Extracellular glucan production by Postia (= Poria) placenta

Transcription

1 Extracellular glucan production by Postia (= Poria) placenta By JESSIE A. MICALES, ADRIAN L. RICHTER, and TERRY L. HIGHLEY Forest Products Laboratory 1 Forest Service, U.S. Department of Agriculture, Madison, Wisconsin, U.S.A K e y w o r d s : Brown-rot fungi, carbohydrate metabolism, glucan, laminarinase, glucanase 1. Introduction Contents 2. Materials and methods 2.1 Culture maintenance and determination of decay ability Glucan production Enzyme activity 3. Results 3.1 Decay capacity of ME20 L8035spA Extracellular glucan production 3.3 Laminarinase and glucan-degrading enzyme activity 4. Discussion 5. Summary References 1. Introduction A common morphological feature of wood-decay fungi in decayed wood is the presence of an extracellular matrix or sheath (J. G. PALMER et al., 1983a, b) that surrounds the hyphae. This matrix is thought to perform several diverse functions, all critical to the wood-decay process. These functions include (1) the concentration of carbohydrate-degrading enzymes and retention of metabolites, (2) nutrient storage, (3) attachment of the hyphae to the substrate, and (4) protection against desiccation and other forms of chemical and physical damage by environmental agents (T. L. HIGHLEY, 1987). The chemical composition of the extracellular matrix produced by Coniophora puteana (Schum. ex Fr.) Karst., a brown-rot fungus, and Trametes lactinea Berk., a white-rot fungus, has been determined by 13C-NMR to consist primarily of glucan (D. M. FRANCIS and L. E. LEIGHTLEY, 1983). 1 The Forest Products Laboratory is maintained in cooperation with the University of Wisconsin. This article was written and prepared by U.S. Government employees on official time, and it is therefore in the public domain and not subject to copyright. 17* In: Material und Organismen. 24(4): ; 1990.

2 260 Jessie A. Micales, Adrian L. Richter, and Terry L. Highley Wood-decay fungi also produce extracellular polysaccharides in liquid media, particularly under certain nutrient conditions. This material has been shown to consist primarily of.3-glucan with single (1-6)-linked -D-glucopyranosyl units and has been identified in culture filtrates of Phanerochaete chrysosporium Burds. (B. BES; et al., 1987; A. J. BUCHALA and M. LEISOLA, 1987), Schizophyllum commune Fr. (W. STEINER et al., 1987; P. VAN DER VALK et al., 1977), and Stereum sanguinolentum (Alb. et Schw.: Fr.) Fr. (K. AXELSSON et al., 1968). Glucan has also been shown to ensheathe the hyphae of S. commune in liquid culture (P. VAN DER VALK et al., 1977; J. G. H. WESSELS et al., 1972). Because of their similarity in chemical composition, the extracellular glucan obtained in liquid culture is most probably identical to the matrix observed on hyphae and wood surfaces in decayed wood. J. A. MICALES and T. L. HIGHLEY (in press) have reported an isolate of the brown-rot fungus Postia placenta (Fr.) M. Lars. and Lomb. that is unable to degrade wood. This isolate, ME20, is able to produce extracellular carbohydrate-degrading enzymes, H 2O 2, and oxalic acid, all thought to be essential to the wood degrading process. Liquid cultures of ME20 are not as viscous as those of P. placenta, which are able to degrade wood (J. A. MICALES, personal observation). Therefore, the objective of the study reported here was to determine whether the production and degradation of extracellular glucan by ATE20 varies from that of other isolates of P. placenta. 2. Materials and methods 2.1 Culture maintenance and determination of decay ability The following isolates of P. placenta were used in this study: MAD698 (dikaryotic). ME20 and L8035spA (monokaryotic), and ME20 L8035spA (dikaryotic, derived by crossing ME20 and L8035spAi All isolates were stored in the culture collection of the Center for Forest Mycology Research, Forest Products Laboratory, Madison, Wisconsin. The wood decay capacity of ME20 L8035spA was determined by the standard ASTM soil-block method (ASTM, 1971) using southern yellow pine blocks (25.4 mm 25.4 mm 3.2 mm, long axis parallel to the grain). Soil-block bottles were incubated at 27 C and 70 percent relative humidity for 12 weeks. At the end of the period, the percentage of weight loss of each block was determined. The decay abilities of MAD698 and ME20 were previously reported (J. A. MICALES and T. L. HIGHLEY, in press).

