CHINESE JOURNAL OF BIOTECHNOLOGY Volume 24, Issue 12, December 2008 Online English edition of the Chinese language journal Cite this article as: Chin J Biotech, 2008, 24(12), 2122−212 4. Received: October 17, 2008; Accepted: November 25, 2008 Supported by: Federal Agency for Science and Innovations, State contract No. 02.512.12.2002. Corresponding author: V.I. Tishkov. Tel: +7-495-9393208; Fax: +7-495-9393208; E-mail: vitishkov@gmail.com Copyright © 2008, Institute of Microbiology, Chinese Academy of Sciences and Chinese Society for Microbiology. Published by Elsevier BV. All rights reserved. RESEARCH PAPER Recombinant D-amino Acid Oxidase with Improved Properties S.V Khoronenkova 1 , and V.I.Tishkov 1, 2 1 Department of Chemical Enzymology, Chemistry Faculty, M.V. Lomonosov Moscow State University, Lenin`s Hills, Moscow 119992, Russian Federation 2 A.N.Bach Institute of Biochemistry, Russian Academy of Scineces, Leninskii pr. 33-2, Moscow 119071, Russian Federation Abstract: D-amino acid oxidase from Trigonopsis variabilis (TvDAAO) was overproduced in Escherichia coli cells, and properties of the recombinant enzyme were studied. Single point mutants of the enzyme with 2.4-fold higher thermal stability or changed spectra of substrate specificity compared with wild-type enzyme were prepared. It was shown that mutant TvDAAO has higher catalytic efficiency in cephalosporin C oxidation in comparison with wild-type enzyme. One mutant of recombinant TvDAAO was crystallized and its structure was solved with resolution 2.8 Å. Keywords: D-amino acid oxidase; Trigonopsis variabilis; directed mutagenesis; X-ray structure Introduction D-amino acid oxidase (EC 1.4.3.3, DAAO) is a FAD-dependent enzyme that catalyses oxidative deamination of D-amino acids yielding the corresponding α-keto acids. The enzyme is of both fundamental and practical value [1,2] . In microorganisms, DAAO allows exogenous D-amino acids to be used as growth substrates providing carbon and nitrogen. In mammals, including humans, the enzyme plays an important role in maintaining the necessary levels of D-amino acids, for example, D-serine in brain tissues. At the moment, it is established that hyperactivity of D-amino oxidase in brain is one of the causes of shizophrenia [3] . Practical interest in DAAO is mainly due to its use in production of 7-aminocephalosporanic acid from the natural antibiotic cephalosporin C [4] . The enzyme can be also used in biosensors [5] for preparation of unnatural L-amino acids [6] and α-keto acids [7] . At the moment, DAAO has been found in different types of organisms [1] but microbial enzymes are the most suitable choice for practical application [8] . D-amino acid oxidase from yeast Trigonopsis variabilis (TvDAAO) shows the best thermal stability [9] and the highest activity with cephalosporin С [10] among DAAOs from other sources. However, at the moment, there are some factors that limit practical application of DAAO. The first one is the high cost for the production of enzyme. The next point is low thermal and operational stability and narrow spectrum of substrate specificity of the enzyme. To solve these problems, it is necessary to develop new recombinant strains for daao overproduction and optimize the cultivation process to obtain mutant enzymes with improved stability and desired substrate specificity. 1 Results and discussions In this laboratory, the gene of DAAO from T. variabilis was cloned [11] and expressed in Escherichia coli (E. coli) cells. After optimization of expression system and cultivation conditions, yield of active and soluble recombinant enzyme