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Keywords:

  • Phanerochaete chrysosporium;
  • Laccase;
  • Lignin degradation

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

Phanerochaete chrysosporium strain ME446 produced laccase when grown in a medium with high nitrogen (24 mM), glucose, acetate buffer and high copper (0.4 mM CuSO4). Copper appeared to serve as an inducer. The highest amount of laccase activity was found in 2–3 day old cultures. Both intracellular and extracellular laccases eluted as four peaks (L1–L4) on a MonoQ (anion exchange) column. All four peaks from intracellular fluid appear to comprise multiple polypeptides that gave rise to a ∼400 kDa peak on a gel filtration column. Extracellular laccases had a non-denaturing Mr of 100 kDa.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

Laccases (benzenediol:oxygen oxidoreductases; EC 1.10.3.2) are copper-containing oxidases that catalyze the oxidation of methoxy-substituted monophenols, ortho and para diphenols, aromatic amines, syringal-dazine and non-phenolic compounds such as 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonate) (ABTS) [1, 2]. Fungal laccases have been implicated in sporulation, rhizomorph formation, pathogenesis and formation of fruity bodies and lignin degradation [1–4]. Thus, laccases appear to have a significant role in fungal biology. Although laccases from many fungi have been studied, the presence of laccase in P. chrysosporium, an extensively studied model lignin degrader [5, 6], has been demonstrated only recently [7]. In a previous study it was shown that in P. chrysosporium laccase activity was present when cellulose was used as a carbon source but absent when it was substituted by glucose [7]. Here, we show that when CuSO4 was added to P. chrysosporium cultures with glucose as a carbon source, multiple laccase isoforms were produced.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

P. chrysosporium strain ME446 (ATCC 34541) was grown in the presence of low (0.02 mM) or high (0.4 mM) copper sulfate medium with sufficient nitrogen (24 mM). Each liter of low copper medium for mycelial growth contained: 10 g glucose, 2 g ammonium tartrate, 1.2 mg thiamine, 1 ml of 400 mM veratryl alcohol, and 100 ml of 0.2 M sodium acetate buffer (pH 4.5), 100 ml of basal medium and 50 ml of trace elements. Each liter of basal medium contained: 20 g KH2PO4, 5 g MgSO4·7H2O, 1 g CaCl2. Each liter of trace element solution contained: 3.0 g MgSO4·7H2O, 0.5 g MnSO4, 1.0 g NaCl, 0.1 g FeSO4·7H2O, 0.1 g CoCl2·6H2O, 0.1 g ZnSO4·7H2O, 0.1 g CuSO4·5H2O, 10 mg KAl(SO4)·12H2O, 10 mg H3BO3, 10 mg Na2MoO4·2H2O, 1.5 g nitrilotriacetate adjusted to pH 6.5 with KOH. Cultures were grown in 500 ml Erlenmeyer flasks, containing 25 ml of medium inoculated with conidiospores to give a final OD600 of 0.3. High copper medium flasks also received 100 μl of 0.1 M CuSO4. Flasks were incubated at 30°C without shaking.

Laccase activity was assayed using 0.65 mM 2,6-dimethoxyphenol (DMOP) as the substrate, in 0.1 M Tris-acetate buffer (pH 7.0) and/or citric acid-Na2HPO4 buffer (pH 3.0) at 25°C. One unit was defined as an increase in absorbance of 1.0 per min at 468 nm and 25°C [8]. Laccase activity was assayed in the presence of catalase (10 U/ml) to ensure the absence of H2O2. LIP and MNP activities were assayed as previously described [9, 10].

The time course of laccase production was determined using triplicate stationary cultures. Flasks were harvested each day over a period of 12 days. Mycelial mats were separated from extracellular fluid, washed with distilled water several times, and suspended in 3 ml of Tris-acetate buffer, pH 7.0. All subsequent manipulations were done on ice. Mycelial mats were homogenized four times for 30 s using a Biospec homogenizer and 1 ml of the homogenate was centrifuged for 1 min at 16.000×g. The supernatant, intracellular fluid (IF), was recovered and 800 μl was used for the laccase assay.

Since copper can interfere with laccase assays when DMOP is used as a substrate, CuSO4 was removed from the extracellular high copper medium by the following method. 2 ml of extracellular fluid was centrifuged in Centriprep 10 (Amicon, Beverly, MA) for the removal of original medium. Resulting samples (∼0.1 ml) were rinsed in sterile distilled water three times by centrifugation. The final volume of each sample was adjusted to 2 ml. Thus no concentration of extracellular fluid resulted. Samples processed in this manner were referred to as EF.

