Assays of D-Amino Acid Oxidase Activity

D-amino acid oxidase (DAAO) is a well-known flavoenzyme that catalyzes the oxidative FAD-dependent deamination of D-amino acids. As a result of the absolute stereoselectivity and broad substrate specificity, microbial DAAOs have been employed as industrial biocatalysts in the production of semi-synthetic cephalosporins and enantiomerically pure amino acids. Moreover, in mammals, DAAO is present in specific brain areas and degrades D-serine, an endogenous coagonist of the N-methyl-D-aspartate receptors (NMDARs). Dysregulation of D-serine metabolism due to an altered DAAO functionality is related to pathological NMDARs dysfunctions such as in amyotrophic lateral sclerosis and schizophrenia. In this protocol paper, we describe a variety of direct assays based on the determination of molecular oxygen consumption, reduction of alternative electron acceptors, or α-keto acid production, of coupled assays to detect the hydrogen peroxide or the ammonium production, and an indirect assay of the α-keto acid production based on a chemical derivatization. These analytical assays allow the determination of DAAO activity both on recombinant enzyme preparations, in cells, and in tissue samples.

INTRODUCTION D-Amino acid oxidase (DAAO,EC 1.4.3.3) is a flavoenzyme containing a non-covalently bound FAD molecule per 40 kDa monomer that belongs to the dehydrogenase/oxidase class of flavoproteins (Pollegioni et al., 2007a). DAAO catalyzes the oxidative deamination of the D-isomer of α-amino acids to the corresponding α-keto acids, according to the scheme reported in Figure 1. It shows a strict selectivity toward D-isomers (i.e., it does not oxidize L-amino acids) and possesses a broad substrate specificity: it oxidizes aliphatic, aromatic and polar α-D-amino acids, whereas D-aspartate and D-glutamate are not substrates for DAAO .
3) The spontaneous hydrolysis of the imino acid to give the corresponding α-keto acid and ammonium [reaction (3) in Figure 1].
DAAO is present in all eukaryotic taxa with the exception of plants (Pollegioni et al., 2007a). All known DAAOs share the same catalytic mechanism, but show important differences in biochemical and structural properties, such as catalytic efficiency, substrate specificity, oligomeric state, stability, kinetic mechanism, and FAD binding. The diverse properties of DAAOs from different sources reflect their physiological role (for an exhaustive review, see Pollegioni et al., 2007a). DAAO is an efficient catabolic enzyme in yeasts where it oxidizes α-D-amino acids, making it possible to use them as carbon, nitrogen, or energy sources. The main role of DAAO in mammals (including humans) is related to its presence in selected brain areas where it is devoted to the catabolism of D-serine. This α-D-amino acid is a neuromodulator acting as a coagonist of the N-methyl-D-aspartate receptors (NMDARs). Dysfunction of this receptor has been correlated to different neurological or psychiatric diseases: an overstimulation of these receptors is involved in stroke, epilepsy, in neurodegenerative pathologies such as Parkinson's and Alzheimer's diseases and amyotrophic lateral sclerosis. An hypostimulation of NMDARs, in contrast, is involved in psychiatric diseases such as schizophrenia, attentiondeficit hyperactivity disorder, and chronic depression (Ross et al., 2006;Wu et al., 2007;Mitchell et al., 2010;Pollegioni and Sacchi, 2010;Nagasawa et al., 2012;Sacchi et al., 2012;Zhou and Sheng, 2013). Thus, by modulating D-serine levels, DAAO plays a key role in regulating NMDARs activation state in mammals. Owing to of its role in human brain, molecules that modulate or inhibit DAAO can act as innovative drugs for the treatment of the many diseases linked to NMDARs dysfunction (Sacchi et al., 2013). Microbial DAAOs possess properties that render them suitable for industrial biotechnological applications: for example, they are stable enzymes and show broad substrate specificity, high turnover number (Table 1), and a tight binding with the FAD cofactor. The production of DAAOs in large amounts as recombinant proteins together with the availability of the 3D-structure (e.g., Mattevi et al., 1996;Mizutani et al., 1996;Umhau et al., 2000;Kawazoe et al., 2006), allowed the design and production by protein engineering techniques of enzyme variants with new and evolved properties (Pollegioni et al., 2007b;Rosini et al., 2008Rosini et al., , 2009Rosini et al., , 2010Wang et al., 2008;Wong et al., 2010;Pollegioni and Molla, 2011;Golubev et al., 2014).
The main biotechnological applications of DAAO have been reviewed in detail in Pollegioni et al. (2007bPollegioni et al. ( , 2008 and Pollegioni and Molla (2011) and comprise: (i) production of 7amino cephalosporanic acid from cephalosporin C, the starting molecule for the production of semi-synthetic cephalosporins; (ii) resolution of racemic mixtures of amino acids to produce enantiomerically pure natural or synthetic amino acids as fine chemicals; (iii) detection and quantification of D-amino acids in biological samples and foodstuff-D-amino acids are components of the bacterial cell wall and thus their presence in food could indicate bacterial contamination and can be used as an indicator of foodstuff aging; and (iv) use as selective marker in plants.
Here, we describe in detail the analytical assays listed above; full lists of materials and equipment required and step-by-step procedures are given, together with examples of application and anticipated results.

