AGRICULTURE AND BIOLOGY JOURNAL OF NORTH AMERICA

ISSN Print: 2151-7517, ISSN Online: 2151-7525

© 2010, ScienceHuβ, http://www.scihub.org/ABJNA

Screening of some marine-derived fungal isolates for lignin degrading

enzymes (LDEs) production
Atalla, M. Mabrouk*; Zeinab, H. Kheiralla**; Eman, R. Hamed*; Amani, A. Youssry** and
Abeer, A. Abd El Aty*
* Chemistry of Natural and Microbial Products Dept. National Research Centre, Cairo, Egypt.
** Botany Dept. Faculty of Girls for Arts, Science and Education, Ain Shams University.
* E.mail: erhamed@yahoo.com
ABSTRACT
A study was carried out to establish the lignin-degrading enzymes (LDEs) activity of a large
number of diverse marine ascomycetes. Eighty eight fungal isolates obtained from different algae,
sea grasses and decaying wood samples collected from Abou-keer, Alexanderia, Egypt, were
screened for the presence of laccase (Lac), lignin-peroxidase (LiP) and manganese-dependent
peroxidase (MnP) activities. Results obtained from both qualitative and quantitative assay
showed that the marine fungal isolate Trematosphaeria mangrovei measured the highest zone
diameter and colony diameter in agar plate screening test with guaiacol and showed a final
specific activity of 82.51 U/mg protein with higher laccase activity 14.03 U/ml when grown in low
nitrogen (LN) medium with half-strength sea water, initial pH 4.5 for 14 days. The other two
ligninolytic activities could not be detected. It was recommended that the marine fungal isolate
Trematosphaeria mangrovei was the most promising one for laccase enzyme production.
Keywords: Marine-derived fungi, Lignin degrading enzymes, ligninases, laccase, bioremediation.
INTRODUCTION invertebrates, especially corals and sponges, fungi in
detritus of marine macrophytes and in marine
Lignin, the second most abundant renewable organic
extreme environments (Kohlmeyer and Kohlmeyer,
polymer on earth, is a major component of wood.
1979 and Raghukumar, 2008).
Because of the importance of wood and other
lignocellulosics as a renewable resource for the The ability of fungi to degrade lignocellulose is due
production of paper products, feeds, chemicals, and to their possession of extracellular enzymes,
fuels, there has been an increasing research mainly lignin peroxidase (LiP), manganese
emphasis on the fungal degradation of lignin peroxidase (MnP) and laccase (Lac). These
(Boominathan and Reddy, 1992). enzymes have been shown to degrade not only
lignocellulose, but also recalcitrant environmental
Fungi play an important role in degradation and
pollutants such as crude oil wastes, textile effluents,
mineralization of lignocellulosic substrates in the
organochloride agrochemicals and pulp effluents
marine environment. These lignicolous fungi
which are a cause of serious environmental pollution
comprising Ascomycetes, Basidiomycetes and
(Mtui and Nakamura, 2004; Kiiskinen et al., 2004).
Deuteromycetes have distinct spore structures that
set them apart from their terrestrial counterparts. Worldwide, there is little published information on
Such fungi have been defined as obligate marine the lignin-degrading ability of the obligate and
fungi “which grow and sporulate exclusively under facultative marine fungi (Mtui and Nakamura,
marine conditions” (Kohlmeyer and Kohlmeyer 1979). 2007). Moreover, the presences of MnPs, LiPs and
In order to accommodate the possibility that Lacs in facultative and marine fungi have not been
terrestrial species might also be active in the sea, investigated except for few reports (Raghukumar et
Kohlmeyer and Kohlmeyer offered a definition for al., 1999).
“facultative marine fungi” as those originating from
Therefore, the major object of this study was to
freshwater or terrestrial environment that are capable
investigate the production of lignin-degrading
of growth and sporulation in the sea.
enzymes by some local marine fungal isolates.
Marine filamentous fungi, obligate and facultative are
known to occur in association with algae, seagrasses
and mangroves, fungi cohabiting with marine

