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A feed-harvest culturing method to improve lignin peroxidase production by Phanerochaete chrysosporium INA-12 immobilized on polyurethane foam
JOURNAL OF FERMENTATIONAND BIOENGINEERING Vol. 68, No. 1, 60-63. 1989
A Feed-Harvest Culturing Method to Improve Lignin Peroxidase Production by Phanerochaete chrysosporium INA-12 Immobilized on Polyurethane Foam CI~CILE CAPDEVILA, GEORGES CORRIEU, AND MARCEL ASTHER* Laboratoire de G~nie des Procddds Biotechnologiques, Agro-Alimentaires, Centre de Biotechnologies A gro-Industrielles, L N.R.A., 78850- Thiverval-Grignon, France Received 28 November 1988/Accepted 25 April 1989
Phanerochaete chrysosporium INA-12 immobilized with polyurethane foam produced high lignin peroxidase levels in 24-h repeated batch culture when the biomass was partially regenerated on day 5 (40.6 nkat. ml-1). The enzyme synthesis continued at a high level for eight batches, with only 7 ~ activity lost every 24 h.
thiamine-HC1, 0.0025g.1-1; yeast extract, l g-l-1; oleic acid 0 . 4 g . l -~, and soybean phospholipids, 0 . 7 5 g . / - l . Oleic acid was emulsified with Tween 80 as described by Asther et al. (7). The culture medium was buffered to pH 6.5 with 1.46 g.l -~ potassium 2,2-dimethylsuccinate. Polyurethane foam (Filtren T45, from Recticel, Wetteren, Belgium) was used as the support. Three polyurethane foam cubes, each 2 cm × 2 cm × 1 cm, were placed in 150-ml Erlenmeyer flasks and autoclaved at 120°C for 20min. Incubation was done in triplicate at 37°C without shaking, in Erlenmeyer flasks containing 30 ml of medium. Experiments were repeated at least twice. After inoculation with 6 × 107 conidiospores, the cultures were flushed with 100% oxygen for 2 min. Conidiospores attached easily to the polyurethane foam and growth took place throughout the support. The enzyme production was activated by adding veratryl alcohol and soybean phospholipids to a final concentration of 2.5 mM and 0.1 g. 1- i respectively, after 2 d of total incubation. At the same time, cultures were flushed with 100% oxygen for 2 min. Growth temperature was held at 37°C for the first 2 d of incubation and then reduced to 30°C for the remainder of the fermentation period. On day 5, the extracellular medium was aseptically decanted and 30 ml of a fresh medium buffered to pH 5.5 with 1.46 g. 1-t potassium 2,2-dimethylsuccinate were added back. This medium contained: glycerol, 2.5 g. l-1; diammonium tartrate, 0,45 g.1-1 and yeast extract, 0.25 g-1-1 for biomass regeneration and veratryl alcohol, 2.5 mM, and soybean phospholipids, 0.1 g . l - i for enzyme activation. Eight successive harvests of lignin peroxidase were obtained every 24, 48, or 72 h. Medium for semi-continuous cultures contained only veratryl alcohol (2.5 mM) and soybean phospholipid (0.1 g.l-t). Upon each addition of fresh medium, cultures were flushed with 100% oxygen for 2 min. Incubation was done at 30°C. Lignin peroxidase activity was measured by the rate of oxidation of veratryl alcohol to veratraldehyde by the method of Tien and Kirk (12), except that 2 mM veratryl alcohol and 0.27 mM H202 were used. Lignin peroxidase activity was expressed in nanokatals (nkat). Growth was measured in terms of dry weight of mycelium after it was filtered and dried overnight at 105°C on glass-fiber filters (GF/D; Whatmann, Inc.). An average
