0 Acad&mie
des
Plant biology
/ Elsevier, Paris
sciences
and pathology
/ Sio/ogie
et pafhologie
vgg6fales
Effects of copper and aromatic on the laccases of the white-rot Marasmius
inducers fungus
quercophilus
Effets du cuivre et de composks aromatiques SW les laccases du cbampignon de la pourriture blancbe Marasmius quercophilus Anne-Marie Laboratoire techniques
Farnet*,
Simone
de microbiologic, de Saint-JerBme,
(Received 23 November Note communicated
Tagger,
Upressa CNRS 6116, lnstitut mediterraneen 13397 Marseille cedex 20, France
1998; accepted
by Henri
Jean Le Petit
1
d’ecologie
et de paleoecologie,
faculte des sciences et
March 1999)
Duranton
Abstract - Marasmius quercophilus is a white-rot fungus producing lactase, a polyphenoloxidase involved in lignin degradation. The 17 strains under study were isolated from an evergreen oak litter (La Gardiole-de-Rians, Var, France). All the isolates synthesised the same constitutive lactase. Copper and various aromatic compounds in the medium enhanced lactase activity to different degrees. A copper concentration of 5 mg L-’ seemed to be the optimal tested. Coniferyl alcohol appeared to have the most extensive inducing potential. Each aromatic compound induced different new isoenzymatic forms in the electrophoretic patterns. These isoenzymes were similar for every strain. 0 Academic des Sciences / Elsevier, Paris evergreen
oak
litter
/ induction
/ polyphenoloxidase
/ white-rot
fungi
R6sumCz- Marasmius quercophilus est un champignon de la pourriture blanche produisant de la lactase, une polyphenoloxydase impliquee dans la degradation de la lignine. Les 17 souches etudiees ont et6 isolees d’une lit&e de chene vert (La Gardiole-de-Rians, Var, France). Tous les isolats synthetisent la meme lactase constitutive. Le cuivre et des composes aromatiques varies augmentent l’activite lactase 2 differents degres. LJne concentration en cuivre de 5 mg L-l semble etre la concentration optimale testee. L’alcool coniferylique para avoir le potentiel inducteur le plus important. Chaque compose aromatique induit de nouvelles isoformes enzymatiques dil&entes dans les profils electrophoretiques. Ces isoenzymes sont identiques pour chaque souche. 0 Academic des Sciences / Elsevier, Paris litPre
du chike
Version
vert
/ induction
/ polyphCnoloxydase
/ champignons
abregee
Marasmius quercophilus est un basidiomycete de la pourriture blanche caracteristique de la for@t de ch@ne vert (Quercus ilex L.). Lors de periodes climatiques favorables (printemps, automne), de nombreux carpophores de ce
* Correspondence C. R. Acad. 1999,322,499-503
Sci.
and
reprints:
Paris,
Sciences
[email protected] de
la vie
/
Life Sciences
de la pourriture
blanche
champignon sont observables dans cet ecosysteme typiquement mediterraneen. Les 17 souches analysees ont 6te recoltees dans la station d’iltude de la fortt domaniale de la Gardiole-de-Rians War, France) qui est compos6e en grande partie de cette essence. Ce basidiomycete semble Ctre implique dans la degradation de la lignine de par sa production de
A.-M.
Farnet
et al.
