In situ localization of manganese peroxidase production in mycelial pellets of Phanerochaete chrysosporium

G. Jiménez-Tobon1, W. Kurzatkowski2, B. Rozbicka2, J. Solecka2, I. Pocsi3 and M. J. Penninckx1

1 Laboratoire de Physiologie et Ecologie Microbienne, Faculté des Sciences, Université Libre de Bruxelles, c/o Institut Pasteur, 642 Rue Engeland, B-1180 Brussels, Belgium
2 Laboratory of Actinomycetes and Fungi imperfecti, National Institute of Hygiene, Warsaw, Poland
3 Department of Microbiology and Biotechnology, Faculty of Sciences, University of Debrecen, Debrecen, Hungary

Correspondence
M. J. Penninckx
upemulb{at}resulb.ulb.ac.be


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The ultrastructure of Phanerochaete chrysosporium hyphae from pellets in submerged liquid cultures was investigated in order to learn more about the interrelation between fungal architecture and manganese peroxidase (MnP) production. At day 2 of cultivation, some subapical regions of hyphae in the outer and middle zones of the pellet initiated differentiation into intercalary thick-walled chlamydospore-like cells of about 10 µm diameter. At the periphery of the cytoplasm of these cells, a large number of mitochondria and Golgi-like vesicles were observed. The sites of MnP production were localized at different stages of cultivation by an immunolabelling procedure. The immunomarker of MnP was mainly concentrated in the chlamydospore-like cells and principally distributed in Golgi-like vesicles located at the periphery of the cytoplasm. The apices of hyphae in the outer layer of the pellets were apparently minor sites of MnP production. Maximal MnP release into the culture supernatant coincided with apparent autolysis of the chlamydospore-like cells. Production of extracellular autolytic chitinase and protease coincided with the disappearance of these structures from the pellets. The chlamydospore-like cells observed in the mycelial pellets of P. chrysosporium could be metabolically active entities operating as an enzyme reservoir, delivering their content into the surrounding medium possibly by an enzyme-mediated autolytic process.


Abbreviations: LiP, lignin peroxidase; MnP, manganese peroxidase


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Lignin peroxidase (LiP: EC 1.11.1.14), manganese peroxidase (MnP: EC 1.11.1.13) and laccase (EC 1.10.3.2) are the most important enzymes associated with the lignin degradative pathway of white rot fungi (Kirk & Farrell, 1987; Leonowicz et al., 2001). These enzymes have several potential applications in bioremediation of recalcitrant compounds including dyes, polyphenols and other xenobiotics (Lamar et al., 1992). However, commercial exploitation of these biocatalysts is often hampered because of the lack of knowledge about their production parameters in industrial conditions. Mycelial morphology in submerged cultures is usually classified into two different forms: individual filamentous mycelia (dispersed form) and spherical colonies called pellets (Prosser, 1995; Cox et al., 1998). The disadvantages of dispersed mycelial growth include increased wall growth and reduction in efficiency of mixing and oxygen supply to the cells due to increased viscosity of the medium (Schügerl et al. 1983). These problems may be solved to some extent by growth in the form of pellets, which also optimizes harvesting owing to the improved filtration characteristics of the broth (Braun & Vecht-Lifshitz, 1991).

The pellets are formed by the development of a spore inoculum into agglomerates of hyphae trapped together during germination (Gerin et al., 1993). The exact mechanism behind pellet formation is not known. The morphology of pellets may depend on many factors including the inoculum level, genetic factors, medium composition, addition of surfactants, shearing forces, etc. (Metz & Kossen, 1977).

Cultivation in the form of pellets was proposed for the industrial production of some secondary metabolites and enzymes (Braun & Vecht-Lifshitz, 1991). Because fungal morphology affects the rheological properties of the fermentation broth, control of morphology is highly desired in industrial fungal fermentation (Park et al., 2002). Phanerochaete chrysosporium grown in agitated liquid culture typically forms pellets, and produces MnP under nitrogen limitation in the presence of Mn2+. The size of the pellets was found to be a crucial factor for LiP and MnP production (Jaspers et al., 1994; Jiménez-Tobon et al., 1997). In order to learn more about the interrelation between fungal architecture and MnP production, we investigated the ultrastructure of P. chrysosporium hyphae from pellets in submerged liquid cultures. The enzyme production was found to be principally associated with chlamydospore-like spherical cells, produced by differentiation of subapical cells located in the outer and middle zones of the pellet. These structures could operate as an enzyme reservoir, delivering their content into the surrounding medium.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Growth conditions and enzyme assay.
Phanerochaete chrysosporium ATCC 24725 was maintained on YMPG agar (Bonnarme & Jeffries, 1990). The strain was grown at 30 °C on MnP producing medium (Bonnarme & Jeffries, 1990). Conidiospore production for inoculation, MnP assay and determination of pellet size were described previously (Jiménez-Tobon et al., 1997). One unit (U) of MnP activity is defined as the amount of enzyme catalysing the oxidation of 1 µmol vanillylacetone min-1.

