A novel fluorimetric method for laccase activities measurement using Amplex Red as substrate

Talanta 162 (2017) 143–150

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A novel fluorimetric method for laccase activities measurement using Amplex Red as substrate Tian Wang, Yuqiang Xiang, Xiaoxiao Liu, Wenli Chen, Yonggang Hu

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State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China

A R T I C L E I N F O

A BS T RAC T

Keywords: Laccase activity Amplex Red Resorufin Fluorescence Soil enzyme

In this paper, a novel fluorescence-based method for laccase assay was presented. The method was based on the transformation of Amplex Red into a highly fluorescent and colored resorufin catalyzed by laccase in the presence of O2. The catalysis and transformation mechanism were investigated in detail. The kinetic parameters of the Amplex Red catalysis by laccase were determined using the Lineweaver–Burk equation. Vmax and Km were estimated to be 15.63 μmol min−1 and 76.88 μmol L−1, respectively. Under optimal conditions, a good linear correlation was found between fluorescence intensity and laccase activities within 5.62–702 U L−1 (r=0.9992), with a detection limit of 1.76 U L−1 (S/N=3). A series of repeatability measurements (351 U L−1 laccase) gave reproducible results with a relative standard deviation (RSD) of 1.9% (n=11). The recoveries ranged from 93.7% to 100.0% after standard additions. Common existing species such as Mg2+, Zn2+, Ni2+, Al3+, Co2+, Cd2+, K+, Ca2+, Na+, Fe3+, Li+, Cu2+, Mn2+, Fe2+, l-lysine, glycine, glucose, phenol, humic acid, lignin peroxidase, manganese peroxidase alkaline phosphatase, cellulose, glucose oxidase, urease, catalase, invertase, and horseradish peroxidase did not significantly exhibit interference. The test solution (i.e., Amplex Red stock solution) could stabilize at least three months via storage in dark at 4 ± 0.1 °C. These results confirmed that the laccase–Amplex Red system was stable and reproducible with strong anti-interference ability and good selectivity, suggesting that this method can has great potential in practical applications for the assay of laccase activity. The proposed method was further successfully used to detect laccase activities in 38 soil samples. We noticed that the laccase activity significantly correlated with total nitrogen content (r=0.559; p < 0.01) of soil, indicating laccase activity assay holds great promise as an index of soil analysis. These findings indicate that this presented method has great perspective in ecological investigation and fundamental research of soil environment.

1. Introduction Soil enzymes are the primary biological mechanism of organicmatter decomposition and nutrient cycling. Enzyme activities can be indicators of microbial activity, decay rates, and substrate availability for microbial or plant uptake. Measuring enzyme activities in soils can be used to understand relationships among microbial community function, resource availability, ecosystem processes, and how ecosystems functionally respond to natural and anthropogenic disturbances [1,2]. Laccases (EC 1.10.3.2) are widely distributed among plants [3], insects [4], and fungi [5], and some bacteria [6]. They have been documented to catalyze the oxidation of various organopollutants, such as polycyclic aromatic hydrocarbons, polychlorinated biphenyls, synthetic dyes, pesticides, and iron-cyanide complexes [7] using oxygen as the final electron acceptor [8]. Thus, laccases play an important role in

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a number of physiological roles, such as in the carbon cycle, morphogenesis, and defense against parasitism [9–11]. The most common methods of measuring laccase activity in complex environments (e.g., soils or litters) are colorimetric and use various substrates such as syringaldazine (N-N′-bis-(3,5-dimethoxy-4-hydroxybenzylidene) hydrazine) and 2,2′-azino-bis-3-ehtylbenzothiazoline-6-sulfonicacid (ABTS) [9]. Syringaldazine has been previously considered as the most common substrate for laccases [7,9,12]. However, the use of syringaldazine-based methods is limited because the quinone produced from syringaldazine is insoluble in aqueous media [13]. Farnet et al. [13] modified the structure of syringaldazine to produce a new watersoluble compound, which has been used to measure the laccase activity of litter extract. However, all reported syringaldazine-based methods suffer from unstable syringaldazine-quinone adsorption [13]. Presently, ABTS is commonly used to quantify laccase activity because of the production of the radical cation ABTS•+. However, in a complex

Correspondence to: College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China. E-mail address: [email protected] (Y. Hu).

http://dx.doi.org/10.1016/j.talanta.2016.10.006 Received 27 June 2016; Received in revised form 18 September 2016; Accepted 2 October 2016 Available online 03 October 2016 0039-9140/ © 2016 Elsevier B.V. All rights reserved.

