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Strategies for improved lignin peroxidase production in agitated pellet cultures of Phanerochaete chrysosporium and the use of a novel inducer
FEMS MicrobiologyLetters 71 (1990) 325-330 Published by Elsevier
325
FEMSLE04157
Strategies for improved ligrfin peroxidase production in agitated pellet cultures of Phanerochaete chrysosporium and the use of a novel inducer M. Liebeskind t, H. HiScker t C. W a n d r e y 2 a n d A.G. J~iger 2 t Institute o/Textile Chemistry and Macromolecular Chemistry, University o/Aachen. Aachen and 2 Institute o/Biotechnology. Research Centre Jiilich (KFA), Jiilich. F..R.G.
Received 12 April 1990 Revisionreceived29 May 1990 Accepted 30 May 1990 Key words: Phanerochaete chrysosporium; Mycelial pellets; 3,4-Dimethoxybenzylamine; Lignin peroxidase
1. SUMMARY
2. INTRODUCTION
The production of extracellular H202-dependent lignin peroxidase by the basidiomycete Phanerochaete chrysosporium in agitated submerged cultures was improved by the addition of a novel inducer and by a particular agitation and temperature program. The combination of an exact shift of agitation rate from higher to lower speed after the onset of secondary metabolism, the simultaneous decrease of the incubation temperature from 39 to 25°C and the supply of appropriate concentrations of dimethoxybenzylamine instead of veratryi alcohol resulted in a 5-fold increase in enzyme production compared to control experiments. ~/lth this method, lignin peroxidase activities at a maximum of 1312 U 1-~ were obtained using glycerol as sole carbon source and nitrogen-limited media.
The ligninolytic enzyme system of the basidiomycete Phanerochaete chrysosporium is synthesized in response to nitrogen starvation [1]. The lack of nitrogen however limits lignin peroxidase production. The low yield of lignin peroxidase has become a problem for the large scale production and the application of these fungal enzymes to biotechnological processes. Several different cultivation methods for the production of lign-.'n peroxidases are presently known, including the use of shallow stationary cultures, agitated submerged cultures [2] and multiple immobilization methods
Correspondence to." M. Liebeskind,Institute of Textile Chemistry and MacromolecularChemistry, Universityof Aachen, D-5100 Aachen,F.R.G.
[31. The use of different detergents (Tween 20, Tween 80, and 3-[(3-colamidopropyl)-dimethylammonio]l-propanesulfonate, CHAPS) [2] made it possible to obtain lignin biodegradation and the formation of the ligninolytic enzyme system in agitated pellet cultures. This enzyme activity can be enhanced by addition of veratryl alcohol (dimcthoxy benzylalcohoi) [4] which is both an irducer and a lignin model compound. Odier et al. [5] were able to circumvent the nitrogen based
0378-1097/90/$03.50 © 1990 Federationof European MicrobiologicalSocieties
326
iCH~
i
CH~
OCHj OCH)
(1)
iCH
+H20 LiP ~ LiP ~ O C H ~ "''~ -NH3 OCH3
(2)
OCH 3 OCH~
(3)
Fig. 1. Effectof lignin peroxidase(LIP)on dimethoxybenzylamine(1) and formationof ver~,irylalcohol(2) and veratrylaldehyde (3). repression of the ligninolytic enzyme system by use of a mutant strain (Phanerochaete chrysosporium INA 12) and thus achieved high yields of lignin peroxidase. The use of amino acids, covalently bonded to lignin model compounds, as growth substrate for Phanerochaete chrysosporium has also been suggested [6] to avoid the nitrogen based repression of the enzyme formation. Other efforts have been focussed on the role of the appropriate pellet size [7], the influence of temperature starts [8] and other cultivation parameters. The high variance of the enzyme yields of more than 25% [2] and the low reproducibility of the results [9], however, could not be explained. In this study the effects upon the lignin peroxidase activity of Phanerochaete chrysosporium BKM-F-1767 in agitated submerged cultures, of addition of dimethoxybenzylamine instead of veratryl alcohol and the use of modified cultivation conditions were investigated. Dimethoxybenzylamine acts as a lignin model compound, as an enzyme inducer and as nitrogen source in the secondary metabofism (Fig. 1).
3. MATERIALS A N D M E T H O D S
3.1. Organism and culture conditions Phanerochaete chrysosporium BKM F-1767 (ATCC 24725) was maintained on 3% malt agar slants, pH 5.2 (B. Braun Melsungen, F.R.G.). The fungus was grown routinely in 150 ml of a chemically defined liquid medium in 500 ml Erlenmeyer flasks containing 1% glycerol, 0.05% Tween 80, and 0.1 M ir~ms-aconitic acid, pH 4.5, as buffer.