Extracellular chitinase (EC 3.2.1.14) was estimated with colloidal crab shell chitin (Xia et al., 2001). One unit (U) of chitinase activity is defined as the amount of enzyme catalysing the release of reducing sugars corresponding to 1 µmol N-acetylglucosamine min-1.

Extracellular protease activity (EC 3.4.–.–) was measured with Azocoll as the substrate (Dosoretz et al., 1990). One enzyme unit (U) is defined as the amount of enzyme which catalyses the release of azo dye causing an A520 change of 0·001 min-1.

Procedure for microscopy.
Pellet formation and growth of mycelium was monitored by phase-contrast microscopy (Docuval microscope, Carl-Zeiss).

For electron microscopy ultrathin sections of hyphae from the periphery and central parts of pellets were prepared by the procedure of Strunk (1978) modified by Kurzatkowski et al. (1991). Immunolabelling on ultrathin sections was carried out basically according to Kurzatkowski et al. (1991). For the immunodetection of MnP, a rabbit antiserum obtained according to Jiménez-Tobon (1999) was raised against a highly purified preparation of the H3 enzyme isoform (Orth et al., 1994). Preimmune serum was obtained from the same rabbit before immunization. Western blot experiments have shown that the antibodies obtained react with purified MnP, although some faint cross-reaction was observed with H8 LiP isoenzyme (Jiménez-Tobon, 1999).

Antigen–antibody complexes were visualized with goat anti-rabbit (IgG)–15 nm gold conjugate (BioCell). Controls were included either by omitting the antibody against MnP, or by adding pre-immune serum (dilution 1 : 400 in PBG; Jiménez-Tobon, 1999), followed by goat anti-rabbit IgG–15 nm gold conjugate. It was checked that the immunomarker was found only at locations where the antigen (MnP)–antibody reaction was effective. The ultrathin sections were examined with a JEM 100 C (JEOL) transmission electron microscope at 80 kV.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
MnP production pattern
A typical curve of MnP production in the supernatant of a P. chrysosporium culture forming pellets is shown in Fig. 1. No extracellular MnP activity was observed before day 4 of cultivation, which corresponded to the end of the trophophase and entry in the idiophase (Jiménez-Tobon et al., 1997). The maximal level of MnP secreted into the extracellular medium was reached between days 7 and 8 of cultivation and thereafter decreased. Neither LiP nor laccase was produced in the cultivation procedure used here (Bonnarme & Jeffries, 1990; Jiménez-Tobon et al., 1997).



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 1. Enzyme and biomass production in a culture of P. chrysosporium. The cultivations were performed at 30 °C in agitated 500 ml shake flasks (150 r.p.m.) containing 160 ml of Bonnarme & Jeffries (1990) medium. The initial inoculum was of 104 conidiospores ml-1. The cultivations were performed in triplicate and each enzyme activity determination was performed at least three times in each culture. Standard deviations never exceeded 10 % of the mean values presented. {lozenge}, Biomass; {bullet}, MnP; {blacklozenge}, chitinase; {square}, protease.

 
Ultrastructure of P. chrysosporium hyphae in pellets
With the conidial inoculation density used here the mean final diameter of the pellets was about 3·5 mm after 4 days of cultivation (Jiménez-Tobon et al., 1997). Pellets of P. chrysosporium typically have three zones: an outer layer, surrounded by its growing boundary, a middle zone with some cells undergoing a lysis process, and a central core filled with senescent cells (Jaspers et al., 1994; Jiménez-Tobon, 1999). This type of structure has also been shown for several other fungal strains forming pellets (Wittler et al., 1986; Braun & Vecht-Lifshitz, 1991).

