Appl Microbiol Biotechnol (1991) 35:674-680

017575989100201A t/ea ., M' robiology B clmotogy © Springer-Verlag 1991

Use of monoclonal antibodies to detect Mn(ll)-peroxidase in birch wood degraded by Phanerochaete chrysosporium

G. Daniel I , J. Jellison z, B. Goodell z, A. Paszczyfiski 3, and R. Crawford 3

1 Department of Forest Products, Box 7008, Swedish University of Agricultural Sciences, S-75007 Uppsala, Sweden 2 Wood Science and Technology, College of Forest Resources, University of Maine, Orono, ME 04469, USA 3 Department of Bacteriology and Biochemistry, College of Agriculture, University of Idaho, Moscow, ID 83843, USA

Received 7 December 1990/Accepted 2 May 1991

Summary. A monoclonal antibody (Mab) produced to purified Mn(II)-peroxidase was visualized on and within cell corners of. birch wood degraded by Phanerochaete chrysosporium using colloidal gold irnmuno-transmis- sion electron microscopy techniques. Labelling of the fungal cell membrane and cell wall was also observed. The same Mab was used to visualize the penetration of extracellular fungal metabolite extracts, infiltrated into previously decayed wood. Binding of antibodies to the lignin-rich cell corner region of the middle lamella in wood decayed by P. chrysosporium was observed in sec- tioned wood blocks and in wood infiltrated with crude extracellular extracts from P. chrysosporium liquid cul- tures. When a control monoclonal antiserum, produced to extracellular metabolites of Postia (Poria) placenta and cross-reactive with fungal cellulase, was used in la- belling, the cellulose rich region of the wood cell walls were labelled. Labelling in the middle lamella cell com- ers was only noted in what has been described as non- or poorly lignified cell corner regions.

Introduction

Although the biochemistry and molecular biology of lignocellulose degradation has been the source of much recent study, less emphasis has been placed on the bio- physical processes involved in the biodegradation of solid wood. One approach for studying degradative mechanisms in decayed wood is to use immunological or cytochemical techniques together with microscopic analyses of the degraded wood and fungal hyphae. A number of researchers have used cytochemical ap- proaches to localize degradation products within the wood cell wall (Murmanis and Highley 1987). These approaches provide information on general degrada- tion patterns but do not provide for any distinction be- tween enzymatic and potential non-enzymatic degrada-

Offprint requests to: G. Daniel

tion (Highley et al. 1988; Illman et al. 1988; Koenigs 1974).

To allow study of enzymatic action within wood, po- lyclonal antibodies to extracellular metabolites have been produced (Goodell and Jellison 1986) and used in fluorescence and transmission electron microscopy (TEM) immunolabelling studies (Goodell et al. 1988). Similarly, purified enzymes have also been used in the production of polyclonal antibodies. Srebotnik et al. (1988b) and Daniel et al. (1988, 1989), for example, used colloidal-gold-labelled polyclonal antibodies to show ligninases located preferentially on the plasmal- emma of the decay fungus Phanerochaete chrysospo- rium. In another approach, Garcia et al. (1987), used enzyme-gold complexes to study ultrastructural aspects of wood decay. No labelling was observed after incuba- tion of thin sections with ligninase-gold complexes and wood partially hydrolysed by ligninolytic fungi was not labelled by the enzyme probe. An antibody to ligninase was also produced and all fungal hyphae of white-rot fungi used in the 1987 study (except Pleurotus ostreatus, Garcia et al. 1987) labelled positively with anti-ligni- nase. The ligninase probe was located near the fungal plasmalemma and was occasionally observed extracel- lularly, associated with fungal slime.

Garcia et al. (1987) also studied the binding of lignin peroxidase-gold probes to native wood. Absence of wood labelling after treatment with ligninase followed by anti-ligninase and gold-labelled Protein A was pos- tulated to be due the to inaccessibility of wood lignin to the active site of the enzyme. Recent immunolabelling studies (Daniel et al. 1988, 1989; Srebotnik et al. 1988b; Blanchette et al. 1989) have shown ligninase to be closely associated with both P. chrysosporium and ex- posed, degraded regions of wood fibres. TEM studies showed labelling of all cell wall layers. Cytochemical studies using 3,3' diaminobenzidine have also sug- gested a localized distribution within the $2 cell wall of degrading wood fibres (Daniel et al. 1988, 1989).

