screening of some marine-derived fungal isolates for lignin degrading enzymes ldes production

8/3/2019 Screening of Some Marine-Derived Fungal Isolates for Lignin Degrading Enzymes LDEs Production

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AGRICULTURE AND BIOLOGY JOURNAL OF NORTH AMERICAISSN Print: 2151-7517, ISSN Online: 2151-7525

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

Screening of some marine-derived fungal isolates for lignin degradingenzymes (LDEs) production

Atalla, M. Mabrouk*; Zeinab, H. Kheiralla**; Eman, R. Hamed*; Amani, A. Youssry** andAbeer, 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: [email protected]

ABSTRACT

A study was carried out to establish the lignin-degrading enzymes (LDEs) activity of a largenumber of diverse marine ascomycetes. Eighty eight fungal isolates obtained from different algae,sea grasses and decaying wood samples collected from Abou-keer, Alexanderia, Egypt, werescreened for the presence of laccase (Lac), lignin-peroxidase (LiP) and manganese-dependentperoxidase (MnP) activities. Results obtained from both qualitative and quantitative assayshowed that the marine fungal isolate Trematosphaeria mangrovei measured the highest zonediameter and colony diameter in agar plate screening test with guaiacol and showed a finalspecific activity of 82.51 U/mg protein with higher laccase activity 14.03 U/ml when grown in lownitrogen (LN) medium with half-strength sea water, initial pH 4.5 for 14 days. The other twoligninolytic activities could not be detected. It was recommended that the marine fungal isolateTrematosphaeria mangrovei was the most promising one for laccase enzyme production.

Keywords: Marine-derived fungi, Lignin degrading enzymes, ligninases, laccase, bioremediation.

INTRODUCTION

Lignin, the second most abundant renewable organicpolymer on earth, is a major component of wood.Because of the importance of wood and other lignocellulosics as a renewable resource for theproduction of paper products, feeds, chemicals, and

fuels, there has been an increasing researchemphasis on the fungal degradation of lignin(Boominathan and Reddy, 1992).

Fungi play an important role in degradation andmineralization of lignocellulosic substrates in themarine environment. These lignicolous fungicomprising Ascomycetes, Basidiomycetes andDeuteromycetes have distinct spore structures thatset them apart from their terrestrial counterparts.Such fungi have been defined as obligate marinefungi “which grow and sporulate exclusively under marine conditions” (Kohlmeyer and Kohlmeyer 1979).In order to accommodate the possibility that

terrestrial species might also be active in the sea,Kohlmeyer and Kohlmeyer offered a definition for “facultative marine fungi” as those originating fromfreshwater or terrestrial environment that are capableof growth and sporulation in the sea.

Marine filamentous fungi, obligate and facultative areknown to occur in association with algae, seagrassesand mangroves, fungi cohabiting with marine

invertebrates, especially corals and sponges, fungi indetritus of marine macrophytes and in marineextreme environments (Kohlmeyer and Kohlmeyer,1979 and Raghukumar, 2008).

The ability of fungi to degrade lignocellulose is dueto their possession of extracellular enzymes,

mainly lignin peroxidase (LiP), manganeseperoxidase (MnP) and laccase (Lac). Theseenzymes have been shown to degrade not onlylignocellulose, but also recalcitrant environmentalpollutants such as crude oil wastes, textile effluents,organochloride agrochemicals and pulp effluentswhich are a cause of serious environmental pollution(Mtui and Nakamura, 2004; Kiiskinen et al ., 2004).

Worldwide, there is little published information onthe lignin-degrading ability of the obligate andfacultative marine fungi (Mtui and Nakamura,2007). Moreover, the presences of MnPs, LiPs andLacs in facultative and marine fungi have not been

investigated except for few reports (Raghukumar et al., 1999).

Therefore, the major object of this study was toinvestigate the production of lignin-degradingenzymes by some local marine fungal isolates.



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MATERIALS AND METHODS

Chemicals: 2-Methoxyphenol (Guaiacol) and 3,4-Dimethoxybenzyl alcohol (Veratryl alcohol) werepurchased 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 woodsamples:

All algae and sea grasses samples were collectedfrom 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 sameplace in 2008. The collected samples were brought to

the laboratory in clean plastic bags and stored in icebox.

Isolation of marine fungi: Samples were cut insmall pieces, washed with sterile sea water, blottedbetween two folds of sterilized filter paper andtransferred 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), potatocarrot agar (KM), glucose peptone yeast extract agar (GPY) and Boyd &Kohlmeyer (B&K) agar]. Plateswere incubated at 25 ºC for 4 days, exposed to dailyexamination to observe the developing growth.

