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An extracellular laccase with potent dye decolorizing ability from white rot fungus Trametes sp. LAC-01
International Journal of Biological Macromolecules 81 (2015) 785–793
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An extracellular laccase with potent dye decolorizing ability from white rot fungus Trametes sp. LAC-01 Zhuo-Ren Ling a , Shan-Shan Wang b , Meng-Juan Zhu c , Ying-Jie Ning a , Shou-Nan Wang a , Bing Li a , Ai-Zhen Yang a , Guo-Qing Zhang a,∗ , Xiao-Meng Zhao a,∗ a Key Laboratory of Urban Agriculture (North) of Ministry of Agriculture, College of Biological Science and Engineering, Beijing University of Agriculture, Beijing 102206, China b 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
a r t i c l e
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Article history: Received 7 April 2015 Received in revised form 28 July 2015 Accepted 6 September 2015 Available online 8 September 2015 Keywords: Laccase Trametes sanguinea Dye decolorizing activity
a b s t r a c t A novel laccase was purified from fermentation broth of white rot fungus Trametes sp. LAC-01 using an isolation procedure involving three ion-exchange chromatography steps on DEAE-cellulose, SP-Sepharose, and Q-Sepharose, and one gel-filtration step. The purified enzyme (TSL) was proved as a monomeric protein with a Mr of 59 kDa based on SDS-PAGE and FPLC. Partial amino acid sequences were obtained by LC–MS/MS sharing considerably high sequence similarity with that of other laccases. It possessed optimal pH of 2.6 and temperature of 60 ◦ C using ABTS as the substrate. The Km of the laccase toward ABTS was estimated to 30.28 M at pH 2.6 and 40 ◦ C. TSL manifested considerably high oxidizing activity toward ABTS, but was avoid of degradative activity toward benzidine, caftaric acid, etc. It was effective in the decolorization of phenolic dyes – Bromothymol Blue and Malachite Green with decolorization rate higher than 60% after 24 h of incubation. Adjunction of Cu2+ with the final concentration of 2.0 mmol/L significantly activated laccase production with a steady high level of 275.8-282.2 U/mL in 96-144 h. The high yield and short production period makes Trametes sp. LAC-01 and TSL potentially useful for industrial and environmental application and commercialization. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Laccase (-diphenol: dioxygen oxidoreductase, EC 1.10.3.2) is a group of blue multicopper oxidases that catalyze the oxidation of a wide variety of organic aromatic substrates concomitantly with the reduction of molecular oxygen to water [1,2]. It was first obtained from the Japanese lacquer tree (Rhus vernicifera) in 1883 and subsequently isolated from fungi in 1896, which makes it one of the oldest enzymes ever described [2]. Laccases are widely distributed in plants, bacteria, insects, and especially fungi [3,4]. They can be roughly divided into two major groups, i.e. laccases from higher plants and those from fungi [2,5]. Plant laccases involve in lignin synthesis and wound healing, whereas fungal laccases probably have more roles and participate in lignin degradation, pigmentation, pathogenesis, fungal plant-pathogen/host interaction, and stress defence, etc. [2,6,7].
∗ Corresponding authors. E-mail addresses: [email protected] (G.-Q. Zhang), [email protected] (X.-M. Zhao). http://dx.doi.org/10.1016/j.ijbiomac.2015.09.011 0141-8130/© 2015 Elsevier B.V. All rights reserved.
At present, more than 100 fungal laccases from Basidiomycota and Ascomycota have been purified and characterized [2,3]. Purification methods described in literature include ion exchange, affinity, and gel filtration chromatography [8–10]. Enzymatic properties including molecular mass, subunit composition, optimal pH and temperature, substrates, and Km values have been widely studied and reviewed [1,2,5]. Nowadays, fungal laccases are being extensively evaluated due to their wide applications in food, pulp and paper, textile, nanobiotechnology, and pharmaceutical industries [1,11]. Trametes sanguinea, called ‘red blood blot fungus’ in Chinese, is a white rot saprobic fungus with synonyms names of Pycnoporus sanguineus and Trametes sanguineus belonging to the family Polyporaceae [12]. It is widely distributed in China, occurs in summer and early autumn, and grows on dead hardwoods. Its fruiting bodies are bright orange to red with a diameter range of 3-10 cm. Previous literature revealed that T. sanguinea was rich in bioactive enzymes, e.g., laccase, protease, and protopectinase [13–16]. A laccase with 62 kDa, optimal pH of 5.0, and optimal temperature of 60 ◦ C was isolated from T. sanguinea M85-2 [16]. In the present study, T. sanguinea strain LAC-01 was isolated from the Baiwangshan National
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Forest Park, Beijing, China. We aim to purify an extracellular laccase from fermentation broth the strain LAC-01. Enzymatic properties and dye decolorizing application are also investigated. 2. Materials and methods 2.1. Fungal strain and culture condition Strain LAC-01 was isolated from the fruiting bodies of Trametes sp., which were collected from a deadwood of Populus tomentosa in the Baiwangshan National Forest Park (Beijing, China). It was cultured at 28 ◦ C, stored at 4 ◦ C, and monthly transferred to fresh potato dextrose agar (PDA) slant medium which contained (g/L): potato, 200; glucose, 20; and agar, 20. To obtain its extracellular laccase, strain LAC-01 was inoculated in the liquid potato dextrose (PD) medium containing (g/L): potato, 200 and glucose, 20, and in an orbital shaking incubator at 180 rpm and 28 ◦ C for 8 days. The fermentation broth was subsequently used as laccase primary extract. 2.2. Effect of Cu2+ on laccase production in liquid fermentation In order to enhance the enzyme yield, effect of Cu2+ on laccase production in liquid fermentation was observed. CuSO4 solution (1 mol/L) was added into the fermentation medium (the PD medium) with the final concentration of 0, 0.25, 0.50, 1.0, and 2.0 mM, respectively. The broth was collected at 72, 96, 120, 144, 168, and 192 h, respectively. Following a centrifugation at 8000 rpm and 4 ◦ C for 15 min, laccase activity in broth was assayed using the standard assay. 2.3. Classification based on rDNA sequence analysis Classification of the strain LAC-01 was analyzed based on PCR amplification and phylogenetic analysis of the internal transcribed spacer (ITS) regions which contained ITS-1, 5.8S, and ITS-2 region in the rDNA Cluster [17,18]. Total genomic DNA of fungal mycelia was extracted using the CTAB method [19]. The ITS region was amplified by PCR using universal primers ITS1 (5 TCCGTAGGTGAACCTGCGG 3 ) and ITS4 (5 TCCTCCGCTTATTGATATGC 3 ) and performed in a volume of 50 L under standard conditions [20]. The PCR production was subsequently sequenced by Shanghai SANGON Biological Engineering Co. (Shanghai, China). Obtained sequences were compared with sequences in the BLAST/NCBI database (http:// blast.ncbi.nlm.nih.gov/Blast.cgi) to determine the nearest matches. Phylogenetic trees were finally constructed according to the Neighbor-Joining (NJ) algorithm using MEGA Software (Version 6.06). Bootstrap analysis was based on 1000 replication. 2.4. Purification of laccase After 8-day fermentation, the broth was initially filtered to remove mycelial debris, followed by centrifugation at 8000 rpm and 4 ◦ C for 15 min. Subsequently, the supernatant were dialyzed with Tris–HCl buffer (10 mM, pH 8.0) at 4 ◦ C overnight. The supernatant was further applied on an anion exchange DEAE-cellulose column (2.5 × 20 cm2 , Bio-Rad) in Tris–HCl buffer (10 mM, pH 8.0) with the flow rate of 2 mL/min. After removal of unadsorbed proteins, adsorbed proteins were desorbed from the column with 100, 300, and 1000 mM NaCl added to the Tris–HCl buffer, respectively. Laccase active fraction was chromatographed on a cation exchange SP-Sepharose column (2.5 × 20 cm2 , Bio-Rad) in HAc–NaAc buffer (10 mM, pH 4.0) with the flow rate of 2 mL/min. After collection of unadsorbed proteins, the column was eluted with 800 mM NaCl added to the HAc–NaAc buffer. Subsequently, enzyme containing fraction was subjected to another anion exchange chromatography
on Q-Sepharose column (1.5 × 10 cm2 , Bio-Rad) in Tris–HCl buffer (10 mM, pH 7.5) with the flow rate of 1 mL/min. Following removal of unadsorbed proteins, adsorbed proteins were desorbed with three gradients of 100, 300, and 500 mM NaCl in Tris–HCl buffer. Laccase rich fractions were pooled, dialyzed and finally purified by fast protein liquid chromatography (FPLC) on a Superdex 75 gel filtration column (0.15 M NH4 HCO3 buffer, pH 8.5) with a flow rate of 0.8 mL/min. 2.5. Assay for laccase activity Laccase activity was spectrophotometrically determined by using 2, 2 -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate [21,22]. Laccase solution (10 L) was mixed with 0.6 mM ABTS solution (190 L, in 50 mM sodium acetate buffer, pH 4.5) at 40 ◦ C for 5 min, followed by an addition of 10% TCA (200 L) to end the reaction. The increase in absorbance was monitored at 405 nm to test enzyme activity. All determinations were performed in triplicate. One enzyme unit (U) was defined as the amount of enzyme required to produce one absorbance increase of the reaction mixture at 405 nm/min/mL [6]. 2.6. Determination of molecular mass The molecular mass (Mr) of the present laccase was determine using FPLC-gel filtration and sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). In FPLC chromatography, a standard curve based on elution volume and Log Mr of molecular mass standards (GE Healthcare) was obtained. Mr of the active laccase can be calculated based on its elution volume [23]. In SDS-PAGE, a 12% resolving gel and a 5% stacking gel were used following the standard procedure [24]. Another standard curve of relative mobility and Log Mr of unstained molecular mass standards (Thermo Scientific) can be used for the Mr evaluation of inactive monomeric protein or subunits. 2.7. LC–MS/MS analysis The protein band of purified laccase on SDS-PAGE was recovered and digested by trypsin. Digestion products were subsequently analyzed by LC–MS/MS for amino acid sequences. The data were acquired using Xcalibur software (Thermo Electron). Sequence homologues were searched using the BLAST/NCBI database [25,26]. 2.8. Effects of pH and temperature on the activity of purified laccase To determine the effect of pH values on laccase activity, a series of ABTS solution in various pH values were used instead of the standard ABTS solution at pH 4.5. The assay buffers were prepared in citric acid–Na2 HPO4 buffers (pH range of 2.2-8.0). In the temperature assay, standard activity assay was measured in different temperature from 20 ◦ C to 90 ◦ C instead of 40 ◦ C in the standard assay. 2.9. Determination of pH stability and thermostability of purified laccase In the pH stability assay, enzyme solutions were previously incubated in 50 mM citric acid–Na2 HPO4 buffers at different pH values (2.2, 2.6, 3.0, and 3.4, respectively) for different durations (10, 20, 30, 40, 50, and 60 min, respectively) at 4 ◦ C. The residual laccase activity was triplicately measured using the standard assay. In the thermostability assay, enzyme solutions were previously incubated at various temperatures (50, 60, 70, and 80 ◦ C, respectively) for 10, 20, 30, 40, 50, and 60 min, respectively. The residual laccase activity
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was assayed in triplicate using the standard assay after the reaction mixture had been cooled down to room temperature [9]. 2.10. Effects of chemical reagents on laccase activity To estimate the influence of metal ions and EDTA on enzyme activity, chemical reagents including CaCl2 , CdCl2 , CoCl2 , CuCl2 , FeCl2 , FeCl3 , KCl, MgCl2 , MnCl2 , NaCl, ZnCl2 , and EDTA (at concentrations of 1.25, 2.5, 5.0, and 10 mM, respectively) were mixed with equal volumes of the purified laccase solution at 4 ◦ C for 1 h. The residual laccase activity was measured in triplicate using the standard assay. Control samples were treated with distilled water instead of the metal ion solution. 2.11. Assay for substrate specificity and kinetic parameter of purified laccase Fig. 1. Effect of Cu2+ on laccase production in liquid fermentation.