The white-rot fungus Phanerochaete chrysosporium secretes an extraceUular hemeprotein (lignin peroxidase) involved in lignin biodegradation which appears during the secondary growth phase upon carbon or nitrogen starvation (1, 2). Faison and Kirk (3) and Leisola et al. (4) showed that addition of veratryl alcohol increased lignin peroxidase synthesis. Kirk et al. (5) observed that an excess of trace elements stimulated enzyme activity. Buswell et al. (6) have demonstrated that P. chrysosporium INA-12 produced a high lignin peroxidase activity under conditions of nitrogen sufficiency when glycerol was used as the carbon source. Asther et al. (7, 8) reported that lignin peroxidase production by strain INA-12 was markedly enhanced and fermentation time for maximum activity was reduced in the presence of exogenous oleic acid emulsified with Tween 80 and soybean phospholipids. Agitation of the culture suppresses both the ligninolytic activity and the synthesis of extracellular lignin peroxidase (3, 9). To overcome this problem, development of suitable growth conditions for enzyme production has been studied. Linko (10) reported that lignin peroxidase can be produced with nylon-web immobilized P. chrysosporium BKM-F1767, with a slight decrease in the enzyme activities in successive batches. Similar results were obtained with polyurethane foam as support by Kirkpatrick and Palmer (11). In these experiments, veratryl alcohol was used as an activator for enzyme production; 12.2 (10) and 1.6 (11) nkat.m1-1 were obtained, respectively. In this paper, we describe a feed-harvest culturing method to improve lignin peroxidase production by P. chrysosporium INA-12 immobilized on polyurethane foam. The microorganism used in this study was Phanerochaete chrysosporium INA-12 (Collection Nationale de Culture de Microorganismes 1-398, Institut Pasteur, Paris, France). Cultures were grown in a synthetic medium containing: K H 2 PO4, 2 g. 1- l; CaCI2.2H20, 0.14 g. l- 1; MgSO4.7H20, 0.7 g . / - i ; FeSO4.7H20, 0.07 g . l - l ; ZnSO4.7H20 ' 0.0462 g. 1-1, MnSO4. H20, 0.035 g. l- J; CuSO4- 5H20, 0.007 g. 1- l; glycerol 10 g . l - I ; diammonium tartrate, 1.84 g.l-~; * Corresponding author. 60
VOL 68, 1989
NOTES
m
It} !
~.1~ OJ ] [ ~
Control
' A
B
I C
a
0 ~
50 ~
I
lo
i~
[
I
I
I
'
'
'
'
zl//l
6
JO ~
I
0
2
61
45
3s
4ncu6l:[on.rSme
FIG. 1. Lignin peroxidase activity ( [] ), specificactivity ( va), and productivity (11) by P. chrysosporium INA-12 in activated batch culture. On day 2 enzyme activity was stimulated by oxygen (A), oxygen and veratryl alcohol (B), oxygen, veratryl alcohol and soybean phospholipids (C), or oxygen, veratryl alcohol, soybean phosphalipids and temperature-shifting (D).
FIG. 2. Lignin peroxidase activity (~) and biomass dry weight (e) produced by P. chrysosporium INA-12 in repeated batch culture. On day 2 enzyme activity was activated by adding veratryl alcohol and soybean phospholipids (J,). On day 5 biomass was partially regenerated by adding glycerol, tartrate diammonium and yeast extract, and enzyme was activated by veratryl alcohol and soybean phospholipids ( ~ ). For semi-continuous culture enzyme activity was activated by veratryl alcohol and soybean phospholipids ( ~ ). Growth temperature (---).
predetermined foam cube dry weight was subtracted from the weight o f foam plus mycelium. The effects o f various growth conditions on lignin peroxidase production by Phanerochaete chrysosporium INA12 immobilized on polyurethane foam were investigated on 2-day-old cultures. The results are summarized in Fig. 1. Maximum lignin peroxidase activity was obtained after 5 d o f incubation. In cultures flushed with 100% oxygen on day 2, enzyme activity was higher (1.3-fold) than in control cultures. Enzyme production was stimulated by adding 2 . S m M veratryl alcohol and 0.1g.1-1 soybean phospholipids; 28.6 and 32.2 nkat.m1-1 were obtained respectively. On the other hand, lignin peroxidase production by P. chrysosporium INA-12 was 2.5-fold enhanced when the temperature was held at 37°C for the first 2 d o f incubation and then reduced to 30°C, compared with a culture grown at 37°C throughout the fermentation period. Specific activity o f lignin peroxidase in the extracellular medium was stimulated in the same way. Changes in growth conditions after that time resulted in lowered enzyme amounts (data not shown).
