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A rapid method to quantify pro-oxidant activity in cultures of wood-decaying white-rot fungi
Journal of Microbiological Methods 61 (2005) 261 – 271 www.elsevier.com/locate/jmicmeth
A rapid method to quantify pro-oxidant activity in cultures of wood-decaying white-rot fungi A.N. Kapicha,b,c,*, B.A. Priorb, T. Lundella, A. Hatakkaa a
Department of Applied Chemistry and Microbiology, University of Helsinki, Viikinkaari 9, Viikki Biocenter 1, P.O.Box 56, 00014 Helsinki, Finland b Department of Microbiology, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa c Department of Immunology, International Sakharov Environmental University, 23 Dolgobrodskaya Str, 220009 Minsk, Belarus Received 5 July 2004; received in revised form 14 December 2004; accepted 14 December 2004 Available online 11 January 2005
Abstract A new, rapid method for evaluation of lipid peroxidation promoting (pro-oxidant) activity in cultures of wood-decaying fungi was developed. The method is based on measurement of the rate of oxygen consumption in the reaction of linoleic acid peroxidation initiated by fungal culture filtrates. The liquid cultures of the white-rot fungi Bjerkandera adusta and Phanerochaete chrysosporium grown on wheat straw-containing glucose–peptone–corn steep liquor medium possessed significant levels of the pro-oxidant activity. Other white-rot fungi producing manganese peroxidase (MnP) were also found to show the activity. MnP demonstrated a crucial role as the major pro-oxidant agent in the fungal cultures. The total pro-oxidant activity may be considered as net result of the peroxidation by MnP and the inhibition by antioxidant compounds present in the fungal culture fluids. D 2004 Elsevier B.V. All rights reserved. Keywords: Lipid peroxidation; Manganese peroxidase; Pro-oxidant activity; Wood-decaying fungi
1. Introduction White-rot basidiomycetous fungi are the only group of microorganisms that are able to degrade lignin efficiently via reactions initiated by oxidative * Corresponding author. Department of Immunology, International Sakharov Environmental University, 23 Dolgobrodskaya Str, 220009 Minsk, Belarus. Tel.: +375 17 2302678; fax: +375 17 2306897. E-mail address: [email protected] (A.N. Kapich). 0167-7012/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2004.12.010
ligninolytic enzymes (Hatakka, 2001). However, these enzymes are apparently too large to penetrate the secondary cell walls of sound wood, and therefore the fungi must employ low molecular weight, oxidative agents to promote lignin decay (Srebotnik et al., 1988; Hammel et al., 2002). Manganese peroxidase (MnP) is the most common extracellular ligninolytic enzyme produced by almost all white-rot fungi (Gold et al., 2000; Hatakka, 2001; Hofrichter, 2002). MnP may oxidize phenolic lignin substructures in wood via diffusible Mn3+–chelator complexes. However, non-
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phenolic units that are the most recalcitrant and abundant in natural lignin are not susceptible to oxidation and degradation by this mechanism. Recently, it has been proposed that MnP may initiate lipid peroxidation thereby generating lipid peroxyl radicals, which in turn can be the primary agents of fungal attack on non-phenolic lignin in wood (Kapich et al., 1999a). It has been shown that lipid peroxidation may be coupled to degradation and mineralization of lignin (Bao et al., 1994; Kapich et al., 1999b). Moreover, MnP-mediated lipid peroxidation may be involved in degradation of xenobiotic compounds and man-made recalcitrant materials (Moen and Hammel, 1994; Bogan et al., 1996; Sato et al., 2003). Thus, the evaluation of the ability of fungal cultures to promote lipid peroxidation is of great importance. Stimulation of peroxidation of unsaturated lipids with concomitant production of lipid radicals is recognized as prooxidant activity (Halliwell and Gutteridge, 1999; Schewe, 2002). The crucial reaction of lipid peroxidation proceeds with the consumption of oxygen and leads to generation of lipid peroxyl radicals (L! +O2 Y LOO!). Additional uptake of oxygen also occurs in subsequent reactions. Therefore, measurement of the rate of oxygen uptake can be an overall indicator of the intensity of lipid peroxidation (Halliwell and Gutteridge, 1999). This approach to measure lipid peroxidation has been applied to reactions involving purified enzyme preparations (Kapich et al., 1999b; Watanabe et al., 2000). Whether such an approach may be applied to measure the pro-oxidant activity in fungal culture fluids where the actual MnP activity may be much lower and antioxidant compounds may also be present was unclear. Moreover, it had not been verified whether only MnP is responsible for the initiation of lipid peroxidation in fungal cultures. It is possible that other oxidative enzymes, like lignin peroxidase (LiP) and laccase, or even non-enzymatic agents are involved in the peroxidation and O2 consuming reactions. The aim of this study was to develop a rapid method for determination of the pro-oxidant activity in liquid cultures of white-rot fungi. The role of the ligninolytic peroxidases (MnP and LiP) as the most probable catalysts of the pro-oxidant activity in fungal cultures was also investigated using this method.
2. Materials and methods 2.1. Fungal strains and chemicals The strains Ceriporiopsis subvermispora FP 90031SP and Phanerochaete chrysosporium ME-446 were obtained from USDA Forest Products Laboratory (Madison, Wisconsin, USA). Bjerkandera adusta SCC 0169, Pycnoporus coccineus SCC 0041, and Pycnoporus sanguineus SCC 0294 were obtained from the SAPPI Culture Collection (University of the Free State, Bloemfontein, South Africa). Fomes fomentarius A71, Panus tigrinus A26, and Phlebia radiata 79 (ATCC 64658) were from the Collection of Wooddecaying Fungi at the Division of Microbiology, Department of Applied Chemistry and Microbiology (University of Helsinki, Finland). Coriolus versicolor K12-1-1, Daedalea quercina K8-3, and P. sanguineus K5-2-3 were isolated from fruiting bodies of the fungi collected near Stellenbosch (South Africa). The strains were subcultured on malt extract agar and maintained at 4 8C. Corn steep liquor was obtained from African Products, Bellville, South Africa and from SigmaAldrich Co. Linoleic acid, veratryl (3,4-dimethoxybenzyl) alcohol, and 2,6-dimethoxyphenol were purchased from Sigma-Aldrich Co. All other chemicals used were of analytical grade. Buffers and solutions were made in Milli-Q ultrapure water. 2.2. Culture conditions The wheat straw-containing glucose–peptone–corn steep liquor (GPCSL) medium previously developed for production of ligninolytic peroxidases by P. chrysosporium ME-446 was used (Kapich et al., 2004). Two batches of wheat straw received from distinct geographical regions (South Africa and Spain, Europe) were applied. The straw was milled and sieved to a maximum particle size of 10 mm before addition to the GPCSL medium. The medium contained (g l 1): wheat straw, 20; glucose, 10; BactokPeptone (Difco), 3; corn steep liquor, 1; KH2PO4, 1; MgSO4d 7H2O, 0.5; initial pH 4.8. Fungal inoculum was prepared as described (Kapich et al., 2004). All cultivations were carried out in 250-ml Erlenmeyer flasks containing 50 ml of the wheat straw-GPCSL medium with agitation (120–140 rpm) on a rotary shaker at 288C except for the strains of P. chrysosporium, P. coccineus, and P.
