APLICAÇÕES DAS ENZIMAS LIGNINOLÍTICAS DE

1
UNIVERSIDADE ESTADUAL DE MARINGÁ
CENTRO DE CIÊNCIAS BIOLÓGICAS
Programa de Pós-Graduação em Ciências Biológicas
APLICAÇÕES DAS ENZIMAS LIGNINOLÍTICAS DE
Pleurotus pulmonarius NA ELIMINAÇÃO DE
POLUENTES AMBIENTAIS
DANIELA FARANI DE SOUZA
Maringá
2008
2
DANIELA FARANI DE SOUZA
APLICAÇÕES DAS ENZIMAS LIGNINOLÍTICAS DE
Pleurotus pulmonarius NA ELIMINAÇÃO DE
POLUENTES AMBIENTAIS
Tese apresentada ao programa de Pós
Graduação em Ciências Biológicas da
Universidade Estadual de Maringá, como
parte dos requisitos para obtenção do título
de Doutor em Ciências Biológicas.
Maringá
2008
3
Orientadora
Profa. Dra. Rosane Marina Peralta
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APRESENTAÇÃO
Esta tese de doutoramento está apresentada na forma de dois artigos científicos
REMOVAL
OF
PENTACHLOROPHENOL
BY
Pleurotus
pulmonarius
IN
SUBMERGED CULTURES UNDER LIGNINOLYTIC AND NON-LIGNINOLYTIC
CONDITIONS. Daniela Farani de Souza, Cristina Giatti Marques de Souza, Maria
Aparecida Ferreira Costa, Cinthia Gandolfi Bôer,
Márcia Fernandes Nishiyama,
Heloísa Bressan Gonçalves, and Rosane Marina Peralta, a ser
submetido ao
periódico científico CHEMOSPHERE.
THE RATIO Mn PEROXIDASE/LACCASE IS HIGHLY AFFECTED BY THE INITIAL
MOISTURE CONTENT ON CORN COB SOLID STATE CULTURES OF Pleurotus
pulmonarius.
Daniela Farani De Souza, Cissa Kelmer Bracht, Ana Maria
Alexandrino, Gisele Pezente Ferreira, Maria Aparecida Ferreira Costa, Cinthia
Gandolfi Boer, Cristina Giatti Marques De Souza, Rosane Marina Peralta, a ser
submetido ao periódico científico BIORESOURCE TECHNOLOGY
5
RESUMO
Os elementos estruturais principais da madeira são celulose, hemicelulose e
lignina. Lignina é um polímero aromático impermeável a água, encontrado em todas
as plantas superiores. A madeira e outros tecidos vasculares geralmente contém 20
a 30% de lignina. Este polímero é sintetizado pela polimerização oxidativa de três
álcoois cinamílicos substituídos (precursores fenólicos) iniciada pelas lacases e/ou
peroxidases. Estas reações inespecíficas criam um biopolímero tridimensional,
heterogêneo e com alta massa molecular. Como uma macromolécula aromática
complexa, a lignina fornece resistência e rigidez às paredes celulares e tecidos de
todas as plantas vasculares por estar em estreita associação com a celulose e as
hemiceluloses. Além disso, a lignina está envolvida no transporte de água nos
vegetais, além de formar uma barreira contra a destruição microbiana por dificultar o
acesso aos polissacarídeos rapidamente assimiláveis.
Devido à sua heterogeneidade e tipos de ligações que contém, a lignina não
pode ser clivada por enzimas hidrolíticas como a maioria dos outros polímeros
naturais (celulose, amido, proteínas, etc). A lignina é, entretanto, degradada pelos
fungos causadores das podridões branca, marrom e macia da madeira. Os fungos
causadores da podridão branca da madeira são capazes de degradar tanto os
carboidratos quanto a lignina, enquanto os causadores das podridões marrom e
macia utilizam preferencialmente a celulose e as hemiceluloses. Fungos causadores
da podridão branca são os únicos organismos capazes de degradar a lignina a CO2
e água. Entretanto, eles não conseguem utilizar este substrato como única fonte de
carbono e energia.
A biodegradação da lignina é um processo oxidativo, que envolve enzimas
como a lignina peroxidase (LiP), manganês peroxidase (MnP), peroxidase versátil
(VP) e lacase (lcc). As peroxidases e lacases possuem a habilidade de catalisar
oxidações envolvendo um elétron, resultando na formação de radicais que sofrem
então numerosas reações espontâneas. Estes radicais, por sua vez, conduzem a
clivagens de diferentes ligações incluindo fissão do anel aromático. Grande ênfase
vem sendo dada ao estudo dos fungos da podridão branca da madeira produtores
de Mn peroxidases e lacases, incluindo as espécies não produtoras de lignina
peroxidases.
6
Manganês peroxidase (MnP, EC 1.11.1.13) é uma heme-proteína glicosilada
extracelular. Esta enzima é expressa em múltiplas formas com massas moleculares
variando entre 40 e 48 kDa e pI entre 2,9 e 7,0. A enzima tem sido descrita em
muitos basidiomicetos causadores da podridão branca. A principal função da MnP é
a oxidação do Mn+2 a Mn+3. MnP utiliza Mn+2 como substrato preferencial (doador
de electrons). O íon Mn+3 gerado pela mnP é um oxidante potente, porém instável
em meio aquoso.
Mn+3 é estabilizado pela formação de complexos com ácidos
orgânicos fisiológicos, oxalatos e malonatos, por exemplo, e difunde-se para longe
do sítio ativo. Estes quelatos Mn+3-ácidos orgânicos são oxidantes potentes capazes
de oxidar grande variedade de substratos incluindo compostos fenólicos e corantes.
As lacases (benzenodiol:oxigênio oxido-redutases, EC 1.10.3.2) são enzimas
multi-cobre pertencentes ao grupo das oxidases azuis, que catalisam a oxidação de
ampla variedade de compostos aromáticos (particularmente fenóis) com a redução
do oxigênio a água. Numa reação típica, um substrato fenólico perde um elétron
dando origem a um radical ariloxil. Esta espécie ativa pode ser convertida a uma
quinona num segundo estágio da oxidação.
Fungos causadores da podridão branca e suas enzimas ligninolíticas,
possuidoras de ampla especificidade pelo substrato, tem grande aplicação potencial
nos processos de biopolpação e biobranqueamento, bem como na bioremediação
de poluentes aromáticos com similaridades estruturais a lignina. O uso de fungos
que degradam a lignina ou de suas enzimas ligninolíticas isoladas no tratamento da
polpa do papel poderia fornecer maior seletividade na remoção da lignina
comparada aos processos químicos.
Outra área onde os fungos ligninolíticos e suas enzimas podem ser úteis é na
bioremediação. Bioremediação é o processo pelo qual os resíduos danosos são
biologicamente convertidos a compostos menos danosos ou reduzidos a níveis
abaixo das concentrações limites. Fungos causadores da podridão branca degradam
e
mineralizam
compostos
xenobióticos
como
hidrocarbonetos
policíclicos
aromáticos, corantes industriais e outros poluentes de solo como atrazina e
pentaclorofenol. A degradação e detoxificação destes poluentes envolvem enzimas
oxidativas inespecíficas. Pelo fato da lignina ser um composto poliaromático natural
degradado pelos fungos da podridão branca usando enzimas inespecíficas, tais
organismos podem degradar compostos danosos utilizando os mesmos sistemas
enzimáticos.