P. chrysosporium INA-12 immobilized on polyurethane foam was able to produce high lignin peroxidase levels in repeated batch culture. Semi-continuous batch cultures were repeated every 24, 48, and 72 h after the addition o f veratryl alcohol and soybean phospholipids (Table 1). Enzyme activity was markedly improved and reached 37.6 nkat. m1-1 for 24-h repeated batch culture. Lignin peroxidase productivity was increased 12.6-fold in comparison with a control culture. In cultures supplemented on day 5 with glycerol (2.5 g. 1-1), diaxnmonium tartrate (0.45 g. l- i), and yeast extract (0.25g./-I), enzyme production was imiaroved (40.6 nkat.m1-1) as compared to cultures only supplemented with veratryl alcohol and soybean phospholipids. Figure 2 shows the courses o f lignin peroxidase production and biomass formation during 24-h repeated batch culture when biomass was partially regenerated on day 5. Enzyme synthesis continued at a high level for several batches. The mean activity of successive harvests declined by 7% every 24 h. After eight repetitions, the initial enzyme production had decreased
TABLE 1. Repeated batch production of lignin peroxidase by P. chrysosporium INA-12" Repeated batch production (h) Controlb 24¢ 48¢ 72c
Without biomass regeneration Lignin peroxidase Productivity Increase activity in productivity (nkat •ml - ]) (nkat. ml - 1.d - l) fold 15.0_+ 1.5 3.0 1.0 37.6_+ 1.4 37.6 12.6 31.5_+ 1.1 15.7 5.3 30.5__-1.9 10.2 3.4
With biomass regeneration Lignin peroxidase Productivity Increase activity in productivity (nkat. ml - i) (nkat. ml - 1.d - 1) fold ---40.6_+ 1.2 40.6 13.6 32.9_+ 1.3 16.5 5.5 30.9_+ 1.8 10.3 3.4
All cultures were grown as described in Materials and Methods. Values are mean ± standard deviation for three replicate cultures. b Maximum activity was at day 5 after inoculation. c Values are means from five repeated batch cultures. a
62
CAPDEVILA ET AL.
by 50%. Biomass was found to decline slightly after 7 d incubation. Enzyme activity in the wild-type strain is secondary metabolism, appearing u p o n limitation of carbon or nitrogen. Linko (10) reported that it is essential to add a suitable carbon source (glucose) for repeated lignin peroxidase production by P. chrysosporium BKM-FI767. Similar results were obtained by Kirkpatrick and Palmer (11). According to our study lignin peroxidase can be produced with P. chrysosporium INA-12 without carbon and nitrogen sources. Enzyme synthesis was markedly stimulated in activated and repeated batch cultures by oxygen and certain additives such as veratryl alcohol and soybean phospholipids. Kirk et al. (9) reported that increased oxygen concentration stimulates lignin peroxidase synthesis. Asada et al. (14) observed that the effect o f polyurethane foam on enzyme production is due to an increase in the mycelial surface exposed to the oxygen-rich atmosphere. Several authors (3, 10, 11, 13) showed that enzyme synthesis was increased when the culture medium was supplemented with veratryl alcohol, a natural secondary metabolite o f P. chrysosporium as well as a lignin peroxidase substrate. T o n o n and Odier have recently demonstrated that veratryl alcohol enhanced lignin peroxidase production by protecting the enzyme against inactivation by the hydrogen peroxide produced by the fungus during the primary growth phase (15). The mechanism by which phospholipids enhanced extracellular enzyme production has not been elucidated. In cultures supplied with soybean phospholipids P. chrysosporium INA-12 was preferentially enriched with phosphatidylcholine and lysophosphatidylcholine (8). Phospholipids are known to be mainly localized within the cell membranes, when they ensure the normal flow o f metabolic processes and cell acclimatization to different environmental conditions. Phosphatidylcholine and lysophosphatidylcholine generally occur in the outer half o f the membrane bilayer (16). Changes in phospholipid composition appear to modify membrane permeability. Rao et al. (17) demonstrated that specific enrichment of phosphatidylcholine, phosphatidylethanolamine, or phosphatidylserine selectively affected the overall sensitivity o f Saccharomyces cerevisiae cells to polyene antibiotics. On the other hand, P. chrysosporium INA-12 requires temperature shifting for maximum lignin peroxidase production. Constantinides et aL (18) demonstrated that when the temperature was deliberately shifted from that best for growth to that optimal for secondary metabolites formation, a 15% increase in penicillin yield was obtained from Penicillium chrysogenum. Naik et al. (19) reported that zearalemore production by Fusarium graminearum was enhanced by reducing the incubation temperature to 10°C following a period o f growth at 25°C. A hypothesis o f low temperature requirement fot toxin biosynthesis was suggested, with the idea that the enzymes responsible for the production of zearalenone may be activated at low temperatures which are not optimum for growth of the microorganism. Results reported in this paper show the possibility of reusing P. chrysosporium INA-12 mycelium immobilized on polyurethane foam for repeated lignin peroxidase batch production. Enzyme amounts in this study were significantly higher (40.6 nkat.m1-1) (Asther, M. et ai., French Patent, 88-12887, 1988) than those obtained by other methods (12.2 (10) and 1.6 (11) nkat. ml-1). However, fur-
J. FERMENT.BIOENG., ther work is required to elucidate whether or not a process using a feed-harvest culturing method can be developed for large scale lignin peroxidase production. The authors thank M. Delattre for excellent technical assistance in FPLC analyses of extracellular proteins. REFERENCES 1. Tien, M. and Kirk, T.K.: Lignin-degrading enzyme from hymenomycete t~hanerochaete chrysosporium Burds. Science, 221,661-663 (1983). 2. Gold, M.H., Kuwahara, M, Chiu, A.A., and Glenn, J.K.:
3. 4. 5.