In the substrate specificity assay, seven aromatic reagents (in concentration of 5.0 mM, 50 mM pH 2.6 citric acid–Na2 HPO4 buffers) were tested as the substrates including ABTS, benzidine, caftaric acid, catechol, guaiacol, o-Toluidines, phenol, and tyrosine. The reaction was started by adding 10 L of laccase solution and 190 L substrate solution, and incubated at 40 ◦ C for 5 min, followed by an addition of 10% TCA (200 L) to end the reaction. The substrate oxidation rate was followed by measuring the absorbance change with the molar extinction coefficients of various substrates [27,28]. Laccase activity toward ABTS was regarded as 100%. All determinations were performed in triplicate. Kinetic studies of the purified laccase were carried out using ABTS as substrate in a series concentration ranging from 12.5 to 800 M at pH 2.6 and 40 ◦ C. All determinations were performed in triplication. The reciprocals of the substrate concentrations and the reciprocals of the corresponding initial velocities were then used to generate a Lineweaver-Burk plot [29]. 2.12. Assay of dye decolorization by purified laccase The decolorization of different dyes used in textile and chemical industries by purified laccase (0.5 U/mL) was determined over a 24 h period. Eight kinds of dyes were used including Bromothymol Blue, Eriochrome Black T, Evans Blue, Fuchsin Basic, Malachite Green, Methylene Blue, Methyl Orange, and Reactive Brilliant Blue R. The test was performed at 40 ◦ C by adding 20 L purified laccase into 380 l citric acid–Na2 HPO4 buffers (pH 2.6) which contains different kinds of dyes, respectively. Initial concentration of dyes was determined based on their maximal absorbance wavelength with an absorbance value range of 0.7-1.3. The decolorizing ability of laccase was determined spectrophotometrically as the relative decrease of absorbance at 4, 12, and 24 h. All reactions were performed in triplicate [28,30,31]. 3. Results 3.1. Effect of Cu2+ on laccase production in liquid fermentation Effect of Cu2+ on laccase production of Strain LAC-01 in liquid fermentation was visualized in Fig. 1. In the negative control group, laccase activity in PD medium without any Cu2+ adjunction reached its highest level of 122.8 U/mL after 8 day liquid fermentation. Adjunction of Cu2+ with the final concentration of 2.0 mmol/L significantly activated laccase production with a steady high level of 275.8-282.2 U/mL in 96-144 h. At 72 h, laccase activity in 2.0 mM Cu2+ group achieved 243.7 U/mL, while enzyme activity was avoid in the control group at the same time. On the other hand, adjunction
of Cu2+ with the final concentration higher than 2.0 mM strongly inhibited the growth of T. sanguinea mycelia (data not shown). 3.2. Classification of the laccase producing fungus Strain LAC-01 was initially identified as species from genus Trametes based on its morphological properties. A 596-bp fragment of ITS region (GenBank accession No. of KP723552) was sequenced and compared to GenBank using BLAST. A phylogenetic tree based on ITS region of Trametes species was constructed (Fig. 2). Strain LAC-01 was closest to T. sanguinea (GenBank accession No. JN164981.1), sharing the query cover of 96% and the sequence identity of 100%. Strain LAC-01 was classified as T. sanguinea based on both morphological and molecular identification [12]. 3.3. Laccase purification and molecular mass determination On DEAE-cellulose anion exchange chromatography, the crude enzyme extract was chromatographed into three unadsorbed fractions (D1, D2, and D3) eluted with Tris–HCl buffer (10 mM, pH 8.0) and three adsorbed fractions (D4, D5, and D6) eluted with 100, 300, and 1000 mM NaCl in Tris–HCl buffer (10 mM, pH 8.0), respectively. Subsequently, fraction D5 possessing laccase activity was fractionated on SP-Sepharose cation exchange chromatography and resolved into an unadsorbed fraction S1 with the laccase activity in starting buffer (10 mM, pH 4.0 HAc–NaAc buffer) and an adsorbed fraction S2 in starting buffer with 800 mM NaCl. Laccase active fraction S1 was further applied on Q-Sepharose anion exchange chromatography and separated into three adsorbed fractions Q1, Q2, and Q3 with three gradients of 100, 300, and 500 mM NaCl in Tris–HCl buffer (10 mM, pH 7.5), respectively. The laccase active fraction Q2 was finally subjected to an FPLC-Superdex 75 HR 10/30 column to yield only one fraction SU1 containing purified laccae and named as T. sanguinea laccase (TSL). SU1 fraction possessed a Mr of 59 kDa as estimated by FPLC based on the standard curve of elution volume-log Mr (Fig. 3A). In SDS-PAGE, SU1 fraction appeared as a single band with a Mr of 59 kDa (Fig. 3B). It suggested that the native laccase is a monomeric protein. The enzyme was purified 78.06-fold from the crude extractive with 5.76% yield. The purified laccase exhibited an activity of 839.16 U/mg toward ABTS at the standard assay condition (Table 1). 3.4. Amino acid sequence LC–MS/MS analysis of the purified laccase disclosed four internal peptides: Seq-01, RSPGTTTADLAVIKVTQGKR, Seq-02, RLVSLSCDPNHTFSIDGHTMTVIEADSVNTQPLEVDSIQIFAAQRY, Seq-03,
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51
96
24 7
Trametes elegans (JN164986.1) 63
Trametes maxima (JN164957.1) Trametes membranacea (JN164956.1) Trametes gibbosa (KJ140582.1)
24
23
38
63
Trametes hirsuta (HQ435869.1) 92
Trametes villosa (KF573031.1)
77
Trametes conchifer (JN164988.1) Trametes junipericola (AY684171.2) Trametes suaveolens (KF573015.1) Trametes velutina (KF573019.1)
54 95 42
46
Trametes sanguinea LAC-01 (This study) Trametes cinnabarina (KF573022.1) Trametes ljubarskii ( AY684174.2)
3
35
Trametes sanguinea (JN164981.1)
43
Trametes ectypa (JN164961.1) Trametes ochracea (JN164976.1)
85
Trametes obstinata (EU661880.1) Trametes purpurea (EU661881.1)
33
Trametes orientalis (EU771082.1) 33
50 83
Trametes pubescens (KJ140686.1) Trametes cubensis (JN164989.1) Trametes trogii (HM989941.1) Trametes ellipsospora (JN048767.1) Trametes hispida (EU661874.1) Trametes robiniophila (EU661883.1)
95
Trametes corrugata (EU661875.1) 98
Trametes palisotii (EU661873.1) Trametes versicolor (EU273523.1)
Fig. 2. Neighbor-joining phylogenetic tree based on ITS sequences showing the distance of strain LAC-01 with the nearest species of the genus Trametes. Bootstrap values at nodes are percentages of 1000 replicates.
RYSFVLDASQPVDNYWIRA, and Seq-04, RSAGSSEYNYDNPIFRD. Based on BLAST results, they all belonged to Cuperdoxin superfamily which were blue copper proteins including laccases. The homology search of the obtained sequences in BLAST showed that they demonstrated considerably high sequence homology to those of previously reported laccases from genus Trametes and other white rot fungi (Table 2).
3.5. Optimal pH and temperature, pH stability, and thermostability of purified laccase The purified laccase achieved its maximal oxidizing activity toward ABTS at pH 2.6 and underwent a gradual decline when the assay pH rose from 2.6 to 5.0. Approximately 60% of total activity survived at pH 5.0. When the pH value further increased, a sharp decrease in enzyme activity was observed with an almost zero level of laccase activity above pH 6.2 (Fig. 4A). The purified laccase demonstrated a considerable high thermostability with an optimal temperature of 60 ◦ C. Oxidizing activity toward ABTS at 60 ◦ C was nearly twice as high as that of at 20 ◦ C. More than 20%
of total enzyme activity remained when it was assayed at 90 ◦ C (Fig. 4B). TSL shows strong stability in the assay pH range of 2.2-3.4 (Fig. 5A). Most of the oxidizing activity remained after 60 min preincubation in the different pH conditions. A slight increase (about 10%) can be observed after 60 min pre-incubation at pH 2.6. TSL possesses considerably high thermostability. Most of the enzyme activity maintains when it underwent 60 min pre-incubation at 5070 ◦ C (Fig. 5B). There are soft but continuous increases in laccase activity when enzyme solution was previously incubated at 50 ◦ C and 60 ◦ C for 0-60 min. When incubated at 70 ◦ C, enzyme activity reaches the highest level with 31.3% of increase rate after 50 min of pre-incubation. TSL is sensitive to temperature as high as 80 ◦ C. During pre-incubation at 80 ◦ C, a sharp and continuous decrease in enzyme activity can be observed. 3.6. Effects of chemical reagents The sensitivity of purified laccase to metal ions and EDTA is summarized in Table 3. TSL was not obviously affected by the existence of K+ at concentration of 1.25-10 mM, Ca2+ at concentration of
Table 1 Summary of purification procedure of TSL laccase (from 1 L fermentation broth). Purification step
Total protein (mg)
Specific activity (U/mg)
Total activity (U)
Recovery of activity (%)
Purification fold
Fermentation broth DEAE-cellulose (D5) SP-Sepharose (S1) Q-Sepharose (Q2) Superdex 75 (SU1)
5805 325.8 143.92 23.03 4.29
10.75 53.01 114.05 430.97 839.16
62,404 17,271 16,414 9925 3600
100 27.7 26.3 15.9 5.76
1 4.93 10.61 40.09 78.06
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Table 2 Comparison of partial amino acid sequence of TSL in this study and other fungal laccases. Species
Amino acid sequence
GenBank No.