7
Pleurotus sp pertence a sub classe dos fungos causadores da podridão
branca produtores de Mn peroxidases e lacases mas não lignina peroxidases. Uma
das espécies mais importantes é Pleurotus pulmonarius (FR) Quélet, que foi utilizada
neste trabalho.
No primeiro artigo foi avaliada a habilidade do P. pulmonarius remover
pentaclorofenol (PCF) em culturas submersas ligninolíticas e não ligninolíticas. Para
se obter uma condição ligninolítica, compostos fenólicos solúveis de sabugo de
milho foram utilizados como indutores das enzimas ligninolíticas. Os compostos
fenólicos aumentaram a produção de lacase de 32,1±3,0 U/L no meio basal para 532
U/L, enquanto nenhuma alteração na produção da Mn peroxidase foi observada
(18,0 U/L). Cerca de 70% da concentração inicial de PCF (25 ppm) foi removida nas
culturas submersas sob condições ligninolíticas, enquanto não mais do que 20% do
PCF foi removido nas culturas sob condições não ligninolíticas. A produção de
lacase parece ser fundamental para a remoção do PCF por P. pulmonarius.
No segundo artigo, a produção de lacase e Mn peroxidase por P. pulmonarius
e a sua capacidade em descolorir os corantes industriais Remazol Brilliant Blue R
(RBBR), vermelho do congo, azul de metileno e violeta de etila foram estudados em
culturas em estado sólido. A condição de alta umidade inicial (80-90%) promoveu
fortemente a expressão da lacase, enquanto a Mn peroxidase foi produzida
principalmente nas culturas desenvolvidas em baixas umidades iniciais (50-70%).
Culturas contendo altas atividades de Mn peroxidase foram mais eficientes na
descoloração dos corantes vermelho do congo, violeta de etila e azul de metileno.
Os filtrados livres de células com alta atividade Mn peroxidase foram mais eficientes
em descolorir vermelho do congo, violeta de etila e azul de metileno, enquanto
RBBR foi igualmente descolorido por extratos com alta atividade Mn peroxidase e
por extratos com alta atividade de lacase. Nenhum extrato conseguiu descolorir Poly
R478. Os dados obtidos sugerem que a Mn peroxidase de P. pulmonarius é mais
eficiente que sua lacase na descoloração dos corantes testados.
Os resultados obtidos neste estudo mostram claramente o potencial de
Pleurotus pulmonarius na remoção de compostos aromáticos e no tratamento de
efluentes contaminados com corantes.
Palavras-chaves:
pentaclorofenol,
bioremediação.
Pleurotus
enzimas
pulmonarius,
ligninolíticas,
descoloração
manganês
de
peroxidase,
corantes,
lacase,
8
SUMMARY
The major structural elements of wood are cellulose, hemicellulose and lignin.
Lignin is a water-impermeable aromatic polymer found in all higher plants. Wood and
other vascular tissues generally are 20-30% lignin. It is synthesized by oxidative
polymerization of the precursors p-coumaryl alcohol, coniferyl alcohol and sinapyl
alcohol, initiated by laccase and/or peroxidase. These unspecific reactions create a
high molecular weight, heterogeneous and three-dimensional biopolymer. As a
complex aromatic macromolecule, it provides strength and rigidity to cell walls and
tissues of all vascular plants by acting as a glue between the polysaccharide
filaments and fibers. In addition, lignin is involved in water transport in plants and
forms a barrier against microbial destruction by protecting the readily assimilable
polysaccharides.
Because of the bond types and their heterogeneity, lignin cannot be cleaved
by hydrolytic enzymes as most other natural polymers (cellulose, starch, proteins,
etc). Lignin is decayed by white-rot, brown-rot and soft-rot fungi. White-rot fungi are
able to degrade both carbohydrates and lignin, whereas brown-rot and soft-rot fungi
prefer cellulose and hemicelluloses as substrates. Fungi causing white-rot are so far
the only organisms known that degrade lignin to CO2 and H2O, but they cannot use
this substrate as sole carbon and energy source.
Lignin biodegradation is an oxidative process, involving enzymes such as
lignin peroxidase (LiP), manganese peroxidase (MnP), versatile peroxidase (VP) and
laccase (lcc). The peroxidases and laccases have the ability to catalyze one-electron
oxidations resulting in the formation of radicals which undergo numerous
spontaneous reactions. These, in turn, lead to various bond cleavages including
aromatic ring fission. In recent years, more emphasis has been placed on analyzing
specially Mn peroxidases and laccases, because these enzymes are produced by
most white-rot fungi, including species whick lack LiP.
Manganese peroxidase (MnP, EC 1.11.1.13) is extracellular, glycosylated and
contains heme as the prosthetic group. The enzyme is expressed in multiple forms
with molecular weights from 40 to 48 kDa and pIs between 2.9 and 7.0. It has been
found in many white-rot and soil litter-decomposing basidiomycetes. The main
function of MnP is the oxidation of Mn2+ to Mn3+. MnP uses Mn2+ as the preferred
substrate (electron donor). Mn3+ generated by MnP is a strong oxidizer but it is quite
9
unstable in aqueous media. Mn3+ is stabilized by forming complexes with
physiologically occurring organic acids, such as malonate or oxalate, and then it
diffuses away from the active site. Mn3+ organic acid chelates are strong diffusible
oxidants capable of oxidizing a large variety of substrates, including phenolic lignin
model compounds and several dyes.
Laccases (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) are multicopper
enzymes belonging to the blue oxidases that catalyse the one-electron oxidation of a
wide variety of aromatic compounds (particularly phenols) with the reduction of
oxygen to water. In a typical laccase reaction, a phenolic substrate is subjected to an
one-electron oxidation giving rise to an aryloxyradical. This active species can be
converted to a quinone in the second stage of oxidation.
White-rot fungi and their ligninolytic enzymes, with broad substrate specificity,
have potential applications in biopulping and biobleaching, as well as in the
bioremediation of aromatic pollutants with structural similarities to lignin. The use of
lignin-degrading fungi or isolated ligninolytic enzymes for the treatment of pulp could
potentially provide greater selectivity than chemical processes in the removal of
lignin.
Other area where white-rot fungi and their ligninolytic enzymes can be useful
is in bioremediation. Bioremediation is the process by which hazardous wastes are
biologically converted to harmless compounds, or to levels that are below
concentration limits. White-rot fungi have been found to degrade and mineralize
xenobiotic compounds, such as polycyclic aromatic hydrocarbons, industrial dyes
and other soil pollutants, such as atrazine and pentachlorophenol. Degradation and
detoxification of these pollutants involve oxidative enzymes which are not very
specific. Since lignin is a natural polyaromatic compound which is degraded by whiterot fungi using ligninolytic extracellular oxidative enzymes, it was naturally assumed
that these organisms could degrade hazardous compounds using the same enzyme
systems.
