The novel strain Fusarium proliferatum LE1 (RCAM02409) produces α- L -fucosidase and arylsulfatase during the growth on fucoidan

Research Paper

The novel strain Fusarium proliferatum LE1 (RCAM02409)produces a-L-fucosidase and arylsulfatase during thegrowth on fucoidan

Svetlana V. Shvetsova1,2, Elena V. Zhurishkina1, Kirill S. Bobrov1, Natalia L. Ronzhina1,Irina M. Lapina1, Dina R. Ivanen1, Tatiana Yu. Gagkaeva3 and Anna A. Kulminskaya1,2

1 National Research Center «Kurchatov Institute», B.P. Konstantinov Petersburg Nuclear Physics Institute,Gatchina, Russia

2 St. Petersburg State Polytechnical University, St. Petersburg, Russia3 All-Russian Institute of Plant Protection, Russian Academy of Agricultural Sciences, St. Petersburg, Pushkin,Russia

Enzymes capable of modifying the sulfated polymeric molecule of fucoidan are mainly producedby different groups of marine organisms: invertebrates, bacteria, and also some fungi. We havediscovered and identified a new strain of filamentous fungus Fusarium proliferatum LE1 (depositionnumber in Russian Collection of Agricultural Microorganisms is RCAM02409), which is a potentialproducer of fucoidan-degrading enzymes. The strain LE1 (RCAM02409) was identified on the basisof morphological characteristics and analysis of ITS sequences of ribosomal DNA. Duringsubmerged cultivation of F. proliferatum LE1 in the nutrient medium containing natural fucoidansources (the mixture of brown algae Laminaria digitata and Fucus vesiculosus), enzymic activities ofa-L-fucosidase and arylsulfatase were inducible. These enzymes hydrolyzed model substrates,para-nitrophenyl a-L-fucopyranoside and para-nitrophenyl sulfate, respectively. However, thea-L-fucosidase is appeared to be a secreted enzymewhile the arylsulfatase was an intracellular one.No detectable fucoidanase activity was found during F. proliferatum LE1 growth in submergedculture or in a static one. Comparative screening for fucoidanase/arylsulfatase/a-L-fucosidaseactivities among several related Fusarium strains showed a uniqueness of F. proliferatum LE1 toproduce arylsulfatase and a-L-fucosidase enzymes. Apart them, the strain was shown to produceother glycoside hydrolyses.

Abbreviations: PSA – potato sucrose agar; SNA – synthetic nutrient agar

Keywords: Fucoidan / a-L-Fucosidase / Arylsulfatase / Fusarium proliferatum

Received: April 18, 2014; accepted: September 7, 2014

DOI 10.1002/jobm.201400309

Introduction

Fucoidans are heterogeneous sulfated polysaccharidesabundant in cell walls of brown algae and someinvertebrates. For the last decades, an increasing numberof reports have been describing a widest range of their

biological activities: antiviral [1, 2], antibacterial [3],anticoagulant [4–6], immunostimulatory [7, 8], antiflam-matory [5, 9], and anticancer [10–13]. Such outstandingrange of fucoidan bioactivity stimulates many investi-gators to seek for a medical use of these sulfatedpolysaccharides and to study the relationships betweentheir structure and functionality. However, the detailedanalysis of fucoidans’ chemical structure and their wideuse are limited by highmolecular weight and complexityof the molecules. Moreover, the type of linkage betweenfucose residues in polysaccharidic chain, type of branch-ing, localization of sulfate groups, composition and

Correspondence: Dr. Anna A. Kulminskaya, Laboratory of Enzymology,Petersburg Nuclear Physics Institute, Gatchina, Orlova Rosha 188300,RussiaE-mail: [email protected]: þ7 81371 32014Fax: þ7 81371 32303

Environment � Health � Techniques

Alfa-L-fucosidase and arylsulfatase from the novel strain Fusarium proliferatum 1

