ИСТИНА

D-amino acid oxidase (DAAO, EC 1.4.3.3) is FAD containing enzyme and catalyses oxidative deamination of D-amino acids to corresponding α-ketoacids. The enzyme plays important role in regulation of many processes and it is widely used in practice. One of the most important aspects of DAAO usage is Cephalosporin C oxidation during the preparation of 7 aminocephalosporanic acid – the key compound for production of semi-synthetic cephalosporin antibiotics. Among all the D-amino acid oxidases known, DAAO from yeast Trigonopsis variabilis (TvDAAO) is the best enzyme for this process due to its high catalytic activity towards cephalosporin C and high thermal stability. Nevertheless its stability needs to be increased. It can be achieved using rational design approach. Gene of TvDAAO was cloned and expressed in E.coli cells earlier in our laboratory. The enzyme was expressed at high level in soluble and active form. 3D structure of mutant TvDAAO was solved and provided powerful background for rational design of the enzyme properties. Hydrophobization of α-helices is one of the common approaches for protein stabilisation. Structure analysis of TvDAAO showed that there are 13 serine residues located in α helices. One residue, Ser44, is conservative and its substitution to any other amino acid residue is unreasonable. Other selected Ser residues can be divided into two groups: 4 residues are located inside the protein globule and 8 ones - on the surface. All four serine residues located inside the protein and four on the surface were substituted to alanine residues. Mutant TvDAAOs were expressed in E.coli cells using standard procedure. The enzymes were expressed at high level in soluble and active form. Thermal stability and substrate specificity profile of mutant TvDAAOs were studied. The role of each residue in TvDAAO stability was discussed.