Pleurotus sp belongs to a subclass of white rot fungus that produces laccases,
Mn peroxidases, but not lignin peroxidase. One of the most important species,
Pleurotus pulmonarius (FR.) Quélet (P. sapidus, P. sajor-caju) was used in this study.
In the first work the removal of pentachlorophenol by Pleurotus pulmonarius
developed under ligninolytic and non-ligninolytic conditions was studied in
submerged conditions. To obtain a ligninolytic condition, corn cob soluble phenolic
compounds were used as inducers of ligninolytic enzymes. The phenolics improved
10
the laccase production from 32.1± 3.0 U/L in basal medium to 532 ± 3.0 U/L U/L,
while no alteration in Mn peroxidase production was observed (18.0 ± 3.0 U/L).
Approximately 70% of the initial PCP (25 ppm) was removed in submerged cultures
under ligninolytic conditions, while not more than 20% of PCP was removed under
non-ligninolytic conditions. The production of laccase appears to be important to
improve the degradation of PCP by P. pulmonarius.
In the second work, the production of laccase and Mn peroxidase by P.
pulmonarius and the capability of cultures to decolourise the industrial dyes Remazol
Brilliant Blue R (RBBR), congo red, methylene blue, Poly R478 and ethyl violet were
studied in solid state conditions. The condition of high initial moisture content (8090%) strongly promoted the expression of laccase, while manganese peroxidase was
produced specially in cultures developed at low initial moisture (50-70%). Cultures
containing high manganese peroxidase activities were more efficient to decolourise
congo red, ethyl violet and methylene blue.
Cell free filtrates with high manganese
peroxidase activity were more efficient to decolourise congo red, ethyl violet and
methylene blue, while RBBR was equally decolourised by Mn peroxidase and
laccase. No cell free extracts from P. pulmonarius was able to decolorize Poly R478.
Environmental pollutants are a serious concern world-wide. The results
obtained in this study showed the potential applicability of Pleurotus pulmonarius for
the removal of aromatic compounds and wasterwater treatment of dye effluents.
Key
words:
Pleurotus
pulmonarius,
dye
decolorisation,
ligninolytic enzymes, manganese peroxidase, bioremediation,
pentachlorophenol,
11
REMOVAL
OF
PENTACHLOROPHENOL
BY
Pleurotus
pulmonarius
IN
SUBMERGED CULTURES UNDER LIGNINOLYTIC AND NON-LIGNINOLYTIC
CONDITIONS
DANIELA FARANI DE SOUZA, CRISTINA GIATTI MARQUES DE SOUZA, MARIA
APARECIDA FERREIRA COSTA, CINTHIA GANDOLFI BOER, MÁRCIA
FERNANDES NISHIYAMA, HELOÍSA BRESSAN GONÇALVES AND ROSANE
MARINA PERALTA*
Departamento de Bioquímica, Universidade Estadual de Maringá, CEP 87015-900,
Maringá– PR, Brazil.
ABSTRACT
The removal of pentachlorophenol (PCP) by Pleurotus pulmonarius grown in
submerged cultures under ligninolytic and non-ligninolytic conditions was studied in
this work.
Under ligninolytic conditions, the fungus was more resistant to PCP,
removing 70% of PCP at an initial concentration of 25 mg/L, against a removal of
20% under non-ligninolytic conditions. PCP acted as a laccase inducer, and the
existence of high activities of enzyme seems to be related to its degradation by P.
pulmonarius.
Key words: laccase, Pleurotus pulmonarius, xenobiotics, pentachlorophenol,
phenolic compounds, ligninolytic enzymes.
INTRODUCTION
Pentachlorophenol (PCP) has been widely used as a wood preservative and
pesticide and it is a xenobiotic compound, toxic to all forms of life. PCP acts by
uncoupling oxidative phosphorylation via making cell membranes permeable to
protons, resulting in dissipation of transmembrane proton gradients and consequent
electrical potentials (Mcallister et al., 1996). Its toxicity against a variety of organisms
is unfavourable for biodegradation (Chiu et al., 1998). Also, its resistance to
degradation is owed to a 3-dimensional configuration of the rich chlorine atoms
(Apajalahti and Salkinoja-Salonen, 1984; Semple et al., 2001). PCP is now banned in
12
most countries. In Brazil, its use as wood preservative was allowed until 2006 and it
appears as one of the most common soil and wastewater pollutant in the country
(Almeida et al., 2007). PCP is persistent with a half-life of up to 178 and 200 days in
soil and water systems, respectively (Larson et al., 1997).
The possibility of using white-rot fungi to degrade PCP and related compounds
has been focus of interest due to the presence of a ligninolytic system containing
unspecific peroxidases (lignin peroxidase and Mn peroxidase) and laccase. Some
fungi produce all these enzymes while other produce only one or two of them. It is
generally believed that the same non-specific system responsible for lignin
degradation by white-rot fungi, is also involved in the oxidation of aromatic pollutants
(Lamar and Dietrich, 1990, Dritsa et al., 2007). The first studies on xenobiotic
degradation by white rot fungi were carried out with Phanerochaete chrysosporium. It
was shown that this basidiomycete is able to degrade PCP and several other
xenobiotic compounds in a nitrogen-limiting medium (ligninolytic condition) while
degradation was reduced in high nitrogen medium (non-ligninolytic conditions)
(McAllister et al., 1996; Mileski et al., 1988; Bending et al., 2002). However, there
has been increasing interest in studying the capability of other white rot fungi to
degrade PCP and other pollutants (Dritsa et al., 2007, Ryu et al., 2000, Marcial et al.,
2005, Levin et al., 2003, Machado et al., 2005, Ullah et al., 2000a,b).
An increasing range of white rot fungi is now being investigated for
bioremediation purposes (Machado et al., 2005; Alleman et al., 1995; Lamar and
Dietrich, 1990, Mileski et al., 1988). Laccase appears as the main enzyme involved
in the removal of xenobiotics including PCP in some white-rot species such as
Pleurotus and Coriolus (Ricotta et al., 1996, Ullah et al., 2000a, Ullah et al., 2000b,
Sedarati et al., 2003, Levin et al., 2003), while in other white-rot fungi such as
Nematoloma frowardii (Hofrichter et al., 1998, Sack et al., 1997), Phanerochaete.
chrysosporium (Moen and Hammel, 1994), Irpex lacteus (Baboravá et al., 2006) and
Bjerkandera sp (Eibes et al., 2007, Rubilar et al., 2007), the oxidation of xenobiotics
is mainly due to the action of Mn peroxidase.