The work presented here is part of a study attempt- ing to localize and characterize Mn(II)-peroxidase, a 46 000 Da glycoprotein isolated from P. chrysosporium:

675

Mn(II)-dependent peroxidase is a lignin-degrading en- zyme with a haem prosthetic group. The enzyme is in- volved in oxidation of mainly phenolic structures in lig- nin and it oxidizes Mn(II) to Mn(III) in the presence of appropriate chelating agents. The complexed Mn(III) can oxidize lignin oligomeric substructures (Shoemaker and Leisola 1990). In contrast to previous studies with polyclonal antibodies, monoclonal antibodies (Mab) were produced to the enzyme and used in this work.

Materials and methods

Organism and culture conditions. P. chrysosporium BKM-F-1767 (ATCC 24725) was maintained and spore suspension for inocula prepared as previously described (Huynh and Crawford 1985). The fungus was grown in defined medium that contained 2.2 mM nitrogen. Mycelia were grown on the roughened interior walls of a 20-1 carboy (Paszczyfiski et al. 1986).

Enzyme purification. A 2-1 quantity of medium from a 5-day-old culture was filtered through glass wool, concentrated to 10 ml us- ing a PM-10 Amicon (Amicon Corp., Lexington, Mass., USA) ul- trafiltration membrane, and dialysed overnight against 10 mM so- dium acetate, pH 5.0. The concentrated solution was centrifuged and 1 ml of clear solution was injected on a HPLC Mono Q HR 5/5 anion exchange column (Pharmacia, Uppsala, Sweden). Be- fore injection the column was equilibrated by washing with 10 mM sodium acetate buffer (pH 5.0). Chromatography was per- formed using an HP 1090 liquid chromatograph equipped with a diode array detector and HP 9000 Series 300 computer that used HP 7995A chemstortion software (Hewlett-Packard, Palo Alto, Calif., USA).

As shown in Fig. 1, the distribution of lignin peroxidase pro- teins was similar to that of earlier work (Kirk et al. 1986), but the ratio of peak areas was different. Fractions harvested between re- tention times 21 to 26 min were pooled, dialysed, and freeze- dried. These fractions contained Mn(II)-peroxidase activity and were free of ligninase activity. Assay methods for the peroxidases have been described previously (Paszczyfiski et al. 1988).

Antibody production. Two interperitoneal injections of the purified enzyme, 50 p.1/injection mixed 1 : 1 with Freunds incomplete adju- vant, were given to balb/cj mice at 2-week intervals. Three days before spleen harvest, mice were given an intravenous tail injec- tion of 50 p.g of purified enzyme without adjuvant. Hybrid cells between spleen cells of immunized mice and myeloma cell line SP 2/0-Ag 14 were created using previously published techniques (Schreier et al. 1980). Hybridoma culturing in hypoxanthine ami- nopterin thymidine (HAT) medium and subsequent expansion of colonies that gave a positive immunoglobulin G (IgG) or immu- noglobulin M (IgM) cross-reaction with the inject antigen was done using standard procedures. Identification of positive colo- nies was done using an enzyme-linked immunosorbent assay (ELISA) (Voller et al. 1977). The clones used in this study (338C and 338D) were the result of two cycles of dilution cloning of cell lines originally from colony 338. Dilution cloning was performed using mouse spleen cells as feeder cells using the procedure of Rener et al. (1985). Production of Mab was done using low pro- tein Ventrix medium according to the manufacturer's instructions, and subsequently via ascites production in balb/cj mice. Standard procedures were used including pristine priming of mice prior to injection of Mab-producing clones to stimulate tumour develop- ment. Partial purification of Mab was achieved by ammonium sulphate precipitation (Johnstone and Thorpe 1982). Although two enzyme fractions were pooled for use as antigen in the study, each Mab clone produces only one site-specific antibody. Mab produced by clones 338C and D was screened by ELISA to check that the antibodies produced were specific to Mn(II)-peroxidase.

0.10"

0.07

uJ 0 Z < ~o ri- O tn ra

0.05 -

I

I i I

o.o2q I I i

3"- I

o

4 1 %

~ - Ligninase H8

17%

13%

, !