3 Extracellular glucan production by Postia Glucan production Cultures were grown in aerated liquid media containing a basal salt solution (T. L. HIGHLEY, 1973) and variable carbohydrates. Carbohydrates were obtained from the following sources: cellobiose - Sigma: xylose, mannose and fructose - Eastman; glucose - Fisher; and galactose - Pfanstiehl Laboratory, Waukegan, Illinois. 2 Two-liter flasks containing 1,500 ml of medium were inoculated with 50 ml hyphal suspension, prepared by blending 2 to 3-week-old cultures of P. placenta grown in 25 ml 1-percent cellobiose with 100 ml sterile distilled water. Inoculum was standardized in large experiments by mixing several suspensions of a given isolate and dispensing the mixture into 50-ml aliquots. Cultures were incubated at 25 C for 10 days unless otherwise specified. Extracellular glucan was collected from culture filtrates by alcohol precipitation as described by W. STEINER et al. (1987). The chemical composition of the glucan isolated from MAD698 was determined by HPLC after- acid hydrolysis using the procedure of R. C. PETTERSEN et al. (1984). Protein concentration was determined (O. H. LOWRY et al., 1951) using bovine serum albumin for the standard curve. 2.3 Enzyme activity Isolates were grown in 25-ml stationary liquid cultures in 250-ml Erlenmeyer flasks with 0.5 percent cellobiose in a basal salts solution (T. L. HIGHLEY, 1973). Each flask was inoculated with a 5-mm-diameter mycelial plug taken from the margin of 7- to 14-day-old colonies grown on 2 percent (w/v) malt extract agar. Cultures were incubated at 25 C for 4 or 8 weeks. Culture filtrates were collected by vacuum filtration through glass-fibered filter paper, dialyzed overnight against deionized water, ph 4.0, and assayed for laminarinase and glucan-degrading enzyme activity by measuring the increase in reducing groups using Nelson's modification of the Somogyi method (N. NELSON, 1944). Laminarin (Sigma) and extracellular glucan, isolated from P. placenta as described above, were used as substrates. Enzyme assays were performed by mixing 1 ml dialyzed culture filtrate with 1 ml substrate (1 percent w/v in 0.1 M McIlvaine buffer, ph 5.0) and incubating at 40 C for 24 hours. One unit of enzyme activity was defined as the amount of enzyme needed to liberate reducing power equivalent to 1 µmol of glucose per 24 hours at 40 C. The ability of a commercial preparation of laminarinase (Sigma L-9259) to degrade the extracellular glucan produced by MAD698 was also estimated by the reducing sugar assay (N. NELSON, 1944). Two units of laminarinase in 100 µ1 0.1 M McIlvaine buffer, ph 5.0, were added to 1 ml glucan (1 percent w/v in 0.1 M McIlvaine buffer, ph 5.0) and incubated as described above. 2 The use of trade or firm names in this publication is for reader information and does not imply endorsement by the U.S. Department of Agriculture of any product or service.

4 262 Jessie A. Micales, Adrian L. Richter, and Terry L Highley 3. Results 3.1 Decay capacity of ME20 L8035spA The weight loss of southern yellow pine test blocks by the hybrid isolate was 55.3 percent (based on an average of seven replications: standard error, 11.0). This value was similar to the percentage of weight loss caused by MAD698 (60.4 percent) (J. A. MICALES and T. L. HIGHLEY, 1989). The weight loss caused by ME20 was 5.1 percent (J. A. MICALES and T. L. HIGHLEY, 1989). 3.2 Extracellular glucan production The quantity of glucan isolated from culture filtrates of MAD698, L8035spA, ME20, and ME20 L8035spA is listed in Table 1. All isolates except ME20 produced extracellular glucan, although the quantities produced by MAD698 and the hybrid were quite variable. The chemical composition of the glucan formed by MAD698 was determined, after acid hydrolysis, as 87 percent glucose, 0.4 percent mannose, and 12.6 percent unidentified material. Xylose, galactose, and arabinose were not present. Less than 10 µg protein was detected per milligram of the dried glucan. A commercial preparation of laminarinase released large quantities of reducing sugars from the glucan. T a b 1 e 1: Glucan production by isolates of Postia placenta 1 1 Isolates grown in aerated culture in 1 percent cellobiose + basal salts for 10 days at 25 C The amount of glucan produced by MAD698 varied with time (Tab. 2). Little or no glucan was isolated early or late in the growth curve; the largest