Laccase is described as the most important ligninolytic enzyme of P.
pulmonarius CCB19 in submerged cultures (Zilly et al., 2002). In submerged cultures
without inducers, the enzyme is produced in small amounts after depletion of carbon
and nitrogen sources. However, its production can be stimulated significantly by the
presence of a wide variety of inducing molecules, mainly aromatic or phenolic
compounds related to lignin or lignin derivatives (Souza et al., 2004). The induction of
13
laccase by phenolic compounds is well studied in several basidiomycetes, including
Pleurotus sajor caju (Lo et al. 2001), Marasmus quercophilus (Farnet et al. 1999),
Pleurotus eryngii (Muñoz et al. 1997), and Trametes modesta (Nyanhongo et al.
2002).
Pleurotus spp are among the easiest mushrooms to cultivate (Cohen et al.,
2002). The specie most studied concerning its application in bioremediation
processes is P. ostreatus. It shows high potential to degrade several organic
compounds including polycyclic aromatic hydrocarbons (PAH) and chlorophenols in
liquid and solid state fermentation cultures (Bezalel et al., 1996a-c, Zeddel et al.,
1993). Pleurotus spp, in contrast to P. chrysosporium, expresses the ligninolytic
system during the growth phase, and the production of ligninolytic enzymes is not
inhibited by high nitrogen concentration. Other Pleurotus species have also
demonstrated potential in bioremediation processes (Rodrigues et al., 2004, Cohen
et al., 2002).
One of these species, Pleurotus pulmonarius (P. sajor-caju) when
cultured under submerged conditions produces laccase as the main extracellular
enzyme (Souza et al., 2004, Zilly et al., 2002). In this type of cultures, it was able to
decolourise textile industrial dyes (Zilly et al., 2002). Additionally, its capability to
degrade atrazine in solid state cultures has also been described (Masaphy et al.,
1996). In the present study, we examined the ability of Pleurotus pulmonarius to
grow in the presence of PCP in submerged cultures under ligninolytic and nonligninolytic conditions. An attempt was done to correlate the removal of PCP with the
production of laccase by the fungus.
2. MATERIALS AND METHODS
2.1. Organism
Pleurotus pulmonarius CCB19 was obtained from the São Paulo Botany
Institute Culture Collection. It was cultured on potato dextrose agar Petri dishes
(PDA) for 2 weeks at 28oC. When the Petri dish was fully covered with the mycelia,
mycelial plugs measuring 10 mm in diameter were made and used as inoculum for
submerged cultures.
2.2. Pre-culture conditions
14
One disk from the growing edge of the mycelium on PDA plates was
transferred to a 250 mL Erlenmeyer flask containing 50 ml of potato-dextrose
medium. The cultures were incubated at 28 °C in a r otary shaker at 120 rpm for 5
days. Homogenized pellets from 5-day-old shaken cultures were used as inocula
(corresponding to 1–2 mg dry weight per mL of culture).
2.3. Submerged ligninolytic and non-ligninolytic culture conditions
Cultures were carried out under ligninolytic and non-ligninolytic conditions. In
non-ligninolytic conditions, homogenized pellets from 5 days-old shaken PD cultures
(1-2 dry weight per ml of culture), were transferred to 25 mL of mineral medium
(Montenecourt and Eveileigh, 1977) supplemented with glucose
(10 g/L) and
ammonium tartrate (0.86 g/L). To prepare the ligninolytic medium, mineral medium
was firstly autoclaved with corn cob power (50 g of corn cob power plus 500 ml of
mineral medium) for 15 min to extract soluble phenolic compounds. The mixture was
filtered and the filtrate was then supplemented with glucose (10/L) and ammonium
tartrate (0.86 g/L).
2.4. Rate of growth and PCP tolerance test
The fungus was tested for its growth rate in solid media containing
pentachlorophenol (PCP). Ligninolytic and non-ligninolytic media (described above)
were supplemented with agar (16 g/L) and PCP (1 to 30 mg/L) from a stock solution
1mg/mL in ethanol. Inocula (10 mm diameter) were cut with a cork borer from fullygrown PDA cultures and transferred into Petri dishes. Three replicates were
performed for each PCP concentration.
2.5. Effect of PCP in submerged cultures
PCP was added in the 5th day of cultivation at a final concentration of 25 mg/L.
The effect of PCP in submerged cultures was estimated by comparison of the dry
biomass weight and the extracellular enzyme activities (Mn peroxidase and laccase)
with control variants without PCP.
2.6. Extraction of PCP adsorbed on the mycelial mass
15
To evaluate the PCP content adsorbed on the mycelia obtained from
submerged cultures, the biomass was filtered from culture, washed with distilled
water, and immediately frozen at -20o C before freeze-drying. The resulting dried
extract was ground to small particle-size before addition to ethanol in a 125 mL flask
that was shaken at 120 rpm in an orbital shaker for 24 h. The filtrate was separated
by centrifugation (15 min at 5000g) and used to quantify PCP by HPLC.
2.7. Analysis of PCP
PCP was measured by HPLC using a reversed phase column 4.6 x 250 mm,
packed with R-Sil C18 (10µM) with a mobile phase of acetonitrile:water:acetic acid
(75:25:0.125). Elution was carried out isocratically at a flow rate of 0.7 mL/L.
Detection of PCP was at 238 nm with a retention time of 11 min. Residual PCP in the
cultures was identified by retention time in comparison to an authentic PCP standard
and co-elution with added PCP. Calibration plots of peak areas of PCP standards
were linear in the range 20-120 µg/mL.
16
2.8. Enzyme assays
The laccase activity was followed spectrophotometrically at 525 nm through
the oxidation of syringaldazine to its quinine form, using a molar absorption
coefficient of 65,000 for the product (Leonowicz and Grzywnowicz, 1981). The
reaction mixture contained 1.5 mL phosphate buffer (0.1 M, pH 6.5), 0.2 mL
syringaldazine (0.5 mM in ethanol solution) and 0.1 mL of culture filtrates. Mn
peroxidase activity was assayed by the oxidation of 1 mM MnSO4 in 0.05 M sodium
malonate , pH 4.5, in the presence of 0.1 mM H2O2. Manganic ions, Mn3+, form a
complex with malonate, which absorbs at 270 nm (ξ270= 11.59 mM-1 cm-1) (Wariishi et
al., 1992). Enzymic activity was expressed as international units (U) defined as the
amount of enzyme required to produce 1 µmol product per min.
2.9. Other chemical analysis
The total phenol content was determined colorimetrically at 750 nm, using the
Folin-Ciocalteau reagent and expressed as ferulic acid equivalents.
Total
carbohydrates were determined by the phenol-sulfuric acid method with glucose as
the standard (Dubois et al., 1956). Total protein content was estimated by the
Bradford method using bovine serum albumin as the standard (Bradford, 1976). Total
nitrogen was determined by the Kjeldahl standard method.
2.10. Statistical analyses
The data obtained were compared using paired t-test, and the level of
significance of p<0.05 was chosen for all statistical comparisons. The data are
presented as mean±SEM. The analysis was done using the statistical program pack
GraphPad Prism® (Graph Pad Software, San Diego, USA).
2.11. Reagents
The enzymatic substrates and PCP were obtained from Sigma Chemical
Corp., St Louis, MO. PDA was obtained from Difco Laboratories, Detroit, MI. The
solvents used in HPLC analysis were of chromatographic grade. All other reagents
were of analytical grade.