Trametes sanguinea Pycnoporus coccineus Trametes cinnabarina Trametes sanguinea Trametes sanguinea Ganoderma lucidum Trametes pubescens Trametes versicolor Ganoderma fornicatum Trametes punicea Trametes hirsuta Trametes gibbosa Trametes sanguinea Pycnoporus coccineus Trametes cinnabarina Trametes sanguinea Trametes sanguinea Trametes gibbosa Trametes hirsute Trametes punicea Trametes versicolor Pleurotus ostreatus Trametes pubescens Ganoderma lucidum Trametes sanguinea Pycnoporus coccineus Trametes cinnabarina Trametes sanguinea Trametes sanguinea Trametes punicea Lentinula edodes Trametes hirsute Trametes gibbosa Ganoderma lucidum Trametes sanguinea Pycnoporus coccineus Trametes cinnabarina Trametes sanguinea Trametes sanguinea Trametes gibbosa Trametes punicea Trametes pubescens Trametes versicolor Ganoderma lucidum
RSPGTTTADLAVIKVTQGKR 113 RSPGTTTADLAVIKVTQGKR 132 73 RSPGTTTADLAVIKVTQGKR 92 113 RSPGTTAADLAVIKVTQGKR 127 113 RTPGTTSADLAVIKVTQGKR 127 197 RSPATPTADLAVISVTQGKR 216 197 RSPSTTTADLAVISVTAGKR 216 197 RSPSTTTADLSVISVTPGKR 216 198 RSTATPTADLAVVNVTQGKR 217 113 RTPGNTTTELAVITVTQGKR 132 197 RAPSDTTAELSVIKVTKGKR 216 74 RSPSTVSADLSVINVTPGKR 93 RLVSLSCDPNHTFSIDGHTMTVIEADSVNTQPLEVDSIQIFAAQRY 136 RLVSLSCDPNHTFSIDGHTMTVIEADSVNTQPLEVDSIQIFAAQRY 181 96 RLVSLSCDPNHTFSIDGHTMTVIEADSVNTQPLEVDSIQIFAAQRY 141 136 RLVSLSCDPNHTFSIDGHTMTIIEADSVNTQPLEVDSIQIFAAQRY 181 136 RLVSLSCDPNHTFSIDGHTMTVIEADSVNTQPLEVDSIQIFAAQRY 181 97 RLVSLSCDPNHTFSIDGHDLTIIETDSVNTQPLVVDSIQIFAAQRY 142 220 RLVSLSCDPNHTFSIDGHNLTIIEVDSVNSQPLEVDSIQIFAAQRY 265 136 RLVSLSCDPNHTFSIDNHTMTIIETDAINTQPLEVDSIQIFAAQRY 181 220 RLVSLSCDPNHTFSIDGHNMTIIETDSINTAPLVVDSIQIFAAQRY 265 96 RLVSISCDPNHVFAIDGHNMTIIEVDSVNTQPLVVDSIQIFPGQRY 141 220 RLVSLSCDPNYVFSIDGHNMTIIETDSINTQPLVVDSIQIFAAQRY 265 220 RLVSLSCDPNFTFSIDGHAMTVIEADAVNHEPLTVDSIQIFAGQRY 265 RYSFVLDASQPVDNYWIRA 180 RYSFVLDASQPVDNYWIRA 198 140 RYSFVLDASQPVDNYWIRA 158 180 RYSFVLDASQPVDNYWIRA 198 180 RYSFVLDASQPVDNYWIRA 198 180 RYSFVLEANQPVDNYWIRA 198 278 RYSFVLDATQPVDNYWVRA 296 264 RYSFVLNANQPVDNYWIRA 282 141 RYSFVLEANQPVDNYWVRA 159 269 RYSFVLEANQPVNNYWIRA 287 RSAGSSEYNYDNPIFRD 345 RSAGSSEYNYDNPIFRD 361 429 RSAGSSEYNYDNPIFRD 445 345 RSAGSSEYNYDNPIFRD 361 345 RSAGSSEYNYDNPVFRD 361 429 RSAGSTEYNYDDPIFRD 445 345 RSAGSTEYNYDNPIFRD 361 429 RSAGSTVYNYDNPIFRD 445 429 RSAGSTVYNYDNPIFRD 445 449 RSAGSSEYNYENPVRRD 465
Seq-01 (This study) ACH61789.1 CAD12769.1 ACG61169.1 ACG61158.1 ABK59822.1 AAM18407.1 AAL07440.1 ABK59827.1 ACR24938.1 AIZ72721.1 AEQ38867.1 Seq-02 (This study) ACH61789.1 CAD12769.1 ACG61169.1 ACG61158.1 AEQ38867.1 AIZ72721.1 ACR24938.1 AAL93622.1 AEQ38864.1 AAM18407.1 AHA83589.1 Seq-03 (This study) ACH61789.1 CAD12769.1 ACG61169.1 ACG61158.1 ACR24938.1 AAT99288.1 AIZ72727.1 AEQ38867.1 AHA83587.1 Seq-04 (This study) ACH61789.1 AAN71597.1 ACG61169.1 ACG61158.1 ADK13091.1 ACR24938.1 AAM18407.1 AAL07440.1 AHA83591.1
Amino acid residues identical to corresponding residues of the purified laccase are underlined.
Table 3 Effect of metal ions and EDTA on laccase activity. Chemical reagent
Residual activity (% of control) 10 mM
+
Na K+ Ca2+ Cd2+ Co2+ Cu2+ Fe2+ Mg2+ Mn2+ Zn2+ Fe3+ EDTA
86.1 105.8 86.6 0.7 76.8 68.4 7.5 80.6 40.6 83.4 0.0 74.9
± ± ± ± ± ± ± ± ± ± ± ±
5.0 mM 1.9 0.6 1.4 1.2 1.1 0.3 0.2 0.9 1.6 0.9 0.0 2.0
89.5 104.4 92.9 73.3 76.9 68.6 26.5 82.2 85.0 87.2 20.8 79.2
± ± ± ± ± ± ± ± ± ± ± ±
2.5 mM 0.5 1.0 1.1 0.7 0.1 0.4 2.6 1.5 2.4 0.3 0.1 1.1
Laccase activity in the absence of metal ions was regarded as 100%. Values represent mean ± standard deviation (n = 3).
92.6 103.6 99.0 74.4 70.2 68.7 71.5 91.0 79.0 95.3 39.9 81.7
± ± ± ± ± ± ± ± ± ± ± ±
1.25 mM 1.5 1.6 0.8 1.5 0.3 0.4 0.3 0.3 0.2 1.7 0.4 0.5
98.3 102.5 99.7 76.3 87.7 77.0 77.7 87.8 68.2 99.3 49.1 74.2
± ± ± ± ± ± ± ± ± ± ± ±
0.9 0.9 0.7 1.6 2.8 0.8 0.4 0.2 1.6 0.4 0.4 0.3
790
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Fig. 3. Molecular mass determination of the purified laccase. (A) FPLC-gel filtration on Superdex 75 HR 10/30 column. Eluent: 0.15 M NH4 HCO3 buffer (pH 8.5). Fraction size: 0.8 mL. Flow rate: 0.8 mL/min. Fraction SU1 represents purified laccase. (B) SDS-PAGE of Fraction SU1 from FPLC. Right lane: Molecular weight markers, from top downwards, -galactosidase (116.0 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45.0 kDa), lactate dehydrogenase (35.0 kDa), REase Bsp98I (25.0 kDa), -lactoglobulin (18.4 kDa), and lysozyme (14.4 kDa).
Fig. 4. Effects of pH and temperature on activity of the purified laccase TSL with ABTS as substrate. (A) Effect of pH on TSL activity. (B) Effect of temperature on TSL activity.
3.8. Decolorization of dyes 1.25-5.0 mM, and Zn2+ at concentration of 1.25-2.5. It was inhibited with about 15-30% loss of total activity by Cd2+ , Co2+ , Cu2+ , Mg2+ , Mn2+ , and EDTA concentration of 1.25-5.0 mM. Addition of Fe2+ and Fe3+ with concentration higher then 5 mM led to more than 50% loss of total activity.
3.7. Substrate specificity and kinetic parameter Although TSL demonstrated considerably high oxidizing activity toward ABTS, it was avoid of degradative activity toward benzidine, caftaric acid, catechol, guaiacol, o-toluidines, and tyrosine. On the other hand, it manifested a very low oxidation activity toward phenol with relative activity of 13.20% comparing with ABTS. After incubation of the purified laccase with a series concentration of ABTS (12.5 to 800 M) at pH 2.6 and 40 ◦ C, the reactions were found to follow Michaelis–Menten kinetics, displaying the Km value of 30.28 M toward ABTS according to the Lineweaver-Burk plots (data not shown).
Decolorizing acitivity of purified laccase on industrial and laboratory dyes was evaluated at a low enzyme activity of 0.5 U/mL (Table 4). TSL possessed considerably high decolorizing activities toward Bromothymol Blue and Malachite Green with efficiencies higher than 50%. After incubating for 4 h, Bromothymol Blue and Malachite Green were decolorized with the rates of 62.0% and 46.1%, respectively. A low decolorizing rate of 8.1% was obtained when TSL was assayed toward Evans Bue. When incubating time prolonged from 12 to 24 h, decolorizing rates of 63.1–65.4% toward Bromothymol Blue and 70.1–75.8% toward Malachite Green were attained. After incubating for 24 h, TSL demonstrated low decolorizing rates of about 10% toward Evans Bue, Fuchsin Basic, and Methylene Blue. It was almost avoid of decolorizing activity toward Methyl Orange and Remazol Brilliant Blue R. 4. Discussion White rot fungi, such as species from genera Coriolus, Ganoderma, Pleurotus, and Trametes, possess remarkable degradation
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Table 4 Decolorization of the dyes by purified laccase TSL. Dye
max
Concentration (g/mL)
Bromothymol Blue Eriochrome Black T Evans Blue Fuchsin Basic Malachite Green Methylene Blue Methyl Orange Remazol Brilliant Blue R
440 540 610 530 614 664 460 605
800 400 28 20 8 8 20 100
Decoloration rate (%) 4h 62.0 0 8.1 1.7 46.1 0 0 0.5
12 h ± ± ± ± ± ± ± ±
0.2 0.1 0.2 0.9 0.5 0.4 0.1 0.1
63.1 0 9.3 5.4 70.1 6.8 0 1.0
24 h ± ± ± ± ± ± ± ±
0.1 0.1 1.0 0.4 0.6 0.5 0.3 0.1
65.4 0 11.2 8.6 75.8 7.9 0.1 3.9
± ± ± ± ± ± ± ±
0.3 0.1 0.3 0.9 0.6 0.4 0.5 1.3
Decolorization efficiencies higher than 50% are highlighted in bold. Values represent mean ± standard deviation (n = 3).
ability toward both lignin and cellulose biopolymers in lignocellulose biomass by producing ligninolytic enzymes including laccase, manganese peroxidase (MnP), lignin peroxidase (LiP) [32–34]. Among them, fungal laccases are of great interest to industry, and have been used in bio-degradation of lignocellulosic
Fig. 5. pH stability and thermostability of purified laccase. (A) pH stability of TSL. Residual activity was measured using the standard assay after incubating at pH values for 10-60 min. The assay pH values were 2.2 (), 2.6 (䊏), 3.0 (×), and 3.4 (). (B) Thermostability of TSL. Residual activity was measured using the standard assay after incubating at various temperatures for 10-60 min. The assay temperatures were 50 ◦ C (×), 60 ◦ C (䊐), 70 ◦ C (), and 80 ◦ C (䊉).
