Appl Biochem Biotechnol DOI 10.1007/s12010-014-1005-0

Advances in Detection Methods of L-Amino Acid Oxidase Activity Zhiliang Yu & Yangsheng Wang & Ning Zhou & Minyan Zhao & Juanping Qiu & Jianxun Lin

Received: 6 March 2014 / Accepted: 26 May 2014 # Springer Science+Business Media New York 2014

Abstract L-Amino acid oxidase (LAAO) is widely distributed in many different organisms and found to play important biological roles, thus attracting a great deal of attention for characterization of its activity. Diverse detection methods with their own properties have been established. This review advanced different LAAO activity assays based on substrate consumption, cofactor amount, and product accumulation. The description of benefits and drawbacks of each method is expected to help researchers find appropriate detection method of LAAO activity for their own purpose. Keywords L-Amino acid oxidase . Activity determination . Substrate-based . Cofactor-based . Product-based

Introduction L-Amino acid oxidase (LAAO; EC 1.4.3.2) is attracting increasing attention due to its diverse and important biological functions, such as antimicrobial [1–3], anti-HIV [4], antitumor cell [5], induction of cell apoptosis [6], etc. It is found in various species, not only in snake and insect venoms [7] but also in sea hare [8], fungi [9], bacteria [10, 11], and algae [12]. Among them, snake venom LAAO is the best characterized member of this enzyme family. So far, almost all the discovered LAAOs are flavoenzymes with a homodimeric structure, in which each subunit contains a non-covalently bound flavin adenine dinucleotide (FAD) molecule as cofactor, except the lysine oxidase of Marinomonas mediterranea [13, 14]. As shown in Fig. 1, LAAO can catalyze the stereospecific oxidation of L-amino acid to form the corresponding α-imino acid and generate hydrogen peroxide (H2O2). Subsequently, the α-imino acid is hydrolyzed to the corresponding α-keto acid along with the release of ammonia (NH4+)

Z. Yu (*) : Y. Wang : N. Zhou : M. Zhao : J. Qiu College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou, China e-mail: [email protected] J. Lin Department of Electrical Engineering, Columbia University, New York, NY, USA

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Fig. 1 Schematic representation of L-amino acid catalyzation by LAAO

via non-enzymatic oxidation [15, 16]. The H2O2 can further cause a decarboxylation of the αketo acid if not degraded by catalase [17, 18]. Different methods have been established for determining the LAAO activity, based on amino acid consumption [19], oxygen change [20, 21], FAD cofactor amount determined by HPLC or coupled with peroxidase [22–25], and accumulation of products including NH4+ [26–32], α-keto acid [33, 34], and H2O2 [35–65]. Among them, the product-based methods have gained dominant popularity due to their relatively high sensitivity and reproducibility. In particular, the fluorometric assay using Amplex Red reagent (10-acetyl-3,7dihydroxyphenoxazine) in combination of horseradish peroxidase (HRP) [66] has been considered as the gold standard in detecting H2O2 released from biological samples. It has been reported that H2O2 can be detected down to nanomolar level, providing almost the best sensitivity (Molecular Probes, Invitrogen). On the other hand, HRP-based assay for H2O2 detection is easily inactivated due to the instability of HRP. Furthermore, most of HRP substrates are highly toxic. The product of α-keto acid is usually measured spectrophotometrically by hydrazine assay using 2, 4-dinitrophenylhydrazine (DNP) [33, 65]. The Berthelot reaction [30] and Nessler’s reagent method [26, 27], or modifications of them, are commonly used for the spectrophotometric determination of NH4+. Each method has its own advantages and disadvantages. This review aims to bring together the increasing body of knowledge concerning pros and cons for each possible alternative of LAAO activity assay. With these in hand, the researchers can choose an appropriate detection method to best suit their needs.