17
3. RESULTS
3.1. Growth of P. pulmonarius in submerged cultures under ligninolytic and
non-ligninolytic conditions.
Previous results have shown that the supplementation of media with corn cob
soluble phenolic compounds resulted in a considerable improvement of both, laccase
production and capability of cultures to decolourise industrial dyes (Tychanowicz et
al., 2004). For this reason, to obtain a ligninolytic condition in submerged culture, the
same strategy was used in this work. The addition of soluble corn cob extract in the
basal medium resulted in an improvement of phenolic content (as ferulic acid
equivalents) from 0 (basal medium, non-ligninolytic condition) to 1.43 µmoles/ml
(ligninolytic condition), without any significant alteration of carbon (determined as
total carbohydrate and total protein contents) and nitrogen sources (determined as
Kjeldahl nitrogen) (data not shown). The production of biomass and enzymes
(laccase and Mn peroxidase) in both media is shown in Fig 1. A slight increase
(statistically non-significant after application of t-test) in the production of biomass
was observed by the addition of soluble corn cob extract. The presence of corn cob
soluble phenolic compounds improved the production of laccase more than 10 times
(from 32.1±3.0 U/mL to 532±28.0 U/mL), while no significant alteration in the
production of Mn peroxidase was observed (18.0±2.0 U/L).
3.2. Growth rates in solid media and PCP tolerance
The growth rates of P. pulmonarius in Petri dishes were tested under
ligninolytic and non-ligninolytic conditions (FIG 2). The dishes were fully grown in 6-7
days. The growth rate measurements were analyzed by the least-squares method.
The correlation coefficients R2 ranged from 0.96 to 0.99 in the three replicates. The
growth rate of P. pulmonarius was 3.17±0.26 mm/d in non-ligninolytic medium and
3.34±0.32 mm/d in ligninolytic medium. In spite of the slight improvement at the
growth rate under ligninolytic conditions, comparison of the growth in both conditions
did not reveal significant differences (P>0.05). This type of cultivation was used to
test the tolerance of P. pulmonarius to PCP. The ligninolytic condition improved the
PCP tolerance. Growth inhibition under the non-ligninolytic condition was more
pronounced and started at PCP concentrations above 10 mg/L (statically significant)
18
whereas under the ligninolytic condition growth inhibition started at concentrations
above 20 mg/L and was much less pronounced.
3.3. Effect of addition of PCP in submerged cultures under ligninolytic and nonligninolytic conditions
An amount of 25 mg/L of PCP (final concentration) was added to five-daysubmerged cultures of P. pulmonarius developed under ligninolytic and nonligninolytic conditions. The addition of PCP in both types of cultures increased about
2 times the levels of laccase (from 600 U/L to 1,200 U/L) (Fig 3A), while no alteration
was observed in the levels of Mn peroxidase (35 U/L) (Fig 3B). In relation to the
biomass, PCP inhibited the growth of the fungus under both conditions, although the
inhibition had been more evident under non-ligninolytic conditions (Fig 3C).
3.4. Evaluation of PCP removal
Figure 4 shows the removal of PCP by P. pulmonarius in ligninolytic and nonligninolytic submerged cultures. After 25 days of cultivation, around 70 and 20% of
the initial PCP were removed in culture filtrates developed under ligninolytic and nonligninolytic conditions, respectively. In both types of culture, less than 8% of PCP was
found adsorbed for the fungal mycelium.
DISCUSSION
In this work, the supplementation of submerged cultures with corn cob extract
had a very pronounced effect on the production of laccase by P. pulmonarius without
improvement in the levels of Mn peroxidase. In both, ligninolytic and non-ligninolytic
media, maximal Mn peroxidase activities were found in 20th day cultures. These
results are in agreement with those obtained in previous studies with the same strain
of P. pulmonarius, when low levels of Mn peroxidase were found in submerged
cultures (Souza et al. 2004). The laccase inducer effect of the soluble plant extract is
considered to be related to its phenolic contents. Cotton stalk extract for example,
acted as inducer of laccase in Pleurotus ostreatus submerged cultures (Platt et al.
1984, Ardon et al. 1996, Ardon et al., 1998). However, this effect was not a general,
considering that the same extract did not stimulate the production of laccase by
19
Ganoderma applanatum, Trametes versicolor and Rhizoctonia solani (Ardon et al.
1996).
Our results also showed that PCP increased the production of laccase by P.
pulmonarius in submerged cultures, while the production of manganese peroxidase
was barely affected by the presence of PCP. In addition to this, the previous
presence of a higher laccase activity apparently increased both the resistance of the
fungus to PCP and its removal from the medium. Inductive aromatic compounds very
often are toxic to fungal growth and metabolism, and it has been proposed that one of
the possible functions of fungal laccase is the polymerization of toxic aromatic
compounds (Thurston 1994). Therefore, laccase may function as a defense
mechanism against oxidative stress. Some potential pollutants of the environment
have been described as laccase inducers. In liquid cultures of Trametes versicolor,
the addition of several xenobiotics, including PCP at 0.5 mM, improved the laccase
production (Mougin et al., 2002). In submerged cultures of T. versicolor, the addition
of 10 mg/L phenol improved 20 fold the production of laccase (Pazarhoglu et al.,
2005).
More recently, the efffects of
paraquat dichloride (1,1′-dimethyl-
4,4′bipyridinium dichloride hydrate, methyl viologen dichloride), an effective contact
herbicide, were examined for its action on the laccase activity of Trametes versicolor
and Abortiporus biennis strains (Jaszek et al., 2006). Because of its prooxidative
character, paraquat is commonly used in laboratories as an oxidative stress
conditioning factor. The addition of 25 µM paraquat to T. versicolor and 20 µM
paraquat to A. biennis cultures significantly stimulated the production of laccase.
CONCLUSION
Environmental pollutants are a serious concern world-wide. Many studies have
shown that these persistent compounds can be degraded by the ligninolytic system
of white-rot fungi. In this paper, a convenient submerged medium to study the
capability of P. pulmonarius to remove PCP is proposed. Apparently, laccase is
important enzyme involved in the degradation of PCP by Pleurotus pulmonarius.
an
20
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25
400
300
200
100
dry biomass (mg)
Mn peroxidase (U/L)
500
laccase (U/L)
80
25
600
20
15
10
5
0
0
0
1
2
3
4
time (d)
5
6
60
40
20
0
0
1
2
3
4
5
6
time (d)
0
1
2
3
4
5
6
time (d)
Figure 1. Production of laccase and Mn peroxidase and biomass by P. pulmonarius. The cultures were developed in ligninolytic (●) and
non-ligninolytic conditions in submerged conditions. Media of three different experiments.
26
growth rate (mm/d)
4
3
2
1
0
0
5
10
15
20
25
30
35
PCP mg/L
Figure 2. Growth rates of P. pulmonarius in ligninolytic (●) and non-ligninolytic (○)
solid media containing the indicated concentrations of pentachlorophenol.
