Characterization of a novel laccase from the isolated Coltricia perennis and its application to detoxification of biomass

Process Biochemistry 47 (2012) 671–678

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Characterization of a novel laccase from the isolated Coltricia perennis and its application to detoxification of biomass Dayanand Kalyani a,b,1 , Saurabh Sudha Dhiman a,b,1 , Hoon Kim c , Marimuthu Jeya a,b , In-Won Kim a,b , Jung-Kul Lee a,b,∗ a

Department of Chemical Engineering, Konkuk University, Seoul 143-701, South Korea Institute of SK-KU Biomaterials, Konkuk University, Seoul 143-701, South Korea c Department of Pharmacy, Sunchon National University, Suncheon 540-742, South Korea b

a r t i c l e

i n f o

Article history: Received 3 December 2011 Received in revised form 10 January 2012 Accepted 11 January 2012 Available online 20 January 2012 Keywords: Detoxification Laccase Phenolic compound Purification

a b s t r a c t A highly efficient laccase-producing fungus was isolated from soil and identified as Coltricia perennis SKU0322 by its morphology and by comparison of its internal transcribed spacer (ITS) rDNA gene sequence. Extracellular laccase (Cplac) from C. perennis was purified to homogeneity by anion-exchange and gel filtration chromatography. Cplac is a monomeric glycoprotein with 12% carbohydrate content and a molecular mass of 66 kDa determined by polyacrylamide-gel electrophoresis. Ultraviolet-visible absorption spectroscopy observed type 1 and type 3 copper signals from Cplac. The enzyme acted optimally at pH 3–4 and 75 ◦ C. Its optimal activity was with 2,2-azino-bis (3-ethylbenzothiazoline6-sulfonate) (ABTS), it also oxidized various lignin-related phenols. The enzyme was characterized as a multi-copper blue laccase by its substrate specificity and internal amino acid sequence. It showed a higher catalytic efficiency towards ABTS (kcat /Km = 18.5 s−1 ␮M−1 ) and 2,6-dimethoxyphenol (kcat /Km = 13.9 s−1 ␮M−1 ) than any other reported laccase. Its high stability and catalytic efficiency suggest its suitability for industrial applications: it detoxified phenolic compounds in acid-pretreated rice straw and enhanced saccharification yield. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Laccases (benzenediol oxygen oxidoreductase, EC 1.10.3.2) are multi-copper oxidases belonging to the group of blue oxidases. Multi-copper oxidases contain three types of copper sites (types 1, 2, and 3). Their catalytic redox site is a cluster of four copper atoms that performs monoelectronic oxidation of suitable substrates at the expense of molecular oxygen. Overall, the catalytic cycle reduces one molecule of oxygen to two molecules of water and oxidizes four substrate molecules to four radicals [1]. Laccases are widely found in nature in higher plants, fungi, insects, and bacteria [2]. Due to their relatively broad substrate specificity, they have wide potential industrial applicability, including pulp bleaching in the paper industry, dye decolorization, oxygen

∗ Corresponding author at: Department of Chemical Engineering, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, South Korea. Tel.: +82 2 450 3505; fax: +82 2 458 3504. E-mail address: [email protected] (J.-K. Lee). 1 These authors contributed equally to this work. 1359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2012.01.013

cathode development for biofuel cells, biosensors, bioremediation, and detoxification of environmental pollutants [3]. Industrial lignocellulose-based bio-ethanol production generally includes four major steps: pretreatment, saccharification, fermentation, and separation [4]. During the pretreatment of lignocellulosic feedstocks to fermentable sugars, a number of byproducts are produced. These include furan derivatives, weak acids, phenolics, and inorganic compounds [5]. Lignin is degraded to aromatic compounds such as the low-molecular weight phenolics syringaldehyde, 4-hydroxybenzaldehyde and vanillin, which inhibit the fermentation of lignocellulosic hydrolyzates [6]. These compounds degrade biological membranes, reducing their ability to serve as selective barriers and enzyme matrices. This adversely affects both cell growth and sugar assimilation [7]. Therefore, the facile removal of these inhibitory compounds would aid pretreatment and improve the overall enzymatic hydrolysis and fermentation process. Each species of white-rot fungus differs in the composition of its ligninolytic enzymes [8], the production of these enzymes needs to be studied in various species with different ecological backgrounds. White-rot fungi have been reported to degrade lignocelluloses effectively, with laccases being important to the degradation [9].

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Therefore, broad study of microorganisms with laccase activity is necessary to identify suitable laccases from various strains. This work reports the isolation of a new fungus, Coltricia perennis, belonging to the family Ectomycorrhizal. It produced a highly efficient laccase that was purified and characterized. A partial gene encoding the enzyme was identified by internal peptide sequencing and subsequent degenerate PCR. The properties of the enzyme, including its substrate specificity and partial amino acid sequence, showed it to be a multi-copper blue laccase. As a demonstration of its industrial application, the enzyme was used to detoxify biomass (rice straw) to enhance its saccharification by cellulase. 2. Materials and methods 2.1. Materials 2,2 -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,6-dimethoxy phenol (DMP), guaiacol, toluidine, 3-aminobenzoic acid, phenyldiamine, and caffeic acid were from Sigma–Aldrich (St. Louis, MO, USA). Celluclast 1.5L from Trichoderma reesei ATCC 26921 and laccase produced by a genetically modified Aspergillus oryzae (Novozym 51003) were purchased from Novozyme (Denmark). Novozym 51003 is a robust stable laccase used for lignin modification in pulp and paper industry. All other chemicals and reagents were of analytical grade from commercial sources, unless otherwise stated.