Mineral salts and trace elements were used as described elsewhere [9] with the exception that 1 g MnSO4. H 2 0 and 0.15 g FeSO4-7HzO were added to the stock solution. For inoculum, cultures of the fungi were grown from conidia [2] in stationary 2.8-1 Fernbach flasks containing 50 ml of the medium described above (without detergent and without inducer). After 48 h the mycelium was blended for 60 s using a Waling blender. This inoculum was added at a concentration of 45 mg mycelium (dry weight) per 1 medium. Aft cultures were grown on a rota.ry shaker RC-TK, lnfors HT, SDM 5.0 cm (Infors Munich, F.R.G.) (agitatk,a rates indicated in the text) and flushed with 100% 02 at the time of inoculation and then every 24 h or when a sample was taken. Incubation temperature was 3 9 ° C when not indicated otherwise in the text. Two different inducers were used in this study: dimethoxybenzylalcohol (veratryl alcohol) and dimethoxybenzylamine (DMBA) (all from Merck Schuchardt, F.R.G.). The compounds were filter sterilized and added to the cultures at the time of L~,,calatlon. S:andard fermentations contained 0.2 mM veratryl alcohol if not indicated otherwise in the text. For pellet size determination and enumeration, the content of an Erlenmeyer flask was poured on to a glass plate. The pellets were counted and their size was determined with a scale. Single hyphae, growing out of the mycelial pellets were not considered. If the flasks contained more than 2000 pellets, only three flasks were counted.
3.2. A ,alyticai methods Lignin peroxidase activity was determined by the method of Tien and Kirk [10]. One unit (U) is defined as 1/~mol of veratryl alcohol oxidized in 1 min, and activities are expressed as U !- i. Glycerol and ammonium were determined by specific tests using enzymes purchased from Boehringer Mannhelm, F.R.G.
3.3. Separation of extraceilular proteins Extracellular culture fluid was concentrated by ultrafiltration washed twice with H 2 0 and then separated directly by FPLC on a Mono Q colunm (Pharmacia Biotechnology, Freiburg, F.R.G.)
using a gradient of acetate buffer, pH 6.0, from 10 m M to 600 mM as described earlier [2.9].
3.4. Product identification Products from lignin peroxidase catalyzed oxidation of dimethoxybenzylamine were identified from reaction mixtures containing 800 /~1 enzyme concentrate (10000 U / l ) , 1 ml D M B A (0.24 mM) in sodium '.artrate pH 3.0, 0.25 M and 100/tl H202 (8 mM). The reaction was started by H202 addition. The reaction mixture was separated by high pressure liquid chromatography on a ODS-1 column with a gradient of CH3CN ( 0 60%) as eluent. The products were identified by their Rf and their UV-absorption spectra (compared with authentic standards) and quantified by their absorbance at 254 nm.
39 C
200
l
!
ZOO
100-
'~
100
0
o ~9 C
2S c
~9 c
300.
3OO
200.
!
i
2OO
100
1oo
,
84
96
108
84
g6
108
120
132
144
o
Fermentot;en time [ h i Fig. 2. Dependence
of lignin peroxidase productivity of
4. RESULTS
Phanerochaete ch~sosporium on cultivation temperature and shaker speed. Arrow indicates the shift of agitation rate and incubation temperature.
4.1. Influence of agitation rate The formation of mycelial pellets was strictly dependent on the speed of the rotary shaker: the pellet number was directly, the pellet diameter was inversely proportional (Table 1). Consequently the surface area of each pellet was large at low agitation rate, but the calculated total pellet surface was rather small (6390 mm2), but it increased up to 23 500 m m 2 per flask at an agitation rate of 240 rpm (the surfaces were calculated from the diameter of the pellets). Under all cultivation conditions the form of the pellets was approximately spherical and uniform when the preculture was blended in a Waring blender for at least 60 s. Shorter periods of disruption resulted in larger and more irregular pellets. The use of spores as inoculum
also resulted in small pellets but earlier findings [2], that the onset of lignin peroxidase production is delayed if spores were used, were confirmed. Therefore subsequent experiments were performed with precultures and agitation rates of 240 rpm. As can be seen in Fig. 2, lignin peroxidase reached a level of about 150 U ! -~ with sharp decline and loss of enzyme activity within hours. This decline could be avoided when the speed of the shaker was decreased to 90 rpm, after the onset of the enzyme production. Usually the shaker speed was decreased after 90 h of fermentation (Fig. 2). In this case the activity increased for more than 24 h and did not decline at least for the next 24 h. This speed shift had no influence on the number and the shape of the pellets. At the onset of secondary
Table 1 Effect of agitation rate on number and surface area of Pkanerochaete chrysosporium pellets Values are means+ S.D. for 3 to 5 replicate cultures. rpm
Pellet number
Pellet diameter (ram)
Individual pellet surface (mm2)
Individual pellet volume (mm3)
Medium area surface area/flask (mm2)
90 144 200 240
120+ 12 395-1- 35 1200+ 80 6300+ 102
4.1 +0.5 2.8+0.5 1.9+0.5 l.l +0.5
52.8 24.0 11.4 3.7
36.1 11.1 3.6 0.7
6400 9500 13700 23500
metabolism the nitrogen source was depleted a n d primary growth had stopped, so no change in the ratio o f pellet surface to pellet n u m b e r could occur. Thus agitation rate had a p r o n o u n c e d influence on the duration and level of lignin peroxidase activity.