In a transmission electron microscopy analysis (not illustrated here) we have observed that the wall of young apical cells present in the outer region of the pellet was typically composed of one thin layer with an electron-opaque cytoplasm densely packed with ribosomes. A large number of elongated mitochondria possessing numerous flat cristae were also present in the cytoplasm. Nuclei in young, growing hyphae were often elongated parallel to the long axis of the hyphae. Nuclear pores were observed in the nuclear envelope. At the nuclear envelope the extensive membrane system of an endoplasmic reticulum was present. Small vacuoles were characteristic for the young cells and the cross-walls were not closed. The cytoplasm of subapical cells was electron-opaque and contained a large number of mitochondria and nuclei. The cell wall was composed of two layers. Small vacuoles surrounded by a tonoplast could also be seen. In ageing cells present in the inner part of the pellet, the cell wall was composed of several sublayers. The cytoplasm was electron-transparent and large vacuoles surrounded by a tonoplast were noted. The developed cross-walls were accompanied by Woronin bodies.

At day 2 of cultivation, some subapical regions of hyphae apparently started a process of differentiation into spherical cells of about 10 µm diameter morphologically similar to chlamydospores (Fig. 2a, b). These intercalary chlamydospore-like cells grew in number to culminate between days 3 and 4 of cultivation. Disappearance of these structures apparently started between days 4 and 5 and was nearly complete after 7 days of cultivation. Chlamydospore-like cells have also been observed in cultures not producing MnP but apparently never engaged in an autolytic process without the addition of extra Mn2+, which is necessary for enzyme induction (not shown).



View larger version (83K):
[in this window]
[in a new window]
 
Fig. 2. Chlamydospore-like cells produced by P. chrysosporium. (a, b) Cells observed by phase-contrast microscopy in subapical regions of hyphae obtained from the outer layer of pellets after 4 days cultivation. Bars, 30 µm (a) and 10 µm (b). (c) Transmission electron micrograph of an ultrathin section of a typical chlamydospore-like cell present in the outer layer of 4 day pellets of P. chrysosporium (cw, cell wall; M, mitochondrion; N, nucleus; v, vesicles; V, vacuoles). Bar, 1 µm.

 
The diameter of the chlamydospore-like spherical cells observed in the outer and middle zones of the pellet varied from 8 to 13 µm. Fig. 2(c) shows a typical cell observed after 4 days cultivation which was surrounded by a thick wall with a cytoplasm densely packed with mitochondria and nuclei. Golgi-like vesicles 50–200 nm in diameter, produced from the endoplasmic reticulum, were visible and apparently had a tendency to be distributed to the periphery of the cell.

Immunolocalization of the sites of MnP production
The sites of MnP production were localized at different stages of cultivation. MnP-reacting antibodies were found concentrated mainly in the chlamydospore-like cells and secondarily at the apical ends (Fig. 3a, b). In chlamydospore-like cells from 3 and 4 day cultures, the immunomarker was found to be predominantly concentrated in the Golgi-derived vesicles located at the peripheral part of the cytoplasm (Figs 3b and 4a). In chlamydospore-like cells from 6 day cultures the immunomarker density was lower and distributed in approximately equal proportions between the cell wall and the Golgi-derived vesicles (Figs 3b and 4b). A much lower density of the immunomarker was always detected in the other cell organelles and in the cytoplasm. Cells in the senescent central core of the pellet exhibited only a very faint immunoreaction (Fig. 3c).



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 3. Quantitative distribution of the MnP immunomarker in organelles of P. chrysosporium cells at different times of cultivation. The mean values reported represent the number of gold-conjugated particles per µm2 of hyphal ultrathin sections from the periphery and central parts of pellets. Five pellet sections were examined. (a) Apical extensions; (b) chlamydospore-like cells; (c) central cores of pellets.

 


View larger version (82K):
[in this window]
[in a new window]
 
Fig. 4. Immunoelectron microscopy of distribution of the MnP immunomarker in chlamydospore-like cells. (a) Distribution pattern of the gold-conjugated particles in a chlamydospore-like cell from the periphery of a 4-day-old pellet. At the cell wall (cw) a vesicle (v) is packed with immunomarker of MnP. Surrounding this vesicle, smaller vesicles are seen showing enzyme immunomarkers in the interior. Bar, 0·5 µm. (b) Distribution pattern for a cell from a 6-day-old pellet. Bar, 0·1 µm.