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sanguineus that were cultivated at 37 8C. Culture fluids were filtered through Miracloth and centrifuged (13,400 g; 6 min, 4 8C) to remove all solid particles and fragments of the mycelium. 2.3. Oxygen consumption experiments Pro-oxidant activity was measured at 25 8C as the maximal rate of oxygen consumption during linoleic acid peroxidation promoted by the fungal culture fluids or ligninolytic enzymes. The rates of oxygen consumption were taken from the linear portions of Oxygraph recordings (Hansatech Instruments Oxygraph, UK) and were calculated using the software of the manufacturer. The procedure included consecutive addition of the components to the reaction chamber containing the oxygen electrode. First, ultrapure water or appropriate buffer solution was placed into the reaction chamber and saturated with dissolved oxygen by intensive stirring. The chamber was closed with a tight-fitting plunger and further additions were made via a small capillary hole in the plunger with a Hamilton syringe. In the optimized method, the components were added in the following order: 620 Al of distilled water, 250 Al of 200 mM sodium acetate buffer (pH 4.5), 10 Al of a freshly prepared 100 mM emulsion of linoleic acid in 10% (wt./vol.) Tween 20, 10 Al of 100 mM MnSO4, 100 Al of fungal culture fluid or 0.015 U of purified enzyme (MnP or LiP), and 10 Al of 10 mM H2O2 (freshly prepared) to give a final reaction volume of 1 ml. For reaction controls, the compounds were stepwise omitted or the culture fluid samples were heat-treated in boiling water bath for 10 min before addition to the reaction mixtures. Every oxygen consumption experiment was repeated at least three times with basically identical outcomes. Occasionally when there was a defect with an electrode, the data from the treatment were discarded and the experiment was repeated. The pro-oxidant activity was expressed as units per milliliter of the culture fluid (U ml 1), where 1 U equals 1 Amol of O2 consumed per minute in the reaction of linoleic acid peroxidation. 2.4. Determination of the thiobarbituric acid (TBA)reactive substances The reactions of linoleic acid peroxidation catalyzed by the culture fluids were first carried out in
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loosely capped sterile 20-ml vials and then assayed for the presence of TBA-reactive products. The complete reaction mixture contained 300 Al of the culture fluid obtained after cultivation of P. chrysosporium ME-446 in the GPCSL medium with South African wheat straw (MnP activity—0.16 U ml 1; LiP activity—0.36 U ml 1), and the following filtersterilized compounds: 50 mM sodium acetate buffer (pH 4.5), 1 mM MnSO4, 1 mM linoleic acid, 0.1% (wt/vol) Tween 20, and 0.1 mM H2O2 in a total volume of 3 ml. Reaction mixtures without linoleic acid, without Mn2+, and without or with temperature inactivated culture fluid were used as controls. The vials were incubated on a rotary shaker at 37 8C in the dark for 1 h. Products of linoleic acid peroxidation were assayed in the reaction mixtures by the TBA method as described previously (Kapich et al., 1999b). 2.5. Enzyme assays All enzymatic activities were determined spectrophotometrically (DU 640 Beckman Coulter, USA, or Shimadzu PharmaSpec UV-1700, Japan) at 25 8C. MnP activity in culture fluids was determined by following the formation of coerulignone (3,3V,5,5Vtetramethoxydiphenoquinone) from 2,6-dimethoxyphenol (DMP) at 469 nm; (molar extinction coefficient=49,600 M 1 cm 1) (Wariishi et al., 1992). The reaction mixture (1 ml) contained 50 mM sodium tartrate (pH 4.5), 1 mM MnSO4, 0.5 mM DMP, and 100 Al cultural fluid. The reaction was initiated by adding 0.1 mM hydrogen peroxide and the resulting MnP activity observed was corrected by subtracting the laccase activity. Laccase activity was determined in a similar manner except that MnSO4 and hydrogen peroxide were excluded from the reaction mixture. MnP activity in the protein fractions separated by anion-exchange chromatography was also estimated by measuring formation of Mn3+– malonate complex at 270 nm (molar extinction coefficient=11,590 M 1 cm 1) (Wariishi et al., 1992). LiP activity was measured by monitoring oxidation of veratryl alcohol to veratraldehyde at 310 nm (molar extinction coefficient=9,300 M 1 cm 1) (Tien and Kirk, 1988). For all the enzyme activities, 1 unit (U) represents 1 Amol of product formed within 1 min of reaction time.
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2.6. Purification and characterization of the ligninolytic peroxidases The ligninolytic peroxidases MnP and LiP were separated by Mono-Q anion-exchange chromatography from 72-h cultures of P. chrysosporium ME-446 grown on wheat straw GPCSL medium (Kapich et al., 2004). Fractions corresponding to separate LiP and MnP isoenzymes were pooled, concentrated, and diafiltrated (10 kDa cut-off, Filtron Microsep) with ultrapure water. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) was performed as described (Laemmli, 1970) using low range molecular weight markers (Amersham Pharmacia Biotech). Isoelectric focusing (IEF) was performed using the Multiphore II electrophoresis system and 0.5 mm manually cast ultrathin polyacrylamide gels with pH range pH 2.5–5 generated by carrier ampholytes according to the manufacturer’s instructions (Amersham Pharmacia Biotech). The pH range was determined using a low pI calibration standard (Amersham Pharmacia Biotech). Protein bands in all PAGE gels were visualized by Coomassie Brilliant Blue stain. 2.7. Analysis of phenolic compounds Total content of phenolic compounds in the wheat straw-containing GPCSL medium was determined by a standard method with 4-aminoantipyrine (APHA, 1995).
3. Results 3.1. Lipid peroxidation initiated by culture fluids of P. chrysosporium ME-446 Aliquots of culture fluids of P. chrysosporium ME446 cultivated on the wheat straw-containing GPCSL medium were used to develop the method for determination of the pro-oxidant activity. Time courses of typical reactions of linoleic acid peroxidation obtained with two different culture fluid samples are shown in Fig. 1. Addition of the linoleic acid emulsion, Mn2+ ions, and culture fluid into the O2 saturated acetate-buffered solution caused no change to the background drift (0.3–0.6 nmol ml 1 min 1) of the oxygen electrode and the O2 consuming
Fig. 1. Consumption of O2 in the reaction of linoleic acid peroxidation obtained with two different culture fluids of P. chrysosporium ME-446. (Curve 1) Reaction with culture fluid sample (1) obtained after cultivation of the fungus in the GPCSL medium with Spanish wheat straw (MnP activity—0.30 U ml 1; LiP activity—0.01 U ml 1); (curve 2) reaction with culture fluid sample (2) obtained after cultivation of the fungus in the GPCSL medium with South African wheat straw (MnP activity—0.16 U ml 1; LiP activity—0.36 U ml 1); (curve 3) reaction with thermally inactivated culture fluid sample (2). Reaction mixtures contained 50 mM sodium acetate buffer (pH 4,5); 1 mM linoleic acid, 0.1% Tween 20; 1 mM MnSO4; 100 Al culture fluid; 0.1 mM H2O2. Arrows indicate the addition of the components to the reaction mixture: C18:2—emulsion of linoleic acid in Tween 20; Mn2+— solution of MnSO4, Cult. fluid—culture fluid sample, H2O2— solution of H2O2.
reaction started only after addition of 0.1 mM H2O2. Two major reaction patterns were observed with culture fluid samples. In the first cases, with culture fluids obtained after cultivation of the fungus in the GPCSL medium with Spanish wheat straw and containing high MnP activity but only traces of LiP activity, a short period of slow increase in the concentration of dissolved O2 was observed and thereafter, slow consumption of O2 commenced (Fig. 1, curve 1). This medium contained a relatively high level of phenolic compounds (19.8F1.2 mg L 1). A second reaction pattern showed no initial generation of O2 and fast oxygen uptake began immediately or shortly after addition of H2O2 (Fig. 1, curve 2). This pattern was prevalent with fungal culture fluids containing both MnP and LiP activities, which were obtained after cultivation in the medium with South African wheat straw. This medium contained a lower amount of phenolic compounds
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(15.4F0.7 mg L 1). With both reaction patterns, the rate of O2 consumption followed first-order saturation kinetics. If linoleic acid or culture fluid was omitted from the reaction mixture, the dissolved O2 concentration remained constant. Moreover, in the absence of linoleic acid some evolution of O2 was observed (data not shown). Heat-treated culture fluid samples lost their ability to initiate peroxidation of linoleic acid with O2 consumption (Fig. 1, curve 3). 3.2. Effect of organic acid buffer solutions Acetate buffer (pH 4.5) supported the highest rate of O2 consumption during peroxidation of linoleic acid catalyzed by the culture fluid, whereas malonate and tartrate both suppressed the reaction considerably (Fig. 2). Even ultrapure water supported the reaction more effectively than malonate or tartrate corroborating the conclusion that these chelating organic acids inhibited the lipid peroxidation reactions. In acetate buffer, peroxidation of linoleic acid strongly depended on the pH value in the reaction mixture and the highest rate of O2 consumption occurred at pH between 4.0 and 4.5 (Fig. 3).
Fig. 2. Influence of organic acids on the O2 consumption in the reaction of linoleic acid peroxidation initiated by culture fluid sample (2) of P. chrysosporium ME-446 (MnP activity—0.16 U ml 1; LiP activity—0.36 U ml 1). Reaction mixtures contained ultrapure water or sodium acetate, malonate or tartrate buffer solutions (50 mM each, pH 4.5) and all the other components described for Fig. 1. The arrow indicates the addition of 0.1 mM H2O2.
Fig. 3. Influence of pH on the maximal rate of O2 consumption in the reaction of linoleic acid peroxidation initiated by culture fluid sample (2) of P. chrysosporium ME-446 (MnP activity—0.16 U ml 1; LiP activity—0.36 U ml 1). The reaction mixtures in 50 mM sodium acetate, adjusted to the exact pH values contained the same components as described for Fig. 1.
