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Suppressive effect of l -phenylalanine on lignin peroxidase in the white-rot fungus Phanerochaete chrysosporium
EISEVIER
FEMS Microbiology Letters 131 (1995) 185-188
Suppressive effect of L-phenylalanine on lignin peroxidase in the white-rot fungus Phanerochaete chrysosporium Yasumi Akamatsu ‘, Mikio Shimada
*
Wood Research Institute, Kyoto Uniuersily, Uji, Kyoto, 611, Japan Received 9 June 1995; revised 4 July 1995; accepted 6 July 1995
Abstract The effects of added L-amino acids and on lignin peroxidase activity in ligninolytic cultures of Phanerochaete chrysosporitun were investigated. Among 11 amino acids tested, including phenylalanine, glutamate, glutamine, histidine, alanine, iso-leucine, omithine, glycine, aspartate, proline, and arginine, phenylalanine was the most effective in suppression of lignin peroxidase synthesis. By contrast, glutamate and NH: enhanced the lignin peroxidase synthesis at lower concentrations (below 1 mM), but suppressed it significantly at higher concentrations. Keywords: White-rot fungus; Phanerochaete chrysosporium; Lignin peroxidase;
Veratryl alcohol; L-Phenylalanine;
Enzyme suppression
* Corresponding author. Tel: +&?l (0774) 32-3111; Fax: +81 (774) 33-3049; E-mail: [email protected] ’ Present address, Fukui General Green Center, Fukui, Maruoka, 910-02, Japan.
hol, which is produced as a secondary metabolite from L-phenylalanine by this fungus was also suppressed by the nitrogenous compounds added [6]. Quite recently, Hattori and Shimada have found that phenylalanine ammonia-lyase, which is involved in the initial step of the veratryl alcohol biosynthesis [7,8], occurred in ligninolytic low nitrogen cultures of P. chrysosporium but not in those containing high nitrogen concentrations [9]. The primary objective of this investigation was to examine the possibility that L-phenylalanine may suppress lignin peroxidase synthesis. This compound is the initial precursor which, after deamination by phenylalanine ammonia-lyase, leads to the secondary metabolite veratryl alcohol that may have a biochemical connection with the ligninolytic system of P. chrysosporium 181.In addition, the effect of phenylalanine on the ligninolytic system has not been previously examined [2,6].
0378-1097/95/$09.50
Societies. All rights reserved
1. Introduction Lignin biodegradation has received widespread interest from fundamental and applied viewpoints [1,2]. Lignin decomposition by the white-rot fungus Phanerochaete chrysosporium has been characterized as a secondary metabolic (idiophasic) event [3], which is triggered by N-, C-or S-nutrient starvation [4]. The ligninolytic systems that decompose lignin to CO2 has been reported to be suppressed by addition of L-glutamate, L-glutamine, L-histidine or NH: [5,6]. Furthermore, the biosynthesis of veratryl alco-
8 1995 Federation
SSDI 0378-1097(95)00257-X
of European
Microbiological
Y. Akamatsu, M. Shimada /FEMS
186
Microbiology Letters 131 (1995) 185-188
I
2. Materials and methods 2.1. Microorganism
and culture conditions
I’
.JJ
,’
= E
$0.5The white-rot fungus, P. chrysosporium (ATCC 24725) was grown without shaking at 37°C in lOO-ml flasks containing 10 ml of Kirk’s medium [lo] under the normal atmosphere. The culture medium was modified using 2% glucose, 1.2 mM ammonium tartrate, 7-fold increase of the minerals, and 10 mM trans-aconitate buffer (pH 4.3). Tween 80 (O.l%, vol/vol) and veratryl alcohol (1.5 mM) were added for enhancing excretion of lignin peroxidase into the medium, as previously reported [ill. On day 3 of the cultivation, 1 ml of the aqueous solution of each of amino acid (2mM), NH:, and cycloheximide (100 pg> or an equal amount of water (control) was added into a set of ligninolytic cultures. Incubation was performed until the specified time of the lignin peroxidase assays. 2.2. Lignin peroxidase
r .Z? > ‘5 : !& _J OL 0
1.