MATERIALS AND EQUIPMENT
Unless otherwise stated, all chemicals and reagents were of analytical grade and were purchased from Sigma-Aldrich (Milan, Italy). Solutions were prepared at room temperature using ultrapure water (e.g., Milli-Q grade water) and assays were carried out at 25 • C.
One DAAO unit is defined as the amount of enzyme that converts 1 µmol of D-amino acid per minute at 25 • C (Molla et al., 1998). O 2 concentration at 25 • C and at air saturation is 0.253 mM. Unless otherwise stated, in order to obtain reliable specific activity values on different D-amino acids, a substrate concentration 10-fold higher than the K m value is used.

Enzymes
Recombinant RgDAAO and hDAAO were expressed and purified from Escherichia coli cells as previously described (Fantinato et al., 2001;Molla et al., 2006). The concentration of purified enzyme preparations was determined by using the extinction coefficient at ∼450 nm (12.6 and 12.2 mM −1 cm −1 for RgDAAO and hDAAO, respectively).

Determination of Molecular Oxygen Consumption: Polarographic Assay
DAAO activity can be assayed polarographically using an oxygen electrode Molla et al., 2006). 1. Oxygraph system (Hansatech Instr. Ltd, Pentney, Norfolk, UK): a highly sensitive S1 Clark-type polarographic oxygen electrode disc is mounted in a DW1/AD electrode chamber and connected to the Oxygraph electrode control unit. The electrode disc comprises a central platinum cathode and a concentric circular silver anode. By using the electrode chamber, dissolved oxygen can be measured in liquid-phase samples in the 0.2-2.5 mL volume range. Precise temperature control of the sample and electrode disc (the oxygen solubility decreases as temperature increases) is achieved by connecting the water jacket of the electrode chamber to a thermoregulated circulating water bath. The sample is continuously stirred in order to ensure that the dissolved oxygen is kept evenly distributed throughout the reaction vessel.

Determination of the Redox Dye Reduction
Different redox dyes are known for their ability to directly react with the reduced form of the cofactor of flavindependent oxidases and dehydrogenases and thus can be used as artificial electron acceptors for the assay of these enzymes. 2,6-Dichlorophenol-indophenol (DCPIP), methylene green (MG), and thionine (Thi) show absorption changes in the visible range between their oxidized and reduced forms and were thus selected to directly detect different enzymatic activities, including DAAO (Brugger et al., 2014).