3.3. Effect of Mn2+ and H2O2 Mn2+ ions were essential for the peroxidation of linoleic acid promoted by culture fluids as seen as O2 consumption in the reactions. Without Mn2+, an extended period (about 10 min) of increase in the concentration of dissolved O2 was observed after addition of hydrogen peroxide (Fig. 4A). When small amounts of Mn2+ ions (0.01–0.05 mM) were introduced into the reaction mixtures, O2 consumption increased accordingly. However, complete depletion of O2 was reached with 0.1 mM or higher concentrations of Mn2+ ions. With 0.5 mM Mn2+, consumption of O2 attained the highest rate, about 90 nmol ml 1 min 1, which was not exceeded by higher concentrations of Mn2+ ions (data not shown). In general, no peroxidation of linoleic acid occurred without the addition of H2O2 when samples of culture fluids of P. chrysosporium ME446 were applied as the pro-oxidant agents. Even with small concentrations of H2O2 (0.01 mM), the reaction started immediately albeit at a very slow rate of O2 consumption (6.4 nmol ml 1 min 1)
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3.4. Accumulation of TBA-reactive substances during peroxidation of linoleic acid To confirm that the consumption of O2 was related to peroxidation of linoleic acid, we determined accumulation of TBA-reactive substances in the reaction mixtures. Peroxidation of linoleic acid as catalyzed by culture fluids in the complete system resulted in the accumulation of TBA-reactive substances (0.073 mM) within 1 h. In the cases when linoleic acid, Mn2+ ions, or culture fluid was omitted or the culture fluid sample was inactivated by heat treatment, the accumulation of TBA-reactive substances was negligible (0.001 mM or less). 3.5. Time course of pro-oxidant, MnP, and LiP activities in submerged culture of P. chrysosporium ME-446
Fig. 4. Influence of Mn2+ (A) and H2O2 (B) on the consumption of O2 in the reaction of linoleic acid peroxidation initiated by the culture fluid sample (2) of P. chrysosporium ME-446 (MnP activity—0.16 U ml 1; LiP activity—0.36 U ml 1). Reaction mixtures contained all the components as described for Fig. 1 with specific concentrations of MnSO4 or H2O2. The arrow indicates the addition of H2O2.
(Fig. 4B). When 0.05 mM H2O2 was added, the consumption of O2 proceeded until exhaustion. The highest rate of oxygen consumption was attained with 0.1 mM H2O2 whereas higher concentrations of H2O2 apparently inhibited the reaction. Moreover, with 0.3 mM of H2O2 initial evolution of O2 was observed that only later turned to oxygen consumption.
The investigation of the influence of different factors on the peroxidation of linoleic acid catalyzed by culture fluids yielded a new method for evaluation of the pro-oxidant activity in cultures of whiterot fungi (see Materials and methods). Using this method we investigated the relationship between the pro-oxidant activity and MnP/LiP activities in submerged culture of P. chrysosporium ME-446 grown in the GPCSL medium supplemented with Spanish wheat straw. MnP activity appeared in culture fluids after 60 h of cultivation and quickly reached a maximum value at 66 h (Fig. 5). However, when MnP activity appeared in the cultures, only very low level of pro-oxidant activity and no LiP activity was detected. Subsequently, concomitant increase of both the pro-oxidant and LiP activities occurred. After 68 h of cultivation, MnP activity began to decrease while the pro-oxidant and LiP activities increased to a maximal level 6 h after the peak of MnP activity was attained. 3.6. Pro-oxidant activities of purified MnP and LiP The possibility that the ligninolytic peroxidases may be major pro-oxidant agents in the fungal culture fluids was investigated using purified MnP P5 and LiP P4 isoenzymes of P. chrysosporium ME-446 (Kapich et al., 2004). The molecular weights of the enzymes determined by SDS–PAGE were 45.5 and 40
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3.7. Pro-oxidant activities in cultures of various wood-decaying fungi Only cultures of white-rot fungi producing MnP showed the pro-oxidant activity (Table 1). However, no direct correlation between the level of MnP and the level of pro-oxidant activity was observed in culture fluid samples. The highest pro-oxidant activity was found in culture fluids of B. adusta SCC 0169,
.
Fig. 5. Time course of MnP ( ), LiP (n), and pro-oxidant (PO, E) activities in the submerged culture of P. chrysosporium ME-446 grown in the GPCSL medium supplemented with Spanish wheat straw. Bars on symbols indicate the standard deviation of means (n=3); where absent, bars fall within symbols. For more details, see Materials and methods.
kDa for MnP P5 and LiP P4, respectively. Both proteins were acidic with values of pI 4.2 for MnP P5 and 3.9 for LiP P4. A possible role of ligninolytic peroxidases in pro-oxidant activity is supported by our observation that the culture fluid filtrates obtained upon ultrafiltration and containing low molecular weight compounds (b10 kDa) showed no pro-oxidant activity. In the O2 consumption assay with linoleic acid, the MnP P5 enzyme showed significant pro-oxidant activity (Fig. 6A). Addition of 0.015 U of the MnP P5 in the acetate-buffered reaction mixture caused immediate O2 uptake even without addition of H2O2. A similar curve was obtained when ultrapure water was used instead of acetate whereas malonate and tartrate as chelating organic acids completely suppressed the ability of MnP P5 enzyme to develop peroxidation of linoleic acid in the absence of hydrogen peroxide, which is seen as O2 consumption in the reaction. When 0.1 mM H2O2 was added into the reaction mixtures containing malonate or tartarate, only low level of O2 consumption was observed (Fig. 6B). Uptake of O2 was not observed when linoleic acid or Mn2+ ions were omitted from the reactions or when the MnP P5 enzyme was thermally inactivated. The LiP P4 enzyme failed to show any pro-oxidant activity under conditions similar to those assessed for MnP P5 (data not shown).
Fig. 6. Influence of organic acids on the consumption of O2 in the reaction of linoleic acid peroxidation catalyzed by MnP P5 enzyme fraction, purified from submerged cultures of P. chrysosporium ME446, in the absence (A) and in the presence (B) of 0.1 mM H2O2. Reaction mixtures contained ultrapure water or sodium acetate, malonate, or tartrate buffer solutions (50 mM each, pH 4.5) and all the other components as described for Fig. 1 except that 0.015 U of MnP P5 was used instead of the culture fluid sample. The arrow indicates addition of the enzyme.
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Table 1 Pro-oxidant activity and activities of ligninolytic enzymes (meansFstandard deviation, n=3) in submerged cultures of wood-decaying fungi grown in wheat straw-containing GPCSL medium Fungus species and straina Bjerkandera adusta SCC 0169 Ceriporiopsis subvermispora FP90031SP Coriolus versicolor K 12-1-1 Daedalea quercina K8-3 Fomes fomentarius A71 Panus tigrinus A26 Phanerochaete chrysosporium ME-446 Phlebia radiata 79 Pycnoporus coccineus SCC 0041 Pycnoporus sanguineus K5-2-3 Pycnoporus sanguineus SCC 0294 a b c d
Cultivation time, days
Pro-oxidant activity, U ml
6 4 4 8 5 10 3 10 10 7 10
2.07F0.08 0.40F0.01 0.15F0.01 ND ND 0.05F0.01 1.48F0.04 0.05F0.01 ND ND ND
1
MnP activity, U ml 1
LiP activity, U ml 1
Laccase activity, U ml 1
0.17F0.02 0.10F0.02 0.02d ND ND 0.03d 0.43F0.02 0.05F0.01 ND ND ND
NDb ND ND ND ND ND 0.23F0.02 ND ND ND ND
TRc 0.05F0.01 0.07F0.01 ND 0.22F0.01 0.11F0.01 TR 0.01d 0.13F0.01 0.25F0.01 0.15F0.01
For strain origin, see Materials and methods. Not detected. Trace, activity lower than 0.01 U ml 1. Standard deviation of means less than 0.01.
although the highest MnP was established in culture fluids of P. chrysosporium ME-446. Only the latter fungus was able to produce LiP in the wheat strawcontaining GPCSL medium. The strains of white-rot fungi F. fomentarius A71, P. coccineus SCC 0041, P. sanguineus K5-2-3, and P. sanguineus SCC 0294 were able to actively produce laccase but did not show any MnP or pro-oxidant activity. The brown rot fungus D. quercina that was used as a control in this study showed no production of the ligninolytic enzymes or the pro-oxidant activity as well.
4. Discussion This is the first attempt to estimate the pro-oxidant (lipid peroxidation promoting) activity in cultures of wood-decaying fungi. Despite the availability of various methods to determine the antioxidant activity (Kapich and Shishkina, 1992; Kapich, 1995; Mau et al., 2002), it is surprising that no methods have been described to detect the pro-oxidant activity in fungal cultures. The reactions of lipid peroxidation possibly play an important role in the physiology and secondary metabolism of wood-decaying fungi, in particular for the degradation of non-phenolic lignin and xenobiotics. Therefore, the evaluation of the ability of fungal cultures to promote lipid peroxidation is of great importance.