5 Extracellular glucan production by Postia 263 quantities were formed during the logarithmic phase. Isolate ME20 failed to produce glucan throughout the growth cycle. The type of carbon source did not greatly influence the amount of glucan produced by MAD698 (Tab. 3). The largest quantities of glucan were formed when the fungus was grown in xylose or fructose; growth on galactose, mannose, cellobiose, and glucose all resulted in equivalent levels of glucan formation. There was little difference in glucan production at different concentrations of glucose and cellobiose. Isolate ME20 failed to produce any glucan regardless of the carbon source or concentration. T a b l e 2 : Dry weight, protein and glucan production by MAD698 and ME20 with time 1 1 Isolates grown in aerated 1 percent cellobiose t basal salts at 25 C. 2 All readings represent the average of two replications. T a b l e 3 : Effect of carbon source on production of extracellular glucan by MAD698 1,2 1 Isolates grown in aerated culture at 25 C for 10 days 2 Isolate ME20 produced no glucan.

6 264 Jessie A. Micales, Adrain L. Richter, and Terry L. Highley 3.3 Laminarinase and glucan-degrading enzyme activity Levels of laminarinase and enzymes specific for the degradation of the glucan produced by P. placenta, are listed in Table 4. Laminarinase production per milligram dry weight was not significantly different between isolates MAD698 and L8035spA. Laminarinase production, by ME20 was three times higher than that of the "normal" strains. The hybrid exhibited an intermediate level of laminarinase formation. Laminarinase represented a larger proportion of total extracellular protein for ME20 and L8035spA than for the other isolates. Table 4: Production of laminarinase and glucan-dgrading enzymes by isolates of Postia placenta after 4 weeks 1 Isolate ME20 and the hybrid also produced larger quantities of enzymes capable of breaking down the glucan per milligram dry weight of mycelium. After 4 weeks of growth, the culture filtrates of MAD698 and L8035spA were unable to break down the glucan into reducing sugars. Of the four isolates, the hybrid devoted the largest proportion of its total extracellular protein production to the formation, of these glucan-degrading enzymes After 2 months in static culture, MAD689 was able to degrade the glucan (Tab. 5) and produced levels of glucan-degrading enzymes equivalent to that produced by ME20. The source of the glucan did not greatly influence the amount of degradation produced by any of the isolates, although the glucan formed by the hybrid was most extensively degraded by MAD698, ME20, and the hybrid. Isolate ME20 continued to produce high levels of glucandegrading enzymes per milligram dry weight of mycelium. Isolate L8035spA did not extensively degrade the glucan, and the hybrid produced intermediate levels of degradation. Of the four isolates, MAD698 devoted the largest portion of its total protien production to the formation of glucan-

7 Extracellular glucan production by Postia 265 Table 5 : Degradation of extracellular glucan by isolates of Postia placenta after 8 weeks 1 1 Isolates groan in nonaerated 1 percent cellobiose + basal salts at 25 C. 2 All readings represent the average of two replications. degrading enzymes. The remaining isolates all produced similar quantities of enzyme per microgram of protein. 4. Discussion The nondegradative isolate of P. placenta, ME20, failed to produce extracellular glucan when grown in liquid culture, regardless of the carbon source or concentration. Isolates of P. placenta that degrade wood formed varying amounts of this polysaccharide. The chemical composition of the glucan produced by MAD698 was 87 percent glucose. This compares well with the chemical composition of glucan produced by other wood-decay fungi, including S. commune (90 percent) (J. G. H. WESSELS et al., 1972) and P. chrysosporium (92 percent) (B. BES et al., 1987). A commercial preparation of laminarinase was able to degrade the glucan. Such activity strongly suggests that the material is indeed a -1,3-glucan, as described for other fungi (K. AXELSSON et al., 1968; B. BES et al., 1987; A. J. BUCHALA and M. LEISOLA, 1987; W. STEINER et al., 1987; P. VAN DER VALK et al., 1977). The glucan-degrading enzyme activity, which was detected in culture filtrates of P. placenta, was probably due to one or more laminarinases. Enzyme purification procedures are currently being used to determine whether these activities are due to the same enzymes.