lactase, polyph&ol oxydase bleue 2 cuivre. Cette enzyme catalyse des coupures C& ainsi que des dkmkthoxylations dans des modPles de lignine. Ces &actions d’oxydation de composks aromatiques sont couplkes avec la reduction de l’oxyggne mokulaire en eau. Dans cette etude, nous nous proposons d’analyser l’influence de certains composks aromatiques ainsi que du cuivre sur l’activite lactase glohale prksente dans les milieux de culture. Des profils electrophoretiques de ces milieux de culture ultrafiltrks ont kgalement &i: realis& afin de mettre en evidence d’eventuelles isoformes nouvelles. Les inducteurs aromatiques test& sont: l’acide phydroxybenzoique (prkcurseur de la lignine), l’alcool conifkrylique (monolignol structural), l’alcool vikatrylique et l’acide vanillique (produits de degradation de la lignine). L’activitk lactase a ktk dktectke de manike significative dans le milieu apr& cinq jours de culture pour chaque experience. Dans le milieu M saris cuivre (CuSO,) et inducteur et dans le milieu minkal avec cellulose, l’activite lactase moyenne ktait de 0,008 U l-1. L’activitk lactase optimum dans le milieu M avec le cuivre a et6 obtenu avec une concentration en cuivre de 5 mg L-l. Pour chaque souche aucune activiti: lactase n’a ktk dktectke et la croissance du mycklium a dimink pour une concentration en cuivre de 7 mg L-l. L’activitk lactase a augment6 en ajoutant les composk aromatiques 5 une concentration finale de 250 mg L-‘. L’acide phydroxybenzoique, l’alcool vkatrylique, l’acide fkrulique, l’acide vanillique et l’alcool conifirilique ont augment6 respectivement l’activiti: lactase de 1,8, 2,3, 34, 5, 7,3 fois en moyenne par rapport ?I l’activitk lactase dans le milieu M additionnk de CuSO, 5 mg L-l. Les profils klectrophorktiques etaient reproductibles pour chaque experience. Toutes les reponses t‘taient identiques pour chaque souche ktudike. Nous avons observe une m?me bande (Rf: 0,681 quel que soit le milieu de culture utilisk et nous en avons conclu que cette bande etait une forme constitutive de la lactase. L’acide phydroxybenzdique et l’alcool vkratrylique ont induit la synth&e de deux isoformes (of: 0,75 et 0,56 respectivement). Une autre isoenzyme posskdant une mobilitk relative de 0,88 a et6 observee avec l’acide fkrulique, l’acide vanillique et l’alcool conif&ylique. En effet, un profil identique de trois bandes (Rf: 0,68; 0,75 et 0,881 avec di%rentes intensitk a et6 obtenu en utilisant ces trois inducteurs. L’augmentation de l’activitk lactase dans un milieu additionni: en cuivre a deja ett? observke chez de nombreux champignons de la pourritutre blanche. Cette induction peut stre expliquke par la pksence de quatre atomes de cuivre dans le site actif de la lactase. Nous avons pu ggalement observer l’effet inducteur des di&rents composk aromatiques sur l’activite lactase globale dans le milieu de culture. Concernant les klectrophor&es, toutes les isoformes mises en evidence etaient monomkriques ce qui represente une propriktir classique des laccases fongiques. De plus, les electrophorkes ont montri: une seule isoforme constitutive (Rf: 0,681. Une seule forme constitutive est relativement peu
500
commune chez les laccases de champignons de la pourriture blanche. Le profil klectrophoretique obtenu a partir du milieu M a r&klk cette unique forme enzymatique. Done ce milieu ne contient pas d’inducteurs pour l’activitk lactase puisque le profil Gctrophoktique obtenu avec le milieu mineral plus cellulose etait identique. L’addition de cuivre dans le milieu a augment6 l’activitk lactase et l’intensitk de la r&Clation sur le gel. L’induction peut done augmenter la production de l’enzyme saris entrainer la prksence d’autres isoformes. Les cinq compos& aromatiques test& ont montre dif&ents effets sur la synthese d’isoformes de lactase. Avec l’alcool vkratrylique et l’acide phydroxybenzo’ique, la rkvelation d’activite enzymatique sur gel de polyacrylamide de la forme constitutive Ctait plus faible. Dans ce cas, la diminution de la production de la lactase constitutive semble etre equilibrke par la synthke des isoformes (Rf 0,56 et 0,75 respectivement) puisque I’activitk lactase globale dans le milieu est toujours plus importante que celle du milieu de base. De plus, le plus important des potentiels d’induction, celui de l’alcool vkratrylique (activitk moyenne de 0,058 U L-l> @tait associe avec la presence de la bande caractkriske par le plus haut poids mokulaire (of: 0,561 et qui montrait une intensitk de r&Clation importante sur gel de polyacrylamide. Cette observation nous a permis de dkduire que cette enzyme avait une influence importante sur l’activite lactase. Avec l’acide ftmlique, I’acide vanillique et l’alcool conifirylique, la synthke en proportions variCes de trois isozymes (Rf 0,68, 0,75 et 0,881 peut expliquer la difference entre les activitk lactase moyennes obserkes (respectivement 0,092, 0,118 et 0,176 U L-l). Toutefois, lors d’etudes ultkieures, les activitCs spkifiques des isozymes