MARINE-DERIVED BASIDIOMYCETES: LIGNINOLYTIC ENZYME PRODUCTION
UNDER LIQUID-STATE FERMENTATION SUPPLEMENTED WITH PYRENE
Mariana J. Magrini1, Rafaella C. Bonugli-Santos1, Marili V. N. Rodrigues2, Sinesio Boaventura
Júnior2, Lara D. Sette1
1Divisão de Recursos Microbianos – CPQBA/UNICAMP, Campinas, SP, Brazil.
2Divisão de Química Orgânica e Farmacêutica – CPQBA/UNICAMP, Campinas, SP, Brazil.
*E-mail: [email protected]
Support:
Fungal strains inoculation:
50 ml 2% malt extract
broth, 72 hours at 28º C
and 140 rpm
2 mg of pyrene
Enzymatic extract achievement:
Addition of 50 ml ethyl acetate
Cell disintegration in disperser
(Polytron) at 14,000 rpm during 1 min
Filtration under cotton
soaked with ethyl acetate
directly engaged in the
funnel of separation
After a vigorous shaking
(1 min) the aqueous
phase was collected
and re-extracted
The organic phase was submitted to
pyrene quantification
Cultures were harvested by
filtration and centrifuged
at 10,000 rpm for 30 min
The supernatant was used
as enzyme source
Crude extract
The aqueous phase was used
as enzyme source Aqueous extract
INTRODUCTION
Polycyclic aromatic hydrocarbons (PAHs) such as pyrene can be found in petroleum and many are known to be carcinogenic or co-carcinogenic. The activities derived from the
oil industry may be responsible for accidental discharges, including oil release into the marine environment, that cause serious environmental, social and economical damages
(Haritash and Kaushik 2009). The inherent ability of white-rot fungi to degrade lignin in wood suggests that such organisms may be useful for the biological decontamination of a
wide range of organopollutants and, in most cases, ligninolytic enzymes have been implicated. Ligninases produced by white-rot fungi include lignin peroxidase (LiP), manganese
peroxidase (MnP) and laccase. In the present study three ligninolytic basidiomycetes (Marasmiellus sp. CBMAI 1062, Tinctoporellus sp. CBMAI 1061 and Peniophora sp. CBMAI
1063) isolated from the Brazilian sponges Amphimedon viridis and Dragmacidon reticulata (Menezes et al. 2009), were used to evaluate the production of LiP, MnP and laccase
in liquid-state fermentation supplemented with pyrene.
Keywords: marine fungi, ligninolytic enzymes, basidiomycetes
RESULTSMATERIALS AND METHODS
Enzymatic activities were determined
by oxidation of:
LiP: veratryl alcohol
MnP: phenol red
Laccase: o-dianisidine, guaiacol,
syringaldazine and ABTS
0
1000
2000
3000
4000
5000
6000
Crude extract
En
zym
e A
cti
vit
y U
L-1
MnP
Tinctoporellus sp. CBMAI 1061
Marasmiellus sp. CBMAI 1062
Peniophora sp. CBMAI 10630
200
400
600
800
1000
1200
Aqueous extract Crude extract
En
zym
e A
cti
vit
y U
L-1
LiP
0
20
40
60
80
100
120
140
160
180
Aqueous extract Crude extract
En
zym
e A
cti
vit
y U
L-1
Laccase - Syringaldazine
0
500
1000
1500
2000
2500
3000
3500
4000
4500
Crude extract
En
zym
e A
cti
vit
y U
L-1
Laccase - ABTS The better production of LiP
(1,039.42 UL-1) and MnP
(5,570.50 UL-1) was obtained by
Marasmiellus sp. CBMAI 1062.
0
5000
10000
15000
20000
25000
30000
Aqueous extract Crude extract
En
zym
e A
cti
vit
y U
L-1
Laccase - Guaiacol
CPPG-GBM
0
5000
10000
15000
20000
25000
30000
Aqueous extract Crude extract
En
zym
e A
cti
vit
y U
L-1
Laccase - O-dianisidine
Laccase was the higher enzyme produced and
guaiacol was found to be the most sensitive substrate:
Peniophora sp. CBMAI 1063 (25,603.13 UL-1),
Tinctoporellus sp. CBMAI 1061 (21,267.77 UL-1) and
Marasmiellus sp. CBMAI 1062 (2,025.77 UL-1).
The fungal strains Peniophora
sp. CBMAI 1063 and
Tinctoporellus sp. CBMAI 1061
produced 184.19 UL-1 and 98.57
UL-1 of LiP and 1,579.47 UL-1
and 3,019.43 of MnP,
respectively.
When syringaldazine was used as substrate,
enzimatic activity was higher in the aqueous
extracts: Marasmiellus sp. CBMAI 1062 (167.81
UL-1), Peniophora sp. CBMAI 1063 (132.76 UL-1)
and Tinctoporellus sp. CBMAI 1061 (113.39 UL-1).
The crude extracts showed higher enzymatic values when compared to aqueous extracts.
Fungal strains cultivation:
2% (w/v) malt extract agar
(MA2) , 10 days at 28º C
3 fungal
culture plugs
Pyrene addition and
incubation for more 7 days
REFERENCES Haritash AK and Kaushik CP. 2009. Biodegradation aspects of polycyclic aromatic hydrocarbons (PAHs): a review.
Journal of Hazardous Materials, 169: 1–15.
Menezes CB, Bonugli-Santos RC, Miqueletto PB, Passarini MRZ, Silva CHD, Justo MR,Leal RR, Fantinatti-
Garboggini F, Oliveira VM, Berlinck RGS, Sette LD. 2009. Microbial diversity associated with algae, ascidians and
sponges from the north coast of São Paulo state, Brazil. Microbiological Research, 165: 466-482.
CONCLUSIONS
The use of different methodologies for evaluating the ligninolytic activity could be considered
relevant.
The strategy of aqueous phase achievement could negatively affect the ligninolytic activity.
Assay sensitivity for ligninolytic enzymes is largely depended upon the efficiency of substrates.
The production of ligninolytic enzymes in the presence of pyrene supports the hypothesis of
PAHs degradation by sponge-derived basidiomycetes.