compounds, wastewater treatment, bio-pulping and bio-bleaching in textile industry [3,11,35]. Nowadays, laccases from genus Trametes have been extensively investigated including screening, purification, and structure analysis of novel enzyme proteins, cloning and heterologous expression of laccase genes, enzymatic engineering and fermentation methods, and potential applications in decolorization and detoxication [36–44]. Although fungal laccases possess a great biotechnological potential, commercial applications are limited by low enzyme yields and high costs. The present laccase produced about 280 U/mL laccase activity after cultivation in PD medium for only 4 day with Cu2+ as the inducer with the final concentration of 2.0 mM. The possible mechanism for this phenomenon is that copper ions enhance the laccase genetic transcription level during the laccase synthesis [45]. Laccase production of Cerrena unicolor strain 137 reached its maximum level of 18.7 U/mL after 10 days in complex tomato juice medium [22]. F. solani MAS2 laccase needed incubation time of 40 days and achieved the maximum activity at day 35 of 575 mU/mg protein [46]. Optimization of laccase production of Trametes trogii S0301 by response surface methodology revealed that the maximum laccase activity of 122.9 U/mL was obtained at day 9 with phenol, CuSO4 and PEG 4000 as inducers [47]. The high laccase yield and short production period of TSL would be advantageous for application and commercialization. The extracellular laccase TSL from fermentation broth of T. sanguinea strain LAC-01 was purified with an isolation procedure involving three steps of ion exchange chromatography and one final FPLC-gel filtration on Superdex 75, resulting in a very low recovery rate of 5.76%. Although many reported fungal laccases possessed relatively high pH stability (e.g. Inonotus baumii and Abortiporus biennis), TSL of present study was very sensitive with a low optimal pH of 2.6 and completely inactivated above pH 6.2 [9,48]. To obtain effective separation results, anion exchange chromatography as DEAE-cellulose and Q-Sepharose often performed in alkaline condition. That is why most enzyme activity loss in the present purification protocol happened at DEAE-cellulose (pH 8.0), Q-Sepharose (pH 7.5), and gel filtration (pH 8.5) steps. On the other hand, it demonstrated a considerably high purification factor of 78.06-fold, which is much higher than that of laccase from Trametes hirsuta with purification fold of 9.51 [49]. TSL is a monomeric protein just like other fungal laccases and demonstrated a Mr of 59 kDa which fell well within the range of molecular masses of most fungal laccases reported (50-90 kDa) [2]. A recent study of a novel laccase from Cerrena sp. possesed a very close Mr of 58.6 kDa and was also monomeric [30]. Two laccases (lcc1 and lcc2) were isolated from Trametes versicolor with significant difference Mrs of 60 and 100 kDa, respectively [36]. Another laccase from T. sanguinea M85-2 manifested a Mr of 62 kDa [16]. Uzan et al. reported three laccases from Pycnoporus coccineus (Trametes coccineus) BRFM 938, P. sanguineus (T. sanguinea) BRFM 902 and P. sanguineus BRFM 66 with the Mr of 61.8, 62.9, and 59.5 kDa, respectively [50]. Other laccases from genus
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Table 5 Characteristic comparison of laccases from T. sanguinea LAC-01 (this study) and M85-2 [16], and P. sanguineus (T. sanguinea) BRFM 902 and 66 [50].
Moleculr mass (kDa) Molecular structure Optimal pH Optimal temperature (◦ C) Km value toward ABTS (M) Recovery of activity (%) Purification fold
LAC-01
M85-2
BRFM 902
BRFM 66
59 Monomeric 2.6 60 30.28 5.76 78.06
62 Monomeric 5.0 60 No database 73 6
62.9 Monomeric 4.5-5 65 32 32 6.5
59.5 Monomeric 4.5 71 33 20 6.5
Trametes also possessed Mr close to 60 kDa including T. gallica (60 kDa) [51], T. pubescens (60 kDa) [52], T. multicolor (63 kDa) [53], T. villosa (63 kDa) [54], and so on. On the other hand, Pleurotus pulmonarius Lcc2 showed a low Mr of 46 kDa, while Mr of Podospora anserine laccase was as high as 383 kDa [2]. A plant laccase from Carica papaya was found to be a hexameric protein of about 260 kDa [8]. Since laccases perform very important roles in the life cycles, many white rot fungi species secrete more than one kind of laccase in different strains or conditions, e.g., Agaricus bisporus, P. sanguineus, and T. versicolor [2,36,50]. A characteristic comparison of TSL of this study and other laccases from T. sanguinea M85-2 [16] and P. sanguineus (T. sanguinea) BRFM 902 and 66 [50] was summarized in Table 5. The four T. sanguinea laccases shared a close Mr of 60 kDa, Km value of 30 M toward ABTS (except strain M85-2), and an optimal temperature higher than 60 ◦ C. The four amino acid sequences of TSL demonstrated great sequence homology to that of P. sanguineus BRFM 902 and 66 laccases (ACG61169.1 and ACG61158.1) as shown in Table 2. On the other hand, TSL possessed a lowest pH optimum of 2.6 and lost most activity above pH 6.2, while laccases from strain BRFM 902 and 66 were stable at pH range of 4-6 and manifested an optimal pH of 4.5 [50]. Laccase from T. sanguinea M85-2 possessed an optimal pH of 5.0 and optimal temperature of 60 ◦ C [16]. TSL was not obviously affected by the presence of K+ at concentration of 1.25-10 mM and continuous reduced by Fe2+ and Fe3+ , which was similar to laccases from I. baumii and L. ventriosospora [6,9]. Many previous studies indicated that laccases can be enhanced by low levels of Cu2+ ions of 0.5-3.5 mM [6,55,56]. In the present study, Cu2+ in the concentration range of 1.25-5.0 mM reduced the enzyme activity of about 15-30%. Similar results were observed in the assay toward laccases from A. placomyces [23], F. solani [46], and Trametes sp. [10]. Another laccase from Cerrena sp. HYB07 was not obviously affected by Cu2+ of 10 mM [30], while laccase from I. baumii was strongly enhanced with a fold of 10.814.6 by Cu2+ in the assay concentration range of 1.25-10 mM [9]. It suggests that Cu2+ play multifarious effects (positive, neutral, or negative) on laccase activities in different species although most of the laccases possess cupredoxin-like domains with copper ions. The Km value is a kinetic parameter that indicates the affinity of an enzyme and its substrate. The higher the value is, the lower the affinity will be. TSL demonstrates a considerably low Km value of 30.28 M toward ABTS among the reported fungal laccaes. Another two laccases from T. sanguinea BRFM 902 and 66 manifest very close Km values toward ABTS of 32 and 33 M, respectively [50]. Many laccases have high Km values toward ABTS, such as those from A. placomyces (392 M) [23], Coprinus comatus (1590 M) [55], I. baumii (1310 M) [9], Trametes polyzona (150 M) [37], etc. Other laccases from Cerrena sp. HYB07 (93.4 M) [30], T. trogii (69 M) [47], and Trametes sp. (50 M) [10] share close but higher Km values. It suggests that laccases from T. sanguinea are much more efficient than other laccases. The role of fungi in dyes decolorization can be divided into three groups: biosorption, biodegradation, and bioaccumulation [57]. TSL was an effective biodegradation enzyme in the decolorization of
phenolic dyes. It can decolor Bromothymol Blue and Malachite Green with decolorization rate higher than 60% after 24 h of incubation. Other fungal laccases also showed decoloring activity toward industrial and laboratory dyes. A novel laccase newly reported from Cerrena sp. HYB07 and laccase from R. virescens aslo demonstrated decoloring activity toward the two dyes [28,30]. T. hirsuta laccase was reported to demonstrate decolorizing ability toward non-phenolic dyes, such as Methyl Red [58]. Laccase from P. sanguineus BRFM 66 also possessed considerably high decolorization activity toward Anthraquinonic Dye Poly R-478 (33%) and Heterocyclic Dye Azure B (21%) after 52 h of incubation [50]. Laccase from T. trogii was effective to decolorize Malachite Green, Bromophenol Blue, Crystal Violet, and Acid Red without the addition of redox mediators [47]. In the present study, the two dyes Bromothymol Blue and Malachite Green share very close structure with three benzene rings. Fuchsin Basic and Methyl Orange are both benzene derivatives. Eriochrome Black T, Evans Blue, Methylene Blue, and Remazol Brilliant Blue R are naphthalene or heterocyclic derivatives. The two active dyes might fit well with the enzyme activity centers and can be degraded. Acknowledgements This work was financially supported by National Grants of China (31200070 and 2012BAD14B09), Beijing NOVA Program (XX2015B025), Beijing Higher Education Young Elite Teacher Project (YETP1714), Undergraduate Research Program of Beijing, and Beijing Innovative Grant of Modern Agricultural Technology System (PXM2013-014207-000096). References [1] P. Giardina, V. Faraco, C. Pezzella, A. Piscitelli, S. Vanhulle, G. Sannia, Cell Mol. Life Sci. CMLS 67 (2010) 369–385. [2] P. Baldrian, FEMS Microbiol. Rev. 30 (2006) 215–242. [3] C.M. Rivera-Hoyos, E.D. Morales-Alvarez, R.A. Poutou-Pinales, A.M. Pedroza-Rodriguez, R. Rodriguez-Vazquez, J.M. Delgado-Boada, Fungal Biol. Rev. 27 (2013) 67–82. [4] M. Jaszek, M. Osinska-Jaroszuk, G. Janusz, A. Matuszewska, D. Stefaniuk, J. Sulej, J. Polak, M. Ruminowicz, K. Grzywnowicz, A. Jarosz-Wilkolazka,BioMed Res. Int. 2013 (2013), 497492. [5] A.M. Mayer, R.C. Staples, Phytochemistry 60 (2002) 551–565. [6] G.Q. Zhang, Q.J. Chen, H.X. Wang, T.B. Ng, J. Mol. Catal. B Enzym. 85–86 (2013) 31–36. [7] U.N. Dwivedi, P. Singh, V.P. Pandey, A. Kumar, J. Mol. Catal. B Enzym. 68 (2011) 117–128. [8] N. Jaiswal, V.P. Pandey, U.N. Dwivedi, Int. J. Biol. Macromol. 72 (2015) 326–332. [9] J. Sun, Q.J. Chen, M.J. Zhu, H.X. Wang, G.Q. Zhang, J. Mol. Catal. B Enzym. 99 (2014) 20–25. [10] D. Daassi, H. Zouari-Mechichi, A. Prieto, M.J. Martinez, M. Nasri, T. Mechichi, World J. Microb. Biot. 29 (2013) 2145–2155. [11] S.R. Couto, J.L.T. Herrera, Biotechnol. Adv. 24 (2006) 500–513. [12] L. Lesage-Meessen, M. Haon, E. Uzan, A. Levasseur, F. Piumi, D. Navarro, S. Taussac, A. Favel, A. Lomascolo, FEMS Microbiol. Lett. 325 (2011) 37–48. [13] S.M. Zhong, N. Sun, H.X. Liu, Y.Q. Niu, Y. Wu, Exp. Ther. Med. 9 (2015) 341–344. [14] M. Sakamoto, Y. Shirane, I. Naribayashi, K. Kimura, N. Morishita, T. Sakamoto, T. Sakai, Eur. J. Biochem. 226 (1994) 285–291. [15] Y. Tohya, S. Sasagawa, Jpn. J. Pharmacol. 18 (1968) 19–29. [16] Y. Nishizawa, K. Nakabayashi, E. Shinagawa, J. Ferment. Bioeng. 80 (1995) 91–93. [17] A. Grajales, C. Aguilar, J.A. Sanchez, BMC Evol. Biol. 7 (2007) 90.
