Abstract
Laccase is a kind of polyphenol oxidase having potential in applications for pulp bleaching, waste water treatment in mills, and removal of phenols in the food industry. The normal laccase from fungus or bacterial contains four copper atoms per protein molecular, imparting a blue color. Here it is reported that a white laccase is produced by a white rot fungus Panus conchatus from its solid-state fermentation. The activity center of this laccase is Cu2FeZn, which lacks the typical type-1 blue copper color. The polyacrylamide gel electrophoresis of purified laccase showed a main polypeptide with a molecular weight of about 60 kDa. Laccase substrate 2,6-dimethoxylphenol and others, such as syringaldazine, o-tolidine, and ABTS, were readily oxidized, among which the Km for syringaldazine was the highest. The isoelectric point of this enzyme was 3.6 and it was stable at temperatures below 45 °C over a wide range of pH (4-12).
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Purification and Characterization of White Laccase from the White-rot Fungus Panus conchatus
Pandeng Zhou,a,b Chaoying Fu,a Shiyu Fu,a,* and Huaiyu Zhan a
Laccase is a kind of polyphenol oxidase having potential in applications for pulp bleaching, waste water treatment in mills, and removal of phenols in the food industry. The normal laccase from fungus or bacterial contains four copper atoms per protein molecular, imparting a blue color. Here it is reported that a white laccase is produced by a white rot fungus Panus conchatus from its solid-state fermentation. The activity center of this laccase is Cu2FeZn, which lacks the typical type-1 blue copper color. The polyacrylamide gel electrophoresis of purified laccase showed a main polypeptide with a molecular weight of about 60 kDa. Laccase substrate 2,6-dimethoxylphenol and others, such as syringaldazine, o-tolidine, and ABTS, were readily oxidized, among which the Km for syringaldazine was the highest. The isoelectric point of this enzyme was 3.6 and it was stable at temperatures below 45 °C over a wide range of pH (4-12).
Keywords: White-rot fungus; Panus conchatus; White laccase; Activity center; Copper; Iron; Zinc
Contact information: a: State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640; b: College of Chemical and Environmental Engineering, Hunan City University, Yiyang Hunan 413000, China; *Corresponding author: shyfu@scut.edu.cn
INTRODUCTION
In recent years, laccase has provoked considerable interest in a wide range of applications such as delignification and pulp bleaching, water or soil bioremediation, dye decolorization, and chemo-enzymatic synthesis (Martínez et al. 2009; Faraco et al. 2009; Sadhasivam et al. 2009; Mikolasch and Schauer 2009). The characteristics and functions of laccase depend on both the fungal species and the cultural conditions (Giardina et al. 1999). Most fungal laccase varieties belong to the family of blue copper phenol oxidases, containing four copper atoms per molecule distributed among three different copper binding sites (Cole et al. 1990; Giardina et al. 1999; Fraterrigo et al. 1999). The type-1 (or blue copper) site is responsible for the blue color of laccase and has a maximum absorbance at 605 nm in the visible spectrum (Giardina et al. 1999). The type-2 copper site exhibits no signal in UV-visible spectrum, but has signals in ESR spectra (Cole et al. 1990; Fraterrigo et al. 1999). The type-3 site is a double-copper center, corresponding to a UV absorbance near 320 nm (Giardina et al. 1999; Fraterrigo et al. 1999). However, yellow laccase from Panus tigrinus or Pleurotus ostreatus has no maximum in the blue region of the absorption spectrum; this can be explained by the fact that the copper is in its reductive (Cu+) valence state (Leontievsky et al. 1997; Pozdnyakova et al. 2004). A white laccase isoenzyme (POXA1) from Pleurotus ostreatus has only one copper, but two zinc and one iron atom per molecule (Palmieri et al. 1997).
Usually, typical blue laccase can use phenolic compounds or phenolic units in lignin as substrates. Natural or synthetic mediators, such as 3-hydroxyanthranilic acid, ABTS, HBT, etc., are often required for promoting catalytic ability and extending the application of laccase in degrading non-phenolic compounds (Camarero et al. 2007; Call and Mücke 1997). A yellow or white laccase may oxidize non-phenolic compounds in the absence of a mediator, thus exhibiting higher redox potential and broader specificity (Leontievsky et al. 1997; Xu et al. 2001).
