Holzforschung / Vol. 50 / 1996 / No.4

Transcription

1 Holzforschung / Vol. 50 / 1996 / No.4

2 312 C. R. Jordan et al.: Oxalic Acid and Brown Rot Decay Holzforschung 50 (1996) Detection and Quantification of Oxalic Acid from the Brown-Rot Decay Fungus, Postia placenta By C. R. Jordan 1, W. V. Dashek 1, and T. L. Highley 2 1 Dept. of Biological Sciences, Clark Atlanta University, Atlanta, GA, U.S.A. 2 USDA-Forest Service, Forest Products Laboratory, Madison, WI, U.S.A. Brown-rot Wood-decay Oxaloacetase Oxalic acid Fenton s reaction Postia placenta The mechanism by which brown-rot fungi initiate depolymerization of holocellulose in wood remains unknown. Recently, oxalic acid has received considerable attention in cellulose breakdown by brown-rot fungi. The oxalic acid could serve as a proton donor for hydrolytic or an eletron donor for oxidative (Fenton s reaction-h 2 O 2 /Fe 2+ ) cleavages of cellulose. The acid may originate via oxaloacetase action upon oxaloacetate. We report the applications of HPLC and oxalic acid kit calorimetry to quantify oxalic acid from hyphae grown on southern pine wood blocks (WBs) or in liquid culture. Oxaloacetase activity was not observed in either Amicon YM10 (MW cut-off, 10,000) filter-retained intra- or extracellular fractions of liquid cultured hyphae or in homogenates from decayed WBs. In contrast, oxalic acid was detected by HPLC in both P. placenta liquid cultures and decayed WBs. The less sensitive, oxalic acid, spectrophotometric kit (mg vs. µg) did not detect oxalic acid in liquid cultures. These results constitute additional evidence for an oxalic acid-fe 2+ /H 2 O 2 -Fenton s mechanism for brown rot-induced cellulose degradation. However, the origin of oxalic acid was not established. Introduction The mechanisms by which brown-rot fungi degrade cellulose may be quite different from those of other cellulolytic organisms (Highley and Flournoy 1994; Flournoy 1994). Jordan et al. (1994, 1996) reviewed the literature which prompted the hypothesis that brown-rot fungi might degrade cellulose via Fenton chemistry, i.e., Fe 2+ /H 2 O 2 yielding an OH radical. Because iron appears to exist within wood as Fe 3+, a mechanism must exist for the conversion of Fe 3+ to Fe 2+ for Fenton chemistry to be relevant to wood decay. In this regard, Schmidt et al. (1981) proposed that oxalic acid, which is secreted by brown-rot fungi (Takao 1965) may reduce Fe 3+ to Fe 2+. Because ocalic acid has been reported to reduce the viscosity of cellulose (Shimada et al. 1991), the acid could mediate a direct depolymerization of wood cellulose (Green et al. 1991; Shimada et al. 1991) and hemicellulose (Bech- Anderson 1987). Furthermore, Green et al. (1992) demonstrated that sufficient oxalic acid is synthesized by P. placenta to decrease the ph of the decaying wood thereby potentiating brown-rot-induced degradation of cellulose. Recently, Dutton et al. (1993) reported oxalate production by whiterot basidiomycetes raising the possibility of oxalic involvement in white-rot induced decay also. A possible regulatory role for oxalic acid in lignin biodegradation by Phanerochaete chrysosporium has been suggested (Shimada et al. 1993). As for the origin of oxalic acid, it appears to arise through the actions of oxaloacetase (Hayishi et al. 1956; Akamatsu et al. 1991, 1992, 1993) and/or glyoxylate oxidase (Akamatsu 1993; Akamatsu and Shimada 1993, 1994) in Tyromyces palustris, In addition, Shimada et al. (1994) were able to detect the two enzymes in the white-rot fungi, Phanerochaete chrysosporium and Coriolus verisicolor. Furthermore, these investigators established the time courses for oxaloacetase and glyoxylate oxidase appearances in relation to that for oxalic acid production during 14 days of T. palustris cultivation. Although oxaloacetase activity appeared earlier than that of glyoxylate oxidase, the time-dependent appearance of oxalic acid suggested that both enzymes contribute to oxalic acid s formation. The present paper reports the HPLC and an oxalic acid kit detection of oxalic acid production by the brown-rot fungus Postia placenta, grown in liquid culture or on southern yellow pine wood blocks and the acid s origin. Thus, this and the accompanying paper (Jordan et al. 1996) sought evidence for an oxaloacetase-oxalic acid-fe 2+ /H 2 O 2 mechanism for Postia placenta induced wood decay. Certain of the results herein have been reported in preliminary form (Jordan et al. 1994). Copyright 1996 Walter de Gruyter Berlin New York