, , , , , ,, II = ojo I \ ,"" 9"/. / / / t ~, / ° 6*/,

-- A o,o L I / / r i V l iL . . . . . " ' " t

I l I I I

10 20 30 40 50 TIME (rain)

Fig. 1. HPLC profiles of filtrate from a 5-day-old culture of Pha- nerochaete chrysosporium strain BKM grown in a 20-1 rotated car- boy: - - , / 1 4 o 9 ; . . . . , A2s0. HPLC peak values represent the per- centage of individual haem protein absorbance as a fraction of the total A4o9. We employed a non-linear gradient of 10 mM to 1 M sodium acetate buffer, pH 5.0:0-5 min, 10 mM sodium acetate; 5- 35 min, 40% 1 M sodium acetate; 35-45 min, 80% 1 M sodium ace- tate; 45-50 min, 100% 1 M sodium acetate

For these screenings all antibody-producing clones were sub- jected to multiple assays in an antigen-specific ELISA. In these assays the antigen to be tested was diluted to the desired concen- tration with Na-carbonate (pH 9.6). Then 200 I.d/well of the an- tigen was added to Immulon II plates (Dynetech Labs, 900 Slat- er's Lane, Alexandria, Va., USA) and incubated for a minimum of 4 h at 37 ° C. For the Mn(II)-peroxidase monoclonals 338C and 338D, crude fungal extracts, purified inject antigen and four com- mercially available horseradish peroxidase (HRP) isozymes were all tested for reactivity with the cloned monoclonals to Mn(II)- peroxidase. After incubation with the antigen, plates were washed and blocked with phosphate-buffered saline (PBS)/I% Tween 20/ 2% fetal calf serum (FCS) and 200 ~d/well of media derived from antibody secreting clones was added. Plates were incubated for a minimum of 4 h at 37 ° C and washed as before. Sigma (St. Louis, Mo., USA) goat-anti-mouse (GAM) IgG and/or (GAM) IgM alkaline phosphatase conjugate was diluted 1:750 in PBS-2% egg albumin or FCS and added at 200 gl/well: Plates were incubated for a minimum of 3 h at 37 ° C. After addition of 1 mg/ml of p-nitrophenol phosphate in pH-9.8 diethanolamine substrate buf- fer, plates were incubated for 15-30 min. Absorbanee at 405 nm (A4o5) was read on a Biotek 407 spectrophotometer.

A Mab produced to metaholites from Postiaplacenta (Goodell et al. 1988) was produced using similar procedures to those de- scribed above and was used as one of the control probes in this study. This Mab was produced to extracellular metabolites of P. placenta grown in liquid culture and was obtained from a clone designated number 103. Clone 103 was selected after being sub- jected to multiple screenings, based on its ability to preferentially react in ELISA with fungal cellulases from seven different fungal sources including a highly purified cellulase from Trichoderma reesei (A. Ayers, Cedar Crest College, Allentown, Pa., USA).

Soil block preparation, fixing and embedding for TEM. Small blocks (3.0 x 1.5 x 0.5 cm) of birch (Betula verrucosa Ehrh.) were

676

placed in 125-ml erlenmeyer flasks containing garden loam. After autoclaving, the wood blocks were inoculated with a spore sus- pension of Phanerochaete chrysosporium P- 127 (= ATCC 28326) and incubated in an environmental chamber (relative humidity 75%, temperature 38 ° C). After 6 weeks growth (corresponding to ca. 60-70% dry weight loss), samples were removed for light mi- croscopy to determine the extent of decay. Other small samples taken for TEM were fixed for 3 h in 4% v/v paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) containing 1% v/v glutaralde- hyde at 4 ° C. Samples were thereafter dehydrated in ethanol and either directly embedded in London resin (London Resin, Basing- Stoke, UK) or embedded after washing in buffer (3 x 15 min) and post-fixed in 2% w/v osmium tetroxide (2 h at RT). Selected ma- terial was sectioned using a diamond knife mounted on a Sorvall Porter Blum Ultramicrotome (RMC, Tucson, Arizona, USA) and sections collected on nickel grids. Post-staining, when performed, was done using uranyl acetate (saturated in 50% ethanol) and Reynolds (1963) lead citrate.

