Amino acid oxidase selectivity

D-Amino acid oxidase (EC 1.4.3.3) extracted from sheep kidney possesses low selectivity and at pH 8-9 will oxidise many D amino acids, whereas L-amino acid oxidase (EC 1.4.3.2) from snake venom (Crotalus adaman-teus) at pH 8-9 catalyses the oxidation of many L amino acids. However, as these enzymes show different reactivity towards different amino acids, the results for a sample that contains several D and L amino acids may be difficult to interpret. The use of these enzymes is therefore only recommended for the measurement of one isomer of an isolated amino acid. They may also be used to remove an unwanted isomer from a sample containing both to allow subsequent measurement of the other. 

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. 

Kinetic measurements of H2O2 formation result in fast electrode responses (less than 12 s) using chemically bound L-amino acid oxidase (LAAO) covering a platinum electrode (170) to assay for cysteine, leucine, tyrosine, phenylalanine, tryptophane, and methionine. Potentiometric-selective electrodes for amino acids are Constructed by immobilizing LAAO on chemically modified graphite... 

The preparation of D-amino acids with the above three-enzyme system requires enzymes with opposite stereochemical selectivity and a suitable amino add as a donor. While D-amino add oxidase is an enzyme, the function of which in Nature is mainly related to the ehmination of D-amino adds, L-amino acid oxidases are usually found in aggressive animals (snakes). Bacterial L-amino acid oxidases often show a specific activity that is too low for preparative purposes [33]. Moreover, D-amino transferases are less common than the L-specific ones and require more expensive D-amino adds as amino donors. 

Another example of a coupled enzyme reaction demonstrates the versatility of the transaminase system in biocatalysis. Using a racemic d,L-amino acid mixture as the starting material, the enzyme D-amino acid oxidase from Trigonopsis mriabilis will convert the D-amino acid in the mixture selectively into the corresponding 2-keto acid. The L-amino acid of the d,l- pair is neither a substrate nor an inhibitor of d-amino acid oxidase. If a transaminase is present in the same reaction mixture, the 2-keto acid can be transaminated in the presence of L-aspartate to the corresponding L-amino acid. The entire reaction can be driven to completion as described previously by decarboxylation of the oxaloacetate. Thus, in a single pot, racemic d,l-amino acids can be convened directly into optically active L-amino acids (Fig. 12.7-11). 

It has been proved that the selectivity of an analytical method is directly connected to the complexity of the matrix from which the analyte must be determined. As a result, the same method can be more selective or less selective, depending on the qualitative and quantitative composition of the matrix from which the analyte must be determined. For example, two types of electrochemical sensors are described for the assay of thyroid hormones L-T3 and L-T4. The first is an amperometric biosensor based on L-amino acid oxidase (l-AAOD),270 whereas the other is an amperometric immunosensor based on anti-L-T3 and anti-L-T4.271 If T3 and T4 have to be determined from phramaceutical products, both types of sensors have the necessary sensitivity and selectivity. When it is required to determine both hormones in biological fluids or in thyroid tissue, the proposed biosensors are not selective enough because l-AAOD catalyzes the reactions of both thyroid hormones. The amperometric biosensors can only make the discrimination between the two thyroid hormones, namely, L-T3 and L-T4, since the specific antibody reacts only with the specific antigen. 

From these and many similar examples it became evident that discrimination between enantiomers is often a matter of degree. Absolute discrimination, however, is shown by specific oxidases like D-amino acid oxidase of mammalian kidney and L-amino acid oxidase of snake venom. "No one [member] of this class of biological catalysts has yet been known to attack measurably an amino acid antipodal to its normally susceptible category of substracts ) [Greenstein and Winitz (1961)] [Zellor and Maritz (1945)]. Equally selective is the phosphorylation of mevalonic acid by the enzyme mevalonic kinase the R- form is phosphorylated, the S- form is unaffected (Tchen 1958). 