MATERIALS AND METHODS
Chemicals: 2-Methoxyphenol (Guaiacol) and 3,4-
Dimethoxybenzyl alcohol (Veratryl alcohol) were
purchased from Fluka Co. 2,2´-Azino-bis(3-
ethylbenzothiazoline-6-sulphonic acid) diammonium
salt (ABTS) was obtained from MP. Bio (LCN) Co,
USA.
Microorganisms.
Source of algae, sea grasses and decayed wood
samples:
All algae and sea grasses samples were collected
from Abou Keer, Alexandria during the 4 seasons
(summer, autumn, winter and spring) at a depth of 3-
5m as described previously by (Atalla et al., 2008) ,
decayed wood samples collected from the same
place in 2008. The collected samples were brought to
the laboratory in clean plastic bags and stored in ice
box.
Isolation of marine fungi: Samples were cut in
small pieces, washed with sterile sea water, blotted
between two folds of sterilized filter paper and
transferred to petri dishes contained suitable medium
(Höller, 1999; Rowley et al., 2003).
Different kinds of media were used in isolation
[biomalt agar (BIO), malt extract agar (ME), potato
carrot agar (KM), glucose peptone yeast extract agar
(GPY) and Boyd &Kohlmeyer (B&K) agar]. Plates
were incubated at 25 ºC for 4 days, exposed to daily
examination to observe the developing growth.
Fungal isolates were picked up under the dissecting
microscope, transferred to biomalt agar (BIO) slants.
Purification was carried out using single spore and
hyphal tip techniques individually and transferred to
suitable medium. Stock cultures were maintained on
biomalt agar (BIO) slants and kept in the refrigerator
at (5-6 ºC) for later use.
Identification of the isolated fungi: Isolated fungi
were identified in the National Research Centre,
Chemistry of Natural and Microbial Products Dept.
[(Microbial Culture Collection Unit (MCCU)] according
to (Pitt and Hocking, 1985 and Kohlmeyer and
Kohlmeyer, 1991).
Media: Different types of media were used in this
study.
Isolation media: Four media were used for isolation,
each contained the following components in 1l of
80% sterile sea water (Höller, 1999). Biomalt agar
(BIO): contained, biomalt 20 g, agar15 g.
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Agric. Biol. J. N. Am., 2010, 1(4): 591-599
Malt extract agar (MEA): contained, malt extract 30 g,
peptone 3 g, agar 12 g.
Potato carrot agar (KM): contained, cooked and
sliced potatoes 20 g, cooked and sliced carrots 20 g,
agar 20g. Glucose peptone yeast extract agar (GPY):
contained, glucose. H2O 1.0 g, peptone 0.5 g, yeast
extract 0.1 g, agar 15 g.
Boyd &Kohlmeyer (B&K) agar: contained, glucose 10
g, peptone 2 g, yeast extract 1 g, agar 18 g in 1l of
50% sea water (Kohlmeyer and kohlmeyer, 1979 and
D'Souza et al., 2006).
Screening media: Two different types of media were
used for qualitative and quantitative assay of lignin-
degrading enzymes production.
Qualitative media (D'Souza et al., 2006): Guaiacol-
supplemented agar: contained, glucose 10 g,
peptone 2 g, yeast extract 1 g, agar 18 g and 4mM
guaiacol in1l of 50% sterile sea water.
Quantitative media (Rigas et al., 2005).
a- Kirk's medium: composed of g/l 50% sterile sea
water; KH2PO4 0.20, CaCl2 0.01, MgSO4.7H2O 0.05,
ammonium tartrate 0.22, 2.2-dimethylsuccinic acid
2.90, glucose 5 , thiamine 0.1, Tween 80 0.10% v/v,
veratryl alcohol 1.5 mM, and a mixture of trace
elements composed of (mg/l): MnSO4 33, Fe2 (SO4)3
50, ZnSO4.7H2O 43, CuSO4.7H2O 80, H2MoO4 50.
b- Malt Extract Broth (MEB): contained g/l 50% sterile
sea water; malt extract 17 and mycological peptone
3.
Qualitative assay for lignin-degrading enzymes
production: The fungal isolates were screened for
LDEs production by growing them on plates of B&K
medium containing 4mM guaiacol (D'Souza et al.,
2006). Petri dishes (15 cm in diameter) each
containing 30 ml of medium were used.
Preparation of fungal isolates inoculum for guaiacol
oxidation test was performed by removing disks 10
mm. in diameter from the edge of expanding colonies
7 days old cultures grown on B&K agar medium.
Then, a disk was placed on the surface of guaiacol-
supplemented agar plates. The inoculated plates
were incubated at 25 ºC in dark for 7 days. The
production of intense brown color under and around
the fungal colony was considered as a positive
reaction resulting from guaiacol oxidation (Okino et
al., 2000). The diameter of coloured zone, growth
rate were measured in mm.