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
An extracellular laccase with potent dye decolorizing ability from white rot fungus Trametes sp. LAC-01 Zhuo-Ren Ling a , Shan-Shan Wang b , Meng-Juan Zhu c , Ying-Jie Ning a , Shou-Nan Wang a , Bing Li a , Ai-Zhen Yang a , Guo-Qing Zhang a,∗ , Xiao-Meng Zhao a,∗ a Key Laboratory of Urban Agriculture (North) of Ministry of Agriculture, College of Biological Science and Engineering, Beijing University of Agriculture, Beijing 102206, China b 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
a r t i c l e
i n f o
Article history: Received 7 April 2015 Received in revised form 28 July 2015 Accepted 6 September 2015 Available online 8 September 2015 Keywords: Laccase Trametes sanguinea Dye decolorizing activity
a b s t r a c t A novel laccase was purified from fermentation broth of white rot fungus Trametes sp. LAC-01 using an isolation procedure involving three ion-exchange chromatography steps on DEAE-cellulose, SP-Sepharose, and Q-Sepharose, and one gel-filtration step. The purified enzyme (TSL) was proved as a monomeric protein with a Mr of 59 kDa based on SDS-PAGE and FPLC. Partial amino acid sequences were obtained by LC–MS/MS sharing considerably high sequence similarity with that of other laccases. It possessed optimal pH of 2.6 and temperature of 60 ◦ C using ABTS as the substrate. The Km of the laccase toward ABTS was estimated to 30.28 M at pH 2.6 and 40 ◦ C. TSL manifested considerably high oxidizing activity toward ABTS, but was avoid of degradative activity toward benzidine, caftaric acid, etc. It was effective in the decolorization of phenolic dyes – Bromothymol Blue and Malachite Green with decolorization rate higher than 60% after 24 h of incubation. Adjunction of Cu2+ with the final concentration of 2.0 mmol/L significantly activated laccase production with a steady high level of 275.8-282.2 U/mL in 96-144 h. The high yield and short production period makes Trametes sp. LAC-01 and TSL potentially useful for industrial and environmental application and commercialization. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Laccase (-diphenol: dioxygen oxidoreductase, EC 1.10.3.2) is a group of blue multicopper oxidases that catalyze the oxidation of a wide variety of organic aromatic substrates concomitantly with the reduction of molecular oxygen to water [1,2]. It was first obtained from the Japanese lacquer tree (Rhus vernicifera) in 1883 and subsequently isolated from fungi in 1896, which makes it one of the oldest enzymes ever described [2]. Laccases are widely distributed in plants, bacteria, insects, and especially fungi [3,4]. They can be roughly divided into two major groups, i.e. laccases from higher plants and those from fungi [2,5]. Plant laccases involve in lignin synthesis and wound healing, whereas fungal laccases probably have more roles and participate in lignin degradation, pigmentation, pathogenesis, fungal plant-pathogen/host interaction, and stress defence, etc. [2,6,7].
∗ Corresponding authors. E-mail addresses: [email protected] (G.-Q. Zhang), [email protected] (X.-M. Zhao). http://dx.doi.org/10.1016/j.ijbiomac.2015.09.011 0141-8130/© 2015 Elsevier B.V. All rights reserved.
At present, more than 100 fungal laccases from Basidiomycota and Ascomycota have been purified and characterized [2,3]. Purification methods described in literature include ion exchange, affinity, and gel filtration chromatography [8–10]. Enzymatic properties including molecular mass, subunit composition, optimal pH and temperature, substrates, and Km values have been widely studied and reviewed [1,2,5]. Nowadays, fungal laccases are being extensively evaluated due to their wide applications in food, pulp and paper, textile, nanobiotechnology, and pharmaceutical industries [1,11]. Trametes sanguinea, called ‘red blood blot fungus’ in Chinese, is a white rot saprobic fungus with synonyms names of Pycnoporus sanguineus and Trametes sanguineus belonging to the family Polyporaceae [12]. It is widely distributed in China, occurs in summer and early autumn, and grows on dead hardwoods. Its fruiting bodies are bright orange to red with a diameter range of 3-10 cm. Previous literature revealed that T. sanguinea was rich in bioactive enzymes, e.g., laccase, protease, and protopectinase [13–16]. A laccase with 62 kDa, optimal pH of 5.0, and optimal temperature of 60 ◦ C was isolated from T. sanguinea M85-2 [16]. In the present study, T. sanguinea strain LAC-01 was isolated from the Baiwangshan National
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Forest Park, Beijing, China. We aim to purify an extracellular laccase from fermentation broth the strain LAC-01. Enzymatic properties and dye decolorizing application are also investigated. 2. Materials and methods 2.1. Fungal strain and culture condition Strain LAC-01 was isolated from the fruiting bodies of Trametes sp., which were collected from a deadwood of Populus tomentosa in the Baiwangshan National Forest Park (Beijing, China). It was cultured at 28 ◦ C, stored at 4 ◦ C, and monthly transferred to fresh potato dextrose agar (PDA) slant medium which contained (g/L): potato, 200; glucose, 20; and agar, 20. To obtain its extracellular laccase, strain LAC-01 was inoculated in the liquid potato dextrose (PD) medium containing (g/L): potato, 200 and glucose, 20, and in an orbital shaking incubator at 180 rpm and 28 ◦ C for 8 days. The fermentation broth was subsequently used as laccase primary extract. 2.2. Effect of Cu2+ on laccase production in liquid fermentation In order to enhance the enzyme yield, effect of Cu2+ on laccase production in liquid fermentation was observed. CuSO4 solution (1 mol/L) was added into the fermentation medium (the PD medium) with the final concentration of 0, 0.25, 0.50, 1.0, and 2.0 mM, respectively. The broth was collected at 72, 96, 120, 144, 168, and 192 h, respectively. Following a centrifugation at 8000 rpm and 4 ◦ C for 15 min, laccase activity in broth was assayed using the standard assay. 2.3. Classification based on rDNA sequence analysis Classification of the strain LAC-01 was analyzed based on PCR amplification and phylogenetic analysis of the internal transcribed spacer (ITS) regions which contained ITS-1, 5.8S, and ITS-2 region in the rDNA Cluster [17,18]. Total genomic DNA of fungal mycelia was extracted using the CTAB method [19]. The ITS region was amplified by PCR using universal primers ITS1 (5 TCCGTAGGTGAACCTGCGG 3 ) and ITS4 (5 TCCTCCGCTTATTGATATGC 3 ) and performed in a volume of 50 L under standard conditions [20]. The PCR production was subsequently sequenced by Shanghai SANGON Biological Engineering Co. (Shanghai, China). Obtained sequences were compared with sequences in the BLAST/NCBI database (http:// blast.ncbi.nlm.nih.gov/Blast.cgi) to determine the nearest matches. Phylogenetic trees were finally constructed according to the Neighbor-Joining (NJ) algorithm using MEGA Software (Version 6.06). Bootstrap analysis was based on 1000 replication. 2.4. Purification of laccase After 8-day fermentation, the broth was initially filtered to remove mycelial debris, followed by centrifugation at 8000 rpm and 4 ◦ C for 15 min. Subsequently, the supernatant were dialyzed with Tris–HCl buffer (10 mM, pH 8.0) at 4 ◦ C overnight. The supernatant was further applied on an anion exchange DEAE-cellulose column (2.5 × 20 cm2 , Bio-Rad) in Tris–HCl buffer (10 mM, pH 8.0) with the flow rate of 2 mL/min. After removal of unadsorbed proteins, adsorbed proteins were desorbed from the column with 100, 300, and 1000 mM NaCl added to the Tris–HCl buffer, respectively. Laccase active fraction was chromatographed on a cation exchange SP-Sepharose column (2.5 × 20 cm2 , Bio-Rad) in HAc–NaAc buffer (10 mM, pH 4.0) with the flow rate of 2 mL/min. After collection of unadsorbed proteins, the column was eluted with 800 mM NaCl added to the HAc–NaAc buffer. Subsequently, enzyme containing fraction was subjected to another anion exchange chromatography
on Q-Sepharose column (1.5 × 10 cm2 , Bio-Rad) in Tris–HCl buffer (10 mM, pH 7.5) with the flow rate of 1 mL/min. Following removal of unadsorbed proteins, adsorbed proteins were desorbed with three gradients of 100, 300, and 500 mM NaCl in Tris–HCl buffer. Laccase rich fractions were pooled, dialyzed and finally purified by fast protein liquid chromatography (FPLC) on a Superdex 75 gel filtration column (0.15 M NH4 HCO3 buffer, pH 8.5) with a flow rate of 0.8 mL/min. 2.5. Assay for laccase activity Laccase activity was spectrophotometrically determined by using 2, 2 -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate [21,22]. Laccase solution (10 L) was mixed with 0.6 mM ABTS solution (190 L, in 50 mM sodium acetate buffer, pH 4.5) at 40 ◦ C for 5 min, followed by an addition of 10% TCA (200 L) to end the reaction. The increase in absorbance was monitored at 405 nm to test enzyme activity. All determinations were performed in triplicate. One enzyme unit (U) was defined as the amount of enzyme required to produce one absorbance increase of the reaction mixture at 405 nm/min/mL [6]. 2.6. Determination of molecular mass The molecular mass (Mr) of the present laccase was determine using FPLC-gel filtration and sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). In FPLC chromatography, a standard curve based on elution volume and Log Mr of molecular mass standards (GE Healthcare) was obtained. Mr of the active laccase can be calculated based on its elution volume [23]. In SDS-PAGE, a 12% resolving gel and a 5% stacking gel were used following the standard procedure [24]. Another standard curve of relative mobility and Log Mr of unstained molecular mass standards (Thermo Scientific) can be used for the Mr evaluation of inactive monomeric protein or subunits. 2.7. LC–MS/MS analysis The protein band of purified laccase on SDS-PAGE was recovered and digested by trypsin. Digestion products were subsequently analyzed by LC–MS/MS for amino acid sequences. The data were acquired using Xcalibur software (Thermo Electron). Sequence homologues were searched using the BLAST/NCBI database [25,26]. 2.8. Effects of pH and temperature on the activity of purified laccase To determine the effect of pH values on laccase activity, a series of ABTS solution in various pH values were used instead of the standard ABTS solution at pH 4.5. The assay buffers were prepared in citric acid–Na2 HPO4 buffers (pH range of 2.2-8.0). In the temperature assay, standard activity assay was measured in different temperature from 20 ◦ C to 90 ◦ C instead of 40 ◦ C in the standard assay. 2.9. Determination of pH stability and thermostability of purified laccase In the pH stability assay, enzyme solutions were previously incubated in 50 mM citric acid–Na2 HPO4 buffers at different pH values (2.2, 2.6, 3.0, and 3.4, respectively) for different durations (10, 20, 30, 40, 50, and 60 min, respectively) at 4 ◦ C. The residual laccase activity was triplicately measured using the standard assay. In the thermostability assay, enzyme solutions were previously incubated at various temperatures (50, 60, 70, and 80 ◦ C, respectively) for 10, 20, 30, 40, 50, and 60 min, respectively. The residual laccase activity
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was assayed in triplicate using the standard assay after the reaction mixture had been cooled down to room temperature [9]. 2.10. Effects of chemical reagents on laccase activity To estimate the influence of metal ions and EDTA on enzyme activity, chemical reagents including CaCl2 , CdCl2 , CoCl2 , CuCl2 , FeCl2 , FeCl3 , KCl, MgCl2 , MnCl2 , NaCl, ZnCl2 , and EDTA (at concentrations of 1.25, 2.5, 5.0, and 10 mM, respectively) were mixed with equal volumes of the purified laccase solution at 4 ◦ C for 1 h. The residual laccase activity was measured in triplicate using the standard assay. Control samples were treated with distilled water instead of the metal ion solution. 2.11. Assay for substrate specificity and kinetic parameter of purified laccase Fig. 1. Effect of Cu2+ on laccase production in liquid fermentation.
