Bioresource Technology 128 (2013) 49–57

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Bioresource Technology

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Purification, biochemical characterization and dye decolorization capacityof an alkali-resistant and metal-tolerant laccase from Trametes pubescens

Jing Si a, Feng Peng b, Baokai Cui a,⇑a Institute of Microbiology, Beijing Forestry University, Beijing 100083, Chinab Institute of Biomass Chemistry and Technology, Beijing Forestry University, Beijing 100083, China

h i g h l i g h t s

" A novel laccase (Tplac) from white rot fungus Trametes pubescens was purified and characterized." Tplac performed better catalytic efficiency toward ABTS with kcat/Km at 8.34 s�1 lM�1." Tplac was highly stable and resistant under alkaline conditions." Tplac was intrinsically highly metal-tolerant by enhancing the affinity toward substrate." Tplac could degrade and detoxify dyes used in textile industries.

a r t i c l e i n f o

Article history:Received 5 July 2012Received in revised form 16 October 2012Accepted 19 October 2012Available online 29 October 2012

Keywords:Trametes pubescens laccasePurificationAlkali-resistant capacityMetal toleranceDye decolorization application

0960-8524/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.biortech.2012.10.085

⇑ Corresponding author. Address: Institute of MUniversity, P.O. Box 61, Qinghuadong Road 35, HaidChina. Tel./fax: +86 10 62336309.

E-mail address: baokaicui@yahoo.com.cn (B. Cui).

a b s t r a c t

Extracellular laccase (Tplac) from Trametes pubescens was purified to homogeneity by a three-stepmethod, which resulted in a high specific activity of 18.543 U mg�1, 16.016-fold greater than that ofcrude enzyme at the same level. Tplac is a monomeric protein that has a molecular mass of 68 kDa.The enzyme demonstrated high activity toward 1.0 mM ABTS at an optimum pH of 5.0 and temperatureof 50 �C, and under these conditions, the catalytic efficiency (kcat/Km) is 8.34 s�1 lM�1. Tplac is highly sta-ble and resistant under alkaline conditions, with pH values ranging from 7.0 to 10.0. Interestingly, above88% of initial enzyme activity was maintained in the presence of metal ions at 25.0 mM, leading to anincrease in substrate affinity, which indicated that the laccase is highly metal-tolerant. These unusualproperties demonstrated that the new fungal laccase Tplac has potentials for the specific industrial orenvironmental applications.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Laccase (benezenediol: oxygen oxidoreductase, EC 1.10.3.2), themost abundant member of the multicopper protein family, iswidely distributed in plants, fungi, insects, and bacteria (Claus,2004). This protein contains four histidine-rich copper binding do-mains, which coordinate copper atoms types I–III that differ intheir environment and spectral properties (Thurston, 1994). Theenzyme can catalyze the oxidation of an array of substrates, suchas mono-, di-, and polyphenols, aromatic amines, methoxyphenols,and ascorbate through a one-electron transfer. The oxidation iscoupled to the reduction of oxygen to H2O (Thurston, 1994). Fur-thermore, laccase is of particular interest with regards to variouscommercial applications because of its ability to oxidize a wide

ll rights reserved.

icrobiology, Beijing Forestryian District, Beijing 100083,

range of reaction capabilities and relevant substrate specificities.Thus, research concerning laccase is being carried out in variousfields of interest: textile, pulp and paper, food, and cosmeticsindustries, as well as in bioremediation, biosensor, biofuel, andorganic synthesis applications (Arora and Sharma, 2010). To date,more than 100 laccases have been isolated from different microor-ganisms. However, most of these laccases are ‘common’ with alower yield of enzymatic activity and tolerance to extreme condi-tions (Kim et al., 2012). This reduced performance hampers theirlarge-scale commercial and industrial use for most applications.Therefore, it is necessary to search for novel laccases with higheryields of activity and versatile properties.

Global industrialization has resulted in the release of largeamounts of potentially toxic compounds into the biosphere (Gomiet al., 2011). Among these compounds, dye-containing effluentsrepresent highly problematic wastewaters due to their higherchemical (COD) and biochemical oxygen demand (BOD), sus-pended solids, and the content of toxic compounds, as well as theircolor, which makes them easily recognized and poses esthetic

50 J. Si et al. / Bioresource Technology 128 (2013) 49–57

problems (Jonstrup et al., 2011). Cleaning up the environment byremoval of hazardous contaminants from textile effluents is a cru-cial and challenging problem that requires numerous approachesto reach long-lasting suitable solutions. Among the various typesof dyes in the textile processing industry, azo dyes are extensivelyused, and they dominate the dyestuff market with a share ofapproximately 70% (Enayatizamir et al., 2011). Physical and chem-ical methods have been adopted in the treatment of azo dyes, butthey have led to the generation of secondary pollution by releasinghazardous byproducts (Kalpana et al., 2011). Thus, microbial treat-ment of dyes has gained popularity due to its safety, efficiency, andability to transform hazardous chemicals into less toxic com-pounds (Asgher et al., 2008).

White rot basidiomycetes are among the most potent organ-isms to biodegrade and detoxify a wide range of wastes and pollu-tants, as carried out by phenol-targeting redox enzymes, namely,laccases and peroxidases (Mendonça et al., 2008). However, waste-water discharged from textile industries characterized by neutralor alkaline pHs and high concentrations of metal ions is causingserious threats and severely damaging the natural habitat (Xiaoet al., 2012). These conditions limit the functions of fungal laccasesand can cause them to lose their activities. Thus, exploring novellaccases that can be directly used under the aforementioned spe-cial conditions is an important job in the area of dye degradation.