purifkes devront @tre calculkes afin d’expliquer le r61e des inducteurs sur l’expression de la lactase. Cette &rie de tests a decrit les conditions permettant d’augmenter la production de lactase de souches de Marasmius quercophilus en utilisant le phenomke d’induction. Nous avons montr& que la concentration optimale en CuSO, pour l’activitk lactase dans notre etude ktait de 5 mg L-’ et que l’alcool conifkylique etait l’inducteur le plus effkace pour augmenter l’activitk lactase. Cette etude a egalement ktabli l’existence de trois nouvelles isozymes en comparaison au profil constitutif, selon l’inducteur utilisk, et qui semblent likes a l’augmentation de l’activitk lactase. Optimiser cette activite est un objectif interessant puisque cette enzyme peut Gtre utiliske en biotechnologie du fait des reactions qu’elle catalyse. En effet, des systemes enzymatiques comme la lactase qui sont impliquks dans la degradation de la lignine peuvent Ptre utilisks dans le blanchiment de la pPte 2 papier ou la degradation de polluants aromatiques. Les potentiels de telles enzymes ont engendrk un grand int&&t pour les pro&d& de catalyse enzymatique. Dans des etudes ultkrieures, la purification de chaque isozyme sera utile afin de determiner leur activite spkcifique respective ainsi que leurs actions sur des composks phknoliques varik.
C. R. Acad.
Sci. Paris, Sciences
de la vie / Life Sciences 1999.322,499-503
Induction
1. Introduction
fresh
Marasmius quercophilus is a white-rot basidiomycete which colonises an evergreen oak litter in the site of La Gardiole-de-Rians (Var, France). This site has been the subject of our interest for many years since evergreen oak represents an important part of Mediterranean ecology [Il. As described by Tagger et al. [2], this fungus seems to be greatly involved in lignin degradation because of its lactase production. This phenomenon is particularly important since lignin is the most prevailing polymer in litter after cellulose. The role of lactase in ligninolysis has already been described by several authors such as Bourbonnais et al. [3] using kraft lignin Lactase (EC 1 .I 0.3.2.) is a blue copper which catalyses C,-C, cleavage and
as a lignin model. polyphenoloxidase demethoxylation of
lignin substructure models. Its action on various substrates (pdiphenols as well as orthoand meta-substituted phenols) is coupled with the reduction of molecular oxygen to water [4]. Our intention was to describe and compare the regulation of the lactase enzymatic system in 17 M. quercophilus isolates. In particular, we wanted to evaluate the effect of inducers on the expression of lactase by measuring total extracellular activity and by comparing the electrophoretic patterns of the studied strains. The inducers studied here were: phydroxybenzoic gnin precursor), coniferyl alcohol (structural ferulic acid, vanillic acid and veratryl alcohol lignin degradation).
2. Materials 2.1.
Isolation
Seventeen from a part
acid monolignol), (products
(li-
2.2.
Cultures
50 mg L-’ (Sigma). Second, the obtained was used to inoculate an wheat flour, 20 g L-‘) which favours Then, one rhizomorph was used
for induction
to obtain
a pure
dicaryotic
assays
Liquid cultures were carried out in 250-mL Erlenmeyer flasks with 50 mL of the following medium: malt extract medium, 20 g LL’ (medium M), cultures containing medium M and CuSO,, 2.5, 5 or 7 mg L-’ as well as cultures with mineral medium [S] plus cellulose 1 g L-‘. Cultures with medium M were also made with CuSO,, 5 mg L-‘, and an aromatic inducer, 250 mg L-’ (veratryl alcohol, phydroxybenzoic acid, ferulic acid, vanillic acid or coniferyl alcohol). Cultures were inoculated with about 1 g of CR. Acad. Sci. Paris, Sciences 1999,322,499-503
de la vie / Life Sciences
an axial
shaking
All the cultures were performed were filtered on a glass microfibre
860. One unit (U) of lactase amount of enzyme that oxidises per minute. 2.3.Electrophoresis
experiments
in
at 30 “C triplicates. filter GF/D,
The
as previously
same
cultures
activity is defined as the 1 umole of the substrate
described
in 1-L Erlenmeyer flasks with 200 were also filtered and concentrated cell, model 8200 (Amicon, Beverly, reached 5 mL. The membrane used
were
grown
mL of medium. They on an ultrafiltration USA) till the volume (Amicon), was rated at
IO MW (10 kD) cut-off. PAGE (polyacrylamide trophoresis) as carried out by Laemmli [7] using king gel and 7.5% separating gel at 220 V
gel elec4% stacwith the
Mini-Protean II electrophoresis cell (Biorad) in denaturing (presence of SDS) or non-denaturing conditions. Gel staining was performed using syringaldazine (dissolved in methanol) at a final concentration of 0.1% (v/v) in phosphate buffer 0.1 M, pH 5.7 for 30 min. Electrophoresis was in triplicates
Lactase
(1 ha) using the rhizomorphic form of the fungus. For each strain, a fungal cap culture was first made on a malt-agar medium, 20 g LL’ (Bio Merieux, Marcy-I’Etoile, France)
plate
under
quercophilus
in each
case.