(B) Horseradish Peroxidase and 4-Aminoantipyrine Coupled Assay
1. UV/Vis spectrophotometer (V-560, Jasco) connected to a water bath for temperature control or a microtiter plate reader (Infinite 200, Tecan). 2. 1.5 mL plastic disposable microcuvettes or 96-well microplates. 3. Automated liquid handling system (epMotion R 5075, Eppendorf). 4. 100 mM disodium pyrophosphate buffer, pH 8.5. 5. 15 mM 4-AAP solution in water. The solution must be freshly prepared every day and kept on ice. 6. 200 mM phenol. 7. 250 U/mL HRP in 100 mM disodium pyrophosphate buffer, pH 8.5. A concentrated stock solution (e.g., 2,500 U/mL) can be stored at 4 • C for several months; the diluted working solution must be freshly prepared every day and kept on ice.  14. Hydrogen peroxide stock solution 30% (w/w) in water (9.8 M): the diluted solutions (10-100 µM, to be used for the calibration curves) must be freshly prepared every day. Determination of α-Keto Acid Production α-Keto acids concentration can be determined by measuring their absorbance in the ultraviolet range (Tedeschi et al., 2012) or following their chemical derivatization with 2,4dinitrophenylhydrazine (DNP) to produce the corresponding 2,4-dinitrophenylhydrazone derivatives, which show an absorption maximum at 445 nm (Figure 4; Nagata et al., 1988

Determination of Ammonium Production: Glutamate Dehydrogenase and α-Ketoglutarate Coupled Assay
The ammonium produced by DAAO reaction on a D-amino acid can be detected by a second enzyme-coupled reaction in which α-ketoglutarate is converted to L-glutamate by L-glutamate dehydrogenase from bovine liver (GDH, EC 1.4.1.3; specific activity ≥35 U/mg protein. One GDH unit is defined as the amount of enzyme that reduces 1 µmol of α-ketoglutarate to Lglutamate per minute at pH 7.3 and 25 • C, in the presence of ammonium ions) and NADH is oxidized to NAD + (Figure 5; Holme and Goldberg, 1975). The decrease of absorbance at 340 nm, due to the conversion of NADH to NAD + , is followed.

STEPWISE PROCEDURES
Determination of Molecular Oxygen Consumption: Polarographic Assay 1. Preparation of the electrode disc: before use, a small drop of electrolyte is placed on top of the dome of the central platinum cathode of the electrode disc and a 1.5 cm 2 cigarette Rizla+ Blue rolling paper spacer is placed over the electrolyte. The paper spacer is covered with a similar sized piece of thin, oxygen-permeable polytetrafluoroethylene membrane. Once prepared, the disc is mounted in the base of the electrode chamber and 1 mL of deionized water is added in the chamber. 2. Calibration: the disc is connected to the electrode control unit, which applies a polarizing voltage (0.7 V) between the platinum and silver electrodes and generates a small current proportional to oxygen concentration in the sample. The amperometric value measured with 1 mL of stirred airsaturated water is considered as 100% of oxygen content, and the amperometric value measured after adding a few crystals of sodium dithionite corresponds to 0% of oxygen content. 3. Measurement of DAAO activity: 1 mL of the substrate solution is placed in the electrode chamber, thermostated at 25 • C, and the registration of amperometric signal is started. Once the recorded signal is stable, 10 µL of DAAO solution (∼0.05 U) is added. The DAAO activity is calculated from the initial (1-2 min) oxygen consumption rate (µmol/min) using the following equation: where mL TOT is the final volume of the reaction mixture (1.01 mL). To accurately determine the DAAO activity, the initial oxygen consumption rate should be in the 0.02-0.05 µmol/min range. The detection range is reported in Table 3.

Application: Determination of the Steady-State Kinetic Parameters and Substrate Specificity
The apparent kinetic parameters are investigated by this assay, employing different concentrations of each D-amino acid as substrate Molla et al., 2006). The initial reaction rates (µmol/min) at different substrate concentrations (in the 0.1-10-fold K m range) are fitted using the Michaelis- where V max and K m are the apparent (i.e., at fixed O 2 concentration, 0.253 mM) maximal velocity and Michaelis-Menten constant for the substrate tested.