27
50
A
1000
800
600
400
C
40
dry biomass (mg)
Mn peroxidase (U/L)
1200
laccase U/L)
200
B
1400
30
20
150
100
50
10
200
0
0
0 2 4
5
10 15 20 25
time course (days)
0
0 2 4
5
10 15 20 25
time course (d)
0 2 4
5
10 15
20 25
time course (d)
Figure 3. Effect of PCP in submerged cultures of P. pulmonarius developed under ligninolytic and non-ligninolytic conditions. The arrow
indicates the time at which PCP was introduced in the cultures at the final concentration of 25 mg/L. (●) ligninolytic cultures with PCP;
(○) ligninolytic cultures without PCP; (■) non-ligninolytic cultures with PCP; (□) non-ligninolytic cultures without PCP.
28
Residual PCP (%)
120
100
80
60
40
20
0
5
10
15
20
25
30
time course (d)
Figure 4. Residual PCP in culture filtrates of P.pulmonarius developed under
ligninolytic (●) and non-ligninolytic conditions (■). (□) amount of PCP adsorbed to the
mycelial mass developed under non-ligninolytic conditions; (○) amount of PCP
adsorbed to the mycelial mass developed under ligninolytic conditions.
29
THE RATIO Mn PEROXIDASE/LACCASE IS HIGHLY AFFECTED BY THE
INITIAL MOISTURE CONTENT ON CORN COB SOLID STATE CULTURES OF
Pleurotus pulmonarius
DANIELA FARANI DE SOUZA, CISSA KELMER BRACHT, ANA MARIA
ALEXANDRINO, GISELE PEZENTE FERREIRA, MARIA APARECIDA FERREIRA
COSTA, CINTHIA GANDOLFI BOER, CRISTINA GIATTI MARQUES DE SOUZA
and ROSANE MARINA PERALTA
Departamento de Bioquímica, Universidade Estadual de Maringá
ABSTRACT
The production of ligninolytic enzymes by the white-rot fungus Pleurotus pulmonarius
(FR.) Quélet and the capability of cultures to decolourise the industrial dyes Remazol
Brilliant Blue R (RBBR), congo red, methylene blue and ethyl violet were studied in
this work. The condition of high initial moisture content (80-90%) strongly promoted
the expression of laccase, while manganese peroxidase was produced specially in
cultures developed at low initial moisture (50-70%). Cultures containing high
manganese peroxidase activities were more efficient to decolorize the four dyes than
those containing high laccase activity. Cell free filtrates with high manganese
peroxidase activity were also more efficient to decolorize methylene blue, ethyl violet
and congo red, while the dye RBBR was efficiently decolorized by both enzymes, Mn
peroxidase and laccase.
Key words: Pleurotus pulmonarius, ligninolytic enzymes, dye decolorization,
industrial dyes
1. INTRODUCTION
Textile industries consume large volumes of water and chemicals for wet
processing of textiles. The chemical reagents used are very diverse in chemical
composition, ranging from inorganic compounds to polymers and organic products
(Mishra and Tripathy, 1993; Banat et al., 1996 and Juang et al., 1996). The presence
of very low concentrations of dyes in effluents is highly visible and undesirable
30
(Nigam et al., 2000). There are more than 100,000 commercially available dyes with
over 7×105 ton of dye-stuff produced annually (Zollinger, 1987). Due to their chemical
structure, dyes are resistant to fading on exposure to light, water and many
chemicals (Poots and McKay, 1976 and McKay, 1979). Many dyes are difficult to
decolourise due to their complex structure and synthetic origin. There are many
structural varieties, such as, acidic, basic, disperse, azo, diazo, anthroquinone based
and metal complex dyes. Decolorization of textile dye effluents does not occur when
they are treated aerobically by municipal sewerage systems (Willmott et al., 1998).
Ligninolytic fungi have been studied as possible agent of biodegradation
because their extracellular degradation systems are basically non-specific, a fact that
allows the degradation of mixtures of refractory substances (Kirk and Farrell, 1987).
The major enzymes associated with the lignin-degrading ability of white-rot fungi are
lignin peroxidase (EC 1.11.1.14), manganese peroxidase (EC 1.11.1.13) and laccase
(EC 1.10.3.2) (Vicuna, 2000). Some white-rot fungi produce all these enzymes while
others produce only one or two of them (Leonowicz et al., 2001). Several strains of
the dye- or colored material-degrading microorganisms have been reported, including
species of Pleurotus, Coriolus, Trametes, Polyporus, Lentinus and Picnoporus
(Leonowicz et al., 2001, Sani et al., 1998, Kahraman and Yesilada, 1999, Das et al.,
2000, Sam and Yesilada, 2001, Zilly et al., 2002). The capability of dye decolorization
may be due to the activities of lignin peroxidase (Young and Yu, 1997, Ollikka et al.,
1993), Mn peroxidase (Boer et al., 2004, Heinfling et al., 1998, Hatvani and Mecs,
2002), and laccase (Wong and Yu, 1999, Zilly et al., 2002, Pointing and Vrjmoed,
2000, Landelbauer et al., 2004, Rodriguez et al., 1999).
Pleurotus spp. are among the easiest mushrooms to cultivate (Cohen et al.
2002). The two most important species cultivated on an industrial scale are P.
ostreatus and P. pulmonarius (P. sajor-caju, P. sapidus). In nature they grow on
wood, usually on dead, standing trees or on fallen logs. Various substrates that
contain lignin and cellulose can be used for Pleurotus cultivation, such as wood
chips, corn wheat, rice straw, cotton stalks, waste hulls, and other agricultural
wastes, some of which can be recycled and upgraded for use as animal feed or for
preparation of other products (Cohen et al. 2002). These substrates are frequently
used to study the production of MnP and laccase by Pleurotus spp in both
submerged and solid state cultures. Solid state culture is defined as a cultivation in
which a microorganism grows on a moist water-insoluble solid material in the
absence or near absence of free water (Mooyoung et al. 1983, Gervais and Molin
31
2003). It mimics the natural environment of the white-rot fungi and holds tremendous
potential for the production of enzymes, including laccase and MnP (Pandey et al.
1999).
Pleurotus pulmonarius (P. sajor-caju) when cultured under submerged and
solid state conditions using wheat bran as substrate produces laccase as the main
extracellular enzyme (Souza et al., 2002). The capability of P. pulmonarius
to
decolourise textile dyes in both types of cultures has already been described and
associated with the production of laccase considering that the production of Mn
peroxidase in these cultures was very low (Zilly et al., 2002, Tychanowicz et al,
2004). More recently however, it has been described that when P. pulmonarius was
cultured in wheat bran solid state medium at low initial moisture content, it produced
both enzymes, Mn peroxidase and laccase, in elevated amounts (Souza et al., 2006).
The objectives of this study were to compare the production of laccase and Mn
peroxidase by P. pulmonarius in solid state cultures using three different substrates,
wheat bran, corn cob and pineapple peel at different initial moisture contents and to
evaluate the capability of these cultures to decolorize some industrial dyes.
