Heterologous expression and characterization of laccase 2 from Coprinopsis cinerea capable of decolourizing different recalcitrant dyes

The gene (CcLcc2) encoding laccase from the basidiomycete Coprinopsis cinerea Okayama-7 #130 was synthesized by polymerase chain reaction-based two-step DNA synthesis, and heterologously expressed in Pichia pastoris. The recombinant protein was purified by ammonium sulphate precipitation and nickel nitrilotriacetic acid chromatography. The molecular mass of CcLcc2 was estimated to be 54 kDa by denaturing polyacrylamide gel electrophoresis. The optimum pH and temperature for laccase catalysis for the oxidation of 2,2ʹ-azino-bis(3-ethylbenzothiazoline-6-sulphonate) (ABTS) were 2.6 and 45 °C, respectively. The Km values of the enzyme towards the substrates ABTS, 2,6-dimethoxyphenol (2,6-DMP) and guaiacol were 0.93, 1.02 and 28.07 mmol·L−1, respectively. The decolourization of methyl orange, crystal violet and malachite green, commonly used in the textile industry, was assessed. The decolourization percentage of crystal violet and malachite green was 80% after 4 h of reaction, and that of methyl orange was 50% at 4 h. These results show that the CcLcc2 has enormous potential for the decolourization of highly stable triphenylmethane dyes.


Introduction
Wastewaters with substantial amounts of dyes from textile, paper or leather industries are often strongly coloured and detrimental to natural environment, even at very low dye concentrations. [1] These wastes reduce dissolved oxygen and light penetration in water and, therefore, affect the survival of aquatic life. Approximately 10% of the total dyes used in various textile processes worldwide are directly discharged without any treatment into the environment. [2,3] Therefore, pretreatment of these industrial effluents before release is of critical importance. Traditional physicalÀchemical processes, such as irradiation, precipitation and membrane-filtration, are usually expensive, inefficient and difficult to use. [4] Interest is now focused on better alternatives such as microbial biodegradation and biodecolourization of dyes. Several fungi and bacteria can degrade and decolourize dyes, especially some basidiomycetes that produce laccase, which is responsible for the degradation of these organic compounds, [5,6] have been isolated and characterized. [7À10] Laccases or benzenediol:oxygen oxidoreductases are polyphenol oxidases that catalyse the oxidation of phenolic substrates and aromatic compounds to form various small molecular products. [11] Laccases show their unique function alone or in concert with other enzymes. [12] High-level laccase expression must be taken into account before it can be used commercially. It is well known that laccase expression level in some isolates is too low for industrial applications. [13] Heterologous expression in Pichia pastoris can meet these requirements, for it enhances the expression levels 10-, 100-, or even 1000-fold compared to the normal ones. [13,14] Coprinopsis cinerea Okayama-7 #130 is an important ink cap basidiomycete that contains a large family of laccases (17 members). Preliminary studies indicate that 8 out of 17 members of this family exhibit enzymatic activity on 2,2ʹ-azino-bis(3-ethylbenzothiazoline-6-sulphonate) (ABTS) and 2,6-dimethoxyphenol (2,6-DMP). [15] However, decolourization of triphenylmethane and azo dyes by any member of this family, with the exception of a single or more species of bacteria, has not been reported. [4,16À20] In the present work, the gene of laccase, CcLcc2, was chemically synthesized and expressed in P. pastoris. After purification, its fundamental enzymological properties and application in decolourization of dyes were investigated.