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medium such as soil or litter, ABTS•+ can oxidize many other molecules and lead to an underestimation of laccase activity [14]. Notably, previously presented methods mainly extract enzymes from soils [15] and the yield is usually very low as only a limited percent of the total soil enzymatic activity is extracted even with procedures lasting several hours [16]. In addition, the efficacy and reliability of the extraction differ regarding the buffer composition and concentration [17,18]. Therefore, the development of a laccase-activity assay method directly applicable to soil or litter homogenates is highly desired. Fluorescence detection is attracting extensive research interest and has been successfully used to trace quantities of analytes in various samples. Fluorescence detection has numerous advantages, such as superior sensitivity, wide linear dynamic range, rapidity, high accuracy, and selectivity [19,20]. Amplex Red (AR; 10-acety-3,7-dihydroxyphenoxazine) is a colorless, nonfluorescencent derivative of resorufin (RSF). Previous researchers have demonstrated that AR could be oxidized by a horseradish peroxidase (HRP)–H2O2 system to the highly fluorescent derivative RSF. As a result, the HRP–H2O2–AR system has been successfully used for the detection of L-asparaginase activity [21], hydrogen peroxide [22–24]. However, the effects of AR oxidation in known assay systems always rely on HRP and H2O2, which largely limits AR application in sensor development [25]. The present work was based on our findings demonstrating the ability and efficacy of laccase to catalyze the oxidation of the nonfluorescent AR to be highly fluorescent and colored RSF without the requirement of any additional oxidizers such as H2O2 with exception of dissolved oxygen. A simple and sensitive AR assay method independence of H2O2 and HRP, for the first time, was established for laccaseactivity test. The possible mechanism of the color reaction catalyzed by laccase was also investigated in detail. The developed method was satisfactorily used to estimate the laccase activity of soil samples and indicated potential practical applications.

Chemical Reagent Co., Ltd. (Shanghai, China). AR was dissolved in DMSO, diluted to 80.0 μmol L−1 with ultrapure water and DMSO. Because AR could be photooxidized to trace RSF [26,27], in order to keep its stability, the AR stock solution was stored in dark at 4 ± 0.1 °C. Humic acid sodium salt was technical grade, and all other chemicals were analytical grade. 2.3. Standard laccase activity assay Laccase was purified according to the protocol described by Blanford [28]. In a typical procedure, 10.0 mg of laccase was dissolved in 1.0 mL of 20.0 mmol L−1 acetate buffer (pH 5.5) and centrifuged at 14,000 rpm and 4 ± 0.1 °C for 1 h. The supernatant was applied to an anion-exchange column (previously equilibrated with buffer) at 4 ± 0.1 °C. Laccase (isoform mixture) was eluted with 60.0 mmol L−1 ammonium sulfate in 20 mmol L−1 sodium acetate buffer (pH 5.5) and stored at 4 ± 0.1 °C. Laccase activity was determined through the oxidation of ABTS at 420 nm (ε420=36,000 L mol−1 cm−1). Enzyme activity was expressed in units (U; defined as 1 μmol of substrate oxidased per minute). The working solutions of laccase for subsequent experiments were prepared by diluting the stock solution with ultrapure water. 2.4. Analysis of laccase–AR reaction

2. Material and methods

The fluorescence spectra of the laccase–AR reaction were recorded on a spectrofluorophotometer. The product of laccase–AR reaction was also analyzed using an ultra-high-definition accurate-mass quadrupole time-of-flight liquid chromatography/mass spectrometry system. Electron spin resonance analysis of laccase–AR reaction was performed at room temperature with an radiation of 9.15 GHz (X band), a modulation frequency of 100 kHz, a sweep width of 10 mT, modulation width of 1 mT, centerfield of 326 mT, scan time of 60 s, time constant of 0.01 s, and micropower of 5 mW.