Substrate-Based Assay Amino Acids The ninhydrin (2,2-dihydroxyindane-1,3-dione) is the most widely used reagent for the colorimetric detection of amino acids [67]. The reaction between ninhydrin and the amine

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group on amino acid will produce intense color which can be both qualitatively and quantitatively measured. The sensitivity of ninhydrin reaction-based assay can reach down to around micromolar level of amino acid [67], which is sufficient for many purposes. In addition, the procedure is simple. However, free α-amines from unwanted peptides and proteins can react with ninhydrin, thus making this assay’s specificity low for quantitative determination of LAAO activity. Fluorescent assay has been developed to detect amino acids to yield nanomolar sensitivity [68]. ο-Diacetylbenzene and o-phthaldialdehyde have been reported to be able to react with amino acids to produce strongly fluorescent compounds [68]. Better results were obtained by using o-phthalaldehyde than ο-diacetylbenzene. This type of fluorescence reaction requires alkaline medium in the presence of a reducing agent such as 2-mercaptoethanol. To avoid the pungent smell from 2-mercaptoethanol, 2-mercaptopropionic acid has been successfully used instead. No heating is necessary and the fluorescence signal may easily be detected in 5 min after mixing of the reagents. Despite of the much improved sensitivity, this type of assay still suffers in the following two aspects. First, pure amino acid substrate is required to compensate its low specificity nature as mentioned above in the colorimetric-based assay; second, though most of amino acids can be detected satisfactorily using the o-phthalaldehyde reaction, imino acid proline and hydroxyproline do not yield fluorescence, and cysteine gives poor signal. High-pressure liquid chromatography (HPLC) has also been reported to characterize LAAO activity based on amino acid consumption [19]. It was known that IL4I1, a secreted LAAO expressed by macrophages and dendritic cells, can catalyze the oxidative deamination of L-Phe and to a lesser extent L-Trp to the corresponding α-keto acids [19]. Briefly, HPLC analysis of L-Phe and L-Trp content was conducted on a Hitachi Diode array C2455 Elite device equipped with a Lachrom pump L2130 and an L2300 oven. The separation was obtained on a mixed-mode Primesep 100 column packed with 5-μm particles by using a linear gradient of acetonitrile (20 to 60 %) in water/trichloroacetic acid with pH 2.5 at a flow rate of 1 mL/min at 30 °C. Dimethylaminobenzoic acid was used as an internal standard. The amounts of L-Phe and L-Trp after oxidation by IL4I1 were quantified as the peak area ratio between each amino acid and the internal standard. Although HPLC-based method is potentially universal for measuring all types of amino acids, its throughput is low and it needs optimization of the HPLC condition for different L-amino acids. Moreover, the results may easily be interfered with other external and internal compounds. Therefore, highly purified sample before analysis is required. In addition, the instrument of HPLC is limited for many laboratories. Oxygen During LAAO-coupled reaction, oxygen is being consumed to yield H2O2. Accordingly, researchers have used a classic Warburg manometer or an oxygen-sensitive electrode to characterize LAAO activity. Oxygen consumption was reported to be determined in Warburg manometer at 30 °C with certain volume of pyrophosphate liquid [69]. The gas phase was oxygen, and the center well contained NaOH and a roll of filter paper for absorption of CO2 and H2O from the gas phase. LAAO and amino acid were first placed in separate compartments and then mixed together after establishment of thermal equilibrium. With the development of the oxidization reaction, oxygen in the confined system was gradually consumed and the changed level of the liquid column in manometer was converted to the oxygen consumption. Subsequently, the LAAO activity can be estimated. The oxygen consumption can also be measured by a Hansatech oxygen-monitoring system [20, 21]. A small volume of LAAO was placed in a cuvette which was made anaerobic by cycling between

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vacuum and argon and sealed with a rubber septum. The substrates of L-amino acids were also prepared anaerobic by bubbling argon. The reaction was started by injecting the substrate solution through the rubber septum. With a computer-interfaced graphical mode or a Yellow Springs Instrument model 5300 oxygen electrode, the oxygen concentration was detected. Then, the LAAO activity was calculated using the previously determined extinction coefficient. Such oxygen consumption-based method requires a well-controlled anaerobic environment and thus its throughput is low.