Determination of the Redox Dye Reduction
This assay is based on the reduction of one of the redox dyes DCPIP, MG, and Thi by DAAO. These dyes show absorption changes between their oxidized (colored) and reduced (colorless) forms in the visible range, with absorption maxima at 520, 655, and 600 nm, respectively (Brugger et al., 2014).
1. The reaction mixture is prepared in a 96-well microplate, mixing the electron acceptor solution (450 µM DCPIP, 75 µM MG, or 100 µM Thi, all final concentrations) and the D-amino acid solution in 50 mM phosphate buffer, pH 8.0, in a total volume of 100 µL. 2. 10-20 µL of DAAO are added (∼0.2 U). 3. The electron acceptor reduction is followed at 520 nm for DCPIP, 655 nm for MG, and 600 nm for Thi (Abs x nm ). 4. The DAAO activity is calculated from the initial absorbance variation at the corresponding wavelength per minute ( Abs x nm /min) using the following equation: where the molar extinction coefficients (ε x nm ) of DCPIP, MG, and Thi are 6.6, 46.6, and 55.4 mM −1 cm −1 , at 520, 655, and 600 nm, respectively. mL TOT is the final volume of the reaction mixture (0.1 mL). For an accurate determination of the DAAO activity, the Abs x nm /min should be in the 0.1-0.3 range. The detection range is reported in Table 3.

A) Horseradish Peroxidase and o-Dianisidine Coupled Assay
The hydrogen peroxide produced by the DAAO reaction is reduced by HRP to water and o-DNS is simultaneously oxidized to give a colored compound showing an absorption maximum at 440 nm with an extinction coefficient of 13 mM −1 cm −1 (see Figures 1, 2A).
1. The reaction mixture is prepared in a plastic disposable microcuvette or in a 96-well microplate, mixing the Damino acid solution, 1 mM o-DNS, and 1 U/mL of HRP, in 75 mM disodium pyrophosphate buffer, pH 8.5 (all final concentrations), in a total volume of 1 mL or 300 µL, for microcuvettes or 96-well microplates, respectively. 2. 10-20 µL of DAAO are added (∼0.01 U). 3. The absorbance at 440 nm (Abs 440 nm ) is monitored. 4. The DAAO activity is calculated from the initial absorbance variation at 440 nm per minute ( Abs 440 nm /min) using the following equation: where the molar extinction coefficient at 440 nm of o-DNS is 13 mM −1 cm −1 and mL TOT is the final volume of the reaction mixture (1 or 0.3 mL). To accurately determine the DAAO activity, the Abs 440 nm /min should be in the 0.1-0.3 range. The detection range is reported in Table 3.

Application: Screening for RgDAAO Variants with Broader Substrate Specificity
The reaction catalyzed by RgDAAO has been exploited in the analytical determination of the D-amino acid content in biological samples . Since the enzyme is not active on acidic D-amino acids, it cannot be used to detect the total amount of D-amino acids. In an attempt to obtain DAAO variants to detect and quantify the total amount of D-amino acids, a directed evolution approach was performed; the wild-type DAAO cDNA was randomly mutated by errorprone PCR and a colorimetric screening procedure was set up to determine any alteration in the substrate specificity of RgDAAO . This screening procedure is performed in an automated liquid handling system (see Figure 6): 1. To the 1 mL saturated E. coli culture 1 mM isopropyl β-Dthiogalactopyranoside (IPTG, final concentration) is added and then incubated at 30 • C for 2 h. 2. 50 µL of the cells culture are transferred to different wells of a 96-well microplate (one microplate for each substrate tested). 3. 150 µL of lysis buffer are added in each well and the plates incubated at 37 • C for 30 min. 4. DAAO activity is assayed on the crude extracts by adding 100 µL of developing solution (100 mM D-amino acid, 1 mM o-DNS and 1 U/mL HRP, in 100 mM disodium pyrophosphate buffer, pH 8.5, all final concentrations). 5. The time course of the absorbance change at 440 nm is followed at room temperature by using a microtiter plate reader and comparing it with that of cells expressing wild-type RgDAAO as control.