Trametes pubescens is a common white-rot fungus, and its crudeenzyme, which was previously extracted and acclimatized, wasused for dye decolorization (Roriz et al., 2009). Accordingly, thepresent paper reports on the purification and biochemical charac-terization of a novel alkali-resistant and metal ion-tolerant laccaseTplac from white rot fungus T. pubescens. The enzyme was purifiedby anionic exchange and Sepharose chromatography and evaluatedfor its potentials for dye decolorization.

2. Methods

2.1. Dyes and chemicals

The dyes used in this study were prepared by being filteredthrough a 0.22-lm membrane to remove bacteria before use. Forthis study, 2,20-Azino-bis(3-ethylbenzothiazoline-6-sulfonate)(ABTS), agar powder, and trypsin were all Sigma–Aldrich products(St. Louis, MO, USA). L-Cysteine, L-3,4-dihydroxyphenylalanine(L-DOPA), 2,6-dimethoxyphenol (2,6-DMP), dithiothreitol (DTT),sodium azide (NaN3), and protein marker were purchased fromTakaRa (Dalian, China). Other chemicals used were of analyticalreagent grade.

2.2. Fungal strain and inocula preparation

T. pubescens Cui 7571 was collected from Chebaling Nature Re-serve of Guangdong Province in China. This strain was maintainedthrough periodic (monthly) transfer on yeast extract glucose agar(YGA) at 4 �C. The YGA medium used for the experiment contained(g L�1 of distilled water): yeast extract 5, glucose 20, agar 20, KH2PO4

1, MgSO4�7H2O 0.5, ZnSO4�7H2O 0.05, and vitamin B1 0.01, and thepH value of the medium was adjusted to 5.0 before sterilization.

Prior to use, the stored fungal strain was inoculated onto newlyprepared YGA plates and grown at 28 �C. Five mycelial disks (1 cmdiameter) were removed from the peripheral region of the 5-day-old YGA plate and used to inoculate into a 250-mL Erlenmeyerflask containing 100 mL of yeast extract glucose medium (YG, iden-tical to YGA without agar). The cultivation was carried out in a darkchamber under 150 rpm shaking speed at 28 �C. After 6 days,mycelia were homogenized using an Ace Homogenizer (Hengao

Co., Tianjin, China) at 5000 rpm for 30 s, and the pellet suspensionswere later prepared as inocula for the next experiment.

2.3. Production and purification of Tplac

An aliquot of 10 mL of the inocula (0.045 g, dry weight) wasinoculated into a 250-mL Erlenmeyer flask containing 100 mL ofYG medium and incubated at 28 �C in a shaking incubator. After6 days, the cultures were centrifuged at 12,000 rpm for 20 min toremove mycelia and medium debris, and the cell-free supernatantwas used as a crude enzyme solution.

The supernatant was salt fractionated with 75% (w/V) ammo-nium sulfate at 4 �C overnight and dialyzed with a 10 kDa cut-offmembrane against 0.1 M citrate–phosphate buffer (pH 5.0) andfurther concentrated by PEG 20000. The resulting solution wasthen loaded onto a DEAE-cellulose DE52 anionic exchange chroma-tography column (30 � 2.6 cm; Pharmacia) pre-equilibrated with0.1 M citrate–phosphate buffer (pH 5.0) overnight at a flow rateof 1.0 mL min�1. The laccase protein was eluted initially with0.1 M citrate–phosphate buffer (pH 5.0) and subsequently with alinear salt gradient of 0–1.0 M NaCl solution in 0.1 M citrate–phos-phate buffer (pH 5.0) at a flow rate of 2.0 mL min�1. Activity frac-tions were assayed for protein contents by the Bradford methodusing bovine serum albumin as standard protein (Bradford,1976), and the laccase activity of each fraction was determinedat 420 nm using 1.0 mM ABTS as substrate (Kalyani et al., 2008).One unit was defined as the amount of enzyme that oxidized1 lmol of substrate per minute. Fractions containing the main lac-case activity were collected, pooled, dialyzed, and concentrated byPEG 20000. Next, the eluted solution was applied to a SepharoseGL-6B chromatography column (60 � 2.6 cm; Pharmacia) pre-equilibrated with 0.1 M citrate–phosphate buffer (pH 5.0) over-night at a flow rate of 1.0 mL min�1. The laccase was re-eluted with1.0 M NaCl in 0.1 M citrate–phosphate buffer (pH 5.0) at1.5 mL min�1 and monitored as mentioned above. Finally, fractionscontaining the main laccase activity were collected, pooled, dia-lyzed, concentrated by PEG 20000, and stored at �20 �C until fur-ther use.

2.4. Biochemical characterization of purified Tplac

2.4.1. Gel electrophoresis and the spectral property of purified TplacThe purified laccase Tplac was subjected to sodium dodecyl sul-

fate–polyacrylamide gel electrophoresis (SDS–PAGE) for molecularmass determination. This assay was performed according to a pre-viously described protocol (Eisenman et al., 2007) with a 5% (w/V)stacking gel and a 12% (w/V) separating gel using a vertical gelelectrophoresis system (Bio-Rad). The sample was dissolved in 2volumes of 4� loading buffer and denatured by incubating at100 �C for 8 min. After electrophoresis, the gel was stained withCoomassie Brilliant Blue R-250 for 2 h at room temperature, andthe molecular mass of Tplac was measured by comparison with aprotein marker. Similarly, protein with laccase activity and its iso-enzyme were evaluated using non-denaturing PAGE (native PAGE)on a 5% stacking gel and a 12% separating gel. The native gel wasstained with 0.1 M citrate–phosphate buffer (pH 5.0) containing1.0 mM ABTS or 1.0 mM guaiacol.

