Amino acid oxidase and

L-phenylalanine L-amino acid oxidase and horseradish peroxidase (E) I-... 

R. Fernandez-Lafuente, V. Rodriguez, and J.M. Guisan, The coimmobilization of D-amino acid oxidase and catalase enables the quantitative transformation of D-amino acids (D-phenylalanine) into alpha-keto acids (phenylpyruvic acid). Enz. Microb. Technol. 23, 28-33 (1998). 

An alternative two-step biocatalytic route, first developed at Glaxo in the 1970s, utilized a D-amino acid oxidase and an amidase to provide 7-ACA under physiological conditions (Scheme 1.12). This process has since been established in several companies, with minor modifications. In fact, 7-ACA was manufactured by GSK at Ulverston (Cumbria, UK) using both the chemical and biocatalytic processes in parallel for a period of 2 years during which time the environmental benefits of the biocatalytic process were assessed (see Section 1.6). 

Dominguez et al. [71] L-Amino acids D-Amino acids Grapes r,-Amino acid oxidase or D-amino acid oxidase and peroxidase/entrapped in electrode composite matrix by physical inclusion Graphite-Teflon-ferrocene composite electrode/OV vs. Ag/AgCl Ferrocene... 

Varadi et al. [72] L-Amino acids D-Amino acids Beers (brewing process) L-Amino acid oxidase and d-amino acid oxidase/in a Plexi-cell thin-layer on natural protein membrane Pt wire electrode/ +100 mV vs. Ag/AgCl ... 

Y. Inaba, K. Mizukami, N. Hamada-Sato, T. Kobayashi, C. Imada and E. Watanabe, Development of a D-alanine sensor for the monitoring of a fermentation using the improved selectivity by the combination of d-amino acid oxidase and pyruvate oxidase, Biosens. Bioelectron., 19(5) (2003) 423-431. 

Figure 2 Chemical conversion of 5-(4-hydroxybutyl)hydantoin (3) to racemic 6-hydroxynorleucine. Enzymatic conversion of racemic 6-hydroxynorleucine to L-6-hydroxy-norleucine (1) by D-amino acid oxidase and glutamate dehydrogenase.

Chemical synthesis and isolation of 2-keto-6-hydroxyhexanoic acid required several steps. In a second, more convenient process (Fig. 2), the ketoacid was prepared by treatment of racemic 6-hydroxynorleucine [produced by hydrolysis of 5-(4-hydroxybutyl)hydantoin (3)] with D-amino acid oxidase and catalase. After the e.e. of the remaining L-6-hydroxynorleucine had risen to >99%, the reductive animation procedure was used to convert the mixture containing 2-keto-6-hydroxyhexanoic acid and L-6-hydroxynorleucine entirely to L-6-hy-droxynorleucine with yields of 91-97% and e.e. of >98%. Sigma porcine kidney D-amino acid oxidase and beef liver catalase or Trigonopsis variabilis whole cells (source of oxidase and catalase) were used successfully for this transformation [22],... 

N.B. Fibroblasts tend to lack D-amino acid oxidase and replacement of L-valine with D-valine in media encourages the growth of epithelial cells. 

N. Yoshida, Y. Sakai, A. Isogai, H. Fukuya, M. Yagi, Y. Tani, and N. Kato, Primary structures of fungal fructosyl amino acid oxidases and their application to the measurement of glycated proteins, Eur. J. Biochem., 1996, 242, 499-505. 

A modification of this reaction concerns the availability of the keto acid substrate. To circumvent its complicated lengthy chemical synthesis, 2-keto-6-hydro-xyhexanoic acid was synthesized by treatment of racemic 6-hydroxynorleucine with D-amino acid oxidase and catalase (Fig. 37). The production of racemic 6-hydroxynorleucine occurs by hydrolysis from 5-(4-hydroxybutyl)hydantoin. d-Amino acid oxidase converts the D-enantiomer of racemic 6-hydroxynorleucine to the corresponding ketoacid which is reductively aminated to l 6-hydroxynorleucine by GluDH. 

Deracemization by Stereoinversion via the Two-enzyme System D-Amino Acid Oxidase and L-Amino Transferase... 