We describe here a new simple method to quantify the pro-oxidant activity. With this method we show that lipid peroxidation promoting activity is found in the culture fluids of the white-rot fungi when grown in the GPCSL medium with wheat straw. The peroxidative mechanism of the reaction was evident from the rapid consumption of dissolved O2 in the course of the reactions with concomitant accumulation of TBA-reactive substances as products. Ultrafiltrates of the culture fluids containing only low molecular weight compounds failed to initiate lipid peroxidation indicating that high molecular weight substances are responsible for the prooxidant activity of the culture fluids. The enzymatic nature of the pro-oxidant agent is confirmed by our observation that heat-treatment of the culture fluid samples completely destroyed their ability to initiate lipid peroxidation. Lipoxygenases are the most commonly recognized enzymes that effectively oxidize unsaturated fatty acids like linoleic acid with concomitant uptake of O2 (Grechkin, 1998; Ku¨hn and Borchert, 2002; Schewe, 2002). However, it is unlikely that fungal lipoxygenases were the pro-oxidant agents in this case due to their intracellular location (Husson et al., 1998; Feussner and Wasternack, 2002). In addition, the conditions that favored extracellular lipoxygenase-like activity of P. chrysosporium ME-446 in our study differed considerably from the optimal
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conditions supporting lipoxygenase activity (Bisakowski and Kermasha, 1998; Kuribayashi et al., 2002). The requirement of Mn2+ ions and H2O2 for the O2 consumption reactions clearly indicated that MnP produced in the fungal cultures may play a pivotal role in the process of linoleic acid peroxidation. Optimum pH for the reactions was also close to the optimum pH of MnP activity (Glenn et al., 1986). However, the chelating organic acids, tartrate and malonate, which have been shown to be important for MnP catalysis (Wariishi et al., 1992), showed only 1/10 of the efficiency in comparison to acetate in these linoleic acid peroxidation reactions initiated by culture fluid (Fig. 2). Moreover, even distilled water supported higher rate of linoleic acid peroxidation than malonate or tartrate. This result clearly indicates that the chelating organic acids inhibited the lipid peroxidation reaction, which is unusual for reactions catalyzed by MnP. Investigation of the relationship between MnP, LiP, and pro-oxidant activities in culture fluids of P. chrysosporium ME-446 revealed their interconnection. Surprisingly, the pro-oxidant activity was related more closely to the level of LiP than MnP activity in the fungal cultures. Indeed, when MnP activity already appeared in the culture fluid, only traces of pro-oxidant activity could be detected and in fact, the peak of pro-oxidant activity coincided with production of LiP rather than MnP (Fig. 5). However, with purified ligninolytic enzymes, only MnP showed strong pro-oxidant activity whereas LiP failed to cause peroxidation of linoleic acid with consumption of oxygen. Mn2+ ions were absolutely necessary for the prooxidant activity of purified MnP. This is typical for MnPs from P. chrysosporium since their catalysis depends on the presence of Mn2+ (Gold et al., 2000). However, the oxidation of linoleic acid by MnP had other unusual features. Hydrogen peroxide was not needed for the reaction in acetate buffered solutions or ultrapure water. Surprisingly, chelating organic acids malonate and tartrate completely suppressed the MnP-catalyzed peroxidation of linoleic acid in the absence of hydrogen peroxide (Fig. 6A). Addition of H2O2 slightly stimulated the prooxidant activity of MnP only in the presence of malonate or tartrate (Fig. 6B). These data point to
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the optimal conditions for evaluation of the prooxidant activity in solutions of purified MnP and in the complex culture fluids of wood-decaying fungi being different. Our results indicate that MnP was a crucial agent that provided pro-oxidant properties to culture fluids of P. chrysosporium ME-446 and other wood-decaying fungi due to its lipoxygenase-like activity. The comparative investigation of pro-oxidant activity and activities of ligninolytic enzymes in submerged cultures of selected wood-decaying fungi unambiguously demonstrated that only culture fluids containing MnP showed the pro-oxidant activity. The laccaseproducing cultures that did not produce MnP were unable to show any pro-oxidant activities. However, as it has been shown earlier under chelator-limited conditions, MnP also exhibits significant catalase-like activity producing O2 from H2O2 (Timofeevski and Aust, 1997). Indeed we could monitor evolution of O2, most probably due to this MnP-associated, catalase-like activity in the beginning of some reactions (see for example Fig. 1, curve 1). The catalase-like activity seen as generation of O2 increased with the increase of H2O2 concentration in the reaction mixture. It seems that O2 producing catalase-like reactions could proceed simultaneously with O2 consuming lipoxygenase-like reactions in the reaction mixtures containing MnP, Mn2+ ions, linoleic acid, and H2O2 in the presence of low-affinity ligands such as acetate. In addition, the pro-oxidant activity of the fungal cultures apparently depended not only on the presence of MnP and some other compounds in the culture fluids also influenced the pro-oxidant activity. Many aromatic and in particular, phenolic compounds are known to possess strong antioxidant properties inhibiting lipid peroxidation (Halliwell and Gutteridge, 1999). These compounds were released from wheat straw into the culture medium and may have inhibited the MnP-catalyzed lipid peroxidation. It is noteworthy that wood-decaying and especially whiterot fungi are also able to produce antioxidant compounds (Kapich and Shishkina, 1992; Kapich, 1995). The trace levels of pro-oxidant activity in culture fluids with high MnP activity could be due to these straw-derived and fungal compounds (Fig 5). Thus, the total pro-oxidant activity may be considered as net result of the action of pro-oxidant factors and
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antioxidant compounds present in the fungal culture fluids. A simple assay described here provides an excellent method to quantify the pro-oxidant activity in cultures of wood-decaying fungi. The method is rapid and easy to use and does not need complex sample processing or time-consuming analysis of the products of lipid peroxidation. It also can serve as an additional indication of the presence of MnP in cultures of wood-decaying fungi.
Acknowledgements Financial support from the Academy of Finland, as particular funding for the Center of Excellence on Microbial Resources (project number 53305), from the University of Stellenbosch and the National Research Foundation of South Africa are gratefully acknowledged.
References APHA, 1995. Standard Methods for the Examination of Water and Wastewater. American Public Health Association, Washington, DC, USA. Bao, W., Fukushima, Y., Jensen, K.A., Moen, M.A., Hammel, K.E., 1994. Oxidative degradation of non-phenolic lignin during lipid peroxidation by fungal manganese peroxidase. FEBS Lett. 354, 297 – 300. Bisakowski, B., Kermasha, S., 1998. Characterization of purified lipoxygenase extracts from Fusarium proliferatum. J. Agric. Food Chem. 46, 2382 – 2388. Bogan, B.W., Lamar, R.T., Hammel, K.E., 1996. Fluorene oxidation in vivo by Phanerochaete chrysosporium and in vitro during manganese peroxidase-dependent lipid peroxidation. Appl. Environ. Microbiol. 62, 1788 – 1792. Feussner, I., Wasternack, C., 2002. The lipoxygenase pathway. Annu. Rev. Plant Biol. 53, 275 – 297. Glenn, J.K., Akileswaran, L., Gold, M.H., 1986. Mn(II) oxidation is the principal function of the extracellular Mn-peroxidase from Phanerochaete chrysosporium. Arch. Biochem. Biophys. 251, 688 – 696. Gold, M.H., Youngs, H.L., Sollewijin Gelpke, M.D., 2000. Manganese peroxidase. In: Sigel, A., Sigel, H. (Eds.), Metal ions in biological systems, vol. 37. Marcel Dekker, New YorkBasel, pp. 559 – 586. Grechkin, A., 1998. Recent developments in biochemistry of the plant lipoxygenase pathway. Prog. Lipid Res. 37, 317 – 352. Halliwell, B., Gutteridge, J.M.C., 1999. Free Radicals in Biology and Medicine. University Press, Oxford.