� 2014 WILEY-VCH Verlag GmbH & Co.KGaA,Weinheim www.jbm-journal.com J. Basic Microbiol. 2014, 54, 1–9

content of another monosaccharides in different fucoi-dans vary significantly depending on the type of algaeand the time of harvesting [10]. In addition, there areseveral reports that describe different biological activitiesfor native and fragmented fucoidan [13–15]. All theseissues require methods enabling cleavage of a bigmolecule of fucoidan to short fragments without lossof labile groups. Among several hydrolytic methodsyielding sulfated polyfucosaccharides with lower molec-ular weight or short fucooligosaccharides, enzymaticmethods are considered to be the mildest ones sincechemical and physical fucoidan-hydrolyzing techniquescause the loss of sulfate groups, which are determinativeto their bioactivity.

By analogy with cellulolytic complex, one may assumethat the complex of fucoidan-hydrolyzing enzymes foundin various organisms will include exo- and endo-activeglycoside hydrolases, namely fucoidanases and fucosi-dases. Additionally, one should expect the presence ofsulfatases in such complexes. Indeed, there are manyreports of fucoidanases, a-L-fucosidases, and sulfatases,which were extracted frommarine invertebrates [16–18],marine bacteria [19–22], and some kinds of fungi [23–26].Traditionally, specific screening procedures are necessaryto find new strains capable of producing enzymes ofinterest like, for example, it was reported for fucoida-nases found in marine bacteria or some fungi [20, 25]. Inour case, the strain of filamentous fungus capable ofgrowing in fucoidan-containing medium was foundaccidentally. Filamentous fungus Fusarium sp. grew outspontaneously in a water solution of this polysaccharide.This work describes the strain Fusarium sp. LE1 (intra-laboratory name) that produces two enzymes of fucoi-dan-hydrolyzing complex, a-L-fucosidase and arylsulfa-tase, during the growth in the medium containing thegrounded powders of brown algae Laminaria digitata andFucus vesiculosus. In addition, we compared its ability toproduce fucoidanase, a-L-fucosidase and arylsulfataseenzymes with several representative Fusarium strains.

Materials and methods

Chemical reagents and substratesMechanically grounded brown algae L. digitata purchasedfrom Verina Company, Ltd (Saint Petersburg, Russia)and the mixture of algae L. digitata and F. vesiculosus(50/50%) produced by Iztselenie Company, Ltd (SaintPetersburg, Russia) were used as the source of fucoidan.Fucoidan fraction was extracted from the mixture ofbrown algae L. digitata/F. vesiculosus according to themethod described in the work [27]. All chemicals for

fucoidan isolation and substrates for glycoside hydro-lases were purchased from the companies Sigma–Aldrich, Acros Organics or AppliChem, unless indicatedotherwise. Substrates p-nitrophenyl a-galactopyranoside,p-nitrophenyl a-glucopyranoside were synthesizedfrom the corresponding monosaccharides according tothe method described for a-glucosides [28]. Substratesp-nitrophenyl b-galactopyranoside, p-nitrophenyl b-glucopyranoside, p-nitrophenyl b-cellobiopyranoside,p-nitrophenyl b-xylopyranoside were synthesized fromthe corresponding monosaccharides according to themethod described for b-glucosides [29]. Substrate p-nitro-phenyl Na sulfate was synthesized according to theprocedure reported in Ref. [30].

Strains of F. proliferatum (Matsush.) Nirenberg # 58510,58524, 81601, 58471 and F. verticillioides (Sacc.)Nirenberg # 58526 are stored in the Russian Collectionof Agricultural Microorganisms (RCAM) in the automatedTube Store (Liconic Instruments, Lichtenstein), strainsF. verticillioides 3621 and 3622 were kindly provided byDr. Antonio Moretti (ISPA-CAN, Bari, Italy).