Quantitative estimation of Lignin-degrading
enzymes: Kirk's medium was used as liquid
fermentation media for quantitative estimation of
enzyme activities from the selected strains (Rigas et
al., 2005).
Inoculum preparation: The inoculum was prepared
by transferring two agar plugs (1cm diameter) from 7
days old cultures grown on (MEA) into 250-ml flasks
containing 50 ml Malt Extract Broth (MEB) media and
incubated at 25 ºC under stationary conditions for 6
days.
After 6 days of growth, the cultures were
homogenized and further used as inoculum for the
estimation of ligninolytic activities.
Liquid medium used for enzyme activities: Ten-
milliliter aliquots (trace elements) were added to 90
ml Kirk's media (in 250-ml Erlenmeyer flasks). Each
flask was inoculated with 1 ml from inoculum and
incubated at 25 ºC in dark under stationary conditions
for 14 days.
Assay for enzyme activities: At the end of each
growth period, inoculated flasks were collected and
centrifugated. The filtrate was tested for lignin-
degrading enzymes activity as follows and enzyme
activity was expressed in international units (Mtui and
Masalu, 2008).
Lignin peroxidase (LiP).
Lignin peroxidase (LiP) (EC 1.11.1.14) activity was
determined spectrophotometrically at 310 nm through
the oxidation of veratryl alcohol to veratryl aldehyde
(molar absorptivity, ε310 = 9300 M-1 cm-1).
The reaction mixture contained 300 µL veratryl
alcohol (8 mM), 600 µl 0.3M citrate/0.4M phosphate
buffer (pH 4.5), 60 µL culture supernatant, and 1890
µL distilled water. The mixture was incubated for 2
min at 30 ºC and the reaction was initiated by
addition of 150 µL H2O2 (5 mM). The absorbance
was immediately measured in one-minute intervals
after addition of H2O2. One unit (U) of LiP activity
was defined as activity of an enzyme that
catalyzes the conversion of 1 µ mole of veratryl
alcohol per minute (Takamiya et al., 2008).
Manganese peroxidase (MnP): Activity of
manganese peroxidase (MnP) (EC 1.11.1.13) was
measured by using guaiacol as a substrate. The
increase in absorbance at 465 nm due to oxidation of
guaiacol (ε465= 12,100 M-1 cm-1) was measured.
The reaction mixture contained 300 µL sodium
succinate buffer (0.5 M, pH 4.5 at 27 ºC), 300 µL
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guaiacol (4 mM), 600 µL manganese sulphate (1
mM), 300 µL culture supernatant and 1200 µL
distilled water. The mixture was incubated for 2
min at 30 ºC and the reaction was initiated by
addition of 300 µL hydrogen peroxide (1mM). The
absorbance was measured in 1 min intervals after
addition of hydrogen peroxide. One unit of MnP
activity was defined as activity of an enzyme that
catalyzes the conversion of 1µ mole of guaiacol per
minute.
Laccase (Lac): Laccase (EC 1.10.3.2) activity was
measured based on the oxidation of the substrate
2,2’-azino–bis (3-ethylbenzothiazoline)-6-sulphonic
acid (ABTS). The rate of ABTS oxidation was
determined spectrophotometrically at 420 nm.
The reaction mixture contained 600 µL sodium
acetate buffer (0.1 M, pH 5.0 at 27 ºC), 300 µL
ABTS (5 mM), 300 µL mycelial liquid fraction and
1400 µL distilled water. The mixture was then
incubated for 2 min at 30 ºC and the reaction was
initiated by addition of 300 µL hydrogen peroxide.
The absorbance was measured immediately in one-
minute intervals after addition of hydrogen peroxide.
One unit of laccase activity was defined as
activity of an enzyme that catalyzes the conversion of
1 mole of ABTS ( ε420= 36,000 M-1 cm-1 ) per minute.
Buffers.
Citrate - Phosphate buffer: 0.3 M citrate/0.4 M
phosphate buffer was prepared as follows:
(a) 0.3 M citric acid and (b) 0.4 M dibasic sodium
phosphate. Known volume of (a) was added to
known volume of (b) until reaching the desired pH.
Succinate buffer: 0.5 M sodium succinate buffer
was prepared as follows:
(a) 0.5 M succinic acid and (b) 0.5 M NaOH. Known
volume of (a) was added to known volume of (b) until
the desired pH was reached.
Acetate buffer: 0.1 M sodium acetate buffer was
prepared as follows:
(a) 0.1 M acetic acid and (b) 0.1 M sodium acetate.
Known volume of (a) was added to known volume of
(b) until reaching the desired pH.
Determination of total proteins: The protein
content of the culture filtrate was estimated according
to the method of Lowry et al. (1951). And bovine
serum albumin (BSA) used as a standard at known
concentrations (20, 40, 80, 100, 150 and 200 µg).
 Agric. Biol. J. N. Am., 2010, 1(4): 591-599