Fungal isolates were picked up under the dissectingmicroscope, transferred to biomalt agar (BIO) slants.Purification was carried out using single spore andhyphal tip techniques individually and transferred tosuitable medium. Stock cultures were maintained onbiomalt agar (BIO) slants and kept in the refrigerator at (5-6 ºC) for later use.

Identification of the isolated fungi: Isolated fungiwere identified in the National Research Centre,Chemistry of Natural and Microbial Products Dept.[(Microbial Culture Collection Unit (MCCU)] accordingto (Pitt and Hocking, 1985 and Kohlmeyer andKohlmeyer, 1991).

Media: Different types of media were used in thisstudy.

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.

Malt extract agar (MEA): contained, malt extract 30 g,peptone 3 g, agar 12 g.

Potato carrot agar (KM): contained, cooked andsliced 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, yeastextract 0.1 g, agar 15 g.

Boyd &Kohlmeyer (B&K) agar : contained, glucose 10g, peptone 2 g, yeast extract 1 g, agar 18 g in 1l of 50% sea water (Kohlmeyer and kohlmeyer, 1979 andD'Souza et al., 2006).

Screening media: Two different types of media wereused 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 4mMguaiacol in1l of 50% sterile sea water.

Quantitative media (Rigas et al ., 2005).

a- Kirk's medium: composed of g/l 50% sterile seawater; KH2PO4 0.20, CaCl2 0.01, MgSO4.7H2O 0.05,ammonium tartrate 0.22, 2.2-dimethylsuccinic acid2.90, glucose 5 , thiamine 0.1, Tween 80 0.10% v/v,veratryl alcohol 1.5 mM, and a mixture of traceelements 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% sterilesea water; malt extract 17 and mycological peptone

3.Qualitative assay for lignin-degrading enzymesproduction: The fungal isolates were screened for LDEs production by growing them on plates of B&Kmedium containing 4mM guaiacol (D'Souza et al.,2006). Petri dishes (15 cm in diameter) eachcontaining 30 ml of medium were used.

Preparation of fungal isolates inoculum for guaiacoloxidation test was performed by removing disks 10mm. in diameter from the edge of expanding colonies7 days old cultures grown on B&K agar medium.Then, a disk was placed on the surface of guaiacol-

supplemented agar plates. The inoculated plateswere incubated at 25 ºC in dark for 7 days. Theproduction of intense brown color under and aroundthe fungal colony was considered as a positivereaction resulting from guaiacol oxidation (Okino et al., 2000). The diameter of coloured zone, growthrate were measured in mm.



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Quantitative estimation of Lignin-degradingenzymes: Kirk's medium was used as liquidfermentation media for quantitative estimation of enzyme activities from the selected strains (Rigas et al ., 2005).

Inoculum preparation: The inoculum was preparedby transferring two agar plugs (1cm diameter) from 7days old cultures grown on (MEA) into 250-ml flaskscontaining 50 ml Malt Extract Broth (MEB) media andincubated at 25 ºC under stationary conditions for 6days.

After 6 days of growth, the cultures werehomogenized and further used as inoculum for theestimation of ligninolytic activities.

Liquid medium used for enzyme activities: Ten-milliliter aliquots (trace elements) were added to 90ml Kirk's media (in 250-ml Erlenmeyer flasks). Each

flask was inoculated with 1 ml from inoculum andincubated at 25 ºC in dark under stationary conditionsfor 14 days.

Assay for enzyme activities: At the end of eachgrowth period, inoculated flasks were collected andcentrifugated. The filtrate was tested for lignin-degrading enzymes activity as follows and enzymeactivity was expressed in international units (Mtui andMasalu, 2008).

Lignin peroxidase (LiP).

Lignin peroxidase (LiP) (EC 1.11.1.14) activity wasdetermined spectrophotometrically at 310 nm through

the oxidation of veratryl alcohol to veratryl aldehyde(molar absorptivity, ε310 = 9300 M

-1cm

-1).

The reaction mixture contained 300 µL veratrylalcohol (8 mM), 600 µl 0.3M citrate/0.4M phosphatebuffer (pH 4.5), 60 µL culture supernatant, and 1890µL distilled water. The mixture was incubated for 2min at 30 ºC and the reaction was initiated byaddition of 150 µL H2O2 (5 mM). The absorbancewas immediately measured in one-minute intervalsafter addition of H2O2. One unit (U) of LiP activitywas defined as activity of an enzyme thatcatalyzes the conversion of 1 µ mole of veratrylalcohol per minute (Takamiya

et al.,2008).