Hammel, K.E., Kapich, A.N., Jensen, K.A., Ryan, Z.C., 2002. Reactive oxygen species as agents of wood decay by fungi. Enzyme Microb. Technol. 30, 445 – 453. Hatakka, A., 2001. Biodegradation of lignin. In: Hofrichter, M., Steinbqchel, A. (Eds.), Biopolymers. Biology, Chemistry, Biotechnology, Applications, Vol. 1. Lignin, Humic Substances and Coal. Wiley-VCH, Weinheim, Germany, pp. 129 – 180. Hofrichter, M., 2002. Review: lignin conversion by manganese peroxidase (MnP). Enzyme Microb. Technol. 30, 454 – 466. Husson, F., Pagot, Y., Kermasha, S., Belin, J.M., 1998. Fusarium proliferatum: induction and intracellular location of a lipoxygenase. Enzyme Microb. Technol. 23, 42 – 48. Kapich, A.N., 1995. Antioxidant activity of mycelium extracts of xylotrophic Basidiomycetes. Mikol. Fitopatol. 29, 35 – 40 (in Russian). Kapich, A.N., Shishkina, L.N., 1992. Antioxidant properties of wood-decaying Basidiomycetes. Mikol. Fitopatol. 26, 486 – 492 (in Russian). Kapich, A.N., Jensen, K.A., Hammel, K.E., 1999a. Peroxyl radicals are potential agents of lignin biodegradation. FEBS Lett. 461, 115 – 119. Kapich, A., Hofrichter, M., Vares, T., Hatakka, A., 1999b. Coupling of manganese peroxidase mediated lipid peroxidation with destruction of nonphenolic lignin model compounds and 14C-labeled lignins. Biochem. Biophys. Res. Commun. 259, 212 – 219. Kapich, A.N., Prior, B.A., Botha, A., Galkin, S., Lundell, T., Hatakka, A., 2004. Effect of lignocellulose-containing substrates on production of ligninolytic peroxidases in submerged cultures of Phanerochaete chrysosporium ME-446. Enzyme Microb. Technol. 34, 187 – 195. Kqhn, H., Borchert, A., 2002. Regulation of enzymatic lipid peroxidation: the interplay of peroxidizing and peroxide reducing enzymes. Free Radic. Biol. Med. 33, 154 – 172. Kuribayashi, T., Kaise, H., Uno, C., Hara, T., Hayakawa, T., Joh, T., 2002. Purification and characterization of lipoxygenase from Pleurotus ostreatus. J. Agric. Food Chem. 50, 1247 – 1253. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 – 685. Mau, J.-L., Lin, H.-C., Chen, C.-C., 2002. Antioxidant properties of several medicinal mushrooms. J. Agric. Food Chem. 50, 6072 – 6077. Moen, M.A., Hammel, K.E., 1994. Lipid peroxidation by the manganese peroxidase of Phanerochaete chrysosporium is the basis for phenanthrene oxidation by the intact fungus. Appl. Environ. Microbiol. 60, 1956 – 1961. Sato, S., Honda, Y., Kuwahara, M., Watanabe, T., 2003. Degradation of vulcanized and nonvulcanized polyisoprene rubbers by lipid peroxidation catalyzed by oxidative enzymes and transition metals. Biomacromolecules 4, 321 – 329. Schewe, T., 2002. 15-lipoxygenase-1: a prooxidant enzyme. Biol. Chem. 383, 365 – 374. Srebotnik, E., Messner, K., Foisner, R., 1988. Penetrability of white-rot degraded pine wood by the lignin peroxidase of Phanerochaete chrysosporium. Appl. Environ. Microbiol. 54, 2608 – 2614.
A.N. Kapich et al. / Journal of Microbiological Methods 61 (2005) 261–271 Tien, M., Kirk, T.K., 1988. Lignin peroxidase of Phanerochaete chrysosporium. Methods Enzymol. 161, 238 – 249. Timofeevski, S.L., Aust, S.D., 1997. Effects of Mn2+ and oxalate on the catalatic activity of manganese peroxidase. Biochem. Biophys. Res. Commun. 239, 645 – 649. Wariishi, H., Valli, K., Gold, M.H., 1992. Manganese (II) oxidation by manganese peroxidase from the basidiomycete Phanero-
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chaete chrysosporium. Kinetic mechanism and role of chelators. J. Biol. Chem. 267, 23688 – 23695. Watanabe, T., Katayama, S., Enoki, M., Honda, Y., Kuwahara, M., 2000. Formation of acyl radical in lipid peroxidation of linoleic acid by manganese-dependent peroxidase from Ceriporiopsis subvermispora and Bjerkandera adusta. Eur. J. Biochem. 267, 4222 – 4231.
A rapid method to quantify pro-oxidant activity in cultures of wood-decaying white-rot fungi A.N. Kapicha,b,c,*, B.A. Priorb, T. Lundella, A. Hatakkaa a
Department of Applied Chemistry and Microbiology, University of Helsinki, Viikinkaari 9, Viikki Biocenter 1, P.O.Box 56, 00014 Helsinki, Finland b Department of Microbiology, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa c Department of Immunology, International Sakharov Environmental University, 23 Dolgobrodskaya Str, 220009 Minsk, Belarus Received 5 July 2004; received in revised form 14 December 2004; accepted 14 December 2004 Available online 11 January 2005
Abstract A new, rapid method for evaluation of lipid peroxidation promoting (pro-oxidant) activity in cultures of wood-decaying fungi was developed. The method is based on measurement of the rate of oxygen consumption in the reaction of linoleic acid peroxidation initiated by fungal culture filtrates. The liquid cultures of the white-rot fungi Bjerkandera adusta and Phanerochaete chrysosporium grown on wheat straw-containing glucose–peptone–corn steep liquor medium possessed significant levels of the pro-oxidant activity. Other white-rot fungi producing manganese peroxidase (MnP) were also found to show the activity. MnP demonstrated a crucial role as the major pro-oxidant agent in the fungal cultures. The total pro-oxidant activity may be considered as net result of the peroxidation by MnP and the inhibition by antioxidant compounds present in the fungal culture fluids. D 2004 Elsevier B.V. All rights reserved. Keywords: Lipid peroxidation; Manganese peroxidase; Pro-oxidant activity; Wood-decaying fungi
1. Introduction White-rot basidiomycetous fungi are the only group of microorganisms that are able to degrade lignin efficiently via reactions initiated by oxidative * Corresponding author. Department of Immunology, International Sakharov Environmental University, 23 Dolgobrodskaya Str, 220009 Minsk, Belarus. Tel.: +375 17 2302678; fax: +375 17 2306897. E-mail address: [email protected] (A.N. Kapich). 0167-7012/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2004.12.010
ligninolytic enzymes (Hatakka, 2001). However, these enzymes are apparently too large to penetrate the secondary cell walls of sound wood, and therefore the fungi must employ low molecular weight, oxidative agents to promote lignin decay (Srebotnik et al., 1988; Hammel et al., 2002). Manganese peroxidase (MnP) is the most common extracellular ligninolytic enzyme produced by almost all white-rot fungi (Gold et al., 2000; Hatakka, 2001; Hofrichter, 2002). MnP may oxidize phenolic lignin substructures in wood via diffusible Mn3+–chelator complexes. However, non-
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phenolic units that are the most recalcitrant and abundant in natural lignin are not susceptible to oxidation and degradation by this mechanism. Recently, it has been proposed that MnP may initiate lipid peroxidation thereby generating lipid peroxyl radicals, which in turn can be the primary agents of fungal attack on non-phenolic lignin in wood (Kapich et al., 1999a). It has been shown that lipid peroxidation may be coupled to degradation and mineralization of lignin (Bao et al., 1994; Kapich et al., 1999b). Moreover, MnP-mediated lipid peroxidation may be involved in degradation of xenobiotic compounds and man-made recalcitrant materials (Moen and Hammel, 1994; Bogan et al., 1996; Sato et al., 2003). Thus, the evaluation of the ability of fungal cultures to promote lipid peroxidation is of great importance. Stimulation of peroxidation of unsaturated lipids with concomitant production of lipid radicals is recognized as prooxidant activity (Halliwell and Gutteridge, 1999; Schewe, 2002). The crucial reaction of lipid peroxidation proceeds with the consumption of oxygen and leads to generation of lipid peroxyl radicals (L! +O2 Y LOO!). Additional uptake of oxygen also occurs in subsequent reactions. Therefore, measurement of the rate of oxygen uptake can be an overall indicator of the intensity of lipid peroxidation (Halliwell and Gutteridge, 1999). This approach to measure lipid peroxidation has been applied to reactions involving purified enzyme preparations (Kapich et al., 1999b; Watanabe et al., 2000). Whether such an approach may be applied to measure the pro-oxidant activity in fungal culture fluids where the actual MnP activity may be much lower and antioxidant compounds may also be present was unclear. Moreover, it had not been verified whether only MnP is responsible for the initiation of lipid peroxidation in fungal cultures. It is possible that other oxidative enzymes, like lignin peroxidase (LiP) and laccase, or even non-enzymatic agents are involved in the peroxidation and O2 consuming reactions. The aim of this study was to develop a rapid method for determination of the pro-oxidant activity in liquid cultures of white-rot fungi. The role of the ligninolytic peroxidases (MnP and LiP) as the most probable catalysts of the pro-oxidant activity in fungal cultures was also investigated using this method.