3. Results
of the strains
a malt-agar
incubated
Marasmius
2.7 pm (Whatman, UK). Enzyme activity was measured by following the oxidation of syringaldazine [N, N’-bis-(3,5dimethoxy-4-hydroxybenzylidene)hydrazine] to quinone (Ed = 65 000 Mm’ cm-‘) at 525 nm in phosphate buffer 0.1 M, pH 5.7 [61 on a spectrophotometer Kontron Uvikon
performed
dicaryons of M. quercophilus were isolated of the site of La Gardiole de Rians, Var, France
to inoculate culture.
and
from
of
and methods
and chloramphenicol, pure mycelial culture agar medium (whole rhizomorph production.
fungi
for 5 d. Cultures
of laccases
activity
was
significantly
detected
in the
me-
dium after 5 days of growth for each experiment (figure 7). In medium M without copper and inducer and in mineral medium plus cellulose the average activity of the lactase (+ 5%) was 0.008 U L-’ of sample. The optimal lactase activity in medium copper concentration 1 g of fresh fungi). detected and the
M plus copper was obtained with a of 5 mg L-’ (5 mg per approximately For each strain no lactase activity was growth of mycelium qualitatively de-
creased when the copper concentration reached 7 mg LL’ (7 mg per approximately 1 g of fresh fungi). Table I shows that for all the strains tested, lactase activity was increased by adding p-hydroxybenzoic, veratryl alcohol, ferulic acid, vanillic acid or coniferyl alcohol at a final concentration of 250 mg L-’ in medium M with CuSO,, 5 mg LL’. These aromatic compounds, respectively, enhanced activity 1.8, 2.3, 3.8, 5 and 7.3 times on average compared to the lactase activity in medium M with CuSO,, 5 mg LL’. The most extensive enhancement was observed with coniferyl tivity:
alcohol which considerably an average activity of 0.170
increased U L-’ was
lactase obtained.
ac-
We also studied the effects of the inducers tested on the electrophoresis patterns. These patterns were reproducible for each experiment (figure 2). All the isoenzyme forms were monomers since the electrophoretic patterns with or without SDS were similar. for each M. quercophilus
All the responses were isolate. We observed
the same the same
501
A.-M.
Farnet
et al.
0,24
-
0,22
--
02
--
0.68
-
v
-
-
0.75
-
2
3
4
5
w
w---
0.88 1
m
m--
6
Incubation time (day)
Figure
1. Average
lactase
activity
(U
L-‘)
in
shaken
cultures
Figure
of
2. SDS-PAGE
lane
band (Rf: 0.68) whatever the culture medium used and we concluded that it was a constitutive isoform. pHydroxybenzoic acid and veratryl alcohol induced the synthesis of two other isoforms with Rf: 0.75 and Rf: 0.56, respectively. Another isoenzyme with a relative mobility of 0.88 was observed with ferulic acid, vanillic acid and coniferyl alcohol. Indeed, an identical pattern of three bands (Rf: 0.68, 0.75, 0.88) with different intensities was obtained using these three aromatic inducers.