Chemical synthesis of CcLcc2, vector construction and transformation
The CcLcc2 gene without the N-terminus signal peptide and an added mating factor a signal sequence was chemically synthesized via the polymerase chain reaction (PCR)-based two-step DNA synthesis (PTDS) method. [21,22] Oligonucleotides of 60 bp lengths were synthesized by the Shanghai Sangon Biological Engineering Technology and Service Co. Ltd, China. The DNA fragment was cloned and sequenced after PCR amplification. The expression vector pYM7898 was constructed in our laboratory after inserting CcLcc2 into a modified pPIC9K (Invitrogen), in which the Xho I site in the G418 resistance cassette and the Sac I site in the AOX1 promoter were removed by site-directed mutagenesis. The 6 Ã His-tag was added after the Kex2 protease cleavage site of the signal sequence. The plasmid pYM7898 (2 mg) was linearized by digestion with enzyme (Sal I) and then transformed into competent P. pastoris GS115 by electroporation (Bio-Rad Genepulser, Hercules, CA, USA). The transformants were screened on selective plates (1.34% yeast nitrogen base (YNB) without amino acids, 0.8 molÁL À1 sorbitol, 5% glucose and 2% agar). The colonies that appeared were subsequently screened by performing direct PCR to confirm integration of CcLcc2 into the P. pastoris GS115 genome.

Expression and purification of recombinant CcLcc2 protein
A single P. pastoris-CcLcc2 colony was incubated into 50 mL of buffered glycerol-complex medium (1% yeast extract, 2% peptone, 1.34% YNB, 0.000004% biotin and 1% glycerol) at 28 C with constant shaking (220 rÁmin À1 ), until the culture reached an OD 600 of 4.0. The cells were then harvested (5000 rÁmin À1 , 3 min), rinsed twice with sterilized water, and then resuspended to an OD 600 of 1.0 in 200 mL of buffered minimal methanol (BMM) medium (100 mmolÁL À1 potassium phosphate, pH 6.0, 1.34% YNB, 0.000004% biotin and 0.5% methanol). To induce the laccase gene expression, methanol was added every 24 h to a final concentration of 0.5%. Recombinant proteins in the culture supernatant were precipitated by 80% ammonium sulphate fractionation. The precipitate was resuspended with 10 mL of BMM solution and dialysed against the same buffer through sephadex G-15. The enzyme solution was applied to a HisTrap HP kit (Amersham Biosciences K.K., Tokyo, Japan) according to the manufacturer's instructions.

Protein analysis and deglycosylation analysis
The laccase obtained after purification was analysed by 12% (w/v) sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), using the mini-protein gel electrophoresis system (Bio-Rad Lab, Hercules, CA, USA). The separated protein bands were stained with 0.2% Coomassie brilliant blue R-250. Protein concentrations were determined by the Bradford method, using bovine serum albumin as standard. [23] Deglycosylation of the natural laccase was performed using an N-glycosidase F deglycosylation kit (Roche, Penzberg, Germany), according to the manufacturer's instructions.

Conditions of the laccase activity assay
Enzyme activity was determined by monitoring the oxidation of ABTS at 420 nm (e mM ¼ 36.0 LÁmmol À1 Ácm À1 ). The reaction mixture for the standard assay contained 1.6 mmolÁL À1 ABTS in 190 mL of McIIvaine buffer (100 mmolÁL À1 citric acidÀNa 2 HPO 4 , pH 2.6) and 10 mL of purified enzyme. Assay mixtures were incubated at 30 C for 10 min after initiating the reaction by the addition of enzyme. The reaction was terminated by the addition of 50 mL of 1.0 molÁL À1 NaF. One unit of enzyme activity is defined as the amount of enzyme that catalysed the oxidation of 1 mmol of ABTS per minute under the assay conditions.