The UV–visible adsorption spectrum of purified Tplac in 0.1 Mcitrate–phosphate buffer (pH 5.0) was recorded between 200 and800 nm with a UV–visible spectrophotometer (UNICO 4802, You-nike Co., Shanghai, China).

2.4.2. Internal amino acid sequence of purified TplacThe purified Tplac was loaded onto SDS–PAGE. After electro-

phoresis and protein visualization, the laccase bands of interestwere cut up from the gel and digested overnight using trypsin as

J. Si et al. / Bioresource Technology 128 (2013) 49–57 51

described earlier (Shevchenko et al., 1996). The cleaved peptideswere eluted and analyzed by nano liquid chromatography coupledwith tandem mass spectrometry (LC–MS/MS) for interest aminoacid sequencing. Amino acid sequences were identified by homol-ogy using an mass spectrometry data analysis program, SEQUEST(Thermo Finnigan, San Jose, CA, USA), against the database of theNational Center for Biotechnology Information (NCBI) fungal lac-case sequence database, and aligned across thirteen laccases byClustalX1.83 algorithm and DNAMAN6.0 software.

2.4.3. Effects of pH and temperature on the activity and stability ofpurified Tplac

The effect of pH value on Tplac activity was determined in thecitrate–phosphate buffer within a pH range of 1.0–13.0 at 25 �Cusing 1.0 mM ABTS as substrate. The pH stability of the enzymewas assessed by pre-incubating the enzyme in citrate–phosphatebuffer with pH values ranging from 1.0 to 13.0 at 25 �C for 72 h,and the residual laccase activities were determined with ABTS assubstrate.

The optimum temperature for Tplac was examined in the cit-rate–phosphate buffer with different temperatures from 10 to90 �C at pH 5.0 using 1.0 mM ABTS as substrate. The thermal stabil-ity of the purified laccase was evaluated by pre-incubating the en-zyme in citrate–phosphate buffer (pH 5.0) with differenttemperatures from 10 to 90 �C for 2 h, and the residual laccaseactivities were determined with ABTS as substrate.

Aliquots of samples were taken at regular intervals and werecentrifuged at 12,000 rpm for 20 min and the supernatant wasused for laccase activity determination. Experiments were all per-formed in triplicate and laccase activities at the optimum pH ortemperature were taken as control (100%).

2.4.4. Substrate specificity and kinetic property of purified TplacVarious substrates, i.e., ABTS, catechol, 2,6-DMP, L-DOPA, ferulic

acid, guaiacol, hydroquinone, phenol, pyrogallol, syringaldazine,tyrosine, and veratryl alcohol, were used to determine the sub-strate specificity of Tplac at 1.0 mM in 0.1 M citrate–phosphatebuffer (pH 5.0). The rate of substrate oxidation was determinedby measuring the absorbance increase at the respective wave-length, and the molar extinction coefficient (em) of each substratewas obtained from the literature (Eisenman et al., 2007; Kalyaniet al., 2012; Litthauer et al., 2007). Experiments were all performedin triplicate.

The apparent Michaelis–Menten constant (Km) and catalyticconstant (kcat) of Tplac were determined using ABTS as substratein 0.1 M citrate–phosphate buffer (pH 5.0) at 50 �C. A Linewe-aver–Burk plot was made from the initial oxidation rates at differ-ent ABTS concentrations ranging from 0.1 mM to 1.0 mM. Thecatalytic efficiency (specificity constant, kcat/Km) of the purified en-zyme was calculated according to Km and kcat data.

2.4.5. Effects of inhibitors and metal ions on the activity of purifiedTplac

The effects of various inhibitors (0.05, 0.1, and 1.0 mM) on puri-fied Tplac activity were investigated using L-cysteine, DTT, EDTA,and NaN3. The remaining laccase activity was measured by pre-incubating the purified enzyme in the presence of each inhibitorat 50 �C for 15 min using ABTS as substrate. Experiments were allperformed in triplicate.

The effects of various metal ions, at concentration of 25.0 mM,on purified Tplac activity were also evaluated by separately addingCu2+ (copper sulfate), K+ (potassium chloride), Na+ (sodium chlo-ride), Mn2+ (manganese sulfate), Ca2+ (calcium chloride), Fe2+ (fer-rous sulfate), Fe3+ (ferric chloride), Mg2+ (magnesium chloride),Zn2+ (zinc sulfate), Ba2+ (barium chloride), or Al3+ (aluminum chlo-ride) to the reaction mixture. Similarly, the enzymatic assays were

conducted under the aforementioned conditions, and experimentswere all performed in triplicate. Laccase activity determined at theoptimum pH and temperature conditions in the absence of anyinhibitor or metal ion was taken as control (100%). Km and kcat val-ues of purified Tplac in the presence of metal ions at 25.0 mM weredetermined using ABTS as substrate.