In this work, another white laccase producer, Panus conchatus, was researched. Compared with the lignocellulolytic enzyme system of the well-known white-rot fungi Phanerochaete chrysosporium, the enzymes produced by P. conchatus contain high levels of extracellular laccase activity with minimum cellulase activity, which suggests their potential application in the selective delignification of pulp (Hu et al. 2001). The major ligninolytic enzymes of this fungus are laccase and manganese peroxidase, with no lignin peroxidase. The crude P. conchatus laccase has been successfully used for pulp bleaching in the presence of some mediators in previous studies (Aniwar et al. 2003; Mo et al. 2006).
The objective of this work was to identify and characterize the white laccase produced by Panusconchatus. The laccase was purified and metal constituents in the protein were determined. This work was able to describe members of the laccase family and provide new insight to an oxidative enzyme.
EXPERIMENTAL
Material and Methods
Fungus and culture conditions
A strain of white-rot fungus, Panus conchatus, was screened and isolated from decayed wood samples (Hu et al. 2001) and was maintained on a malt agar slant at 4 °C in the laboratory.
The fungus was cultured in 30 mL of malt juice medium in a rotating flask at 40 °C for 7 days. Freshly harvested 7-day-old mycelia of fungus were mixed with 100 mL sterilized water and homogenized in a high-speed blender at 10000 rpm for 2 min. Ten mL of homogeneous mycelial suspension was inoculated in a 500 mL Erlenmeyer flask containing 10 grams (o.d. weight) sterilized wet rice straw with a moisture content of 65%. Solid-state fermentation was carried out at 40 °C for 28 days. The laccase activity of the crude cultivation extract was determined every two days.
Laccase assays
Laccase activity was measured by monitoring the oxidation of 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) at 420 nm (ε420nm = 36,000 M-1·cm-1). Measurements were performed in triplicate for each sample analysis. One international unit of enzyme activity is defined as the amount of enzyme required to oxidize 1 µmol substrate in one minute (Bourbonnais and Paice 1990).
Enzyme purification
The crude enzyme was harvested from the fermentation culture of P. conchatus after 24 days. Ammonium sulphate precipitation was used as the initial step in enzyme purification. Solid ammonium sulphate was added to the crude enzyme to 30% saturation under constant stirring at 4 °C for 3 h. The precipitate was removed by centrifuging at 8000 g for 5 min at 4 °C. Supplemental ammonium sulphate was added into the supernatant to 80% saturation. The precipitated proteins were collected by centrifuging and were re-dissolved in deionized water. Buffer exchange and further concentration against 50 mM NaH2PO4 buffer (pH 5.0) were performed in an Amicon stirred ultrafiltration cell (Amicon, Beverly, MA) with an YM-10 membrane (molecular weight cut-off 10,000 Da). Then, the enzyme concentrate was loaded on a DEAE-cellulose DE-32 column, which was pre-equilibrated with 200 mL phosphate buffer (50 mM, pH 5.0), and was eluted with a linear gradient of 0-0.5 M NaCl in 400 mL phosphate buffer (50 mM, pH 5.0). Fractions containing laccase activity were pooled and concentrated by ultrafiltration through an Amicon membrane YM-10. The concentrated protein was finally loaded on a Sephadex G-100 column and eluted with water to obtain purified laccase. Fractions containing laccase were pooled, concentrated, and freeze-dried.
SDS-PAGE
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using 11% polyacrylamide gel as described by Laemmli (1970). Markers with molecular masses ranging from 14.4 to 97.4 kDa (Bio-Rad) were used as standards and were run with each gel. Protein bands were stained with Coomassie brilliant blue G-250. Native polyacrylamide gel electrophoresis (Native-PAGE) was performed following the same method as described above without sodium dodecyl sulfate. Proteins with laccase activity on Native-PAGE gels were visualized by staining with ABTS at pH 4.0. Isoelectric focusing (IEF) was performed on 5% polyacrylamide gels containing ampholytes covering a pH range from 2.5 to 5.5. The anode solution was 1 M phosphoric acid and the cathode solution was 1 M sodium hydroxide.