Cultivation media and conditionsFilamentous fungus Fusarium sp. LE1 grew up spontane-ously within amonth at 4 °C on the surface of 1% purifiedfucoidan water solution. Once detected, the microorgan-ism was transferred onto the malt-agar medium to bemaintained onto Petri dishes at the temperature ofþ23 °C. Morphological features of Fusarium culture weredescribed after 10th day of the growth at the temperatureof þ23 °C on three nutrient media with modifications:potato sucrose agar (PSA), malt extract-agar, and SNA [31].Micromorphological features were studied with a CarlZeiss Axio Imager M1 microscope after 14th growth dayon SNA under the same conditions.

To induce fucoidan-hydrolytic activities of the investi-gated strain Fusarium sp., specific media were used(g per 100ml of the artificial sea water prepared withmodification according to the recipe described at theweb-site of the Collection of Algae and Protozoa Cultures(http://www.ccap.ac.uk/media/documents/MASM_000.pdf), excluding soil extract): mediumA contained powderof brown algae L. digitata, 2; wheat bran, 1, pH wasadjusted at 7.5; medium B: medium Aþ peptone, 0.5;medium C: medium B, pH adjusted at 5.0; medium D:powder L. digitata, 2, peptone 1, pH 7.5; medium E:powder L. digitata, 2, yeast extract, 0.5, pH 7.5; mediumF: mixture of algae L. digitata/F. vesiculosis, 2, peptone, 0.5,wheat bran, 1, pH 5.0. Cultivation was carried out within10 days in Erlenmeyer flasks (400ml) containing 100mlof the corresponding medium at 23 °C with shaking(125 rpm).

2 Svetlana V. Shvetsova et al.


http://www.ccap.ac.uk/media/documents/MASM_000.pdf

http://www.ccap.ac.uk/media/documents/MASM_000.pdf

In order to screen the ability of the strain Fusarium sp.LE1 and other listed above Fusarium strains to producea-L-fucosidase and arylsulfatase activities, agarizedCzapekmedium consisting p-nitrophenyl a-L-fucopyrano-side or p-nitrophenyl Na sulfate, respectively, was usedfor incubation of all used microorganisms at 23 °C for10 days.

Enzyme assaysSpecific activities of a-L-fucosidase, arylsulfatase, andfucoidanase were assayed during the culture growth inthe A–F media. Aliquots of the culture liquids (5ml) werewithdrawn daily during the culture growth in eachmedium. Every sample was centrifuged, the resultedsupernatant (100ml) was adjusted to pH 6.5 by addition of50ml of 0.5M K-phosphate buffer solution (pH 6.5) and20ml of 10mM of the corresponding substrate solution(p-nitrophenyl a-L-fucopyranoside or p-nitrophenyl Nasulfate, respectively), were added. The reaction mixturewas incubated at 37 °C overnight; the reaction wasstopped by adding of 10% solution of Na2CO3 and opticaldensity was measured at 400 nm. One unit (IU) of thea-L-fucosidase activity was defined as the quantity ofthe enzyme contained in 1ml of the culture liquidand capable of hydrolyzing 1mmol of p-nitrophenyla-L-fucopyranoside per minute under the above con-ditions. One unit (IU) of the arylsulfatase activity wasdefined as the quantity of the enzyme contained in 1mlof the culture liquid and capable of hydrolyzing 1mmolof p-nitrophenyl Na sulfate per minute under the aboveconditions.

Arylsulfatase activity was tested during the growthof Fusarium sp. LE1 in culture liquid as well as insidethe cells. For this, 2-ml aliquots of culture liquid withcells were withdrawn daily and centrifuged for 10min;supernatant was removed and cells were washedseveral times with 10–15ml of 20mM Tris HCL buffersolution, pH 7.4. Resulted cells were melted in liquidnitrogen for 10min to homogeneous slurry, redissolvedin 1.5ml of the same buffer and centrifuged before use.Reaction mixtures consisted of 70ml of the culture liquidor cells content, 30ml of 1M Tris HCl, pH 7.4, and 10ml ofp-nitrophenyl Na sulfate were incubated for 120min at37 °C before stopping the reaction by Na2CO3 solutionand measurement of the optical density.