2.5. Dye decolorization capacity of purified Tplac

2.5.1. Dye decolorizationThe decolorization capacity of the purified laccase Tplac for

structurally various dyes was monitored by the decrease in absor-bance at the wavelength of each dye. The 10.0 mL reaction mix-tures for dye decolorization contained 50.0 mg L�1 dye in 0.1 Mcitrate–phosphate buffer (pH 5.0) and 1.0 U mL�1 pure enzymesolution. In all the cases, the mixtures were incubated in a darkchamber under 150 rpm shaking speed at 50 �C. In parallel, thenegative control contained all components except enzyme, andexperiments were all performed in triplicate. Aliquots of sampleswere taken at regular intervals and were centrifuged at12,000 rpm for 20 min and the supernatant was used for decolor-ization determination. Decolorization rate was expressed in termsof percentage and calculated as follows:

Decolorization rate ð%Þ ¼ Initial absorbance� Final absorbanceInitial absorbance

� 100

2.5.2. Effects of heavy metal ions on the dye decolorization capacity ofpurified Tplac

The effects of heavy metal ions on Congo Red biodegradationcapacity of purified Tplac were studied by separately adding Cu2+

(copper sulfate), Zn2+ (zinc sulfate), or Fe3+ (ferric chloride) intothe dye decolorization reaction mixtures and their concentrationswere varied in the range of 20.0–60.0 mM. Similarly, experimentswere all performed in triplicate, and the decolorization rate deter-mination was conducted under the aforementioned conditions.Dye decolorization rate determined in the absence of any heavymetal ion was taken as control.

2.5.3. Degraded metabolites identificationOnce complete dye decolorization was achieved, the metabo-

lites formed after biodegradation of Congo Red were extractedthree times with an equal volume of ethyl acetate with vigorousshaking. The combined organic phase was filtered over Na2SO4

on filter paper and concentrated in a rotary vacuum evaporator.GC–MS analysis was carried out using a QP 2010 mass spectropho-tometer (Shimadzu model No. U-2800). Ionization voltage was70 eV and the temperature of the injection port was 280 �C. Gaschromatography was conducted in temperature programmingmode with a Resteck column (0.25 � 30 mm, XTI-5). Initial columntemperature was 80 �C for 2 min, which was later increased line-arly at 10 �C per min up to 280 �C and held for 7 min. GC–MS inter-face was maintained at 290 �C and helium was used as the carriergas at a flow rate of 1.0 mL min�1 with a 30 min run time.

2.5.4. Phytotoxicity testThe ethyl acetate extracted metabolites of Congo Red formed

after biodegradation by Tplac were dried and dissolved in 50 mLof distilled water to a final concentration of 2.0 g L�1. Seeds ofPhaseolus mungo, Sorghum vulgare, and Triticum aestivum wereused for phytotoxicity tests, and the experiments were carriedout at room temperature by placing ten seeds in separate 5.0-mLof solutions containing either dye, metabolites, or distilled water.

52 J. Si et al. / Bioresource Technology 128 (2013) 49–57

Germination (%) and plumule (cm) and radicle (cm) lengths wererecorded after 7 days.

2.6. Statistical data analysis

The results obtained during experimentation were expressed interms of mean values and standard error means. Data weresubjected to statistical analysis of one-way analysis of variance(ANOVA) and Tukey–Kramer comparison test by using SPSS18.0software. Probability (P value) less than 0.05 or 0.01 (⁄P < 0.05 or⁄⁄P < 0.01) was considered significant or highly significantrespectively.

Fig. 1. Molecular mass determination of purified Tplac from Trametes pubescensthrough SDS–PAGE (a) (Lane 1 crude culture filtrate; Lane 2 purified laccase fromammonium sulfate precipitation; Lane 3 purified laccase from DEAE-cellulose DE52anionic exchange chromatography; Lane 4 purified laccase from Sepharose GL-6Bchromatography; M protein molecular mass marker) and zymogram analysis (b)with native PAGE (Lane 1 ABTS staining; Lane 2 guaiacol staining).

3. Results and discussion

3.1. Purification of laccase Tplac

Since the laccase constitutively produced by basidiomycete fun-gi can be used in many fields, it is necessary to develop an effectivelarge-scale, high-purity production process. In the present study,laccase Tplac of T. pubescens obtained from a 6-day culture wasused for subsequent purification. The purification of this laccasewas performed using a three-step method of ammonium sulfateprecipitation, anionic exchange, and Sepharose chromatography.After ammonium sulfate precipitation, a total amount of about8.390 mg mL�1 of protein, corresponding to approximately36.253 U mL�1 of laccase activity, was loaded onto DEAE-celluloseDE52 anionic exchange chromatography column eluted initiallywith buffer and subsequently with 0–1.0 M NaCl solution. Supple-mentary Fig. S1 depicts that there were two apparent fractionscontaining laccase activity during the elution procedure. Interest-ingly, the fraction containing the higher activity was observedwhen the eluent was 0.1 M citrate–phosphate buffer (pH 5.0),amounting to a specific activity of 5.798 U mg�1, which was5.008 times higher than that of crude enzyme at identical experi-mental conditions. Furthermore, the fraction was pooled and con-centrated and applied to Sepharose GL-6B chromatography columnfor further purification (Supplementary Fig. S2). Table 1 lists thepurification data of Tplac. Overall, the three-step procedure re-sulted in a high specific activity of 18.543 U mg�1, 16-fold greaterthan that of crude enzyme at the same level.