Manganese peroxidase (MnP): Activity of manganese peroxidase (MnP) (EC 1.11.1.13) wasmeasured by using guaiacol as a substrate. Theincrease in absorbance at 465 nm due to oxidation of guaiacol (ε465= 12,100 M

-1cm

-1) was measured.

The reaction mixture contained 300 µL sodiumsuccinate buffer (0.5 M, pH 4.5 at 27 ºC), 300 µL

guaiacol (4 mM), 600 µL manganese sulphate (1mM), 300 µL culture supernatant and 1200 µLdistilled water. The mixture was incubated for 2min at 30 ºC and the reaction was initiated byaddition of 300 µL hydrogen peroxide (1mM). The

absorbance was measured in 1 min intervals after addition of hydrogen peroxide. One unit of MnPactivity was defined as activity of an enzyme thatcatalyzes the conversion of 1µ mole of guaiacol per minute.

Laccase (Lac): Laccase (EC 1.10.3.2) activity wasmeasured based on the oxidation of the substrate2,2’-azino–bis (3-ethylbenzothiazoline)-6-sulphonicacid (ABTS). The rate of ABTS oxidation wasdetermined spectrophotometrically at 420 nm.

The reaction mixture contained 600 µL sodiumacetate buffer (0.1 M, pH 5.0 at 27 ºC), 300 µLABTS (5 mM), 300 µL mycelial liquid fraction and1400 µL distilled water. The mixture was thenincubated for 2 min at 30 ºC and the reaction wasinitiated 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 asactivity of an enzyme that catalyzes the conversion of 1 mole of ABTS ( ε420= 36,000 M

-1cm

-1) per minute.

Buffers.

Citrate - Phosphate buffer: 0.3 M citrate/0.4 Mphosphate buffer was prepared as follows:

(a) 0.3 M citric acid and (b) 0.4 M dibasic sodiumphosphate. Known volume of (a) was added toknown 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. Knownvolume of (a) was added to known volume of (b) untilthe desired pH was reached.

Acetate buffer: 0.1 M sodium acetate buffer wasprepared 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 proteincontent of the culture filtrate was estimated accordingto the method of Lowry et al. (1951). And bovineserum albumin (BSA) used as a standard at knownconcentrations (20, 40, 80, 100, 150 and 200 µg).



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RESULTS AND DISCUSSION

Isolation of marine fungi from algae and seagrasses samples: Forty-four fungal isolatesbelonging to ten fungal genera and twenty-threespecies have previously been isolated from six

different algae samples, brown alga Paduia sp .,Sargassum sp.and Cystoseira sp ., red alga Corallina

sp. and Pterocladia sp., green alga Ulva (Enteromorpha sp .) collected from Abou-keer,Alexanderia, Egypt, during the four seasons asdescribed previously by (Atalla et al., 2008). Themarine fungal genera were Acremonium, Alternaria,

Aspergillus, Cladosporium, Dendryphiopsis,Fusarium, Moniliella, Penicillium, Scopulariopsis, andVerticillium .

Table 1. fungal isolates belonging to ten fungal genera

Sea grasses collected during the four seasons of 2004 showed, nine fungal isolates belonging to fivefungal genera, Aspergillus, Cladosporium,Geotrichum, Penicillium, and Verticillium .Mtui and Nakamura (2008) isolated a basidiomycetefungus Flavodon flavus from decayed sea grassleaves of Thallasodendon ciliatum collected about 50m off the Coast Mjimwema, 20 km South of Dar-ELSalaam, Tanzania. Raghukumar et al. (1995) found that the sea grassesand mangrove plants are the major contributors of lignocellulose in the highly productive costal marineenvironments, and fungi are believed to be importantin 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 marinefungal 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 decayedwood pieces of mangrove swamps from ChoraoIsland in Goa, India. But a white-rot basidiomycete

Trametes trogii was isolated from decayed acaciawood (from North West of Tunisia) (Zouari-Mechichiet al ., 2006)These results agreed with those of (Sarma and Hyde,2001) who stated that the lignocellulosic substrates inthe marine environment, particularly mangrove wood,support a diverse mycota.