(B) Horseradish Peroxidase and 4-Aminoantipyrine Coupled Assay
This assay determines the amount of hydrogen peroxide produced by the reaction catalyzed by DAAO coupled to the oxidation of 4-AAP by HRP in the presence of phenol. The resulting quinoneimine product shows an absorption maximum at 505 nm with an extinction coefficient of 6.58 mM −1 cm −1 (see Figure 2B).
1. The reaction mixture is prepared in a plastic disposable microcuvette or in a 96-well microplate, mixing the D-amino acid solution, 1.5 mM 4-AAP, 2 mM phenol, and 2.5 U/mL HRP, in 75 mM disodium pyrophosphate buffer, pH 8.5 (all final concentrations), in a total volume of 1 mL or 300 µL, for microcuvettes or 96-well microplates, respectively. 2. 10-20 µL of DAAO are added (∼0.05 U). 3. The absorbance at 505 nm (Abs 505 nm ) is monitored. 4. The DAAO activity is calculated from the initial absorbance variation at 505 nm per minute ( Abs 505 nm /min) using the following equation: U mL DAAO = 2 × Abs 505 nm /min ε 505 nm (quinoneimine) × mL DAAO × mL TOT where the molar extinction coefficient at 505 nm of quinoeimine is 6.58 mM −1 cm −1 and mL TOT is the final volume of the reaction mixture (1 or 0.3 mL). To accurately determine the DAAO activity, the Abs 505 nm / min should be in the 0.1-0.3 range. The detection range is reported in Table 3. Owing to the stoichiometry of the reaction, in which two molecules of hydrogen peroxide react with 4-AAP to produce one molecule of quinoneimine (see Figure 2B), a factor 2 is added at the numerator.

C) Horseradish Peroxidase and Amplex R UltraRed Coupled Assay
This assay is based on the reaction catalyzed by HRP on the fluorogenic substrate Amplex R UltraRed with hydrogen peroxide in a 1:1 stoichiometric ratio to give the brightly fluorescent product Amplex R UltroxRed (see Figure 2C).
General procedure: 1. The reaction mixture, as described for each application below, is prepared and set up in a 96-well microplate. 2. The microplate is incubated for 30-60 min in the dark at 25 • C under agitation. 3. The Stop Reagent solution is added to each well and the microplate is incubated for 5 min in the dark at 25 • C under agitation. This step allows the fluorescence signal-generating reaction to be terminated at a user-determined time point, ensuring that the timing of the standard and unknown samples measurements is the same. After reagent addition, the fluorescence signal is stable for at least 3 h. 4. The fluorescence intensity is recorded using a fluorimeter microplate reader: λ exc = 535 nm (slit = 25 nm); λ em = 595 nm (slit = 20 nm). 5. The enzymatic activity is calculated on the basis of a calibration curve obtained using known amounts of purified recombinant DAAO (0.02-0.4 mU range). The detection range is reported in Table 3. Due to the response variability of the fluorescence signal, the calibration curve must be carried out on each 96-well microplate, and triplicates for each standard and sample are recommended.

(b) Detection of DAAO Activity in U87 Cells
The enzymatic assay is performed on U87 glioblastoma control (not transfected) cells and on the same cells transfected with the plasmid expressing hDAAO .

(c) Identification of hDAAO Inhibitors
The effect of small molecules on the enzymatic activity of hDAAO is evaluated by measuring the activity of purified recombinant hDAAO at different concentrations of inhibitors, in the presence of D-serine as substrate (Hopkins et al., 2013;Terry-Lorenzo et al., 2014, 2015. where Bottom is the fluorescence value at the bottom plateau and Top is the fluorescence value at the top plateau (Terry-Lorenzo et al., 2015).

