An extracellular yellow laccase from white rot fungus Trametes sp. F1635 and its mediator systems for dye decolorization

Biochimie 148 (2018) 46e54

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Research paper

An extracellular yellow laccase from white rot fungus Trametes sp. F1635 and its mediator systems for dye decolorization Shou-Nan Wang a, Qing-Jun Chen b, Meng-Juan Zhu c, Fei-Yang Xue a, Wei-Cong Li a, Tian-Jian Zhao a, Guang-Dong Li a, Guo-Qing Zhang a, * a

Key Laboratory of Urban Agriculture (North China) of Ministry of Agriculture, College of Biological Science and Engineering, Beijing University of Agriculture, Beijing 102206, China Beijing Key Laboratory for Agricultural Application and New Technique, College of Plant Science and Technology, Beijing University of Agriculture, Beijing 102206, China c Department of Fungal Resource, Shandong Agricultural University, Taian, Shandong 271018, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 December 2017 Accepted 23 February 2018

A novel extracellular laccase was purified from fermentation broth of the white rot fungus Trametes sp. F1635 by a three-step protocol including two consecutive ion-exchange chromatography steps on DEAESepharose and SP-Sepharose, and a final gel-filtration on Superdex 75. The purified laccase (TsL) was a monomeric protein with the molecular mass of 64.8 kDa. It demonstrated high oxidation activity of 4.00
104 U/mg towards ABTS. Its N-terminal amino acid sequence was AIGPVADLTIINNAV which was unique and sharing high similarity of other fungal laccases. TsL was a yellow laccase based on absorption spectrum analysis. It demonstrated an acidic pH optimum of 2.6 and temperature optimum of 50  C towards ABTS. The Km and Vmax values towards ABTS were estimated to 18.58 mM and 1.35 mmol/min, respectively. TsL manifested effective decolorization activity towards eriochrome black T (EBT), remazol brilliant blue R (RBBR), malachite green (MG), and eriochrome black T (EBT) (over 60%). Violuric acid (VA) and acetosyringone (AS) were the optimal mediators for the laccase in dye decolorization. Results suggest that TsL demonstrates great potential for dye decolorization and water treatment. © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Keywords: Trametes Laccase Purification Laccase-mediator system Dye decolorization

1. Introduction The white rot fungi (WRF) can efficiently decay the lignin in wood [1]. They cause the rotted wood to feel moist, soft, or stringy and appear white or yellow. In resent years, accumulating literature suggest that there are great potential for WRF application since they can degrade not only lignin but also a wide variety of environmentally pollutants, such as synthetic dyes, industrial wastewater and aromatic pesticides [2,3]. There are many different ligninolytic enzymes that are involved in the decay of wood by white rot fungi, including laccases (Lac, E.C. 1.10.3.2), lignin peroxidases (LiP, E.C. 1.11.1.14), manganese-dependent peroxidases (MnP, E.C. 1.11.1.13) and versatile peroxidases (VP, E.C. 1.11.1.16), etc [4]. It has been reported that among various enzymes, laccases represent great potential for biotechnological and environmental

* Corresponding author. E-mail address: [email protected] (G.-Q. Zhang).

applications [3,5]. Laccases (benzenediol: oxygen oxidoreductase) are multicopper oxidase enzymes belonging to the superfamily of multicopper oxidases. They are widely distributed in bacteria, plants and especially fungi [3]. In general, laccases are extracellular glycoproteins and present mostly as monomers, dimers or tetramers with a typical molecular weight range of 60e80 kDa for the monomer and a carbohydrate content of 15e20% [6e8]. Typical laccases (blue laccases) have four copper atoms which are distributed in three different copper centers (Type 1, 2 and 3): Type 1 (T1) or blue copper center, Type 2 (T2) or normal copper center, and Type 3 (T3) or coupled binuclear copper center. Spectral characteristic studies reveal that the T1 site of laccases imparts a light blue color to the enzyme solutions confirms optic absorption at around 600 nm, T2 site is invisible in electron absorption spectra, and T3 site can be identified by the presence of a shoulder close to 330 nm [7,9]. The copper atom in T1 site is responsible for the color of the enzyme (blue), while copper atoms in the type 2 and 3 centers are involved in the catalytic reactions with various substrates [5,10]. Non-typical

https://doi.org/10.1016/j.biochi.2018.02.015 0300-9084/© 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

S.-N. Wang et al. / Biochimie 148 (2018) 46e54

laccases lacking of T1 site are described as yellow laccases. Laccases exhibit various functions including cross-linking of monomers, degradation of polymers and ring cleavage of aromatic compounds. They are green catalysts and can oxidize a variety of compounds like carbohydrates, aromatic and non-aromatic compounds by a one-electron transfer mechanism using molecular oxygen as the electron acceptor [11]. On the other hand, recalcitrant substrates with high redox potentials are hard to oxidize by laccases alone. Therefore, some redox mediators, such as 2, 20 -azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), are added with laccases to enhance the oxidation activities. These mediators act as electrons shuttles involving in the oxidation of complex substrates (such as lignin polymers) that can not enter the active site of laccases [11]. Laccases and laccase-mediator systems (LMS) possess great potentials in decolorization and detoxification of textile industry wastewater and environmental remediation applications [12,13]. WRF species from genus Trametes have been well studied since they are good laccase producers such as T. hirsute, T. trogii, and T. versicolor [3,14e16]. In the present study, we aimed to purify a novel extracellular laccase with different enzymatic properties from fermentation broth of our newly isolated Trametes sp. F1635. Enzymatic properties and LMS for dye decolorizing application were also investigated. 2. Materials and methods 2.1. Fungal strain Fruiting bodies of a wild Trametes mushroom were collected from Mount Wutai, Shanxi province, China. Pure mycelial culture, numbered as strain F1635, was obtained in Petri dishes containing potato dextrose agar (PDA) medium at 28  C. The fungus was cultured at 28  C, stored at 4  C, and monthly transferred to fresh PDA slants, in the Key Laboratory of Urban Agriculture (North China) of Ministry of Agriculture, Beijing University of Agriculture. Fresh culture was raised for the experimentation. 2.2. Identification of strain F1635 Taxonomical identification was carried out based on morphological and molecular characteristics. Molecular identification was based on Internal Transcribed Spacer (ITS) region (ITS1-5.8S-ITS2) analysis using ITS1 (50 TCCGTAGGTGAACCTGCGG 30 ) and ITS4 (50 TCCTCCGCTTATTGATATGC 30 ) primers [17]. Trametes sp. F1635 was cultured in Petri dishes containing PDA medium at 28  C for 5 d, followed by total genomic DNA extraction of mycelia using a genomic DNA extraction kit (TIANGEN, China). The ITS region was amplified by PCR in a volume of 25 mL under standard conditions [18]. Subsequently, the PCR production was sequenced by Sangon Biotech (Shanghai) Co., Ltd. (China), and compared with ITS sequences obtained from GenBank using the Blast tool in NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The phylogenetic tree was made by the MEGA 6.0 software using the neighbor-joining (NJ) method with the bootstrap value 1000. 2.3. Laccase production and purification Laccase production was carried out using potato dextrose medium with the fed-batch fermentation method in a fermentation tank (5 L) at 28  C for 100 h. The fermentation broth was initially filtered to remove mycelial debris using cotton gauze filter. After centrifugation at 4  C and 9000 rpm for 15 min, the supernatant was dialyzed in distilled water overnight. Subsequently, the crude laccase solution was applied to anion exchange chromatography on