2.1. Apparatus

2.5. Soil sample collection and preparation

A RF-5301PC fluorescence spectrophotometer (Shimadzu, Kyoto, Japan) equipped with 1 cm quartz cells was used to record the fluorescence spectra. A multi-microplate reader (Fluoroskan Ascent FL, Thermo Fisher Scientific Instruments Co., Ltd., Shanghai, China) was used for fluorescence measurements. An ultra-high-definition accurate-mass quadrupole time-of-flight liquid chromatography/mass spectrometry system (Agilent 6540, Agilent Technologies Inc., CA, USA) was used to analyze the product of laccase–AR reaction. A spectrometer (JES–FA200, JEOL Ltd., Tokyo, Japan) was used for the Electron spin resonance analysis of laccase–AR reaction. A UV–vis spectrophotometer (Beckman Coulter, Brea, CA, USA) was used for colorimetric analysis. A DEAE–SephadexA–50 anion-exchange column (BioMan, Shanghai, China) was used for laccase purification. A vacuum freeze drier (ALPHA 1–4 LSC, Marin Christ, Osterode, Germany) was used for soil samples drying. An elemental analyzer (Vario MAX-CN, Elementar, Hanau, Germany) was used for analysis of total N content of soil samples. A high-speed refrigerated centrifuge (CR21GII) was purchased from Hitachi Ltd. (Kyoto, Japan). Ultrapure water was purified with a Millipore Simplicity System (Pall Co., Ltd., Washington, NY, USA) and had an electrical resistance of 18 MΩ cm. The pH of all buffer solutions was measured by a PB-10 pH meter (Sartorius, Gottingen, Germany).

Soils from evergreen pine forest, camphor forest, willow forest, tea plantation, grassland, and vegetable field were collected (5 cm depth) from Shizishan, Huazhong Agricultural University, Hongshan District, Wuhan City (30°28ʹN, 114°21ʹE). These samples were then sieved ( < 2 mm) and dried in a vacuum freeze drier at −55 ± 0.1 °C for 24 h. All dried samples were well sealed and stored at 4 ± 0.1 °C before use. 2.6. Fluorimetric assay of laccase activity in soil samples The laccase activities of soil samples were analyzed in batch experiments within 2 weeks. In brief, 800 μL of phosphate buffer (pH 6.4), 100 μL of soil suspension (50.0 mg mL−1) and 100 μL of AR (80.0 μmol L−1) were sequentially dispensed into a 2 mL centrifuge tube. The fluorescence signal was then determined after 10 min incubation at 25 ± 0.1 °C using a multi-microplate reader. 2.7. Colorimetric assay of laccase activity in soil samples The laccase activities of the same soil samples were also analyzed via the ABTS and syringaldazine methods by using a UV–vis spectrophotometer. The ABTS method was used according to the procedures described by Floch et al. [29], in which laccase activities were determined without the need for laccase extraction from soil samples. Briefly, 0.1 g of soil and 200 μL of 0.1 mol L−1 ABTS solution were added into 10.0 mL of buffer (tris(hydroxymethyl)aminomethane, maleic acid, citric acid, boric acid, sodium hydroxide) with pH of 2.0. After incubation at 30 ± 0.1 °C for 5 min, the mixture was centrifuged under 12,000 rpm at 4 ± 0.1 °C for 2 min, and the oxidation rate of ABTS to ABTS•+ released into the supernatant was measured at

2.2. Materials Syringaldazine and ABTS, AR, RSF, and laccase (EC 1.10.3.2; pdiphenol: dioxygen oxidoreductases from Trametes versicolor, catalog number 38429) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Dimethylsulfoxide (DMSO) was purchased from Sinopharm 144