ae
~e
sa;
4
4.2. Influence of incubation temperature The level o f ligmn peroxidase production was also d e p e n d e n t on the incubation temperature. A decrease from 39 to 25 ° C resulted in an increase of lignin peroxidase production from 180 to 220 U l - t (Fig. 2). A shift of temperature together with a simultaneous shift o f shaking speed increased the enzyme production by about 50% (Fig. 2) in c o m parison to control experiments without shifts. All these cultivations were p e r f o r m e d with 0.2 m M of veratryl alcohol as enzyme inducer.
*e'
4-
)e.
o
Fig. 3. Effect of lignin peroxidase on dimethoxybenzylamine, HPLC-profile of the reaction products: ! = dimethoxybenzylamine: 2 = veratryl alcohol: 3 = veratrylic acid: 4 = veratrylaldehyde; detector wave length 254 nm (mAU = milliampere unit).
4.3. Effect of inducers To investigate the influence of different inducers on lignin peroxidasv p r o d u c t i o n various c o n c e n t r a t i o n s o f veratryl alcohol a n d dimethoxybcnzylamin¢ were a d d e d to the agitated pellet cultures. The results (Table 2) indicate that the optimal concentration of veratryl alcohol is between 1.0 and 3.0 m M as d e m o n s t r a t e d earli,:r [4]. Cultures with D M B A showed higher enzyme activities than those with veratryl alcohol at all tested concentrations with an average m a x i m u m activity of 1233 U l - t lignin peroxidas¢ at a concentration of 5.0 m M D M B A . E n z y m e activity appeared earlier with increasing concentrations of veratryi alcohol, whereas higher concentrations of
d i m e t h o x y b e n z y l a m i n e delayed the appearance of enzyme activity. This fact indicates that during the fermentation, a m m o n i u m might be released from dimethoxybenzylamine. The peak-delay effect of a m m o n i u m ions o n lignin biodcgradation by Phanerochaete chrysosporium has previously been reported [11]. To c o n f i r m these findings, D M B A was inc u b a t e d with lignin peroxidase in high concentrations (see MATERIALS AND METHODS) and the solution was analyzed for possible produts. Both veratryl alcohol a n d veratrylaldehyde were detected (Fig. 3). O b v i o u s l y the a m i n e was d e a m i n a t e d to the c o r r e s p o n d i n g alcohol and sub-
Table 2 Effect of concentration of veratryl alcohol and dimethoxybenzylamine on lignin peroxidase production of Phanef~ho@te chr~,-
sosporium Values are means + S.D. for 4 to 5 replicate cultures (cultures were grown with a temperature shift from 39 to 30°C and a speed shift from 240 to 90 rpm). n,d,, not determined. Cone.
Veratryl alcohol
(raM)
Lignin peroxidase activity (U I- I)
Time of maximum activity (h)
DMBA Lignin peroxidase activity (U I- I)
Time of maximum activity (h)
0.2 0.5 1.0 3.0 5.0
330 5:35 500 ± 66 874 4-96 808 + 93 n.d.
104 104 97 84 -
n.d. 780 :i:93 n.d, 912 =l=30 1233 + 92
110 144
96
A g409
jl~,
t--I
i
-800
. ;
"
J"
.
,
.
lo
20
30
-600
•
-4oo
" e u
-200
'4
oxidase production by Phanerochaete chrysosporium in agitated pellet cultures and the shape,
I!
i
5. DISCUSSION This study has demonstrated that lignin per-
o
t (mtn)
-000
|
i
.
zyme-time programs of two not exactly synchronized cultures. Farell et al, [12] had shown earlier that the different extraceUular proteins of Phanerochaete chrysosporium appeared in agitated pellet cultures after different times.