2 3 4 Culture days
5
6
Fig. 1. Effects of amino acids on lignin peroxidase production during cultivation of P. chtysosporium: 0, L-Phenylalanine (0.2 mM); 0, Glutamate (0.2 mM1; 0, and n , Control (H,O). Lignin peroxidase (Lip) activities were assayed for each set of 3 cultures. The enzyme activities were assayed on day 2 and day 3 before the addition of the compounds. The reaction mixtures for lignin peroxidase assays contained veratryl alcohol (3.3 mM), hydrogen peroxide (0.25 mM), 50 mM tartrate buffer (pH 4.5), and appropriate amounts of the culture filtrate. The lignin peroxidase activity was assayed as described in the text at the times of 14, 25, 47, and 68 h after addition of the compounds.
assay
Three flasks were tested for measurement of lignin peroxidase activity. The cultures were mixed for averaging enzyme activities, and filtered through nylon cloth. Lignin peroxidase activity in the filtrate was assayed twice at pH 4.5 and 30°C by measurement of the increase in absorbance at 340 nm due to veratraldehyde formed [ 121.
3. Results and discussion
3.1. Comparison of suppressive effects of phenylalanine and other amino acids
Fig. 1 shows the kinetics of lignin peroxidase activity after addition of phenylalanine and glutamate at the time indicated by the arrow. The results clearly indicate that phenylalanine caused a delay of about 3 days in the appearance of lignin peroxidase activity, as compared with the control. Glutamate did not cause such an effect, but rather enhanced enzyme activity to a higher level until day 6. Addition of cycloheximide, an inhibitor of protein synthesis, caused a delay of one day in the appearance of lignin peroxidase (data not shown), which is consistent
with the finding that the ligninolytic system was inhibited by addition of cycloheximide [6]. Thus the expression of lignin peroxidase synthesis triggered by nitrogen starvation may be regulated at the transcription level, as suggested by Li et al. [13]. Possi-
Table 1 Effects of various amino acids on repression of lignin peroxidase activity in the ligninolytic cultures of P. chrysosporium Amino acids added
Lignin peroxidase
Control (Hz LO) Arginine Proline Aspartate Glutamine Glycine Omithine Isoleucine Glutamate Alanine Histidine Phenylalanine
100 137 119 107 106 104 101 82 80 75 57 0
activity (o/o)
Final concentrations of the amino acids added were 0.2 mM. Each amino acid was added 67 h of cultivation and further incubated for 25 h. Cultures were then harvested and the lignin peroxidase activities were determined and expressed as the relative value based on the control (42.4 nkat/culture).
Y. Akomatsy M. Shimada /FEMS Microbiology Letters 131 (1995) 185-188
ble lignin peroxidase-suppressing effects of the other L-amino acids were examined. Table 1 shows that no lignin peroxidase activity was detected in the cultures 25 h after addition of phenylalanine. In contrast, the other amino acids such as glutamine, alanine, arginine, aspartate, glycine, isoleucine, ornithine, and proline as well as glutamate did not show any significant inhibition of the lignin peroxidase synthesis at this concentration (0.2 mM). Only histidine reduced the enzyme activity by 43%. It was alternatively confirmed that L-phenylalanine (0.2-l mM) did not inhibit lignin peroxidase activity on the oxidation of veratryl alcohol in the in vitro system (data not shown). Thus, L-phenylalanine appeared to be the strongest suppressor in vivo at this concentration.
187
3.2. Comparison of the suppressive eflects by Lphenylalanine and NH,+
Fig. 2. Comparison of the suppressions of lignin peroxidase (Lip) activity caused by phenylalanine (0) and NH: (0) which had been added to the ligninolytic cultures of P. chrysosporium at different concentrations. Lignin peroxidase activities were assayed 23 h after addition of each amino acid at the final concentrations indicated. The lignin peroxidase activity in the control was 20.7 nkat/culture.