A combination of D-amino acid oxidase and L-amino transferase is an example of a deracemization by stereoinversion. The product is an L-amino acid. The reaction catalyzed by amino transferase has an equilibrium constant close to unity, a very unpractical situation leading to uncomplete transformation and to the production of almost inseparable mixtures of amino acids (at least two, the amino acid product and the amino add used as an amino donor). For preparative purposes it is therefore mandatory to shift the equihbrium to the product side. A recent example of a deracemization procedure based on this coupled enzymatic system is the preparation of L-2-naphthyl-alanine 6 as illustrated in Scheme 13.9 [28]. The reaction occurs in one pot with initial oxidation of the D-amino acid catalyzed by D-amino acid oxidase from Rhodotonda gracilis. The hydrogen peroxide that is formed in stoichiometric amounts is decomposed by catalase. The a-keto add is the substrate for L-aspartate amino transferase (L-Asp amino transferase), which is able to use L-cysteine sulfinic acid 7 as an amino donor. 

Amino Acid Oxidase and Chemical In Situ Reduction of the Initially Formed Imino Compound A simple and interesting procedure for the deracemization of a-amino acids was introduced by Soda [48], who combined the oxidation of the D-enantiomer of D,L-proline to dehydro-prohne with a chemical reduction of the imine 21 in on -pot, thereby restoring the racemic mixture. If the reaction in the first step is completely enantioselective, the e.e. of the amino acid after one cycle is 50%. Repeating the reaction in successive cycles raises the e.e. close to 100% (Scheme 13.19). 

Scheme 13.19 Chemoenzymatic deracemization with D-amino acid oxidase and chemical reduction.

Scheme 13.21 Reaction catalyzed by D-amino acid oxidase and L-amino acid oxidase.

The X-ray crystallographic structure of Callosdasma rhodostoma L-amino acid oxidase indicates that it is functionally a dimer in which each subunit is constituted by three domains an FAD-binding domain, a substrate-binding domain, and a helical domain [58]. A funnel is formed at the interface between the latter domains and provides substrate access to the active site. Thus, the mode of substrate binding (Figure 13.1b) and access significantly differs between L-amino acid oxidase and D-amino acid oxidase. 

Flavin oxidases include d- and l-amino acid oxidases, and some amine oxidases, although others are quinoproteins (Section 9.8.3). In these enzymes, the flavin is reduced by dehydrogenation of the substrate, byway of an intermediate substrate-flavin adduct, as occurs in the dehydrogenases (Section 7.3.3). 

For these reasons, progress has been obtained with model, rather than physiological, substrates. In particular, recent studies of the reaction of the amino acid oxidases with )8-halogenated-a-amino acids and of D-amino acid oxidase and glucose oxidase with nitroalkanes and their carbanions have begun to clarify the chemical mechanism of these reactions. The results and interpretations of these studies are discussed briefly below. 

There are also a number of enzymes that degrade organic N compounds, inside or outside the cell, thereby making N available for assimilation or uptake, respectively. Examples we will consider include urease, amino acid oxidases, and extracellular peptidases (MulhoUand et al., 2002 MulhoUand and Lee, in revision. Chapter 7 by MulhoUand and Lomas, Fig. 2, this volume Table 32.1). In addition, for heterotro-phic organisms, digestive enzymes, especially proteases, are important for internal N cycling, recouping cellular N, and excretion. 

Luo H, Yu H, Li Q, Shen Z. Cloning and co-expression of D-amino acid oxidase and glutaryl-7-aminocephalosporanic acid acylase genes in Escherichia coli. Enz. Microb. Technol. 2004 35(6-7) 514-518. 

Amino acid oxidase and Oamino acid oxidase are oxygen-requiring enzymes that catalyze the conversion of amino acids to the corresponding keto acids and ar ammonium ion. The oxygen cosubstrate is converted to hydrogen peroxide. Flavin adenine dinucleotide (FAD) is required as a cofactor. Both enzymes are of minor importance in amino acid catabolism. Note that all of the amino acids described in this text are L-amino acids. D-Amino acids are minor components of... 

Ammonium ions are produced by the catabolism of a number of amino acids. Glutamate dehydrogenase is the major source of ammonium ions in the body. Ammonium ions are also produced from the catabolic pathways of serine, histidine, tryptophan, glycine, glutamine, and asparagine. L-Amino acid oxidase and... 

---