-~oo
; ,G
.2O0
4
4" Fig. 4. Fast pl,~zin liquid chromatography profile(absorbancy at 409 nm) o( the extracellular fluid from a 5-day culture of PhanerochaetechrysosporiumATCC 2472S,grownwith veratryl alcohol (A) and dimethoxybenzylamine(B) ~dotted line represents the acetate gradient from 10 mM to 600 mM).
sequently oxidized to the aldehyde (Fig. 1). Veratrylic acid was identified as a third main reaction ~product. The comparison of veratryl alcohol and dimethoxybenzylamine as substrates for lignin peroxidase showed that, under the same conditions, the conversion rate of DMBA was 5.5% of the conversion rate of veratryl alcohol.
4.4. Effect of dimethoxybenzylamine on the profile of the extracellular proteins of Phanerochaete chrysosporium Fig. 4 shows the extraeellular haemoproteins (409 nm absorbing material) of 6-day-old cultures. The enzyme profile of cultures supplied with DMBA and with veratryl alcohol were almost identical. The difference in peak height may reflect the temporal differences of the genetic isoen-
size, and number of pellets can be controlled by variation of the agitation rate. Although the importance of agitation rates has been described previously [4] it was never examined systematically. Small mycelial pellets have the advantage of short diffusion paths and are therefore a good nutrient and oxygen supply of the cells in the centre of the pellets. The small pellet form can be achieved with high agitation rates. High agitation rates however produce shear forces, which might repress the formation of the iigninolytic enzyme system [13]. This repression can be avoided by a significant decrease of the agitation rate after the onset of ligninolytic activity. It is suggested that not only the agitation rate of the shaker is important for ~he enzyme production, but also the size and diameter of the flasks used and the amplitude of the respective shaker. For that reason the given speeds have not to be seen as absolute values but have to be adjusted (o an optimal shaker type-flask size-speed ratio for each laboratory. If the culture conditions are reproduced exactly the standard deviations of enzyme productivities are lower than 10% (Table 2) compared to over 30% in earlier reports [2] and the results were more reliable than reported by other authors [9], A speed shift in combination with a simultaneous temperature shift resulted in a more than 2-fold increase in enzyme activity compared to control experiments. A temperature shift from 39 to 30 *C has already been described by Asther et al. [8]. To obtain reliable results the precultures should be thoroughly blended. The addition of dimethoxybenzylamine instead of veratryl alcohol significantly increased lignin
330 peroxidase production. The results show that it was not D M B A itself, but its cleavage p r o d u c t veratryl alcohol which is the real inducer o f the enzyme activity. (1) Veratryl alcohol is a cleavage product of D M B A . (2) The extracellular enzyme profile of cultures supplied with the two different substances were almost identical. (3) The enzyme production in cultures with high concentrations of D M B A was delayed, probably due to the repressive effect of ~Lmmonium on lignin peroxidase production. It was calculated that one unit of enzyme released 0.0546 # m o l a m m o n i u m from D M B A . It can be assumed that this slowly released a m m o n i u m enables the fungus to continue with metabolism and resulted therefore in the very high enzyme activity.
REFERENCES [l] Fenn, P. and Kirk. T.K. (1981) Arch. Microbiol. 130, 59-65. [2] J~tger,A., Croan. S. and Kirk, T.K. (1985) Appl. Environ. Microbiol. 50. 1274-1278. i3] Jitger, A., Kern. H. and Wandrey. C. (1989) 4th Int. Conf. Biotechnology in the Pulp and Paper Industry, Raleigh NC. pp. 142-143, Tappi Press Atlanta. [4] Leisola, M. and Fiechter, A. ~2985) FEMS MicrobioL Len. 29, 33-36. [5] Asther, M., Corrieu, O., Drapon, R. and Odier, E. (1987) Enzyme Microb. Technol, 9, 245-249. [6] Tien, M., Kersten, P. and Kirk, T.K. (1987) Appl. Environ. Microbiol. 53, 242-245. [7] Leisola, M.. Thanei-Wyss. U. and Fiechter, A. (1985) J. Biotechnol. 3, 97-107. [8] Asther, M., Capdevila, C. and Corrieu, G. (1988) Appl. Environ. Microbiol. 54, 3194-3196. [9] Tien, M. and Kirk, T,K. (1988) Methods Enzymol. 161, 238-249. [~0] Tien, M. and Kirk, T,K. (1984) Proc. N:ltl. Acad. Sci. U.S.A. 81, 2280-2284. [11] Keyser, P., Kirk, T.K. and Zeikus, J. (1978) J. Bacteriol. 135, 790-797. [12] Farrell, R., Murtagh, K., Tien, M., Mnzuch, M. and Kirk, T.K. (1989) enmzyme Microb. Technol. ! z, 322-328. [13] Kirk, T.K., Schultz, E., Connors, W.J., Lorenz~ L.F. and Zeikus, .LG. (1978) Arch. Microbiol. 117, 277-285.
