134 S.Y. Fu et al. / FEMS Microbiology Letters 147 (1997) 133^137 zymes including lignin peroxidase (LiP) [3], manganese-dependent peroxidase (Mn-P) [

Transcription

1 FEMS Microbiology Letters 147 (1997) 133^137 E ect of nutrient nitrogen and manganese on manganese peroxidase and laccase production by Pleurotus sajor-caju Shi Yu Fu a, Hui-sheng Yu a, John A. Buswell b; * a Guangzhou Institute of Chemistry, Chinese Academy of Sciences, Guangzhou , People's Republic of China b Department of Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China Received 18 November 1996; revised 3 December 1996; accepted 4 December 1996 Abstract Pleurotus sajor-caju, strain Pl-27, produces manganese-dependent peroxidase (MnP) and laccase, but not lignin peroxidase, when grown on a defined medium with glucose as sole carbon source. MnP activity was detected in fungal cultures supplemented with both high (26 mm-n) and low (2.6 mm-n) nutrient nitrogen although higher specific activity values were recorded under the latter conditions. Conversely, laccase production was not influenced by nutrient nitrogen levels under the growth conditions adopted. Both the titre and time of appearence of MnP were also affected by the concentration of Mn in the culture medium with highest enzyme levels recorded in cultures supplemented with 15 ppm Mn. Two MnP and five laccase isoforms were identified by FPLC and gel electrophoresis. Keywords: Manganese peroxidase; Laccase; Lignin degradation; Pleurotus sajor-caju 1. Introduction The cultivation of edible mushrooms is a prime example of the conversion of low-value waste, primarily from agricultural practices, into a highervalue commodity which can serve as food material for humans and as a source of commercially important metabolites [1]. Among the major types of nonagaric mushrooms which are cultivated on an industrial scale is the oyster mushroom group which includes Pleurotus ostreatus and P. sajor-caju. These species are highly adaptable and able to growand * Corresponding author. Tel.: ; fax: ; b202768@mailserv.cuhk.hk fruit on a wide variety of lignocellolosic wastes e.g. cereal straws, sawdust, bagasse and banana leaves. Fungal growth on lignocellulosic residues and subsequent fruit body yields are dependent upon the mushroom's ability to synthesise the relevant hydrolytic and oxidative enzymes which convert the individual components of the substrate (i.e. cellulose, hemicellulose, lignin) into lowmolecular weight compounds that can be assimilated for nutrition. Since lignin degradation appears to be the rate-limiting step in substrate colonisation [2], the development of strategies for improving mushroom yields requires a thorough understanding of the ligninolytic system(s) maintained by the fungus and the physiological factors a ecting activity. Fungal attack on the lignin polymer appears to involve several en / 97 / $17.00 Copyright ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S (96)

2 134 S.Y. Fu et al. / FEMS Microbiology Letters 147 (1997) 133^137 zymes including lignin peroxidase (LiP) [3], manganese-dependent peroxidase (Mn-P) [4] and laccase [5], and ligninolytic enzyme activity in several nonedible fungi is regulated by various nutrients including nitrogen, carbon, and manganese [6^8]. As part ofour research aimed at elucidating the lignocellulolytic enzyme systems ofedible mushroom species, we have examined the lignin-degrading enzymes of P. sajor-caju, and also report on the effects of nitrogen and manganese on enzyme production. 