represents 1 ␮mol of oxidized guaiacol per minute. All assays were carried out in triplicate. Protein concentration was determined by Bradford’s method [15] with a bovine serum albumin standard. 2.5. Purification of Cplac Liquid cultures were harvested and pooled after 7 days. Cells were removed by centrifugation at 9500 rpm for 20 min. The supernatant (1 l) was filtered through a 0.22 ␮m membrane (Millipore, Bedford, MA, USA) and then concentrated by ultrafiltration (Viva-flow, Vivascience, Hannover, Germany) with a 30 kDa cut-off membrane. The fluid was then further concentrated to 5 ml by ultrafiltration with a 10 kDa cutoff (Ultra-4, Amicon, Bedford, MA, USA). This was applied to a DEAE Sepharose TM Fast Flow column (1.6 cm × 10 cm, Amersham Biosciences) equilibrated with 50 mM sodium acetate buffer (pH 4.8). The column was washed with the same buffer, and absorbed proteins were eluted by a linear concentration of gradient NaCl (0–1 M) at a flow rate of 1 ml min−1 . The fractions containing laccase activity were pooled, dialyzed, and concentrated to ca. 2 ml by ultrafiltration with 30 kDa cutoff (Ultra-4, Amicon, Bedford, MA, USA). The concentrated enzyme solution was applied to a Hiload 16/60 Superdex 200 pg column equilibrated with 25 mM sodium acetate containing 15 mM NaCl at pH 4.8. Protein was eluted with the same buffer at a flow rate of 0.5 ml min−1 . Laccase-rich fractions were pooled and stored at 4 ◦ C for further use. Protein in the column’s effluent was monitored by measuring absorbance at 280 nm. Chromatographic separation was performed using a BioLogic FPLC system (Bio-Rad, CA, USA). All procedures were performed at 4 ◦ C. 2.6. Determination of molecular mass, carbohydrate and copper contents and spectral analysis

2.2. Isolation of the microorganisms Soil was collected from Sorak Mountain, Republic of Korea, by the capillary tube method and diluted in sterile dilution solution (0.9% saline). Aliquots were spread on potato-dextrose agar (PDA) plates containing a laccase indicator (0.02% ABTS). To avoid bacterial growth, the plates were supplemented with chloramphenicol (0.01%). Plates were incubated at 30 ◦ C until positive halos of laccase-producing microorganisms formed green zones. The size ratios of the colored halos to the colony were used to indicate suitable isolates for further investigation [10]. The isolated strain was identified by ITS rDNA sequence analysis. For sequence analysis, the ITS1-5.8 S-ITS2 rDNA region of the fungus was amplified by polymerase chain reaction using the primer set pITS1 (5 -TCCGTAGGTGAACCTGCCG-3 ) and pITS4 (5 TCCTCCGCTTATTGAT-ATGC-3 ). A 426-bp amplicon was obtained. It was then cloned and sequenced. The sequence was submitted to the GenBank with accession number (JN211120). Pairwise evolutionary distances and a phylogenetic tree were constructed with MEGA 4 software [11]. The identified strain SKU0322 was deposited at the Korean Culture Center of Microorganisms (KCCM) with the accession number KCCM11199P. 2.3. Media and culture conditions The stock culture of the isolated strain was maintained on potato dextrose agar (PDA) plates at 4 ◦ C with periodic transfer. To produce inoculum, five mycelia disks (5 mm diameter) were removed from the peripheral region of the PDA plates and inoculated in 500 ml flasks containing 50 ml potato dextrose broth (PDB). After 5 days’ incubation, 5 ml preculture was inoculated into Erlenmeyer flasks (250 ml) containing 50 ml of basal medium. The basal medium used for the experiment contained (g l−1 ): glucose, 10; peptone, 2; yeast extract, 1; ammonium tartrate, 2; KH2 PO4 , 1; MgSO4 ·7H2 O, 0.5; KCl, 0.5; trace element solution, 1 ml. The trace element solution composition was as follows (g l−1 ): B4 O7 Na2 ·10H2 O, 0.1; CuSO4 ·5H2 O, 0.01; FeSO4 ·7H2 O, 0.05; MnSO4 ·7H2 O; 0.01; ZnSO4 ·7H2 O, 0.07; (NH4 )6 Mo7 O24 ·4H2 O, 0.01. The medium pH was adjusted to 5.0. Cultures were incubated at 30 ◦ C on a rotary shaker (180 rpm). In order to determine the point of maximal laccase production, laccase activities were measured over a 13-day period with the addition of different inducers; 1 mM of vanillic acid, veratryl alcohol, guaiacol, veratraldehyde, and FeSO4 ; 0.5 mM of xylidine, and 2 mM of CuSO4 to the basal medium.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as reported by Laemmli [16] with 5% stacking gel and 10% resolving gel using a Biorad mini VE vertical electrophoresis system (Bio-Rad, CA, USA). Native PAGE was performed as described by Telke et al. [17]. Staining was carried out after native PAGE by incubating the gel at room temperature with 50 mM sodium acetate buffer (pH 4.8) containing 0.1 mM ABTS or 2,6-DMP and with gentle shaking until green or brown bands were clearly visible. The glycoprotein nature of the protein in polyacrylamide gel was detected using a GelCode glycoprotein staining kit (Pierce Biotechnology, USA) following the manufacturer’s instructions. The carbohydrate content of the laccase was determined by the phenol sulfuric acid method using a glucose standard [18]. Its copper content was determined by inductively coupled plasma mass spectroscopy (ICPMS) (PlasmaQuad 3, VG Elemental, Thermo Jarrell Ash Corp., U.S.A.). A spectrum of Cplac solution (0.7 mg ml−1 ) was obtained by scanning the sample between 200 and 800 nm. 2.7. Effects of pH and temperature on laccase activity and stability The effects of pH on laccase activity towards ABTS substrate were tested at pH 2–6 in 50 mM citrate-phosphate buffer (pH 2–4) or 50 mM sodium-acetate buffer (pH 4–6). The purified laccase’s activity towards ABTS substrate was examined at 25–85 ◦ C at the optimal pH value. The thermal stability of the laccase was determined by incubating it at 60, 65, 70, and 75 ◦ C and determining its activity at various time intervals. 2.8. Substrate specificity and enzyme kinetics The substrate specificity of the purified laccase was tested using ABTS, 2,6-DMP, l-DOPA, 3-aminobenzoic acid, phenyldiamine, toluidine, guaiacol, veratryl alcohol, and caffeic acid substrates. Rates of substrate oxidation were determined by measuring absorbance increases at respective wavelengths. Molar extinction coefficients (ε) were obtained from the literature [19]. Km and Vmax values of the purified Cplac were determined by measuring the activity of the enzyme with various concentrations (0.01–0.1 mM) of ABTS and 2,6-DMP substrates at the optimal pH in each case. Kinetic constants were calculated by the Michaelis–Menten method using the Graphpad prism 5 program.