47

a column of DEAE-Sepharose (2.5 cm
30 cm) previously eluted with the starting buffer (10 mM Tris-HCl buffer, pH 8.5). After sampling, the solution was eluted successively with 0, 100, and 300 mM NaCl in the same buffer. All obtained fractions were monitored for laccase activity, and laccase rich fraction (D2) were pooled and dialyzed for further purification on cation exchange chromatography of SP-Sepharose (10 mM HAc-NaAc, pH 3.8). After removal of an unadsorbed fraction (SP1), two adsorbed fractions (SP2 and SP3) were eluted with 50 and 150 mM NaCl in the starting buffer, respectively. Laccase active fraction SP3 was finally applied to gel filtration by fast protein liquid chromatography (FPLC, GE Healthcare, USA) on a Superdex 75 gel filtration column (0.15 M NH4HCO3 buffer, pH 8.5) using an AKTA Purifier (GE Healthcare, USA). The second fraction (SU2) eluted constituted the purified laccase from Trametes sp. F1635 (abbreviated as TsL).

2.4. Assay for laccase activity Laccase activity was determined spectrophotometrically using 2, 20 -azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate [19]. Enzyme solution (5 mL) was mixed with 0.6 mM ABTS solution (195 mL, in 50 mM sodium acetate buffer, pH 4.5) at 37  C (water bath) for 5 min, followed by an addition of 10% TCA (190 mL) to end the reaction. One enzyme unit (U) was defined as the amount of enzyme required to produce an increase of one absorbance unit at 420 nm per minute per milliliter of the reaction mixture under the assay conditions. All treatments were performed in triplicate [20].

2.5. Determination of molecular mass FPLC-gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were performed for molecular mass (Mr) determination. During the purification protocol, molecular mass standards (GE Healthcare, USA) were applied to the FPLC chromatography. The standard curve of Log Mr vs elution volume was obtained. Mr of the active enzyme can be calculated based on the curve. SDS-PAGE was performed using the standard procedure with a 12% resolving gel and a 5% stacking gel [20]. After the electrophoresis, the gel was stained with Coomassie brilliant blue (CBB) R-250. Another standard curve of Log Mr vs relative mobilities of molecular mass standards (Genview, USA) was obtained. Mr of the present purified laccase was evaluated based on the two curves.

2.6. N-terminal and inner amino acid sequencing After SDS-PAGE, the purified enzyme on the gel was transferred to a polyvinylidenedifluoride (PVDF, Bio-Rad, USA) membrane by electro-blotting and stained with CBB R-250. The stained band was then excised and analyzed by the automated Edman degradation method using an HP G1000A Edman degradation unit (Hewlett Packard Company, USA) and an HP1000 HPLC system (Hewlett Packard Company, USA) [21]. After electrophoresis and visualization, the protein band of purified laccase TsL on SDS-PAGE was recovered and digested overnight using trypsin. Subsequently, digestion products were eluted and analyzed by nano liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS, Agilent, USA) for inner amino acid sequencing. The data were acquired using Xcalibur software (Thermo Electron, USA). Sequence homologues were searched using the BLAST/NCBI database [19].

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2.9. Effect of chemical reagents

Table 1 Characteristics of the LMS assays. Dye

lmax (nm)

Concentration (mg/L)

Eriochrome black T (EBT) Evans blue (ET) Methyl orange (MO) Bromothymol blue (BMB) Fuchsin basic (FB) Malachite green (MG) Remazol brilliant blue R (RBBR) Methylene blue (MB)

540 610 460 440 530 614 605 664

400 28 20 800 20 8 100 8

The effects of chemical reagents on the purified laccase were investigated using NaCl, KCl, CaCl2, CdCl2, CoCl2, CuCl2, MgCl2, MnCl2, and EDTA in the final concentrations of 1.25, 2.5, 5.0, and 10 mM, respectively. The purified enzyme solution was preincubated with the assay reagents (1:1, v/v) at 4  C for 1 h before the standard laccase assay was performed. The residual laccase activity was measured in triplicate using the standard assay. Enzyme treated with distilled water was used as the control [20,21].

2.7. Absorption spectrum

2.10. Enzyme kinetics

To find out the copper catalytic centers, the UVevisible absorption spectrum of the purified laccase was recorded in 50 mM sodium acetate buffer (pH 4.5) within the range of 200e800 nm using a UVeVis spectrophotometer (Shanghai Puyuan, 19000 PC, China) [22].

To estimate the kinetic constants (Km and Vmax), a series enzymatic reactions of purified laccase were performed using ABTS as substrate in a series ABTS concentration range from 12.5 to 800 mM at pH 2.6 (citric acid-Na2HPO4 buffer, 50 mM) and room temperature (25  C). The constants were evaluated using a Lineweaver-Burk plot [18].

