ИСТИНА

D-amino acids oxidase (DAAO) catalyzes oxidation of D-amino acids with strict stereo specificity. It is used in analytical biotechnology and in fine organic synthesis of optical pure unnatural L-amino acids and α keto acids. The most prominent biotechnological process involving DAAO is the two-step enzymatic conversion of cephalosporin C (CephC ) to 7 aminocephalosporanic acid (7 ACA). Currently 7 ACA is the key precursor for synthesis of cephalosporanic antibiotics of different generations. Over 50 percent of annual production of cephalosporins is based on 7 ACA. DAAO from the yeast Trigonopsis variabilis (TvDAAO) is the excellent candidate for creation highly efficient biocatalysts for CephC oxidation, because it possesses the highest catalytic activity with CephC among known DAAOs. Nevertheless, to reduce the cost of whole biotechnological process properties of TvDAAO need optimizing in terms of catalytic activity with CephC as well as thermal stability. Moreover, the enzyme with narrow substrate specificity spectrum is needed for medical diagnostics of neurodegenerative disorders related to D amino acids derangements in human brain. The latest results of improving catalytic activity with cephalosporin C and thermal stability of TvDAAO using rational protein design will be presented. Analysis of structure and cephalosporin C docking to the active site of TvDAAO were performed. Residues Phe54 and Phe258 were identified as interacting with substrate molecule. Site-directed mutagenesis of the residues concerned to small one led to significant changing of substrate specificity spectrum and decreasing of thermal stability. Mutants of Phe54 showed the highest catalytic activity with cephalosporin C and were combined with previously studied substitution Cys108Phe showing higher thermal stability and activity with CephC in comparison to wild type enzyme. Additive effect of combined mutations was observed. As a result, stable mutant TvDAAOs with higher catalytic activity with cephalosporin C and narrow substrate specificity spectrum were obtained. New mutant TvDAAOs are highly promising for production of 7-ACA, chiral synthesis and medical diagnostic of neurodegenerative disorders and other biotechnological applications.