to titrate active sites of the mutants; 4) some of the
tested enzyme mutants are characterized by higher specificity constants in
hydrolysis of colorimetric substrate, however they are not as effective as a
wild type penicillin acylase in biocatalytic ampicillin synthesis by acyl
transfer.
Copyright (C) Chemistry Dept., Moscow State University, 2002
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