For initial HPLC analysis 2 ml of IF and EF were directly applied to a MonoQ (Pharmacia) column. Elution was accomplished by using a 0.01–1 M sodium acetate buffer (pH 6.0) gradient with a flow rate of 1 ml min−1. The absorbance of the eluent was monitored at 280 nm or 600 nm (for protein and copper enzymes, respectively) and 1 ml fractions were collected. These fractions were assayed for laccase activity by adding 0.1 ml of DMOP and recording the absorbance at 468 nm after 2 h.

For molecular mass determination EF was concentrated 50-fold (CEF) and IF was concentrated 20-fold (CIF) by ultrafiltration through a PM 10 (Amicon) membrane and equilibrated with 10 mM sodium acetate buffer pH 6.0. Concentration of CEF led to 20–60% loss in activity, hence 40 flasks were used for large culture batches. CEF and CIF were fractionated on a MonoQ column and the fractions representing copper enzyme peaks were pooled. Each pool was desalted and concentrated by Centriprep 10 and stored at −20°C. These pools (L1–L4) were then injected on a gel filtration column 300 SW (Waters) containing 10 μm particles and eluted with a phosphate buffer (0.06 M Na2HPO4, 0.04 M NaH2PO4, 0.01 M KCl, and 0.001 M MgSO4, pH 7.0) at a flow rate of 0.4 ml min−1. Elution was monitored at OD600 and/or OD280 and 0.4 ml fractions were collected for laccase activity assays. The void volume was determined using blue dextran and the non-denaturing molecular mass was calculated using protein standards (Sigma Chemical Co., Cat. # MW-GF-1000 kit). Gel filtration fractions containing laccase activity were concentrated, analyzed on sodium dodecyl sulfate (SDS) polyacrylamide gels, and protein bands were stained with 0.05% Coomassie brilliant blue R and dried [11]. Molecular masses of the proteins were determined using protein standards (Boehringer Mannheim, Cat. # 1317474).

The effect of pH on laccase activity was measured over the range of 2.2–7.0 using a glycine-HCl buffer (pH 2.2–3.0), citric acid-Na2HPO4 buffer (pH 3.0–5.0), and sodium phosphate buffer (pH 5.0–7.0).

3Results and discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

The time course for laccase production by P. chrysosporium revealed that laccase activity was absent from EF and IF of cultures grown in low copper medium with glucose. The lack of laccase activity in low copper medium is consistent with previous reports [5, 7, 12]. However, laccase activity was detected in cultures with high levels of copper. Thus, copper appears to induce laccase production even when glucose is used as a carbon source. Because DMOP can also undergo peroxidative oxidation, all laccase assays were performed in the presence of catalase which ensured the absence of hydrogen peroxide. Furthermore, no LIP or MNP activities were found in any samples that showed laccase activity. Hence, the activity monitored was attributed to laccases. Because LIP and MnP were not produced under the conditions used in this study it might be possible to use these conditions for investigating the role of laccases in lignin degradation by P. chrysosporium. In high copper medium, laccase activity reached its peak on day 2 in IF and on day 3 in EF (Fig. 1). Flask to flask variations were high in EF between days 2 and 4. Additionally, flasks with lower intracellular activity showed lower extracellular activity. Flask to flask variations in metabolism are inherent in P. chrysosporium cultures [13].

image

Figure 1. Time course of laccase production by P. chrysosporium grown in high copper medium (0.4 mM) containing 24 mM nutrient nitrogen and glucose. Mean and standard error of triplicate cultures are shown.

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The pH optimum for laccase activity in EF appeared to be 3.0. This falls in the range commonly reported for fungal laccases and is consistent with the optimum reported for P. chrysosporium laccase produced in the presence of cellulose [7].

Interestingly, when IF and EF were applied to a MonoQ column without being concentrated, both samples showed four laccase activity peaks (L1–L4). L1 was the major activity peak in both IF and EF and it corresponded to the highest OD600 peak (Fig. 2). Activity peaks L2, L3 and L4 were also associated with absorbance at 600 nm. However, it was necessary to use CIF and CEF for obtaining L1–L4 samples for further purification. When fractionated individually on a gel filtration (GF) column, all four samples (L1–L4) from CEF displayed one OD600 peak which was coincidental with the laccase activity peak and had Mr of 100 kDa (data not shown). Thus, all four extracellular laccase isoforms which were separated by charge appeared to have similar non-denaturing molecular masses. All four intracellular samples (L1–L4) had one major and one minor activity peak when fractionated individually on a GF column. The major laccase activity peak was coincidental with the major OD600 peak at the exclusion limit of the column (Fig. 3). From sample profiles shown in Fig. 3 it can be seen that a significant amount of absorbance at 280 nm was associated with this peak. Based on the extrapolation of the standard curve, the non-denaturing molecular mass of the major CIF laccase peaks was ∼400 kDa. The minor peak showed a negligible amount of activity and absorbance at 600 nm. However, this minor peak corresponded to the 100 kDa laccase activity peak found in the extracellular samples. Hence, this peak could represent a form of laccase that is secreted.