5; coniferyl
property
of copper
and
aromatic
inducers
on the
lactase
for
(U L-‘)
of the
Malt extract
cuso, 2.5 mg L-’
1
0.017 0.011 0.010 0.005 0.011 0.006 0.006 0.009 0.005 0.007 0.005 0.010 0.006 0.008 0.011 0.007 0.007 0.008 0.003
0.028 0.015 0.019 0.009 0.017 0.011 0.010 0.012 0.009 0.011 0.008
0.040 0.025 0.023 0.015 0.025 0.020 0.021 0.023 0.017 0.019 0.018
0.076
0.034
0.057 0.055 0.033 0.041 0.035 0.031 0.034 0.044 0.035 0.051
0.013 0.008 0.012 0.015 0.010 0.011 0.013 0.004
0.024 0.015 0.021 0.025 0.015 0.018 0.021 0.005
0.033 0.030 0.042 0.040 0.045 0.055 0.043 0.008
0.040 0.033 0.043 0.050 0.045 0.035 0.038 0.054 0.080 0.082 0.055 0.062 0.058 0.065 0.075 0.062 0.054 0.016
502
activity deviation
alcohol,
lane
lactase:
6; vanillic
acid,
a tetrameric
17 M. quercophilus
Strain
2 3 4 5 6 7 a 9 10 11 12 13 14 15 16 17 Average Standard
cuso, 5 mg LL’
activity
isoenzymes
obtained
for all
in malt extract 5 mg L-‘: lane at 250 mg L-’ 4; ferulic acid,
lane
7.
enzyme
has
been
obser-
ved only in the case of the genus Pocfospora [I 01. Electrophoretic experiments also showed a unique constitutive
The increase of lactase activity when copper is added to the culture medium is consistent with numerous previous I. Effects
of lactase
studies. It has already been described by Huber and Lerch [8] in the case of Neurospora crassa as well as Dittmer et al. [9] with Phanerochaete chrysosporium. The active site of lactase contains four copper atoms and this can explain the action of copper on lactase activity. The aromatic compounds also had an inducing effect on global lactase activity in the culture medium. in the electrophoresis experiments, all the isoenzyme forms were monomers since the electrophoretic patterns with or without SDS were similar. This is a quite common
4. Discussion
Table
patterns
the strains tested (Rf are specified on the left side). Electrophoresis was carried out from cultures grown medium (medium M): lane 1; medium M and CuSO,, 2; medium M, CuSO,, 5 mg L-’ and aromatic inducers veratryl alcohol, lane 3; p-hydroxybenzoic acid, lane
Marasmius quercophilus after 5 d of growth: in malt extract (medium M) and CuSO,, 5 mg L-’ (---I, in medium M, CuSO, 5 mg L-’ and different inducers (250 mg L-‘): p-hydroxybenzoic acid (-), veratryl alcohol (- -), ferulic acid (---), vanillic acid (- -), coniferyl alcohol (-).
p-Hydroxybenzoic acid
C. R. Acad.
studied,
grown
extract
medium.
Ferulic acid
Vanillic acid
0.082 0.075 0.090 0.088 0.070 0.095 0.072 0.074
0.104 0.122 0.095 0.105 0.099 0.100 0.112 0.102 0.098 0.111 0.085
0.222 0.212 0.173 0.078 0.094 0.087 0.068 0.055 0.203 0.183 0.154
0.095 0.117 0.119 0.096 0.089 0.125 0.104 0.011
0.164 0.182 0.173 0.187 0.192 0.210 0.155 0.055
0.068 0.097 0.077 0.082 0.091 0.098 0.071 0.068 0.076 0.081 0.010
Sci.