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cence was turned on with an emission wavelength at around 583 nm (Fig. 1A), and AR oxidation resulted in significantly increased fluorescence from a nearly zero background level. These properties agreed with that of RSF [27]. This finding was further confirmed by the peak at 1.5 min with a molecular weight of 214.05, which were responsible for RSF (Fig. 1B). When the dissolved O2 was removed by bubbling N2 into the AR solution, the significant decrease in fluorescence signal demonstrated O2 dependence (Fig. 1C1). Fluorescence intensity was almost unchanged by the introduction of superoxide dismutase (SOD, 0.1 g L−1) and HO• quenchers (10.0 mmol L−1 n-butanol and isobutanol). Consequently, we assumed that oxygen was directly involved in the reaction without generating O2•− and HO•. As previously reported, AR radicals are formed during the process of AR conversion to RSF [31]. No electron spin resonance signal was obtained during our experiments at room temperature and pH 7.0 (Fig. 1C2) probably because this free radical is unstable under these conditions [32]. Based on the above results, the entire mechanism was deduced and is depicted in Scheme 1. First, AR was oxidized to AR radical by dissolved oxygen (O2). Then, AR radical rapidly decomposed to RSF or recovered to AR undergoing a dismutation reaction [31]. However, the reaction of AR with dissolved oxygen at room temperature and neutral pH was relatively slow in the absence of laccase, resulting in too low concentrations of AR radicals that led to rapid RSF formation. The reaction of AR–O2 was catalyzed and accelerated by laccase addition. More ARs were then oxidized to form sufficient AR radicals, thereby producing the highly fluorescent and colored RSF. To study the kinetics of AR transformation catalyzed by laccase, initial reaction rates were determined with 1.0–50 μmol L−1 AR. The kinetic parameters of the laccase catalysis of AR were determined with the Lineweaver–Burk equation [33] represented as follow:

420 nm. The syringaldazine method was used according to the method described by Farnet et al. [13,30]. Briefly, laccase extractions were obtained using 10 g of soil in a 1.0 L flask containing 200 mL of an extraction solution (Polyvinylpolypyrolidone 5.7 g, CaCl2 0.2 mol L−1, Tween 80 0.05%). These samples were subjected to axial shaking for 1 h at 120 rpm at room temperature. Solids were eliminated by centrifugation at 10,000 rpm for 15 min and filtrated through Whatman GF/D (2.7 μm) and Whatman GF/C filters (1.7 μm). The filtrates of each extract were concentrated using polyethyleneglycol to a final volume of 10% of the initial volume. The concentrated soil extract (500 μL) and syringaldazine (10 μL, 0.6% in methanol) were dispensed into 2.5 mL of acetate buffer (0.1 mol L−1, pH 4.0). After incubation at 25 ± 0.1 °C for 2 min, enzyme activity was measured by monitoring the oxidation of syringaldazine to quinone at 525 nm. 2.8. Assay of total nitrogen content in soil samples The total N content in 500 mg of dry sample was analyzed using an elemental analyzer. Each measurement was conducted in five replicates. Linear regression was subsequently analyzed to obtain r value and associated p value and to investigate the dependence of laccase activities on total N content of the soil samples. 3. Results and discussion 3.1. Characterization of laccase–AR reaction The colorless AR was rapidly oxidized to a red product (Fig. 1A, inset) after mixing laccase with AR solution. Meanwhile, AR fluores-

Fig. 1. (A) Fluorescence spectra of laccase–Amplex-Red (AR) reactions (excitation 530 nm, emission 583 nm). Inset: colorless AR (a) was catalyzed by laccase and converted to red resorufin (RSF) (b). (B) HPLC–MS chromatograms of the product of laccase–AR. Inset: peak at 1.5 min. (C1) Effects of quenchers on laccase–AR fluorescence intensity. SOD: superoxyde dismutase, O2: oxygen, N2: nitrogen. (C2) Electron spin resonance signal of AR solution in the presence and absence of laccase. Error bars indicate standard deviations from seven independent measurements.