2. Materials and methods 2.1. Fungal strains and chemicals The strains Ceriporiopsis subvermispora FP 90031SP and Phanerochaete chrysosporium ME-446 were obtained from USDA Forest Products Laboratory (Madison, Wisconsin, USA). Bjerkandera adusta SCC 0169, Pycnoporus coccineus SCC 0041, and Pycnoporus sanguineus SCC 0294 were obtained from the SAPPI Culture Collection (University of the Free State, Bloemfontein, South Africa). Fomes fomentarius A71, Panus tigrinus A26, and Phlebia radiata 79 (ATCC 64658) were from the Collection of Wooddecaying Fungi at the Division of Microbiology, Department of Applied Chemistry and Microbiology (University of Helsinki, Finland). Coriolus versicolor K12-1-1, Daedalea quercina K8-3, and P. sanguineus K5-2-3 were isolated from fruiting bodies of the fungi collected near Stellenbosch (South Africa). The strains were subcultured on malt extract agar and maintained at 4 8C. Corn steep liquor was obtained from African Products, Bellville, South Africa and from SigmaAldrich Co. Linoleic acid, veratryl (3,4-dimethoxybenzyl) alcohol, and 2,6-dimethoxyphenol were purchased from Sigma-Aldrich Co. All other chemicals used were of analytical grade. Buffers and solutions were made in Milli-Q ultrapure water. 2.2. Culture conditions The wheat straw-containing glucose–peptone–corn steep liquor (GPCSL) medium previously developed for production of ligninolytic peroxidases by P. chrysosporium ME-446 was used (Kapich et al., 2004). Two batches of wheat straw received from distinct geographical regions (South Africa and Spain, Europe) were applied. The straw was milled and sieved to a maximum particle size of 10 mm before addition to the GPCSL medium. The medium contained (g l 1): wheat straw, 20; glucose, 10; BactokPeptone (Difco), 3; corn steep liquor, 1; KH2PO4, 1; MgSO4d 7H2O, 0.5; initial pH 4.8. Fungal inoculum was prepared as described (Kapich et al., 2004). All cultivations were carried out in 250-ml Erlenmeyer flasks containing 50 ml of the wheat straw-GPCSL medium with agitation (120–140 rpm) on a rotary shaker at 288C except for the strains of P. chrysosporium, P. coccineus, and P.
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sanguineus that were cultivated at 37 8C. Culture fluids were filtered through Miracloth and centrifuged (13,400 g; 6 min, 4 8C) to remove all solid particles and fragments of the mycelium. 2.3. Oxygen consumption experiments Pro-oxidant activity was measured at 25 8C as the maximal rate of oxygen consumption during linoleic acid peroxidation promoted by the fungal culture fluids or ligninolytic enzymes. The rates of oxygen consumption were taken from the linear portions of Oxygraph recordings (Hansatech Instruments Oxygraph, UK) and were calculated using the software of the manufacturer. The procedure included consecutive addition of the components to the reaction chamber containing the oxygen electrode. First, ultrapure water or appropriate buffer solution was placed into the reaction chamber and saturated with dissolved oxygen by intensive stirring. The chamber was closed with a tight-fitting plunger and further additions were made via a small capillary hole in the plunger with a Hamilton syringe. In the optimized method, the components were added in the following order: 620 Al of distilled water, 250 Al of 200 mM sodium acetate buffer (pH 4.5), 10 Al of a freshly prepared 100 mM emulsion of linoleic acid in 10% (wt./vol.) Tween 20, 10 Al of 100 mM MnSO4, 100 Al of fungal culture fluid or 0.015 U of purified enzyme (MnP or LiP), and 10 Al of 10 mM H2O2 (freshly prepared) to give a final reaction volume of 1 ml. For reaction controls, the compounds were stepwise omitted or the culture fluid samples were heat-treated in boiling water bath for 10 min before addition to the reaction mixtures. Every oxygen consumption experiment was repeated at least three times with basically identical outcomes. Occasionally when there was a defect with an electrode, the data from the treatment were discarded and the experiment was repeated. The pro-oxidant activity was expressed as units per milliliter of the culture fluid (U ml 1), where 1 U equals 1 Amol of O2 consumed per minute in the reaction of linoleic acid peroxidation. 2.4. Determination of the thiobarbituric acid (TBA)reactive substances The reactions of linoleic acid peroxidation catalyzed by the culture fluids were first carried out in
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loosely capped sterile 20-ml vials and then assayed for the presence of TBA-reactive products. The complete reaction mixture contained 300 Al of the culture fluid obtained after cultivation of P. chrysosporium ME-446 in the GPCSL medium with South African wheat straw (MnP activity—0.16 U ml 1; LiP activity—0.36 U ml 1), and the following filtersterilized compounds: 50 mM sodium acetate buffer (pH 4.5), 1 mM MnSO4, 1 mM linoleic acid, 0.1% (wt/vol) Tween 20, and 0.1 mM H2O2 in a total volume of 3 ml. Reaction mixtures without linoleic acid, without Mn2+, and without or with temperature inactivated culture fluid were used as controls. The vials were incubated on a rotary shaker at 37 8C in the dark for 1 h. Products of linoleic acid peroxidation were assayed in the reaction mixtures by the TBA method as described previously (Kapich et al., 1999b). 2.5. Enzyme assays All enzymatic activities were determined spectrophotometrically (DU 640 Beckman Coulter, USA, or Shimadzu PharmaSpec UV-1700, Japan) at 25 8C. MnP activity in culture fluids was determined by following the formation of coerulignone (3,3V,5,5Vtetramethoxydiphenoquinone) from 2,6-dimethoxyphenol (DMP) at 469 nm; (molar extinction coefficient=49,600 M 1 cm 1) (Wariishi et al., 1992). The reaction mixture (1 ml) contained 50 mM sodium tartrate (pH 4.5), 1 mM MnSO4, 0.5 mM DMP, and 100 Al cultural fluid. The reaction was initiated by adding 0.1 mM hydrogen peroxide and the resulting MnP activity observed was corrected by subtracting the laccase activity. Laccase activity was determined in a similar manner except that MnSO4 and hydrogen peroxide were excluded from the reaction mixture. MnP activity in the protein fractions separated by anion-exchange chromatography was also estimated by measuring formation of Mn3+– malonate complex at 270 nm (molar extinction coefficient=11,590 M 1 cm 1) (Wariishi et al., 1992). LiP activity was measured by monitoring oxidation of veratryl alcohol to veratraldehyde at 310 nm (molar extinction coefficient=9,300 M 1 cm 1) (Tien and Kirk, 1988). For all the enzyme activities, 1 unit (U) represents 1 Amol of product formed within 1 min of reaction time.
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2.6. Purification and characterization of the ligninolytic peroxidases The ligninolytic peroxidases MnP and LiP were separated by Mono-Q anion-exchange chromatography from 72-h cultures of P. chrysosporium ME-446 grown on wheat straw GPCSL medium (Kapich et al., 2004). Fractions corresponding to separate LiP and MnP isoenzymes were pooled, concentrated, and diafiltrated (10 kDa cut-off, Filtron Microsep) with ultrapure water. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) was performed as described (Laemmli, 1970) using low range molecular weight markers (Amersham Pharmacia Biotech). Isoelectric focusing (IEF) was performed using the Multiphore II electrophoresis system and 0.5 mm manually cast ultrathin polyacrylamide gels with pH range pH 2.5–5 generated by carrier ampholytes according to the manufacturer’s instructions (Amersham Pharmacia Biotech). The pH range was determined using a low pI calibration standard (Amersham Pharmacia Biotech). Protein bands in all PAGE gels were visualized by Coomassie Brilliant Blue stain. 2.7. Analysis of phenolic compounds Total content of phenolic compounds in the wheat straw-containing GPCSL medium was determined by a standard method with 4-aminoantipyrine (APHA, 1995).
3. Results 3.1. Lipid peroxidation initiated by culture fluids of P. chrysosporium ME-446 Aliquots of culture fluids of P. chrysosporium ME446 cultivated on the wheat straw-containing GPCSL medium were used to develop the method for determination of the pro-oxidant activity. Time courses of typical reactions of linoleic acid peroxidation obtained with two different culture fluid samples are shown in Fig. 1. Addition of the linoleic acid emulsion, Mn2+ ions, and culture fluid into the O2 saturated acetate-buffered solution caused no change to the background drift (0.3–0.6 nmol ml 1 min 1) of the oxygen electrode and the O2 consuming
Fig. 1. Consumption of O2 in the reaction of linoleic acid peroxidation obtained with two different culture fluids of P. chrysosporium ME-446. (Curve 1) Reaction with culture fluid sample (1) obtained after cultivation of the fungus in the GPCSL medium with Spanish wheat straw (MnP activity—0.30 U ml 1; LiP activity—0.01 U ml 1); (curve 2) reaction with culture fluid sample (2) obtained after cultivation of the fungus in the GPCSL medium with South African wheat straw (MnP activity—0.16 U ml 1; LiP activity—0.36 U ml 1); (curve 3) reaction with thermally inactivated culture fluid sample (2). Reaction mixtures contained 50 mM sodium acetate buffer (pH 4,5); 1 mM linoleic acid, 0.1% Tween 20; 1 mM MnSO4; 100 Al culture fluid; 0.1 mM H2O2. Arrows indicate the addition of the components to the reaction mixture: C18:2—emulsion of linoleic acid in Tween 20; Mn2+— solution of MnSO4, Cult. fluid—culture fluid sample, H2O2— solution of H2O2.