in malt
Paris,
Sciences
de
la vie / Life Sciences 1999.322.499-503
Induction
isoform (Rf: 0.68). Only one constitutive lactase is relatively uncommon in the case of lactase from white-rot fungi. The electrophoretic pattern obtained with medium M showed only the constitutive band. Thus, this medium did not contain inducers for lactase activity since the electrophoretic pattern observed with mineral medium associated with cellulose was the same. Adding CuSO, in the medium increased the constitutive lactase activity and the intensity of activity staining of this constitutive isozyme on polyacrylamide gel. It means that induction may also increase the production of the enzyme induced without leading to the presence of other isoforms. The five aromatic compounds tested led to different effects on the synthesis of lactase isozymes and on total lactase activity. With veratryl alcohol and phydroxybenzoic acid, the activity staining of the constitutive isozyme decreased on polyacrylamide gel (figure 2). In this case, the decrease in constitutive lactase production seemed to be balanced by the synthesis of the isozymes, with Rf 0.56 and 0.75, respectively, since total lactase activity was still higher than the activity in the basal medium (table /). Furthermore, the most important stimulating potential, that of veratryl alcohol (average activity: 0.058 U L-‘) was associated with the presence of the band characterised by the highest molecular weight (Rf: 0.56) and which showed an extensive staining activity on the gel. This observation led us to deduce that this isozyme seemed to have an extensive influence on lactase activity. With ferulic acid, vanillic acid and coniferyl alcohol the synthesis in various proportions of the three isozymes with Rf: 0.68, 0.75 and 0.88 may explain the differences between the average lactase activity (respectively, 0.092, 0.118 and 0.176 U L-l). However, in further studies, the specific activities of the isozymes purified have to be calculated to explain the role of the inducers on lactase expression.
This series of tests described the experimental conditions to increase lactase production of M. quercophilus strains using induction. We have shown that the optimal copper concentration fdr lactase activity in our study was
CR. Acad. 1999.322,49X03
Sci. Paris,
Sciences
de la vie
/ Life Sciences
from
Marasmius
quercophilus
5 mg L-’ and that coniferyl alcohol was the most important inducer for lactase enhancement. It also established the existence of three new isozymes compared to the constitutive pattern, depending on the inducer used, which seemed to be linked to the increase of lactase activity. Optimising lactase activity is an interesting aim since this enzyme can be used in biotechnology because of the reactions it catalyses. Indeed, enzymatic systems such as lactase which are involved in ligninolysis can be used in pulp bleaching or aromatic pollutant degradation [2]. The potentials of such enzymes have provoked considerable interest in enzyme-catalysed processes. In further studies, the purification of each isozyme will be useful to determine their respective specific activity and their effects on various phenolic structures.
References [l] Barber0 mediterraneenne potentielles,
M., Loisel R., Les chCnaies vertes : valeurs phytosociologiques, Phytocoenologia 11 (1983) 225-244.
du sud-est de la France dynamiques et
[21 Tagger S., Perissol C., Gil G., Vogt G., Le Petit J., Phenoloxidases ofthe white-rot fungus Marasmius quercophilus isolated from an evergreen oak litter (Quercus ibex, L.), Enzyme Microb. Technol. 23 (1998) 372-379. [31 Bourbonnais R., Paice M.G., Reid I.D., Lanthier P., Yaguchi M., Lignin oxidation by lactase isozymes from Trametes versicolor and role of the mediator 2,2’-Azinobis (3-ethylbenzthiazoline-6-sulfonate) in kraft lignin depolymerization, Appl. Env. Microbial. 61 (1995) 1876-l 880. [4] Linder M.C., of the Elements,
Biochemistry of copper, in: Frieden E. (Ed.), Biochemistry Plenum Press, New York, 1990, pp. 392-404.
[51 Kirk T.K., Schultz culture parameters chrysosporium, Arch. 161 Harkin of lactase 469-476.
E., Connors W.J., Lorenz L.F., Zeikus on lignin metabolism by Microbial. 11 (1978) 277-285.
J.M., Larsen M.J., ObstJ.R., in sporophores of wood
C.T., Lerch and lactase
J., Influence Phanerochaete
of
Use of syringaldazinefordetection rotting fungi, Mycologia 66 (1974)
I71 Laemmli U.K., Cleavage of structural proteins the head of bacteriophage T4, Nature 227 (1970) [81 Huber tyrosinase 335-338.
5. Conclusion
of laccases
K., The influence in Neurospora
during the assembly 680-685.
of
of copper in the induction of crassa, FEBS Lett. 219 (1987)
191 Dittmer J.K., Pate1 N.J., Dhawale S.W., Dhawale S.S., Production of multiple lactase by Phanerochaete chrysosporium grown under nutrient sufficiency, FEMS Microbial. Lett. 149 (1997) 65-70. [lo] Reinhammar Copper Enzymes,
B., Lactase, in: Lontie CRC Press, Boca Raton,
R. (Ed.), FL, 1984,
Copper Protein pp. l-35.
and