We further examined the possibility that NH: rather than L-phenylalanine might be more directly involved in the suppression of lignin peroxidase synthesis, because it could be liberated from this aromatic amino acid by phenylalanine ammonia-lyase which was markedly activated along with appearance of lignin peroxidase activity in the ligninolytic cultures [9]. Fig. 2 shows the comparison of different effects of phenylalanine and ammonia. The results obtained with NH: added in the range from 0.01 mM to 3 mM indicate that NH: impaired strongly lignin peroxidase synthesis at 3 mM and rather moderately (about 75% of the control) around 2 mM. However, a 40% increase was observed around 0.5 mM in sharp contrast with the pattern exhibited by phenylalanine. Further alternative experiments with glutamate added at various concentrations showed that this amino acid also slightly enhanced lignin peroxidase activity, but impaired it significantly in the range from 1.5 to 3 mM (15 to 25% activity of the control). The observed suppression of the lignin peroxidase synthesis with both glutamate and NH: at higher concentrations are in good agreement with the previous report that ligninolytic activity (14Clignin + 14COZ) was reduced to 17% by addition of 2.8 mM r_-glutamate [5]. According to the central role of L-glutamate in the primary amino acid
metabolism of living cells, it is not surprising that the suppressive effect of NH: on lignin peroxidase activity is very similar to that of glutamate in ligninolytic culture of P. chrysosporium. This assumption is reasonably supported by the previous finding that addition of NH: to ligninolytic cultures enhanced L-glutamate dehydrogenase and L-glutamine synthetase activities, resulting in an increase of the intracellular glutamate level [6]. Accordingly, the idiophasic phase of the ligninolytic culture is switched back to the primary (tropho) phase at greater concentrations of glutamate. However, a moderately higher concentration of L-glutamate or ammonium nitrogen are beneficial for the lignin peroxidase synthesis, whereas phenylalanine impaired it more strongly at the lower concentrations. In conclusion, this investigation shows that Lphenylalanine added to the ligninolytic cultures of P. chrysosporium strongly suppressed lignin peroxidase synthesis. Thus, r_-phenylalanine plays a key role, as does L-glutamate in the regulation of secondary metabolism of this fungus. Furthermore, the two amino acids impaired the lignin peroxidase synthesis somewhat differently. However, the molecular mechanisms remain to be elucidated.
188
Y. Akamatsy
M. Shimada / FEMS Microbiology Letters 131 (1995) 185-188
References [l] Ander, P. and Eriksson K.E. (1978) Lignin degradation and utilization by microorganisms. Prog. Ind. Microbial. 14, l58. [2] Leisola, M.S.A. and Fiechter, A. (19851 New trends in lignin biodegradation. Adv. Biotech. Process. 5, 59-89. [3] Kirk, T.K. and Farrell, R.L. (19871 Enzymatic ‘Combustion’:The microbial degradation of lignin. Ann. Rev. Microbial. 41, 465-505. [4] Jeffries, T.W., Choi, S. and Kirk, T.K. (1981) Nutritional regulation of lignin degradation by Phanerochaete chrysosporium. Appl. Environ. Microbial. 42, 290-296 [5] Fenn, P. and Kirk, T.K. (1981) Relationship of nitrogen to the onset and suppression of ligninolytic activity and secondary metabolism in Phanerochaete chrysosporium. Arch. Microbial. 130, 59-65. [6] Fenn, P., Choi, S. and Kirk, T.K. (19811 Ligninolytic activity of Phanerochaete chrysosporium: Physiology of suppression by NH: and L-glutamate. Arch. Microbial. 130, 66-71. [7] Jensen, K.A., Evans, Jr.K.M.C. Evans, Kirk, T.K. and Hammel, K.E. (1994) Biosynthetic pathway for veratryl alcohol in
[8]
[9]
[lo]
[ll]
[12] [13]
the ligninolytic fungus Phanerochaete chrysosporium. Appl. Environ. Microbial. 60, 709-714. Shimada, M., Nakatsubo, F., Higuchi, T. and Kirk, T.K. (1981) Biosynthesis of the secondary metabolic veratryl alcoho1 in relation to lignin degradation in Phanerochaete chrysosporium. Arch. Microbial. 129, 321-324. Hattori, T. and Shimada, M. (1995) The detection of PAL activity in the culture of Phanerochaete chrysosporium. Proc. 40th Ann. Conf. Jpn. Wood Res. Soc.(Tokyo) p. 599. Kirk, T.K., Schulz, E., Connors, W.J., Lorenz, L.F. and Zeikus, J.G. (19781 Influence of culture parameters on lignin metabolism by Phanerochaete chrysosporium. Arch. Microbiol. 117, 277-285. Asther,M., Corrieu, G., Drapon, R. and Odier, E. (19871 Effects of Tween 80 and oleic acid on ligninase production by Phanerochaete chrysosporium INA-12. Enzyme Microb. Technol. 9, 245-249. Tien, M. and Kirk, T.K. (1988) Lignin peroxidase of Phanerochaete chrysosporium. Methods. Enzymol. 161, 238-249. Li, D., Alit, M. and Gold, M.H. (1994) Nitrogen regulation of lignin peroxidase gene transcription. Appl. Environ. Microbiol. 60, 3447-3449.