UV-visible spectroscopy
The UV-visible absorption spectrum of the purified Panus conchatus laccase was determined at wavelengths between 200 and 700 nm at room temperature in a phosphate buffer (50 mM, pH 6.0) using a SHIMADZU 2550 spectrophotometer.
Amino acid sequence analysis
The N-terminal amino acid sequence of purified laccase was determined by step-wise Edman degradation as described by Palmieri et al. (1997). A Perkin-Elmer applied biosystem 477A protein sequencer was used and was equipped with a 120A HPLC apparatus for on-line phenylthiohydantoin-amino acid analysis. Proteins isolated by SDS-PAGE were electroblotted onto a polyvinylidene difluoride membrane. The excised bands were directly analyzed. The constituents of amino acids were determined using a Hitachi 835-50 amino acid analyzer after hydrolysis of 100 mg laccase in 6 M hydrochloride at 110 °C for 24 h.
Atomic absorption spectroscopy
The zinc, copper, and iron contents in laccase were determined with a Perkin-Elmer 5100 atomic absorption spectrometer equipped with a Zeeman graphite furnace module and an autosampler.
Substrate specificity and stability
The compounds listed in Table 5 were tested as electron-donor substrates of laccase. Substrate specificity was qualitatively studied by measuring the initial velocity of the catalytic reaction at 25 °C. Initial rates were calculated from the linear portion of the progress curve. The wavelengths used for measuring oxidation kinetics were 420 nm for ABTS (ε = 36 000 M-1· cm-1), 590 nm for o-tolidine (ε = 6 340 M-1·cm-1), 530 nm for syringaldazine (ε = 65 000 M-1· cm-1), and 468 nm for 2,6-dimethoxylphenol (ε = 49 600 M-1 cm-1) (Xiao et al. 2004; Di Nardo et al. 2004).
The stability was studied by incubating 4.0 U purified laccase in 1,000 mL of buffer under a variety of pH (diluted sulfuric acid 2 to 4, sodium acetate 4 to 6, phosphoric buffer 6 to 9, sodium bicarbonate/sodium hydroxide 10 to 11, potassium chloride/sodium hydroxide 12 to 13), temperature, and incubation time conditions. The relative activity was calculated by normalizing the activity measured at pH 4.0 at room temperature without incubation to 100%.
RESULTS
Production and Purification of Laccase
Laccase and manganese peroxidase are the two ligninolytic enzymes that are extracellularly excreted by Panus conchatus in solid-state fermentation (Fig. 1) and submerged fermentation (data not shown). During solid-state fermentation, laccase activity reached a maximum of 33 IU/g straw after 22 days cultivation and maintained a high level of activity for at least 3 days. Meanwhile, manganese peroxidase activity was very slight, and not even detectable after 24 days. No lignin peroxidase activity was detected at any time in the cultures. Therefore, the crude enzyme from solid-state fermentation was harvested after 24 days, which contained only laccase as a ligninolytic enzyme.
Fig. 1. Profile of ligninolytic enzymes produced by P. conchatus during solid-state fermentation (mean of triplet data)
Ammonium sulfate-precipitated enzyme was loaded on a DEAE-cellulose column and eluted with a continuous gradient 0 to 0.5 M NaCl in phosphate. The elution profile is shown in Fig. 2. One peak of laccase activity was eluted from DEAE-cellulose column. The fractions with high laccase activity were pooled and loaded onto a Sephadex G-100 column. A colorless fraction with laccase activity was obtained and a white laccase protein was obtained after being lyophilized. The separated colorful compounds, probably from the degraded lignin in straw, were retained on the column without collection.