RESULTS AND DISCUSSION sp. and Pterocladia sp., green alga Ulva
(Enteromorpha sp.) collected from Abou-keer,
Isolation of marine fungi from algae and sea
Alexanderia, Egypt, during the four seasons as
grasses samples: Forty-four fungal isolates
described previously by (Atalla et al., 2008). The
belonging to ten fungal genera and twenty-three
marine fungal genera were Acremonium, Alternaria,
species have previously been isolated from six
Aspergillus, Cladosporium, Dendryphiopsis,
different algae samples, brown alga Paduia sp.
Fusarium, Moniliella, Penicillium, Scopulariopsis, and
,Sargassum sp.and Cystoseira sp., red alga Corallina
Verticillium.
Table 1. fungal isolates belonging to ten fungal genera

Marine algae
Fungal isolates Paduia sp. Pterocladi Cystoseira Sargassum Corallina Ulva Total
a sp. sp. sp. sp.
Acremonium charticola 1 0 0 1 0 0 2
Alternaria alternata 0 1 0 0 0 0 1
Aspergillus candidus 1 0 0 0 1 0 2
A. flavus 0 1 0 1 0 1 3
A. niger 0 0 0 1 1 1 3
A. oryzae 0 0 0 1 0 0 1
A. parasiticus 0 1 1 0 1 0 3
A. terreus 1 0 0 0 1 0 2
A. versicolor 0 1 0 1 1 1 4
Cladosporium cladosporioides 0 1 0 0 0 1 2
C. macrocarpum 0 1 0 0 0 0 1
Dendryphiopsis atra 0 0 0 0 0 1 1
Fusarium solani 0 0 0 0 0 1 1
Moniliella suaveolens 0 1 0 0 0 0 1
Penicillium brevicompactum 0 1 1 0 0 0 2
P. camemberti 1 0 1 1 0 1 4
P. chrysogenum 0 1 0 0 0 1 2
P. citrinum 0 0 0 1 0 1 2
P. echinulatum 0 0 0 1 0 0 1
P. expansum 0 0 0 0 1 1 2
P. nalgiovense 0 0 0 0 1 1 2
Scopulariopsis brevicaulis 0 0 1 0 0 0 1
Verticillium lecanii 0 1 0 0 0 0 1
Total 4 10 4 8 7 11 44

Sea grasses collected during the four seasons of
2004 showed, nine fungal isolates belonging to five
fungal genera, Aspergillus, Cladosporium,
Geotrichum, Penicillium, and Verticillium.
Mtui and Nakamura (2008) isolated a basidiomycete
fungus Flavodon flavus from decayed sea grass
leaves of Thallasodendon ciliatum collected about 50
m off the Coast Mjimwema, 20 km South of Dar-EL
Salaam, Tanzania.
Raghukumar et al. (1995) found that the sea grasses
and mangrove plants are the major contributors of
lignocellulose in the highly productive costal marine
environments, and fungi are believed to be important
in lignocellulose degradation in these ecosystems
(Raghukumar, et al., 1994).
Isolation of marine fungi from decayed wood
samples: Out of thirty five fungal isolates isolated
from different decayed wood samples, seven marine
fungal isolates were identified as, Alternaria alternata,
Alternaria solani, Epicoccum purpurascens,
Gonatorrhodiella parasitica, Monascus rubber,
Trematosphaeria mangrovei and Ulocladium
chartarum Table (2).
D’Souza et al. (2006) isolated 40 fungi from decayed
wood pieces of mangrove swamps from Chorao
Island in Goa, India. But a white-rot basidiomycete
Trametes trogii was isolated from decayed acacia
wood (from North West of Tunisia) (Zouari-Mechichi
et al., 2006)
These results agreed with those of (Sarma and Hyde,
2001) who stated that the lignocellulosic substrates in
the marine environment, particularly mangrove wood,
support a diverse mycota.