FAD Cofactor-Based Assay With the exception of lysine oxidase from M. mediterranea [13, 14], all LAAOs are flavoenzymes with a homodimeric structure in which each subunit contains a non-covalently bound FAD molecule as cofactor. FAD is very tightly bound to LAAO. It has been reported that the amount of LAAO can be measured based on its FAD cofactor content [22, 25]. Before detection, the sample of LAAO needed heating and centrifugation for dissociation of FAD from LAAO. Absorption spectra of sample were recorded using a Beckman DU-640 spectrophotometer. To determine the level of FAD cofactor in LAAO, the observed absorbance changes were used and the calculations were made using values determined from the extinction coefficient of free standard FAD [22]. To improve the accuracy and sensitivity, HPLC, mass spectrometry (MS) and nuclear magnetic resonance (NMR) can be used to replace the spectrophotometer for performing the identification of FAD cofactor and its amount. Similarly, FAD standard as a comparison is needed. All these spectroscopy-involved methods can only be applied to the detection of FAD cofactor in LAAO samples devoid of other flavoenzymes. In addition, they can only be used to characterize the LAAO content, but not its activity. Among them, the spectrophotometer-based FAD detection is much simpler and more achievable, but its sensitivity and accuracy is lower. In contrast, other three methods of FAD detection are much more sensitive and accurate, but much more complicate and need more expensive instruments. Moreover, the throughputs of the latter three are lower.

Product-Based Assay Ammonia The most common ammonia detection methods are the Nessler reaction and Berthelot reaction. The Nessler reagent consists of dipotassium tetraiodomercurate (II) in a diluted alkaline solution, normally NaOH, which can produce an aurantium compound after reacting with NH4+ [26, 27]. The resultant color is measured at 490 nm. The LAAO activity is assessed as deaminase. One unit of LAAO is expressed as the amount of enzyme which releases 1 μmol of ammonium per minute under standard condition [26, 27]. There are only a few literatures about quantitative measurement of LAAO activity with Nessler reaction [29], probably due to the high toxicity of Nessler reagent. Besides, it can be interfered with other amino compounds. In Berthelot reaction, the combination of ammonia, phenol, and hypochlorite will result in a blue coloration. The detection limit is about 5 μM of ammonia in water or 90 ppb [30], which is comparable to other colorimetric assays. Compared with the Nessler reaction, this Berthelot reaction involves less dangerous chemicals and the products are more soluble in water. In addition, the Berthelot reagent is more specific to ammonia as against other nitrogen-containing

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compounds (e.g., urea, amino acids), and more sensitive than the Nessler one. However, one drawback of this technique is its slow reaction kinetics. A commercial assay kit (EnzyChrom ammonia/ammonium kit, Bioassay system, USA) is available for measuring NH4+/NH3. In this method, NADH is converted to NAD+ in the presence of NH4+/NH3, ketoglutarate, and glutamate dehydrogenase. The decrease in optical density at 340 nm or fluorescence intensity at 450 nm of emission and 360 nm of excitation is directly proportionate to the NH4+/NH3 concentration in samples. Puiffe et al. reported to use this method for fluorimetric detection of NH4+/NH3 concentration from L-Phe oxidation by LAAO [19]. This method is simple, direct, and automation-ready. However, the linear detection limit is only in a range of 24 to 1,000 μM NH4+/NH3. Moreover, the working reagents are unstable and the kit is expensive, mostly due to the use of dehydrogenase. If strong base such as NaOH is added to NH4+ solution generated by LAAO activity, NH3 will be released, which can be sensed based on the color change of paper pH indicator above the NH4+ solution [31, 65]. The results in Fig. 2 showed that paper pH indicator of negative control maintained yellow as the original without any color change. In contrast, both positive control and the test with LAAO activity have caused the color change of paper indicator from yellow to absinthe-green, meaning NH4+ was generated by LAAO activity in the test. This pH paper method is useful for preliminary qualitative screening, but not quantitative detection, due to its low sensitivity and accuracy. The ammonia generated by L-lysine oxidase can be detected by potentiometric biosensor [32]. This system consisted of a chemically immobilized lysine oxidase membrane and an allsolid-state ammonium selective electrode. The lysine oxidase membrane was attached to the surface of the ammonium selective electrode by means of a Viton O-ring and a dialysis membrane. When in use, the enzymatic degradation of lysine will produce ammonia which is then detected by the ammonium electrode. Therefore, the lysine oxidase activity can be evaluated. Although the stability and sensitivity of this method are high, the equipment is too complicate and its throughput is low. In addition, it requires strict control of the reaction conditions, such as temperature and pH, in order to obtain meaningful result. α-Keto Acid Like amino acids, α-keto acids can be directly measured with HPLC analysis [19]. After oxidation of L-Phe by LAAO, the solution was filtered through a 0.22-μm disposable unit. Twomillimolar internal standard of dimethylaminobenzoic acid were added and sample was precipitated for 30 min with 10 % trichloroacetic acid (TCA) at 4 °C. Precipitate was centrifuged at 10,000×g for 10 min at 4 °C, and supernatant was injected to a Hitachi Diode