Immunolabelling of samples. Tagging of antigenic sites was done using specific antisera tagged with colloidal gold (Janssen Life Science Products, Auroprobe EM GAM IgG and lgM G15). Im- munoelectron microscopy was as outlined by Horisberger and Rosset (1977), Goodman et al. (1980) and Daniel et al. (1989). U1- trathin sections of non-osmium-treated material were first pre- treated with 10% H202 (10 min), washed in PBS (pH 7.2), and then incubated in 0.1 M aqueous glycine (30 min). After washing in drops of 0.1% bovine serum albumin (BSA)-TRIS-hydrochlo- ride (containing 20 mM TRIS, 0.5 M NaCI, 0.05% Tween 20, 20 mM NaN3 and 1 mg/ml of BSA) sections were incubated in drops of 5% normal goat serum for 15 mins. Sections were then incubated in drops of the specific Mab (1:10 in PBS - 1% normal goat serum) for 1 h at RT. Sections were then washed in 0.1% BSA-TRIS (4 x 5 min) and transferred to drops of the GAM col- loidal gold conjugate diluted 1 : 10 in 0.1% BSA for 30 rain. Grids were then washed in 0.1% BSA-TRIS (2 x 5 min) and subsequently washed four times in distilled H20. Controls included omission of the specific antisera stage and use of antisera preadsorbed with purified manganese peroxidase. Staining when performed was as described above. Sections were viewed using a Philips (Eindhov- en, The Netherlands) 201 transmission electron microscope oper- ated at 60 kV.

Immunolabelling of wood infiltrated with fungal extracts. The abil- ity of Mn(II)-peroxidase to penetrate wood samples of birch was studied by vacuum immersion of small, highly degraded wood pieces removed from soil block samples, in the extract from liquid cultures of P. chrysosporium. This extract prossessed both Mn(II) and lignin peroxidase activity. The concentrated extracts was di- luted in 0.33 M Na tetraborate buffer (pH 3.0) (ca. 7 mg/ml pro- tein) and the wood samples infiltrated for 1 h under slight va- cuum. Thereafter samples were washed briefly in the same buffer (3 x 5 min) and processed as outlined for TEM. The conditions for immunolabelling were as above, except that sections after specific antibody incubation were washed successively in PBS-BSA Tween 20 (pH 7.2); TRIS-hydrochloride-BSA-Tween 20 (pH 8.2); and then incubated with the gold-labelled anti-mouse IgG/IgM conjugate (goat anti-mouse G15) in TRIS-hydrochloride-BSA- Tween 20 (pH 8.4) for 1 h.

Results

Enzyme-linked immunosorbent assay (ELISA)

Clonal hybr idoma colonies 338C and 338D were se- lected based on strong positive reactions in a general screening of hybr idoma clones for IgG production. Colony 338 had been selected for cloning based upon

the strong reaction shown between antibodies secreted in the colony media and the purified Mn(II) -peroxidase inject antigen. After two cycles of dilution cloning, sub- clones 338C and 338D were selected for further exami- nation based on relatively high levels of IgG secretion in low protein Ventrix media. The ELISA/t4o5 was 0.44-0.48 and 0.76-0.81 above the control for 338C and 338D, respectively. Clones 338C and D were shown to produce antibodies that reacted in ELISA with the ori- ginal inject antigen, purified Mn(lI)-peroxidase. Clone 338, however, even though produced to purified Mn(II) -peroxidase was not specific to Mn(II)-peroxi- dase and reacted strongly with purified P. chrysospo- rium ligninase 1 (Paszczyfiski et al. 1986) in ELISA cross-reactivity studies. The area of serological homo- logy between these two enzymes was not determined.

The Mabs of 338C and 338D also showed a cross- reaction in ELISA with fungal extracts o f P. chrysospo- rium, P. sordida and Trametes (Coriolus) versicolor. The Aaos readings generally ranged f rom 0.29-0.49 vs. con- trol levels of 0.0-0.05. It is not surprising that a Mab produced to Mn(II)-peroxidase from P. chrysosporium should show cross-reaction with extracts f rom other white-rot species. T. versicolor for example is known to produce both Mn(II ) and lignin peroxidases (Johans- son and Nyman 1987; JSnsson et al. 1987). Blanchette et al. (1989) has also noted cross-reactivity of antisera f rom lignin peroxidase of P. chrysosporium with other white-rot fungi. The specificity of a Mab will depend upon the nature of the antigen to which the ant ibody is produced, and the degree of homology between the an- tigen and competing metabolites produced by the fun- gus. Because Mab can be produced to specific regions of enzymes, they may be selected for b road or narrow antigen specificity, dependent upon the homology that exists between different enzymes.