4#"
ACKNOWLEDGEMENTS W e are grateful to H. Kneifel for the H P L C analysis o f the D M B A reaction products a n d to H. ~erndt for valuable discussion.
~[
......
al.L
325
FEMSLE04157
Strategies for improved ligrfin peroxidase production in agitated pellet cultures of Phanerochaete chrysosporium and the use of a novel inducer M. Liebeskind t, H. HiScker t C. W a n d r e y 2 a n d A.G. J~iger 2 t Institute o/Textile Chemistry and Macromolecular Chemistry, University o/Aachen. Aachen and 2 Institute o/Biotechnology. Research Centre Jiilich (KFA), Jiilich. F..R.G.
Received 12 April 1990 Revisionreceived29 May 1990 Accepted 30 May 1990 Key words: Phanerochaete chrysosporium; Mycelial pellets; 3,4-Dimethoxybenzylamine; Lignin peroxidase
1. SUMMARY
2. INTRODUCTION
The production of extracellular H202-dependent lignin peroxidase by the basidiomycete Phanerochaete chrysosporium in agitated submerged cultures was improved by the addition of a novel inducer and by a particular agitation and temperature program. The combination of an exact shift of agitation rate from higher to lower speed after the onset of secondary metabolism, the simultaneous decrease of the incubation temperature from 39 to 25°C and the supply of appropriate concentrations of dimethoxybenzylamine instead of veratryi alcohol resulted in a 5-fold increase in enzyme production compared to control experiments. ~/lth this method, lignin peroxidase activities at a maximum of 1312 U 1-~ were obtained using glycerol as sole carbon source and nitrogen-limited media.
The ligninolytic enzyme system of the basidiomycete Phanerochaete chrysosporium is synthesized in response to nitrogen starvation [1]. The lack of nitrogen however limits lignin peroxidase production. The low yield of lignin peroxidase has become a problem for the large scale production and the application of these fungal enzymes to biotechnological processes. Several different cultivation methods for the production of lign-.'n peroxidases are presently known, including the use of shallow stationary cultures, agitated submerged cultures [2] and multiple immobilization methods
Correspondence to." M. Liebeskind,Institute of Textile Chemistry and MacromolecularChemistry, Universityof Aachen, D-5100 Aachen,F.R.G.
[31. The use of different detergents (Tween 20, Tween 80, and 3-[(3-colamidopropyl)-dimethylammonio]l-propanesulfonate, CHAPS) [2] made it possible to obtain lignin biodegradation and the formation of the ligninolytic enzyme system in agitated pellet cultures. This enzyme activity can be enhanced by addition of veratryl alcohol (dimcthoxy benzylalcohoi) [4] which is both an irducer and a lignin model compound. Odier et al. [5] were able to circumvent the nitrogen based
0378-1097/90/$03.50 © 1990 Federationof European MicrobiologicalSocieties
326
iCH~
i
CH~
OCHj OCH)
(1)
iCH
+H20 LiP ~ LiP ~ O C H ~ "''~ -NH3 OCH3
(2)
OCH 3 OCH~
(3)
Fig. 1. Effectof lignin peroxidase(LIP)on dimethoxybenzylamine(1) and formationof ver~,irylalcohol(2) and veratrylaldehyde (3). repression of the ligninolytic enzyme system by use of a mutant strain (Phanerochaete chrysosporium INA 12) and thus achieved high yields of lignin peroxidase. The use of amino acids, covalently bonded to lignin model compounds, as growth substrate for Phanerochaete chrysosporium has also been suggested [6] to avoid the nitrogen based repression of the enzyme formation. Other efforts have been focussed on the role of the appropriate pellet size [7], the influence of temperature starts [8] and other cultivation parameters. The high variance of the enzyme yields of more than 25% [2] and the low reproducibility of the results [9], however, could not be explained. In this study the effects upon the lignin peroxidase activity of Phanerochaete chrysosporium BKM-F-1767 in agitated submerged cultures, of addition of dimethoxybenzylamine instead of veratryl alcohol and the use of modified cultivation conditions were investigated. Dimethoxybenzylamine acts as a lignin model compound, as an enzyme inducer and as nitrogen source in the secondary metabofism (Fig. 1).
3. MATERIALS A N D M E T H O D S
3.1. Organism and culture conditions Phanerochaete chrysosporium BKM F-1767 (ATCC 24725) was maintained on 3% malt agar slants, pH 5.2 (B. Braun Melsungen, F.R.G.). The fungus was grown routinely in 150 ml of a chemically defined liquid medium in 500 ml Erlenmeyer flasks containing 1% glycerol, 0.05% Tween 80, and 0.1 M ir~ms-aconitic acid, pH 4.5, as buffer.