Test

Positive control Negative control

Fig. 2 Determination of NH4+ by adding NaOH with paper pH indicator [65]. Test: LAAO with substrate L-Leu; positive control: ammonia solution; negative control: LAAO without substrate L-Leu

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array C2455 Elite device equipped with a Lachrom pump L2130 and an L2300 oven. The amount of phenylpyruvate was quantified as the peak area ratio between the compound and internal standard using the EZchrom Elite software (Hitachi). In theory, this HPLC-involved method is almost applicable to all different α-keto acids, but different α-keto acids have different HPLC conditions. In addition, this method is complicate and its throughput is low. Besides direct detection, α-keto acids can be measured in indirect ways after reacting with other compounds to yield more easily detectable products. DNP can react with carbonyl group to form brown-red dinitrophenylhydrazone. Therefore, as carbonyl derivative, α-keto acid generated from LAAO catalytic reaction can be measured using DNP [33, 65]. In general, this method includes five steps. Firstly, LAAO is added to L-amino acid solution to produce the corresponding α-keto acid. Secondly, the oxidization reaction will be terminated by adding TCA. Thirdly, DNP is added to react with the produced α-keto acid. Fourthly, NaOH is added to terminate the reaction. Finally, after centrifugation, the supernatant with dinitrophenylhydrazone will be measured at 520 nm. As shown in Fig. 3, neither the test tube of LAAO without L-Leu nor the one with denatured LAAO in the presence of L-Leu yields visible color change, while both the tube with α-keto acid (positive control) and the oxidation solution of L-Leu by LAAO develop brown-red color. The LAAO activity can be quantitatively determined by measuring the absorbance at 520 nm and using standard α-keto acid curve. This method is sensitive and can accurately quantify the LAAO activity. However, DNP is toxic and poorly water-soluble. Besides, this compound is difficult to be stored. More importantly, the generated H2O2 has to be removed by catalase to prevent α-keto acid from being decarboxylated. Semicarbazide has been used to measure L-lysine oxidase activity. Specifically, the α-keto acid generated from oxidation of L-Lys by LAAO can react with semicarbazide to form αketo-ε-aminocaproate semicarbazone, which is spectrophotometrically monitored at 248 nm [34]. Like the above-mentioned DNP probe, this method is also limited by the need of catalase to remove H2O2. Moreover, it has been found that semicarbazide is a weak inhibitor of some LAAOs, thus lowering the LAAO activity. In 2002, Geueke et al. described a lactate dehydrogenase-coupled assay to measure α-keto acid for characterization of LAAO activity [56]. In this method, catalase, L-Ala, NADH, lactate

Fig. 3 Detection of a-keto acid production using 2, 4-dinitrophenylhydrazine (DNP) [65]. positive control: αketo leucine (OD520 =0.232); LAAO with substrate L-Leu (OD520 =0.198); LAAO without substrate L-Leu (OD520 =0.032); boiling-denatured LAAO with substrate L-Leu (OD520 =0.045)