In a subsequent study, concentrated preparat ions of 338C and D monoclonals prepared f rom ascites fluid reacted strongly to Mn(II ) -peroxidase/ l igninase and also to four isoenzymes of H R P used as test antigens at 11.5 Ixg/ml when tested for specificity using ELISA. When tested prior to concentrating, clone 338 showed cross-reaction with A4o5 values above negative controls o f 0.15 and 0.17 for acidic H R P isoenzyme RZ 3,0 Type VI I I (Sigma PO889), and H R P acidic isoenzyme RZ3.0 Type I I (Sigma PO889), respectively, vs./14o5 =0.19 for the Mn(II ) -peroxidase/ l igninase positive control.

Labelling of cell corners of degraded wood

Figures 2 and 3 show localization of the Mn(II)-peroxi- dase monoclonal within P. chrysosporium hyphae which had colonized wood. Labelling varied in overall inten- sity according to the hyphae sectioned, but generally showed localization in the region of the cell membrane and fungal cell wall (Figs. 2, 3). In some spores (Fig. 4) and hyphae, a more general labelling of the cytoplasm was obtained, which may be the result o f the ability of 338C and 338D to cross-react with general peroxidases, as noted in the previous section.

677

Fig. 2-8. Cytoplasmic localization of Mn(II)-peroxidase in P. chrysosporium and differential binding of monoclonal antibodies to degraded wall regions of birch. 2, 3 The Mn(II)-peroxidase was localized within the intracellular hyphal cytoplasm and at the cell membrane-cell wall (cw) region (arrows). 4 General labelling within the cytoplasm and outer cell walls of a spore. 5 Degraded cell wall corner of birch labelled with a monoclonal antibody to a partially purified cellulase from Postia placenta. Preferential bind- ing to the lighter secondary walls and within the electron-lucent middle lamella corner was apparent. 6 Highly degraded cell wall corner of birch labelled with anti-Mn(lI)-peroxidase. Labelling

was restricted to the more lignified middle lamella (m/) (darker areas) regions and almost absent from the secondary ($1, $2) wall layers and electron-lucent (elr) area within the middle lamella cell comer (m/c). Figs. 7-8. Localization of Mn(II)-peroxidase in highly degraded cell walls of birch subjected to the degradative action of peroxidase extracts from Phanerochaete chrysosporium. 7 Anti-Mn(II)-peroxidase shows preferential localization in the highly lignified middle lamella (m/) and cell comer (talc) regions. 8 Labelling also occurred in highly degraded fibre wall regions, but was restricted to the lumena wall (I/w) of undegraded fibres (arrows). Bars = 1.0 ~tm

Figure 6 shows b ind ing o f the Mab p robe to a h ighly degraded cell corner middle lamella region. A charac- teristic feature with the Mn(I I ) -pe rox idase p robe local i - zat ion was the lack o f labelling in the e lect ron- lucent areas in the interior o f the degraded middle lamella cell corner, Label was concent ra ted in the l ignin-rich (dark-

er) areas. Previous work using specific chemical and immunolog ica l markers (Daniel and Ni lsson 1987; Danie l et al. 1991) indicated that these areas in birch and other w o o d species are non- or poor ly lignified. The reference Mab p r o d u c e d to extracel lular metabol - ires o f P. placenta was used as a control to el iminate the

678

possibility that the lignin-rich areas had a non-specific affinity for immunogloblins resulting in the illusion of specificity.

Mabs from clone 103, previously shown to bind var- ious fungal cellulases but not to bind ligninase, in con- trast to the probe made to Mn(II)-peroxidase (Fig. 6), were observed to bind only in the electron-lucent areas in degraded middle lamella cell corners (Fig. 5) The cellulose-rich cell walls were also preferentially la- belled. Significant labelling was not apparent in the lig- nin-rich middle lamellar region. Recent work by Blan- chette et al. (1989) using cellulase and xylanase gold conjugates has also shown preferential binding to the electron-lucent areas within the middle lamella cell corner regions of Betula papyrifera.