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Scheme 1. Schematic of AR-fluorescence induction by laccase.

Fig. 2. (A) Effect of pH. (B) Effect of DMSO volume in the reaction system. (C) Limit of detection (LOD) versus AR concentration. (D) Linear-regression analysis of laccase activity on fluorescence intensity. Error bars indicate standard deviations from seven independent measurements.

is the Michaelis constant, and Vmax is the maximum initial rate. The Vmax and Km values were estimated to be 15.63 μmol min−1 and 76.88 μmol L−1, respectively.

Table 1 Comparison of analytical features of laccase-activity detection among reported assays. Method

Corresponding regression equation

Linear range (U L−1)

LOD (U L−1)

Reference

Resonancescattering assay Resonancescattering assay Glassy-carbon electrode Flow-injection spectrophotometry AR method

ΔI468 nm=88.8U−1.9 r=0.9964 ΔI=734.0Ulaccase−9.7 r=0.9920 y=0.473x+4.184 r=0.9835 y (mA s−1) =3.05x (U mL−1)+1.18 r=0.988 F=0.1434U+0.0151 r=0.9992

80–960

20

[34]

100– 1200 1.37– 33.79 600– 24,000

50

[35]

/

[36]

200

[37]

1.76

This work

1 K 1 1 = m ⋅ + V0 Vmax [S ] Vmax

5.62– 702

3.2. Fluorescence method for laccase activity assay The effects of pH and concentration of DMSO on the laccase–AR reaction were investigated. The experiment of pH optimization was conducted under conditions: 1.0 μmol L−1 AR, 6.5 U L−1 laccase, 0.5% DMSO, and 10 min incubation time at 25 ± 0.1 °C. Fig. 2A shows that fluorescence intensity was increased with increased pH from 5.6 to 6.4, and the highest fluorescence intensity was obtained at pH 6.4. With further increased pH, fluorescence intensity decreased. This phenomenon can be explained by the fact that the optimum pH of laccase for the catalysis of AR is around 6.4; too high or too low pH affects the active center of laccase, causing laccase to exhibit low activity [38]. Thus, pH 6.4 was selected for subsequent experiments. At pH 6.4, concentration of DMSO was optimized subsequently. Fig. 2B shows that fluorescence intensity increased with increased DMSO concentrations from 0.01% to 0.5% (v/v), and then decreased with further

(1)

where V0 is the initial reaction rate, [S] is the concentration of AR, Km 146

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Fig. 3. (A) Effects of various coexistent substances on laccase activity. (B) Selectivity of the fluorometric AR assay on soil enzymes. (C) Effect of soil on fluorescent signal of RSF. (D1) Fluorescence signals detected by mixture (a) and supernatant (b) of soil and AR after incubation. (D2) Relationship between fluorescence intensity and concentration of soil. Error bars indicate standard deviations from five independent measurements.

conditions: 8.0 μmol L−1 AR, 0.5% DMSO, pH 6.4, and 10 min incubation time at 25 ± 0.1 °C. A good linear correlation was found between fluorescence intensity and laccase activities within 5.62– 702 U L−1 (r=0.9992). The linear fitting equation was F=0.1434U +0.0151, where F is fluorescence intensity, and U is laccase activity (Fig. 2D). Eleven measurements of 351 U L−1 laccase yielded a relative standard deviation of 1.9%. This result guaranteed stability and repeatability. The limit was further estimated to be 1.76 U L−1 (Table 1), suggesting that this method can efficiently assay laccase

increased DMSO concentration. This phenomenon can be explained by the fact that too low DMSO concentration resulted in insoluble AR in the reaction system, and too high resulted in inhibited laccase activity [39]. Thereby, 0.5% DMSO was used for subsequent experiments. Limits of detection (LODs) for laccase assay at different AR concentrations within 1.0–50.0 μmol L−1 were detected under optimum conditions (Fig. 2C). LOD had the lowest value of 1.76 U L−1 (S/ N=3) when the concentration of AR was increased to 8.0 μmol L−1. The working curve for determination of laccase activity was obtained under