FEMS Microbiology Letters 131 (1995) 185-188
Suppressive effect of L-phenylalanine on lignin peroxidase in the white-rot fungus Phanerochaete chrysosporium Yasumi Akamatsu ‘, Mikio Shimada
*
Wood Research Institute, Kyoto Uniuersily, Uji, Kyoto, 611, Japan Received 9 June 1995; revised 4 July 1995; accepted 6 July 1995
Abstract The effects of added L-amino acids and on lignin peroxidase activity in ligninolytic cultures of Phanerochaete chrysosporitun were investigated. Among 11 amino acids tested, including phenylalanine, glutamate, glutamine, histidine, alanine, iso-leucine, omithine, glycine, aspartate, proline, and arginine, phenylalanine was the most effective in suppression of lignin peroxidase synthesis. By contrast, glutamate and NH: enhanced the lignin peroxidase synthesis at lower concentrations (below 1 mM), but suppressed it significantly at higher concentrations. Keywords: White-rot fungus; Phanerochaete chrysosporium; Lignin peroxidase;
Veratryl alcohol; L-Phenylalanine;
Enzyme suppression
* Corresponding author. Tel: +&?l (0774) 32-3111; Fax: +81 (774) 33-3049; E-mail: [email protected] ’ Present address, Fukui General Green Center, Fukui, Maruoka, 910-02, Japan.
hol, which is produced as a secondary metabolite from L-phenylalanine by this fungus was also suppressed by the nitrogenous compounds added [6]. Quite recently, Hattori and Shimada have found that phenylalanine ammonia-lyase, which is involved in the initial step of the veratryl alcohol biosynthesis [7,8], occurred in ligninolytic low nitrogen cultures of P. chrysosporium but not in those containing high nitrogen concentrations [9]. The primary objective of this investigation was to examine the possibility that L-phenylalanine may suppress lignin peroxidase synthesis. This compound is the initial precursor which, after deamination by phenylalanine ammonia-lyase, leads to the secondary metabolite veratryl alcohol that may have a biochemical connection with the ligninolytic system of P. chrysosporium 181.In addition, the effect of phenylalanine on the ligninolytic system has not been previously examined [2,6].
0378-1097/95/$09.50
Societies. All rights reserved
1. Introduction Lignin biodegradation has received widespread interest from fundamental and applied viewpoints [1,2]. Lignin decomposition by the white-rot fungus Phanerochaete chrysosporium has been characterized as a secondary metabolic (idiophasic) event [3], which is triggered by N-, C-or S-nutrient starvation [4]. The ligninolytic systems that decompose lignin to CO2 has been reported to be suppressed by addition of L-glutamate, L-glutamine, L-histidine or NH: [5,6]. Furthermore, the biosynthesis of veratryl alco-
8 1995 Federation
SSDI 0378-1097(95)00257-X
of European
Microbiological
Y. Akamatsu, M. Shimada /FEMS
186
Microbiology Letters 131 (1995) 185-188
I
2. Materials and methods 2.1. Microorganism
and culture conditions
I’
.JJ
,’
= E
$0.5The white-rot fungus, P. chrysosporium (ATCC 24725) was grown without shaking at 37°C in lOO-ml flasks containing 10 ml of Kirk’s medium [lo] under the normal atmosphere. The culture medium was modified using 2% glucose, 1.2 mM ammonium tartrate, 7-fold increase of the minerals, and 10 mM trans-aconitate buffer (pH 4.3). Tween 80 (O.l%, vol/vol) and veratryl alcohol (1.5 mM) were added for enhancing excretion of lignin peroxidase into the medium, as previously reported [ill. On day 3 of the cultivation, 1 ml of the aqueous solution of each of amino acid (2mM), NH:, and cycloheximide (100 pg> or an equal amount of water (control) was added into a set of ligninolytic cultures. Incubation was performed until the specified time of the lignin peroxidase assays. 2.2. Lignin peroxidase
r .Z? > ‘5 : !& _J OL 0
1.
