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
 Agric. Biol. J. N. Am., 2010, 1(4): 591-599

MATERIALS AND METHODS Malt extract agar (MEA): contained, malt extract 30 g,
peptone 3 g, agar 12 g.
Chemicals: 2-Methoxyphenol (Guaiacol) and 3,4-
Dimethoxybenzyl alcohol (Veratryl alcohol) were Potato carrot agar (KM): contained, cooked and
purchased from Fluka Co. 2,2´-Azino-bis(3- sliced potatoes 20 g, cooked and sliced carrots 20 g,
ethylbenzothiazoline-6-sulphonic acid) diammonium agar 20g. Glucose peptone yeast extract agar (GPY):
salt (ABTS) was obtained from MP. Bio (LCN) Co, contained, glucose. H2O 1.0 g, peptone 0.5 g, yeast
USA. extract 0.1 g, agar 15 g.
Microorganisms. Boyd &Kohlmeyer (B&K) agar: contained, glucose 10
g, peptone 2 g, yeast extract 1 g, agar 18 g in 1l of
Source of algae, sea grasses and decayed wood
50% sea water (Kohlmeyer and kohlmeyer, 1979 and
samples:
D'Souza et al., 2006).
All algae and sea grasses samples were collected
Screening media: Two different types of media were
from Abou Keer, Alexandria during the 4 seasons
used for qualitative and quantitative assay of lignin-
(summer, autumn, winter and spring) at a depth of 3-
degrading enzymes production.
5m as described previously by (Atalla et al., 2008) ,
decayed wood samples collected from the same Qualitative media (D'Souza et al., 2006): Guaiacol-
place in 2008. The collected samples were brought to supplemented agar: contained, glucose 10 g,
the laboratory in clean plastic bags and stored in ice peptone 2 g, yeast extract 1 g, agar 18 g and 4mM
box. guaiacol in1l of 50% sterile sea water.
Isolation of marine fungi: Samples were cut in Quantitative media (Rigas et al., 2005).
small pieces, washed with sterile sea water, blotted
a- Kirk's medium: composed of g/l 50% sterile sea
between two folds of sterilized filter paper and
water; KH2PO4 0.20, CaCl2 0.01, MgSO4.7H2O 0.05,
transferred to petri dishes contained suitable medium
ammonium tartrate 0.22, 2.2-dimethylsuccinic acid
(Höller, 1999; Rowley et al., 2003).
2.90, glucose 5 , thiamine 0.1, Tween 80 0.10% v/v,
Different kinds of media were used in isolation veratryl alcohol 1.5 mM, and a mixture of trace
[biomalt agar (BIO), malt extract agar (ME), potato elements composed of (mg/l): MnSO4 33, Fe2 (SO4)3
carrot agar (KM), glucose peptone yeast extract agar 50, ZnSO4.7H2O 43, CuSO4.7H2O 80, H2MoO4 50.
(GPY) and Boyd &Kohlmeyer (B&K) agar]. Plates
b- Malt Extract Broth (MEB): contained g/l 50% sterile
were incubated at 25 ºC for 4 days, exposed to daily
sea water; malt extract 17 and mycological peptone
examination to observe the developing growth.
3.
Fungal isolates were picked up under the dissecting
microscope, transferred to biomalt agar (BIO) slants. Qualitative assay for lignin-degrading enzymes
Purification was carried out using single spore and production: The fungal isolates were screened for
hyphal tip techniques individually and transferred to LDEs production by growing them on plates of B&K
suitable medium. Stock cultures were maintained on medium containing 4mM guaiacol (D'Souza et al.,
biomalt agar (BIO) slants and kept in the refrigerator 2006). Petri dishes (15 cm in diameter) each
at (5-6 ºC) for later use. containing 30 ml of medium were used.
Identification of the isolated fungi: Isolated fungi Preparation of fungal isolates inoculum for guaiacol
were identified in the National Research Centre, oxidation test was performed by removing disks 10
Chemistry of Natural and Microbial Products Dept. mm. in diameter from the edge of expanding colonies
[(Microbial Culture Collection Unit (MCCU)] according 7 days old cultures grown on B&K agar medium.
to (Pitt and Hocking, 1985 and Kohlmeyer and Then, a disk was placed on the surface of guaiacol-
Kohlmeyer, 1991). supplemented agar plates. The inoculated plates
were incubated at 25 ºC in dark for 7 days. The
Media: Different types of media were used in this
production of intense brown color under and around
study.
the fungal colony was considered as a positive
Isolation media: Four media were used for isolation, reaction resulting from guaiacol oxidation (Okino et
each contained the following components in 1l of al., 2000). The diameter of coloured zone, growth
80% sterile sea water (Höller, 1999). Biomalt agar rate were measured in mm.
(BIO): contained, biomalt 20 g, agar15 g.