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dehydrogenase, and LAAO were mixed together. Then, L-Ala was oxidized to pyruvate by LAAO activity. Subsequently, the formation of lactate produced by the lactate dehydrogenase from pyruvate and NADH was spectrophotometrically measured at 340 nm (ε = 6.22 mM−1 cm−1). Based on the lactate concentration, the amount of pyruvate can be calculated. Therefore, the LAAO activity in reaction will be determined. Catalase is also needed to remove H2O2. This method is simple and sensitive. However, oxygen should be absent or in short supply. In addition, LAAO should be in limiting amounts. Moreover, the activity of lactate dehydrogenase and its enzymatic efficiency are crucial to detection. H2O2 As mentioned briefly in the introduction, HRP-involved measurement of H2O2 formation is the most popular LAAO activity assay and remains as the gold standard so far. Using the H2O2 generated by LAAO as oxidizing agent, HRP can oxidize a broad range of H2O2-sensitive probes including 4-aminoantipyrine [35], o-dianisidine (ODA) [23, 36–40], 2,2′-azino-bis(3ethylbenzthiazoline-6) sulfonic acid (ABTS) [41], o-phenylenediamine (OPD) [42–44], 3,3′,5,5′-tetramethylbenzidine (TMB) [45], and guaiacol [46] to form compounds with distinctive colors. These developed colors can be spectrophotometrically measured at respective wavelengths for quantitative analysis. This type of method gains its popularity due to simplicity, reasonable sensitivity, and high reproducibility. However, most of these chemicals including ABTS, OPD, and ODA are mutagenic, carcinogenic, or extremely toxic. It is worth noting that TMB is neither mutagenic nor carcinogenic, can yield products with high absorption coefficients, and is more sensitive than OPD and ABTS [47]. However, it is poorly watersoluble and decomposes rapidly in aqueous solutions. In addition, TMB stock prepared in dimethyl sulfoxide (DMSO) or methanol/acetone is only stable for a few weeks [48]. In the pH from 5.5 to 6.0, TMB is only soluble to a concentration of 0.42 mM, 45 times less than OPD. The oxidation of guaiacol with H2O2 gave an undefined product mixture, and absolute molar absorptivity cannot be determined [50]. Fluorometric measurement is commonly known to have very high detection sensitivity. Therefore, Amplex Red has been applied to commercial Amplex® Red Hydrogen Peroxide/ Peroxidase Assay Kit (Molecular Probes, Invitrogen) for detection of H2O2 [52, 53]. This assay reaction can be done in 100 μL within 30 min in 96-well plate at room temperature. The redfluorescent oxidation product will be fluorometrically measured by using excitation filter at around 540 nm and emission filter at 590 nm. The detection limit can be down to 10 pmol of H2O2 in a 100-μL volume, which is 100 nM. Therefore, this method is ultrasensitive and its throughput is high, as compared with other mentioned assays. Although fluorometric measurement is more sensitive than spectrophotometric one, absorption-detecting microplate readers are more achievable than fluorescence-detecting ones in many laboratories. Thus, one can alternatively measure the oxidation product at 560 nm. Overall, Amplex Red kit assay is almost the most widely used method with high sensitivity, reliability, and simplicity. However, the kit is expensive and the working stock is unstable, which is mainly resulted from HRP. An amperometric biosensor based on electron transfer between an electrode and immobilized peroxidase, which catalyzes the reduction of H2O2, is highly promising to determine the LAAO activity [54, 55]. Based on the difference of electron transfer, amperometric biosensor method can be divided into two different ways. The first is mediator-free H2O2 sensor where electron directly transfers between HRP and electrode. Spectrographic graphite, carbon black, and glassy carbon have been used to fabricate mediatorless H2O2 sensors. However, its detection limit can only reach micromolar level of H2O2 due to its slow electron transfer rate. The second one involves mediator materials such as ferrocene and its derivatives,