2.4. Enzyme assay

2.9. Effects of metal ions and inhibitors on Cplac

Laccase activity was determined spectrophotometrically (Varian Cary 100 Bio UV–vis spectrophotometer, Palo Alto, CA), using ABTS as the substrate [12]. The oxidation of ABTS was observed by measuring absorbance increases at 420 nm (εmax = 3.6 × 104 M−1 cm−1 ). Enzyme activity was expressed as international units (IU), where 1 IU represents the amount of enzyme that forms 1 ␮mol product per minute. Lignin peroxidase activity was assayed by veratryl alcohol oxidation [13]. The enzyme was incubated with 10 mM veratryl alcohol, 250 mM d-tartaric acid (pH 2.5), and 500 ␮M H2 O2 . The extinction coefficient of veratryl aldehyde (oxidized veratryl alcohol) at 310 nm is 9300 M−1 cm−1 . One unit represents 1 ␮mol of veratryl alcohol oxidized to veratryl aldehyde per minute. Manganes peroxidase activity was assayed by guaiacol oxidation [14]. The enzyme was incubated with 400 ␮M guaiacol, 50 mM sodium acetate buffer (pH 4.5), 200 ␮M MnSO4 , and 100 ␮M H2 O2 . The extinction coefficient of oxidized guaiacol at 465 nm is 12,100 M−1 cm−1 . One unit

The effects of various metal ions and inhibitors on the purified Cplac were determined using assay mixtures (2 ml) comprising appropriately diluted enzyme, 50 mM sodium acetate buffer (pH 4.8), and 0.1 mM ABTS with various concentrations of inhibitors and 5 mM metal ions. Laccase activity in the absence of inhibitor or metal ions was defined as 100%. Measurements were carried out in triplicate. 2.10. Internal amino acid sequence of Cplac Protein bands of interest were cut from polyacrylamide gels and digested overnight using trypsin (Sigma) as described earlier [20]. The cleaved peptides were eluted and analyzed by nano LC–MS/MS for internal amino acid sequencing. Protein sequences were identified by homology searching using an MS data analysis program, SEQUEST (Thermo Finnigan, San Jose, CA, USA), against the fungi protein

D. Kalyani et al. / Process Biochemistry 47 (2012) 671–678 database of the National Center for Biotechnology Information protein sequence database.

673

17

Coltricia perennis strain GEL3915 (AJ406472)

6

Coltricia perennis (AF026583) Coltricia perennis strain CH08526 (HQ534102)

2.11. Preparation of genomic DNA and PCR amplification

Coltricia perennis isolate AFTOL-ID 447 (AY218526)

Genomic DNA was isolated using a Wizard nucleic acid purification kit (Promega), according to manufactures instructions. Degenerate primers (Cplac HWHGFFQ 5 -CAYTGGCAYGGNTTYTTYCA-3 ) and (Cplac QRYSFVL 5 CAGCACAAAGCTATAACGCTG-3 ) were designed based on the partial peptide sequences and the conserved sequences of the copper-binding regions. PCR was performed as follows: all PCRs contained 1× buffer, 4 ␮l 10 mM dNTPs, 10 pmol/␮l each primer, and 5 units Taq DNA polymerase in 100 ␮l. Amplification was performed with one cycle of denaturation at 94 ◦ C for 5 min followed by 35 cycles of 60 s at 94 ◦ C, annealing for 60 s at 45–55 ◦ C, and extension for 180 s at 72 ◦ C. A final extension was then conducted at 72 ◦ C for 10 min. For analysis, 10 ␮l reaction mixture was electrophoresed on 1% agarose gel and stained with ethidium bromide solution (5 ␮g ml−1 ). The purified PCR products were ligated to TA cloning 1 vector (RBC Bioscience, Taipei, Taiwan) and sequenced with M13 primers from both strands at Macrogen (Seoul, South Korea).