3.2. Gel electrophoresis and the spectral property of purified Tplac

As demonstrated in Fig. 1a, the homogeneity of the purifiedTplac was verified by SDS–PAGE with Coomassie Brilliant Blue R-250 staining analysis. A unique protein band was obtained forTplac, with a mobility corresponding to a molecular mass of68 kDa, which was higher than that of Trametes versicolor, whichhad a molecular mass of 60 kDa (Zhu et al., 2011). This could be ex-plained that various compositions of subunits exist in differentfungal laccases (Fang et al., 2012). Activity staining of Tplac also re-vealed a single band corresponding to the position of laccase activ-

Table 1Purification of laccase Tplac from the crude culture of Trametes pubescens.

Purification step Total activity(U mL�1)

Crude culture filtrate 44.253Ammonium sulfate precipitation 36.253DEAE-cellulose DE52 anionic exchange chromatography

column27.372

Sepharose GL-6B chromatography column 21.836

ity, as visualized with ABTS or guaiacol as substrate (Fig. 1b). Theseobservations suggested that the purified laccase from T. pubescensis a typical fungal laccase in molecular mass and a monomeric pro-tein in composition.

The nature of the catalytic center was determined by spectralproperty of the purified laccase (data were not shown). Tplac’sUV–vis spectrum exhibited a shoulder at 340 nm, typical of a typeIII binuclear copper center. An absorption peak at 610 nm indicatedthe presence of a type I copper center, which is considered to beresponsible for the enzyme’s blue color (Sadhasivam et al., 2008).

3.3. Internal amino acid sequence of purified Tplac

Internal peptide sequencing of the purified Tplac exhibited sev-eral fragments, such as HWHG, GTFWYHSHLSTQYCDGLRG,KRYRFRLVS, and NSAILRY, identical to those of published fungallaccases from Coriolopsis gallica, Dichomitus squalens, Lentinus tigri-nus, Polyporus brumalis, Polyporus ciliatus, Trametes sp. 420, Tra-metes sp. AH28-2, Trametes trogii, T. versicolor, and Trametesvillosa respectively, which belong to the multicopper oxidase fam-ily (Fig. 2). Additionally, results in Fig. 2 show that Tplac had twocopper binding domains (type I and II) and shared three potentialN-glycosylation sites (Thurston, 1994). A homology search re-vealed that the deduced gene product had 78.01%, 76.76%,82.16%, 82.16%, 80.50%, 82.16%, 76.35%, 79.67%, 76.76%, 76.35%,77.18%, or 77.18% amino acid identify with PDB: 2VDZ (C. gallica),EJF60081 (D. squalens), AAX07469.1 (L. tigrinus), PDB: 2QT6 (L.tigrinus), ABN13591.1 (P. brumalis), AAG09231.1 (P. ciliatus),AAW28936.1 (Trametes sp. 420), PDB: 3KW7 (Trametes sp. AH28-2), PDB: 2HRG (T. trogii), CAA77015 (T. versicolor), EIW62366 (T.versicolor), or AAB47735 (T. villosa) respectively. These results im-plied that Tplac from T. pubescens is a typical laccase with the con-served copper binding sites and it has some differences from otherfungal laccases, but a new protein.

Total protein(mg mL�1)

Specific activity(U mg�1)

Purificationfold

Yield(%)

38.223 1.158 1.000 1008.390 4.321 3.732 81.924.721 5.798 5.008 61.85

1.178 18.543 16.016 49.34

Fig. 2. Multiple amino acid sequences alignments of purified Tplac from Trametes pubescens with other fungal laccases. The accession numbers were: PDB: 2VDZ (Coriolopsisgallica), EJF60081 (Dichomitus squalens), AAX07469.1 (Lentinus tigrinus), PDB: 2QT6 (Lentinus tigrinus), ABN13591.1 (Polyporus brumalis), AAG09231.1 (Polyporus ciliatus),AAW28936.1 (Trametes sp. 420), PDB: 3KW7 (Trametes sp. AH28-2), PDB: 2HRG (Trametes trogii), CAA77015 (Trametes versicolor), EIW62366 (Trametes versicolor), andAAB47735 (Trametes villosa). The ClustalX1.83 algorithm and DNAMAN6.0 software were used for alignment. Residue positions identical in all thirteen sequences wereindicated with a black background. Conserved copper binding residues were boxed in red. Potential N-glycosylation sites of Tplac were marked with red arrows. Theunderline suggested the amino acids obtained from nano liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) sequencing.

J. Si et al. / Bioresource Technology 128 (2013) 49–57 53

3.4. Effects of pH and temperature on the activity and stability ofpurified Tplac

As shown in Fig. 3a, purified laccase Tplac from T. pubescens dis-played activity over a broad pH range of 4.5–11.0 with an optimum

pH at 5.0, and under this condition, the laccase activity was up to22.157 U mL�1. The pH stability profile shows that Tplac washighly stable over a broad range of pH values ranging from 4.5–10.0, maintaining 75% of its original activity after incubation at25 �C for 72 h. When the pH at 5.0, the laccase activity was

0

20

40

60

80

100

120

0 2 4 6 8 10 12 14

pH value

Rel

ativ

e la

ccas

e ac

tivi

ty (

%)

pH optimum pH stability

0

20

40

60

80

100

120

0 20 40 60 80 100Temperature (°C)

Rel

ativ

e la

ccas

e ac

tivi

ty (

%)

Temperature optimum Temperature stability(a) (b)

Fig. 3. Effects of pH (a) and temperature (b) on the activity and stability of purified Tplac from Trametes pubescens. The values of 100% relative laccase activity toward ABTS forpH and temperature were 22.157 and 28.687 U mL�1 respectively. Each value is the mean value ± standard error mean of triplicate.