Fig. 2. DEAE-cellulose chromatography of P. conchatus crude enzymes
Fig. 3. Electrophoresis images of purified Panus conchatus laccase: A) Native-PAGE of crude enzyme (i), enzymes treated by both DEAE-cellulose and Sephadex chromatography (ii and iii, two vials from Sephadex separation), enzymes treated by DEAE-cellulose (iv and v, two vials from DEAE-cellulose separation); B) SDS-PAGE of purified laccase (ii in A) and DEAE-laccase
Either crude or purified enzyme preparations were run on native-PAGE media. The laccase protein bands can be observed by staining with ABTS solution because of the formation of green-colored oxidative products. At the beginning of the reaction with ABTS, the green band was narrow but easy to diffuse, resulting in wide bands in the PAGE gels (Fig. 3a). Figure 3a also shows that there were two laccase isoenzymes in the crude enzyme preparation (Fig. 3a-i) extracted from solid-state fermentation, as visualized on Native-PAGE gel. One enzyme had higher activity and was purified further. The other had minor activity with no further purification. In partially purified enzyme fractions by DEAE-cellulose chromatography, there were still two isoenzymes with laccase activity, as shown in Figs. 3a-iv and v. When these fractions were concentrated and passed through gel filtration chromatography, the purified protein contained only one laccase isoenzyme and only one protein band on Native-PAGE (Figs. 3a-ii, iii) and SDS-PAGE gel (Fig. 3b). The DEAE purified enzyme (DEAE-Lac in Fig 3 B) has two protein bands also, one of them is strong and another is weak. The crude enzyme have many bands that didn’t separate well. The molecular mass of the purified laccase determined by SDS-PAGE analysis was about 60 kDa. The isoelectric point of laccase was 3.6.
Molecular Mass Measured by MALDI-MS
More accurate determination of molecular mass of laccase was carried out using MALDI-mass spectrometry (Fig. 4). The measured molecular mass of purified P. conchatus laccase was 56.1 kDa, which is close to that of other white-rot fungi, but less than that of plant laccase such as sycamore. The molecular mass of this laccase is 60 kDa based on SDS-PAGE. In general, the molecular mass of laccases from groups of terrestrial fungi was obtained based on PAGE in the range of 60 to 80 kDa, including laccase from Armillaria mellea (80 kDa, Curir et al. 1997), Pycnoporus cinnabarinus (74 kDa, Eggert et al. 1996), Trametes sanguinea (62 kDa, Nishizawa et al. 1995), Trametes villosa (63 kDa, Xu et al. 1999), Schizophyllum commune (62, 64, 72 kDa, Phillips and Leonard 1976), Pleurotus ostreatus (59 kDa, Sannia et al. 1986), Rigidoporus lignosus (52, 55 kDa, Geiger et al. 1986), Phellinus noxius (70 kDa, Driouich et al. 1992), Botrytis cinerea (74 kDa, Slomczynski et al. 1995). The laccase from wood, Sycamore (95-100 kDa, Thurston 1994) is bigger than that from fungi.
Fig. 4. MALDI TOF spectrum of Panus conchatus laccase
*The analyte mixed with a saturated sinipinic acid solution.
UV-visible Absorption Characteristics
The UV-visible spectrum of purified laccase shown in Fig. 5 differs from typical blue laccase as widely reported (Giardina et al. 1999; Fraterrigo et al. 1999). There is a shoulder of absorbance at 320 nm, corresponding to the two-copper center of a type-3 site. However, there was no absorbance peak around 600 nm, which indicates that the Panus conchatus laccase does not exhibit an apparent color, that means no type 1 copper.
Fig. 5. UV-visible spectra of laccase produced by Panus conchatus
Metal Composition
Three kinds of metal elements, copper, iron, and zinc, were found in purified laccase (0.1 mg/mL) as determined by atomic absorption spectroscopy. The content of each element was 2.0 mol/mol copper/protein, 1.0 mol/mol zinc/protein, and 1.0 mol/mol iron/protein. The metal composition suggests that the ratio of copper/zinc/iron is 2:1:1 for the P. conchatus laccase. This result is in accordance with the UV-visible spectrum, in which no blue copper in type-1 site appears, since type-1 and 2 sites are taken by zinc and iron. However, the metal composition of P. conchatus laccase was different from the earlier reported Pleurotus ostreatus laccase isoenzyme, which contained copper, iron, and zinc in a ratio of 1:1:2 (Palmieri et al. 1997).
The constitution of metal atoms in laccase may be dependent on the culture conditions. Thurston (1994) proposed that the typical laccases with Cu-I and Cu-II are produced from submerged culture. Iimura et al. (1995) got a colorless laccase from solid-state fermentation of Coriolus versicolor, and Leonitievsky got a yellow laccase from solid-state culture of fungus (Leonitievsky et al. 1997). The reason for this laccase was regarded as the peptide of laccase was combined with degraded lignin derivatives (Mot et al. 2012). However, the present laccase produced by P. conchatus was colorless in solid-state fermentation.