2 3 4 Culture days
5
6
Fig. 1. Effects of amino acids on lignin peroxidase production during cultivation of P. chtysosporium: 0, L-Phenylalanine (0.2 mM); 0, Glutamate (0.2 mM1; 0, and n , Control (H,O). Lignin peroxidase (Lip) activities were assayed for each set of 3 cultures. The enzyme activities were assayed on day 2 and day 3 before the addition of the compounds. The reaction mixtures for lignin peroxidase assays contained veratryl alcohol (3.3 mM), hydrogen peroxide (0.25 mM), 50 mM tartrate buffer (pH 4.5), and appropriate amounts of the culture filtrate. The lignin peroxidase activity was assayed as described in the text at the times of 14, 25, 47, and 68 h after addition of the compounds.
assay
Three flasks were tested for measurement of lignin peroxidase activity. The cultures were mixed for averaging enzyme activities, and filtered through nylon cloth. Lignin peroxidase activity in the filtrate was assayed twice at pH 4.5 and 30°C by measurement of the increase in absorbance at 340 nm due to veratraldehyde formed [ 121.
3. Results and discussion
3.1. Comparison of suppressive effects of phenylalanine and other amino acids
Fig. 1 shows the kinetics of lignin peroxidase activity after addition of phenylalanine and glutamate at the time indicated by the arrow. The results clearly indicate that phenylalanine caused a delay of about 3 days in the appearance of lignin peroxidase activity, as compared with the control. Glutamate did not cause such an effect, but rather enhanced enzyme activity to a higher level until day 6. Addition of cycloheximide, an inhibitor of protein synthesis, caused a delay of one day in the appearance of lignin peroxidase (data not shown), which is consistent
with the finding that the ligninolytic system was inhibited by addition of cycloheximide [6]. Thus the expression of lignin peroxidase synthesis triggered by nitrogen starvation may be regulated at the transcription level, as suggested by Li et al. [13]. Possi-
Table 1 Effects of various amino acids on repression of lignin peroxidase activity in the ligninolytic cultures of P. chrysosporium Amino acids added
Lignin peroxidase
Control (Hz LO) Arginine Proline Aspartate Glutamine Glycine Omithine Isoleucine Glutamate Alanine Histidine Phenylalanine
100 137 119 107 106 104 101 82 80 75 57 0
activity (o/o)
Final concentrations of the amino acids added were 0.2 mM. Each amino acid was added 67 h of cultivation and further incubated for 25 h. Cultures were then harvested and the lignin peroxidase activities were determined and expressed as the relative value based on the control (42.4 nkat/culture).
Y. Akomatsy M. Shimada /FEMS Microbiology Letters 131 (1995) 185-188
ble lignin peroxidase-suppressing effects of the other L-amino acids were examined. Table 1 shows that no lignin peroxidase activity was detected in the cultures 25 h after addition of phenylalanine. In contrast, the other amino acids such as glutamine, alanine, arginine, aspartate, glycine, isoleucine, ornithine, and proline as well as glutamate did not show any significant inhibition of the lignin peroxidase synthesis at this concentration (0.2 mM). Only histidine reduced the enzyme activity by 43%. It was alternatively confirmed that L-phenylalanine (0.2-l mM) did not inhibit lignin peroxidase activity on the oxidation of veratryl alcohol in the in vitro system (data not shown). Thus, L-phenylalanine appeared to be the strongest suppressor in vivo at this concentration.
187
3.2. Comparison of the suppressive eflects by Lphenylalanine and NH,+
Fig. 2. Comparison of the suppressions of lignin peroxidase (Lip) activity caused by phenylalanine (0) and NH: (0) which had been added to the ligninolytic cultures of P. chrysosporium at different concentrations. Lignin peroxidase activities were assayed 23 h after addition of each amino acid at the final concentrations indicated. The lignin peroxidase activity in the control was 20.7 nkat/culture.