2. MATERIAL AND METHODS
2.1. Microorganism
Pleurotus pulmonarius CCB-19 was obtained from the Culture Collection of
the Botany Institute of São Paulo. It was cultured on potato dextrose agar (PDA)
medium for 2 weeks at 28 °C. When the plates were f ully covered with the mycelia,
mycelial plugs measuring 10 mm in diameter were made and used as inocula.
2.2. Enzyme production on SSC
P. pulmonarius CCB-19 was cultivated in 250 ml Erlenmeyer flasks containing
5 g of wheat bran, corn cob or pineapple peel. The following salts were added to give
a final salt concentration of (mg/g): K2HPO4, 1; MgSO4·7H2O, 0.2 and CaCl2 · 2 H2O,
0.1. Five to 50 ml of distilled water was added to adjust the moisture content. Dry
weight of the substrate and moisture content were determined gravimetrically, after
drying the samples at 60 °C. The pH of the media wa s approximately 6.0. The
32
cultures were inoculated aseptically by using three mycelial plugs obtained from the
colony edge of a colony grown on PDA medium. Incubation was at 28 °C.
2.3. Enzyme extraction
Crude extract was obtained by adding 50 ml of cold water to the contents of
each flasks, stirring for 1 h at 4 °C, and using fi ltration and centrifugation. The
supernatant was stored at –20 °C until assay.
2.4. Dye decolorization experiments on SSC
To test the ability of cultures to decolourise industrial dyes, each dye was membranefiltered through a 0.45 µm cellulose nitrate filter and mixed with the corn cob medium,
previously autoclaved, to a final concentration of 200 ppm. After 15 days, the residual
dyes in the cultures were extracted firstly with 50 ml of water followed by 50 ml of a
mixture of methanol:acetone:water (1:1:1). Dye disappearance was determined
spectrophotometrically by monitoring the absorbance at the wavelength of maximum
absorbance for each dye. In control cultures, either dye or the fungus (abiotic control)
was omitted. To calculate the residual dye in the cultures, the total dye extracted with
water and organic mixture in the abiotic control was considered as 100%. The
amount of adsorbed dye on corn cob medium after growth of the fungus was always
less than 10%.
2.5. Dye decolourisation by culture filtrates
A volume of 0.5 ml of each dye to give a final concentration of 100 ppm and
0.5 ml of cell-free culture filtrates previously dialyzed against water to remove small
molecules, were added to 4.0 ml of 50 mM malonate buffer, pH 4.5 containing 1 mM
MnSO4 and 0.1 mM H2O2 or 4.0 ml of 50 mM phosphate buffer, pH 6.5. The
mixtures were incubated on a rotary shaker at 40o C for 2 h. Dye disappearance was
determined spectrophotometrically by monitoring the absorbance at the wavelength
of maximum absorbance for each dye. Boiled crude culture filtrates were used as
negative controls.
33
2.6. Enzyme assays
All enzyme assays were performed at 40 °C. Laccase activity was followed
spectrophotometrically at 525 nm, by the oxidation of syringaldazine to its quinone
form, using a molar absorptivity of 65,000 for the product (Leonowicz and
Grzywnowicz 1981). The reaction mixture contained 1.5 ml phosphate buffer (0.1 M,
pH 6.5), 0.2 ml syringaldazine (0.5 mM in ethanol solution), and 0.1 ml of culture
filtrates. The MnP activity was assayed by the oxidation of 1 mM MnSO4 dissolved in
0.05 M sodium malonate, pH 4.5, with 0.1 mM H2O2. The absorption of the complex
resulting from this reaction was measured at 270 nm (ε270 = 11.59 mM–1cm–1)
(Wariishi et al. 1997).
2.7. Dyes
The following dyes were used in this work: one anthracene derivative,
Remazol brilliant blue (RBBR); one
triphenylmethane dye, Ethyl violet;
one
heterocyclic dye, Methylene blue; one polymeric dye, Poly R-478 and one azo dye,
Congo red
2.8. Statistical analysis
The data obtained were compared using paired t-test, and the level of
significance of p<0.05 was chosen for all statistical comparisons. The data are
presented as mean ± SEM. The analysis was done using the statistical program pack
GraphPad Prim® (Graph Pad Software, San Diego, USA).
2.9. Chemicals
The enzymatic substrates were obtained from SIGMA Chemical Corp., St
Louis, MO. PDA was obtained from DIFCO Laboratories, Detroit, MI. All other
reagents were of analytical grade.
34
3. RESULTS
3.1. Effect of substrate and initial moisture content (IMC) in the production of P.
pulmonarius enzymes
The production of ligninolytic enzymes by P. pulmonarius CCB-19 was
determined using three different substrates at different initial moisture contents (Fig.
1). Laccase was the main ligninolytic enzyme produced by the fungus in wheat bran
cultures and its production was positively affected by increases in initial moisture
content (Fig. 1B). In pineapple peel cultures both enzymes were produced at high
amounts, being high initial moisture content (80-90%) the best condition to produce
laccase, and low initial moisture content (70-75%), the best condition to produce Mn
peroxidase. The substrate where the initial moisture content had the strongest effect
in the production of enzymes was corn cob (Fig 1A). Initial moisture content of 8590% was the best condition to produce laccase and very low Mn peroxidase activity
was detected in these filtrates. The best initial moisture contents to produce Mn
peroxidase changed from 50 to 65%, a condition where the production of laccase
was very low (Fig 1A).
The impact of initial moisture content on the relative
production of both enzymes in these cultures can be best observed in Fig 2. The Mn
peroxidase/laccase ratio changed from 9.93 (at IMC of 60%) to less than 0.01 (at
IMC of 90%) in corn cob cultures. The lowest MnP/laccase ratio was found in wheat
bran cultures (ranged from 0.37 to 0.002) followed by pineapple peel cultures
(ranged from 2.23 to 0.37).
3.2. Effect of initial moisture content in capability to decolorize industrial dyes
by corn cob cultures of P. pulmonarius
The industrial dyes used in this work were selected on the basis of their
stability over a wide range of pH (3-11), thermostability, stability under culture
conditions in non-inoculated flasks and they were representative for each dye
chemical category (anthracene derivative, azo, heterocyclic, polymeric and
triphenylmethane dyes).
The fungal cultures were able to decolorize completely RBBR, and partially
ethyl violet, congo red and methylene blue (Table 1). The cultures with 60% of initial
moisture content were more efficient in the dye decolorization of these three last dyes
35
than the cultures developed at 85% of initial moisture content (p<0.05). The dye Poly
R478 was barely decolorized by cultures in both moisture content cultures. Alcoholic
extracts from mycelia and corn cob showed that less than 10% of the dyes were
adsorbed by the mixture of fungi plus corn cob. RBBR was equally decolourized by
cultures developed at initial moisture contents of 60 and 85%.