 
Extracellular autolytic enzymes produced by P. chrysosporium
From the above-described results it appears that maximal MnP secretion coincided with apparent autolysis of the chlamydospore-like cells. These could thus act as an enzyme reservoir, delivering their content into the culture supernatant by a process of autolysis. In order to support this assumption we examined the extracellular enzyme profile of autolytic hydrolases involved in disruption of organelle and cell wall structure (White et al., 2002; Pocsi et al., 2003) (Fig. 1). Extracellular chitinase activity started to increase at day 3, which corresponded to the deceleration phase preceding the end of the trophophase. The enzyme reached a plateau at day 5 of cultivation. Proteolytic activity started to increase during the trophophase at day 2, reached a maximum on day 4 and decreased to a lower level on day 7. A second smaller peak of proteolytic activity was attained between days 9 and 10.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The relationships between culture morphology and LiP and MnP production by P. chrysosporium have not as yet been well documented. Using a sandwiched mode of solid culture, and an immunodetection procedure, Moukha et al. (1993) suggested that peroxidases (LiP and MnP) were initially secreted only at the apex of secondary growing hyphae and later slowly released into the surrounding medium. No other sites of enzyme production were detected in their study. An apical mode of enzyme production was also shown here in P. chrysosporium pellets, but was apparently minor compared to production by the chlamydospore-like cells. The mycelial morphology in a submerged culture is nevertheless different from that of a plate culture. Particular situations of nutrient and oxygen concentrations in a pellet might create a physiological context encouraging a structural differentiation of the idiophasic hypha of the fungus. Fragmentation of idiophasic fungal hyphae is under metabolic and environmental control (Gow, 1994) and may result in differentiated cell structures having particular metabolic properties: for example yeast-like cells synthesizing the antibiotic cephalosporin in Acremonium chrysogenum (Bartoshevich et al., 1990; Sandor et al., 1998). In the case of P. chrysosporium it has been reported that a hyperoxidant state of growth, caused by limiting respirable carbon, induces a disorganization in the intracellular architecture of LiP-secreting hyphae in mycelial pellets, which in turn may promote enzyme production (Zacchi et al., 2000). Production of metabolically active chlamydospore-like cells by P. chrysosporium might therefore result from a particular physiological context in the MnP-producing pellet medium. However, the exact nature of these chlamydospore-like cells remains undefined at present. Chlamydospore-like cells and ‘pseudochlamydospores’, distinct from ‘true’ chlamydospores, have been reported occasionally in fungi, for example in Fusarium (Leslie, 2000) and Tremella sp. (Chee-Jen & Oberwinkler, 2000). Further studies are required to elucidate their structure, such as transmission electron microscopy to determine the thickness of their outer cell walls and whether they contain reserves of glucans and lipids. P. chrysosporium and its anamorph Sporotrichum pulverentulum are known to produce several differentiated structures, including typical chlamydospores (Larone, 1995; St-Germain & Summerbell, 1996; Wu, 1998). P. chrysosporium ‘true’ chlamydospores typically have diameters of up to 60 µm and contain abundant reserves of glucans and lipids (St-Germain & Summerbell, 1996; de Hoog et al., 2000). This was apparently not the case for the chlamydospore-like cells, which have a mean diameter of about 10 µm and several characteristics of metabolically active structures. These cells might be related to yeast-like forms and intermediate structures between hyphae and chlamydospores in other fungi (Bermejo et al., 1981; Dominguez et al., 1978). Moreover in certain fungal species, chlamydospores were shown to be fully functional cells, morphologically and physiologically active and independent from mycelium (Vidotto et al., 1996).

MnP production in P. chrysosporium chlamydospore-like cells was found mainly associated with Golgi-derived vesicles produced from the endoplasmic reticulum and located in the peripheral part of the cytoplasm. Mitochondria and nuclei were characteristically co-located with the vesicles, which suggests a cooperative metabolic activity. LiP and MnP were also found mainly associated with Golgi-derived vesicles in apical cells of secondary growing hyphae (Bonnarme et al., 1994).