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Table 2. Fungal isolates associated with sea grasses as an aromatic model compound. Results showed
and decayed wood samples that none of the fungi isolated from algae and sea
Fungal isolates Sea Decayed Total grasses samples exhibited an ability to oxidize
grasses wood guaiacol, no halo of brown colour was formed under
samples
and around the fungal colonies (Negative for guaiacol
Alternaria 0 1 1
alternata
oxidation), indicating the lack of ligninolytic enzymes.
A. solani 0 1 1 Positive results for guaiacol oxidation appeared only
Aspergillus 1 0 1 with the seven fungal isolates obtained from decayed
candidus wood samples, where a halo of intense brown color
A. flavus 1 0 1 was formed under and around the fungal colonies
A. niger 1 0 1 (Positive for guaiacol oxidation), indicating the
A. versicolor 1 0 1 presence of ligninolytic enzymes Fig. (1).
Cladosporium 1 0 1
sphaerospermum These results agreed with D’Souza et al. (2006) who
n s 0 1 1 stated that, out of 40 fungi isolated from decayed
Geotrichum 1 0 1 mangrove wood, 3 isolates showed positive reaction
candidum for laccase activity when grown in the presence of
Gonatorrhodiella 0 1 1 guaiacol.
parasitica Mtui and Masalu (2008) demonstrated the guaiacol
Monascus rubber 0 1 1 oxidation by mycelial cultures of a marine fungal
Penicillium 1 0 1 isolate Laetiporus sulphureus isolated from mangrove
camemberti forests of coastal Tanzania after 7 days of incubation.
P. echinulatum 1 0 1 The ability of L. sulphureus enzymes to degrade the
Trematosphaeria 0 1 1 aromatic model compound indicated that they are the
mangrovei main decomposers of cellulose, hemicellulose and
Verticillium 1 0 1 lignin contained in the mangrove trees, this implies
lecanii that the enzymes can also be used in detoxification of
Ulocladium 0 1 1 aromatic pollutants such as agrochemicals and
chartarum industrial effluents.
Total 9 7 16 Growth and oxidation characteristics: The white
Survey of some marine fungal isolates for rot fungus Pleurotus ostreatus was used as a
Lignin-degrading enzyme activities: All fungal positive control to differentiate between growth and
isolates were screened for the presence of oxidation characteristics of the selected positive
laccase, lignin-peroxidase and manganese- marine fungal isolates: Alternaria alternata, Alternaria
dependent peroxidase activities by using an agar solani, Epicoccum purpurascens, Gonatorrhodiella
plate assay as a qualitative method for the parasitica, Monascus rubber, Trematosphaeria
determination of lignocellulolytic enzyme mangrovei and Ulocladium chartarum, Results
production. showed that, the marine fungal isolate
Pointing (1999) showed that qualitative assays are Trematosphaeria mangrovei measured about 26 mm
powerful tools used in screening fungi for colour zone diameter and 33.5 mm growth colony
lignocellulose degrading enzyme production. Such diameter on the 7th day of cultivation, followed by the
tests give a positive or negative indication of enzyme fungus Epicoccum purpurascens which has 22.5 mm
production. They are particularly useful in screening colour zone diameter and 28 mm growth colony
large numbers of fungal isolates for several classes diameter. Both Alternaria alternata, Alternaria solani
of enzyme, where definitive quantitative data are not similar in the oxidation scale and growth, they have
required. 20, 19 mm in colour zone diameter and 17, 20 mm in
growth colony diameter respectively. The fungal
Lignin modifying enzymes assay (Guaiacol isolates Gonatorrhodiella parasitica and Ulocladium
oxidation): Guaiacol oxidation is one of the most chartarum similar in the oxidation scale 14, 13 mm as
convenient qualitative assay for LMEs production well as in the growth rate 19.5, 19 mm, Monascus
among fungi. Eighty eight of marine fungal isolates rubber has the lowest diameter 10mm and 17 mm
were screened for guaiacol oxidation and radial either colour zone or growth.
growth rate on agar plates containing 4mM guaiacol

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Monascus rubber

Ulocladium chartarum Trematosphaeria mangrovei

Pleurotus ostreatus
Alternaria alternata
(control)

Epicoccum purpurascens

Alternaria solani

Gonatorrhodiella parasitica

Fig. 1: Photo of B&K agar plate showing positive guaiacol oxidation by seven fungal isolates obtained from
decayed wood samples after 7 days of inoculation.