2.8. Effects of pH and temperature

2.11. Dye decolorization by LMS

The effect of pH on laccase activity was determined in a pH range from 2.0 to 8.0 using the citric acid-Na2HPO4 buffers (50 mM, pH range of 2.2e8.0). The effect of temperature on laccase activity was examined from 20  C to 90  C. ABTS was used as the substrate [20]. In the pH stability assay, the purified laccase was preincubated at 4  C and in citric acid-Na2HPO4 buffers (50 mM) at pH 2.2, 2.6, 3.0, and 3.4 for 0, 10, 20, 40, and 60 min, respectively. In the thermo stability assay, the purified laccase was pre-incubated at 40, 45, 50, 55, and 60  C for 0, 10, 20, 40, and 60 min, respectively. Subsequently, the residual activity was measured using the standard assay [18].

Decolorization activities of the purified laccase and its mediator systems were evaluated towards eight kinds of textile or chemical dyes including: three azo dyes of eriochrome black T (EBT), evans blue (EB), and methyl orange (MO), three triphenylmethane dyes of bromophenol blue (BMB), fuchsin basic (FB), and malachite green (MG), one anthraquinone dye of remazol brilliant blue R (RBBR), and one thiazine dye of methylene blue (MB) [20,21]. Four mediators were assayed including acetosyringone (AS), 1hydroxybenzotriazole (HBT), (2, 2, 6, 6-tetramethylpiperidin-1-yl) oxidanyl (TEM), and violuric acid (VA) [11]. The reaction mixture containing the purified enzyme solution (7.5 mL, 2 U/mL), dye

100 73

Polyporus melanopus (JQ964423.1) Polyporus tubiformis (KC572035.1)

63

Polyporus submelanopus (JQ964425.1) Polyporus tuberaster (KC572037.1)

97

F1635 81

100 Trametes trogii (HQ000043.1) Trametes pavonia (KF573032.1)

92

Trametes gibbosa (KC525203.1)

95

Trametes cinnabarina (KF573022.1)

46 98

Trametes sanguinea (JX082366.1) Cerrena unicolor (KX527879.1) Cerrena aurantiopora (KJ668561.1)

100 99

Cerrena consors (KJ957779.1) Inonotus hispidus (JX501315.1) Inonotus linteus (JX985739.1) Inonotus baumii (JN642566.1)

100 93

Inonotus sanghuang (JQ860316.1)

0.05 Fig. 1. Neighbor-joining phylogenetic tree based on ITS sequences showing the distance of strain F1635 with species of Polyporus species. Bootstrap values at nodes are percentages of 1000 replicates.

S.-N. Wang et al. / Biochimie 148 (2018) 46e54

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Table 2 Summary of purification procedure of the purified laccase TsL (from 1 L fermentation broth). Purification step

Yield (mg)

Total activity (U)

Specific activity (U/mg)

Recovery of activity (%)

Purification fold

Fermentation broth DEAE-cellulose (D3) SP-Sepharose (SP2) FPLC (SU2)

1451 470 5.4 1.2

9.33
105 3.68
105 1.05
105 4.80
104

642.8 782.2 1.94
104 4.00
104

100 39.4 11.3 5.14

1 1.22 30.2 62.2

solution (292.5 mL, 50 mM citric acid-Na2HPO4 buffer, pH 4.2), and mediator solution (with the final concentration of 0.1 mM) were incubated at room temperature and dark condition for 4, 12, and 24 h, respectively. Subsequently, the decolorizing abilities were determined by a microplate reader (Bio-rad, USA) [23e25]. Active and inactive enzyme solutions were used as the positive and negative control, respectively. The characteristics of the LMS assays were provided in Table 1. 3. Results

were obtained. By blasting (blastp) in the BLAST/NCBI database, they all demonstrated high similarity with fugal laccases from Polyporus such as Coriolopsis gallica, Trametes gibbosa, T. trogii (Table 4). 3.4. Absorption spectrum UVevisible absorption spectrum of the purified laccase TsL possessed a slight shoulder at about 340 nm while lacked any peak at around 600 nm (Fig. 3).

3.1. Classification of strain F1635 Fruiting bodies of strain F1635 were collected from Wutai Mountain, Shanxi, China. The hymenia were auricular, leathery, and possessed massive vertical pores on the underside of the caps. Based on morphological properties, strain F1635 was preliminarily classified as a species from the genus Trametes. A partial sequence fragment of ITS region was obtained and contained a 622 bp segment (GenBank accession No. of KY234237). A phylogenetic tree was established based on ITS region of Polyporus species including Trametes, Polyporus, and Cerrena spp. from family Polyporaceae, and Inonotus spp. from family Hymenochaetaceae (Fig. 1). Strain F1635 was closest to Trametes trogii (HQ000043.1), sharing the query cover of 98% and the sequence identity of 99%. Strain F1635 was classified as T. trogii based on both morphological and molecular identification. 3.2. Purification of laccase The purified extracellular laccase (TsL) was obtained from fermentation broth of Trametes sp. F1635 following an isolation protocol that entailed two consecutive steps of ion exchange chromatography and a final gel-filtration step of FPLC. The results of laccase purification at different steps were summarized in Table 2. The purified laccae TsL possessed an oxidative activity of 4.00
104 U/mg, a 5.14% recovery of activity and a purification factor of 62.2fold (Fig. 2A, Table 2). Enzyme sample of each steps were collected and applied to SDS-PAGE (Fig. 2B). 3.3. Molecular mass and amino acid sequencing Trametes sp. F1635 laccase TsL appeared as a single band with a molecular mass (Mr) of 64.8 kDa in SDS-PAGE (Fig. 2B) and gel filtration yielded the same estimate of Mr (Fig. 2A), from which we could deduce that TsL was a monomeric protein with a Mr of 64.8 kDa. The N-terminal amino acid sequence of TsL was AIGPVADLTI INNAV which manifested considerably high homology with other fungal laccases (Table 3). When comparing the N-terminal ten amino acid sequence, TsL shared the 100% similarity of laccases from T. trogii SYBC-LZ, Coriolopsis gallica (AAW65489.1), and Abortiporus biennis which are also Polyporales species [26,27]. Results from LC-MS/MS analysis showed that five inner peptide sequences LVSLSCDPNHTFSIDGHSLTVIEADSVNLKPHTVDSIQIFAAQR (lac1), QAILVNDVFPSPLITGNKGDR (lac2), SLYDVDDDSTVITLADWYHLAAR (lac3), SINTLNADLAVITVTK (lac4), and GPIVVYDPQDPHK (lac5)

Fig. 2. (A) FPLC-gel filtration on Superdex 75 HR 10/30 column. Eluent: 0.15 M NH4HCO3 buffer (pH 8.5). Fraction size: 0.8 mL. Flow rate: 0.8 mL/min. Fraction SU2 represents purified laccase. (B) SDS-PAGE of each purification steps. M: Marker, A: Fermentation broth, B: fraction D3 from DEAE-cellulose, C: fraction SP2 from SPSepharose, D: fraction SU2 from FPLC.