To detect fucoidanase activity, all available Fusariumstrains were grown in the medium consisting of sterile1% solution of fucoidan, artificial sea water with orwithout the addition of 0.5% peptone (pH 5). Each samplewas incubated without shaking at 28 °C during 30 days.

Aliquots were withdrawn daily in order to monitorfucoidanase activity by Somogyi–Nelson method [32] and

by carbohydrate-polyacrylamide gel electrophoresis(C-PAGE) as described by Colin et al. [33] with modifica-tions. For analytical runs, the 3-ml aliquots of cultureliquids that initially contained fucoidan were electro-phoresed through a 27% running 1-mm-thick polyacryl-amide gel in 50mM Tris–HCl, 2mM EDTA buffer (pH 8.7),and stained with 0.025% toluidine blue O (Sigma–Aldrich) in 10% solution of acetic acid. Fucanolyticactivity was expected to be detectable by the occurrenceof clear anionic oligosaccharide bands in the gel.

Routine exo-glycoside hydrolase assaysExo-glycoside hydrolase activities in the cultural liquid ofFusarium sp. LE1 grown during the appropriate timein the medium F were determined in the followingway: each sample containing 100ml of centrifugedculture liquid, 20ml of the solution (2mgml�1) ofthe corresponding p-nitrophenyl glycoside (p-nitrophenyla-galactoside, p-nitrophenyl a-glucoside, p-nitrophenyla-D-mannopyranoside, o-nitrophenyl b-glucopyranoside,p-nitrophenyl b-L-fucopyranoside, p-nitrophenyl b-cello-bioside, p-nitrophenyl b-xylopyronoside, p-nitrophenylb-galactopyranoside, o-nitrophenyl b-D-glucopyranoside,p-nitrophenyl N-acetyl-b-D-glucosaminide, or p-nitro-phenyl a-L-fucopyranoside), and 20ml 0.5MNa phosphatebuffer (pH 6.5) was incubated at 37 °C for 90min. Theabsorbency of the released nitrophenol was measuredspectrophotometrically at 400nm after stopping thereaction by 360ml of 10% solution of Na2CO3. One unit(IU) of the corresponding exo-glycoside hydrolase activitywas defined as the quantity of the enzyme contained in1ml of the culture liquid and capable of hydrolyzing1mmol of the corresponding p-nitrophenyl glycoside perminute under the above conditions.

Routine endo-glycoside hydrolase assaysThe following substrates were used for determination ofendo-glycoside hydrolase activities: potato starch, AvicelPH-101 cellulose, laminarin from L. digitata, b-glucan,xylan from Beechwood, and fucoidan from L. digitata. Analiquot of the culture liquid of Fusarium sp. LE1 grownin the medium F within 5 days was dialyzed against20mM Na-acetate buffer solution, pH 5.5. To determinean endo-glycoside hydrolase activity, each sample con-taining 100ml of the corresponding substrate solution(10mg/ml) and 100ml of dialyzed culture liquid wasincubated overnight at 37 °C. Amount of reducing sugarsreleased during hydrolysis of the corresponding poly-saccharides was detected by Somogyi–Nelson method[32]. One unit of an endo-glycoside hydrolase activity wasdefined as the quantity of the enzyme contained in 1mlof the culture liquid and capable of releasing 1mmol of



the reducing groups equivalent to 1mmol of glucoseunder the described conditions.

Protein content was determined according to Lowrymethod using BSA as a standard [34].