reaction started only after addition of 0.1 mM H2O2. Two major reaction patterns were observed with culture fluid samples. In the first cases, with culture fluids obtained after cultivation of the fungus in the GPCSL medium with Spanish wheat straw and containing high MnP activity but only traces of LiP activity, a short period of slow increase in the concentration of dissolved O2 was observed and thereafter, slow consumption of O2 commenced (Fig. 1, curve 1). This medium contained a relatively high level of phenolic compounds (19.8F1.2 mg L 1). A second reaction pattern showed no initial generation of O2 and fast oxygen uptake began immediately or shortly after addition of H2O2 (Fig. 1, curve 2). This pattern was prevalent with fungal culture fluids containing both MnP and LiP activities, which were obtained after cultivation in the medium with South African wheat straw. This medium contained a lower amount of phenolic compounds
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(15.4F0.7 mg L 1). With both reaction patterns, the rate of O2 consumption followed first-order saturation kinetics. If linoleic acid or culture fluid was omitted from the reaction mixture, the dissolved O2 concentration remained constant. Moreover, in the absence of linoleic acid some evolution of O2 was observed (data not shown). Heat-treated culture fluid samples lost their ability to initiate peroxidation of linoleic acid with O2 consumption (Fig. 1, curve 3). 3.2. Effect of organic acid buffer solutions Acetate buffer (pH 4.5) supported the highest rate of O2 consumption during peroxidation of linoleic acid catalyzed by the culture fluid, whereas malonate and tartrate both suppressed the reaction considerably (Fig. 2). Even ultrapure water supported the reaction more effectively than malonate or tartrate corroborating the conclusion that these chelating organic acids inhibited the lipid peroxidation reactions. In acetate buffer, peroxidation of linoleic acid strongly depended on the pH value in the reaction mixture and the highest rate of O2 consumption occurred at pH between 4.0 and 4.5 (Fig. 3).
Fig. 2. Influence of organic acids on the O2 consumption in the reaction of linoleic acid peroxidation initiated by culture fluid sample (2) of P. chrysosporium ME-446 (MnP activity—0.16 U ml 1; LiP activity—0.36 U ml 1). Reaction mixtures contained ultrapure water or sodium acetate, malonate or tartrate buffer solutions (50 mM each, pH 4.5) and all the other components described for Fig. 1. The arrow indicates the addition of 0.1 mM H2O2.
Fig. 3. Influence of pH on the maximal rate of O2 consumption in the reaction of linoleic acid peroxidation initiated by culture fluid sample (2) of P. chrysosporium ME-446 (MnP activity—0.16 U ml 1; LiP activity—0.36 U ml 1). The reaction mixtures in 50 mM sodium acetate, adjusted to the exact pH values contained the same components as described for Fig. 1.
3.3. Effect of Mn2+ and H2O2 Mn2+ ions were essential for the peroxidation of linoleic acid promoted by culture fluids as seen as O2 consumption in the reactions. Without Mn2+, an extended period (about 10 min) of increase in the concentration of dissolved O2 was observed after addition of hydrogen peroxide (Fig. 4A). When small amounts of Mn2+ ions (0.01–0.05 mM) were introduced into the reaction mixtures, O2 consumption increased accordingly. However, complete depletion of O2 was reached with 0.1 mM or higher concentrations of Mn2+ ions. With 0.5 mM Mn2+, consumption of O2 attained the highest rate, about 90 nmol ml 1 min 1, which was not exceeded by higher concentrations of Mn2+ ions (data not shown). In general, no peroxidation of linoleic acid occurred without the addition of H2O2 when samples of culture fluids of P. chrysosporium ME446 were applied as the pro-oxidant agents. Even with small concentrations of H2O2 (0.01 mM), the reaction started immediately albeit at a very slow rate of O2 consumption (6.4 nmol ml 1 min 1)
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3.4. Accumulation of TBA-reactive substances during peroxidation of linoleic acid To confirm that the consumption of O2 was related to peroxidation of linoleic acid, we determined accumulation of TBA-reactive substances in the reaction mixtures. Peroxidation of linoleic acid as catalyzed by culture fluids in the complete system resulted in the accumulation of TBA-reactive substances (0.073 mM) within 1 h. In the cases when linoleic acid, Mn2+ ions, or culture fluid was omitted or the culture fluid sample was inactivated by heat treatment, the accumulation of TBA-reactive substances was negligible (0.001 mM or less). 3.5. Time course of pro-oxidant, MnP, and LiP activities in submerged culture of P. chrysosporium ME-446
Fig. 4. Influence of Mn2+ (A) and H2O2 (B) on the consumption of O2 in the reaction of linoleic acid peroxidation initiated by the culture fluid sample (2) of P. chrysosporium ME-446 (MnP activity—0.16 U ml 1; LiP activity—0.36 U ml 1). Reaction mixtures contained all the components as described for Fig. 1 with specific concentrations of MnSO4 or H2O2. The arrow indicates the addition of H2O2.
(Fig. 4B). When 0.05 mM H2O2 was added, the consumption of O2 proceeded until exhaustion. The highest rate of oxygen consumption was attained with 0.1 mM H2O2 whereas higher concentrations of H2O2 apparently inhibited the reaction. Moreover, with 0.3 mM of H2O2 initial evolution of O2 was observed that only later turned to oxygen consumption.
The investigation of the influence of different factors on the peroxidation of linoleic acid catalyzed by culture fluids yielded a new method for evaluation of the pro-oxidant activity in cultures of whiterot fungi (see Materials and methods). Using this method we investigated the relationship between the pro-oxidant activity and MnP/LiP activities in submerged culture of P. chrysosporium ME-446 grown in the GPCSL medium supplemented with Spanish wheat straw. MnP activity appeared in culture fluids after 60 h of cultivation and quickly reached a maximum value at 66 h (Fig. 5). However, when MnP activity appeared in the cultures, only very low level of pro-oxidant activity and no LiP activity was detected. Subsequently, concomitant increase of both the pro-oxidant and LiP activities occurred. After 68 h of cultivation, MnP activity began to decrease while the pro-oxidant and LiP activities increased to a maximal level 6 h after the peak of MnP activity was attained. 3.6. Pro-oxidant activities of purified MnP and LiP The possibility that the ligninolytic peroxidases may be major pro-oxidant agents in the fungal culture fluids was investigated using purified MnP P5 and LiP P4 isoenzymes of P. chrysosporium ME-446 (Kapich et al., 2004). The molecular weights of the enzymes determined by SDS–PAGE were 45.5 and 40
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3.7. Pro-oxidant activities in cultures of various wood-decaying fungi Only cultures of white-rot fungi producing MnP showed the pro-oxidant activity (Table 1). However, no direct correlation between the level of MnP and the level of pro-oxidant activity was observed in culture fluid samples. The highest pro-oxidant activity was found in culture fluids of B. adusta SCC 0169,
.
Fig. 5. Time course of MnP ( ), LiP (n), and pro-oxidant (PO, E) activities in the submerged culture of P. chrysosporium ME-446 grown in the GPCSL medium supplemented with Spanish wheat straw. Bars on symbols indicate the standard deviation of means (n=3); where absent, bars fall within symbols. For more details, see Materials and methods.
kDa for MnP P5 and LiP P4, respectively. Both proteins were acidic with values of pI 4.2 for MnP P5 and 3.9 for LiP P4. A possible role of ligninolytic peroxidases in pro-oxidant activity is supported by our observation that the culture fluid filtrates obtained upon ultrafiltration and containing low molecular weight compounds (b10 kDa) showed no pro-oxidant activity. In the O2 consumption assay with linoleic acid, the MnP P5 enzyme showed significant pro-oxidant activity (Fig. 6A). Addition of 0.015 U of the MnP P5 in the acetate-buffered reaction mixture caused immediate O2 uptake even without addition of H2O2. A similar curve was obtained when ultrapure water was used instead of acetate whereas malonate and tartrate as chelating organic acids completely suppressed the ability of MnP P5 enzyme to develop peroxidation of linoleic acid in the absence of hydrogen peroxide, which is seen as O2 consumption in the reaction. When 0.1 mM H2O2 was added into the reaction mixtures containing malonate or tartarate, only low level of O2 consumption was observed (Fig. 6B). Uptake of O2 was not observed when linoleic acid or Mn2+ ions were omitted from the reactions or when the MnP P5 enzyme was thermally inactivated. The LiP P4 enzyme failed to show any pro-oxidant activity under conditions similar to those assessed for MnP P5 (data not shown).
Fig. 6. Influence of organic acids on the consumption of O2 in the reaction of linoleic acid peroxidation catalyzed by MnP P5 enzyme fraction, purified from submerged cultures of P. chrysosporium ME446, in the absence (A) and in the presence (B) of 0.1 mM H2O2. Reaction mixtures contained ultrapure water or sodium acetate, malonate, or tartrate buffer solutions (50 mM each, pH 4.5) and all the other components as described for Fig. 1 except that 0.015 U of MnP P5 was used instead of the culture fluid sample. The arrow indicates addition of the enzyme.