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Table 3 Comparison of the N-terminal sequence of TsL with other fungal laccases. Species

N-terminal sequence

Max ident

Accession number/Ref.

Trametes sp. F1635 T. trogii SYBC-LZ Coriolopsis gallica Coriolopsis trogii T. trogii strain B6J LacI T. versicolor Abortiporus biennis Coprinus comatus Russula virescens Pleurotus nebrodensis Coriolopsis gallica Ganoderma lucidum Trametes cinnabarina Trametes ochracea T. sanguinea T. hirsuta Polyporus brumalis Polyporus ciliatus Polyporus grammocephalus C. unicolor

1 AIGPV ADLTI INNAV 15 1 AIGPV ADLTI SNGAV 15 1 AIGPV ADLTI SNGAV 15 1 AIGPV ADLTI SNGAV 15 1 SIGPV ADLTI SNGAV 15 1 GIGPV ADLTI TNAAV 15 1 AIGPV ADLTI 10 1 AIGPV ADLKV 10 1 AIGPT AELVV 10 1 AIGPD DTINF 10 22 AIGPV ADLTI SNGAV 36 22 AIGPV TDLTI SNAAV 36 22 AIGPV ADLTL TNAAV 36 21 GIGPV ADLTI TNAAV 35 22 AIGPV ADLTL TNAAV 36 22 AVGPV ADLTI TDAAV 36 24 AIGPV ADLTI SNADI 38 24 AIGPV ADLTI TNADI 38 22 AIGPV ADLTL VNDVI 36 22 AVGPV ADIHI TDDTI 36

100% 87% 87% 87% 80% 80% 100% 80% 60% 40% 87% 80% 80% 80% 80% 73% 73% 73% 67% 53%

Present study [31] AAW65489.1 2HRG_A [27] AAB35061.1 [26] [47] [36] [35] ABD93940.1 AHA83595.1 AAN71597.1 ALT22024.1 ACN69056.1 ACC43989.1 ABN13592.1 AAG09230.1 ACR24358.1 ALE66001.1, [20]

Amino acid residues identical to corresponding residues of TsL are underlined.

3.5. Effects of pH and temperature The optimal pH value of the purified laccase TsL was 2.6 towards ABTS at 37  C, with about 50% activity loss at pH 4.6 and 95% activity loss at pH 6.2. The oxidizing activity almost vanished when the assay pH was higher pH 7.0 (Fig. 4A). The optimal temperature of TsL was 50  C. About 40% of total laccase activity remained when it was assayed at 80  C (Fig. 4B). In the pH stability assay, TsL showed strong stability in the assay pH range of 2.2e3.4. More the 70% of total activity remained after 60 min incubation (Fig. 4C). At pH 2.6, the oxidizing activity of TsL underwent about 40% raise after incubating for 10e30 min, followed by continuous decline. In the thermo stability assay, Tsl also manifested considerably high stability in the assayed temperature range of 40e60  C for 60 min incubation (Fig. 4D). After incubated at 40e50  C for 60 min, most of oxidizing activity of TsL maintained. When incubated at 60  C, it underwent a slight decrease of oxidizing activity with the residual activity of about 65% of the total.

3.6. Effects of chemical reagents Effects of metal ions and EDTA towards the purified laccase TsL

are shown in Table 5. TsL was not obviously affected by the existence of Naþ and Kþ at concentration of 1.25e10 mM. However, all the assayed divalent metal ions showed dose dependent inhibitory activity of varying degrees. About 40% of the total enzyme activity lost when the assayed Cd2þ concentration was as high as 10 mM. Furthermore, EDTA at concentration of 5e10 mM moderately depressed enzyme activity with inhibitory ratio of about 20%. 3.7. Determination of enzyme kinetics According to Michaelis-Menten kinetics, the kinetic constants of TsL were tested using ABTS as substrate at pH 2.6 and 25  C. The Km and Vmax values were 18.58 mM and 1.35 mmol/min, respectively (data not shown). 3.8. Dye decolorization by LMS Industrial and laboratory dye wastes are the main pollutants pigments in wastewaters and widely used in the world. They can be divided into many types based on their chemical structure, such as azo, triphenylmethane, anthraquinone, and thiazine dyes, etc. The present laccase was an effective decomposer towards EB, RBBR,

Table 4 Comparison of the Peptide sequences of TsL with other fungal laccases. Peptide fragment

Peptide sequence

Lac1

LVSLSCDPNHTFSIDGHSLTVIEADSVNLKPHTVDSIQIFAAQR

Lac2

QAILVNDVFPSPLITGNKGDR

Lac3

SLYDVDDDSTVITLADWYHLAAR

Lac4

SINTLNADLAVITVTK

Lac5

GPIVVYDPQDPHK

Microorganism containing similar sequence Microorganism

Similarity

Accession number

Coriolopsis gallica Trametes gibbosa Trametes trogii Coriolopsis gallica Trametes versicolor Ganoderma lucidum Coriolopsis gallica Grifola frondosa Coriolopsis trogii Coriolopsis gallica Coriolopsis caperata Trametes hirsuta Coriolopsis gallica Coriolopsis rigida Coriolopsis trogii

100% 82% 95% 100% 90% 80% 100% 95% 91% 100% 88% 85% 100% 92% 85%

AJV90966.1 AEQ38868.1 2HRG_A ABD93940.1 AFM31222.1 AHA83586.1 AJV90967.1 OBZ65410.1 AMJ39539.1 ABD93940.1 AGE13770.1 AOX15704.1 ABD93940.1 ADK13098.1 AMJ39540.1