Genomic DNA extractionThe strain under investigation was grown in the mediumcontaining malt extract:water (1:5 v/v) for 48 h withshaking. Cellular biomass was filtered through Bunsenfunnel and washed out in 1 L of the 15mM of Tris–HClbuffer solution containing 150mM NaCl, pH 7.0.Resulted cell suspension was grounded up in liquidnitrogen. Genomic DNA was extracted according to theprotocol described in Ref. [35]. The internal transcribedspacer (ITS) region was amplified by PCR with the use ofstandard primers ITS1 (50-TCCGTAGGTGAACCTGCGG-30)and ITS4 (50-TCCTCCGCTTATTGATATGC-30) [36]. Theresulted product was purified by means of Qiagen gelextraction kit and sequenced by Evrogen Company(Moscow, Russia). The resulted sequence, 558 bp inlength, was checked for similarities to DNA sequencesin the GenBank using a homology search programpackage, the BLAST program [37] on the NCBI server(www.ncbi.nlm.nih.gov/blast). The multiple alignmentand phylogenetic analysis was carried out with twosoftware packages, CLUSTALW2 [38, 39] and TREEVIEW [40].

Homology search for a potential fucoidanase genewithin known genome of F. verticillioides 7600 (CM000578-88.1) was carried out using programs BLASTN 2.2.29þand TBLASTN 2.2.29þ based on nucleotide and/or aminoacid sequences of known fucoidanases: FcnA from themarine bacterium Mariniflexile fucanivorans (AJ877239.1and CAI47003.1), from Alteromonas sp. SN-1009 (Fda1(AAO00508.1) and Fda2 (AAO00509.1) [41], and fromShewanella violacea DSS12 (AP011177.1 and BAJ00350.1).

Results

Identification of the strain Fusarium sp. LE1All morphological characteristics of the Fusarium strainLE1 were determined during its growth on three specificmedia described in Materials and Methods section. Shapeof conidia, length, and type of conidial chains or heads,degree of conidiophore branching and whether phialidesof the aerial conidiophores are monophialidic or poly-phialidic have been used formorphological identificationof the fungal culture. The ITS region of the rDNA (558 bp)was sequenced and compared with the known nucleotidesequences from GenBank database. It was found out thatthe DNA site had 100% identity and maximum score

(1031) with the following sequences: Gibberella intermedia(Kuhlmann) Samuels et al. (teleomorph: F. proliferatum) –strain ZA (JQ690083.1), F. proliferatum (FJ040179.1),F. proliferatum strain CE1 (EU151486.1), F. proliferatumstrain D1 (EU151484.1), Fusarium sp. CID124 haplotypefus124 (EF589878.1). Phylogenetic position of the strainLE1 shown on the picture was established on the basis ofthe comparative analysis of nucleotide sequences (Fig. 1).According to the analysis of macro- and micromorpho-logical characteristics as well as ITS region sequenceanalysis, the fungal strain under study was classified asF. proliferatum [42, 43]. The F. proliferatum strain LE1 hasbeen deposited under the number RCAM02409 in theRussian Collection of Agricultural Microorganisms(RCAM).

Screening of Fusarium strains for fucoidan-hydrolyzing enzymesHaving discovered the spontaneous growth of thefilamentous fungus on the surface of fucoidan solution,we assumed the fungus could produce enzymes thathydrolyze fucoidans. Particularly, we expected to findat least three enzymes: fucoidanase, a-L-fucosidase,and arylsulfatase. Our expectations were based on twoissues: (a) the growth of microorganism in the mediumcontaining fucoidan as a sole nutrient source and thus,activation of necessary enzymes for culture survival; (b) aFusarium strain LD8 was recently reported to producefucoidanase [26] and a-L-fucosidases were described inseveral strains of Fusarium genus [23].