Labelling of degraded wood infiltrated with fungal extract

Figures 7-10 show diffusion of the concentrated ex- tracts from P. chrysosporium into the cell wall regions of partially degraded birch wood samples; Preferential lo- calization of the 338 probe is seen on lignin-rich cell corner and middle lamella areas. Penetration of the ex- tracts into, and along middle lamella/primary cell wall regions was indicated for fibre cell corners showing ad- vanced attack (Fig. 7) and where adjacent secondary cell walls had been degraded (Fig. 8). Structurally, these middle lamella regions often appeared swollen and reticulate in nature. Concentration of extracts on exposed and degraded secondary cell walls was also noted, although in comparison, middle lamella and ex- posed cell corner regions were more intensely labelled (Figs. 9, 10).

Discussion

Despite substantial progress in recent years in elucidat- ing the biochemistry and molecular biology of enzymic wood degradation, the mechanisms by which fungal metabolites penetrate and degrade wood cell walls are still incompletely understood. There remains a need to understand how metabolites such as enzymes act in concert to degrade the complex wood lignocellulose matrix. Work presented here has focused on the use of Mab to a Mn(II)-peroxidase from P. chrysosporium. A1. though there is the possibility that peroxidases other than Mn(II)-peroxidase were labelled in the wood, the labelling pattern closely followed the degradation ob- served by TEM, suggesting that non-specific labelling was limited. Labelling with Mn(II)-peroxidase Mab in the extensively degraded cell corners sectioned from birch blocks colonized by P. chrysosporium or in highly degraded wood infiltrated with fungal extracts, showed similar patterns of preferential binding to the degraded lignin-rich areas of the cell corners and middle lamella regions.

Labelling of these regions by the Mn(II)-peroxidase probe was not a general affinity reaction of the anti- body to lignin or phenolic moieties in wood, because this labelling was not observed in undegraded control samples or with milled birch wood lignin (not shown). In addition, Mab produced to cellulase labelled only the cellulose-rich regions of the degraded wood cell walls. The present work suggests that the enzymes to which the antibodies were produced were capable of infiltrating the wood cell at later stages of degradation to attack specific wood substrates. Given the size of known Mn(II)-peroxidase (ca. 46000 Da) and endo- and exoglucanases (30-50000 Da), enzymes from P. chrysosporium (Eriksson and Wood 1985), and our cur- rent understanding of the pores in intact wood cell walls, it is unlikely that enzymes would penetrate the

Figs. 9, 10. Detection of Mn(II)-peroxidase in highly degraded cell walls of birch sub- jected to the degradative action of per- oxidase extracts from P. chrysosporium. 9. Penetration of extracts into highly degraded fibre cell walls and middle iamella (m/) re- gions. 10. Labelling of middle lamella cell corners (mlc) was intense but generally less for electron-lucent regions (elf) (inset). Bars = 1.0 ~tm

679

wood cell wall in early stages of decay. In highly de- graded cell wall material, such as used in this work,.i t i s likely that much of the wood cell wall structure would have been modif ied in earlier stages of degradat ion (e.g. through non-enzymic attack). Chemical modifica- tion of wood cell wall components by fungi before sig- nificant morphological changes are apparent is also well known and has been reported for both white- and brown-rot decay fungi (Bauch et al. 1976; Koenigs 1974; Yamashi ta et al. 1978).

In the degraded material examined in our work, dif- fusion of enzymes into cell corner regions was possible via both secondary cell walls and middle lamella re- gions, the latter exposed after advanced decay and rup- ture of tangential and radial fibre secondary cell walls. The observed attack of lignin-rich middle lamella re- gions before total secondary cell wall dissolution may reflect differences in chemistry, accessibility and affin- ity of the Mn(II ) -peroxidase for its substrate. Specific at tack and enzymic diffusion along attacked middle la- mella regions has also been observed during selective lignin degradat ion of birch by Lentinula edodes (Daniel et al. 1990). Recent work has further shown narrowly configured, low molecular mass ( > 12000 Da) cellu- losic degrading enzymes (Schmuck et al. 1986) to be produced by some fungal species, opening the possibil- ity that proper ly configured enzymes could penetrate into structurally modif ied (e.g. enlarged micropores) wood cell walls. The present work therefore suggests that ligninolytic and cellulolytic enzymes are released extracellularly during native wood cell wall degrada- tion and that these enzymes may be not only superfi- cially associated with degrading cell wall regions but also have the capacity to diffuse into swollen and par- tially degraded cell wall middle lamella regions. Evi- dence for the presence and activity of wood-degrading enzymes at sites remote f rom fungal hyphae is impor- tant for explaining how cell wall degradat ion occurs at a distance f rom hyphae, as is particularly the case in advanced stages of white rot decay.