Effect of different parameters on dye decolourization
The decolourization of methyl orange, crystal violet and malachite green was assessed using the purified enzyme. The reaction mixture for the standard assay contained 10 mmolÁL À1 dye, McIlvaine buffer and 50 mL of purified enzyme with or without a laccase mediator. Reaction was initiated by adding enzyme to the assay mixtures which was incubated at 30 C for 10 min before, and then subsequently incubated in the dark. The time course of decolourization was determined at 20 min intervals in the first 2 h, and at 1 h intervals in the next 2 h by measuring the abosorbance at 470 nm for methyl orange, 590 nm for crystal violet and 620 nm for malachite green. Decolourization is defined as: Decolourization (%) ¼ 100 Â (Absorbance t 0 À Absorbance t f ) / Absorbance t 0 , where Absorbance t 0 is the absorbance of the assay mixture before incubation; Absorbance t f is the absorbance after incubation. The amount of dye was calculated from standard curves of absorbance versus dye concentration. Control solutions contained heat-inactivated enzyme and blanks contained all of the components except the dye, which was substituted with the same volume of McIIvaine buffer. All the assays were carried out in triplicate.
The effect of pH on dye decolourization was studied by incubating the purified enzyme mixture in McIIvaine buffer (with 0.5 mmolÁL À1 ABTS, 30 C) adjusted to pH 2, 3, 4, 5, 6, 7 and 8. The effect of temperature was assayed by incubating the mixture at different temperatures from 20 to 80 C at 10 C increments at the optimum pH for each dye. To study the effect of dye concentration, 2.5, 5, 10, 20, 30 or 40 mmolÁL À1 of final dye concentration was added in the reaction mixture, along with 0.5 mmolÁL À1 ABTS at the optimum temperature and pH for each dye. The effect of ABTS as laccase mediator on dye decolourization was analysed using 0, 0.5, 1, 2.5, 5 or 10 mmolÁL À1 ABTS in the reaction mixture at the optimum temperature and pH for each dye. To determine the effect of metal ions and laccase inhibitors on the decolourization, metal ions and inhibitors (at the same concentrations described above) were added to the reaction mixture. The mixture was incubated at the optimum temperature and pH for each dye, along with 0.5 mmolÁL À1 ABTS as mediator.

Sequence and data analysis
The Open Reading Frame (ORF) sequence of CcLcc2 was obtained from National Center for Biotechnology Information (NCBI) GenBank (Accession number BK004112) and analysed using the DNAMAN bioinformatics tool (version 6) (Lynnon Corporation, Quebec, Canada). The coding protein sequence of novel synthesized DNA sequence is the same as the protein sequence obtained from GenBank. Codon-usage processing was carried out using the database at http://www.kazusa.or.jp/codon. All graphs were constructed by OriginPro 7.5 (OriginLab, Northampton, MA, USA). All enzymological assays and dye decolourization tests were carried out at least in triplicate. Enzymological data in the graphs represent the averages of three replicates, and the error bars indicate the ranges of the values. Analytical results on dye decolourization varied by <5%.

Purification of CcLcc2
Most white-rot fungi are capable of degrading or oxidizing a range of aromatic organic compounds with the aid of some enzymes, such as lignin peroxidase, manganese peroxidase, versatile peroxidase and laccase. [24] However, white-rot basidiomycetes possessing the same function only secrete one enzyme (laccase). [25] This indicates that laccase from basidiomycetes alone can decompose organic compounds. In addition, these laccases are available in edible mushrooms. These enzymes may be used in environmental bioremediation, as they are safe for humans, and remaining waste from mushroom cultivation after harvesting can be used as a source of the enzyme. Due to these potential applications, laccases have been studied most thoroughly in basidiomycetes. [26,27] In this work, laccase from C. cinerea was synthesized chemically and heterologously expressed in P. pastoris in active form. Laccases are not easily produced in large amounts as recombinant proteins. [13] In this work, the use of the a-factor signal peptide induced the extracellular secretion of recombinant proteins and facilitated subsequent recovery.
After three days of culture, the supernatant exhibited the maximum laccase activity (data not shown). The purification steps for laccase are summarized in Table 1. The laccase was purified by (NH 4 ) 2 SO 4 fractionation from 10% to 80% and nickel nitrilotriacetic acid (Ni-NTA) affinity chromatography. The procedure resulted in $4.5fold purification with 8.4% yield. The final specific activity of laccase was $27 UÁmg À1 . The purified enzyme showed a single protein band in SDS-PAGE (Figure 1). This indicates that no other byproduct proteins and laccase were extracellularly expressed by P. pastoris (lane E, Figure 1).