Results presented in Table (3) showed that the
marine fungal isolate Trematosphaeria mangrovei
measured the highest colour zone diameter and
radial growth. Since is a positive correlation between
the radial growth and the oxidation rate. it was the
best selected strain in the qualitative assay.
Enzymes production in liquid medium: Seven
fungi showed positive reaction for LDEs activity when
grown in the presence of guaiacol were grown in
liquid culture medium (Kirk´s medium) for enzyme
activities. Where laccase, lignin-peroxidase,
manganese-dependent peroxidase, protein and final
pH of the culture filtrate were measured.
The results summarized in Table (4) showed that,
when the selected fungal isolates grown in low
nitrogen (LN) medium with half-strength sea water,
initial pH 4.5 for 14 days, only laccase activity was
detectable in the supernatant and the other two
ligninolytic enzyme activities could not be detected. It
was also noticed that the pH values of the culture
filtrates lied in the acidic range.

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Table (3): Qualitative assay for Lignin-degrading enzymes production.

Mycelial growth and oxidation characteristics

Fungal isolates
Colour zone diameter Oxidation scaleb Fungal colony diameter
a c
(mm) (mm)
Pleurotus ostreatus* 32 +++++ 20

Alternaria alternata 20 +++ 17

Alternaria solani 19 +++ 20

Epicoccum purpurascens 22.5 ++++ 28

Gonatorrhodiella parasitica 14 ++ 19.5

Monascus ruber 10 + 17

Trematosphaeria mangrovei 26 ++++ 33.5

Ulocladium chartarum 13 ++ 19

*(Pleurotus ostreatus) a well known lignin-degrading white rot fungus used as a positive control.
a th
Diameter of the oxidized zone in mm (measured on the 7 day of cultivation).
b
Oxidation scale measured on the 7 th day of cultivation on B&K medium containing 4mM guaiacol: + diameter of the oxidized zone 0-
10mm, ++ zone diameter 11-15 mm, +++ zone diameter 16-20 mm, ++++ zone diameter 21-30 mm, +++++ zone diameter up to 31mm.
c th
Diameter of the mycelial colony in mm measured on the 7 day of cultivation (the initial disc 10 mm diameter).

Table (4): Quantitative estimation of Lignin-degrading enzymes produced by the selected marine fungal isolates.
LDE activities.
Protein Specific Final
Lignin content activity
Fungal isolates Laccase Mn dependent
peroxidase (mg/ml) (U/mg) pH
(U/ml) peroxidase (U/ml)
(U/ml)
Alternaria
3.94 ND ND 0.164 24.04 4.5
alternata

Alternaria solani 3.76 ND ND 0.202 18.59 4.1

Epicoccum
10.85 ND ND 0.183 59.30 4.0
purpurascens
Gonatorrhodiella
1.41 ND ND 0.225 6.27 3.9
parasitica

Monascus ruber 1.85 ND ND 0.194 9.52 3.9

Trematosphaeria
14.03 ND ND 0.17 82.51 3.9
mangrovei
Ulocladium
3.05 ND ND 0.143 21.33 4.6
chartarum
* The values are mean of three replicates.
* ND: not detected