In the substrate specificity assay, seven aromatic reagents (in concentration of 5.0 mM, 50 mM pH 2.6 citric acid–Na2 HPO4 buffers) were tested as the substrates including ABTS, benzidine, caftaric acid, catechol, guaiacol, o-Toluidines, phenol, and tyrosine. The reaction was started by adding 10 L of laccase solution and 190 L substrate solution, and incubated at 40 ◦ C for 5 min, followed by an addition of 10% TCA (200 L) to end the reaction. The substrate oxidation rate was followed by measuring the absorbance change with the molar extinction coefficients of various substrates [27,28]. Laccase activity toward ABTS was regarded as 100%. All determinations were performed in triplicate. Kinetic studies of the purified laccase were carried out using ABTS as substrate in a series concentration ranging from 12.5 to 800 M at pH 2.6 and 40 ◦ C. All determinations were performed in triplication. The reciprocals of the substrate concentrations and the reciprocals of the corresponding initial velocities were then used to generate a Lineweaver-Burk plot [29]. 2.12. Assay of dye decolorization by purified laccase The decolorization of different dyes used in textile and chemical industries by purified laccase (0.5 U/mL) was determined over a 24 h period. Eight kinds of dyes were used including Bromothymol Blue, Eriochrome Black T, Evans Blue, Fuchsin Basic, Malachite Green, Methylene Blue, Methyl Orange, and Reactive Brilliant Blue R. The test was performed at 40 ◦ C by adding 20 L purified laccase into 380 l citric acid–Na2 HPO4 buffers (pH 2.6) which contains different kinds of dyes, respectively. Initial concentration of dyes was determined based on their maximal absorbance wavelength with an absorbance value range of 0.7-1.3. The decolorizing ability of laccase was determined spectrophotometrically as the relative decrease of absorbance at 4, 12, and 24 h. All reactions were performed in triplicate [28,30,31]. 3. Results 3.1. Effect of Cu2+ on laccase production in liquid fermentation Effect of Cu2+ on laccase production of Strain LAC-01 in liquid fermentation was visualized in Fig. 1. In the negative control group, laccase activity in PD medium without any Cu2+ adjunction reached its highest level of 122.8 U/mL after 8 day liquid fermentation. Adjunction of Cu2+ with the final concentration of 2.0 mmol/L significantly activated laccase production with a steady high level of 275.8-282.2 U/mL in 96-144 h. At 72 h, laccase activity in 2.0 mM Cu2+ group achieved 243.7 U/mL, while enzyme activity was avoid in the control group at the same time. On the other hand, adjunction
of Cu2+ with the final concentration higher than 2.0 mM strongly inhibited the growth of T. sanguinea mycelia (data not shown). 3.2. Classification of the laccase producing fungus Strain LAC-01 was initially identified as species from genus Trametes based on its morphological properties. A 596-bp fragment of ITS region (GenBank accession No. of KP723552) was sequenced and compared to GenBank using BLAST. A phylogenetic tree based on ITS region of Trametes species was constructed (Fig. 2). Strain LAC-01 was closest to T. sanguinea (GenBank accession No. JN164981.1), sharing the query cover of 96% and the sequence identity of 100%. Strain LAC-01 was classified as T. sanguinea based on both morphological and molecular identification [12]. 3.3. Laccase purification and molecular mass determination On DEAE-cellulose anion exchange chromatography, the crude enzyme extract was chromatographed into three unadsorbed fractions (D1, D2, and D3) eluted with Tris–HCl buffer (10 mM, pH 8.0) and three adsorbed fractions (D4, D5, and D6) eluted with 100, 300, and 1000 mM NaCl in Tris–HCl buffer (10 mM, pH 8.0), respectively. Subsequently, fraction D5 possessing laccase activity was fractionated on SP-Sepharose cation exchange chromatography and resolved into an unadsorbed fraction S1 with the laccase activity in starting buffer (10 mM, pH 4.0 HAc–NaAc buffer) and an adsorbed fraction S2 in starting buffer with 800 mM NaCl. Laccase active fraction S1 was further applied on Q-Sepharose anion exchange chromatography and separated into three adsorbed fractions Q1, Q2, and Q3 with three gradients of 100, 300, and 500 mM NaCl in Tris–HCl buffer (10 mM, pH 7.5), respectively. The laccase active fraction Q2 was finally subjected to an FPLC-Superdex 75 HR 10/30 column to yield only one fraction SU1 containing purified laccae and named as T. sanguinea laccase (TSL). SU1 fraction possessed a Mr of 59 kDa as estimated by FPLC based on the standard curve of elution volume-log Mr (Fig. 3A). In SDS-PAGE, SU1 fraction appeared as a single band with a Mr of 59 kDa (Fig. 3B). It suggested that the native laccase is a monomeric protein. The enzyme was purified 78.06-fold from the crude extractive with 5.76% yield. The purified laccase exhibited an activity of 839.16 U/mg toward ABTS at the standard assay condition (Table 1). 3.4. Amino acid sequence LC–MS/MS analysis of the purified laccase disclosed four internal peptides: Seq-01, RSPGTTTADLAVIKVTQGKR, Seq-02, RLVSLSCDPNHTFSIDGHTMTVIEADSVNTQPLEVDSIQIFAAQRY, Seq-03,
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51
96
24 7
Trametes elegans (JN164986.1) 63
Trametes maxima (JN164957.1) Trametes membranacea (JN164956.1) Trametes gibbosa (KJ140582.1)
24
23
38
63
Trametes hirsuta (HQ435869.1) 92
Trametes villosa (KF573031.1)
77
Trametes conchifer (JN164988.1) Trametes junipericola (AY684171.2) Trametes suaveolens (KF573015.1) Trametes velutina (KF573019.1)
54 95 42
46
Trametes sanguinea LAC-01 (This study) Trametes cinnabarina (KF573022.1) Trametes ljubarskii ( AY684174.2)
3
35
Trametes sanguinea (JN164981.1)
43
Trametes ectypa (JN164961.1) Trametes ochracea (JN164976.1)
85
Trametes obstinata (EU661880.1) Trametes purpurea (EU661881.1)
33
Trametes orientalis (EU771082.1) 33
50 83
Trametes pubescens (KJ140686.1) Trametes cubensis (JN164989.1) Trametes trogii (HM989941.1) Trametes ellipsospora (JN048767.1) Trametes hispida (EU661874.1) Trametes robiniophila (EU661883.1)
95
Trametes corrugata (EU661875.1) 98
Trametes palisotii (EU661873.1) Trametes versicolor (EU273523.1)
Fig. 2. Neighbor-joining phylogenetic tree based on ITS sequences showing the distance of strain LAC-01 with the nearest species of the genus Trametes. Bootstrap values at nodes are percentages of 1000 replicates.
RYSFVLDASQPVDNYWIRA, and Seq-04, RSAGSSEYNYDNPIFRD. Based on BLAST results, they all belonged to Cuperdoxin superfamily which were blue copper proteins including laccases. The homology search of the obtained sequences in BLAST showed that they demonstrated considerably high sequence homology to those of previously reported laccases from genus Trametes and other white rot fungi (Table 2).
3.5. Optimal pH and temperature, pH stability, and thermostability of purified laccase The purified laccase achieved its maximal oxidizing activity toward ABTS at pH 2.6 and underwent a gradual decline when the assay pH rose from 2.6 to 5.0. Approximately 60% of total activity survived at pH 5.0. When the pH value further increased, a sharp decrease in enzyme activity was observed with an almost zero level of laccase activity above pH 6.2 (Fig. 4A). The purified laccase demonstrated a considerable high thermostability with an optimal temperature of 60 ◦ C. Oxidizing activity toward ABTS at 60 ◦ C was nearly twice as high as that of at 20 ◦ C. More than 20%
of total enzyme activity remained when it was assayed at 90 ◦ C (Fig. 4B). TSL shows strong stability in the assay pH range of 2.2-3.4 (Fig. 5A). Most of the oxidizing activity remained after 60 min preincubation in the different pH conditions. A slight increase (about 10%) can be observed after 60 min pre-incubation at pH 2.6. TSL possesses considerably high thermostability. Most of the enzyme activity maintains when it underwent 60 min pre-incubation at 5070 ◦ C (Fig. 5B). There are soft but continuous increases in laccase activity when enzyme solution was previously incubated at 50 ◦ C and 60 ◦ C for 0-60 min. When incubated at 70 ◦ C, enzyme activity reaches the highest level with 31.3% of increase rate after 50 min of pre-incubation. TSL is sensitive to temperature as high as 80 ◦ C. During pre-incubation at 80 ◦ C, a sharp and continuous decrease in enzyme activity can be observed. 3.6. Effects of chemical reagents The sensitivity of purified laccase to metal ions and EDTA is summarized in Table 3. TSL was not obviously affected by the existence of K+ at concentration of 1.25-10 mM, Ca2+ at concentration of
Table 1 Summary of purification procedure of TSL laccase (from 1 L fermentation broth). Purification step
Total protein (mg)
Specific activity (U/mg)
Total activity (U)
Recovery of activity (%)
Purification fold
Fermentation broth DEAE-cellulose (D5) SP-Sepharose (S1) Q-Sepharose (Q2) Superdex 75 (SU1)
5805 325.8 143.92 23.03 4.29
10.75 53.01 114.05 430.97 839.16
62,404 17,271 16,414 9925 3600
100 27.7 26.3 15.9 5.76
1 4.93 10.61 40.09 78.06
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Table 2 Comparison of partial amino acid sequence of TSL in this study and other fungal laccases. Species
Amino acid sequence
GenBank No.