The research literature features many examples of this basic strategy. Different enzymes allow selective determinations of single species, such as glucose (with glucose oxidase), or groups of substances such as the L-amino acids (with L-amino acid oxidase). Recent reviews should be consulted for a more complete view of the field (66-68). Amperometric enzyme electrodes are discussed in Sections 14.2.5 and 14.4.2(c). 

In 1935, Krebs published a technique for Isomer analysis that employs D-amino acid oxidase, an enzyme which selectively deaminates D-isomers. This method allowed measurement of D-amino acids via the resultant keto acids that were formed or by recovery of intact L-amino acids (28). Other enzymatic methods based on L-amino acid decarboxylases (25,43), L-amino acid acylases and amidases (44) also have been used. Other biologically-based techniques employing selective utilization of L-amino acids by microorganisms appeared as early as 1949 (29,30), but due to their complexity, have not been used widely. 

Femmidez-Lafuente R, Cowan D, Wood A (1995) Hyperstabilization of a thermophilic esterase by multipoint covalent attachment. Enzyme Microb Technol 17 366-372 Femmidez-Lafuente R, Armisen P, Sabuquillo P et al. (1998) Immobilization of lipases by selective adsorption on hydrophobic supports. Chem Phys Lipids 93 185-197 Femmidez-Lafuente R, Rodriguez V, Mateo C et aL (1999) Stabilization of enzymes (D-amino acid oxidase) against hydrogen peroxide via immobilization and post-immobihzation techniques. J Mol Catal B Enzym 7 173-179... 

Specifically, a mutant design process for D-amino acid oxidase was described based on downloaded stereostructures of this chirally selective enzyme. The evaluation method for the process was also presented. ... 

Others, such as penicillinase, will promote the reaction of many substrates. Thus, ampicillin, naficillin, penicillin G, penicillin V, cyclibillin, and dicloxacillin can be determined with a penicillinase electrode. D- and L-amino acid oxidases are even less selective. The former, when coupled to an electrode, responds to D-phenylalanine, D-alanine, o-valine, D-methionine, D-leucine, o-norleucine, and o-isoleucine, while the latter responds to L-leucine, L-tyrosine L-phenylala-nine, L-tryptophan, and methionine. 

Scheme 4.37 Deracemization of racemic a-amino acids by combining an enantioselective amino acid oxidase with a non-selective chemical reducing agent.

A common method for the deracemization of a-amino acids has been to employ amino acid oxidases along with a non-selective reduction of the intermediate imine by hydride-reducing agents (e.g., sodium boro hydride or sodium cyanoborohydride) or amine boranes [99, 100]. 

A further exciting development in the field of chemoenzymatic one-pot synthesis is the integration of artificial metalloproteins (which can then be regarded as the chemocatalytic component ) in such processes. Such a concept was successfully realized by HoUmann, Turner, and Ward et al. in the combination of an artificial transfer hydrogenase with various redox biocatalysts, comprising NADH-, FAD, and heme-dependent enzymes [47]. A selected example is shown in Scheme 19.18. Therein, readily available L-lysine is oxidized by an L-amino acid oxidase toward Al-piperidine<arboxylic acid (52), which is then reduced by the iridium complex-containing metalloprotein to racemic pipecohc acid (rac-53). 

Guilbault and Hrabankova (1970, 1971) have described electrodes that are specific for L-amino acids and D-amino acids. Enzymes were immobilized by gel techniques (using acrylamide gel) on the tips of commercial cation-selective electrodes. L-Amino acid oxidase (l-AAO) was used for L-amino-acid-selective electrodes with the reaction... 

D-Amino acid oxidase (d-AAO) has been immobilized in an acrylamide gel to produce D-amino-acid-selective electrodes. The reaction is... 

Scheme 4.9 Deracemisations of a-amino acids using (a) a o-selective amino acid dehydrogenase and an t-selective a-transaminase, or (b) a o-selective amino acid oxidase and an L-selective a-transaminase.

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