3 C. R. Jordan et al.: Oxalic Acid and Brown Rot Decay 313 Materials and Methods Mycelia of a Postia placenta isolote (MAD 698, Forest Products Laboratory, Madison, WI) maintained upon an agar slant were aseptically transferred to sterile Petri plates containing sterilebasal salts medium (Highley 1973) supplemental with 1% glucose (w/v) solidified with 2% Bacto-Agar (Difco, Detroit, MI). These plates were incubated at C and 70% relative humidity for 5 days in the dark within a Labline gyrotory shaker at 100rpm. Thereafter, 20.5mm mycelial discs were aseptically excised from the plates and homogenized into 11 sterile basal medium containing 1% glucose. Then, 40ml of the homogenized mycelia were inoculated into sterile 11 Erlenmeyer flasks containing 11 sterile basal medium. These flasks were swirled and divided into 500ml volumes for each 11 flask. The flasks were incubated in the Lab-line gyrotory shaker at rpm and C for 25 days in the dark. Southern yellow pine blocks ( nm); the small dimension in the fiber direction) were decayed by Postia placenta by the Standard American Society for Testing and Materials soil-block method (ASTM 1991) or an agar block method. Wood decay chambers for the agar-block procedure were constructed by placing sterile southern yellow pine wafer feeders upon agar in Petri plates and inoculating with P. placenta Decay chambers were incubated at C and 70% relative humidity in the dark until the wood wafer feeder was covered with hyphae. Next, five sterile southern yellow pine sapwood test blocks were placed upon each wood wafer feeder and allowed to incubate at C and 70% relative humidity in the dark for 3 weeks in the Labline gyrotory shaker. oxaloacetic acid standard curve. The 280nm contents of the fractions were quantified by UV spectroscopy at 280nm with a bovine serum albumin (BSA) standard curve. Both high and low molecular weight fractions from intracellular and extracellular P. placenta liquid cultures and 1 N HCl extracts of fungal-inoculated wood blocks were subjected to high performance liquid chromatography (HPLC) utilizing a Bio-Rad Aminex HPX-87 strong cation-exchange resin column, H + (particle size 9µm, mm I. D.) for organic acid analysis (Bio-Rad Labs, Richmond, CA) and the Beckman Systems Gold HPLC model with a 166 Detector and a disc integrator (Beckman Instruments, Palo Alto, CA). Organic acid standards were prepared in HPLC grade water (Aldrich Chemical CO., Milwaukee, WI) as follows: oxalic acid (0.4mg ml -1 ), citric acid (3.5 mg ml -1 ), tartaric acid (4.0mg ml -1 ), malic acid (10.0mg ml -1 ). succinic acid (20.0mg ml -1 ), formic acid (10.0mg ml -1 ) and acetic acid (20.0mg ml -1 ) (Fisher Scientific Co., Fair Lawn, NJ). These organic acid standards were chromatographed both separately and combined in order to determine the efficiency of the column and the retention time for each acid. The samples were filtered through a 0.45 µm Millipore membrane. Five µl aliquots of the sample solutions were injected into the chromatographic system. The cluting solvent was N H 2 SO 4 and organic acid detection was carried out at 210nm and 35 C. Further determinations of oxalic acid were made with a Sigma oxalate kit (Sigma Chemical Co., St. Louis, MO). These determinations were made on the same samples as mentioned above. Results The 500ml contents of the 11 Erlenmeyer flasks were collected by gentle vacuum filtration (on ice) onto Whatman No. 1 quantitative 9.0cm filter papers (Whatman Paper Ltd., Maidstone, England). The fungal hyphae and medium were stored on ice for further analysis. To obtain mycelial homogenates, the collected semi-wet hyphae were homogenized in ph 7.6, imidazole buffer with a mortar and pestle for about 15 min. Intracellular: TO remove the cell wall, the fungal homogenate was centrifuged at 500xq for 15 min. at 4 C. Wherein the resultant pellet was frozen in liquid nitrogen and stored at 20 C, the supernatant was centrifuged at 15,000xq for 30min. and 4 C. The 15,000xq supernatant was subsequently subjected to Amicon ultrafiltration (Amicon, Beverly, MA) using YM10 (molecular weight cut-off 10,000DA) Diaflo ultrafiltration membranes. The molecular weight fraction greater than 10,000 was considered to contain oxaloacetase and oxalic acid less than 10,000Da. Extracellular: The cell free culture filtrate was also subjected to Amicon ultrafiltration employing YM10 Diaflo ultrafiltration membranes to yield molecular weight fractions above and below 10,000Da. An objective of this investigation was to ascertain whether oxaloacetase was synthesized and secreted by P. placenta in liquid culture. Initially, attempts were made to assay A 280nm absorbing substances in both intracellular and extracellular fractions of P. placenta following Amicon ultrafiltration (MW cutoff, 10,000). The data are expressed as YM10 retained as well as YM10 filtrates of intracellular and extracellular fractions (Table 1). The YM10 retained and filtrate of the intracellular fraction contained mg and mg A 280nm absorbing units, respectively. The YM10 retained and YM10 filtrate extracellular fraction exhibited a marked reduction in A 280nm absorbing substances, with the YM Table 1. Summary of A 280nm absorbing substances contents of Amicon ultrafiltrated hyphal mycelial homogenates (intracellular) and growth medium (extracellular) for 21 day liquid cultured Postia placenta a The oxaloacetase assay utilized a 1.25ml reaction mixture consisting of 1.0mM oxaloacetic acid (approx. 98% pure, Sigma Chemical Co., St. Louis, MO) in ph 7.6, 0.2 M imidazole buffer and 0.25 ml ( nm absorbing units) of ultrafiltrated intracellular and extracellular fractions. The activity of oxaloacetase was quantified as the time-dependent disappearance of oxoloacetic acid at 255nm (Lenz et al. 1976) emplying an