Marine algaeFungal isolates Paduia sp. Pterocladi

a sp.Cystoseira

sp. Sargassum

sp.Corallina

sp.Ulva Total

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 3A. 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. macrocarpu m 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. chrysogenu m 0 1 0 0 0 1 2

P. citrinum 0 0 0 1 0 1 2

P. echinulatu m 0 0 0 1 0 0 1

P. expansum 0 0 0 0 1 1 2

P. nalgiovense 0 0 0 0 1 1 2Scopulariopsis 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



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Table 2. Fungal isolates associated with sea grassesand decayed wood samples

Fungal isolates Seagrasses

Decayedwood

samples

Total

Alternaria

alternata

0 1 1

A. solani 0 1 1

Aspergillus candidus

1 0 1

A. flavus 1 0 1

A. niger 1 0 1

A. versicolor 1 0 1

Cladosporium sphaerospermu m

1 0 1

s n 0 1 1

Geotrichum candidum

1 0 1

Gonatorrhodiella

parasitica

0 1 1

Monascus rubber 0 1 1

Penicillium camemberti

1 0 1

P. echinulatu m 1 0 1

Trematosphaeria mangrovei

0 1 1

Verticillium lecanii

1 0 1

Ulocladium chartarum

0 1 1

Total 9 7 16

Survey of some marine fungal isolates for

Lignin-degrading enzyme activities: All fungalisolates were screened for the presence of laccase, lignin-peroxidase and manganese-dependent peroxidase activities by using an agar plate assay as a qualitative method for thedetermination of lignocellulolytic enzymeproduction.

Pointing (1999) showed that qualitative assays arepowerful tools used in screening fungi for lignocellulose degrading enzyme production. Suchtests give a positive or negative indication of enzymeproduction. They are particularly useful in screeninglarge numbers of fungal isolates for several classes

of enzyme, where definitive quantitative data are notrequired.

Lignin modifying enzymes assay (Guaiacoloxidation): Guaiacol oxidation is one of the mostconvenient qualitative assay for LMEs productionamong fungi. Eighty eight of marine fungal isolateswere screened for guaiacol oxidation and radialgrowth rate on agar plates containing 4mM guaiacol

as an aromatic model compound. Results showedthat none of the fungi isolated from algae and seagrasses samples exhibited an ability to oxidizeguaiacol, no halo of brown colour was formed under and around the fungal colonies (Negative for guaiacol

oxidation), indicating the lack of ligninolytic enzymes.Positive results for guaiacol oxidation appeared onlywith the seven fungal isolates obtained from decayedwood samples, where a halo of intense brown color was formed under and around the fungal colonies(Positive for guaiacol oxidation), indicating thepresence of ligninolytic enzymes Fig. (1).

These results agreed with D’Souza et al. (2006) whostated that, out of 40 fungi isolated from decayedmangrove wood, 3 isolates showed positive reactionfor laccase activity when grown in the presence of guaiacol.

Mtui and Masalu (2008) demonstrated the guaiacoloxidation by mycelial cultures of a marine fungalisolate Laetiporus sulphureus isolated from mangroveforests of coastal Tanzania after 7 days of incubation.The ability of L. sulphureus enzymes to degrade thearomatic model compound indicated that they are themain decomposers of cellulose, hemicellulose andlignin contained in the mangrove trees, this impliesthat the enzymes can also be used in detoxification of aromatic pollutants such as agrochemicals andindustrial effluents.

Growth and oxidation characteristics: The whiterot fungus Pleurotus ostreatus was used as a

positive control to differentiate between growth andoxidation characteristics of the selected positivemarine fungal isolates: Alternaria alternata, Alternaria solani, Epicoccum purpurascens, Gonatorrhodiella parasitica , Monascus rubber , Trematosphaeria mangrovei and Ulocladium chartarum , Results showed that, the marine fungal isolateTrematosphaeria mangrovei measured about 26 mmcolour zone diameter and 33.5 mm growth colonydiameter on the 7

thday of cultivation, followed by the

fungus Epicoccum purpurascens which has 22.5 mmcolour zone diameter and 28 mm growth colonydiameter. Both Alternaria alternata, Alternaria solani

similar in the oxidation scale and growth, they have20, 19 mm in colour zone diameter and 17, 20 mm ingrowth colony diameter respectively. The fungalisolates Gonatorrhodiella parasitica and Ulocladium chartarum similar in the oxidation scale 14, 13 mm aswell as in the growth rate 19.5, 19 mm, Monascus rubber has the lowest diameter 10mm and 17 mmeither colour zone or growth.