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and water-soluble dyes to shutter and enhance the electron transfer between electron and enzyme. It has been reported that LAAO activity can be amperometrically measured with 4aminoantipyrine as a mediator [70]. Although the detection limit of some mediated sensors can reach as low as 10−7 ~10−8 M, it is inconvenient to add a mediator. In addition, some mediator materials are easily to diffuse from enzyme layer and will pollute the electrode system [54]. Recently, sol-gel was used to entrap methylene green, a dye mediator for amperometric measurement of H2O2 [54]. Although this modification of biosensor can enhance the method stability, the condition of sol-gel-enzyme coating needs to be well-controlled. To minimize the effect of mediator material on LAAO activity and simplify the detection method, Rau and Fischer used polyacrylamide gel (PAGE) to entrap LAAO [63]. After electrophoresis without heating, PAGE containing LAAO was soaked into the detection assay mixture mainly containing L-amino acids, HRP, 4-aminoantipyrine, and phenol. Because of catalyzation by HRP, the H2O2 produced by the entrapped LAAO activity will cause the formation of the dye (pale pink) from 4-aminoantipyrine which can be documented immediately over a period of 15~45 min in intervals of 2 min, depending on the LAAO concentration. For direct in-gel detection of LAAO activity, analytical electrophoresis should be carried out under non-denaturing condition (without heating) to maintain enzyme activity. This method is simple and practicable. However, this novel assay has a few drawbacks to prevent its widespread use. First, the assay solution has a short shelf life. Second, the concentrated sample in the gel will appear as smeared band. Third and most importantly, this method cannot be used to quantify the exact LAAO activity. Although HRP is the most widely used to measure H2O2 due to its high sensitivity, it is expensive and unstable. Chen et al. used an HRP-free method to determine LAAO activity [61, 62]. In brief, samples with LAAO were electrophoresed in SDS-PAGE without heating. After staining with Coomassie brilliant blue (CBB), the protein bands were cut out for antimicrobial activity test on solid culture medium plate with L-amino acids. Since the tested microbes are sensitive to the H2O2 produced by LAAO activity, antimicrobial zones can be used to determine the LAAO activity. The bigger the zone diameter, the higher the LAAO activity. This method is very simple and costs much less than HRP-based assay. However, it is laborintensive and time-consuming, due to the requirement of microbial incubation, which usually takes overnight. Besides, LAAO may lose partial activities during long-term multiple treatments. Moreover, this method is unsuitable to quantify the LAAO activity. Prussian blue assay has been widely used in sensors to detect the presence of H2O2 in electrochemical reactions because iron (III) and potassium hexacyanoferrate (III) can be oxidized to yield the blue precipitate of Prussian blue through the sequential reactions as shown below. In this assay, H2O2 acts as electron donor [58].   Fe3þ þ Fe3þ ðCNÞ6 3− →Fe3þ Fe3þ ‐‐‐ðCNÞ6  3þ    Fe ‐‐‐Fe3þ ðCNÞ6 → Fe3þ ‐‐‐Fe3þ ðCNÞ6 ads ðPrussian brownÞ   3þ  ‐ Fe ‐‐‐Fe3þ ðCNÞ6 ads þ e‐ → Fe3þ ‐‐‐Fe2þ ðCNÞ6 ads  3þ ‐  2þ  3þ 2þ 3þ Fe þ 3 Fe ‐‐‐Fe ðCNÞ6 ads →Fe 4 Fe ðCNÞ6 3 ↓ ðPrussian blueÞ This color change is fast, sensitive, and reproducible. Based on this scheme, we developed a new application of Prussian blue assay for quantitative in-gel determination of the LAAO activity by detecting the produced H2O2 concentration [58]. In brief, this Prussian blue agar assay included the following steps: (1) prepare Prussian blue agar plate mainly consisting of 1.0 g/L FeCl3 ·6H2O, 1.0 g/L potassium hexacyanoferrate (III), and 1.5 % agar with pH 7.5. If