Release of MnP in the culture supernatant coincided with the phase of disappearance of the chlamydospore-like cells from the mycelial pellets of P. chrysosporium, and with maximal activity of extracellular chitinase and protease. Chitinases and proteases in fungi were identified as autolytic enzymes involved in a variety of functions (Gooday, 1997a; Rao et al., 1998), including disruption of organelle and cell wall structure (Gooday, 1997b; White et al., 2002; Pocsi et al., 2003). Production of proteases is a common feature among fungi including Basidiomycetes (Rao et al., 1998). Chitinases have apparently been reported only occasionally in Basidiomycetes (Tracey, 1955; Ohtakara, 1988) but, as far we are aware, not previously in P. chrysosporium. The presence of protease and chitinase activities in the culture supernatant of P. chrysosporium might reflect their participation in hyphal autolysis steps, possibly including the disappearance of chlamydospore-like cells. For example, age-related extracellular chitinases have been shown to play a crucial role in both autolysis and fragmentation of Penicillium chrysogenum (Sami et al., 2001). In this context, the first peak of extracellular protease produced by P. chrysosporium could be involved in combination with chitinases (and possibly other enzymes) in the lysis of the chlamydospore-like cells, whereas the protease produced during late idiophase would play a role in the decline of MnP, as was suggested for LiP (Dosoretz, 1990; Dass, 1995). The exact role of these lytic enzymes has to be demonstrated in future in vivo enzyme inhibition experiments using for example allosamidin (Sami et al. 2001). From all the various data collected in this study, we conclude the chlamydospore-like structures of P. chrysosporium apparently show the characteristics of metabolically active entities acting as an enzyme reservoir and delivering their content into the surrounding medium by an autolytic process.


   ACKNOWLEDGEMENTS
 
The authors wish to thank the European Community AIR, INCO-Copernicus and ERASMUS programmes. Thanks are also addressed to the Hungarian Scientific Research Fund (grant reference numbers T034315 and T037473) and the ‘Research in Brussels Action’ for financial support.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bartoshevich, Y. E., Zaslavskaya, P. L., Novak, M. J. & Yudina, O. D. (1990). Acremonium chrysogenum differentiation and biosynthesis of cephalosporin. J Basic Microbiol 30, 313–320.[Medline]

Bermejo, J. M., Dominguez, J. B., Goni, F. M. & Uruburu, F. (1981). Influence of carbon and nitrogen sources on the transition from yeast-like to chlamydospores in Aureobasidium pullulans. Antonie van Leeuwenhoek 47, 107–119.[Medline]

Bonnarme, P. & Jeffries, W. (1990). Mn(II) regulation of lignin peroxidase and manganese-dependent peroxidase from lignin-degrading white rot fungi. Appl Environ Microbiol 56, 210–217.

Bonnarme, P., Moukha, S., Moreau, P., Record, E., Lesage, L., Cassagne, C. & Asther, M. (1994). Fractionation of subcellular membranes of the secretory pathway from the peroxides-producing white-rot fungus Phanerochaete chrysosporium. FEMS Microbiol Lett 120, 155–161.[CrossRef]

Braun, S. & Vecht-Lifshitz, S. E. (1991). Mycelial morphology and metabolite production. Trends Biotechnol 9, 63–68.

Chee-Jen, C. & Oberwinkler, F. (2000). Heteromycophaga tremellicola found in the neotype specimen of Tremella brasiliensis. Mycotaxon 76, 163–169.

Cox, P. W., Paul, G. C. & Thomas, C. R. (1998). Image analysis of the morphology of filamentous micro-organisms. Microbiology 144, 817–827.[Free Full Text]

Dass, S. B., Dosoretz, C. G., Reddy, C. A. & Grethlein, H. E. (1995). Extracellular proteases produced by the wood-degrading fungus Phanerochaete chrysosporium under lignolytic and non-lignolytic conditions. Arch Microbiol 163, 254–258.[CrossRef][Medline]

de Hoog, G. S., Guarro, J., Gene, J. & Figueras, M. J. (2000). Atlas of Fungi, 2nd edn, vol 1. Utrecht, The Netherlands: Centraalbureau vor Schimmelcultures.

Dominguez, J. B., Goni, F. M. & Uruburu, F. (1978). The transition from yeast-like to chlamydospore cells in Pullularia pullulans. J Gen Microbiol 108, 111–117.