4 314 C.R. Jordan et al.: Oxalic Acid and Brown Rot Decay filtrate containing mg and the YM10 retained fraction only mg. Because of the occurence of A 280nm absorbing substances, attempts were then performed to quantify oxaloacetase activity in these same fractions. The oxaloacetase assay was carried out at 255nm. Neither the extracellular YM10 retained nor the YM10 filtrate assays were linear over time indicating a lack of detectable enzyme activity. The intracellular YM10 filtrate also lacked detectable enzyme activity. Detection and quantification of oxalic acid To both detect and quantify oxalic acid, both HPLC and a Sigma calorimetric oxalate assay kit were employed. An HPLC elution profile of a mixture of seven authentic organic acids is presented in Figure 1. The retention time of oxalic acid was between 5.91 and 6.46 min. Figure 2 illustrates that the retention time for authentic oxalic acid alone was approximately Both the Amicon YM10 retained (Fig. 3) and filtrate (Fig. 4) of ultrafiltrated mycelial homogenates possessed peaks with retention times of 6.25 and min., respectively approximating that characteristic of oxalic acid. In addition, both the YM10 retained and filtrate fractions of ultrafiltrated growth medium exhibited peaks with retention times of min. (Fig. 5) and min. (Fig. 6), respectively, suggestive of oxalic acid. Furthermore, a similar peak was observed for four replicate ASTM southern yellow pine wood blocks decayed to 25% weight loss by P. placenta. The retention times were 5.89 (Fig. 7), 5.88, 5.93 and 5.89min. for the samples. In contrast, a peak with a retention time representative of oxalic acid (Fig. 8) was not observed for uninoculated control wood blocks. A comparison of the amounts of putative oxalic acid as quantified by HPLC and the Sigma oxalate calorimetric assay kit is depicted in Table 2. Putative oxalic acid was detected in decayed blocks by both methods, but the kit did not detect oxalic acid in aliquots of either mycelial homogenates or growth medium.

