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V. I. Tishkov, A. A. Pometun, A. V. Stepashkina, V. V. Fedorchuk, S. A. Zarubina, I. S. Kargov, D. L. Atroshenko, P. D. Parshin, M. D. Shelomov, R. P. Kovalevski, K. M. Boiko, M. A. Eldarov, E. D’Oronzo, S. Facheris, F. Secundo, S. S. Savin
Rational design
of practically important enzymes
Abstract
Native enzymes
usually do not have properties which are fully suitable for application in
biotechnological processes. That is why that practically all enzymes are
subjected to directed modification of properties before practical application.
In presnt report we will present examples of rational design of some
practically important enzymes. Formate dehydrogenase (FDH, EC 1.2.1.2). We
cloned genes of new FDHs from bacterium Staphylococcus aureus,
thermotolerant yeast Ogataea parapolymorpha, backery yeast Saccharomyces
cerevisiae and moss Physcomitrella patens (SauFDH, OpaFDH, SceFDH
and PpaFDH, respectively). It was found that SauFDH has the highest value of
specific activity compared to other FDHs. OpaFDH shows the same low KM
values with formate and NAD+ as plant FDHs but it has much higher
thermal stability. PpaFDH is the first case when active recombinant enzyme was
obtained with full length signal peptide. Chemical stability of FDH from Pseudomonas
sp. 101 was improved at least by four orders. Wild-type SauFDH and mutant
PseFDH show the best chemical stability compared to all described FDHs.
Wild-type and mutant FDHs from bacteria, yeasts and plants were tested for
activity and stability in ionic liquids. There were different effects of amino
acid changes on surface of protein globule on stability and activity FDHs in
water and ionic liquids. New highly efficient mutant PseFDHs with changed
coenzyme specificity from NAD+ to NADP+ were obtained.
D-amino acid oxidase (DAAO, EC 1.4.3.3). Different multi-points mutants
(mpmDAAO) of yeast DAAO have been prepared. mpmDAAO showed better chemical
stability and higher activity with cephalosporin C compared to wild-type
enzyme. α-Amino acid esters hydrolase (AEH, EC
3.1.1.43). Gene of AEH from Xanthomonas rubrilineans (XrAEH) has been
cloned and overexpressed in E. coli. It was shown that recombinant XrAEH
is more efficient compared to native XrAEH. Structure of single-chain
penicillin acylase (EC 3.5.1.11) from Alcaligenes faecalis (scAfPA) has
been modeled and gene of the enzyme was prepared with PCR. scAfPA was expressed
in E. coli cells and recombinant enzyme was purified and characterized.
Key words: D-amino acid oxidase, Cephalosporin C,
site-directed mutagenesis, rational design, kinetic properties, thermal
stability.
Copyright (C) Chemistry Dept., Moscow State University, 2002
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