54 J. Si et al. / Bioresource Technology 128 (2013) 49–57

20.218 U mL�1 after reaction for 72 h. By contrast, the enzyme wasunstable at lower pH values, exhibiting approximately 5% of its ori-ginal activity after incubation at pH 1.0–4.0 for 72 h. Most fungallaccases are functional at acidic or neutral pH values but lose theiractivities under alkaline conditions (Zhang et al., 2009; Zou et al.,2012). Thus, the high activity, stability, and resistant ability ofTplac under alkaline conditions made this enzyme suitable for spe-cial applications.

As displayed in Fig. 3b, Tplac showed its maximal activity at50 �C, amounting to a laccase activity of 28.687 U mL�1, and dis-played more than 60% of the maximal activity at temperature rang-ing from 25 to 75 �C. After incubation at 50 �C for 2 h, the laccaseactivity was 20.744 U mL�1. The enzyme was stable at relativelymoderate temperatures, i.e., 25–60 �C, which indicated that Tplachas no special requirement for the operational capital and appara-tus. However, the laccase lost almost 50% of its original activityafter incubation at 75 �C for 2 h and was completely inactive at80 �C. The moderate or high temperature-dependent activity ofpurified laccase Tplac from T. pubescens could be due to the highertemperature environment of southern China, where the fungalstrain Cui 7571 was collected.

3.5. Substrate specificity and kinetic property of purified Tplac

Purified laccase Tplac from T. pubescens displayed high activitytoward a wide range of substrates, including phenolic substrates,such as catechol, 2,6-DMP, L-DOPA, ferulic acid, guaiacol, and

Table 2Effects of various substrates and inhibitors on the activity of purified Tplac from Trametes

Substrate Wavelength (nm) Relative activitya (%)

ABTS 420 100 ± 8.37Catechol 400 77.36 ± 10.052,6-DMP 470 98.36 ± 9.37

L-DOPA 280 95.05 ± 9.46

Ferulic acid 287 87.34 ± 8.86Guaiacol 470 91.19 ± 9.37Hydroquinone 248 82.32 ± 10.35Phenol 270 60.34 ± 8.37Pyrogallol 450 72.23 ± 9.58Syringaldazine 525 89.37 ± 10.12Tyrosine 280 0Veratryl alcohol 280 85.51 ± 8.87

a The value of 100% relative laccase activity toward ABTS was 28.687 U mL�1 obtained aerror mean of triplicate.

hydroquinone, as well as non-phenolic substrate, such as ABTS.However, no activity was detected with tyrosine (Table 2). Tplac’sactivity to the various substrates was ranked as follows:ABTS > 2,6-DMP > L-DOPA > guaiacol > syringaldazine > ferulic acid >veratryl alcohol > hydroquinone > catechol > pyrogallol > phenol.Meanwhile, the Km, kcat, and kcat/Km values for ABTS were 105.0lM, 876 s�1, and 8.34 s�1 lM�1 respectively, which were higherthan those of certain fungal laccases (Guo et al., 2011; Zhu et al.,2011). The relatively low Km value for ABTS also indicated a highaffinity of the enzyme toward this substrate.

3.6. Effects of inhibitors on the activity of purified Tplac

Laccase was inhibited to various extents by the usual inhibitors,such as L-cysteine, DTT, EDTA, and NaN3, which indicated the keyroles of the thiol groups and metal-binding active sites on laccaseactivity (Johannes and Majcherczyk, 2000; Lorenzo et al., 2005).The effects of various inhibitors on Tplac activity are summarizedin Table 2. A significant reduction in laccase activity was observedin the presence of 0.05 mM NaN3, 0.1 mM DDT, or 0.1 mM L-cys-teine, whereas no inhibition was assayed with 0.1 mM metal ionchelator EDTA under the same experimental conditions. Even ahigher concentration (1.0 mM) of EDTA did not inhibit ABTS oxida-tion by Tplac, which was similar to the results obtained by Shinand Lee (2000). It seems to be that the inhibitory effect of EDTA de-pends on the substrate, and many substrates can alter the activityof an enzyme by influencing the binding of substrate and/or its

pubescens.

Inhibitor Concentration (mM) Relative activitya (%)

L-Cysteine 0.05 89.75 ± 8.540.1 78.74 ± 9.021.0 38.89 ± 9.14

Dithiothreitol 0.05 79.14 ± 8.94

0.1 19.78 ± 7.861.0 5.66 ± 8.17

EDTA 0.05 1000.1 1001.0 97.85 ± 8.67

Sodium azide 0.05 33.27 ± 9.340.1 2.12 ± 9.131.0 0

t optimum pH and temperature conditions. Each value is the mean value ± standard

Table 4Dye decolorization capacity of purified Tplac from Trametes pubescens.

Dye Chemical class Colorindexname

Wavelength(nm)

Decolorizationratea (%)

Congo Red Azo Directred 25

497 80.53 ± 9.34

Crystal Violet Triphenylmethane Basicviolet 3

595 34.21 ± 8.34

Neutral Red Heterocycle Basic red5

553 75.15 ± 7.38

MethyleneBlue

Thiazine Basicblue 9

661 50.24 ± 8.63

ReactiveBrilliantBlue X-BR

Anthraquinone ReactiveBlue 4

603 66.57 ± 9.57

a Each value is the mean value ± standard error mean of triplicate.