2. Materials and methods 2.1. Organism and culture conditions Pleurotus sajor-caju, strain Pl-27 (CMB025), obtained from the culture collection of the Centre for International Services to Mushroom Biotechnology (CISMB), was cultivated in stationary 250 ml Erlenmeyer asks containing 50 ml ofa de ned medium, ph 6.0 [9]. Nitrogen was added as NH 4 NO 3 and L-asparagine at concentrations of2.6 mm-n and 26 mm-n for low nitrogen (LN) and high nitrogen (HN) media, respectively [10] Enzyme assays Manganese peroxidase, lignin peroxidase and laccase activities were assayed at 37³C as described previously [9] FPLC Culture supernatants concentrated by ultra ltration were applied to a Mono-Q (HR 5/5) column pre-equilibrated with 20 mm KH 2 PO 4^KOH bu er (ph 6.2) and bound protein eluted with a linear gradient of0^2.0 M NaCl in the same bu er (25 ml) at a ow rate of1 ml/min (1 ml fractions). Fractions exhibiting both MnP and laccase activity were di erentiated further by hydrophobic interaction chromatography using a phenylsuperose HR-5/5 column (Pharmacia) equilibrated with 20 mm KH 2 PO 4 - KOH bu er (ph 6.2) containing 2 M ammonium sulphate. Bound protein was eluted with a reverse linear gradient ofammonium sulphate (2.0^0 M) in the same bu er (25 ml) at a ow rate of0.5 ml/min (0.5 ml fractions) Polyacrylamide gel electrophoresis (PAGE) and activity staining of gels Native PAGE was performed on samples following FPLC using the Mini-Protean II system (Bio- Rad). Protein bands (stained with Coumassie Blue) exhibiting laccase activity stained green with 2,2P-azino-bis-ethylbenthiazoline (ABTS) (0.03% w/v) in 125 mm acetate bu er, ph 5.0. MnP bands were detected using ABTS (0.075% w/v) in 55.6 mm acetate bu er, ph 4.5, containing 40 mm MnSO 4 and 0.04 mm H 2 O Isoelectric focusing Isoelectric focusing was carried out on freeze-dried samples (from FPLC steps) dissolved in 25 Wl sample bu er (15% glycerol, 6% ampholyte) using the Mini- Protean II system and Pharmalyte 3.0^10.0 (Pharmacia) as ampholyte Protein Protein was measured by the method ofbradford using bovine serum albumin as standard Growth yields Mycelia were ltered through tared Whatman No. 1 lter paper, washed with distilled water and dried at 100³C to constant weight. 3. Results and discussion Fig. 1 shows time courses for MnP production by P. sajor-caju grown in stationary culture under LN and HN conditions on a de ned medium containing glucose as the sole carbon source. Enzyme levels in LN cultures reached a maximum (66 U/ml) after 12 days with a smaller activity peak evident after 18 days, compared with a single peak (47 U/ml) observed after 14 days in HN cultures. However, highest speci c enzyme activities were recorded after 8 days growth under both sets ofconditions, corre-

3 S.Y. Fu et al. / FEMS Microbiology Letters 147 (1997)133^ Fig.1.E ect of nutrient nitrogen levels on manganese peroxidase production by Pleurotus sajor-caju, Pl-27.Values represent the mean of three replicates. a, low nitrogen (2.6 mm-n); b, high nitrogen (26 mm-n). sponding to 64 and 38 U/mg dry weight fungal biomass in LN and HN cultures, respectively.a second, smaller speci c-activity peak (26 U/mg dry weight fungal biomass) was observed in LN cultures after 18 days.therefore, although MnP production in P. sajor-caju is partially suppressed by high nutrient nitrogen levels, the e ect is not nearly as marked as in another edible mushroom, Lentinula edodes, where no MnP activity was recorded over a 13- week experimental period under the HN conditions adopted in this study [9].Conversely, although peak levels of total laccase activity were considerably higher in HN cultures of P. sajor-caju, increased fungal biomass production accounted for this di erence and enzyme production based on speci c activity (i.e. U/mg protein) was similar under both sets of growth conditions (Fig.2).The di erent