5 C.R. Jordan et al.: Oxalic Acid and Brown Rot Decay 315 Figs Elution profiles from an HPLC resin of Amicon YM10 retained (5) and filtrate (6) of growth medium from liquid cultured P placenta. Figs Elution profiles of 1 N HCI extracts of P. placenta inoculated and uninoculated southern pine wood blocks. Discussion This investigation was concerned with gaining additional insight (Highley and Flournoy 1994) into the putative agents involved in brown-rot decomposition of wood through possible biochemical detections and quantifications of oxaloacetase and oxalic acid. Oxaloacetase detection and quantification Although oxaloacetase of Aspergillus niger was reported to catalyze the hydrolysis of oxaloacetate to

6 316 C.R. Jordan et al.: Oxalic Acid and Brown Rot Decay oxalate and acetate as early as 1956 (Hayishi et al. 1956), the enzymatic mechanism for oxalate generation in wood-rotting basidiomycetes has not been fully elucidated even though oxaloacetase was partially purified by Lenz et al. (1976). In this connection, Akamatsu (1993) provided evidence that oxalate could be derived by the action of glyoxylic acid oxidase upon glyoxylic acid in the brown-rot fungus, Tyromyces palustris. Akamatus et al. (1991, 1992, 1993) were the first to report oxaloacetase activity in cell-free extracts of T. palustris. The occurrence of oxaloacetase could be induced through the addition of sodium bicarbonate about 6 days into the stationary culture. These investigators reported that extracts containing the enzyme consisted of 20 30mg protein ml -1. Therefore, the current investigation employed cultures large enough to approximate 20-30mg protein ml -1 combined Amicon Diaflo YM10 (> 10,000MW) intracellular and extracellular retained fractions (Table 1). However, it is possible that the extracellular fraction contained peptides conjugated to carbohydrates as glycoproteins. Thus, the presence of the enzyme may differ under different cultivation conditions. Preliminary attempts to detect the occurrence of oxaloacetase by following the disappearance of oxaloacetate spectrophotometrically at 255nm revelaed an absorbance increase rather than a decrease over short time periods for YM10 retained hyphal homogenates. In contrast, the absorbance at 255nm of the YM10 retained growth medium remained essentially unchanged as did the YM10 filtrate intracellular and extracellular fractions. The latter is to be expected as the MW of the filtrate s constituents would be <10,000. To assess whether the above increase was enzymatic, various controls were performed utilyzing P. placenta cultured 21 days in liquid medium. These included employing boiled and unboiled YM10 retained hyphal homogenates and growth medium as well as performing oxaloacetase assays with mixtures lacking the substrate, oxaloacetate. The resultant data (not shown) do not support a demonstration of either intracellular or extracellular oxaloacetase in liquid cultured Postia placenta. There are several possible explanations for this. For example, sodium bicarbonate, a known inducer of oxaloacetase (Shimada et al. 1991; Akamatsu et al. 1991) was not added to the cultures in the present investigation. In addition, protease inhibitors such as phenyl methyl sulfonyl fluoride were not incorporated into the hyphal homogenization medium. Also, the current investigation used ultrafiltration procedures as a substitute for dialysis. In this regard, Akamatsu et al. (1993) observed a reduction in T. palustris oxaloacetase activity following overnight dialysis. Therefore, Akamatsu et al. (1993) utilyzed cell-free extracts without dialysis. In the present investigation, oxaloacetase was assayed spectrophotometrically by following the disappearance of oxaloacetate at 255nm (Lenz et al. 1976). The possible occurrence of oxaloacetase in liquid and/or wood block P. placenta cultures could be explored via the application of an oxalic acid commercial assay kit. For example, Akamatsu et al. (1991) used the kit to quantify oxalic acid derived from catalysis of oxaloacetate by oxaloacetase. The kit converts enzymatically formed oxalic acid to formic acid with oxalate decarboxylase and the resulting formate is quantified by an increase in NADH at 340nm. The reduced pyridine nucleotide is formed from NAD by formate dehydrogenase. In summary, with regard to oxaloacetase, supplementation of cultures with sodium bicarbonate may be critical to obtain significant production of oxaloacetase in liquid culture for assay. Additionally, the detection of oxaloacetase may be dependent upon the incorporation of protease inhibitors into the homogenization medium. Furthermore, investigations comparing oxaloacetase in crude, dialyzed and ultrafiltrated extracts seem warranted. Finally, the two oxaloacetase procedures, i.e., following the direct degradation of oxaloacetate or assaying enzymatically derived oxalic acid by the more indirect malate dehydrogenase/formate dehydrogenase kit could both be employed. ldentification of oxalic acid The attempts to establish oxaloacetase activity could have established the presence and origin of P. placenta oxalic acid had they been more conclusive. Therefore, other more direct attempts were performed to detect oxalic acid. High pressure liquid chromatography appeared to detect oxalic acid in both mycelial homogenates and growth medium of P. placenta liquid cultures and P. placenta decayed wood blocks. However, the amplitudes of the putative oxalate peaks in Figures 4, 5, and 6 exceeded the scale raising the possibility that the single peaks may in fact be multiple peaks. To rule out this possibility, sample dilution could be performed to yield peak amplitudes which remain on scale. Other parameters which could be considered include: pre-clean-up of extracts via TLC with authentic oxalic acid standards prior to HPLC as well as spiking extracts with known amounts of oxalic acid. However, oxalic acid was detected by the Sigma oxalate colormetric kit in P. placenta decayed pine blocks, but not in P. placenta liquid cultures. In this connection, the oxalic acid detection level for HPLC was 2µg whereas that of the oxalate calorimetric assay was mg. The quantitative differences

