L-amino acid oxidase (LAAO) is a flavoenzyme containing non-covalently bound flavin adenine dinucleotide, which catalyzes the stereospecific oxidative deamination of l-amino acids to α-keto acids and also produces ammonia and hydrogen peroxide via an imino acid intermediate. LAAOs purified from snake venoms are the best-studied members of this family of enzymes, although a number of LAAOs from bacterial and fungal sources have been also reported. From a biochemical point of view, LAAOs from different sources are distinguished by molecular mass, substrate specificity, post-translational modifications and regulation. In analogy to the well-known biotechnological applications of d-amino acid oxidase, important results are expected from the availability of suitable LAAOs; however, these expectations have not been fulfilled yet because none of the "true" LAAOs has successfully been expressed as a recombinant protein in prokaryotic hosts, such as Escherichia coli. In enzyme biotechnology, recombinant production of a protein is mandatory both for the production of large amounts of the catalyst and to improve its biochemical properties by protein engineering. As an alternative, flavoenzymes active on specific l-amino acids have been identified, e.g., l-aspartate oxidase, l-lysine oxidase, l-phenylalanine oxidase, etc. According to presently available information, amino acid oxidases with "narrow" or "strict" substrate specificity represent as good candidates to obtain an enzyme more suitable for biotechnological applications by enlarging their substrate specificity by means of protein engineering.

L-Amino acid deaminase from Proteus myxofaciens (Pma-LAAD) is a membrane flavoenzyme that catalyzes the deamination of neutral and aromatic L-amino acids into ␣-keto acids and ammonia. PmaLAAD does not use dioxygen to re-oxidize reduced FADH 2 and thus does not produce hydrogen peroxide; instead, it uses a cytochrome b-like protein as an electron acceptor. Although the overall fold of this enzyme resembles that of known amine or amino acid oxidases, it shows the following specific structural features: an additional novel ␣؉␤ subdomain placed close to the putative transmembrane ␣-helix and to the active-site entrance; an FAD isoalloxazine ring exposed to solvent; and a large and accessible active site suitable to bind large hydrophobic substrates. In addition, PmaLAAD requires substrate-induced conformational changes of part of the active site, particularly in Arg-316 and Phe-318, to achieve the correct geometry for catalysis. These studies are expected to pave the way for rationally improving the versatility of this flavoenzyme, which is critical for biocatalysis of enantiomerically pure amino acids.A variety of enzymes has been used to prepare chiral pharmaceutical and agricultural compounds containing enantiomeric amine or amino acid groups. Among these are aminotransferases (EC 2.6.4.X), lipases (EC 3.1.1.X), amine oxidases (EC 1.4.3.22), amino acid dehydrogenases (EC 1.4.1.X), and amino acid oxidases (EC 1.4.3.X) (1, 2).The deracemization of a racemic amino acid to obtain the L-configuration was achieved by using a stereoselective Damino acid oxidase (DAAO, 5 EC 1.4.3.3) followed by chemical reduction. The second step iteratively converts the imino acid produced (from the D-amino acid) back into a DL-mixture to obtain the full resolution of the racemic mixture into the L-enantiomer (1). This approach requires stable recombinant DAAOs possessing wide substrate specificity as well as variants engineered to act on synthetic amino acids (3). Amino acid oxidases with reverse stereoselectivity are also well known flavooxidases, mainly produced by snakes or by microorganisms. In particular, L-amino acid oxidases (LAAO, EC 1.4.3.2) catalyze the stereoselective oxidative deamination of L-amino acids into the corresponding ␣-keto acids and ammonia; the re-oxidation of FADH 2 by dioxygen then generates H 2 O 2 (4). These flavoenzymes catalyze an irreversible reaction (differently from aminotransferases) and do not require a specific step of cofactor regeneration, as otherwise required by the NAD-dependent dehydrogenases. However, because of the problems associated with overexpression of snake venom LAAOs in recombinant hosts and the limited substrate acceptance of the microbial counterparts, no appropriate LAAOs for biocatalysis are available (4).L-Amino acid deaminases (LAADs) represent a suitable alternative to LAAOs. LAAD (first identified in the genera Proteus, Providencia, and Morganella) catalyzes the deamination of the L-isomer of amino acids, yielding the corresponding ␣-keto acids and ammonia without any evide...

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