To check the hypothesis Fusarium strains couldproduce given enzymes, four strains of F. proliferatumand three F. verticillioides ones in addition to F. proliferatumLE1 (RCAM02409) were taken for the screening. After thegrowth of each culture in 1% fucoidan solution for30 days, no detectable activity was found in any Fusariumstrain by C-PAGE (data not shown) and by Somogyi–Nelson method. We failed to observe any new bands in agel that should have indicated the appearance of shorterfucooligosaccharides in the reaction mixture and thus,the fucoidanase activity like it was shown earlier forseveral fucoidanases [21, 33]. We did not observe theappearance of reducing sugars in the culture liquidsduring the growth thatmay indicate the hydrolysis of thepolysaccharide presented in the medium.

Moreover, no significant similarity was found whennucleotide sequences of known fucoidanase geneswere compared with F. verticillioides 7600 genome byBLAST2.2.29þ program. Amino acid sequences of thefucoidanases from GH107 family (The Carbohydrate-Active Enzymes database (CAZy; http://www.cazy.org) [44]) were also aligned with translated nucleotide



http://www.ncbi.nlm.nih.gov/blast

http://www.cazy.org/


sequence of F. verticillioides 7600 genome. The programTBLAST2.2.29þ found short similar sites in differentchromosomes with practically equal and low identities:maximal query cover for Shewanella violacea DSS12sequence with F. verticillioides 7600 genome was 26%with 34% identity. The results of the above search did notallow us to determine the potential fucoidanase gene.

Usage of model substrates with chromophoric groupfor glycoside hydrolases is the simplest way to detectand monitor their activities during the fungal growth.Therefore, available Fusarium strains were grown inagarized Czapek medium containing p-nitrophenyla-L-fucopyranoside or p-nitrophenyl Na sulfate. After5-days growth, visible yellow halo was detected onlyaround the growth area of F. proliferatum LE1 (RCAM02409)with p-nitrophenyl Na sulfate (Fig. 2) indicating thepresence of arylsulfatase activity. No other strains gave

Figure 1. Neighbour-joining tree showing the phylogenetic position of the strain F. proliferatum LE1 (RCAM02409) and relative strains of theFusarium genus. GenBank accession numbers are given in parentheses. Phylogenetic tree was constructed using the software packagesClustalW and TreeView.

Figure 2. Growth of representative Fusarium strains on agarizedCzapek medium containing p-nitrophenyl sulfate.



the same result. The absence of a-L-fucosidase activitywithin this screening procedure can be explained by lowsolubility of p-nitrophenyl a-L-fucopyranoside and thus,low rate of enzymatic reaction due to small concentra-tion of the substrate in the medium.

Alpha-L-fucosidase, arylsulfatase, and other glycosidehydrolase activities of F. proliferatum LE1(RCAM02409)We attempted to select a liquid medium compositionwhere the manifestation of activities of fucoidan-hydrolyzing complex would be maximal. Groundedpowder of brown algae L. digitata or the mixture of algaeL. digitata/F. vesiculosus were added to the cultural mediaas a source of sulfated fucose-containing polysaccharide.To provide nutritionmedia with the additional sources ofcarbon and nitrogen, peptone or yeast extract of differentquantities were added. Slightly basic or slightly acidic pHvalues (7.5 and<5.0, respectively) of the culturalmediumwere used. As a result, F. proliferatum LE1 (RCAM02409)demonstrated maximal a-L-fucosidase activity in themedium F (Fig. 3), containing the mixture of brown algaeL. digitata/F. vesiculosus, wheat bran, peptone, and adjustedat pH 5.0. On the 5th day of cultivation, specific activityof the a-L-fucosidase was 13- and 6-fold higher in themedium F than in the medium B and C, correspondingly.The maximal specific activity of the arylsulfatase was onthe 8th day in the media B and F, minimal one wasestimated in the medium D, and was not detected inthe medium A (Fig. 3). During the cultivation of thestrain F. proliferatum LE1 (RCAM02409) in the medium Fthe a-L-fucosidase activity started growing up on the2nd day of the cultivation and reached its maximum on

the 4–5th day (Fig. 4) slowly decreasing for the nextfew days. The activity of the arylsulfatase of F. proliferatumLE1 (RCAM02409) appeared to be detectable when thea-L-fucosidase activity was falling down and reachedthe maximum on the 8–10th day. During the period ofculture growth, values for pH in the culture mediumchanged from 5 to 8 (Fig. 4, insert).