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ZnSO4, 1.0×10−6 mol L−1 MnSO4, 1.0×10−5 mol L−1 FeSO4, 1.0×10−4 mol L−1 Fe2(SO4)3, 1.0×10−3 mol L−1 CaCl2, 1.0×10−4 mol L−1 NaCl, 1.0×10−2 mol L−1 KCl, 1.0×10−4 mol L−1 Al2(SO4)3, 1.0×10−3 mol L−1 LiCl, 1.0×10−6 mol L−1 NiCl2, 1.0×10−6 mol L−1 CdCl2, 1.0×10−5 mol L−1 CoCl2, 1.0×10−4 mol L−1 L–lysine, 1.0×10−1 mol L−1 glycine, 1.0×10−1 mol L−1 glucose, 1.0×10−4 mol L−1 phenol, 0.02 mg mL−1 humic acid. Results in Fig. 3A indicate that the tested species had no significant influences on the signal of 0.35 U mL−1 laccase, with the tolerable limit of the foreign substance defined as the relative error produced by concomitant species not exceeding ± 5%. In addition, the effects of some soilderived enzymes such as lignin peroxidase and manganese peroxidase [2], alkaline phosphatase and cellulose [42], glucose oxidase [7], urease [43], catalase [44], invertase [45], and horseradish peroxidase [46] were tested under conditions: 0.6 U mL−1 enzyme, 8.0 μmol L−1 AR; 0.5% DMSO; pH 6.4, and 10 min incubation time at 25 ± 0.1 °C. As shown in Fig. 3B, the coexistence of these enzymes with the same laccase concentration did not significantly affect the fluorescence signal caused by laccase. The AR stock solution could stabilize at least three months in dark at 4 ± 0.1 °C. These results indicate that the laccase–AR system was stable and reproducible with strong anti-interference ability and good selectivity, suggesting that it has great potential in practical applications for the assay of laccase activity. The soil samples were suspended with ultrapure water, and the supernatants were obtained and incubated with AR. No significant fluorescent signal caused by the product of RSF was detected, meaning that laccases were mainly absorbed by the soil as the formation of an immobilized enzyme. Moreover, a soil sample was divided into two parts; one was dried only in a vacuum freeze drier, and the other was inactivated with an autoclave. These two parts of soil samples within 0– 40 mg mL−1 were added to the RSF solution to investigate the effect of slurry on the fluorescence detection of RSF under conditions: 8.0 μmol L−1 RSF, 0.5% DMSO, pH 6.4, and 10 min incubation time at 25 ± 0.1 °C. Results in Fig. 3C indicated no significant influence on the fluorescence signal of RSF with the addition of these soil samples. In other words, the slurry in the tested range had no quenching effect on the fluorescence of RSF. These results suggested that the laccase– AR system had a strong anti-interference ability and good selectivity. Accordingly, after the soil samples was incubated with AR solution (8.0 μmol L−1 AR, 0.5% DMSO, pH 6.4, and 10 min incubation time at 25 ± 0.1 °C), the fluorescence signals of the mixture containing slurry did not significantly differ from that of the supernatant of the soil–AR reaction (Fig. 3D1). Thus, we can directly detect the RSF created by the AR-based reaction for the determination of laccase activity without slurry removal from the reaction system. Furthermore, we found that the fluorescence signals of mixture increased with increased soil within 0–10.0 mg mL−1 (Fig. 3D2). However, when the concentration of soil exceeded 10.0 mg mL−1, the increasing tendency of fluorescence signal decreased, which may result from excessive interferences such as phenolic leached from soil [47], which may interfere with the reaction between laccase and AR. Control experiments were based on AR and inactivated soil. Consequently, 5.0 mg mL−1 soil was used to measure the laccase activity of soil.

Fig. 4. Comparative assay of laccase activity from different derived soils through Amplex Red (AR), ABTS, and syringaldazine methods.