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Table 5 Effect of chemical reagents on laccase activity. Chemical reagent

NaCl KCl MgCl2 CaCl2 CuCl2 CoCl2 MnCl2 CdCl2 EDTA

Relative laccase activity (% of control) 10 mM

5 mM

2.5 mM

1.25 mM

104.7 ± 5.6 103.2 ± 2.3 82.0 ± 3.2 68.0 ± 2.5 67.0 ± 3.1 66.9 ± 2.6 60.6 ± 0.6 59.6 ± 2.1 79.7 ± 4.7

101.8 ± 2.6 103 ± 6.3 89.1 ± 6.1 74.9 ± 5.4 74.8 ± 14 71.3 ± 1.2 77.2 ± 1.9 65.3 ± 1.6 83.5 ± 2.8

103.6 ± 1.1 106.4 ± 5.9 92.8 ± 12.6 94.2 ± 6.3 78.4 ± 3.3 81.2 ± 4.0 97.2 ± 0.7 73.8 ± 4.7 91.8 ± 2.0

103.2 ± 2.6 105.3 ± 0.8 97.4 ± 6.7 84 ± 1.9 77.1 ± 0.5 81.7 ± 0.6 100.4 ± 5.0 78.5 ± 3.3 98.1 ± 2.2

Laccase activity in the absence of metal ions was regarded as 100%. Results represent mean ± standard deviation (n ¼ 3).

Fig. 3. UVevisible spectroscopic analysis of the purified laccase TsL.

MG, and EBT, with the highest decolorization rate at 24 h of 89.0%, 76.1%, 61.9%, and 61.0%, respectively. It possessed a decolorization rate at 24 h of 24.8% towards BMB, but very little decolorization activity towards MO, FB, and MB (Fig. 5). The mediators can efficiently enhance the decolorization activity of TsL. VA was the best candidate for LMS of TsL towards EB (95.2%), FB (94.0%), RBBR (90.1%), MO (88.9%), and MG (85.6%). AS can also increase the decolorization activity of TsL towards EB (93.5%), MO (87.0%), MG (77.9%), RBBR (77.7%), FB (76.5%), and EBT (75.7%). TEM was the third enhancer for TsL during the decolorization of MG (84.6%), FB (67.4%), and MO (62.2%). The best mediators were VA for EB, FB,

RBBR, MO, and MG, and AS for EBT, respectively. However, HBT was inefficient in the LMS of TsL towards the assayed dyes. In addition, they were nearly devoid of decolorization activity towards MB.

4. Discussion It is well known that laccases from many fungal species often occur as groups of isoenzymes which are encoded by gene families, e.g., Agaricus bisporus, Cerrena unicolor, Trametes villosa, and Trametes sanguinea [3,6,28]. Laccase isoenzymes demonstrate physiological functions in different physiological states among the total life cycle including spore, mycelia, and fruiting bodies [3,9]. They share a highly similar primary structure but demonstrate differences in physico-chemical characteristics and expression level,

Fig. 4. Effect of pH and temperature on activity and stability of the purified laccase TsL: (A) optimal pH; (B) optimal temperature; (C) pH stability on pH 2.2 (△), pH 2.6 (:), pH 3.0 (A), and pH 3.4 (,); (D) thermal stability at 40  C (-), 45  C (△), 50  C (C), 55  C (A)and 60  C (>).

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Fig. 5. Dye decolorization by LMS: (A) eriochrome black T (EBT), (B) evans blue (EB), (C) methyl orange (MO), (D) bromophenol blue (BMB), (E) fuchsin basic (FB), (F) malachite green (MG), (G) remazol brilliant blue R (RBBR), (H) methylene blue (MB). L, laccase; VA, violuric acid; HBT, 1-hydroxybenzotriazole; TEM, (2, 2, 6, 6-tetramethylpiperidin-1-yl) oxidanyl; AS, acetosyringone.

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which makes difficult to purify individual isoenzymes from fungal cultures for analysis and application [28,29]. In the present study, we purified an extracellular laccase from T. trogii strain F1635 with enzymatic and application characteristics different from those reported. During the purification process, TsL was effectively adsorbed on anion exchange chromatography of DEAE-Sepharose and QSepharose, and strong cation exchange chromatography of SPSepharose, but unadsorbed on weak cation exchange chromatography of CM-cellulose (data not shown). Many fungal laccases commonly interact with anion exchange chromatography rather than cation ones, for instance, laccases from T. sanguinea, C. unicolor, and Leucoagaricus naucinus [18e20]. However, TsL was not adsorbed on CM-cellulose, which is different from laccases from Lepista nuda and Hericium coralloides [21,30]. TsL was determined to be a monomeric protein with the Mr of 64.8 kDa which falls well within the molecular mass range of most of the fungal laccases reported (50e90 kDa) [3,6]. Laccase isoenzymes LacI and LacII from T. trogii strain B6J, Lac1 from T. trogii BAFC 463, and lac from T. trogii SYBC-LZ possess Mr of 62, 62, 60, and 65 kDa, respectively [27,31,32]. They demonstrate similar Mr and subunit composition characteristics close to those of TsL. Nevertheless, Lac 2 of T. trogii BAFC 463 possesses a Mr of 38 kDa [32]. Just like TsL, most fungal laccases are monomeric proteins besides few ones which are dimeric, trimeric, and tetrameric proteins. Laccase from Trametes sp. AH28-2 is a dimeric protein of about 110 kDa, and laccase from T. versicolor is a four-subunit protein of 222 kDa [33,34]. It is worth mentioning that subunits of these multimeric laccases possess similar Mr in the molecular mass range of most fungal laccases. N-terminal and inner amino acid sequences of the purified laccase were analyzed using Edman degradation and LC-MS/MS, respectively. A homology search based on N-terminal amino acid sequence reveals that TsL possesses high identify with laccases from Polyporus species (e.g., A. biennis, C. gallica, G. lucidum, and T. versicolor), but low identify with those from other taxa (e.g. Russula virescens and Pleurotus nebrodensis) [26,35,36]. Furthermore, N-terminal amino acid sequence (1e15) of TsL obtained from Edman degradation analysis demonstrates about 80% similarity with inner amino acid sequence (about 21e35) of Trametes spp. laccases obtained from gene cloning, suggesting that the present laccase from T. trogii strain F1635 possesses a signal peptide sequence of about 20 bp. Inner amino acid sequences lac1 and lac3 of TsL are highly conserved sequences in the second cupredoxin domain of T. versicolor laccase, while lac2 is highly conserved sequences in the first cupredoxin domain of T. versicolor laccase [28,37]. The N-terminal and inner peptide sequences represent the unique partial sequences of the purified laccase, which can ensure that TsL is novel and encoded by different laccase genes. Based on the UVevisible absorption spectrum study of the purified laccase, TsL possesses a slight shoulder at 340 nm, but is devoid of any peak near 600 nm. Accordingly, the purified laccase lacks of the T1 site copper atom, suggesting that TsL is not a typical blue laccase and is a so-called yellow laccase just like laccases from L. naucinus and Panus tigrinus [19,38]. On the other hand, most of the laccase isoenzymes from T. trogii are typical blue laccases. For example, an isoenzyme from T. trogii YDHSD is a blue laccase with an absorption peak at 610 nm for T1 site [15]. Just like many other fungal laccases, TsL possesses an acidic pH optimum of 2.6 and a considerably high temperature optimum of 50  C towards ABTS. A 56 kDa laccase isoenzyme from T. trogii S0301 exhibits an optimal pH of 3.0 and an optimal temperature of 45  C using ABTS as substrate [39]. Another isoenzyme from T. trogii YDHSD is a 64 kDa blue laccase and acts optimally at pH of 2.2e4.5 and temperature of 70  C [15]. TsL demonstrates considerably high