Dosoretz, C. G., Chen, H.-C. & Grethlein, H. E. (1990). Effect of environmental conditions on extracellular protease activity of lignolytic cultures of Phanerochaete chrysosporium. Appl Environ Microbiol 56, 395–400.

Gerin, P. A., Dufrene, Y., Bellon-Fontaine, M. N., Asther, M. & Rouxhet, P. G. (1993). Surface properties of the conidiospores of Phanerochaete chrysosporium and their relevance to pellet formation. J Bacteriol 175, 5135–5144.[Abstract]

Gooday, G. W. (1997a). The ever-widening diversity of chitinase. Carbohydr Eur 19, 18–22.

Gooday, G. W. (1997b). The many use of chitinases in nature. Chitin Chitosan Res 3, 233–243.

Gow, N. A. R. (1994). Yeast–hyphal dimorphism. In The Growing Fungus, pp. 403–422. Edited by N. A. R. Gow & G. M. Gadd. London: Chapman & Hall.

Jaspers, Ch., Jimenez-Tobon, G. A. & Penninckx, M. J. (1994). Evidence for a role of manganese peroxidase in the decolorisation of Kraft pulp bleach effluent by Phanerochaete chrysosporium. Effects of initial culture conditions on enzyme production. J Biotechnol 37, 229–234.[CrossRef]

Jiménez-Tobon, G. A. (1999). A study of cultivation parameters conditioning manganese peroxidase production by Phanerochaete chrysosporium. PhD thesis, Université Libre de Bruxelles.

Jiménez-Tobon, G. A., Penninckx, M. J. & Lejeune, R. (1997). The relationship between pellet size and production of Mn(II) peroxidase by Phanerochaete chrysosporium ATCC 24725 in submerged culture. Enzyme Microb Technol 21, 537–542.[CrossRef]

Kirk, T. K. & Farrell, R. L. (1987). Enzymatic "combustion": the microbial degradation of lignin. Annu Rev Microbiol 41, 465–505.[CrossRef][Medline]

Kurzatkowski, W., Palissa, H., Limpt H., van Doehren H., von Kleinkauf, H., Wolf, W. P. & Kurylowicz, W. (1991). Localization of isopenicillin N synthase in Penicillium chrysogenum PQ-96. Appl Microbiol Biotechnol 35, 517–520.

Lamar, R. T., Glaser, J. A. & Kirk, T. K. (1992). White rot fungi in the treatment of hazardous chemicals and wastes. In Frontiers in Industrial Mycology, pp. 127–143. Edited by G. F. Leatham. New York: Chapman & Hall.

Larone, D. H. (1995). Medically Important Fungi – a Guide to Identification, 3rd edn. Washington, DC: American Society for Microbiology.

Leonowicz, A., Cho, N.-S., Luterek, J., Wilkolazka, A., Wojtas-Wasilewska, M., Matuszewska, A., Hofrichter, M., Wesenberg, D. & Rogalski, J. (2001). Fungal laccase: properties and activity on lignin. J Basic Microbiol 41, 185–227.[CrossRef][Medline]

Leslie, J. F. (2000). Fusarium species associated with sorghum. In Compendium of Sorghum Diseases, pp. 30–31. Edited by R. A. Frederiksen & N. Ovvody. St Paul, MN: APS Press.

Metz, B. & Kossen, N. W. F. (1977). Biotechnology review: the growth of molds under the form of pellets. A literature review. Biotechnol Bioeng 19, 781–799.

Moukha, S. M., Wösten, H. A. B., Asther, M. & Wessels, J. G. H. (1993). In situ localization of the secretion of lignin peroxidases in colonies of Phanerochaete chrysosporium using a sandwiched mode of culture. J Gen Microbiol 139, 969–978.[Medline]

Ohtakara, A. (1988). Chitinase and {beta}-N-acetylhexosaminidase from Pycnoporus cinnabarinus. Methods Enzymol 161, 462–470.

Orth, A. B., Rzhetskaya, M., Cullen, D. & Tien, M. (1994). Characterization of a cDNA encoding a manganese peroxidase from Phanerochaete chrysosporium: genomic organization of lignin and manganese peroxidase-encoding genes. Gene 148, 161–165.[CrossRef][Medline]

Park, J. P., Kim, Y. M., Kim, S. W., Hwang, H. J., Cho, Y. J., Lee, Y. S., Song, C. H. & Yund, J. W. (2002). Effect of aeration on the mycelial morphology and exo-biopolymer production in Cordyceps militaris. Process Biochem 37, 1257–1262.[CrossRef]

Pocsi, I., Pusztahelyi, T., Sami, L. & Emri, T. (2003). Autolysis of Penicillium chrysogenum - a holistic approach. Indian J Biotechnol 2, 293–301.