Unlike polyclonal antibodies, which by their nature are a heterologous mixture of antibodies, monoclonal antibodies provide assurance that the ant ibody pro- duced is specific to the purified antigen. However, there is still no assurance that a homologous site on any one antigen will not also be present on other molecules present in the assay environment. Because screening procedures allow selection of clones with either b road or narrow specificity to the antigen of interest, Mab have a distinct advantage over polyclonal antibodies. In future, immunolocal izat ion of degradative enzymes using Mab should allow us to better assess the role of individual fungal enzymes in pathogenicity. This should also allow us to improve our basic understand- ing of the biological degradat ion of wood.

Acknowledgements. This work was supported partly by a fellow- ship from the Organisation for Economic Co-operation and De- velopment (OECD) (Co-operative Research Project on Food Pro- duction and Preservation) and by McIntire-Stennis Funds.

References

Bauch J, Seehan G, Fitzner H (1976) Microspectrophotometrical investigations on lignin of decayed wood. Mater Org (Bed) 3:141-152

Blanchette B, Abad AR, Farrell RL, Leathers TD (1989) Detection of lignin peroxidase and xylanase by immunocytochemical la- belling in wood decayed by basidiomycetes. Appl Environ Mi- erobiol 55:2293-2301

Daniel G, Nilsson T (1987) Comparative studies on the distribu- tion of lignin and CCA elements in Birch using electron mi- croscopy X-ray microanalysis. Int Res Group on Wood Pre- servation. Doe. no. IRG/WP/1328. Obtained from IRG Secre- tariat, Box 5607, Drottning Kristinas v~ig 67B, S-11486 Stock- holm, Sweden

Daniel G, Nilsson T, Pettersson B (1988) Immunolabelling studies on the detection of enzymes during the degradation of wood by Phanerochaete chrysosporium. Int Res Group on Wood Pre- servation. Doe. no. IRG/WP/1364. Obtained from IRG Secre- tariat, Box 5607, Drottning Kristians v~ig 67B, S-11486 Stock- holm, Sweden

Daniel G, Nilsson T, Pettersson B (1989) Intra- and extracellular localization of lignin peroxidase during degradation of solid wood and wood fragments by Phanerochaete chrysosporium by using transmission electron microscopy and immuno-gold la- belling. Appl Environ Microbiol 55:871-881

Daniel G, Pettersson B, Nilsson T, Vole J (1990) Use of immuno- gold cytochemistry to detect MnlI-dependent and lignin pe- roxidases in wood degraded by the white rot fungi Phanero- chaete chrysosporium and Lentinula edodes. Can J Bot 68:920- 933

Daniel G, Nilsson T, Pettersson B (1991) Poorly and non-lignified regions in the middle lamella cell comers of birch (Betula ver- rucosa) and other wood species. Int Assoc Wood Anat Bull 12:70-83

Eriksson K-E, Wood TM (1985) Biodegradation of cellulose. In: Higuchi T (ed) Biosynthesis and biodegradation of wood com- ponents. Academic Press, New York, pp 469-503

Garcia S, Large JP, Prevost MC, Leisola M (1987) Wood degrad- ation by white rot fungi: cytochemical studies using lignin pe- roxidase-immunoglobulin-gold complexes. Appl Environ Mi- crobiol 53:2384-2387

Goodell B, Jellison J (1986) Detection of a brown-rot fungus us- ing serological assays. Int Res Group on Wood Preservation. Doc. no. IRG/WP/1305. Obtained from IRG Secretariat, Box 5607, Drottning Kristinas v~ig 67B, S-11486 Stockholm, Sweden

Goodell B, Daniel G, Jellison J, Nilsson T (1988) Immunolocali- zation of extracellular metabolites from Poriaplacenta. Int Res Group on Wood Preservation. Doc. no. IRG/WP/1361. Ob- tained from IRG Secretariat, Box 5607, Drottning K_ristinas v/~g 67B, S-11486 Stockholm, Sweden

Goodman SL, Hodges GM, Livingston DC (1980) A review of the colloidal gold marker system. In: Johari O (ed) Scanning elec- tron microscopy II. SEM Inc, AMF O'Hare, Chicago, p 133- 146