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pH and thermo stability at pH of 2.2e3.4 and temperature of 40e50  C. Two isoenzymes of around 62 kDa from T. trogii B6J show their highest oxidation activity towards ABTS at pH 2 and are stable up to 50  C for 24 h [27]. Laccase from Trametes sp. LAC-01 is stable also at pH 2.2e3.4 and maintains most of enzyme activity after incubated at 50e70  C for 60 min [18]. Oxidation activity of the purified laccase is inhibited by most of the assayed divalent metal ions of concentration of 10 mM including Ca2þ, Cd2þ, Co2þ, Cu2þ, Mg2þ, and Mn2þ. In general, fungal laccases exhibit widely divergent reflections by metal ions, especially Ca2þ and Cu2þ. Laccase from Trametes pubescens demonstrates metal-tolerant to Ca2þ, Mg2þ, and Mn2þ, and about 10% increase by Cu2þ at concentration of 25.0 mM [40]. Laccase from T. trogii YDHSD is decreased by Kþ, Naþ, Ca2þ, Cu2þ, Mn2þ, and Mg2þ of different degrees with 78.5%, 83.1%, 68.8%, 80.9%, 91.8%, and 95.1% relative activity remained, respectively [15]. Based on the UVevisible absorption spectrum study, TsL is a yellow laccase lacking of the T1 site copper ion. Metal ions (e.g. Ca2þ, Cu2þ and Mg2þ) can bind near the T1 site of laccase and act as competitive inhibitors electron donors by blocking the access of substrates to the T1 site or inhibiting the electron transfer at the T1 active site [41,42]. The purified laccase shows efficient oxidation activity towards ABTS with Km and Vmax of 18.58 mM and 1.35 mmol/min, respectively. Laccase isoenzymes from T. trogii strain S0301, SYBC-LZ, 201, B6J (LacI and LacII) possess much higher Km values towards ABTS of 69, 42, 30, 50, and 53 mM, respectively [27,31,39,43]. It suggests that TsL has a higher affinity to ABTS than other isoenzymes reported. Fungal laccases and their LMS can catalyse oxidation of various phenolic and aromatic compounds to products that very often are colourful and may be used as dyes, especially in the textile industry [12,13,44]. Among them, T. trogii laccases are quite effective in dyedecolorizing or biomechanical pulping. Crude laccase from T. trogii SYBC-LZ in solid substrate fermentation possesses good decolorizing activity towards RBBR (85.2%), reactive blue 4 (69.6%), acid blue 129 (45.6%), acid red 1 (90.2%), and reactive black 5 (65.4%) [45]. Laccase isoenzyme from T. trogii BAFC 463 is capable of decolorizing activity of 50e100% towards indigoid, triarylmethane, azoic and anthraquinonic synthetic dyes [46]. In the present study, TsL possesses effective decolorization activity towards EB, RBBR, MG, and EBT (over 60%) after incubating for 24 h when it is used alone. VA and AS are good candidates for LMS of TsL in dye treatment. They can not only improve the decolorization efficiency but also shorten the decolorization time. Since laccases and their LMS demonstrate great potentials in industrial and environmental applications, optimal mediators and their applying conditions have been widely investigated [5,13,24,25]. ABTS and VA are optimal mediators to oxidize sulfonamide antibiotics [24]. Jin et al. (2016) reported that VA, HBT, and vanillin were optimal mediators of T. versicolor laccase for pesticide degradation such as pyrimethanil, isoproturon, chlorothalonil, ect [13].

5. Conclusion In summary, a novel extracellular laccase TsL was purified from fermentation broth of the white rot fungus Trametes sp. F1635. It was a yellow laccase with considerably high pH and temperature stabilities. As expected, TsL manifested effective decolorization activity towards industrial dyes EB, RBBR, MG, and EBT. The mediators VA and AS can be used as good candidates for LMS of TsL in dye treatment towards EBT, EB, MO, etc. Both TsL and its LMS were nearly devoid of decolorization activity towards MB. The study indicates that TsL possesses potential application for decolorization of synthetic dyes and waste water treatment.

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Competing financial interests The authors declare no competing financial interests. Acknowledgements This work was financially supported by Beijing Nova Program (XX2015B025), National Grant of China (31501813), Opening Foundation of Key Laboratory of Urban Agriculture (North China) of Ministry of Agriculture (kf2017012), and Undergraduate Research Program of Beijing University of Agriculture.