Table 2 Recoveries for laccase activity of five soil samples. Sample

Before added (U L−1; n=5)

Added (U L−1)

Found (U L−1; n=5)

Recovery (%; n=5)

Soil Soil Soil Soil Soil

29.9 ± 1.3 26.7 ± 1.5 29.3 ± 0.2 20.1 ± 1.2 17.8 ± 0.8

25.5 25.5 25.5 25.5 25.5

54.1 ± 0.3 51.5 ± 0.3 54.8 ± 1.0 45.1 ± 0.5 41.7 ± 0.6

94.9 ± 1.1 97.3 ± 1.4 100.0 ± 4.1 98.0 ± 2.1 93.7 ± 2.4

1 2 3 4 5

Fig. 5. Linear regression analysis of total nitrogen (N) content via laccase activity in soil. Error bars indicate standard deviations from five independent measurements.

3.3. Application for real samples

activity. Common existing interferences of inorganic metal ions such as Mg2+, Zn2+, Ni2+, Al3+, Co2+, Cd2+, K+, Ca2+, Na+, Fe3+, Li+, Cu2+, Mn2+, and Fe2+ were also investigated to test the selectivity of laccase assay. Low-molecular-mass organic compounds, such as l-lysine, glycine, glucose, phenol, and humic acid have been previously demonstrated to influence the ABTS-based laccase assay [40,41]. The effects of these organic compounds were also assessed. The experiment was conducted under conditions: 8.0 μmol L−1 AR, 0.35 U mL−1 laccase, 0.5% DMSO, pH 6.4, and 10 min incubation time at 25 ± 0.1 °C, 1.0×10−3 mol L−1 MgCl2, 1.0×10−4 mol L−1 CuSO4, 1.0×10−4 mol L−1

The laccase activities in six soil samples (Fig. 4) were detected using AR, ABTS, and syringaldazine methods. The high laccase activity is attributed to the excellent anti-interference ability of the proposed method, and laccase extraction was not required. The underestimation of laccase activity by the ABTS method is possibly attributed to the oxidation of many molecules, such as phenol compounds in soil, by ABTS•+ radicals [14]. The lowest laccase activities were estimated using syringaldazine method. In fact, the laccase activities obtained using syringaldazine method were zero, because a limited percentage of the total enzymatic activity was extracted from the soil samples [15,16]. 148

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The recoveries in five soil samples were further investigated (Table 2). The recoveries ranging from 93.7% to 100.0% after standard additions are satisfactory, indicating the good accuracy of the proposed method. The laccase activities of 38 soil samples were analyzed at pH 6.4, 5.0 mg mL−1 soil, 8.0 μmol L−1 AR, 0.5% DMSO, and 10 min incubation time at 25 ± 0.1 °C. Moreover, their total N contents were analyzed using 500 mg soil of each sample (dry weight). We found for the first time that laccase activity of soil was primarily correlated with its total N content (r =0.559, p < 0.01) (Fig. 5). That is, the laccases may play an important role in N cycle, and laccase activity can be used as an index to assess soil quality or the functional capacities of soil ecosystem [48]. These results demonstrated that the proposed method can offer a great potential in practical applications.

[14]

[15] [16]

[17]

[18] [19]

4. Conclusion

[20]

A novel fluorescence method for laccase assay based on the color reaction of AR was demonstrated. Compared with existing methods, the proposed one had the advantages of high sensitivity, wide linear range, stability, repeatability, strong anti-interference ability, and good selectivity. More importantly, the developed method could be directly used for laccase assay in soil without needing enzyme extraction, which is complex and time consuming, recovers only a small part of the total enzyme activity, and has an extractable amount that varies among soils. Our method was also successfully used to detect laccase activities in soil samples and thus has potential practical applications.

[21]

[22]

[23]

[24] [25]

Acknowledgments This work was supported by the National Natural Science Foundation of China Grants (No. 21675057), the National Basic Research Program of China (973 Program Grant no. 2015CB150504), and the State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University (Grant no. AMLKF201405).

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