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 Agric. Biol. J. N. Am., 2010, 1(4): 591-599

Quantitative estimation of Lignin-degrading guaiacol (4 mM), 600 µL manganese sulphate (1

enzymes: Kirk's medium was used as liquid mM), 300 µL culture supernatant and 1200 µL
fermentation media for quantitative estimation of distilled water. The mixture was incubated for 2
enzyme activities from the selected strains (Rigas et min at 30 ºC and the reaction was initiated by
al., 2005). addition of 300 µL hydrogen peroxide (1mM). The
absorbance was measured in 1 min intervals after
Inoculum preparation: The inoculum was prepared
addition of hydrogen peroxide. One unit of MnP
by transferring two agar plugs (1cm diameter) from 7
activity was defined as activity of an enzyme that
days old cultures grown on (MEA) into 250-ml flasks
catalyzes the conversion of 1µ mole of guaiacol per
containing 50 ml Malt Extract Broth (MEB) media and
minute.
incubated at 25 ºC under stationary conditions for 6
days. Laccase (Lac): Laccase (EC 1.10.3.2) activity was
measured based on the oxidation of the substrate
After 6 days of growth, the cultures were
2,2’-azino–bis (3-ethylbenzothiazoline)-6-sulphonic
homogenized and further used as inoculum for the
acid (ABTS). The rate of ABTS oxidation was
estimation of ligninolytic activities.
determined spectrophotometrically at 420 nm.
Liquid medium used for enzyme activities: Ten-
The reaction mixture contained 600 µL sodium
milliliter aliquots (trace elements) were added to 90
acetate buffer (0.1 M, pH 5.0 at 27 ºC), 300 µL
ml Kirk's media (in 250-ml Erlenmeyer flasks). Each
ABTS (5 mM), 300 µL mycelial liquid fraction and
flask was inoculated with 1 ml from inoculum and
1400 µL distilled water. The mixture was then
incubated at 25 ºC in dark under stationary conditions
incubated for 2 min at 30 ºC and the reaction was
for 14 days.
initiated by addition of 300 µL hydrogen peroxide.
Assay for enzyme activities: At the end of each The absorbance was measured immediately in one-
growth period, inoculated flasks were collected and minute intervals after addition of hydrogen peroxide.
centrifugated. The filtrate was tested for lignin- One unit of laccase activity was defined as
degrading enzymes activity as follows and enzyme activity of an enzyme that catalyzes the conversion of
activity was expressed in international units (Mtui and 1 mole of ABTS ( ε420= 36,000 M-1 cm-1 ) per minute.
Masalu, 2008).
Buffers.
Lignin peroxidase (LiP).
Citrate - Phosphate buffer: 0.3 M citrate/0.4 M
Lignin peroxidase (LiP) (EC 1.11.1.14) activity was phosphate buffer was prepared as follows:
determined spectrophotometrically at 310 nm through
(a) 0.3 M citric acid and (b) 0.4 M dibasic sodium
the oxidation of veratryl alcohol to veratryl aldehyde
phosphate. Known volume of (a) was added to
(molar absorptivity, ε310 = 9300 M-1 cm-1).
known volume of (b) until reaching the desired pH.
The reaction mixture contained 300 µL veratryl
Succinate buffer: 0.5 M sodium succinate buffer
alcohol (8 mM), 600 µl 0.3M citrate/0.4M phosphate
was prepared as follows:
buffer (pH 4.5), 60 µL culture supernatant, and 1890
µL distilled water. The mixture was incubated for 2 (a) 0.5 M succinic acid and (b) 0.5 M NaOH. Known
min at 30 ºC and the reaction was initiated by volume of (a) was added to known volume of (b) until
addition of 150 µL H2O2 (5 mM). The absorbance the desired pH was reached.
was immediately measured in one-minute intervals
Acetate buffer: 0.1 M sodium acetate buffer was
after addition of H2O2. One unit (U) of LiP activity
prepared as follows:
was defined as activity of an enzyme that
catalyzes the conversion of 1 µ mole of veratryl (a) 0.1 M acetic acid and (b) 0.1 M sodium acetate.
alcohol per minute (Takamiya et al., 2008). Known volume of (a) was added to known volume of
(b) until reaching the desired pH.
Manganese peroxidase (MnP): Activity of
manganese peroxidase (MnP) (EC 1.11.1.13) was Determination of total proteins: The protein
measured by using guaiacol as a substrate. The content of the culture filtrate was estimated according
increase in absorbance at 465 nm due to oxidation of to the method of Lowry et al. (1951). And bovine
guaiacol (ε465= 12,100 M-1 cm-1) was measured. serum albumin (BSA) used as a standard at known
concentrations (20, 40, 80, 100, 150 and 200 µg).
The reaction mixture contained 300 µL sodium
succinate buffer (0.5 M, pH 4.5 at 27 ºC), 300 µL

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

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

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These results are in consistent with Hou et al. (2004) U/mg protein with higher laccase activity 14.03 U/ml.
findings, who demonstrated that laccase was the only and it was recommended that the marine fungal
ligninolytic enzyme activity detected in the isolate Trematosphaeria mangrovei was the most
supernatant when the fungus was grown in liquid promising one for laccase enzyme production.
culture with or without shaking. Also agreed with
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