2.12. Total RNA preparation, cDNA synthesis and reverse transcription (RT)-PCR Mycelia from cultures were separated from culture fluid, washed twice with distilled water, quick frozen, and ground to a powder with a mortar and pestle. RNA extraction was performed by using the Qiagen RNeasy Kit, following the manufacturer’s instructions (QIAGEN, Valencia, CA). Total RNA concentration was determined spectrophotometrically. In order to remove contaminating DNA, 1 unit per l ␮g of RNA of RQ1 DNAse enzyme (Promega) was added to each RNA sample followed by incubation for 30 min at 37 ◦ C. The RNA was phenol/chloroform extracted, chloroform extracted, precipitated with isopropanol, washed with 70% ethanol, and dissolved in sterile water. The integrity of the RNA was verified by electrophoresis on 1% agarose gels followed by ethidium bromide staining. First-strand cDNA synthesis was carried out using 2 ␮g of total RNA as template and the cDNA synthesis kit from RT-PCR PreMix Kit (iNtRON, Korea), used according to the manufacturer’s instructions. The RT-PCR mixtures containing cDNA and each primer (forward (5 CAYTGGCAYGGNTTYTTYCA-3 ) and reverse (5 -CAGCACAAAGCTATAACGCTG-3 )) were heated at 95 ◦ C for 5 min, followed by 30 cycles of 95 ◦ C for 45 s, annealing at 60 ◦ C for 30 s, and 72 ◦ C for 2 min and, subsequently, a final extension step at 72 ◦ C for 10 min. The housekeeping gene tub1, encoding ␤-tubulin, was chosen as the expression reference [21]. The same procedure was performed to amplify a ␤-tubulin fragment from the RT reaction. 2.13. Pretreatment of rice straw Rice straw (Oryza sativa L.) was obtained from a company in Seoul, Korea. It was cut and dried at 100 ◦ C overnight to ensure low moisture content prior to treatment. The straw was determined to comprise 16% moisture, 34.25% cellulose, 26.14% hemicellulose, and 18.24% lignin. 20 g (dry weight) straw biomass was suspended in 30 ml 0.5% sulfuric acid (w/w) and held at 121 ◦ C for 15 min. It was not washed after the steam explosion pretreatment. The pretreated rice straw was dried at 100 ◦ C to until constant weight before further use.

2.14. Detoxification of pretreated rice straw using Cplac Crude Cplac and commercial laccase Novozym 51003 were used to treat the acid-pretreated rice straw. Only laccase activity was present and other ligninolytic enzymes, such as manganese peroxidase or lignin peroxidase were not detected in crude Cplac. Preliminary assays were performed to optimize the pH, incubation time, and laccase dosage of the detoxification treatment. Assays were performed in 100 ml Erlenmeyer flasks containing 2 g (dry weight) pretreated rice straw in 25 ml 50 mM buffer (pH 2.5–6) at 30 ◦ C in a rotary shaker (150 rpm). Supernatants were periodically analyzed for total phenols by the Folin–Ciocalteau method [22], and Results were expressed as grams of catechol equivalents (CE) per liter of liquid phase.

2.15. Saccharification experiments A typical hydrolysis mixture containing 0.2 g substrate, 25 FPU (Celluclast 1.5L) enzyme, and 10 ml sodium acetate buffer (pH 4.5) was supplemented with the antibiotics tetracycline (40 ␮g/ml) and cycloheximide (30 ␮g/ml) to prevent microbial contamination. The mixture was incubated at 37 ◦ C in a rotary shaker at 150 rpm. Samples were taken from the reaction mixture at different time intervals and immediately heated to 100 ◦ C to denature the enzymes. They were then cooled and centrifuged for 10 min at 8000 rpm. The supernatant was used in reducing sugar analysis. The reducing sugar content was multiplied by 0.9 to calculate saccharification yield [23].

Coltricia perennis strain 92-96 (AF311004)

42

Coltricia perennis SKU0322 (JN211120) Coltricia perennis isolate DSH93-198 (EU339273)

93 53

Coltricia perennis voucher MLS012 (GQ397996)

0.01

Fig. 1. The evolutionary history was inferred using the Neighbor-Joining method; the sequences have been retrieved from NCBI database, showing the phylogenetic relationships of Coltricia perennis SKU0322 and other species of other genus. Numbers at nodes shows the level of bootstrap support based on data for 1000 replication. Bar, 0.01 substitutions per nucleotide position and numbers in parenthesis represent GenBank accession numbers.