597

These results are in consistent with Hou et al. (2004)
findings, who demonstrated that laccase was the only
ligninolytic enzyme activity detected in the
supernatant when the fungus was grown in liquid
culture with or without shaking. Also agreed with
Zouari-Mechichi et al. (2006) who found that the sole
ligninolytic activity detected in liquid cultures using a
glucose-peptone medium was laccase. The hard
wood-colonizing ascomycete Xylaria polymorpha,
lacking peroxidases and produces laccase as sole
ligninolytic oxidoreductase (Liers et al., 2007).
These results lind with the work by Mtui and Masalu
(2008) who demonstrated the ability of Laetiporus
sulphureus to produce MnP and LiP but showed no
laccase (Lac) activity.
Out of seven fungi Trematosphaeria mangrovei
showed a final specific activity of 82.51 U/mg protein
with higher laccase activity 14.03 U/ml. followed by
Epicoccum purpurascens which gave specific activity
of 59.30 U/mg protein with laccase activity of 10.85
U/ml.
Alternaria alternata, Ulocladium chartarum and
Alternaria solani produced levels of laccase activities
as following, 3.94, 3.05 and 3.76 U/ml with specific
activity of 24.04, 21.33 and 18.59U/mg protein.
Lower laccase activities of 1.85 and 1.41U/ml were
observed with the fungal isolates Monascus rubber
and Gonatorrhodiella parasitica.
The amount of laccase produced by Trematosphaeria
mangrovei (14.03 U/ml) was nearly comparable to
the amount produced by the marine basidiomycetes
Flavodon flavus (15 U/ml) (Mtui and Nakamura,
2008). This appreciable amount of laccase can be
used for application in several bioprocesses, such as
biopulping, biobleaching, bioremediation, food
technological uses, and treatment of industrial waste
water (Hublik and Schinner 2000; Robinson et al.,
2001). So that fungi producing laccase are currently
the focus of much attention (Pointing et al., 2000) and
this places a high value on the importance of fungi in
coastal ecology (Bucher et al., 2004).
CONCLUSIONS
It can be concluded that, from a high number of
fungal strains isolated from different algae, sea
grasses and decaying wood samples, the marine
fungal isolate Trematosphaeria mangrovei was
chosen because it exhibited a fast and large
oxidation of guaiacol on agar plates (26 mm), and
Liquid cultivation of the fungus in low nitrogen (LN)
medium with half-strength sea water, initial pH 4.5 for
14 days showed that, only laccase of the three major
ligninolytic enzymes was detected in culture
supernatant. Showed a final specific activity of 82.51
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Agric. Biol. J. N. Am., 2010, 1(4): 591-599
U/mg protein with higher laccase activity 14.03 U/ml.
and it was recommended that the marine fungal
isolate Trematosphaeria mangrovei was the most
promising one for laccase enzyme production.
REFERENCES
Atalla, M. Mabrouk., Zeinab, H. Kheiralla., Eman, R.
Hamed., Amani, A. Youssry and Abeer, A. Abd El Aty
(2008). Isolation, Identification and Antimicrobial
Activities of Some fungi Associated with Marine Algae.
Egypt. Pharm. J. (NRC). 7(1), 85-104.
Boominathan, K. and Reddy, C.A. (1992). Fungal
degradation of lignin: biotechnological applications,
p.763-822. In D. K. Arora, R. P. Elander, and K. G.
Mukerji (ed.), Handbook of applied mycology, vol. 4.
Marcel Dekker, Inc., NewYork, N.Y.
Bucher, V.V.C.; Hyde, K.D.; Pointing, S.B. and Reddy, C.A.
(2004). Production of wood decay enzymes, mass loss
and lignin solubilization in wood by marine
ascomycetes and their anamorphs. Fungal Diversity,
15, 1-14.
D’Souza, D.T.; Tiwari, R.; Sah, A.K. and Raghukumar, C.
(2006). Enhanced production of laccase by a marine
fungus during treatment of colored effluent and
synthetic dyes. Enzyme and Microbial Technology, 38,
504-511.
Höller, U.; König, G.M. and Wright, A.D. (1999). A new
tyrosin kinase inhibitor from a marine isolate of
Ulocladium botrytis and new metabolites from the
marine fungi Asteromyces cruciatus and Varicosporina
ramulosa. Eur. J.Org. Chem., 2949-2955.
Hou, H.; Zhou, J.; Wang, J.; Du, C. and Yan, B. (2004).
Enhancement of laccase production by Pleurotus
ostreatus and its use for the decolorization of
anthraquinone dye. Process Biochemistry, 39, 1415–
1419.
Hublik, G. and Schinner, F. (2000). Characterization and
immobilization of the laccase from Pleurotus ostreatus
and its use for the continuous limination of phenolic
pollutant. Enzyme Microb. Technol., 27, 330-336.
Kiiskinen, L.L.; Rättö M. and Kruus, K. (2004). Screening
for novel laccase-producing microbes. J. Appl.
Microbiol., 97(3), 640-646.
Kohlmeyer, J. and Kohlmeyer, E. (1979). Marine Mycology
- The Higher Fungi, Academic Press, New York, San
Francisco, London.
Kohlmeyer, J. and Kohlmeyer, B.V. (1991). Illustrated key
to the filamentous higher marine fungi. Botanica
Marina, 34, 1-61.
Liers, C.; Ullrich, R.; Pecyna, M.; Schlosser, D. and
Hofrichter, M. (2007). Production, purification and
partial enzymatic and molecular Characterization of a