Trametes sanguinea Pycnoporus coccineus Trametes cinnabarina Trametes sanguinea Trametes sanguinea Ganoderma lucidum Trametes pubescens Trametes versicolor Ganoderma fornicatum Trametes punicea Trametes hirsuta Trametes gibbosa Trametes sanguinea Pycnoporus coccineus Trametes cinnabarina Trametes sanguinea Trametes sanguinea Trametes gibbosa Trametes hirsute Trametes punicea Trametes versicolor Pleurotus ostreatus Trametes pubescens Ganoderma lucidum Trametes sanguinea Pycnoporus coccineus Trametes cinnabarina Trametes sanguinea Trametes sanguinea Trametes punicea Lentinula edodes Trametes hirsute Trametes gibbosa Ganoderma lucidum Trametes sanguinea Pycnoporus coccineus Trametes cinnabarina Trametes sanguinea Trametes sanguinea Trametes gibbosa Trametes punicea Trametes pubescens Trametes versicolor Ganoderma lucidum
RSPGTTTADLAVIKVTQGKR 113 RSPGTTTADLAVIKVTQGKR 132 73 RSPGTTTADLAVIKVTQGKR 92 113 RSPGTTAADLAVIKVTQGKR 127 113 RTPGTTSADLAVIKVTQGKR 127 197 RSPATPTADLAVISVTQGKR 216 197 RSPSTTTADLAVISVTAGKR 216 197 RSPSTTTADLSVISVTPGKR 216 198 RSTATPTADLAVVNVTQGKR 217 113 RTPGNTTTELAVITVTQGKR 132 197 RAPSDTTAELSVIKVTKGKR 216 74 RSPSTVSADLSVINVTPGKR 93 RLVSLSCDPNHTFSIDGHTMTVIEADSVNTQPLEVDSIQIFAAQRY 136 RLVSLSCDPNHTFSIDGHTMTVIEADSVNTQPLEVDSIQIFAAQRY 181 96 RLVSLSCDPNHTFSIDGHTMTVIEADSVNTQPLEVDSIQIFAAQRY 141 136 RLVSLSCDPNHTFSIDGHTMTIIEADSVNTQPLEVDSIQIFAAQRY 181 136 RLVSLSCDPNHTFSIDGHTMTVIEADSVNTQPLEVDSIQIFAAQRY 181 97 RLVSLSCDPNHTFSIDGHDLTIIETDSVNTQPLVVDSIQIFAAQRY 142 220 RLVSLSCDPNHTFSIDGHNLTIIEVDSVNSQPLEVDSIQIFAAQRY 265 136 RLVSLSCDPNHTFSIDNHTMTIIETDAINTQPLEVDSIQIFAAQRY 181 220 RLVSLSCDPNHTFSIDGHNMTIIETDSINTAPLVVDSIQIFAAQRY 265 96 RLVSISCDPNHVFAIDGHNMTIIEVDSVNTQPLVVDSIQIFPGQRY 141 220 RLVSLSCDPNYVFSIDGHNMTIIETDSINTQPLVVDSIQIFAAQRY 265 220 RLVSLSCDPNFTFSIDGHAMTVIEADAVNHEPLTVDSIQIFAGQRY 265 RYSFVLDASQPVDNYWIRA 180 RYSFVLDASQPVDNYWIRA 198 140 RYSFVLDASQPVDNYWIRA 158 180 RYSFVLDASQPVDNYWIRA 198 180 RYSFVLDASQPVDNYWIRA 198 180 RYSFVLEANQPVDNYWIRA 198 278 RYSFVLDATQPVDNYWVRA 296 264 RYSFVLNANQPVDNYWIRA 282 141 RYSFVLEANQPVDNYWVRA 159 269 RYSFVLEANQPVNNYWIRA 287 RSAGSSEYNYDNPIFRD 345 RSAGSSEYNYDNPIFRD 361 429 RSAGSSEYNYDNPIFRD 445 345 RSAGSSEYNYDNPIFRD 361 345 RSAGSSEYNYDNPVFRD 361 429 RSAGSTEYNYDDPIFRD 445 345 RSAGSTEYNYDNPIFRD 361 429 RSAGSTVYNYDNPIFRD 445 429 RSAGSTVYNYDNPIFRD 445 449 RSAGSSEYNYENPVRRD 465
Seq-01 (This study) ACH61789.1 CAD12769.1 ACG61169.1 ACG61158.1 ABK59822.1 AAM18407.1 AAL07440.1 ABK59827.1 ACR24938.1 AIZ72721.1 AEQ38867.1 Seq-02 (This study) ACH61789.1 CAD12769.1 ACG61169.1 ACG61158.1 AEQ38867.1 AIZ72721.1 ACR24938.1 AAL93622.1 AEQ38864.1 AAM18407.1 AHA83589.1 Seq-03 (This study) ACH61789.1 CAD12769.1 ACG61169.1 ACG61158.1 ACR24938.1 AAT99288.1 AIZ72727.1 AEQ38867.1 AHA83587.1 Seq-04 (This study) ACH61789.1 AAN71597.1 ACG61169.1 ACG61158.1 ADK13091.1 ACR24938.1 AAM18407.1 AAL07440.1 AHA83591.1
Amino acid residues identical to corresponding residues of the purified laccase are underlined.
Table 3 Effect of metal ions and EDTA on laccase activity. Chemical reagent
Residual activity (% of control) 10 mM
+
Na K+ Ca2+ Cd2+ Co2+ Cu2+ Fe2+ Mg2+ Mn2+ Zn2+ Fe3+ EDTA
86.1 105.8 86.6 0.7 76.8 68.4 7.5 80.6 40.6 83.4 0.0 74.9
± ± ± ± ± ± ± ± ± ± ± ±
5.0 mM 1.9 0.6 1.4 1.2 1.1 0.3 0.2 0.9 1.6 0.9 0.0 2.0
89.5 104.4 92.9 73.3 76.9 68.6 26.5 82.2 85.0 87.2 20.8 79.2
± ± ± ± ± ± ± ± ± ± ± ±
2.5 mM 0.5 1.0 1.1 0.7 0.1 0.4 2.6 1.5 2.4 0.3 0.1 1.1
Laccase activity in the absence of metal ions was regarded as 100%. Values represent mean ± standard deviation (n = 3).
92.6 103.6 99.0 74.4 70.2 68.7 71.5 91.0 79.0 95.3 39.9 81.7
± ± ± ± ± ± ± ± ± ± ± ±
1.25 mM 1.5 1.6 0.8 1.5 0.3 0.4 0.3 0.3 0.2 1.7 0.4 0.5
98.3 102.5 99.7 76.3 87.7 77.0 77.7 87.8 68.2 99.3 49.1 74.2
± ± ± ± ± ± ± ± ± ± ± ±
0.9 0.9 0.7 1.6 2.8 0.8 0.4 0.2 1.6 0.4 0.4 0.3
790
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Fig. 3. Molecular mass determination of the purified laccase. (A) FPLC-gel filtration on Superdex 75 HR 10/30 column. Eluent: 0.15 M NH4 HCO3 buffer (pH 8.5). Fraction size: 0.8 mL. Flow rate: 0.8 mL/min. Fraction SU1 represents purified laccase. (B) SDS-PAGE of Fraction SU1 from FPLC. Right lane: Molecular weight markers, from top downwards, -galactosidase (116.0 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45.0 kDa), lactate dehydrogenase (35.0 kDa), REase Bsp98I (25.0 kDa), -lactoglobulin (18.4 kDa), and lysozyme (14.4 kDa).
Fig. 4. Effects of pH and temperature on activity of the purified laccase TSL with ABTS as substrate. (A) Effect of pH on TSL activity. (B) Effect of temperature on TSL activity.
3.8. Decolorization of dyes 1.25-5.0 mM, and Zn2+ at concentration of 1.25-2.5. It was inhibited with about 15-30% loss of total activity by Cd2+ , Co2+ , Cu2+ , Mg2+ , Mn2+ , and EDTA concentration of 1.25-5.0 mM. Addition of Fe2+ and Fe3+ with concentration higher then 5 mM led to more than 50% loss of total activity.
3.7. Substrate specificity and kinetic parameter Although TSL demonstrated considerably high oxidizing activity toward ABTS, it was avoid of degradative activity toward benzidine, caftaric acid, catechol, guaiacol, o-toluidines, and tyrosine. On the other hand, it manifested a very low oxidation activity toward phenol with relative activity of 13.20% comparing with ABTS. After incubation of the purified laccase with a series concentration of ABTS (12.5 to 800 M) at pH 2.6 and 40 ◦ C, the reactions were found to follow Michaelis–Menten kinetics, displaying the Km value of 30.28 M toward ABTS according to the Lineweaver-Burk plots (data not shown).
Decolorizing acitivity of purified laccase on industrial and laboratory dyes was evaluated at a low enzyme activity of 0.5 U/mL (Table 4). TSL possessed considerably high decolorizing activities toward Bromothymol Blue and Malachite Green with efficiencies higher than 50%. After incubating for 4 h, Bromothymol Blue and Malachite Green were decolorized with the rates of 62.0% and 46.1%, respectively. A low decolorizing rate of 8.1% was obtained when TSL was assayed toward Evans Bue. When incubating time prolonged from 12 to 24 h, decolorizing rates of 63.1–65.4% toward Bromothymol Blue and 70.1–75.8% toward Malachite Green were attained. After incubating for 24 h, TSL demonstrated low decolorizing rates of about 10% toward Evans Bue, Fuchsin Basic, and Methylene Blue. It was almost avoid of decolorizing activity toward Methyl Orange and Remazol Brilliant Blue R. 4. Discussion White rot fungi, such as species from genera Coriolus, Ganoderma, Pleurotus, and Trametes, possess remarkable degradation
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Table 4 Decolorization of the dyes by purified laccase TSL. Dye
max
Concentration (g/mL)
Bromothymol Blue Eriochrome Black T Evans Blue Fuchsin Basic Malachite Green Methylene Blue Methyl Orange Remazol Brilliant Blue R
440 540 610 530 614 664 460 605
800 400 28 20 8 8 20 100
Decoloration rate (%) 4h 62.0 0 8.1 1.7 46.1 0 0 0.5
12 h ± ± ± ± ± ± ± ±
0.2 0.1 0.2 0.9 0.5 0.4 0.1 0.1
63.1 0 9.3 5.4 70.1 6.8 0 1.0
24 h ± ± ± ± ± ± ± ±
0.1 0.1 1.0 0.4 0.6 0.5 0.3 0.1
65.4 0 11.2 8.6 75.8 7.9 0.1 3.9
± ± ± ± ± ± ± ±
0.3 0.1 0.3 0.9 0.6 0.4 0.5 1.3
Decolorization efficiencies higher than 50% are highlighted in bold. Values represent mean ± standard deviation (n = 3).
ability toward both lignin and cellulose biopolymers in lignocellulose biomass by producing ligninolytic enzymes including laccase, manganese peroxidase (MnP), lignin peroxidase (LiP) [32–34]. Among them, fungal laccases are of great interest to industry, and have been used in bio-degradation of lignocellulosic
Fig. 5. pH stability and thermostability of purified laccase. (A) pH stability of TSL. Residual activity was measured using the standard assay after incubating at pH values for 10-60 min. The assay pH values were 2.2 (), 2.6 (䊏), 3.0 (×), and 3.4 (). (B) Thermostability of TSL. Residual activity was measured using the standard assay after incubating at various temperatures for 10-60 min. The assay temperatures were 50 ◦ C (×), 60 ◦ C (䊐), 70 ◦ C (), and 80 ◦ C (䊉).