[23]

[24]

[25]

[26]

[27]

References [1] C. Sanchez, Lignocellulosic residues: biodegradation and bioconversion by fungi, Biotechnol. Adv. 27 (2009) 185e194. [2] C.A. Ottoni, C. Santos, Z. Kozakiewicz, N. Lima, White-rot fungi capable of decolourising textile dyes under alkaline conditions, Folia Microbiol. (Praha) 58 (2013) 187e193. [3] C.M. Rivera-Hoyos, E.D. Morales-Alvarez, R.A. Poutou-Pinales, A.M. PedrozaRodriguez, R. Rodriguez-Vazquez, J.M. Delgado-Boada, Fungal laccases, Fungal Biol. Rev. 27 (2013) 67e82. [4] M. Vrsanska, S. Voberkova, V. Langer, D. Palovcikova, A. Moulick, V. Adam, P. Kopel, Induction of laccase, lignin peroxidase and manganese peroxidase activities in white-rot fungi using copper complexes, Molecules 21 (2016) 1553. [5] T. Senthivelan, J. Kanagaraj, R.C. Panda, Recent trends in fungal laccase for various industrial applications: an eco-friendly approach-a review, Biotech. Bioproc. E 21 (2016) 19e38. [6] P. Baldrian, Fungal laccases - occurrence and properties, FEMS Microbiol. Rev. 30 (2006) 215e242. [7] O.V. Morozova, G.P. Shumakovich, M.A. Gorbacheva, S.V. Shleev, A.I. Yaropolov, “Blue” laccases, Biochemistry (Moscow) 72 (2007) 1396e1412. [8] C. Hautphenne, M. Penninckx, F. Debaste, Product formation from phenolic compounds removal by laccases: a review, Environ. Technol. Innov. 5 (2016) 250e266. [9] P. Giardina, V. Faraco, C. Pezzella, A. Piscitelli, S. Vanhulle, G. Sannia, Laccases: a never-ending story, CMLS 67 (2010) 369e385. [10] U.N. Dwivedi, P. Singh, V.P. Pandey, A. Kumar, Structureefunction relationship among bacterial, fungal and plant laccases, J. Mol. Catal. B Enzym. 68 (2011) 117e128. [11] A.I. Canas, S. Camarero, Laccases and their natural mediators: biotechnological tools for sustainable eco-friendly processes, Biotechnol. Adv. 28 (2010) 694e705. [12] R. Khlifi, L. Belbahri, S. Woodward, M. Ellouz, A. Dhouib, S. Sayadi, T. Mechichi, Decolourization and detoxification of textile industry wastewater by the laccase-mediator system, J. Hazard Mater. 175 (2010) 802e808. [13] X. Jin, X. Yu, G. Zhu, Z. Zheng, F. Feng, Z. Zhang, Conditions optimizing and application of laccase-mediator system (LMS) for the laccase-catalyzed pesticide degradation, Sci. Rep. 6 (2016) 35787. [14] F. Martinez-Morales, B. Bertrand, A.A. Pasion Nava, R. Tinoco, L. AcostaUrdapilleta, M.R. Trejo-Hernandez, Production, purification and biochemical characterization of two laccase isoforms produced by Trametes versicolor grown on oak sawdust, Biotechnol. Lett. 37 (2015) 391e396. [15] M.Q. Ai, F.F. Wang, F. Huang, Purification and characterization of a thermostable laccase from Trametes trogii and its ability in modification of kraft lignin, J. Microbiol. Biotechnol. 25 (2015) 1361e1370. [16] A. Kocyigit, M.B. Pazarbasi, I. Yasa, G. Ozdemir, I. Karaboz, Production of laccase from Trametes trogii TEM H2: a newly isolated white-rot fungus by air sampling, J. Basic Microbiol. 52 (2012) 661e669. [17] J. Sun, Q.J. Chen, M.J. Zhu, H.X. Wang, G.Q. Zhang, An extracellular laccase with antiproliferative activity from the sanghuang mushroom Inonotus baumii, J. Mol. Catal. B Enzym. 99 (2014) 20e25. [18] Z.R. Ling, S.S. Wang, M.J. Zhu, Y.J. Ning, S.N. Wang, B. Li, A.Z. Yang, G.Q. Zhang, X.M. Zhao, An extracellular laccase with potent dye decolorizing ability from white rot fungus Trametes sp. LAC-01, Int. J. Biol. Macromol. 81 (2015) 785e793. [19] Y.J. Ning, S.S. Wang, Q.J. Chen, Z.R. Ling, S.N. Wang, W.P. Wang, G.Q. Zhang, M.J. Zhu, An extracellular yellow laccase with potent dye decolorizing ability from the fungus Leucoagaricus naucinus LAC-04, Int. J. Biol. Macromol. 93 (2016) 837e842. [20] S.S. Wang, Y.J. Ning, S.N. Wang, J. Zhang, G.Q. Zhang, Q.J. Chen, Purification, characterization, and cloning of an extracellular laccase with potent dye decolorizing ability from white rot fungus Cerrena unicolor GSM-01, Int. J. Biol. Macromol. 95 (2017) 920e927. [21] M. Zhu, G. Zhang, L. Meng, H. Wang, K. Gao, T. Ng, Purification and characterization of a white laccase with pronounced dye decolorizing ability and HIV-1 reverse transcriptase inhibitory activity from Lepista nuda, Molecules 21 (2016) 415. [22] S. Sondhi, P. Sharma, S. Saini, N. Puri, N. Gupta, Purification and