6.
7.
Purification and characterization of an extracellular H202-requiring diarylpropane oxygenase from the white-rot-basidiomycete Phanerochaete chrysosporium. Arch. Biochem. Biophys., 234, 353-362 (1984). Faison, B. D. and Kirk, T. K.: Factors involved in the regulation of a ligninase activity in Phanerochaetechrysosporium. Appl. Environ. Microbiol., 49, 299-304 (1985). Leisola, M. S. A., Thanci-Wyss, U., and Ficehter, A.: Strategies for production of high ligninase activities by Phanerochaete chrysosporium. J. Biotechnol., 3, 97-107 (1985). Kirk, T.K., Croan, S., Tien, M., Mnrtagh, K. E., and Farrell, R.L.: Production of a multiple ligninases by Phanerochaete chrysosporium: effect of selected growth conditions and use of a mutant strain. Enzyme Microbial. Technol., 8, 27-32 (1986). Buswell, J. A., Moiler, B., and Odler, E.: Ligninolytic enzyme production by Phanerochaete chrysosporium under conditions of nitrogen sutficiency. FEMS Microbiol. Lett., 25, 295-299 (1984). Asther, M., Corrieu, G., Drapron, R., and Odier, E.: Effect of Tween 80 and oleic acid on ligninase production by Phanerochaete chrysosporium INA-12. Enzyme Microbial. Technol., 9, 245-249 (1987).
8. Asther, M., Lesage, L., Drapron, R., Corrieu, G., and Odier, E.:
9.
10.
11.
12.
Phospholipid and fatty acid enrichment of Phanerochaete chrysosporium INA-12 in relation to ligninase production. Appl. Microbiol. Biotechnol., 24, 393-398 (1987). Kirk, T.K., Schultz, E., Connors, W.J., Lorenz, L.F., and Zeikus, Z.J.G.: Influence of culture parameters on lignin metabolism by Phanerochaetechrysosporium. Arch. Microbiol., 117, 277-285 (1978). Linko, S.: Production and characterization of extracellular lignin peroxidase from immobilized Phanerochaetechrysosporium in a 10-liter bioreactor. Enzyme Microbial. Technol., 10, 410-417 (1987). Kirkpatrick, N. and Palmer, J.M.: Semi-continuous ligninase production using foam-immobilized Phanerochaete chrysosporium. Appl. Microbiol. Biotechnol., 27, 129-133 (1987). Tlen, M. and Kirk, T.K.: Lignin-degrading enzyme from Phanerochaete chrysosporium: purification, characterization and catalytic properties of a unique H202-requiring oxygenase. Proc. Natl. Acad. Sci. USA, 81, 2280-2284 (1984).
13. Leisola, M. S. A., Meussdaerffer, F., Waldner, R., and Fiechter, A.: Production and identification of extracellular oxidases of
Phanerochaetechrysosporium. J. Biotechnol., 2, 379-382 (1985). 14. Asada, Y., Miyabe, M., Kikkawa, M., and Knwahara, M.: An extracellular NADH-oxidizing peroxidase produced by a lignindegrading basidiomycete, Phanerochaetechrysosporium. J. Ferment. Technol., 65, 483--487 (1987). 15. Tonon, F. and Odler, E.: Influence of veratryl alcohol and hydrogen peroxide on ligninase activity and ligninase production by Phanerochaete chrysosporium. Appl. Environ. Microbiol., 54, 466-472 (1988). 16. Chopra, A. and Khuller, G.K.: Lipids of pathogenic fungi. Prog. Lipid. Res., 22, 189-220 (1983). 17. Rao, T. V. G., Trlvedi, A., and Prasad, R.: Phospholipid enrichment of Saccharomyces cerevisiae and its effect on polyene sensitivity. Can. J. Microbiol., 31, 322-326 (1985). 18. Constanlinides, A., Spencer, J. L., and Gaden, E. L.: Optimization of batch fermentation processes: optimum temperature pro-
VoL. 68, 1989 files for batch penicillin fermentation. Biotechnol. Bioeng., 12, 1081-1098 (1970). 19. Nalk, D.M., Bush, L.V., and Barton, G.L.: Influence of temperature on the strain Fusarium graminearum Schwabe in zearalenone production. Can. J. Plant. Sci., 58, 1095-1097
NOTES
63
(1978). 20. Leisola, M. S. A., Kozulic, B., Meussdaerffer F., and Fiechter, A.: Homology among multiple extraceUular peroxidases from Phanerochaete chrysosoorium. J. Biol. Chem., 262, 419-424 (1987).