responses shown by P. sajor-caju and L. edodes with respect to nutrient nitrogen concentration and levels of MnP activity may be explained in part by the contrasting abilities of the two mushrooms to grow on di erent lignocellulosic residues.under both natural and cultivated conditions, L. edodes grows well on woody substrates which are relatively poor in nitrogen [11], whereas P. sajor-caju is much more adaptable and able to grow and fruit on a wide variety of lignocellulosic wastes in which the nitrogen content may be highly variable. The concentration of Mn in the culture medium also a ected the amounts of Mn-P produced in LN cultures of P. sajor-caju (Fig.3).Highest enzyme titres and speci c activities were recorded in cultures containing 15 and 30 ppm added Mn.Readily detectable levels of MnP were also observed at all other Mn concentrations tested (ranging from zero to 100 ppm) although enzyme titres were much lower and the appearence of the enzyme was considerably delayed in cultures supplemented with 100 ppm.fungal growth was only marginally a ected over a Mn concentration range of 0^15 ppm but some inhibitory e ects were evident at 30 ppm and above.after 21 days growth, the amount of fungal biomass produced in cultures containing 30 and 100 ppm was 58% and 26%, respectively, compared with unsupplemented cultures.mnp production in L. edodes was also reported to be manganese dependent although maximum levels of enzyme production in this mushroom were recorded at Mn concentrations much lower than those found for P. sajor-caju [9].More- Fig.2.E ect of nutrient nitrogen levels on laccase production by Pleurotus sajor-caju, Pl-27.Values represent the mean of three replicates. a, low nitrogen (2.6 mm-n); b, high nitrogen (26 mm-n).

4 136 S.Y. Fu et al. / FEMS Microbiology Letters 147 (1997) 133^137 protein and one laccase protein were detected in crude culture supernatants of L. edodes grown in submerged culture with 2.6 mm-n [9]. Forrester et al. [15] also detected only one major MnP when L. edodes was grown on a commercial oak-wood substrate. No LiP activity was detected in cultures of P. sajor-caju grown under the range of conditions used in this study. Thus, P. sajor-caju resembles L. edodes and other ligninolytic fungi such as Dichomitus squalens [16] and Rigidoporus lignosus [17] in apparently utilising MnP and laccase for lignin degradation. Acknowledgments Fig. 3. E ect of manganese concentration on manganese peroxidase production by Pleurotus sajor-caju, Pl-27. Values represent the mean of three replicates. W, 0 ppm; E, 15 ppm; F, 0.3 ppm; R, 30 ppm; j, 1.1 ppm; a, 100ppm; b, 3.0 ppm. This work was supported by Research Grant CUHK18/92M from the Research Grants Council of Hong Kong, and a Strategic Research Grant from The Chinese University of Hong Kong. over, no MnP activity was detectable in cultures of L. edodes without added Mn or supplemented with concentrations of Mn of 30 ppm and above [9]. P. sajor-caju is also more tolerant of Mn since growth of L. edodes was completely inhibited by Mn concentrations in excess of 30 ppm [9]. In the white-rot fungus P. chrysosporium, Mn regulates the expression of MnP by activating the transcription of the mnp gene via a growth-stage-speci c and concentration-dependent mechanism [12]. P. sajor-caju and L. edodes also di er in the number of isoforms of MnP and laccase produced. Five laccase and two MnP peaks were detected by FPLC in culture uids of P. sajor-caju grown under both HN and LN conditions. Two discrete protein peaks exhibiting laccase activity, and a third broader peak with both laccase and MnP activity, were initially resolved by FPLC using a Mono-Q column. The broad peak was further