8 266 Jessie A. Micales, Adrian L. Richter, and Terry L. Highley The production of glucan varied over the life cycle of MAD698, with the largest accumulations occurring during the logarithmic growth phase. Little glucan was harvested from 20-day-old cultures. Such a distribution agrees with reports for P. chrysosporium, where extracellular glucan is considered a storage material that is synthesized when glucose is excessive and metabolized when glucose becomes limiting (B. BES et al., 1937). In whiterot fungi, excess glucose represses the induction of sugar oxidizing enzymes that produce the hydrogen peroxide necessary for lignin degradation (B. BES et al., 1987). Cellulase and hemicellulase production by white-rot fungi is also repressed by growth on simple sugars (K. E. ERIKSSON and G. GOODELL, 1974; T. L. HIGHLEY, 1977), so the removal of excess glucose by conversion to glucan allows decay to proceed. Although the production of carbohydratedegrading enzymes by many brown-rot fungi is not repressed by the presence of glucose (T. L. HIGHILEY, 1977), the formation of extracellular glucan as a storage material would still be an important survival mechanism. Isolate ME20 and the hybrid produced much higher levels of glucan-degrading enzymes than MAD698 and L8035spA after 4 weeks in culture: MAD698 produced equivalent levels much later in the growth cycle. The regulation of the glucan-degrading enzyme may be faulty in ME20 and the hybrid, and the matrix may be prematurely degraded. However, the hybrid is able to accumulate extracellular glucan and to decay wood, so some other factor must also be involved. Diversity in polysaccharide production has also been observed among isolates of S. commune certain strains derived from Kniep stock (J. G. H. WESSELS, 1965) do not secrete carbohydrates into the medium although low levels of polysaccharides may be associated with the hyphal surface (J. G. H. WESSELS and D. J. NIEDERPRUEM, 1967; J. G. H. WESSELS et al., 1972). The decay capacity of these isolates has not been reported. Another characteristic shared by ME20 and the S. commune isolates is the absence of aerial hyphae. In S. commune both traits have been associated with mutations in the A and E mating factors that relate directly to cell wall synthesis (J. G. H. WESSELS and D. J. NIEDEPRUEM. 1967; J. G. H. WESSELS, 1969). Common A heterokaryons and homokaryons of S. commune with primary mutations in the B mating factor grow little or no aerial hyphae and produce low levels of extracellular carbohydrates. The composition of the glucan in the fungal cell walk of these isolates is also aberrant. These isolates exhibit a much higher ratio of alkali-soluble glucan (S-glucan) to alkali-resistant glucan (R-glucan) than true heterokaryons. and they also produce much higher levels of R-glucanases (enzymes that degrade the alkali-resistant glucan) during growth. High levels of R-glucanase may result in the formation of certain morphological features, including appressed mycelial growth in agar culture (J. G. H. WESSELS and D. J. NIEDERPREUM 1967), which are also observed in ME20. Isolate ME20 produced extremely high levels of