3. Results and discussion 3.1. Isolation and identification of laccase-producing fungi In a preliminary screening of soil samples, 25 microbial strains were found to produce colored halos on PDA plates containing laccase indicator ABTS. The isolate with the biggest colored zone around its colony after 5 days of incubation was selected for further study. To analyze the phylogenetic position, the ITS rDNA sequence of the strain SKU0322 was determined. Fig. 1 shows the phylogenetic relationship between the strain SKU0322 and other related microorganisms found in the GenBank database. The homology assay result indicated that the strain belonged to the phylogenetic branch of Coltricia. The strain exhibited a maximum identity (99%) with C. perennis isolate DSH93-198 and was identified as C. perennis. C. perennis is an ectomycorrhizal fungus that exists naturally in soil and is associated with forming symbiotic relationships with the roots of host plants [24]. 3.2. Effect of inducer and purification of laccase from C. perennis An appropriate inducer can enhance laccase production, and is necessary for effective large-scale production [25]. In most basidiomycete fungi, extracellular laccases are constitutively produced in small amounts and many different inducers have been widely used to stimulate laccase production [26]. In the present work, the effect of various inducers on Cplac activity was studied. On the basis of preliminary studies, inducers were added on the third day of incubation to the actively growing fungal cultures. Supplementary Fig. S1 depicts the effect of various inducers on laccase production. The highest increase of Cplac activity was observed with CuSO4 on the seventh day (2.5-fold), followed by veratryl alcohol (2.1fold) and vanillic acid (1.7-fold) compared to the control without supplementation of inducers. No induction was observed after the addition of veratraldehyde and xylidine. Extracellular laccase production by C. perennis was stimulated significantly by supplemental copper ions. CuSO4 at 2 mM resulted in a high laccase yield of 3.1 U ml−1 , 2.5-fold greater than the control. Laccase activity first occurred on day 4 and reached its maximum on day 7 of culture. Therefore, laccase obtained from a 7-day culture was used for subsequent purification. It was purified by ultrafiltration, ion-exchange, and gel filtration chromatography. Table 1 lists results after the different steps of laccase purification. Overall, Cplac was purified 113-fold. The specific activity of the purified enzyme was 177.5 U mg protein−1 . The purified Cplac showed as a single band on SDS-PAGE, with a mobility

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D. Kalyani et al. / Process Biochemistry 47 (2012) 671–678

Table 1 Purification of laccase from the culture broth of C. perennis SKU0322. Purification step

Total protein (mg)

Total activity (U)

Specific activity (U mg−1 )

Purification fold

Yield (%)

Crude culture filtrate 30 kDa DEAE-cellulose Gel filtration

952.3 344.2 6.2 1.0

1501 1142 436.6 186.4

1.5 3.3 69.8 177.5

1.0 2.1 44.5 113.0

100.0 76.1 29.0 12.3

corresponding to a molecular mass of 66 kDa, as visualized by Coomassie Brilliant Blue staining (Fig. 2a). Activity staining of the laccase, using ABTS and 2,6-DMP substrates, revealed a single protein band corresponding to the position of the laccase activity (Fig. 2b). The laccase was found to be a glycoprotein (Fig. 2b), with an estimated carbohydrate content of 12%. The carbohydrate content of Cplac was within the range (10–20%) reported for laccases from other basidiomycetes, including Coriolus hirsutus, Marasmius quercophilus, and Trametes pubescens [27–29]. Size exclusion chromatography on a Sephacryl S-300 high-resolution column resulted in elution of enzyme activity as a symmetric peak corresponding to a Mr of ca. 65 kDa.

initial activity up to 360 min, 187 min, 56 min, and 34 min, respectively (Fig. 5). Cplac showed activity towards a wide range of substrates, including phenolics (e.g., 2,6-DMP, guaiacol, toluidine, and lDOPA) and a non-phenolic substrates (ABTS). Cplac’s activity to the different substrates was ranked: ABTS > toluidine > 2,6DMP > guaiacol > l-DOPA. No activity was detected with caffeic acid or veratryl alcohol (Table 2). Kinetic constants of Cplac were determined for ABTS and 2,6-DMP. The catalytic efficiency (kcat /Km ) was slightly higher for ABTS (18.5 s−1 ␮M−1 ) than for 2,6-DMP (13.9 s−1 ␮M−1 ). Most notably, Cplac showed higher catalytic activity than any other reported laccase (Table 3).

3.3. Physical and chemical properties of Cplac

3.4. Effects of inhibitors and metal ions

The nature of the catalytic center was determined by spectrophotometry of the purified Cplac. Cplac’s UV–vis spectrum showed an absorption peak at 610 nm, typical of a type I Cu2+ center, that was responsible for the enzyme’s blue color [30]. The shoulder at around 329 nm suggested the presence of a type III binuclear Cu2+ center (Fig. 3). ICP-MS results showed that Cplac contained ca. 3.9 mol Cu per mol of protein, suggesting the presence of 4 Cu molecules per protein molecule, similar to laccases from other white-rot fungi [31]. Cplac activity as a function of pH was determined. The optimum pHs were different for each individual substrate assayed; 3, 3, 3.5, and 4 for ABTS, tolidine, guaiacol, and 2,6-DMP, respectively (Fig. 4a). Cplac demonstrated an acidic pH optimum with all substrates tested. Activity declined sharply to an almost undetectable level as pH approached neutral, possibly due to hydroxide ion binding to the enzyme’s T2/T3 site [30]. At pH 3.0, Cplac had highest activity at 75 ◦ C (Fig. 4b). The stability of purified Cplac was assessed at 60, 65, 70, and 75 ◦ C; the enzyme retained at least 50% of its