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Table 1 Pro-oxidant activity and activities of ligninolytic enzymes (meansFstandard deviation, n=3) in submerged cultures of wood-decaying fungi grown in wheat straw-containing GPCSL medium Fungus species and straina Bjerkandera adusta SCC 0169 Ceriporiopsis subvermispora FP90031SP Coriolus versicolor K 12-1-1 Daedalea quercina K8-3 Fomes fomentarius A71 Panus tigrinus A26 Phanerochaete chrysosporium ME-446 Phlebia radiata 79 Pycnoporus coccineus SCC 0041 Pycnoporus sanguineus K5-2-3 Pycnoporus sanguineus SCC 0294 a b c d
Cultivation time, days
Pro-oxidant activity, U ml
6 4 4 8 5 10 3 10 10 7 10
2.07F0.08 0.40F0.01 0.15F0.01 ND ND 0.05F0.01 1.48F0.04 0.05F0.01 ND ND ND
1
MnP activity, U ml 1
LiP activity, U ml 1
Laccase activity, U ml 1
0.17F0.02 0.10F0.02 0.02d ND ND 0.03d 0.43F0.02 0.05F0.01 ND ND ND
NDb ND ND ND ND ND 0.23F0.02 ND ND ND ND
TRc 0.05F0.01 0.07F0.01 ND 0.22F0.01 0.11F0.01 TR 0.01d 0.13F0.01 0.25F0.01 0.15F0.01
For strain origin, see Materials and methods. Not detected. Trace, activity lower than 0.01 U ml 1. Standard deviation of means less than 0.01.
although the highest MnP was established in culture fluids of P. chrysosporium ME-446. Only the latter fungus was able to produce LiP in the wheat strawcontaining GPCSL medium. The strains of white-rot fungi F. fomentarius A71, P. coccineus SCC 0041, P. sanguineus K5-2-3, and P. sanguineus SCC 0294 were able to actively produce laccase but did not show any MnP or pro-oxidant activity. The brown rot fungus D. quercina that was used as a control in this study showed no production of the ligninolytic enzymes or the pro-oxidant activity as well.
4. Discussion This is the first attempt to estimate the pro-oxidant (lipid peroxidation promoting) activity in cultures of wood-decaying fungi. Despite the availability of various methods to determine the antioxidant activity (Kapich and Shishkina, 1992; Kapich, 1995; Mau et al., 2002), it is surprising that no methods have been described to detect the pro-oxidant activity in fungal cultures. The reactions of lipid peroxidation possibly play an important role in the physiology and secondary metabolism of wood-decaying fungi, in particular for the degradation of non-phenolic lignin and xenobiotics. Therefore, the evaluation of the ability of fungal cultures to promote lipid peroxidation is of great importance.
We describe here a new simple method to quantify the pro-oxidant activity. With this method we show that lipid peroxidation promoting activity is found in the culture fluids of the white-rot fungi when grown in the GPCSL medium with wheat straw. The peroxidative mechanism of the reaction was evident from the rapid consumption of dissolved O2 in the course of the reactions with concomitant accumulation of TBA-reactive substances as products. Ultrafiltrates of the culture fluids containing only low molecular weight compounds failed to initiate lipid peroxidation indicating that high molecular weight substances are responsible for the prooxidant activity of the culture fluids. The enzymatic nature of the pro-oxidant agent is confirmed by our observation that heat-treatment of the culture fluid samples completely destroyed their ability to initiate lipid peroxidation. Lipoxygenases are the most commonly recognized enzymes that effectively oxidize unsaturated fatty acids like linoleic acid with concomitant uptake of O2 (Grechkin, 1998; Ku¨hn and Borchert, 2002; Schewe, 2002). However, it is unlikely that fungal lipoxygenases were the pro-oxidant agents in this case due to their intracellular location (Husson et al., 1998; Feussner and Wasternack, 2002). In addition, the conditions that favored extracellular lipoxygenase-like activity of P. chrysosporium ME-446 in our study differed considerably from the optimal
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conditions supporting lipoxygenase activity (Bisakowski and Kermasha, 1998; Kuribayashi et al., 2002). The requirement of Mn2+ ions and H2O2 for the O2 consumption reactions clearly indicated that MnP produced in the fungal cultures may play a pivotal role in the process of linoleic acid peroxidation. Optimum pH for the reactions was also close to the optimum pH of MnP activity (Glenn et al., 1986). However, the chelating organic acids, tartrate and malonate, which have been shown to be important for MnP catalysis (Wariishi et al., 1992), showed only 1/10 of the efficiency in comparison to acetate in these linoleic acid peroxidation reactions initiated by culture fluid (Fig. 2). Moreover, even distilled water supported higher rate of linoleic acid peroxidation than malonate or tartrate. This result clearly indicates that the chelating organic acids inhibited the lipid peroxidation reaction, which is unusual for reactions catalyzed by MnP. Investigation of the relationship between MnP, LiP, and pro-oxidant activities in culture fluids of P. chrysosporium ME-446 revealed their interconnection. Surprisingly, the pro-oxidant activity was related more closely to the level of LiP than MnP activity in the fungal cultures. Indeed, when MnP activity already appeared in the culture fluid, only traces of pro-oxidant activity could be detected and in fact, the peak of pro-oxidant activity coincided with production of LiP rather than MnP (Fig. 5). However, with purified ligninolytic enzymes, only MnP showed strong pro-oxidant activity whereas LiP failed to cause peroxidation of linoleic acid with consumption of oxygen. Mn2+ ions were absolutely necessary for the prooxidant activity of purified MnP. This is typical for MnPs from P. chrysosporium since their catalysis depends on the presence of Mn2+ (Gold et al., 2000). However, the oxidation of linoleic acid by MnP had other unusual features. Hydrogen peroxide was not needed for the reaction in acetate buffered solutions or ultrapure water. Surprisingly, chelating organic acids malonate and tartrate completely suppressed the MnP-catalyzed peroxidation of linoleic acid in the absence of hydrogen peroxide (Fig. 6A). Addition of H2O2 slightly stimulated the prooxidant activity of MnP only in the presence of malonate or tartrate (Fig. 6B). These data point to
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the optimal conditions for evaluation of the prooxidant activity in solutions of purified MnP and in the complex culture fluids of wood-decaying fungi being different. Our results indicate that MnP was a crucial agent that provided pro-oxidant properties to culture fluids of P. chrysosporium ME-446 and other wood-decaying fungi due to its lipoxygenase-like activity. The comparative investigation of pro-oxidant activity and activities of ligninolytic enzymes in submerged cultures of selected wood-decaying fungi unambiguously demonstrated that only culture fluids containing MnP showed the pro-oxidant activity. The laccaseproducing cultures that did not produce MnP were unable to show any pro-oxidant activities. However, as it has been shown earlier under chelator-limited conditions, MnP also exhibits significant catalase-like activity producing O2 from H2O2 (Timofeevski and Aust, 1997). Indeed we could monitor evolution of O2, most probably due to this MnP-associated, catalase-like activity in the beginning of some reactions (see for example Fig. 1, curve 1). The catalase-like activity seen as generation of O2 increased with the increase of H2O2 concentration in the reaction mixture. It seems that O2 producing catalase-like reactions could proceed simultaneously with O2 consuming lipoxygenase-like reactions in the reaction mixtures containing MnP, Mn2+ ions, linoleic acid, and H2O2 in the presence of low-affinity ligands such as acetate. In addition, the pro-oxidant activity of the fungal cultures apparently depended not only on the presence of MnP and some other compounds in the culture fluids also influenced the pro-oxidant activity. Many aromatic and in particular, phenolic compounds are known to possess strong antioxidant properties inhibiting lipid peroxidation (Halliwell and Gutteridge, 1999). These compounds were released from wheat straw into the culture medium and may have inhibited the MnP-catalyzed lipid peroxidation. It is noteworthy that wood-decaying and especially whiterot fungi are also able to produce antioxidant compounds (Kapich and Shishkina, 1992; Kapich, 1995). The trace levels of pro-oxidant activity in culture fluids with high MnP activity could be due to these straw-derived and fungal compounds (Fig 5). Thus, the total pro-oxidant activity may be considered as net result of the action of pro-oxidant factors and
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antioxidant compounds present in the fungal culture fluids. A simple assay described here provides an excellent method to quantify the pro-oxidant activity in cultures of wood-decaying fungi. The method is rapid and easy to use and does not need complex sample processing or time-consuming analysis of the products of lipid peroxidation. It also can serve as an additional indication of the presence of MnP in cultures of wood-decaying fungi.
Acknowledgements Financial support from the Academy of Finland, as particular funding for the Center of Excellence on Microbial Resources (project number 53305), from the University of Stellenbosch and the National Research Foundation of South Africa are gratefully acknowledged.
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