Rational Design of Practically Important Enzymes
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 K<sub>M </sub>values with formate and NAD<sup>+</sup> 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. Wildtype 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<sup>+</sup> to NADP<sup>+</sup> 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. ?-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.