Various environmental compounds can affect the stability of ligninolytic enzymes. Inhibitors such as EDTA, l-cysteine, sodium azide, thiourea, SDS, and dithiothreitol generally inhibit laccase activity by forming complexes with its copper ions, modifying the active site [32]. The effects of various compounds are listed in Table 4. The purified enzyme was strongly inhibited by 0.1 mM sodium azide, 1 mM l-cysteine, DTT, and thiourea, whereas the metal ion chelator EDTA was not an efficient inhibitor of Cplac at 1 mM. Even higher concentration (5 mM) of EDTA did not inhibit ABTS oxidation by a laccase produced by C. hirsutus [27]. Recently, Lorenzo et al. [32] found that the inhibitory effect of EDTA on laccase activity was dependent on the substrate used. These authors observed a significant reduction in laccase activity in the presence of EDTA when syringaldazine or dimethoxyphenol was used, whereas no inhibition of ABTS oxidation was detected under the same experimental conditions. The interactions of metals with extracellular laccase are particularly important for understanding the biotechnological processes of xenobiotic degradation [33]. The effects of common environmental heavy metal cations on laccase activity were tested by adding

0.20

Absorbance

0.15

0.10

0.05

0.00 200 Fig. 2. Determination of molecular mass of purified Cplac by (a) SDS-PAGE of laccase (M – marker, L1 – crude protein, L2 – ultrafiltration, L3 – DEAE cellulose purification, L4 – Biogel Hiload 16/60 Superdex 200 chromatography) and (b) Zymogram analysis and glycoprotein staining of purified laccase enzyme with native PAGE (L1 – ABTS, L2 – 2,6-DMP, L3 – glycoprotein staining).

300

400

500

600

700

800

Wavelength (nm) Fig. 3. Absorbance spectrum of Cplac (0.7 mg ml−1 in 50 mM sodium acetate buffer, pH 4.8).

D. Kalyani et al. / Process Biochemistry 47 (2012) 671–678

675

Table 2 Substrate oxidizing activity of the purified Cplac. Relative activities (%) were measured using ABTS as substrate after adding each substrate to assay mixture. Assays were done in 50 mM sodium acetate buffer (pH 4.8). Substrate (1 mM)

Absorbance

- max M−1 cm−1 ) Molar extinction coefficient (C

Specific activity (U mg protein−1 )

ABTS Toluidine 2,6-DMP Guaiacol l-DOPA 3-Aminobenzoic acid Phenyldiamine Caffeic acid Veratryl alcohol

420 366 470 436 460 410 515 420 310

36,000 31,000 35,645 6400 38,000 29,000 43,100 – –

100 ± 1.2 85.9 ± 1.7 74.4 ± 2.4 61.0 ± 5.4 49.6 ± 7.9 15.5 ± 3.1 10.7 ± 1.2 ND ND

ND: not detected. Table 3 Kinetic parameters of various laccases. Fungal strain

Substrate

Km (␮M)

Coriolus hirsutus

ABTS 2,6-DMP

56.7 53.0

Cyathus bulleri Pleurotus sajor-caju Pycnoporus sanguineus (SSC 108)

ABTS ABTS ABTS 2,6-DMP

37.0 56.0 130 52.0

Pycnoporus sanguineus

ABTS 2,6-DMP

77.0 203

Panus tigrinus CBS 577.79

ABTS 2,6-DMP

31.0 119

Trichoderma harzianum Aspergillus oryzae Fomitella fraxinea

ABTS ABTS ABTS 2,6-DMP

180 110 270 426

Paraconiothyrium variabile

ABTS 2,6-DMP

203 1176

Coltricia perennis SKU0322

ABTS 2,6-DMP

119.6 72.8

Fe2+ , Cu2+ , Zn2+ , Mg2+ , Mn2+ , Co2+ , Cr3+ Ba2+ , and Ca2+ to Cplac. All the tested metal ions partially inhibited laccase activity between 10 and 26% at 5 mM concentration. Cplac showed a resistance to heavy metals, supporting its potential for use in industrial applications such as bioremediation. Table 4 Effect of typical laccase inhibitors on the Cplac activity. Relative activities (%) were measured using ABTS as substrate after adding each inhibitor to assay mixture (purified Cplac in 50 mM sodium acetate buffer pH 4.8) to reach the final concentrations of inhibitor. Compound

Concentration (mM)

Relative activity (%)

None Sodium azide

– 0.1

100 0

l-Cysteine

0.1 0.5 1

91.8 ± 0.7 57.6 ± 1.5 20.2 ± 4.3

SDS

0.1 0.5 1

97.4 ± 0.5 95.8 ± 0.6 84.9 ± 1.0

EDTA

0.1 0.5 1

100 100 98.5 ± 0.5

Dithiothreitol

0.1 0.5 1

45.7 ± 1.1 2.0 ± 0.4 0

Thiourea

0.1 0.5 1

69.7 ± 1.6 24.4 ± 2.4 0

kcat (s−1 ) 260 126

kcat /Km (s−1 ␮M)

References

4.5 2.3

[27]

54.0 52.0 48.0 21.0

1.4 0.92 0.36 0.40

[44] [45] [19]

68.0 6.92

0.08 0.03

[46]