compounds, wastewater treatment, bio-pulping and bio-bleaching in textile industry [3,11,35]. Nowadays, laccases from genus Trametes have been extensively investigated including screening, purification, and structure analysis of novel enzyme proteins, cloning and heterologous expression of laccase genes, enzymatic engineering and fermentation methods, and potential applications in decolorization and detoxication [36–44]. Although fungal laccases possess a great biotechnological potential, commercial applications are limited by low enzyme yields and high costs. The present laccase produced about 280 U/mL laccase activity after cultivation in PD medium for only 4 day with Cu2+ as the inducer with the final concentration of 2.0 mM. The possible mechanism for this phenomenon is that copper ions enhance the laccase genetic transcription level during the laccase synthesis [45]. Laccase production of Cerrena unicolor strain 137 reached its maximum level of 18.7 U/mL after 10 days in complex tomato juice medium [22]. F. solani MAS2 laccase needed incubation time of 40 days and achieved the maximum activity at day 35 of 575 mU/mg protein [46]. Optimization of laccase production of Trametes trogii S0301 by response surface methodology revealed that the maximum laccase activity of 122.9 U/mL was obtained at day 9 with phenol, CuSO4 and PEG 4000 as inducers [47]. The high laccase yield and short production period of TSL would be advantageous for application and commercialization. The extracellular laccase TSL from fermentation broth of T. sanguinea strain LAC-01 was purified with an isolation procedure involving three steps of ion exchange chromatography and one final FPLC-gel filtration on Superdex 75, resulting in a very low recovery rate of 5.76%. Although many reported fungal laccases possessed relatively high pH stability (e.g. Inonotus baumii and Abortiporus biennis), TSL of present study was very sensitive with a low optimal pH of 2.6 and completely inactivated above pH 6.2 [9,48]. To obtain effective separation results, anion exchange chromatography as DEAE-cellulose and Q-Sepharose often performed in alkaline condition. That is why most enzyme activity loss in the present purification protocol happened at DEAE-cellulose (pH 8.0), Q-Sepharose (pH 7.5), and gel filtration (pH 8.5) steps. On the other hand, it demonstrated a considerably high purification factor of 78.06-fold, which is much higher than that of laccase from Trametes hirsuta with purification fold of 9.51 [49]. TSL is a monomeric protein just like other fungal laccases and demonstrated a Mr of 59 kDa which fell well within the range of molecular masses of most fungal laccases reported (50-90 kDa) [2]. A recent study of a novel laccase from Cerrena sp. possesed a very close Mr of 58.6 kDa and was also monomeric [30]. Two laccases (lcc1 and lcc2) were isolated from Trametes versicolor with significant difference Mrs of 60 and 100 kDa, respectively [36]. Another laccase from T. sanguinea M85-2 manifested a Mr of 62 kDa [16]. Uzan et al. reported three laccases from Pycnoporus coccineus (Trametes coccineus) BRFM 938, P. sanguineus (T. sanguinea) BRFM 902 and P. sanguineus BRFM 66 with the Mr of 61.8, 62.9, and 59.5 kDa, respectively [50]. Other laccases from genus
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Table 5 Characteristic comparison of laccases from T. sanguinea LAC-01 (this study) and M85-2 [16], and P. sanguineus (T. sanguinea) BRFM 902 and 66 [50].
Moleculr mass (kDa) Molecular structure Optimal pH Optimal temperature (◦ C) Km value toward ABTS (M) Recovery of activity (%) Purification fold
LAC-01
M85-2
BRFM 902
BRFM 66
59 Monomeric 2.6 60 30.28 5.76 78.06
62 Monomeric 5.0 60 No database 73 6
62.9 Monomeric 4.5-5 65 32 32 6.5
59.5 Monomeric 4.5 71 33 20 6.5
Trametes also possessed Mr close to 60 kDa including T. gallica (60 kDa) [51], T. pubescens (60 kDa) [52], T. multicolor (63 kDa) [53], T. villosa (63 kDa) [54], and so on. On the other hand, Pleurotus pulmonarius Lcc2 showed a low Mr of 46 kDa, while Mr of Podospora anserine laccase was as high as 383 kDa [2]. A plant laccase from Carica papaya was found to be a hexameric protein of about 260 kDa [8]. Since laccases perform very important roles in the life cycles, many white rot fungi species secrete more than one kind of laccase in different strains or conditions, e.g., Agaricus bisporus, P. sanguineus, and T. versicolor [2,36,50]. A characteristic comparison of TSL of this study and other laccases from T. sanguinea M85-2 [16] and P. sanguineus (T. sanguinea) BRFM 902 and 66 [50] was summarized in Table 5. The four T. sanguinea laccases shared a close Mr of 60 kDa, Km value of 30 M toward ABTS (except strain M85-2), and an optimal temperature higher than 60 ◦ C. The four amino acid sequences of TSL demonstrated great sequence homology to that of P. sanguineus BRFM 902 and 66 laccases (ACG61169.1 and ACG61158.1) as shown in Table 2. On the other hand, TSL possessed a lowest pH optimum of 2.6 and lost most activity above pH 6.2, while laccases from strain BRFM 902 and 66 were stable at pH range of 4-6 and manifested an optimal pH of 4.5 [50]. Laccase from T. sanguinea M85-2 possessed an optimal pH of 5.0 and optimal temperature of 60 ◦ C [16]. TSL was not obviously affected by the presence of K+ at concentration of 1.25-10 mM and continuous reduced by Fe2+ and Fe3+ , which was similar to laccases from I. baumii and L. ventriosospora [6,9]. Many previous studies indicated that laccases can be enhanced by low levels of Cu2+ ions of 0.5-3.5 mM [6,55,56]. In the present study, Cu2+ in the concentration range of 1.25-5.0 mM reduced the enzyme activity of about 15-30%. Similar results were observed in the assay toward laccases from A. placomyces [23], F. solani [46], and Trametes sp. [10]. Another laccase from Cerrena sp. HYB07 was not obviously affected by Cu2+ of 10 mM [30], while laccase from I. baumii was strongly enhanced with a fold of 10.814.6 by Cu2+ in the assay concentration range of 1.25-10 mM [9]. It suggests that Cu2+ play multifarious effects (positive, neutral, or negative) on laccase activities in different species although most of the laccases possess cupredoxin-like domains with copper ions. The Km value is a kinetic parameter that indicates the affinity of an enzyme and its substrate. The higher the value is, the lower the affinity will be. TSL demonstrates a considerably low Km value of 30.28 M toward ABTS among the reported fungal laccaes. Another two laccases from T. sanguinea BRFM 902 and 66 manifest very close Km values toward ABTS of 32 and 33 M, respectively [50]. Many laccases have high Km values toward ABTS, such as those from A. placomyces (392 M) [23], Coprinus comatus (1590 M) [55], I. baumii (1310 M) [9], Trametes polyzona (150 M) [37], etc. Other laccases from Cerrena sp. HYB07 (93.4 M) [30], T. trogii (69 M) [47], and Trametes sp. (50 M) [10] share close but higher Km values. It suggests that laccases from T. sanguinea are much more efficient than other laccases. The role of fungi in dyes decolorization can be divided into three groups: biosorption, biodegradation, and bioaccumulation [57]. TSL was an effective biodegradation enzyme in the decolorization of
phenolic dyes. It can decolor Bromothymol Blue and Malachite Green with decolorization rate higher than 60% after 24 h of incubation. Other fungal laccases also showed decoloring activity toward industrial and laboratory dyes. A novel laccase newly reported from Cerrena sp. HYB07 and laccase from R. virescens aslo demonstrated decoloring activity toward the two dyes [28,30]. T. hirsuta laccase was reported to demonstrate decolorizing ability toward non-phenolic dyes, such as Methyl Red [58]. Laccase from P. sanguineus BRFM 66 also possessed considerably high decolorization activity toward Anthraquinonic Dye Poly R-478 (33%) and Heterocyclic Dye Azure B (21%) after 52 h of incubation [50]. Laccase from T. trogii was effective to decolorize Malachite Green, Bromophenol Blue, Crystal Violet, and Acid Red without the addition of redox mediators [47]. In the present study, the two dyes Bromothymol Blue and Malachite Green share very close structure with three benzene rings. Fuchsin Basic and Methyl Orange are both benzene derivatives. Eriochrome Black T, Evans Blue, Methylene Blue, and Remazol Brilliant Blue R are naphthalene or heterocyclic derivatives. The two active dyes might fit well with the enzyme activity centers and can be degraded. Acknowledgements This work was financially supported by National Grants of China (31200070 and 2012BAD14B09), Beijing NOVA Program (XX2015B025), Beijing Higher Education Young Elite Teacher Project (YETP1714), Undergraduate Research Program of Beijing, and Beijing Innovative Grant of Modern Agricultural Technology System (PXM2013-014207-000096). References [1] P. Giardina, V. Faraco, C. Pezzella, A. Piscitelli, S. Vanhulle, G. Sannia, Cell Mol. Life Sci. CMLS 67 (2010) 369–385. [2] P. Baldrian, FEMS Microbiol. Rev. 30 (2006) 215–242. [3] C.M. Rivera-Hoyos, E.D. Morales-Alvarez, R.A. Poutou-Pinales, A.M. Pedroza-Rodriguez, R. Rodriguez-Vazquez, J.M. Delgado-Boada, Fungal Biol. Rev. 27 (2013) 67–82. [4] M. Jaszek, M. Osinska-Jaroszuk, G. Janusz, A. Matuszewska, D. Stefaniuk, J. Sulej, J. Polak, M. Ruminowicz, K. Grzywnowicz, A. Jarosz-Wilkolazka,BioMed Res. Int. 2013 (2013), 497492. [5] A.M. Mayer, R.C. Staples, Phytochemistry 60 (2002) 551–565. [6] G.Q. Zhang, Q.J. Chen, H.X. Wang, T.B. Ng, J. Mol. Catal. B Enzym. 85–86 (2013) 31–36. [7] U.N. Dwivedi, P. Singh, V.P. Pandey, A. Kumar, J. Mol. Catal. B Enzym. 68 (2011) 117–128. [8] N. Jaiswal, V.P. Pandey, U.N. Dwivedi, Int. J. Biol. Macromol. 72 (2015) 326–332. [9] J. Sun, Q.J. Chen, M.J. Zhu, H.X. Wang, G.Q. Zhang, J. Mol. Catal. B Enzym. 99 (2014) 20–25. [10] D. Daassi, H. Zouari-Mechichi, A. Prieto, M.J. Martinez, M. Nasri, T. Mechichi, World J. Microb. Biot. 29 (2013) 2145–2155. [11] S.R. Couto, J.L.T. Herrera, Biotechnol. Adv. 24 (2006) 500–513. [12] L. Lesage-Meessen, M. Haon, E. Uzan, A. Levasseur, F. Piumi, D. Navarro, S. Taussac, A. Favel, A. Lomascolo, FEMS Microbiol. Lett. 325 (2011) 37–48. [13] S.M. Zhong, N. Sun, H.X. Liu, Y.Q. Niu, Y. Wu, Exp. Ther. Med. 9 (2015) 341–344. [14] M. Sakamoto, Y. Shirane, I. Naribayashi, K. Kimura, N. Morishita, T. Sakamoto, T. Sakai, Eur. J. Biochem. 226 (1994) 285–291. [15] Y. Tohya, S. Sasagawa, Jpn. J. Pharmacol. 18 (1968) 19–29. [16] Y. Nishizawa, K. Nakabayashi, E. Shinagawa, J. Ferment. Bioeng. 80 (1995) 91–93. [17] A. Grajales, C. Aguilar, J.A. Sanchez, BMC Evol. Biol. 7 (2007) 90.
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