(D) Catalase and Purpald R Coupled Assay
The hydrogen peroxide produced by the DAAO reaction is reduced to water by catalase in the presence of methanol as hydrogen donor, giving formaldehyde that, in turn, reacts with the chromogenic compound Purpald R to give the colored product 6-mercapto-s-triazolo-[4,3-b]-s-tetrazine showing an absorption maximum at 550 nm with an extinction coefficient of 7.74 mM −1 cm −1 (Dickinson and Jacobsen, 1970;Watanabe et al., 1978;Sasabe et al., 2014; see Figure 3).
1. The reaction mixture is prepared in a plastic tube mixing 300 µL of the D-amino acid solution, 200 µL of 0.1 mM FAD, 300 µL of 700 U/mL catalase, in 133 mM sodium pyrophosphate buffer, pH 8.3, and 100 µL of 70% methanol (final volume: 900 µL). 2. 100 µL of DAAO solution are added (∼0.02 U). 3. The reaction mixture is incubated at 37 • C for 15 min under agitation. 4. The reaction is stopped by adding 1 mL of 10% (w/v) trichloroacetic acid (final volume: 2 mL) followed by centrifugation at 700 × g for 20 min. 5. A blank solution is prepared, adding 1 mL of 10% (w/v) trichloroacetic acid before the DAAO solution is added. 6. To 1 mL of the supernatant from step 4, 1 mL of 5 M KOH and 1 mL of 34 mM Purpald R in 0.5 M HCl are added. 7. The solution from step 6 is incubated at room temperature for 15 min, followed by the addition of 1 mL of 0.75% (w/v) KIO 4 in 0.2 M KOH with vigorous shaking. 8. 1 mL of the solution from step 7 is transferred in a plastic disposable microcuvette and the absorbance at 550 nm (Abs 550 nm ) is measured. 9. The absorbance at 550 nm is corrected by subtracting the absorbance value of a blank solution and the DAAO activity is calculated from the Abs 550 nm using the following equation (Watanabe et al., 1978): U mL DAAO = 2 × Abs 550 nm /15 min ε 550 nm (chromogen) × mL DAAO × mL TOT where the molar extinction coefficient at 550 nm of 6-mercapto-s-triazolo-[4,3-b]-s-tetrazine is 7.74 mM −1 cm −1 and mL TOT is the final volume of the solution from step 7 (4 mL). The detection range is reported in Table 3. In this assay an average velocity corresponding to the first 15 min of the reaction is determined. A factor 2 is added at the numerator because only half of the initial sample (from step 4) is reacted with Purpald R .

Application: Detection of DAAO Activity in Tissues
The enzymatic assay is performed on post mortem human brain tissues dissected after perfusion from the thoracic aorta with ice-cold phosphate buffer saline (PBS), pH 7.4 (Sasabe et al., 2014).
1. The tissue is homogenized in 7 mM sodium pyrophosphate buffer, pH 8.3, at 3,500 rpm for 2 min and centrifuged at 5,500 × g for 10 min at 4 • C. The supernatant (tissue lysate) is recovered and the pellet discarded. 2. 50 µL tissue lysate is added to the reaction mixture containing 150 µL of 100 mM D-alanine, 100 µL 0.1 mM FAD, 150 µL of 700 U/mL catalase in 133 mM sodium pyrophosphate buffer, pH 8.3, and 50 µL of 70% (v/v) methanol. 3. The reaction mixture is incubated at 37 • C for 60 min under stirring. 4. Reaction is stopped by adding 500 µL of 10% (w/v) trichloroacetic acid and the reaction mixture is centrifuged at 700 × g for 20 min. The supernatant is recovered and the pellet discarded. 5. To 250 µL of the supernatant solution 250 µL of 5 M KOH and 250 µL of 34 mM Purpald R in 0.5 M HCl are added. 6. The reaction mixture is incubated at room temperatures for 15 min to which 250 µL 0.75% (w/v) KIO 4 in 0.2 M KOH is added under vigorous shaking. 7. The DAAO activity is measured on the supernatant and expressed as the amount of D-alanine oxidized per min per milligram of total proteins. Total protein concentration is determined by the method of (Lowry et al., 1951).