185 366

5.9 3.0

[47]

5.20 63.3 208 130.2

0.02 0.5 0.77 0.24

[30] [48] [49]

56 17.6

0.27 0.01

[10]

2217 1012

18.5 13.9

This study

3.5. Identification of the partial gene product Internal peptide sequencing of the purified Cplac showed fragments, AGTFWYHSHLSTQ, PLWYHWHGFFQK, and IYAGQRYSFVL, identical to the laccases of Polyporus brumalis, Lentinus tigrinus, and Trametes sp. 420, respectively, that belong to the multi copperoxidase superfamily. A partial 687 bp amplicon was obtained by PCR using a degenerate primer pair, Cplac HWHGFFQ and Cplac QRYSFVL, which was designed based on the peptide sequences identified by nano LC–MS/MS sequencing. The partial primary structure of Cplac is significantly similar to those of other multicopper oxidases. A homology search revealed that the deduced gene product had 73%, 71%, 70%, and 68%, amino acid identity with AAG09231.1 (P. ciliatus), ADI70681.1 (T. versicolor), AAX07469.1 (L. tigrinus), ABN13591.1 (P. brumalis), and AAW28936.1 (Trametes sp. 420), respectively (Fig. 6). The partial gene product amplified by PCR from the fragment sequences contained the copper-binding sites (I and II) present in multi-copper oxidases [34,35]. 3.6. Regulation of laccase by copper Copper, an essential micronutrient for fungal growth, functions as a metal activator of several fungal enzymes, e.g. oxidases, and in the synthesis of pigments [36]. The induction of Cplac expression by copper in C. perennis is shown in Supplementary Fig. S2. Cultures were grown in medium containing copper at various concentrations, and the effects of copper on Cplac mRNA transcript levels and laccase activities were determined. PCR amplifications of RT reaction products proceeded at uniform efficiencies in all cases, as

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Temperature ( C) Fig. 4. (a) Effect of pH on the activity of purified Cplac towards: (䊉) ABTS, () toluidine, () guaiacol, () 2,6-DMP. Reactions were at room temperature for 3 min in citrate/acetate/phosphate buffer. (b) Effect of temperature on the activity of purified laccase from C. perennis assessed by the standard assay method. Activities are expressed as percentages of maximum activity; error bars do not exceed the dimensions of the symbols.

indicated by the constant level of control ␤-tubulin gene amplification observed in each reaction (Supplementary Fig. S2a). In the absence of copper, a low level of Cplac transcripts was detected in fungal cells; it corresponded to a low level of laccase activity in the culture medium. The addition of 2 mM CuSO4 to the growth medium resulted in increased levels of both Cplac transcripts and enzyme activity (Supplementary Fig. S2b). However, when the concentration of CuSO4 was higher than 3 mM, a significant reduction in laccase activity was observed (Supplementary Fig. S2b). The laccase activity measured in cultures grown in the presence of 2 mM CuSO4 was approximately 2.5-fold greater than that in cultures grown in the absence of CuSO4 . The correlation observed between copper concentration and Cplac transcription indicates that copper plays a role in the regulation of Cplac expression. It is well known that copper is involved in regulation of laccase at the transcriptional level in a number of basidiomycetes such as Trametes versicolor [37], T. pubescens [29], Pleurotus sajor-caju [38], and Coriolopsis rigida [39]. However, to date it is not completely understood how copper activates transcription of laccase genes.

Fig. 6. Multiple sequence alignment of Cplac with other laccases. Amino acid sequences were aligned across six laccases. Residue positions identical in all six sequences are indicated with light green. The accession numbers are: AAG09231.1 (P. ciliatus), ADI70681.1 (T. versicolor), AAX07469.1 (L. tigrinus), ABN13591.1 (P. brumalis), and AAW28936.1 (Trametes sp. 420). The CLUSTAL X algorithm was used for alignment. Conserved Cu-binding residues are boxed. The underline indicates the amino acids obtained from LC–MS/MS sequencing.

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3.7. Cplac treatment of phenolic compounds Biofuels such as bioethanol are being researched as alternatives to petroleum-based fuels to avoid the economic and environmental concerns surrounding the use of oil [40]. Phenolic compounds in biomass hydrolyzates are known to inhibit saccharifying enzymes and alcohol fermentation [6]. Here, a sample biomass, rice straw, was treated with Cplac and Novozym 51003 for 2 h to reduce the presence of phenolic compounds. The treatment with 5 U ml−1 of Cplac at pH 3.5, 4, 4.5, and 5 removed 37%, 45%, 76%, and 68% of phenolic compounds from acid-pretreated rice straw, respectively. However, 5 U ml−1 of Novozym 51003 treatment at pH 3.5, 4, 4.5, and 5 removed 22%, 31%, 52%, and 42% of the phenolic compounds, respectively. Cplac showed maximum 76% of phenolic compound removal, while the commercial laccase provided only 52% of removal. Since the subsequent enzymatic hydrolysis with cellulase was performed at pH 4.5, the same pH 4.5 was used for the detoxification to avoid readjusting the pH condition. The effects of Cplac and Novozym 51003 dosage on the reduction of phenolic compounds were also tested. 1.5 U ml−1 of Cplac and 3.0 U ml−1 of Novozym 51003 were optimal to detoxify acid-pretreated rice straw. Further increasing Cplac or Novozym 51003 dosage did not improve detoxification (data not shown). Cplac could detoxify acid-pretreated rice straw at lower dosage as compared to commercial laccase Novozym 51003, an important property for its industrial-scale application. Chandel and co-workers [41] studied detoxification of wood sugarcane bagasse hydrolysate with 100 U ml−1 of laccase from the fungus Cyathus stercoreus. The result showed 77.5% removal of phenolic compounds after 5 h reaction. However, reaction media employed in the present work contained only 1.5 U ml−1 of Cplac, 1.5% dosage of that reported in the previous study [41]. Polymeric and monomeric phenols have been reported to inhibit and deactivate various cellulases from microorganisms commonly used to produce commercial enzymes [42]. Many studies have shown that laccases are able to detoxify lignocellulosic hydrolysates and after detoxification ethanol productivity has been subsequently increased by 2–3 times [5,41,43]. Till date, no reports are available regarding the effect of phenolic compounds on saccharification process. In this report, we compared the detoxification efficiency of Cplac and commercial laccase on the basis of saccharification yield. Rice straw was pretreated with laccases, followed by hydrolysis using a Celluclast 1.5L, which acted on the solid fraction of the substrate. Treatment of rice straw with Cplac and Novozym