image

Figure 2. MonoQ separation profiles of intracellular and extracellular fluids from P. chrysosporium cultures grown in high copper medium. A linear 0.01–1.0 M sodium acetate (pH 6.0) gradient and a flow rate of 1 ml/min were used. Absorbance was monitored at 600 nm for the detection of copper metalloproteins. IF and EF were not concentrated and 1 ml IF and 2 ml of EF were injected.

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image

Figure 3. Gel filtration profiles for MonoQ peak L2 from concentrated intracellular fluid. The same sample was monitored at OD600 and OD280. The major activity peak was at the exclusion limit of the column while the minor activity peak was found in fractions 19 and 20 (∼100 kDa).

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The gel filtration fractions containing the major peaks (∼400 kDa) derived from the four CIF MonoQ peaks were designated GF400L1, GF400L2, GF400L3 and GF400L4. Because these major activity peaks displayed significant absorbance at 280 nm and eluted away from other proteins, fractions containing these peaks were selected for SDS-PAGE analysis. Each of these samples (GF400L1–GF400L4) had two common bands with Mr values of 68 and 58 kDa on SDS-PAGE gels (Fig. 4). Molecular masses of these two polypeptides fall within the range reported for many fungal laccases [1, 2]. While GF400L2 showed 68 and 58 kDa bands, GF400L1, GF400L3 and GF400L4 had additional protein bands with Mr values of 54, 31, and 16 kDa (Fig. 4). Based on band intensities the 54, 31 and 16 kDa bands appear to be present in lower proportions than the 68 and 58 kDa bands. The fact that all of these polypeptides elute as one peak at the exclusion limit (>300 kDa) of the GF column suggests that these are a part of an intracellular protein complex. Additional experiments are necessary to determine the exact nature of this protein complex.

image

Figure 4. SDS-PAGE analysis of laccase peaks from concentrated intracellular fluid. Lane 2 from panel A and lane 5 from panel B show GF400L4 and GF400L2 respectively. Lane 1 on both panels displays molecular weight markers. Arrows point to various bands found in the samples while the bands visible at the bottom of the gel represent dye front that comigrates with 14 kDa molecular weight marker.

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The GF fractions containing the 100 kDa extracellular laccase peaks derived from L1–L4 MonoQ peaks were designated GF100L1, GF100L2, GF100L3 and GF100L4. GF100L1 showed a smear suggesting that proteases also elute in these fractions. GF100L3 and GF100L4 had numerous proteins suggesting that further purification was necessary. However, GF100L2 showed only two protein bands at 48 and 53 kDa on denaturing gels. Here we suggest that 58 and 68 kDa intracellular bands are precursors to 48 and 53 kDa extracellular bands and that GF100L2 is a heterodimer of 48 and 53 kDa bands. However, it is also likely that 100 kDa non-denaturing molecular mass results from homodimerization of the 48 kDa or the 53 kDa bands. In any case L2 has one band with the molecular mass which is close to the previously reported 46.5 kDa isoform of P. chrysosporium laccase [7]. Further research is necessary for characterizing four isoforms (L1–L4) of laccase produced under these conditions.

In a previous study on P. chrysosporium two laccase isoforms were observed by activity staining of non-denaturing gels [7]. However, in our study we observed four laccase peaks on a MonoQ column suggesting that there are four isoforms. This difference in the number of isoforms between studies could be due to the differences in methods, strains or growth conditions. Whether the multiple peaks observed on the MonoQ column represent separate isozymes or are allozymes remains to be determined. Similarly, exact polypeptide compositions of these isoforms needs to be investigated. Nevertheless, this study shows that P. chrysosporium can produce at least four laccase isoforms and that a considerable amount of laccase activity is associated with mycelia. It also shows that the induction/derepression of laccases is possible when high amounts of copper are added to the medium with glucose.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

This work was partially supported by sabbatical leaves granted to S.S. Dhawale and S.W. Dhawale by Indiana University Purdue University at Fort Wayne and Indiana University East respectively.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References
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