The same work was repeated in submerged cultivation (not showed here), and the result was the same. The reason is not the reduction of copper in protein by reductant, but rather replacement by other metal atoms. The freeze-dried enzyme is white, so we named it the white laccase.
Table 1. Amino Acids in Purified Laccase from P. conchatus
N-terminus Amino Acid Sequences
The constituents of amino acids in purified laccase from P. conchatus are listed in Table 1. The content of tyrosine is the highest, contributing 17% of the dry mass. The second most abundant is valine, at levels as high as 10%.
The N-terminus peptide sequences of purified P. conchatus laccase are given and compared with the sequences of other known laccases in Table 2. It was found that the N-terminus peptide sequences of laccase from P. conchatus were close to that of Basidiomycete species such as P. cinnabarinus, T. versicolor, Basidiomycete PM, C. hirsutus, C. subvermispora, Phlebia radiata, etc. (Bourbonnais et al. 1995; Eggert et al. 1996; Coll et al. 1993; Kojima et al. 1990; Fukushima and Kirk 1995; Saloheimo et al. 1991), but much different from laccase produced by Ascomycete fungi (Perry et al. 1993; Germann et al. 1988; Williamson 1994).
Table 2. N-terminal Amino Sequences of Laccase from P. conchatus and Other Fungi
Substrate Specificity and Stability
Laccase catalyzes the oxidation of a variety of phenolic compounds and aromatic amine substrates and reduces molecular oxygen to water as a co-product. Several substituted phenols and aromatic amines were studied as possible substrates in this work. The relationship between initial reaction rate and initial substrate concentrations produced typical Michaelis-Menten curves. The parameters Km and Vmax for different substrates were calculated and are listed in Table 3. The value of Km for syringaldazine was the highest.
Table 3. Kinetic Parameters of Catalytic Oxidation by Panus conchatus Laccase
Note: The Km and Vmax were calculated according to Cornish-Bowden (1974).
The activity of laccase was stable and could be kept for long periods of time at room temperature. Figure 6a shows that the relative activity of laccase decreased when incubated in higher temperatures (45 to 70 °C). Half of the laccase activity was lost when incubated for 24 h at 45 °C and for only 10 h at 60 °C. Activity was lost very quickly at 70 °C; only 5% activity was left after 4 h incubation. The sensitivity of laccase activity to pH is shown in Fig. 6b. It was found that laccase is stable over a wide range of pH (4 to 12) after being kept at room temperature for 24 h, which suggests potential application in mildly alkali bio-pulping or bio-bleaching.
Fig. 6. Stability of Panus conchatus laccase under: A) different temperature, B) different pH
CONCLUSIONS
- The purified enzyme from Panus conchatus belongs to the laccase family due to the observations that: (1) the enzyme exhibited a broad pattern of substrate (Table 4), (2) oxygen was used as an oxidative substrate, while no H2O2 was required to start the catalytic oxidation, and (3) the determined N-terminal primary structure of the enzyme exhibited a high degree of similarity with the corresponding sequences of known Basidiomycete laccases (Table 3).
- The Panus conchatus laccase exhibited white rather than the typical blue color, as evidenced by UV-visible spectroscopy and atomic absorption spectroscopy. The ratio of constituents Cu to Zn to Fe was 2:1:1. The two copper atoms constitute the double copper center, which is the same as type-3 (Cu-III) in typical blue laccase. The type-1 and 2 copper atoms were substituted by metal atoms (Zn and Fe). The laccase with 4 coppers per protein has different redox potential from plants and fungi because of the different ligands in the type-1 site. Similarly, the redox potential might be different when type-1 copper is substituted by another metal atom. Further study is now under way to prove this assumption.
- The laccase produced by P. conchatus was colorless. The reason is not the reduction of copper in protein by reductant, rather replacement by other metal atoms.
ACKNOWLEDGMENTS
This research was supported by the National Natural Science Foundation of China (No. 30771689). The authors thank Professor Qizhi Yao for her guidance and constructive comments. The authors thank Mr. Hao Liu for his editorial work.
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Article submitted: February 26, 2013; Peer review completed: May 26, 2013; Revised version received and accepted: February 10, 2014; Published: February 12, 2014.