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51003 before enzymatic hydrolysis increased the saccharification yield of rice straw by 48% and 28%, respectively, compared with untreated (only acid-treated) rice straw (Fig. 7). The lower saccharification yield from the untreated biomass may have been due to the release of phenolic compounds that can inhibit cell wall-degrading enzymes [6]. It may also have been due to decreased unproductive binding of the cellulase to lignin after the laccase treatment [5]. Cplac was able to remove phenolic compounds more efficiently than the commercial laccase, leading to significant increase in reducing sugar production from rice straw by Celluclast 1.5L (Fig. 7). Further work is necessary to explore these findings, for example quantification and identification of inhibitory phenolic compounds, optimization of the removal of inhibitory compounds, and optimization of hydrolysis conditions. In summary, a novel laccase, Cplac, from isolated C. perennis was purified and characterized. It exhibited better catalytic efficiency towards non-phenolic and phenolic substrates than any other fungal laccase. The tolerance of Cplac towards extreme conditions, including various heavy metal ions, low pH, and high temperature, demonstrated its potential suitability for use in such as bioremediation, textile, paper and pulp industries. The removal of free phenolic compounds by Cplac reduced the toxic effects of biomass hydrolyzate and enhanced saccharification yield. Acknowledgments This work was supported by the 21C Frontier Microbial Genomics and Applications Center Program, Ministry of Education, Science & Technology (MEST). This research was supported by the Converging Research Center Program through the National Research Foundation of Korea (NRF) funded by the MEST (2011-50210). This study wa s also supported by a grant (PJ007449201006) from Biogreen 21 Program. This research was supported by the 2010 KU Brain Pool of Konkuk University. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.procbio.2012.01.013. References [1] Frasconi M, Favero G, Boer H, Koivula A, Mazzei F. Kinetic and biochemical properties of high and low redox potential laccases from fungal and plant origin. Biochim Biophys Acta 2010;1804:899–908. [2] Giardina P, Faraco V, Pezzella C, Piscitelli A, Vanhulle S, Sannia G. Laccases: a never-ending story. Cell Mol Life Sci 2010;67:369–85. [3] Rodriguez Couto S, Toca Herrera JL. Industrial and biotechnological applications of laccases: a review. Biotechnol Adv 2006;24:500–13. [4] Wang A, Gao L, Ren N, Xu J, Liu C, Cao G, et al. Isolation and characterization of Shigella flexneri G3, capable of effective cellulosic saccharification under mesophilic conditions. Appl Environ Microbiol 2011;77:517–23. [5] Jurado M, Prieto A, Martinez-Alcala A, Martinez AT, Martinez MJ. Laccase detoxification of steam-exploded wheat straw for second generation bioethanol. Bioresour Technol 2009;100:6378–84. [6] Cantarella M, Cantarella L, Gallifuoco A, Spera A, Alfani F. Comparison of different detoxification methods for steam-exploded poplar wood as a substrate for the bioproduction of ethanol in SHF and SSF. Process Biochem 2004;39:1533–42. [7] Palmqvist E, Hahn-Hägerdal B. Fermentation of lignocellulosic hydrolysates. I: inhibition and detoxification. Bioresour Technol 2000;74:17–24. [8] Mikolasch A, Schauer F. Fungal laccases as tools for the synthesis of new hybrid molecules and biomaterials. Appl Microbiol Biotechnol 2009;82:605–24. [9] Lundell TK, Makela MR, Hilden K. Lignin-modifying enzymes in filamentous basidiomycetes—ecological, functional and phylogenetic review. J Basic Microbiol 2010;50:5–20. [10] Forootanfar H, Faramarzi MA, Shahverdi AR, Yazdi MT. Purification and biochemical characterization of extracellular laccase from the ascomycete Paraconiothyrium variabile. Bioresour Technol 2011;102:1808–14. [11] Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 2007;24:1596–9. [12] Palmieri G, Giardina P, Bianco C, Scaloni A, Capasso A, Sannia G. A novel white laccase from Pleurotus ostreatus. J Biol Chem 1997;272:31301–7.

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