We further examined the possibility that NH: rather than L-phenylalanine might be more directly involved in the suppression of lignin peroxidase synthesis, because it could be liberated from this aromatic amino acid by phenylalanine ammonia-lyase which was markedly activated along with appearance of lignin peroxidase activity in the ligninolytic cultures [9]. Fig. 2 shows the comparison of different effects of phenylalanine and ammonia. The results obtained with NH: added in the range from 0.01 mM to 3 mM indicate that NH: impaired strongly lignin peroxidase synthesis at 3 mM and rather moderately (about 75% of the control) around 2 mM. However, a 40% increase was observed around 0.5 mM in sharp contrast with the pattern exhibited by phenylalanine. Further alternative experiments with glutamate added at various concentrations showed that this amino acid also slightly enhanced lignin peroxidase activity, but impaired it significantly in the range from 1.5 to 3 mM (15 to 25% activity of the control). The observed suppression of the lignin peroxidase synthesis with both glutamate and NH: at higher concentrations are in good agreement with the previous report that ligninolytic activity (14Clignin + 14COZ) was reduced to 17% by addition of 2.8 mM r_-glutamate [5]. According to the central role of L-glutamate in the primary amino acid
metabolism of living cells, it is not surprising that the suppressive effect of NH: on lignin peroxidase activity is very similar to that of glutamate in ligninolytic culture of P. chrysosporium. This assumption is reasonably supported by the previous finding that addition of NH: to ligninolytic cultures enhanced L-glutamate dehydrogenase and L-glutamine synthetase activities, resulting in an increase of the intracellular glutamate level [6]. Accordingly, the idiophasic phase of the ligninolytic culture is switched back to the primary (tropho) phase at greater concentrations of glutamate. However, a moderately higher concentration of L-glutamate or ammonium nitrogen are beneficial for the lignin peroxidase synthesis, whereas phenylalanine impaired it more strongly at the lower concentrations. In conclusion, this investigation shows that Lphenylalanine added to the ligninolytic cultures of P. chrysosporium strongly suppressed lignin peroxidase synthesis. Thus, r_-phenylalanine plays a key role, as does L-glutamate in the regulation of secondary metabolism of this fungus. Furthermore, the two amino acids impaired the lignin peroxidase synthesis somewhat differently. However, the molecular mechanisms remain to be elucidated.
188
Y. Akamatsy
M. Shimada / FEMS Microbiology Letters 131 (1995) 185-188
References [l] Ander, P. and Eriksson K.E. (1978) Lignin degradation and utilization by microorganisms. Prog. Ind. Microbial. 14, l58. [2] Leisola, M.S.A. and Fiechter, A. (19851 New trends in lignin biodegradation. Adv. Biotech. Process. 5, 59-89. [3] Kirk, T.K. and Farrell, R.L. (19871 Enzymatic ‘Combustion’:The microbial degradation of lignin. Ann. Rev. Microbial. 41, 465-505. [4] Jeffries, T.W., Choi, S. and Kirk, T.K. (1981) Nutritional regulation of lignin degradation by Phanerochaete chrysosporium. Appl. Environ. Microbial. 42, 290-296 [5] Fenn, P. and Kirk, T.K. (1981) Relationship of nitrogen to the onset and suppression of ligninolytic activity and secondary metabolism in Phanerochaete chrysosporium. Arch. Microbial. 130, 59-65. [6] Fenn, P., Choi, S. and Kirk, T.K. (19811 Ligninolytic activity of Phanerochaete chrysosporium: Physiology of suppression by NH: and L-glutamate. Arch. Microbial. 130, 66-71. [7] Jensen, K.A., Evans, Jr.K.M.C. Evans, Kirk, T.K. and Hammel, K.E. (1994) Biosynthetic pathway for veratryl alcohol in
[8]
[9]
[lo]
[ll]
[12] [13]
the ligninolytic fungus Phanerochaete chrysosporium. Appl. Environ. Microbial. 60, 709-714. Shimada, M., Nakatsubo, F., Higuchi, T. and Kirk, T.K. (1981) Biosynthesis of the secondary metabolic veratryl alcoho1 in relation to lignin degradation in Phanerochaete chrysosporium. Arch. Microbial. 129, 321-324. Hattori, T. and Shimada, M. (1995) The detection of PAL activity in the culture of Phanerochaete chrysosporium. Proc. 40th Ann. Conf. Jpn. Wood Res. Soc.(Tokyo) p. 599. Kirk, T.K., Schulz, E., Connors, W.J., Lorenz, L.F. and Zeikus, J.G. (19781 Influence of culture parameters on lignin metabolism by Phanerochaete chrysosporium. Arch. Microbiol. 117, 277-285. Asther,M., Corrieu, G., Drapon, R. and Odier, E. (19871 Effects of Tween 80 and oleic acid on ligninase production by Phanerochaete chrysosporium INA-12. Enzyme Microb. Technol. 9, 245-249. Tien, M. and Kirk, T.K. (1988) Lignin peroxidase of Phanerochaete chrysosporium. Methods. Enzymol. 161, 238-249. Li, D., Alit, M. and Gold, M.H. (1994) Nitrogen regulation of lignin peroxidase gene transcription. Appl. Environ. Microbiol. 60, 3447-3449.