A Feed-Harvest Culturing Method to Improve Lignin Peroxidase Production by Phanerochaete chrysosporium INA-12 Immobilized on Polyurethane Foam CI~CILE CAPDEVILA, GEORGES CORRIEU, AND MARCEL ASTHER* Laboratoire de G~nie des Procddds Biotechnologiques, Agro-Alimentaires, Centre de Biotechnologies A gro-Industrielles, L N.R.A., 78850- Thiverval-Grignon, France Received 28 November 1988/Accepted 25 April 1989
Phanerochaete chrysosporium INA-12 immobilized with polyurethane foam produced high lignin peroxidase levels in 24-h repeated batch culture when the biomass was partially regenerated on day 5 (40.6 nkat. ml-1). The enzyme synthesis continued at a high level for eight batches, with only 7 ~ activity lost every 24 h.
thiamine-HC1, 0.0025g.1-1; yeast extract, l g-l-1; oleic acid 0 . 4 g . l -~, and soybean phospholipids, 0 . 7 5 g . / - l . Oleic acid was emulsified with Tween 80 as described by Asther et al. (7). The culture medium was buffered to pH 6.5 with 1.46 g.l -~ potassium 2,2-dimethylsuccinate. Polyurethane foam (Filtren T45, from Recticel, Wetteren, Belgium) was used as the support. Three polyurethane foam cubes, each 2 cm × 2 cm × 1 cm, were placed in 150-ml Erlenmeyer flasks and autoclaved at 120°C for 20min. Incubation was done in triplicate at 37°C without shaking, in Erlenmeyer flasks containing 30 ml of medium. Experiments were repeated at least twice. After inoculation with 6 × 107 conidiospores, the cultures were flushed with 100% oxygen for 2 min. Conidiospores attached easily to the polyurethane foam and growth took place throughout the support. The enzyme production was activated by adding veratryl alcohol and soybean phospholipids to a final concentration of 2.5 mM and 0.1 g. 1- i respectively, after 2 d of total incubation. At the same time, cultures were flushed with 100% oxygen for 2 min. Growth temperature was held at 37°C for the first 2 d of incubation and then reduced to 30°C for the remainder of the fermentation period. On day 5, the extracellular medium was aseptically decanted and 30 ml of a fresh medium buffered to pH 5.5 with 1.46 g. 1-t potassium 2,2-dimethylsuccinate were added back. This medium contained: glycerol, 2.5 g. l-1; diammonium tartrate, 0,45 g.1-1 and yeast extract, 0.25 g-1-1 for biomass regeneration and veratryl alcohol, 2.5 mM, and soybean phospholipids, 0.1 g . l - i for enzyme activation. Eight successive harvests of lignin peroxidase were obtained every 24, 48, or 72 h. Medium for semi-continuous cultures contained only veratryl alcohol (2.5 mM) and soybean phospholipid (0.1 g.l-t). Upon each addition of fresh medium, cultures were flushed with 100% oxygen for 2 min. Incubation was done at 30°C. Lignin peroxidase activity was measured by the rate of oxidation of veratryl alcohol to veratraldehyde by the method of Tien and Kirk (12), except that 2 mM veratryl alcohol and 0.27 mM H202 were used. Lignin peroxidase activity was expressed in nanokatals (nkat). Growth was measured in terms of dry weight of mycelium after it was filtered and dried overnight at 105°C on glass-fiber filters (GF/D; Whatmann, Inc.). An average
The white-rot fungus Phanerochaete chrysosporium secretes an extraceUular hemeprotein (lignin peroxidase) involved in lignin biodegradation which appears during the secondary growth phase upon carbon or nitrogen starvation (1, 2). Faison and Kirk (3) and Leisola et al. (4) showed that addition of veratryl alcohol increased lignin peroxidase synthesis. Kirk et al. (5) observed that an excess of trace elements stimulated enzyme activity. Buswell et al. (6) have demonstrated that P. chrysosporium INA-12 produced a high lignin peroxidase activity under conditions of nitrogen sufficiency when glycerol was used as the carbon source. Asther et al. (7, 8) reported that lignin peroxidase production by strain INA-12 was markedly enhanced and fermentation time for maximum activity was reduced in the presence of exogenous oleic acid emulsified with Tween 80 and soybean phospholipids. Agitation of the culture suppresses both the ligninolytic activity and the synthesis of extracellular lignin peroxidase (3, 9). To overcome this problem, development of suitable growth conditions for enzyme production has been studied. Linko (10) reported that lignin peroxidase can be produced with nylon-web immobilized P. chrysosporium BKM-F1767, with a slight decrease in the enzyme activities in successive batches. Similar results were obtained with polyurethane foam as support by Kirkpatrick and Palmer (11). In these experiments, veratryl alcohol was used as an activator for enzyme production; 12.2 (10) and 1.6 (11) nkat.m1-1 were obtained, respectively. In this paper, we describe a feed-harvest culturing method to improve lignin peroxidase production by P. chrysosporium INA-12 immobilized on polyurethane foam. The microorganism used in this study was Phanerochaete chrysosporium INA-12 (Collection Nationale de Culture de Microorganismes 1-398, Institut Pasteur, Paris, France). Cultures were grown in a synthetic medium containing: K H 2 PO4, 2 g. 1- l; CaCI2.2H20, 0.14 g. l- 1; MgSO4.7H20, 0.7 g . / - i ; FeSO4.7H20, 0.07 g . l - l ; ZnSO4.7H20 ' 0.0462 g. 1-1, MnSO4. H20, 0.035 g. l- J; CuSO4- 5H20, 0.007 g. 1- l; glycerol 10 g . l - I ; diammonium tartrate, 1.84 g.l-~; * Corresponding author. 60
VOL 68, 1989
NOTES
m
It} !