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necessary, the substrates of L-amino acids can be added; (2) dissolve the mixture completely at 100 °C for 5 min and pour into glass Petri dish to make agar plate; (3) make circular wells with diameter of 6 mm on agar plate; (4) carry out stereospecific oxidation of amino acids by LAAO activity; (5) add 50 μL oxidation solution to each well on Prussian blue agar plate and wait for 30 min at room temperature for color change; (6) visualize the Prussian blue forming and measure the blue halo size; (7) extract the H2O2 concentration and characterize the LAAO activity. It was found that the diameters of the formed Prussian blue halos were perfectly a function of logarithm of H2O2 concentrations varying from 0.5 to 20 mM with a linear fit, thus allowing Prussian blue agar assay for quantitative determination of the LAAO activity described as H2O2 concentration-based style. Coupled with SDS-PAGE, Prussian blue agar assay can be directly used to not only sense the presence of LAAO activity but also determine the numbers and approximate molecular weights of the LAAO protein in one assay [58], showing significant advantages in comparison to usual spectrophotometric and fluorometric detection methods of LAAO. Although this Prussian blue agar assay is more cost-effective and convenient than the HRP-based assays, several drawbacks may limit its further application. Firstly, its quantitative detection limit is only down to 0.5 mM of H2O2. Secondly, potassium hexacyanoferrate (III) itself is safe, but it may degrade and release extremely toxic CN− under the peracidic condition. Thirdly, Prussian blue is a complicate class of chemical compounds containing Prussian blue, Prussian brown, Prussian white, and Berlin green, which are sensitive to pH condition. Therefore, extremely careful pH adjustment in the Prussian agar preparation is required to produce reproducible result of color formation. An assay combining the advantages of HRP-based and Prussian blue-based measurement for detection of LAAO activity is highly promising (Fig. 4). As an important color material, xylenol orange (XO, 3,3′-bis[N,N-bis(carboxymethyl)aminomethyl]-o-cresolsulfonephthalein; Fig. 5) has been demonstrated for detection of H2O2 concentration through the sequential reactions as shown below. •

Fe2þ þ H2 O2 þ Hþ →Fe3þ þ H2 Oþ OH • • OH þ xylenol þ Hþ →xylenol • 2þ xylenol þ Fe →xylenol þ Fe3þ • OH þ Fe2þ þ Hþ →Fe3þ þ H2 O Fe3þ þ xylenol→Fe3þ ‐xylenol ðPurplish redÞ

Fig. 4 Quantitative determination of H2O2 with Prussian blue agar assay [58]. a Dependence of the diameters of Prussian blue halo on the detected H2O2 concentrations in a range of 0.5 mM to 20 mM; b perfect distribution with a linear fit between the diameter of the Prussian blue halo (x axis) and logarithm of the corresponding H2O2 concentration (y axis)

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Fig. 5 Quantitative measurement of H2O2 with FeIII-xylenol orange (FeIII-XO) agar assay [72]. a Dependence of the diameters of purplish red FeIII-XO halo on the detected H2O2 concentrations from 5 to 160 μM; b perfect distribution with a linear fit between the diameter of the purplish red halo (x axis) and logarithm of the corresponding H2O2 concentration (y axis)

These reactions are very fast, sensitive, and reproducible. Usually, the formation of purplish red FeIII-XO complex can be spectrophotometrically measured [71]. On the basis of this scheme, we recently developed a new application of the FeIII-XO formation for quantitative determination of the LAAO activity by in-gel extracting H2O2 concentration [72]. In brief, this FeIII-XO agar assay was performed as follows: (1) prepare solution of ferrous-XO (FeIIXO) with 0.25 mM FeSO4, 6 mM H2SO4, 0.15 mM XO, 0.1 mM D-sorbitol, and 1.5 % agar; (2) dissolve the mixture completely at 100 °C for 5 min and pour into glass Petri dish to make agar plate; (3) make circular wells on agar plate with a hole puncher whose diameter is 6 mm; (4) carry out stereospecific oxidation of amino acids by LAAO activity; (5) add 50 μL of oxidation solutions to each well and wait for 60 min at room temperature for color change; (6) visualize the FeIII-XO formation and measure the size of purplish red halo; (7) extract the H2O2 concentration and characterize the LAAO activity. It was demonstrated that the diameters of the purplish red halos of FeIII-XO complex driven by H2O2 were a linear function of logarithm of H2O2 concentrations from 5 to 160 μM, thus allowing this assay to quantitatively determine the LAAO activity with a similar sensitivity as the HRP-involved methods [72]. In contrast, our FeIII-XO agar assay does not require any detection instrument, and it is more simple, stable, and cost-effective. Compared with Prussian blue agar assay [58], FeIII-XO agar assay is more environmentally friendly. More importantly, it gives two orders of magnitude improvement in sensitivity (5 μM vs. 0.5 mM). Considering its high sensitivity, cost-effectiveness, and convenience, this FeIII-XO agar assay can be used to differentiate the mutants with slight difference in LAAO activity and screen the desired clones with high throughput from a mutant library with altered expression of LAAO, saving a great number of workload for the investigation of the involved regulation mechanisms underlying the LAAO production. Like Prussian blue agar assay [58], SDS-PAGE coupled FeIII-XO agar assay can directly be used to determine LAAO molecular weights and its numbers in one assay, giving crucial advantages over conventional spectrophotometric or fluorometric measurement. On the other hand, Fe III -XO agar assay is easily saturated by H2 O 2 (160 μM). Therefore, to quantify H2O2 with high concentration generated by LAAO, the sample needs to be properly diluted. In addition, since the nature color of assay medium is shallow orange red, the shallow purplish red of FeIII-XO formed by H2O2 with low concentration (5 μM) will be blurred.