3.3. Capability of crude dialyzed cell free extracts from P. pulmonarius solid
state cultures decolorize industrial dyes with high and low MnP/lcc ratio
To test the ability of crude-dialysed cell free extracts to decolourise the
industrial dyes used in this study, the extracts were incubated with 100 ppm of dyes
under two different conditions, to obtain the best condition for Mn peroxidase activity
(50 mM malonate buffer pH 4.5 with 1 mM MnSO4 and 0.1 mM H2O2 ) and the best
condition for laccase activity (50 mM phosphate buffer, pH 6.5). The results are
shown in Figure 3. Both cell free extracts (from 60% and 85% initial moisture content
cultures) efficiently decolourised RBBR.
No cell free extracts were able to
decolourize Poly R478, while the cell free extracts obtained from cultures developed
with initial moisture content of 60% were more efficent to decolourize the dyes
methylene blue, ethyl Violet and congo red.
DISCUSSION
Many white rot fungi have been intensively studied in connection with their
ligninolytic enzyme production and their decolorization ability (Boer et al., 2004,
Pointing and Vrjmoed, 2000, Kasinath et al., 2003, Chagas and Durrant, 2001,
Jarosz-Wilkolazka et al., 2002). However, most studies on dye decolorization have
been carried out using liquid or solid cultures on agar plates, which do not reflect the
natural living conditions (i.e. in wood and other lignocellulosic substrates) of white-rot
fungi. Solid state culture was chosen here because it mimics the natural environment
of the white-rot fungi and high levels of ligninolytic enzymes are generally produced
in this type of cultivation. The list of different substrates used for the cultivation of
Pleurotus sp is long, including several agricultural materials, such as wheat bran,
wheat straw, sugar cane bagasse and corn cob. More recently several fruit wastes
have been described as excellent substrates to produce elevated levels of laccases
36
and Mn peroxidases (Rosales et al., 2002, Songulashvili et al., 2006, Alexandrino et
al., 2007).
In this work, pineapple peel, a yet not used substrate in this type of study was
introduced and tested as substrate for P. pulmonarius. In pineapple peel solid state
cultures both, laccase and Mn peroxidase were produced at levels higher than in
corn cob and wheat bran cultures. A previous study has demonstrated the ability of
P. pulmonarius CCB19 to grow and produce laccase as the main ligninolytic enzyme
on SSC using wheat bran with a moisture content of 75% as the substrate (Souza et
al. 2002). In the present work, we showed that the cultivation of P. pulmonarius
under solid state conditions using corn cob and pineapple peel as substrates resulted
in a convenient condition to produce also high amounts of Mn peroxidase. By varying
only the initial moisture content, the use of corn cob as substrate allowed the
obtainment of cell free extracts rich either in laccase either in Mn peroxidase. In
addition to this,
the low amounts of natural colored pigments in this material,
permitted the use of its cell free extracts in experiments of decolorization without any
additional treatments, because the color of the extracts do not interfere with the
determination of residual dyes. From the results obtained in this work, it is possible to
conclude that Mn peroxidase was the main enzyme responsible for the
decolourisation of congo red, ethyl violet and methylene blue by P. pulmonarius. Our
data suggest that both enzymes laccase and Mn peroxidase from P. pulmonarius are
equally efficient in decolorize RBBR. To detect the real potential of enzymes to
decolorize industrial dyes, it is necessary to obtain isolated fractions of these
enzymes. The main laccase from P. pulmonarius was recently purified (Souza et al.,
2003). Purification of Mn peroxidase is in progress in the laboratory.
ACKNOWLEDGEMENTS
This
work
was
supported
by
grants
from
Conselho
Nacional
de
Desenvolvimento Científico e Tecnológico (CNPq) and Fundação Araucária. R. M.
PERALTA is research fellow of CNPq. D.F.S. is the recipient of a CNPq Fellowship.
We thank A. CHAVES for technical assistance.
37
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40
3200
corn cob
2800
400
200
50
.0
55
.0
60
.0
65
.0
70
.0
75
.0
80
.0
85
.0
90
.0
0
initial moisture content (%)
enzyme activity (U/L)
600
2400
2400
2000
1600
1200
800
pineapple peel
2000
1600
1200
800
400
400
0
0
50
.0
55
.0
60
.0
65
.0
70
.0
75
.0
80
.0
85
.0
90
.0
Enzyme activity (U/L)
Enzyme activity (U/L)
1000
800
2800
wheat bran
50
.0
55
.0
60
.0
65
.0
70
.0
75
.0
80
.0
85
.0
90
.0
1200
initial moisture content (%)
initial moisture content (%)
Figure 1. Effect of initial moisture content in the production of laccase and Mn peroxidase by P. pulmonarius in solid state cultures. The
cultures were developed for 10 days at 28oC, using three different substrates. Laccase Activity;
MnP Activity.
MnP/laccase ration
41
10
9
8
7
6
5
4
3
2
1
0
WB
CC
PP
45
50
55
60
65
70
75
80
85
90
% initial moisture content
Figure 2. Effect of initial moisture content in the ratio Mn peroxidase/laccase of P.
pulmonarius cell free culture filtrates
42
Table 1. Degree of decolorization of industrial dyes by solid state cultures of P.
pulmonarius at two initial moisture contents.
Synthetic dye common name
Λ
(nm)
Anthracene derivative dye
Residual dye
after 15 days of cultivation
60% IMC cultures
85% IMC cultures
MnP
Laccase
595
3.7±2.8(a)
4.4±2.2(a)
497
22.0±5.0(a)
47.3±5.6(b)
665
52.1±5.7(a)
67.9±7.3(b)
596
39.5±6.2(a)
62.5±2.3(b)
530
83.8±6.8(a)
91.8±7.2(a)
RBBR
Azo dyes
Congo red
Heterocyclic dye
methylene blue
Triphenylmethane dye
ethyl violet
Polymeric dye
Poly R478
Dye disappearance was determined spectrophotometrically by monitoring the
absorbance at the wavelength of maximum absorbance of each dye. The extraction
of residual dye was carried out firstly with water followed by extraction with a 1:1:1
mixture of methanol:acetone:water. To calculate the residual dyes in the cultures, the
total dye extracted with water and organic mixture in the abiotic control was
considered as 100%. Values labeled with different lowercase letters in each line are
significantly different (p < 0.05).
43
Residual dye (%)
100
Low MnP/lccpH 4.5
Low MnP/lcc pH 6.5
High MnP/lcc pH 6.5
High MnP/lcc pH 4.5
80
60
40
20
47
8
R
re
d
Po
ly
co
ng
o
io
le
t
et
yl
v
R
BB
R
m
et
hy
le
ne
bl
ue
0
Figure 3. In vitro decolorization of industrial dyes by crude dialyzed cell free extracts
from P. pulmonarius solid state cultures. The dyes were added to the reaction
medium to give a final concentration of 100 ppm. The mixtures were maintained at
40º C for 2 h. The reaction mixture with low MnP/lcc ratio contained 2.1 U/L of
manganese peroxidase activity and 97.0 U/L of laccase activity. The reaction mixture
with high MnP/lcc ratio contained 81.2 U/L of manganese peroxidase activity and 8.0
U/L of laccase activity.