separated by hydrophobic interaction chromatography into ve distinct protein peaks, two of which exhibited only MnP activity and three only laccase activity. The presence of these isoforms was con rmed by PAGE. Multiple forms of MnP [13] and laccase [14] have also been reported in other ligninolytic fungi. Previously, only one MnP References [1] Buswell, J.A. and Chang, S.T. (1993) Edible mushrooms: attributes and applications. In: Genetics and Breeding of Edible Mushrooms (Chang, S.T., Buswell, J.A. and Miles, P.G., Eds.), pp. 297^324. Gordon and Breach Scienti c Publ., Philadelphia, PA. [2] Kirk, T.K. (1983) Degradation and conversion of lignocelluloses. In: The Filamentous Fungi (Smith, J.E., Berry, J.R. and Kristiansen, B. Eds.), Vol. 4, pp. 266^295, Edward Arnold, London. [3] Tien, M. and Kirk, T.K. (1984) Lignin-degrading enzyme from Phanerochaete chrysosporium: puri cation, characterization, and catalytic properties of a unique H 2 O 2 -requiring enzyme. Proc. Natl. Acad. Sci. 81, 2280^2284. [4] Glenn, J.K. and Gold, M.H. (1985) Puri cation and characterization of an extracellular Mn(II)-dependent peroxidase from the lignin-degrading basidiomycete, Phanerochaete chrysosporium. Arch. Biochem. Biophys. 242, 329^341. [5] Thurston, C.F. (1994) The structure and function of fungal laccases. Microbiology 140, 19^26. [6] Keyser, P., Kirk, T.K. and Zeikus, J.G. (1978) Ligninolytic enzyme system of Phanerochaete chrysosporium: synthesized in the absence of lignin in response to nitrogen starvation. J. Bacteriol. 135, 790^797. [7] Je ries, T.W., Choi, S. and Kirk, T.K. (1981). Nutritional regulation of lignin degradation by Phanerochaete chrysosporium. Appl. Environ. Microbiol. 42, 290^296. [8] Bonnarme, P. and Je ries, T.W. (1990). Mn(II) regulation of lignin peroxidases and manganese-dependent peroxidases

5 S.Y. Fu et al. / FEMS Microbiology Letters 147 (1997) 133^ from lignin-degrading white-rot fungi. Appl. Environ. Microbiol. 56, 210^217. [9] Buswell, J.A., Cai, Y.J. and Chang, S.T. (1995) E ect of nutrient nitrogen and manganese on manganese peroxidase and laccase production by Lentinula edodes. FEMS Microbiol. Lett. 128, 81^88. [10] Buswell, J.A., Mollet, B. and Odier, E. (1984) Ligninolytic enzyme production by Phanerochaete chrysosporium under conditions of nitrogen su ciency. FEMS Microbiol. Lett. 25, 295^299. [11] Cowling, E.B. and Merrill, W. (1966) Nitrogen in wood and its role in wood deterioration. Can. J. Bot. 44, 1539^1554. [12] Brown, J.A., Alic, M. and Gold, M.H. (1991) Manganese peroxidase gene transcription in Phanerochaete chrysosporium: activation by manganese. J. Bacteriol. 173, 4101^4106. [13] Ruttimann-Johnson, C., Cullen, D. and Lamar, R.T. (1994) Manganese peroxidases of the white rot fungus Phanerochaete sordida. Appl. Environ. Microbiol. 60, 599^605. [14] Morohoshi, N., Wariishi, N., Muraiso, C., Nagai, T. and Haraguchi, T. (1987) Degradation of lignin by the extracellular enzymes of Coriolus versicolor. IV. Properties of three laccase fractions fractionated from the extracellular enzymes. Mokuzai Gakkaishi. 33, 218^225. [15] Forrester, I.T., Grabski, A.C., Burgess, R.R. and Leatham, G.F. (1988) Manganese, Mn-dependent peroxidases, and the biodegradation of lignin. Biochem. Biophys. Res. Comm. 157, 992^999. [16] Perie, F.H. and Gold, M.H. (1991) Manganese regulation of manganese peroxidase expression and lignin degradation by the white rot fungus Dichomitus squalens. Appl. Environ. Microbiol. 57, 2240^2245. [17] Galliano, H., Gas, G., Seris, J.L. and Boudet, A. (1991) Lignin degradation by Rigidoporus lignosus involves synergistic action of two enzymes: Mn peroxidase and laccase. Enzyme Microb. Technol. 13, 478^482.