Prosser, J. I. (1995). Mathematical modelling of fungal growth. In The Growing Fungus, pp. 319–335. Edited by N. A. R. Gow & G. M. Gadd. London: Chapman & Hall.

Rao, M. B., Tanksale, A. M., Chatge, M. S. & Deshpande, V. S. (1998). Molecular and biotechnological aspects of microbial proteases. Microbiol Mol Biol Rev 62, 597–635.[Abstract/Free Full Text]

Sami, L., Pusztahelyi, T., Emri, T., Varecza, Z., Fekete, A., Grallert, A., Karanyi, Z., Kiss, L. & Pocsi, I. (2001). Autolysis and aging of Penicillium chrysogenum cultures under carbon starvation: chitinase production and antifungal effect of allosamidin. J Gen Appl Microbiol 47, 201–211.[Medline]

Sandor, E., Pusztahelyi, T., Karaffa, L., Karanyi, Z., Pocsi, I., Biro, S., Szentirmai, A. & Pocsi, I. (1998). Allosamidin inhibits the fragmentation of Acremonium chrysogenum but does not influence the cephalosporin-C production of the fungus. FEMS Microbiol Lett 164, 231–236.[CrossRef][Medline]

Schügerl, K., Wittler, R. & Lorenz, T. (1983). The use of molds in pellet form. Trends Biotechnol 1, 120–123.

St-Germain, G. & Summerbell, R. (1996). Identifying Filamentous Fungi. A Clinical Laboratory Handbook, 1st edn. Belmont, CA: Star Publishing.

Strunk, C. A. (1978). A possibility in the preparation of certain regions of Streptomycetes colonies for electron microscopy. In Proceedings of the International Symposium on Nocardia and Steptomycetes, Zentbl Bakteriol Parasitenkd, Infektkrankh Hyg I Abt, Suppl. 6. Edited by M. Mordarski, W. Kurylowicz & J. Jeljaszewicz. Stuttgart & New York: Gustav Fischer.

Tracey, M. V. (1955). Chitinase in some basidiomycetes. Biochem J 61, 579–586.[Medline]

Vidotto, V., Bruatto, M., Accattatis, G. & Caramello, S. (1996). Observation on the nucleic acids in the chlamydospores of Candida albicans. New Microbiol 19, 327–334.[Medline]

White, S., McIntyre, M., Berry, D. R. & McNeil, B. (2002). The autolysis of industrial filamentous fungi. Crit Rev Biochem 22, 1–14.

Wittler, R., Baumgartl, H., Lubbers, D. W. & Schugerl, K. (1986). Investigation of oxygen transfer into Penicillium chrysogenum pellets by microprobe measurements. Biotechnol Bioeng 28, 1024–1036.

Wu, S. H. (1998). Nine new species of Phanerochaete from Taiwan. Mycol Res 102, 1126–1132.[CrossRef]

Xia, G., Jin, C., Zhou, J., Yang, S., Zang, S. & Jin, C. (2001). A novel chitinase having a unique mode of action from Aspergillus fumigatus YJ-407. Eur J Biochem 268, 4079–4085.[Abstract/Free Full Text]

Zacchi, L., Morris, I. & Harvey, P. J. (2000). Disordered ultrastructure in lignin-peroxidase secreting hyphae of the white-rot fungus Phanerochaete chrysosporium. Microbiology 146, 759–765.[Abstract/Free Full Text]

Received 30 April 2003; revised 22 July 2003; accepted 22 August 2003.



This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Jiménez-Tobon, G.
Articles by Penninckx, M. J.
Articles citing this Article
PubMed
PubMed Citation
Articles by Jiménez-Tobon, G.
Articles by Penninckx, M. J.
Agricola
Articles by Jiménez-Tobon, G.
Articles by Penninckx, M. J.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
INT J SYST EVOL MICROBIOL MICROBIOLOGY J GEN VIROL
J MED MICROBIOL ALL SGM JOURNALS
Copyright © 2003 Society for General Microbiology.