Mineral salts and trace elements were used as described elsewhere [9] with the exception that 1 g MnSO4. H 2 0 and 0.15 g FeSO4-7HzO were added to the stock solution. For inoculum, cultures of the fungi were grown from conidia [2] in stationary 2.8-1 Fernbach flasks containing 50 ml of the medium described above (without detergent and without inducer). After 48 h the mycelium was blended for 60 s using a Waling blender. This inoculum was added at a concentration of 45 mg mycelium (dry weight) per 1 medium. Aft cultures were grown on a rota.ry shaker RC-TK, lnfors HT, SDM 5.0 cm (Infors Munich, F.R.G.) (agitatk,a rates indicated in the text) and flushed with 100% 02 at the time of inoculation and then every 24 h or when a sample was taken. Incubation temperature was 3 9 ° C when not indicated otherwise in the text. Two different inducers were used in this study: dimethoxybenzylalcohol (veratryl alcohol) and dimethoxybenzylamine (DMBA) (all from Merck Schuchardt, F.R.G.). The compounds were filter sterilized and added to the cultures at the time of L~,,calatlon. S:andard fermentations contained 0.2 mM veratryl alcohol if not indicated otherwise in the text. For pellet size determination and enumeration, the content of an Erlenmeyer flask was poured on to a glass plate. The pellets were counted and their size was determined with a scale. Single hyphae, growing out of the mycelial pellets were not considered. If the flasks contained more than 2000 pellets, only three flasks were counted.
3.2. A ,alyticai methods Lignin peroxidase activity was determined by the method of Tien and Kirk [10]. One unit (U) is defined as 1/~mol of veratryl alcohol oxidized in 1 min, and activities are expressed as U !- i. Glycerol and ammonium were determined by specific tests using enzymes purchased from Boehringer Mannhelm, F.R.G.
3.3. Separation of extraceilular proteins Extracellular culture fluid was concentrated by ultrafiltration washed twice with H 2 0 and then separated directly by FPLC on a Mono Q colunm (Pharmacia Biotechnology, Freiburg, F.R.G.)
using a gradient of acetate buffer, pH 6.0, from 10 m M to 600 mM as described earlier [2.9].
3.4. Product identification Products from lignin peroxidase catalyzed oxidation of dimethoxybenzylamine were identified from reaction mixtures containing 800 /~1 enzyme concentrate (10000 U / l ) , 1 ml D M B A (0.24 mM) in sodium '.artrate pH 3.0, 0.25 M and 100/tl H202 (8 mM). The reaction was started by H202 addition. The reaction mixture was separated by high pressure liquid chromatography on a ODS-1 column with a gradient of CH3CN ( 0 60%) as eluent. The products were identified by their Rf and their UV-absorption spectra (compared with authentic standards) and quantified by their absorbance at 254 nm.
39 C
200
l
!
ZOO
100-
'~
100
0
o ~9 C
2S c
~9 c
300.
3OO
200.
!
i
2OO
100
1oo
,
84
96
108
84
g6
108
120
132
144
o
Fermentot;en time [ h i Fig. 2. Dependence
of lignin peroxidase productivity of
4. RESULTS
Phanerochaete ch~sosporium on cultivation temperature and shaker speed. Arrow indicates the shift of agitation rate and incubation temperature.
4.1. Influence of agitation rate The formation of mycelial pellets was strictly dependent on the speed of the rotary shaker: the pellet number was directly, the pellet diameter was inversely proportional (Table 1). Consequently the surface area of each pellet was large at low agitation rate, but the calculated total pellet surface was rather small (6390 mm2), but it increased up to 23 500 m m 2 per flask at an agitation rate of 240 rpm (the surfaces were calculated from the diameter of the pellets). Under all cultivation conditions the form of the pellets was approximately spherical and uniform when the preculture was blended in a Waring blender for at least 60 s. Shorter periods of disruption resulted in larger and more irregular pellets. The use of spores as inoculum
also resulted in small pellets but earlier findings [2], that the onset of lignin peroxidase production is delayed if spores were used, were confirmed. Therefore subsequent experiments were performed with precultures and agitation rates of 240 rpm. As can be seen in Fig. 2, lignin peroxidase reached a level of about 150 U ! -~ with sharp decline and loss of enzyme activity within hours. This decline could be avoided when the speed of the shaker was decreased to 90 rpm, after the onset of the enzyme production. Usually the shaker speed was decreased after 90 h of fermentation (Fig. 2). In this case the activity increased for more than 24 h and did not decline at least for the next 24 h. This speed shift had no influence on the number and the shape of the pellets. At the onset of secondary
Table 1 Effect of agitation rate on number and surface area of Pkanerochaete chrysosporium pellets Values are means+ S.D. for 3 to 5 replicate cultures. rpm
Pellet number
Pellet diameter (ram)
Individual pellet surface (mm2)
Individual pellet volume (mm3)
Medium area surface area/flask (mm2)
90 144 200 240
120+ 12 395-1- 35 1200+ 80 6300+ 102
4.1 +0.5 2.8+0.5 1.9+0.5 l.l +0.5
52.8 24.0 11.4 3.7
36.1 11.1 3.6 0.7
6400 9500 13700 23500
metabolism the nitrogen source was depleted a n d primary growth had stopped, so no change in the ratio o f pellet surface to pellet n u m b e r could occur. Thus agitation rate had a p r o n o u n c e d influence on the duration and level of lignin peroxidase activity.