[28] [29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

characterization of an extracellular, thermo-alkali-stable, metal tolerant laccase from Bacillus tequilensis SN4, PLoS One 9 (2014), e96951. S. Ostadhadi-Dehkordi, M. Tabatabaei-Sameni, H. Forootanfar, S. Kolahdouz, M. Ghazi-Khansari, M.A. Faramarzi, Degradation of some benzodiazepines by a laccase-mediated system in aqueous solution, Bioresour. Technol. 125 (2012) 344e347. S.S. Weng, S.M. Liu, H.T. Lai, Application parameters of laccase-mediator systems for treatment of sulfonamide antibiotics, Bioresour. Technol. 141 (2013) 152e159. N. Anders, M. Schelden, S. Roth, A.C. Spiess, Automated chromatographic laccase-mediator-system activity assay, Anal. Bioanal. Chem. 409 (2017) 4801e4809. G.Q. Zhang, T. Tian, Y.P. Liu, H.X. Wang, Q.J. Chen, A laccase with antiproliferative activity against tumor cells from a white root fungus Abortiporus biennis, Process Biochem. 46 (2011) 2336e2340. H. Zouari-Mechichi, T. Mechichi, A. Dhouib, S. Sayadi, A.T. Martinez, M.J. Martinez, Laccase purification and characterization from Trametes trogii isolated in Tunisia: decolorization of textile dyes by the purified enzyme, Enzym. Microb. Technol. 39 (2006) 141e148. U. Kues, M. Ruhl, Multiple multi-copper oxidase gene families in basidiomycetes - what for? Curr. Genet. 12 (2011) 72e94. C. Gu, F. Zheng, L. Long, J. Wang, S. Ding, Engineering the expression and characterization of two novel laccase isoenzymes from Coprinus comatus in Pichia pastoris by fusing an additional ten amino acids tag at N-terminus, PLoS One 9 (2014), e93912. Y.J. Zou, H.X. Wang, T.B. Ng, C.Y. Huang, J.X. Zhang, Purification and characterization of a novel laccase from the edible mushroom Hericium coralloides, J. Microbiol. 50 (2012) 72e78. X. Zeng, Y. Cai, X. Liao, X. Zeng, S. Luo, D. Zhang, Anthraquinone dye assisted the decolorization of azo dyes by a novel Trametes trogii laccase, Process Biochem. 47 (2012) 160e163. L. Levin, F. Forchiassin, A.M. Ramos, Copper induction of lignin-modifying enzymes in the white-rot fungus Trametes trogii, Mycologia 94 (2002) 377e383. H. Ge, Y. Gao, Y. Hong, M. Zhang, Y. Xiao, M. Teng, L. Niu, Structure of native laccase B from Trametes sp. AH28-2, Acta Cryst. Sect. F Struct. Biol. Commun. 66 (2010) 254e258. T. Bertrand, C. Jolivalt, P. Briozzo, E. Caminade, N. Joly, C. Madzak, C. Mougin, Crystal structure of a four-copper laccase complexed with an arylamine: insights into substrate recognition and correlation with kinetics, Biochemistry 41 (2002) 7325e7333. G.T. Tian, G.Q. Zhang, H.X. Wang, T.B. Ng, Purification and characterization of a novel laccase from the mushroom Pleurotus nebrodensis, Acta Biochim. Pol. 59 (2012) 407e412. M.J. Zhu, F. Du, G.Q. Zhang, H.X. Wang, T.B. Ng, Purification of a laccaseexhibiting dye decolorizing ability from an edible mushroom Russula virescens, Int. Biodeter. Biodegr. 82 (2013) 33e39. A. Marchler-Bauer, Y. Bo, L. Han, J. He, C.J. Lanczycki, S. Lu, F. Chitsaz, M.K. Derbyshire, R.C. Geer, N.R. Gonzales, M. Gwadz, D.I. Hurwitz, F. Lu, G.H. Marchler, J.S. Song, N. Thanki, Z. Wang, R.A. Yamashita, D. Zhang, C. Zheng, L.Y. Geer, S.H. Bryant, CDD/SPARCLE: functional classification of proteins via subfamily domain architectures, Nucleic Acids Res. 45 (2017) D200eD203. A. Leontievsky, N. Myasoedova, N. Pozdnyakova, L. Golovleva, 'Yellow' laccase of Panus tigrinus oxidizes non-phenolic substrates without electron-transfer mediators, FEBS Lett. 413 (1997) 446e448. J. Yan, D. Chen, E. Yang, J. Niu, Y. Chen, I. Chagan, Purification and characterization of a thermotolerant laccase isoform in Trametes trogii strain and its potential in dye decolorization, Int. Biodeter. Biodegr. 93 (2014) 186e194. J. Si, F. Peng, B. Cui, Purification, biochemical characterization and dye decolorization capacity of an alkali-resistant and metal-tolerant laccase from Trametes pubescens, Bioresour. Technol. 128 (2013) 49e57. N. Hakulinen, M. Andberg, J. Kallio, A. Koivula, K. Kruus, J. Rouvinen, A near atomic resolution structure of a Melanocarpus albomyces laccase, J. Struct. Biol. 162 (2008) 29e39. Z.M. Fang, T.L. Li, F. Chang, P. Zhou, W. Fang, Y.Z. Hong, X.C. Zhang, H. Peng, Y.Z. Xiao, A new marine bacterial laccase with chloride-enhancing, alkalinedependent activity and dye decolorization ability, Bioresour. Technol. 111 (2012) 36e41. A.M. Garzillo, M.C. Colao, C. Caruso, C. Caporale, D. Celletti, V. Buonocore, Laccase from the white-rot fungus Trametes trogii, Appl. Microbiol. Biotechnol. 49 (1998) 545e551. E.M. Cadena, T. Vidal, A.L. Torres, Can the laccase mediator system affect the chemical and refining properties of the eucalyptus pulp? Bioresour. Technol. 101 (2010) 8199e8204. X. Zeng, Y. Cai, X. Liao, X. Zeng, W. Li, D. Zhang, Decolorization of synthetic dyes by crude laccase from a newly isolated Trametes trogii strain cultivated on solid agro-industrial residue, J. Hazard Mater. 187 (2011) 517e525. P.A. Campos, L.N. Levin, S.A. Wirth, Heterologous production, characterization and dye decolorization ability of a novel thermostable laccase isoenzyme from Trametes trogii BAFC 463, Process Biochem. 51 (2016) 895e903. S. Zhao, C.B. Rong, C. Kong, Y. Liu, F. Xu, Q.J. Miao, S.X. Wang, H.X. Wang, G.Q. Zhang, A novel laccase with potent antiproliferative and HIV-1 reverse transcriptase inhibitory activities from mycelia of mushroom Coprinus comatus, BioMed Res. Int. 2014 (2014) 417461.