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: mamagrini@gmail.com.br

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.