~.1~ OJ ] [ ~
Control
' A
B
I C
a
0 ~
50 ~
I
lo
i~
[
I
I
I
'
'
'
'
zl//l
6
JO ~
I
0
2
61
45
3s
4ncu6l:[on.rSme
FIG. 1. Lignin peroxidase activity ( [] ), specificactivity ( va), and productivity (11) by P. chrysosporium INA-12 in activated batch culture. On day 2 enzyme activity was stimulated by oxygen (A), oxygen and veratryl alcohol (B), oxygen, veratryl alcohol and soybean phospholipids (C), or oxygen, veratryl alcohol, soybean phosphalipids and temperature-shifting (D).
FIG. 2. Lignin peroxidase activity (~) and biomass dry weight (e) produced by P. chrysosporium INA-12 in repeated batch culture. On day 2 enzyme activity was activated by adding veratryl alcohol and soybean phospholipids (J,). On day 5 biomass was partially regenerated by adding glycerol, tartrate diammonium and yeast extract, and enzyme was activated by veratryl alcohol and soybean phospholipids ( ~ ). For semi-continuous culture enzyme activity was activated by veratryl alcohol and soybean phospholipids ( ~ ). Growth temperature (---).
predetermined foam cube dry weight was subtracted from the weight o f foam plus mycelium. The effects o f various growth conditions on lignin peroxidase production by Phanerochaete chrysosporium INA12 immobilized on polyurethane foam were investigated on 2-day-old cultures. The results are summarized in Fig. 1. Maximum lignin peroxidase activity was obtained after 5 d o f incubation. In cultures flushed with 100% oxygen on day 2, enzyme activity was higher (1.3-fold) than in control cultures. Enzyme production was stimulated by adding 2 . S m M veratryl alcohol and 0.1g.1-1 soybean phospholipids; 28.6 and 32.2 nkat.m1-1 were obtained respectively. On the other hand, lignin peroxidase production by P. chrysosporium INA-12 was 2.5-fold enhanced when the temperature was held at 37°C for the first 2 d o f incubation and then reduced to 30°C, compared with a culture grown at 37°C throughout the fermentation period. Specific activity o f lignin peroxidase in the extracellular medium was stimulated in the same way. Changes in growth conditions after that time resulted in lowered enzyme amounts (data not shown).
P. chrysosporium INA-12 immobilized on polyurethane foam was able to produce high lignin peroxidase levels in repeated batch culture. Semi-continuous batch cultures were repeated every 24, 48, and 72 h after the addition o f veratryl alcohol and soybean phospholipids (Table 1). Enzyme activity was markedly improved and reached 37.6 nkat. m1-1 for 24-h repeated batch culture. Lignin peroxidase productivity was increased 12.6-fold in comparison with a control culture. In cultures supplemented on day 5 with glycerol (2.5 g. 1-1), diaxnmonium tartrate (0.45 g. l- i), and yeast extract (0.25g./-I), enzyme production was imiaroved (40.6 nkat.m1-1) as compared to cultures only supplemented with veratryl alcohol and soybean phospholipids. Figure 2 shows the courses o f lignin peroxidase production and biomass formation during 24-h repeated batch culture when biomass was partially regenerated on day 5. Enzyme synthesis continued at a high level for several batches. The mean activity of successive harvests declined by 7% every 24 h. After eight repetitions, the initial enzyme production had decreased
TABLE 1. Repeated batch production of lignin peroxidase by P. chrysosporium INA-12" Repeated batch production (h) Controlb 24¢ 48¢ 72c
Without biomass regeneration Lignin peroxidase Productivity Increase activity in productivity (nkat •ml - ]) (nkat. ml - 1.d - l) fold 15.0_+ 1.5 3.0 1.0 37.6_+ 1.4 37.6 12.6 31.5_+ 1.1 15.7 5.3 30.5__-1.9 10.2 3.4
With biomass regeneration Lignin peroxidase Productivity Increase activity in productivity (nkat. ml - i) (nkat. ml - 1.d - 1) fold ---40.6_+ 1.2 40.6 13.6 32.9_+ 1.3 16.5 5.5 30.9_+ 1.8 10.3 3.4
All cultures were grown as described in Materials and Methods. Values are mean ± standard deviation for three replicate cultures. b Maximum activity was at day 5 after inoculation. c Values are means from five repeated batch cultures. a
62
CAPDEVILA ET AL.