Physical characterization of CcLcc2
The molecular mass of CcLcc2 was around 54 kDa, as determined by SDS-PAGE assay. When the enzyme was treated with endoglycosidase H, there was no change in band size (result not shown). CcLcc2 has one potential Nglycosylation site in the sequence (Asn438), which is predicted by the NetNGlyc 1.0 server. This prediction proves that the extent of glycosylation of CcLcc2 is low.

Laccase activity assay
Effects of pH and temperature on enzyme activity and stability The pH profile for laccase activity against ABTS showed a peak at pH 2.6, corresponding to the maximum activity ( Figure 2(A)). When the effect of pH on enzyme stability was determined at 30 C after different pH treatments at 4 C overnight, the enzyme was found to be stable at pH 2.6À10.6 ( Figure 2(B)). The optimum temperature of CcLcc2 was 45 C (Figure 2(C)), and 90% activity was maintained at 40À70 C. The thermal stability of laccase was determined by incubating the enzyme at pH 2.6 for 1 h and detecting its activity every 10 min. Laccase activity was hardly lost after incubating at 40 C, except when the treatment temperature was increased to 60 C, which caused 90% loss of laccase activity (Figure 2(D)). These results indicate that the purified enzyme has a wide optimum temperature range and good pH stability, which is usually required for industrial applications and is favourable for the development of biotechnological tools.

Substrate specificity
The conventional substrates oxidized by laccase, ABTS, 2,6-dimethoxyphenol and guaiacol, were used in the experiments designed to determine the enzymatic properties of CcLcc2. The kinetic parameters (Table 2) showed that CcLcc2 exhibited high activity with ABTS. The apparent Km value of this enzyme for ABTS was estimated to be 0.93 mmolÁL À1 . The apparent Km values for 2,6-DMP and guaiacol were 1.02 and 28.07 mmolÁL À1 , respectively. The relatively high Kcat value for ABTS (846.78 s À1 ) suggests that ABTS is a preferred substrate for this enzyme.

Decolourization of dyes
The purified enzyme prepared from the third day supernatant was evaluated for enzymatic decolourization activity.

Effect of pH
The optimum pH values for the decolourization of methyl orange, crystal violet and malachite green was 4.0, 3.0 and 4.0, respectively ( Figure 5), which are slightly different from the optimum pH for oxidation of ABTS (Figure 2(A)). This result demonstrated that the pH optimum for laccase is dependent on the substrate. The purified enzyme mixture showed strong decolourization in the acidic pH range and weak decolourization at netural and alkaline pH. This result agrees well with the generally higher activity of laccase at low pH. [1] Effect of temperature The influence of temperature on dye decolourization was investigated by incubating the reaction mixture at various temperatures. The optimum temperature of decolourization ranged from 50 to 70 C. In the case of  methyl orange, 15% dye decolourization was achieved at 2 h of incubation. When the temperature was >50 C, the degree of decolourziation decreased to 50% of the maximum value ( Figure 6(A)). With crystal violet, maximum decolourization of 80% was observed at 60 C and it decreased to 40% at 30 C ( Figure 6 (B)). Malachite green showed properties similar to those of crystal violet: the decolourization increased to 80% at 70 C ( Figure 6(C)).