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Conclusions and Perspectives In this communication, we summarized a variety of detection methods of LAAO activity according to the measurement of substrates, cofactor, and products. LAAO-coupled oxidation reaction is involved in two different substrates, L-amino acid and oxygen. The change of L-amino acid in reaction is mainly determined with fluorometric assay due to its high sensitivity. Almost all common amino acids except proline and cysteine can react with o-phthalaldehyde to give strong fluorescence. In fact, L-amino acids can also be directly measured using HPLC with reasonable sensitivity and good reproducibility. However, for different amino acids, the HPLC conditions are probably different. In addition, these methods may easily be interfered with external amino acids and their derivatives. At present, oxygen consumption is mainly monitored with a manometer or oxygen-sensitive electrode, which requires a well-controlled assay environment. Therefore, it is unsuitable for rapidly processing numerous samples. Most of LAAOs bear two FAD molecules as cofactor. In theory, the amount of detected FAD can be considered to be proportional to the LAAO activity. However, the accuracy of this method is low, probably due to the following reasons. Firstly, it is difficult to completely separate FAD from LAAO for detection. Secondly, the amount of detected FAD is only proportional to all LAAOs both with and without activity. Thirdly, other flavoenzymes tend to affect the detection of LAAO activity. Therefore, this FAD-based method is not very desirable for quantitatively detecting LAAO activity, but for qualitatively determining LAAO activity. Compared with substrate consumption and cofactor content, the product accumulation is more suitable to the determination of LAAO activity. In particular, H2O2 is considered as the most ideal one. Under the involvement of HRP, H2O2 can specifically react with a variety of chemicals, dyes, and their derivatives to give rise of easily detectable compounds. They are the most popular methods, due to their acceptable simplicity and ultrasensitivity. However, HRP is unstable and expensive, and its substrate is usually toxic. Therefore, an HRP-free method is highly desirable. Prussian blue agar assay and FeIII-XO agar assay have been established with several advantages of extraordinary convenience, cost-effectiveness, reliability, accuracy, and stability for determining LAAO activity, ideally serving as sensitive procedures. However, their sensitivity is lower than Amplex Red-based assay. Another product in LAAO-involved reaction is α-keto acid which can be measured using DNP, giving reasonable sensitivity and simplicity. Unfortunately, DNP is toxic and poorly water-soluble. Besides, this detection is easily interfered with H2O2. The last product is ammonia which can be sensed with paper pH indicator. However, it is only applicable to qualitative measurement. Nessler reaction and Berthelot reaction can be used for quantitatively detecting ammonia with high sensitivity and simplicity, but the reaction reagents are toxic and their specificity is low. To date, very little is known about LAAO structural properties and the involved regulation mechanisms underlying its transcription and production. Although during the past dozens of years mutagenesis techniques have become prevalent, tapping the detection method cheaply and efficiently for large-scale screening has proven challenging. Considering the properties of all detection methods for determining the LAAO activity, both Amplex Red-based assay and FeIII-XO agar assay are promisingly suitable for this purpose. However, in the future, the cost and stability of Amplex Red-based assay and sensitivity of FeIII-XO agar assay need to be improved for better properties. Acknowledgments This work was supported by Regional Demonstration of Marine Economy Innovative Development Project, China (No. 12PYY001SF08).

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