A) Direct Spectrophotometric Assay
1. Preparation of reaction mixture: 990 µL of D-amino acid solution in 75 mM disodium pyrophosphate buffer, pH 8.5, are transferred into a quartz microcuvette. 2. The initial absorbance value at the wavelength corresponding to the maximum absorption peak of the α-keto acid produced from the starting amino acid is recorded. 3. Measurement of DAAO activity: 10 µL of DAAO (∼0.01 U) are added and the increase of absorbance at the selected wavelength, due to α-keto acid production, is recorded. The DAAO activity is calculated from the Abs/min using the following equation: where ε x nm is the extinction coefficient at the selected wavelength of the α-keto acid produced and mL TOT is the final volume of the reaction mixture (1 mL). The detection range is reported in Table 3. 4. When the extinction coefficient of the α-keto acid is unknown, a calibration curve is set up by recording the absorbance values at known concentrations of the α-keto acid.

(B) Indirect Assay with 2,4-Dinitrophenylhydrazine
1. Preparation of DAAO reaction mixture: 10 µL of DAAO (∼0.05 U) is added to the D-amino acid solution in 75 mM disodium pyrophosphate buffer, pH 8.5 (final volume = 0.3 mL), and the reaction mixture is incubated at 25 • C for 10 min. 2. Derivatization of α-keto acid produced by DAAO reaction with DNP (Figure 4): 0.15 mL of 1 mM DNP, solubilized in 1 M HCl, are added to the reaction mixture. 3. The reaction is stopped by adding 1.05 mL of 0.6 M NaOH, followed by incubation at 25 • C for 5 min to allow color development. 4. A blank solution is prepared using the same procedure, but without substrate (D-amino acid). where ε 445 nm is the molar extinction coefficient at 445 nm of the α-keto acid derivative and mL TOT is the final volume of the reaction mixture. In this assay an average velocity corresponding to the first 10 min of the reaction is determined. The detection range is reported in where the molar extinction coefficient at 340 nm of NADH is 6.3 mM −1 cm −1 and mL TOT is the final volume of the reaction mixture. The detection range is reported in Table 3.

Determination of the Steady-State Kinetic Parameters and Substrate Specificity
The apparent (i.e., at fixed O 2 concentration) kinetic parameters (k cat,app and K m,app values) at air saturation ([O 2 ] = 0.253 mM at 25 • C) are determined for the wildtype RgDAAO enzyme on various D-amino acids using the oxygen consumption assay . As shown in Figure 7, the chemical nature of the substrates profoundly influences the kinetic parameters; a 6-and 60-fold lower activity is apparent on basic and acidic substrates (D-lysine and D-glutamate, respectively) in comparison to the value obtained on the neutral D-alanine (see Table 2).

Screening for RgDAAO Variants with Broader Substrate Specificity
In an attempt to evolve the substrate specificity of RgDAAO, five rounds of random mutagenesis (under different PCR reaction conditions) are performed : a rapid and sensitive assay is required to select the clone expressing the variant with the activity of interest. By using the colorimetric assay based on the reaction of RgDAAO on the D-amino acids of interest coupled to HRP and o-DNS, RgDAAO variants showing an altered substrate specificity could be identified . The crude extract of E. coli culture expressing clones 1-7 and 2-41, deriving from the first and the second set of PCR conditions, respectively, showed a similar activity on D-alanine, but an altered activity on D-arginine with respect to the wild-type enzyme (Figure 8). Furthermore, clones 4-903 and 5-249, deriving from the fourth and the fifth set of PCR conditions, respectively, expressed RgDAAO variants showing a higher activity on at least two of the three substrates tested (D-alanine, D-aspartate, and D-arginine) compared to the wild-type enzyme (see Figure 8). These four RgDAAO variants possess a broader substrate specificity and thus are useful for analytically determining the total content of D-amino acids.