J. Si et al. / Bioresource Technology 128 (2013) 49–57 55

turnover number (Johannes and Majcherczyk, 2000). Lorenzo et al.(2005) found that the laccase activity was strongly inhibited byEDTA using syringaldazine or 2,6-DMP as substrate, whereas EDTAwas not an efficient inhibitor using ABTS as substrate.

3.7. Effects of metal ions on the activity and ABTS oxidation of purifiedTplac

Another remarkable property of Tplac versus other laccases isits higher tolerance toward metal ions. One of the major obstaclesretarding the development of the practical application in biotech-nological industries is the restricted ability of laccase in the pres-ence of various metal ions (Murugesan et al., 2009). As displayedin Table 3, the effects of common environmental metal ions on lac-case activity were tested by adding Cu2+, K+, Na+, Mn2+, Ca2+, Fe2+,Fe3+, Mg2+, Zn2+, Ba2+ or Al3+ respectively. Interestingly, above88% of initial enzyme activity was maintained in the presence ofthese metal ions at their final concentrations of 25.0 mM afterincubation at 50 �C for 15 min. Meanwhile, the enzyme activitywas enhanced by approximately 111.32% with Cu2+, 106.93% withMn2+, 104.90% with Na+, 104.08% with Zn2+, or 100.35% with Mg2+

respectively. These results were much better than those reportedby previous studies (Guo et al., 2011; Zhu et al., 2011). It was worthnoting that in terms of consideration for potentially industrialapplication, the highly tolerant activity of Tplac toward metal ionswas a highly valued property.

To further understand the effects of metal ions on Tplac activity,the kinetic constants of ABTS oxidation were determined in thepresence of various metal ions. Table 3 shows that the kcat/Km val-ues of Tplac toward ABTS in the presence of metal ions from Cu2+ toMg2+ were higher than that of the control at the same level, sug-gesting that these metal ions at 25.0 mM could enhance the affinityof Tplac toward substrate, thus stimulating the enzyme activity.However, the kcat/Km values of Tplac toward ABTS gradually de-creased following the separate addition of metal ions from Ca2+

to Al3+. Because the dramatic variations of kcat/Km data were ob-served in the presence of metal ions from Cu2+ to Al3+, two theoriesare proposed to explain the metal effects regarding laccase activity.One theory is that the binding of several metal ions, i.e., Cu2+, Mn2+,Na+, Zn2+, or Mg2+, induces conformational modification of theenzyme and stimulates decomposition of the trimer complex con-taining substrate, enzyme and metal ion, as evidenced by noncom-petitive inhibition model (Duggleby, 1979). It is well-known thatthe laccase contains three types of copper sites (type I, II, and III),and its catalytic site is a cluster of four copper atoms, which per-forms monoelectronic oxidation of suitable substrates (Frasconiet al., 2010). Therefore, the other theory is that the metal ion, i.e.,

Table 3Effects of metal ions on the activity and ABTS oxidation of purified Tplac fromTrametes pubescens.

Metalion

Relative activitya

(%)Km

(lM�1)kcat

(s�1)kcat/Km

(s�1 lM�1)

Control 100 105.0 876 8.34Cu2+ 111.32 ± 10.23 85.5 1056 12.35Mn2+ 106.93 ± 9.63 88.6 978 11.04Na+ 104.90 ± 9.54 91.0 963 10.58Zn2+ 104.08 ± 9.84 99.0 924 9.33Mg2+ 100.35 ± 8.31 103.0 904 8.78Ca2+ 97.34 ± 7.86 107.7 866 8.04K+ 97.11 ± 9.21 107.3 842 7.85Fe2+ 95.88 ± 9.05 105.2 814 7.74Fe3+ 93.40 ± 10.22 109.5 792 7.23Ba2+ 89.88 ± 9.34 117.2 763 6.50Al3+ 88.42 ± 8.37 114.7 711 6.20

a The value of 100% relative laccase activity toward ABTS was 28.687 U mL�1

obtained at optimum pH and temperature conditions. Each value is the meanvalue ± standard error mean of triplicate.

Ca2+, K+, Fe2+, Fe3+, Al3+, or Ba2+, binds near the T I site of laccaseand acts as a competitive inhibitor of electron donors by blockingthe access of substrates to the T I site or inhibiting the electrontransfer at the T I active site, thereby leading to inhibition of lac-case activity (Fang et al., 2012).

3.8. Dye decolorization capacity of purified Tplac

3.8.1. Dye decolorizationIn textile industry, the decolorization of synthetic textile dyes

and effluents by physical or chemical methods is financially and of-ten not very effective, therefore, enzyme-based decolorization iscurrently of great interest in this field (Arora and Sharma, 2010).In this study, five representatives of structurally various dyes wereused to evaluate the decolorization capacity of purified laccaseTplac. As shown in Table 4, the enzyme from T. pubescens per-formed high decolorization capacity toward all of the selectivedyes, with 80.53% of Congo Red (50.0 mg L�1) being decolorizedby 1.0 U mL�1 pure enzyme after incubation for 72 h. It was sug-gested that the Tplac has potentials for use in various dyes biore-mediation. Based on the dye decolorization results, the azo dyeCongo Red was selected as the model dye for the followingexperiments.