9 Extracellular glucan production by Postia 267 laminarinase, a glucanase that breaks down -1,3-glucan, but J. G. H. WESSELS and D. J. NIEDERPRUEM (1967) demonstrated that laminarin cannot be used as a substrate to estimate R-glucanase activity. Studies are being conducted to examine R- and S-glucanase production and cell wall composition of ME20 and other isolates of P. placenta. Although similar enzymatic mechanisms may be responsible for the abnormal morphology of ME20 and the S. commune isolates, the genetics of the organisms must be expressed quite differently since P. placenta exhibits unifactorial, rather than bifactorial, sexual incompatibility (J. G. H. WESSELS, 1978). The proposed roles of the extracellular matrix -nutrient storage, concentration of enzymes and metabolites, attachment to substrate, and protection of hyphae (T. L. HIGHLEY, 1987) - explain why a culture that is unable to form adequate quantities of matrix material is unable to degrade wood. Electron microscopy is currently being used to determine whether ME20 actually fails to produce a matrix when in contact with wood or whether this phenomenon is associated only with liquid culture. The inhibition of matrix formation could be an effective means of preventing the damage caused by wood-decay fungi. 5. Summary A monokaryotic strain of Postia (= Poria) placenta, ME20, which is unable to degrade wood, also failed to produce extracellular polysaccharide when grown in liquid culture, regardless of carbon source or concentration. Other isolates of P. placenta, including another monokaryon and a hybrid of this monokaryon and ME20, produced large quantities of this material. The polysaccharide consisted primarily of glucose upon acid hydrolysis and resembled the glucan reported in culture filtrates of other wood-decay fungi. It was produced primarily during the logarithmic phase of growth. Isolate ME20 formed high levels of laminarinase and glucan-degrading enzymes compared to the other isolates; the glucan of ME20 may be prematurely degraded. This study supports the importance of the extracellular matrix in the wood-decay process. Zusammenfassung Extracelluläre Glucan-Produktion von Postia (= Poria) placenta Ein monokaryotischer Stamm von Postia (= Poria) placenta, ME20, der nicht in der Lage war, Holz abzubauen, konnte auch keine extracellulären Polysaccharide in Flüssigkultur bilden, unabhängig von einer Kohlenstoffquelle oder -konzentration. Andere Isolierungen von P. placenta, einschließlich eines anderen Monokaryonten und eines Hybriden dieses Monokaryonten und ME20 produzierten große Mengen dieses Materials. Das Polysaccharid bestand hauptsächlich aus Glucose nach Säurehydrolyse und ähnelte dem Glucan, das aus Kulturfiltraten anderer holzzerstörender Pilze beschrieben ist. Es wurde im wesentlichen während der logarithmischen Wachstumsphase produziert. Verglichen mit anderen Isolierungen bildete ME20 große Mengen an Laminarinasen und Glucan-abbauenden Enzymen; von ME20 gebildetes Glucan

10 268 Jessie A. Micales, Adrian L. Richter, and Terry L. Highley kann vorzeitig abgebaut werden Diese Untersuchung unterstreicht die Bedeutung der extracellulären Matrix im Hoizzerstöungsprozeß. Résumé Production de glucane extracellulaire chez Postia (= Poria) placenta Une souche monochariotique de Postia (= Poria) placenta, ME20, qui n'était pas en mesure d'altérer le bois, n'a pa pu, non plus, produire de polysaccharides extracellulaires en milieu de culture liquide, indépendamment d'une struche de carbone et de sa concentration. D'autres isolats de P. placenta y compris un autre monocharion avec ME20, produisirent une forte quantite de cette matiére. Ce polysaccharide était composé principalement de glucose issu d'hydrolyse acide et ressemblant au glucane décrit comme provenant des filtrats de culture d'autres champignons d'altération du bois. La production se réalisait surtout durant la phase logarithmique de la croissance. Comparé à d'autres isolats. ME20 fournit de grandes quantités de laminarinases et d'enzymes destructeurs de glucane. Le glucane produit par ME20 peut donc être détruit prématurement. Cette etude montre l'mportance de la matrix extracellulaire dans le processus de la dégradation du bois. References American Society for Testing and Materials: Standard method fur accelerated laboratory test of natural decay resistance of woods. ASTM D Philadelphia, Pa., AXELSSON, K., BJORNDAL. H. and ERIKSSON, K. E,: An extracellular glucan produced by the rot fungus Stereum sanguinolentum. Acta Chem. Scand. 22 (1968) BES, B., PETTERSSON, B., LENNHOLM, H., IVERSON, T. and ERIKSSON, K. E.: Synthesis, structure, and enzymatic degradation of an extracellular glucan produced in nitrogen-starved cultures of the white rot fungus Phanerochaete chrysosporium. Biotechnol. and Appl. Biochem. 9 (1997) BUCHALA, A. J. and LEISOLA. M.: Structure of the -D-glucan secreted by Phanerochaete chrysosporium in continuous culture. Carbohydr. Res. 165 (1987) ERIKSSON, K. E. and GOODELL. G.: Pleiotropic mutants of the wood-rotting fungus Polyporus adustus lacking cellulase, mannanase and xylanase. Canad. J. Microbiol. 20 (1974) FRANCIS, D. M. and LEIGHTLEY, L. E.: Estracellular layers of wood decay fungi and copper tolerance. Internat. Res. Group on Wood Pres. Doc. No. IRG/NP/1180, 1983, 7 pp. HIGHLEY, T. L.: Influence of carbon source on cellulase activity of white-rot and brown-rot fungi Wood Fiber 5 (1973) HIGHLEY, T. L.: Hemicellulases of white- and brown-rot fungi in relation to host preferences. Material u. Organismen 11 (1977) HIGHLEY, T. L.: Biochemical aspects of white-rot and brown-rot decay. Internat. Res. Group on Wood Pres. Doc. No. IRG/WP/1319, 1987, 22 pp.