To determine the localization of the arylsulfatase inF. proliferatum LE1 (RCAM02409), we measured theactivity in culture supernatant and inside cells duringthe growth of the culture within several days. Resultspresented in Fig. 5 clearly show that arylsulfatase is an

Figure 3. Specific activities of the a-L-fucosidase and arylsulfatasefrom F. proliferatum LE1 (RCAM02409) measured on the 5th day ofgrowth in the media A–F. Values for the a-L-fucosidase activity areshown in gray; shaded columns in the histogram indicate arylsulfataseactivities.

Figure 4. Specific activities of the a-L-fucosidase (gray columns) andarylsulfatase (shaded columns) fromF. proliferatum LE1 (RCAM02409)during the growth in the medium F for 10 days. Insert, changes of pHvalues are shown.

Figure 5. Intracellular and extracellular specific activities of arylsul-fatase from F. proliferatum LE1 (RCAM02409) measured duringthe culture growth on the 3rd, 5th, and 10th days in the medium F (seedetails in Materials and Methods section). White columns refer to theintracellular activity, gray columns refer to the arylsulfatase activity inthe culture liquid.



intracellular enzyme: on third day of the growth itsspecific activity was 32 times higher inside cells then inculture liquid. The difference decreased to 3.5 times by10th day.

The strain F. proliferatum LE1 (RCAM02409) was testedfor the presence of another glycoside hydrolase activitiesduring the cultivation under submerged conditionsin the medium F. It is appeared F. proliferatum LE1(RCAM02409) was able to secrete cellulases and hemi-cellulases in detectable quantities: b-glycosidase, cello-biohydrolase, cellulases, endo-b-glucanases as well asb-xylanases and b-xylosidases (Fig. 6). a-Galactosidaseand a-glycosidase were also detected in predominateamounts. Low level of the b-L-fucosidase activity indicat-ed the absence or a small content of the proper bond typethe given enzyme can hydrolyze under the particularconditions and consequently its synthesis for the givenstrain was reduced.

Discussion

The culture of a filamentous fungus grown up spontane-ously on the solution of purified fucoidan exhibitedthe a-L-fucosidase and arylsulfatase activities during thegrowth in the medium with fucoidan-containing algaeL. digitata/F. vesiculosus. Based on the results of morpho-logical analysis and comparison of ITS region of rDNAfragment (558 bp) with the known nucleotide sequences

from GenBank database, we concluded the fungusbelongs to F. proliferatum.

There is a wide special and intraspecial diversityamong Fusarium fungi. Fusarium genus consists of fungivaried according to the type of nutrition (saprotrophes,necrotrophes, biotrophic pathogens, and endophytes)and the ability of formation of different secondarymetabolites including high toxic ones for mammals. Theability of fungi to adjust to diverse environmentalconditions is caused by their genetic flexibility andmetabolic variety [45]. F. proliferatum is widely spreadin the world as pathogen of cultivated and wild plants.F. proliferatum strains are known to produce mycotoxinssuch as fumonisins, fusaproliferin, and beauvecirin.Some strains of F. proliferatum were reported to becommercial producers of b-glycosidases and b-xylosi-dases, laccases, and other relative enzymes [46–51].