Highley TL, Ibach R, Kirk TK (1988) Properties of cellulose de- graded by the brown rot fungus Postiaplacenta. Int Res Group on Wood Preservation. Doc. no. IRG/WP/1350. Obtained from IRG Secretariat, Box 5607, Drottning Kristinas v~ig 67B, S-11486 Stockholm, Sweden

Horisberger M, Rosset J (1977) Colloidal gold, a useful marker for transmission and scanning electron microscopy. J Histo- chem Cytochem 25:295-305

Huynh V-B, Crawford RL (1985) Novel extracellular enzymes (ligninases) of Phanerochaete chrysosporium. FEMS Microbiol Lett 28:119-123

Illman BL, Meinholtz DC, Highley TL (1988) Generation of hy- droxyl radicals by the brown rot fungus Postia placenta. Int Res Group on Wood Preservation. Doc. no. IRG/WP/1360.

680

Obtained from IRG Secretariat, Box 5607, Drottning Kristinas v~ig 67B, S-11486 Stockholm, Sweden

Johansson T, Nyman PO (1987) A manganese(II)-dependent ex- tracellular peroxidase from the white rot fungus Trametes ver- sicolor. Acta Chem Stand B41:762-765

Johnstone A, Thorpe R (1982) Immunochemistry in practice. Blackwell Scientific Publications, Oxford

Jrnsson L, Johansson T, Sjrstrrm K, Nyman PO (1987) Purifica- tion of ligninase isozymes from the white rot fungus Trametes versicolor. Acta Chem Scand 41:766-769

Kirk TK, Croan S, Tien M, Murtagh KE, Farrell RL (1986) Pro- duction of multiple ligninases by Phanerochaete chrysospo- rium: effect of selected growth conditions and use of a mutant strain. Enzyme Microb Technol 8:27-32

Koenigs JW (1974) Hydrogen peroxide and iron: a proposed sys- tem for decomposition of wood by brown rot basidiomycetes. Wood Fiber 6:66-80

Murmanis L, Highley TL (1987) Cytochemical localization of eel- lulases in decayed and nondecayed wood. Wood Sci Technol 21:101-109

Paszczyfiski A, Huynh V-B, Crawford R (1986) Comparison of lig- ninase-1 and peroxidase-M2 from the white rot fungus Phane- rochaete chrysosporium. Arch Biochem Biophys 244:750-765

Paszczyfiski A, Crawford RL, Huynh V-B (1988) Manganese per- oxidase of the Phanerochaete chrysosporium. Methods Enzy- mol 161B:264-270

Rener JC, Brown BL, Nardone RM (1985) Cloning hybridoma cells by limiting dilution. J Tissue Cult Methods 9:175-177

Reynolds ES (1963) The use of lead citrate at high pH as an elec- tron opaque stain in electron microscopy. J Cell Biol 17:208- 213

Schmuck M, Pilz I, Hayn M, Esterbauer H (1986) Investigation of cellobiohydrolase from Trichoderma reesei by small angle X- ray scattering. Biotechnol Lett 86:397-402

Shoemaker HE, Leisola MSA (1990) Degradation of lignin by Phanerochaete chrysosporium. J Biotechnol 13:101-109

Schreier M, Kohler G, Hengartner H, Berek C, Trucco M, Forni L (1980) Hybridoma Techiques. Cold Spring Harbor Laborato- ry, N.Y. 64pp

Srebotnik E, Messner K, Foisner R (1988a) Penetrability of white rot-degraded pine wood by the lignin peroxidase of Phanero- chaete chrysosporium. Appl Environ Microbiol 54:2608-2614

Srebotnik E, Messner K, Foisner R, Pettersson B (1988b) Ultras- tuctural localization of ligninases of Phanerochaete chrysospo- rium by immunogold labelling. Curr Microbiol 16:221-227

VoUer A, Bidwell DE, Bartlett A (1977) The enzyme-linked immu- nosorbent assays (ELISA): a review with a bibliography of mi- croplate applications. Flowline, Guernsey

Yamashita Y, Fukazawa K, Ishida S (1978) Histochemical obser- vations of wood attacked by white rot fungi, Coriolus versicolor and Cryptoderma yamanoi. Res Bull Coil Exp For Hokkaido Univ 35:109-119