11 Extracellular glucan production by Postia 269 LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. J.. and RANDALL, R. J.: Protein measurements with the folin phenol reagent. J. Biol. Chem. 193 (1951) MICALES, J. A. and HIGHLEY, T. L.: Some physiological characteristics of a nondegradative strain of Postia (= Poria) placenta. Mycologia 81 (1989) NELSON, N.: A photometric adaptation of the Somogyi method for determination of glucose. J. Biol. Chem. 153 (1944) PALMER, J. G., MURMASIS, L. and HIGHLEY, T. L.: Visualization of hyphal sheath in wood-decay hymenomycetes. I. Brown-rotters. Mycologia 75 (1983 a) PALMER, J. G., MURMANIS, L. and HIGHLEY, T. L.: Visualization of hyphal sheath in wood-decay hymenomycetes. II. White-rotters. Mycologia 75 (1983 b) PETTERSEN, R. C., SCHWANDT, V. H. and EFFLAND, M. J.: An analysis of the wood sugar assay using HPLC: A comparison with paper chromatography. J. Chromatogr. Sci. 22 (1984) STEINER, W., LAFFERTY, R. M., GOMES, I. and ESTERBAUER, H.: Studies on a wild strain of Schizophyllum commune: cellulase and xylanase production and formation of the extracellular polysaccharide schizophyllan. Biotechnol. Bioeng. 30 (1987) VAN DER VALK, P., MARCHANT, R. and WESSELS, J. G. H.: Ultrastructural localization of polysaccharides in the wall and septum of the Basidiomycete Schizophyllum commune. Exper. Mycol. 1 (1977) WESSELS, J. G. H.: Morphogenesis and biochemical processes in Schizophyllum commune Fr. Wentia 13 (1965) WESSELS, J. G. H.: Biochemistry of sexual morphogenesis in Schizophyllum commune: Effect of mutations affecting the incompatibility system on cell-wail metabolism. J. Bacteriol. 98 (1969) WESSELS, J. G. H.: Incompatibility factors and the control of biochemical processes. In: SCHWALB, M. N. and MILES, P. G. (Eds.): Genetics and Morphogenesis in the Basidiomycetes. New York (Acad. Press) 1978, WESSELS, J. G. H., KREGER, D. R., MARCHANT, R., REGENSBURG, B. A. and DEVRIES, O. M. H.: Chemical and morphological characterization of the hyphal wall surface of the basidiomycete Schizophyllum commune. Biochem. et Biophys. Acta 273 (1972) WESSELS, J. G. H. and NIEDERPRUEM, D. J.: Role of a cell-wall glucan-degrading enzyme in mating of Schizophyllum commune. J. Bacteriol. 94 (1967) Address of the authors: J. A. MICALES A. L. RICHTER Dr. T. L. HIGHLEY Forest Products Laboratory Forest Service U.S. Department of Agriculture One Gifford Pinchot Drive Madison, WI U.S.A.