7 C.R. Jordan et al.: Oxalic Acid and Brown Rot Decay 317 between HPLC and the Sigma oxalate kit detected oxalic acid (Table 2) are not readily explainable. The quantification of oxalic acid could be verified by other methods. For example, Huang and Tanudjaja (1992) developed an anion-exchange column procedure for measuring oxalate. The procedure involves developing the column with phthalic acid and subsequent assay of column eluates with conductivity detection. The method is considered to be more simple and accurate than the HPLC procedure. Akamatsu et al. (1991) suggested that the accumulation of oxalic acid in the cultures of T. palustris could be explained by the presence of oxaloacetase and the absence of a oxalic acid decomposing system. Oxalic acid can be decarboxylated (Shimazono and Hayaishi 1957; Espejo and Agosin 1991; Shimada et al. 1992). In this regard, the possibility that P. placenta possesses oxalate decarboxylase activity has been reported (Micales 1994). Thus, the demonstration of P. placenta oxalic acid in the present investigation and the report of Micales need to be reconciled. For example, Micales reported that the ph optimum of oxalate decarboxylase was 2.2. However, oxalic acid accumulates in culture with a ph 5.0 or above. Micales suggested that oxalic acid may lower the ph of the medium to within the ph optimum of the enzyme. Thus, the time courses for the appearances of oxalic acid and oxalate decarboxylase need to be performed in relation to the ph of the medium. In conclusion, this paper provided combined HPLC/colorimetric evidence for the occurrence of oxalic acid in P. placenta liquid cultures and P. placenta infected southern yellow pine. This evidence together with the ESI Vocalizations of iron presented in the accompanying paper constitute additional evidence for an involvement of oxalic acid, Fenton chemistry mechanism in brown-rot decay, Acknowledgment We thank Dr. R. Thedford and Mr. Reynold Murray for advice regarding HPLC protocols. The technical assistance of Ms. C. Hutto is appreciated. This work was supported by USDA Forest Service Co-operative Agreements and in part by Clark Atlanta University s NIH-RCMI Grant no. G12RR Dashek s current address is Visiting Scientist, Forest Products Laboratory.

8 318 C. R. Jordan et al.: Oxalic Acid and Brown Rot Decay Env. Microbiol. 13, Received April 7th, 1995 C. R. Jordan W. V. Dashek Department of Biological Sciences Clark Atlanta University J.P. Brawley at Fair St. Atlanta, GA U.S.A. T. L. Highley USDA-Forest Service Forest Products Laboratory One Gifford Pinchot Drive Madison, WI U.S.A.