ae
~e
sa;
4
4.2. Influence of incubation temperature The level o f ligmn peroxidase production was also d e p e n d e n t on the incubation temperature. A decrease from 39 to 25 ° C resulted in an increase of lignin peroxidase production from 180 to 220 U l - t (Fig. 2). A shift of temperature together with a simultaneous shift o f shaking speed increased the enzyme production by about 50% (Fig. 2) in c o m parison to control experiments without shifts. All these cultivations were p e r f o r m e d with 0.2 m M of veratryl alcohol as enzyme inducer.
*e'
4-
)e.
o
Fig. 3. Effect of lignin peroxidase on dimethoxybenzylamine, HPLC-profile of the reaction products: ! = dimethoxybenzylamine: 2 = veratryl alcohol: 3 = veratrylic acid: 4 = veratrylaldehyde; detector wave length 254 nm (mAU = milliampere unit).
4.3. Effect of inducers To investigate the influence of different inducers on lignin peroxidasv p r o d u c t i o n various c o n c e n t r a t i o n s o f veratryl alcohol a n d dimethoxybcnzylamin¢ were a d d e d to the agitated pellet cultures. The results (Table 2) indicate that the optimal concentration of veratryl alcohol is between 1.0 and 3.0 m M as d e m o n s t r a t e d earli,:r [4]. Cultures with D M B A showed higher enzyme activities than those with veratryl alcohol at all tested concentrations with an average m a x i m u m activity of 1233 U l - t lignin peroxidas¢ at a concentration of 5.0 m M D M B A . E n z y m e activity appeared earlier with increasing concentrations of veratryi alcohol, whereas higher concentrations of
d i m e t h o x y b e n z y l a m i n e delayed the appearance of enzyme activity. This fact indicates that during the fermentation, a m m o n i u m might be released from dimethoxybenzylamine. The peak-delay effect of a m m o n i u m ions o n lignin biodcgradation by Phanerochaete chrysosporium has previously been reported [11]. To c o n f i r m these findings, D M B A was inc u b a t e d with lignin peroxidase in high concentrations (see MATERIALS AND METHODS) and the solution was analyzed for possible produts. Both veratryl alcohol a n d veratrylaldehyde were detected (Fig. 3). O b v i o u s l y the a m i n e was d e a m i n a t e d to the c o r r e s p o n d i n g alcohol and sub-
Table 2 Effect of concentration of veratryl alcohol and dimethoxybenzylamine on lignin peroxidase production of Phanef~ho@te chr~,-
sosporium Values are means + S.D. for 4 to 5 replicate cultures (cultures were grown with a temperature shift from 39 to 30°C and a speed shift from 240 to 90 rpm). n,d,, not determined. Cone.
Veratryl alcohol
(raM)
Lignin peroxidase activity (U I- I)
Time of maximum activity (h)
DMBA Lignin peroxidase activity (U I- I)
Time of maximum activity (h)
0.2 0.5 1.0 3.0 5.0
330 5:35 500 ± 66 874 4-96 808 + 93 n.d.
104 104 97 84 -
n.d. 780 :i:93 n.d, 912 =l=30 1233 + 92
110 144
96
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i
-800
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.
,
.
lo
20
30
-600
•
-4oo
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-200
'4
oxidase production by Phanerochaete chrysosporium in agitated pellet cultures and the shape,
I!
i
5. DISCUSSION This study has demonstrated that lignin per-
o
t (mtn)
-000
|
i
.
zyme-time programs of two not exactly synchronized cultures. Farell et al, [12] had shown earlier that the different extraceUular proteins of Phanerochaete chrysosporium appeared in agitated pellet cultures after different times.
-~oo
; ,G
.2O0
4
4" Fig. 4. Fast pl,~zin liquid chromatography profile(absorbancy at 409 nm) o( the extracellular fluid from a 5-day culture of PhanerochaetechrysosporiumATCC 2472S,grownwith veratryl alcohol (A) and dimethoxybenzylamine(B) ~dotted line represents the acetate gradient from 10 mM to 600 mM).
sequently oxidized to the aldehyde (Fig. 1). Veratrylic acid was identified as a third main reaction ~product. The comparison of veratryl alcohol and dimethoxybenzylamine as substrates for lignin peroxidase showed that, under the same conditions, the conversion rate of DMBA was 5.5% of the conversion rate of veratryl alcohol.