by 50%. Biomass was found to decline slightly after 7 d incubation. Enzyme activity in the wild-type strain is secondary metabolism, appearing u p o n limitation of carbon or nitrogen. Linko (10) reported that it is essential to add a suitable carbon source (glucose) for repeated lignin peroxidase production by P. chrysosporium BKM-FI767. Similar results were obtained by Kirkpatrick and Palmer (11). According to our study lignin peroxidase can be produced with P. chrysosporium INA-12 without carbon and nitrogen sources. Enzyme synthesis was markedly stimulated in activated and repeated batch cultures by oxygen and certain additives such as veratryl alcohol and soybean phospholipids. Kirk et al. (9) reported that increased oxygen concentration stimulates lignin peroxidase synthesis. Asada et al. (14) observed that the effect o f polyurethane foam on enzyme production is due to an increase in the mycelial surface exposed to the oxygen-rich atmosphere. Several authors (3, 10, 11, 13) showed that enzyme synthesis was increased when the culture medium was supplemented with veratryl alcohol, a natural secondary metabolite o f P. chrysosporium as well as a lignin peroxidase substrate. T o n o n and Odier have recently demonstrated that veratryl alcohol enhanced lignin peroxidase production by protecting the enzyme against inactivation by the hydrogen peroxide produced by the fungus during the primary growth phase (15). The mechanism by which phospholipids enhanced extracellular enzyme production has not been elucidated. In cultures supplied with soybean phospholipids P. chrysosporium INA-12 was preferentially enriched with phosphatidylcholine and lysophosphatidylcholine (8). Phospholipids are known to be mainly localized within the cell membranes, when they ensure the normal flow o f metabolic processes and cell acclimatization to different environmental conditions. Phosphatidylcholine and lysophosphatidylcholine generally occur in the outer half o f the membrane bilayer (16). Changes in phospholipid composition appear to modify membrane permeability. Rao et al. (17) demonstrated that specific enrichment of phosphatidylcholine, phosphatidylethanolamine, or phosphatidylserine selectively affected the overall sensitivity o f Saccharomyces cerevisiae cells to polyene antibiotics. On the other hand, P. chrysosporium INA-12 requires temperature shifting for maximum lignin peroxidase production. Constantinides et aL (18) demonstrated that when the temperature was deliberately shifted from that best for growth to that optimal for secondary metabolites formation, a 15% increase in penicillin yield was obtained from Penicillium chrysogenum. Naik et al. (19) reported that zearalemore production by Fusarium graminearum was enhanced by reducing the incubation temperature to 10°C following a period o f growth at 25°C. A hypothesis o f low temperature requirement fot toxin biosynthesis was suggested, with the idea that the enzymes responsible for the production of zearalenone may be activated at low temperatures which are not optimum for growth of the microorganism. Results reported in this paper show the possibility of reusing P. chrysosporium INA-12 mycelium immobilized on polyurethane foam for repeated lignin peroxidase batch production. Enzyme amounts in this study were significantly higher (40.6 nkat.m1-1) (Asther, M. et ai., French Patent, 88-12887, 1988) than those obtained by other methods (12.2 (10) and 1.6 (11) nkat. ml-1). However, fur-
J. FERMENT.BIOENG., ther work is required to elucidate whether or not a process using a feed-harvest culturing method can be developed for large scale lignin peroxidase production. The authors thank M. Delattre for excellent technical assistance in FPLC analyses of extracellular proteins. REFERENCES 1. Tien, M. and Kirk, T.K.: Lignin-degrading enzyme from hymenomycete t~hanerochaete chrysosporium Burds. Science, 221,661-663 (1983). 2. Gold, M.H., Kuwahara, M, Chiu, A.A., and Glenn, J.K.:
3. 4. 5.