Currently, the information about endo-active enzymescapable of degrading fucoidan onto smaller fragments isvery scarce. Although fucoidanases have been isolatedin hepatopancreas of the invertebrates (marine mollusk)[16], marine bacteria [19–22], and some types of fungi[24–26], the particular 107th glycoside hydrolase familyin CAZy classification consists only of four members[CAZy; http://www.cazy.org, 44]. Only one of them, asulfated fucan a-1,4-endohydrolase from Mariniflexilefucanivorans, was characterized properly according toits genetic information resulting in the gene cloning andexpression in Escherichia coli. A structure of the enzymewas predicted using computer programs and preliminarymechanistic aspects of the action of the enzyme werediscussed [33]. Much more information can be foundabout a-L-fucosidases and sulfatases in literature due tothe ability of these exo-active enzymes to split offterminal residues of fucose and sulfates from variousnatural poly- and oligosaccharides besides fucoidans. Itresults in a presence of a wider range of organisms as asource of these enzymes. Generally, fucoidan-hydrolyz-ing enzymes are found among organisms of marineorigin, namely bacteria, fungi, and invertebrates whilethere is only one report on a screening for fucoidanaseactivity among non-marine filamentous fungi [25]. Ourprimary aim was to detect a fucoidanase activity duringthe growth of F. proliferatum LE1 (RCAM02409) in differentmedia. We assumed the presence of such an activitybased on the ability of the strain to grow in a fucoidansolution and a finding of a similar enzyme in Fusarium sp.LD8 [26]. However, we succeeded to observe relativelyhigh levels of a-L-fucosidase and arylsulfatase but notfucoidanase. It should be noted that the only Fusariumstrain reported to have a fucoidanase activity was isolatedfrom sand of North Sea in Germany [26]. Marine origin of

Fig. 6. Specific activities of O-glycoside hydrolases measured duringthe growth of the F. рroliferatum LE1 (RCAM02409) in the medium F for5 days: (1) a-galactosidase; (2) a-glucosidase; (3) a-D-mannosidase;(4) b-glucosidase; (5) b-L-fucosidase; (6) b-cellobiohydrolase; (7) b-xylosidase; (8) b-galactosidase; (9) b-D-glucosidase; (10) b-D-gluco-saminidase; (11) arylsulfatase; (12) a-L-fucosidase; (13) amylase;(14) cellulase; (15) laminarinase; (16) b-glucanase; (17) b-xylanase.




this particular strain and necessity to adapt marineenvironment may be reasons for appearance of a newgene coding fucoidanase in this case. More detailedstudies of F. proliferatum LE1 (RCAM02409) are necessaryto know about mechanism of synthesis of differentenzymes and particularly of fucoidan-degrading complexin this strain. Strains of Fusarium genus have beenreported to produce a-L-fucosidases. Genes coding thesynthesis of these enzymes were described and theenzymes were extracted from the F. oxysporum Schltdl.and F. graminearium Schwabe [23]. To the best of ourknowledge, there are no reports on sulfatase inductionamong Fusarium strains. This is the first strain wherearylsulfatase activity was detected. Analyzing the growthof the F. proliferatum LE1 (RCAM02409) (Fig. 4), weobserved a successive appearance of a-L-fucosidase andarysulfatase activities in the culture medium F. Arylsul-fatase appeared to be an intracellular protein and itsdetection in the media is possible only after putativenatural cell lysis and/or secretion in the stationary phaseof the culture (Fig. 5).

In conclusion, the novel strain F. рroliferatum LE1(RCAM02409) is a potential producer of many valuableglycoside hydrolases and enzymes, which can hydrolyzesulfated fucoidans of brown algae. In the future, we planto find out the conditions for maximal production offucoidan-degrading complex, isolate and describe indetails most of potential enzymes.

Acknowledgment

The investigation was partially supported by the GrantNo. 14-26-00067 of the Russian Scientific Foundation(RSF) “Polyphasic approach as a modern base for revisionof biodiversity of plant pathogenic fungi.”

Conflict of interest statement

The authors declare no commercial or financial conflictof interest.

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The novel strain Fusarium proliferatum LE1 (RCAM02409) produces α- L -fucosidase and arylsulfatase during the growth on fucoidan