4.4. Effect of dimethoxybenzylamine on the profile of the extracellular proteins of Phanerochaete chrysosporium Fig. 4 shows the extraeellular haemoproteins (409 nm absorbing material) of 6-day-old cultures. The enzyme profile of cultures supplied with DMBA and with veratryl alcohol were almost identical. The difference in peak height may reflect the temporal differences of the genetic isoen-
size, and number of pellets can be controlled by variation of the agitation rate. Although the importance of agitation rates has been described previously [4] it was never examined systematically. Small mycelial pellets have the advantage of short diffusion paths and are therefore a good nutrient and oxygen supply of the cells in the centre of the pellets. The small pellet form can be achieved with high agitation rates. High agitation rates however produce shear forces, which might repress the formation of the iigninolytic enzyme system [13]. This repression can be avoided by a significant decrease of the agitation rate after the onset of ligninolytic activity. It is suggested that not only the agitation rate of the shaker is important for ~he enzyme production, but also the size and diameter of the flasks used and the amplitude of the respective shaker. For that reason the given speeds have not to be seen as absolute values but have to be adjusted (o an optimal shaker type-flask size-speed ratio for each laboratory. If the culture conditions are reproduced exactly the standard deviations of enzyme productivities are lower than 10% (Table 2) compared to over 30% in earlier reports [2] and the results were more reliable than reported by other authors [9], A speed shift in combination with a simultaneous temperature shift resulted in a more than 2-fold increase in enzyme activity compared to control experiments. A temperature shift from 39 to 30 *C has already been described by Asther et al. [8]. To obtain reliable results the precultures should be thoroughly blended. The addition of dimethoxybenzylamine instead of veratryl alcohol significantly increased lignin
330 peroxidase production. The results show that it was not D M B A itself, but its cleavage p r o d u c t veratryl alcohol which is the real inducer o f the enzyme activity. (1) Veratryl alcohol is a cleavage product of D M B A . (2) The extracellular enzyme profile of cultures supplied with the two different substances were almost identical. (3) The enzyme production in cultures with high concentrations of D M B A was delayed, probably due to the repressive effect of ~Lmmonium on lignin peroxidase production. It was calculated that one unit of enzyme released 0.0546 # m o l a m m o n i u m from D M B A . It can be assumed that this slowly released a m m o n i u m enables the fungus to continue with metabolism and resulted therefore in the very high enzyme activity.
REFERENCES [l] Fenn, P. and Kirk. T.K. (1981) Arch. Microbiol. 130, 59-65. [2] J~tger,A., Croan. S. and Kirk, T.K. (1985) Appl. Environ. Microbiol. 50. 1274-1278. i3] Jitger, A., Kern. H. and Wandrey. C. (1989) 4th Int. Conf. Biotechnology in the Pulp and Paper Industry, Raleigh NC. pp. 142-143, Tappi Press Atlanta. [4] Leisola, M. and Fiechter, A. ~2985) FEMS MicrobioL Len. 29, 33-36. [5] Asther, M., Corrieu, O., Drapon, R. and Odier, E. (1987) Enzyme Microb. Technol, 9, 245-249. [6] Tien, M., Kersten, P. and Kirk, T.K. (1987) Appl. Environ. Microbiol. 53, 242-245. [7] Leisola, M.. Thanei-Wyss. U. and Fiechter, A. (1985) J. Biotechnol. 3, 97-107. [8] Asther, M., Capdevila, C. and Corrieu, G. (1988) Appl. Environ. Microbiol. 54, 3194-3196. [9] Tien, M. and Kirk, T,K. (1988) Methods Enzymol. 161, 238-249. [~0] Tien, M. and Kirk, T,K. (1984) Proc. N:ltl. Acad. Sci. U.S.A. 81, 2280-2284. [11] Keyser, P., Kirk, T.K. and Zeikus, J. (1978) J. Bacteriol. 135, 790-797. [12] Farrell, R., Murtagh, K., Tien, M., Mnzuch, M. and Kirk, T.K. (1989) enmzyme Microb. Technol. ! z, 322-328. [13] Kirk, T.K., Schultz, E., Connors, W.J., Lorenz~ L.F. and Zeikus, .LG. (1978) Arch. Microbiol. 117, 277-285.
4#"
ACKNOWLEDGEMENTS W e are grateful to H. Kneifel for the H P L C analysis o f the D M B A reaction products a n d to H. ~erndt for valuable discussion.
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