Identification of hDAAO Inhibitors
The Amplex R UltraRed-based fluorescence enzyme assay is a useful and adequate screening technique to identify novel inhibitors of hDAAO. As stated in the Introduction section, this flavoenzyme controls the levels of D-serine in the brain, the coagonist of the NMDARs, a central regulator of synaptic plasticity, learning, and memory (Panatier et al., 2006;Zhou and Sheng, 2013): the inhibitors of hDAAO have the potential to treat nervous system disorders such as schizophrenia (Sacchi et al., 2013). Computational tools are used to identify libraries of compounds, then screened for hDAAO inhibition. The affinity of the different molecules is based on IC 50 values (i.e., the compound concentration at which hDAAO activity is halved). As shown in Figure 9, all compounds tested inhibited the enzymatic FIGURE 9 | Enzyme inhibition assays performed by the Amplex ® UltraRed method using recombinant hDAAO, D-serine as substrate and three different compounds (1, black; 2, gray; and 3, light gray) as potential inhibitors. The plot displays the mean values ± SD, n = 4. activity, although compound 1 was a more potent inhibitor compared to compounds 2 and 3 (IC 50 = 1.7 ± 0.1, 10.6 ± 0.9 and 43.5 ± 2.3 µM, respectively).

Detection of DAAO Activity in Tissues
Owing to the involvement of hDAAO in neuropsychiatric disorders (see Introduction), the investigation of its distribution based on its activity in the human CNS is of utmost importance. Thus, DAAO activity in post mortem samples from different regions of the CNS of mice and humans was determined using a coupled assay with catalase and Purpald R (Sasabe et al., 2014). DAAO activity was detected in all the human specimens examined; significant activity was found along the corticospinal tract, rubrospinal tract, nigrostriatal sytem, ponto-/olivo-cerebellar fibers, and in the anterolateral system. DAAO activity was also detected in human primary somatosensory cortex (PSC), in primary motor cortex (PMC), and in the internal capsule, whereas in mice there was no such activity in the cerebral cortex and in the striatum (Figure 10).

CONCLUSIONS
It is useful to determine DAAO activity for different biotechnological and medical applications (Pollegioni et al., 2007bPollegioni and Molla, 2011): (i) investigation of kinetic parameters on different substrates in order to identify the optimized enzyme for a specific industrial process (i.e., synthesis of semi-synthetic antibiotics, resolution of racemic mixtures, etc.); (ii) identification of novel inhibitors of the human DAAO to be used as potential drugs for the treatment of neuropsychiatric disorders; (iii) evolution of DAAO variants more active at low oxygen concentration for the application in anticancer enzyme therapy; and (iv) development of enzyme-based biosensors for the analytical determination of D-amino acids content in biological fluids. DAAO activity can be determined using direct or coupled assays which differ in terms of sensitivity, required instrumentation and cost, as described in this paper and summarized in Table 3.

NOTES
-Assay buffers must be pre-equilibrated at the working temperature. -Instruments with similar specifications of the ones listed in "Materials and Equipment" section can be used. -The reaction rate can be increased by incubating the reaction mixtures at higher temperature (e.g., 37 • C instead of 25 • C), taking in account the thermal stability of the enzymes in the experimental conditions. -In absorbance and fluorescence assays, a sample containing buffer instead of the substrate and another one containing buffer instead of the enzyme must be performed as negative controls to exclude the presence of artifacts. The presence of contaminants in the sample might affect the absorbance or fluorescence signals (e.g., the presence of nucleic acids and/or proteins can increase the absorbance intensity in the UV region). -In coupled assays with HRP or GDH the production of the expected α-keto acid is not directly monitored but it can be verified by different analytical methods (e.g. HPLC, NMR, and MS). -As a general rule, continuous assays should be preferred (if possible), since the initial rate estimation is easier, due to the fact that the shape of the progress curve is visible all along the assay time and any discrepancy from the linearity is thus promptly detected. On the contrary, in discontinuous assays, to assess the linearity of the initial rate, withdrawal of different samples at fixed incubation times is necessary. Furthermore, the termination of the reaction and withdrawal of samples at fixed-time can introduce time and volume inaccuracy.