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

Results presented in Table (3) showed that themarine fungal isolate Trematosphaeria mangrovei measured the highest colour zone diameter andradial growth. Since is a positive correlation betweenthe radial growth and the oxidation rate. it was thebest selected strain in the qualitative assay.

Enzymes production in liquid medium: Sevenfungi showed positive reaction for LDEs activity whengrown in the presence of guaiacol were grown inliquid culture medium (Kirk´s medium) for enzyme

activities. Where laccase, lignin-peroxidase,manganese-dependent peroxidase, protein and finalpH of the culture filtrate were measured.The results summarized in Table (4) showed that,when the selected fungal isolates grown in lownitrogen (LN) medium with half-strength sea water,initial pH 4.5 for 14 days, only laccase activity wasdetectable in the supernatant and the other twoligninolytic enzyme activities could not be detected. Itwas also noticed that the pH values of the culturefiltrates lied in the acidic range.

leurotus ostreatus

(control)

Epicoccum purpurascens

Monascus rubber


Alternaria alternata


Gonatorrhodiella parasitica

Alternaria solani



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

Mycelial growth and oxidation characteristicsFungal isolates

Colour zone diameter(mm)a Oxidation scale

b

Fungal colony diameter(mm)c

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. aDiameter of the oxidized zone in mm (measured on the 7

thday of cultivation).

bOxidation 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

Diameter of the mycelial colony in mm measured on the 7th

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.

Fungal isolates Laccase(U/ml)

Ligninperoxidase

(U/ml)

Mn dependentperoxidase (U/ml)

Proteincontent(mg/ml)

Specificactivity(U/mg)

Final

pH

Alternaria alternata

3.94 ND ND 0.164 24.04 4.5

Alternaria solani 3.76 ND ND 0.202 18.59 4.1

Epicoccum purpurascens

10.85 ND ND 0.183 59.30 4.0

Gonatorrhodiella parasitica

1.41 ND ND 0.225 6.27 3.9

Monascus ruber 1.85 ND ND 0.194 9.52 3.9


14.03 ND ND 0.17 82.51 3.9


3.05 ND ND 0.143 21.33 4.6

* The values are mean of three replicates.* ND: not detected



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These results are in consistent with Hou et al. (2004)findings, who demonstrated that laccase was the onlyligninolytic enzyme activity detected in thesupernatant when the fungus was grown in liquidculture with or without shaking. Also agreed with

Zouari-Mechichi et al. (2006) who found that the soleligninolytic activity detected in liquid cultures using aglucose-peptone medium was laccase. The hardwood-colonizing ascomycete Xylaria polymorpha ,lacking peroxidases and produces laccase as soleligninolytic 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 nolaccase (Lac) activity.Out of seven fungi Trematosphaeria mangrovei showed a final specific activity of 82.51 U/mg proteinwith higher laccase activity 14.03 U/ml. followed byEpicoccum purpurascens which gave specific activityof 59.30 U/mg protein with laccase activity of 10.85U/ml.Alternaria alternata, Ulocladium chartarum andAlternaria solani produced levels of laccase activitiesas following, 3.94, 3.05 and 3.76 U/ml with specificactivity of 24.04, 21.33 and 18.59U/mg protein.Lower laccase activities of 1.85 and 1.41U/ml wereobserved with the fungal isolates Monascus rubber and Gonatorrhodiella parasitica .The amount of laccase produced by Trematosphaeria mangrove i (14.03 U/ml) was nearly comparable tothe amount produced by the marine basidiomycetesFlavodon flavus (15 U/ml) (Mtui and Nakamura,

2008). This appreciable amount of laccase can beused for application in several bioprocesses, such asbiopulping, biobleaching, bioremediation, foodtechnological uses, and treatment of industrial wastewater (Hublik and Schinner 2000; Robinson et al .,2001). So that fungi producing laccase are currentlythe focus of much attention (Pointing et al ., 2000) andthis places a high value on the importance of fungi incoastal ecology (Bucher et al ., 2004).CONCLUSIONSIt can be concluded that, from a high number of fungal strains isolated from different algae, seagrasses and decaying wood samples, the marine

fungal isolate Trematosphaeria mangrovei waschosen because it exhibited a fast and largeoxidation of guaiacol on agar plates (26 mm), andLiquid 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 culturesupernatant. Showed a final specific activity of 82.51

U/mg protein with higher laccase activity 14.03 U/ml.and it was recommended that the marine fungalisolate Trematosphaeria mangrovei was the mostpromising one for laccase enzyme production.

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