laccase from the wood-rotting ascomycete Xylaria
polymorpha. Enzyme and Microbial Technology ,
41,785–793
Lowry, O.H.; Rosebrough, N.J.; Farr, A.L. and Randal, R.J.
(1951). Protein measurement with the folin phenol
reagent. J. Biol. Chem., 193, 262.
Mtui, G. and Nakamura, Y. (2004). Lignin-degrading
enzymes from mycelial cultures of basidiomycetes
fungi isolated in Tanzania. J. Chem. Eng. Jpn., 37(1),
113-118.
Mtui, G. and Nakamura, Y. (2007). Characterization of
lignocellulosic enzymes from white-rot fungus
Phlebia crysocreas isolated from a marine habitat.
J. Eng. Appl. Sci., 2(10), 1501-1508.
Mtui, G. and Nakamura, Y. (2008). Lignocellulosic
enzymes from Flavodon flavus, a fungus isolated from
Western Indian Ocean off the
coast of Dar es Salaam, Tanzania. African Journal of
Biotechnology, 7 (17), 3066-3072.
Mtui, G. and Masalu, R. (2008). Extracellular enzymes
from brown-rot fungus Laetiporus sulphureus isolated
from mangrove forests of coastal Tanzania. Academic
Journals, 3 (4), 154-161.
Okino, L.K.; Machado, K.M.G.; Fabric, C. and Bonomi,
V.L.R. (2000). Ligninolytic activity of tropical rainforest
basidiomycetes. World J. of Microbiol. Biotech., 16,
889-893.
Pitt, J.I. and Hocking, A.D. (1985). Fungi and food spoilage.
Academic Press (Pub.) Sydney, New York, London.
Pointing, S.B. (1999). Qualitative methods for the
determination of lignocellulolytic enzyme production by
tropical fungi. Fungal Diversity, 2, 17-33.
Pointing, S.B.; Jones, E.B.G. and Vrijmoed, L.L.P.
(2000).Optimization of laccase production by
Pycnoporus sanguineus in submerged liquid culture.
Mycologia, 92, 139-144.
Raghukumar, C.; Raghukumar, S.; Chinnaraj, A.;
Chandramohan, D.; D’souza, T.M. and Reddy C.A.
(1994). Laccase and other lignocellulose modifying
enzymes of marine fungi isolated from the coast of
India. Bot. Mar., 37, 515-523.
599
Agric. Biol. J. N. Am., 2010, 1(4): 591-599
Raghukumar, S.; Sathe-Pathak, V.; Sharma, S. and
Raghukumar, C. (1995). Thraustochytrid and fungal
component of marine detritus. ΙΙΙ. Field studies on
decoposition of leaves of the mangrove Rhizophora
apiculata. Aquat. Microb. Ecol., 9, 117-125.
Raghukumar, C.; D’souza, T.M.; Thorn, R.G. and Reddy
C.A. (1999). Lignin modifying enzymes of Flavodon
flavus, a basidiomycete isolated from a coastal marine
environment. Appl. Environ. Microbiol., 65(5), 2103-
2111.
Raghukumar, C. (2008). Marine fungal biotechnology: an
ecological perspective. Fungal Diversity, 31, 19-35.
Rigas, F.; Dritsa, V.; Marchant, R.; Papadopoulou, K.;
Avramides, E.J.; and Hatzianestis, I. (2005).
Biodegradation of lindane by Pleurotus ostreatus via
central composite design. Environment International,
31, 191–196.
Robinson, T.; Chandran, B. and Nigam, P. (2001).Studies
on the production of enzymes by white-rot fungi for the
decolourisation of textile dyes. Enzyme
Microb.Technol., 29,575-579.
DrivenbyNature:PlantLitterQualityandDecomposition.Wallin
g-
Rowley, D.C.; Kelly, S.; Kauffman, C.A.; Jensen, P.R. and
Fenical, W. (2003). Halovirs A-E, new antiviral agents
from a marine-derived fungus of the genus
Scytalidium. Bioorganic & Medical Chemistry, 11,
4263-4274.
Sarma, V.V. and Hyde, K.D. (2001). A review on frequently
occurring fungi in mangroves. Fungal Diversity, 8, 1-
34.
Takamiya, M.; Magan, N. and Warner, P.J. (2008). Impact
assessment of bisphenol A on lignin-modifying
enzymes by basidiomycete Trametes versicolor.
Journal of Hazardous Materials, 154, 33–37.
Zouari-Mechichi, H.; Mechichi, T.; Dhouib, A.; Sayadi, S.;
Martinez, A.T. and Martinez, M.J. (2006). Laccase
purification and characterization from Trametes trogii
isolated in Tunisia: decolorization of textile dyes by the
purified enzyme. Enzyme and Microbial Technology
,39, 141–148.