3.8.2. Effects of heavy metal ions on the dye decolorization capacity ofpurified Tplac

Effects of heavy metal ions on the dye decolorization capacity ofpurified Tplac were analyzed on the basis of time taken for 100%

0

20

40

60

80

100

120

140

Tim

e (h

)

0 20 25 30 35 40 50 60

Concentration (mM)

Cu Zn Fe

Fig. 4. Effects of heavy metal ions on the dye decolorization capacity of purifiedTplac from Trametes pubescens. Time required for 100% decolorization rate of azodye Congo Red by Tplac was obtained in the presence of various heavy metal ionsCu2+, Zn2+, and Fe3+ at different concentrations ranging from 20.0 to 60.0 mM.

Table 5Phytotoxicity test of azo dye Congo Red and its metabolites formed after biodegradation by purified Tplac from Trametes pubescens.

Parameters studied Phaseolus mungo Sorghum vulgare Triticum aestivum

Water Congo Red Metabolites Water Congo Red Metabolites Water Congo Red Metabolites

Germination (%) 100 75 100 90 70 100 100 65 100Plumulea (cm) 15.63 ± 0.55⁄⁄ 6.52 ± 0.32⁄⁄ 14.52 ± 0.32⁄ 15.76 ± 0.32⁄ 7.15 ± 0.37⁄⁄ 13.87 ± 0.23 14.35 ± 0.41⁄⁄ 6.35 ± 0.45⁄⁄ 12.78 ± 0.35⁄

Radiclea (cm) 2.32 ± 0.11⁄⁄ 0.68 ± 0.11⁄ 2.20 ± 0.18⁄ 8.57 ± 0.31 1.42 ± 0.12⁄⁄ 7.18 ± 0.22⁄ 3.70 ± 0.23⁄⁄ 1.40 ± 0.08⁄⁄ 2.81 ± 0.23⁄

Based on the statistical analysis of one-way analysis of variance (ANOVA) and Tukey–Kramer comparison test by using SPSS18.0 software, probability (P value) less than 0.05or 0.01 (⁄P < 0.05 or ⁄⁄P < 0.01) was considered significant or highly significant respectively.

a Each value is the mean value ± standard error mean of triplicate.

56 J. Si et al. / Bioresource Technology 128 (2013) 49–57

decolorization rate of azo dye Congo Red. The values obtained dur-ing experiments conducted in the presence of three metal ions atdifferent concentrations are shown in Fig. 4. The time taken forcomplete decolorization of azo dye Congo Red by purified Tplacfrom T. pubescens was 78 h in the absence of any heavy metalion. Noteworthy was that, the time required for 100% decoloriza-tion rate was decreased by approximately 21 h in the presence ofCu2+ at 30.0 mM and returned to its original level at 40.0 mM,which could be attributed to the highly valued property of metaltolerance of Tplac, thus stimulating the applicability of the enzymefor metal containing effluents removal (Kalpana et al., 2011). Formetal ion Zn2+, a slight reduction in the time taken for 100% decol-orization rate of Congo Red was observed as its concentration from20.0 to 30.0 mM. However, a vivid increase in the time required forcomplete decolorization occurred with continuously increasingconcentrations of Zn2+, and the maximum time taken for 100%decolorization rate of Congo Red was achieved at 60.0 mM, whichwas 1.46-fold higher than that of the control in the absence of Zn2+.Meanwhile, the time taken for complete decolorization by purifiedTplac in the presence of Fe3+ increased dramatically with the in-crease in metal ion concentration even at low levels. When theconcentration of metal ion Fe3+ was 60.0 mM, the time requiredfor 100% dye decolorization was 133 h, which indicated that thepresence of some heavy metal ions in textile effluents may createthe problem of low biodegradability which increases the biologicaltreatment time (Lorenzo et al., 2005). In the light of these results itcan be concluded that this suggested laccase Tplac is found to beefficient for the remediation of heavy metal ions containing dyeeffluents.

3.8.3. Degraded metabolites identificationBy organic solvent extraction and GC–MS analysis, the degraded

metabolites of Congo Red were identified as naphthalene amine(molecular mass 143, m/z 143, retention time 19.657, area18.31%), biphenyl amine (molecular mass 169, m/z 169, retentiontime 10.856, area 4.56%), biphenyl (molecular mass 154, m/z 154,retention time 17.753, area 9.37%), and naphthalene diazonium(molecular mass 156, m/z 156, retention time 14.287, area14.22%). According to the identified metabolites, the degradedpathway was proposed and is shown in Supplementary Fig. S3.The first step was the reduction of the –N@N– bond, which re-sulted in the formation of two reactive intermediates (A and B),thereby leading to several reactions that lead to the productionof stable intermediate.

3.8.4. Phytotoxicity testAs demonstrated in Table 5, the germination percentage and

lengths of the plumule and radicle of P. mungo, S. vulgare, and T.aestivum seeds were greater with Congo Red’s degradation metab-olites or with water treatment when compared to untreated CongoRed treatment. Hence, phytotoxicity studies revealed that the puri-fied laccase Tplac from T. pubescens can detoxify azo dyes used intextile industries.

4. Conclusion

In summary, a novel laccase, Tplac, from white rot fungus T.pubescens was purified and characterized. The enzyme performedwith greater catalytic efficiency toward ABTS than did certain otherfungal laccases, with the catalytic efficiency (kcat/Km) of 8.34 s�1 -lM�1 being observed. Furthermore, Tplac exhibited alkali-resistantactivity, metal tolerance, and dye decolorization capacity at roomtemperature. These unusual properties demonstrated this laccase’spotential suitability for use in such industries as bioremediation,textile, paper, and pulp.

Acknowledgement

This study was supported by the Program for New CenturyExcellent Talents in University (NCET-11-0585).

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.biortech.2012.10.085.

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