6.
7.
Purification and characterization of an extracellular H202-requiring diarylpropane oxygenase from the white-rot-basidiomycete Phanerochaete chrysosporium. Arch. Biochem. Biophys., 234, 353-362 (1984). Faison, B. D. and Kirk, T. K.: Factors involved in the regulation of a ligninase activity in Phanerochaetechrysosporium. Appl. Environ. Microbiol., 49, 299-304 (1985). Leisola, M. S. A., Thanci-Wyss, U., and Ficehter, A.: Strategies for production of high ligninase activities by Phanerochaete chrysosporium. J. Biotechnol., 3, 97-107 (1985). Kirk, T.K., Croan, S., Tien, M., Mnrtagh, K. E., and Farrell, R.L.: Production of a multiple ligninases by Phanerochaete chrysosporium: effect of selected growth conditions and use of a mutant strain. Enzyme Microbial. Technol., 8, 27-32 (1986). Buswell, J. A., Moiler, B., and Odler, E.: Ligninolytic enzyme production by Phanerochaete chrysosporium under conditions of nitrogen sutficiency. FEMS Microbiol. Lett., 25, 295-299 (1984). Asther, M., Corrieu, G., Drapron, R., and Odier, E.: Effect of Tween 80 and oleic acid on ligninase production by Phanerochaete chrysosporium INA-12. Enzyme Microbial. Technol., 9, 245-249 (1987).
8. Asther, M., Lesage, L., Drapron, R., Corrieu, G., and Odier, E.:
9.
10.
11.
12.
Phospholipid and fatty acid enrichment of Phanerochaete chrysosporium INA-12 in relation to ligninase production. Appl. Microbiol. Biotechnol., 24, 393-398 (1987). Kirk, T.K., Schultz, E., Connors, W.J., Lorenz, L.F., and Zeikus, Z.J.G.: Influence of culture parameters on lignin metabolism by Phanerochaetechrysosporium. Arch. Microbiol., 117, 277-285 (1978). Linko, S.: Production and characterization of extracellular lignin peroxidase from immobilized Phanerochaetechrysosporium in a 10-liter bioreactor. Enzyme Microbial. Technol., 10, 410-417 (1987). Kirkpatrick, N. and Palmer, J.M.: Semi-continuous ligninase production using foam-immobilized Phanerochaete chrysosporium. Appl. Microbiol. Biotechnol., 27, 129-133 (1987). Tlen, M. and Kirk, T.K.: Lignin-degrading enzyme from Phanerochaete chrysosporium: purification, characterization and catalytic properties of a unique H202-requiring oxygenase. Proc. Natl. Acad. Sci. USA, 81, 2280-2284 (1984).
13. Leisola, M. S. A., Meussdaerffer, F., Waldner, R., and Fiechter, A.: Production and identification of extracellular oxidases of
Phanerochaetechrysosporium. J. Biotechnol., 2, 379-382 (1985). 14. Asada, Y., Miyabe, M., Kikkawa, M., and Knwahara, M.: An extracellular NADH-oxidizing peroxidase produced by a lignindegrading basidiomycete, Phanerochaetechrysosporium. J. Ferment. Technol., 65, 483--487 (1987). 15. Tonon, F. and Odler, E.: Influence of veratryl alcohol and hydrogen peroxide on ligninase activity and ligninase production by Phanerochaete chrysosporium. Appl. Environ. Microbiol., 54, 466-472 (1988). 16. Chopra, A. and Khuller, G.K.: Lipids of pathogenic fungi. Prog. Lipid. Res., 22, 189-220 (1983). 17. Rao, T. V. G., Trlvedi, A., and Prasad, R.: Phospholipid enrichment of Saccharomyces cerevisiae and its effect on polyene sensitivity. Can. J. Microbiol., 31, 322-326 (1985). 18. Constanlinides, A., Spencer, J. L., and Gaden, E. L.: Optimization of batch fermentation processes: optimum temperature pro-
VoL. 68, 1989 files for batch penicillin fermentation. Biotechnol. Bioeng., 12, 1081-1098 (1970). 19. Nalk, D.M., Bush, L.V., and Barton, G.L.: Influence of temperature on the strain Fusarium graminearum Schwabe in zearalenone production. Can. J. Plant. Sci., 58, 1095-1097
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(1978). 20. Leisola, M. S. A., Kozulic, B., Meussdaerffer F., and Fiechter, A.: Homology among multiple extraceUular peroxidases from Phanerochaete chrysosoorium. J. Biol. Chem., 262, 419-424 (1987).