Effect of dye concentration
The effect of dye concentration on the decolourization efficiency was determined with different initial dye concentrations (2.5À40 mmolÁL À1 ) with a constant amount of purified enzyme. The decolourization decreased with the increase in dye concentration from 2.5to 40 mmolÁL À1 for all dyes (Table 3). The behaviour observed here is similar to that of laccase oxidation of substrates, in which the rate of substrate oxidation increased with the substrate concentration until saturation. The maximum decolourization of methyl orange, crystal violet and malachite green at 4 h was 47.6%, 78.6% and 84.1%, respectively. The most effective decolourization was observed using crystal violet and malachite green as the substrate. The decolourization of methyl orange occurred more slowly than that of the other dyes during the initial period of the reaction, which indicates that CcLcc2 had more difficulty catalyzing the oxidation of azo dyes than of triphenylmethane dyes. The latter may need relatively strong laccase activity or a longer reaction time.
Although the two triphenylmethane dyes were oxidized in the presence of recombinant laccase to the same extent, the decolourization processes were different. Crystal violet was more resistant to decolourization than malachite green. At the start, the decolourization of crystal violet was slow and increased with time, whereas malachite green was decolourized rapidly and barely any decolourization took place in the next reaction ( Figure 6 (B) and 6(C)). This may be due to differences in the chemical structure of the dyes. Moreover, the decolourization activity towards crystal violet was inhibited when the dye concentration was 20 mmolÁL À1 , whereas 30 mmolÁL À1 malachite green resulted in inhibition. This result further indicates that malachite green is more easily decolourized than crystal violet. A similar result with other bacterial species has been observed. [28] On the contrary, there are certain bacterial species that can decolourize crystal violet more easily than malachite green. [18] Effect of redox mediator All tested dyes were poorly decolourized ($10%) by purified enzyme alone. However, in the presence of a redox mediator (ABTS), the purified enzyme achieved efficient decolourization, >50% in 4 h (Figure 7). These results agree with previous studies on other mediators. [7,29] Small increases in the rate of decolourization of dye were observed (Figure 7). With the increase in ABTS concentration up to the dye concentration, decolourization rates did not decrease, except that of malachite green (Figure 7(C)). The inhibition of decolourization of malachite green could be explained by the high concentration of ABTS combining with malachite green. Many investigations have demonstrated that adding certain redox-active compounds to the reaction system can enhance the decolourization of dyes effectively and efficiently. The divalent cation ABTS 2þ (the oxidation product of laccase) may have a role in the oxidation of the dyes as an intermediate oxidant.
Thus, it could be suggested that the decolourization of the tested dyes was indirect and the process was caused by the oxidizing intermediates produced by the laccase enzyme.

Effect of metal ions and inhibitors
Industrial wastewaters are complex mixtures that may contain dyes, salts, metal ions, chelators, precursors and so on. [30] Some of these substances may deactivate laccase. Previous studies have demonstrated that there are limitations to the ability of laccase to decolourize effluents. Therefore, the effects of metal ions and laccase inhibitors on laccase decolourization were investigated in our work.
Metal ions and some potential laccase inhibitors (e.g., DTT, SDS, L-cysteine, p-cumaric acid, NaN 3 and EDTA) were tested for their effect on dye decolourization. The effects of the metal ions are shown in Figure 5. Decolourization of methyl orange (Figure 3(B)) was severely impacted by Fe 2þ and Fe 3þ . However, the same ions had little effect on the decolourization of crystal violet (Figure 3(C)) and malachite green (Figure 3(D)).  The effects of putative laccase inhibitors on dye decolourization are shown in Figure 4. Decolourization of methyl orange (Figure 4(B)) in the presence of NaN 3 was strongly inhibited even at 1 mmolÁL À1 NaN 3 . The effect of each inhibitor was generally proportional to its concentration. The effect of other inhibitors on the decolourization of crystal violet (Figure 4(C)) and malachite green (Figure 4(D)) in the presence of NaN 3 was generally different from that on the decolourization of methyl orange. Other inhibitors (except SDS) slightly inhibited the  decolourization of crystal violet and malachite green. SDS severely inhibited the decolourization of crystal violet and malachite green and had relatively little effect on methyl orange decolourization.

Conclusions
In this work, we demonstrated that the expression of the laccase gene (CcLcc2) from the basidiomycete C. cinerea Okayama-7 in the methylotrophic yeast P. pastoris produces an extracellular laccase which is stable and active in the presence of moderate amounts of metal ions and organic solvents. This enzyme has a good decolourization capacity towards three textile dyes. This capacity can be enhanced by the addition of mediators such as ABTS. In view of the results obtained in the present work, it is clearly indicated that this laccase possesses important properties for industrial applications. Characterization of other functional members of this laccase family is underway and the catalytic mechanism will be subject to future studies.