Biochemical Characteristics and Substrate Degradation Pattern of aNovel Exo-Type �-Agarase from the Polysaccharide-Degrading MarineBacterium Flammeovirga sp. Strain MY04

Wenjun Han,a Yuanyuan Cheng,a,b Dandan Wang,a Shumin Wang,a Huihui Liu,a Jingyan Gu,a,c Zhihong Wu,a Fuchuan Lia

National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, and State Key Laboratory of MicrobialTechnology, Shandong University, Jinan, Chinaa; Department of Food Science and Engineering, Shandong Agriculture and Engineering University, Jinan, Chinab; JinanFruit Research Institute, All-China Federation of Supply and Marketing Co-operatives, Jinan, Chinac

ABSTRACT

Exo-type agarases release disaccharide units (3,6-anhydro-L-galactopyranose-�-1,3-D-galactose) from the agarose chain and, incombination with endo-type agarases, play important roles in the processive degradation of agarose. Several exo-agarases havebeen identified. However, their substrate-degrading patterns and corresponding mechanisms are still unclear because of a lackof proper technologies for sugar chain analysis. Herein, we report the novel properties of AgaO, a disaccharide-producing aga-rase identified from the genus Flammeovirga. AgaO is a 705-amino-acid protein that is unique to strain MY04. It shares sequenceidentities of less than 40% with reported GH50 �-agarases. Recombinant AgaO (rAgaO) yields disaccharides as the sole finalproduct when degrading agarose and associated oligosaccharides. Its smallest substrate is a neoagarotetraose, and its disaccha-ride/agarose conversion ratio is 0.5. Using fluorescence labeling and two-stage mass spectrometry analysis, we demonstrate thatthe disaccharide products are neoagarobiose products instead of agarobiose products, as verified by 13C nuclear magnetic reso-nance spectrum analysis. Therefore, we provide a useful oligosaccharide sequencing method to determine the patterns of en-zyme cleavage of glycosidic bonds. Moreover, AgaO produces neoagarobiose products by gradually cleaving the units from thenonreducing end of fluorescently labeled sugar chains, and so our method represents a novel biochemical visualization of theexolytic pattern of an agarase. Various truncated AgaO proteins lost their disaccharide-producing capabilities, indicating a strictstructure-function relationship for the whole enzyme. This study provides insights into the novel catalytic mechanism and enzy-matic properties of an exo-type �-agarase for the benefit of potential future applications.

IMPORTANCE

Exo-type agarases can degrade agarose to yield disaccharides almost exclusively, and therefore, they are important tools for di-saccharide preparation. However, their enzymatic mechanisms and agarose degradation patterns are still unclear due to the lackof proper technologies for sugar chain analysis. In this study, AgaO was identified as an exo-type agarase of agarose-degradingFlammeovirga bacteria, representing a novel branch of glycoside hydrolase family 50. Using fluorescence labeling, high-perfor-mance liquid chromatography, and mass spectrum analysis technologies, we provide a useful oligosaccharide sequencingmethod to determine the patterns of enzyme cleavage of glycosidic bonds. We also demonstrate that AgaO produces neoagaro-biose by gradually cleaving disaccharides from the nonreducing end of fluorescently labeled sugars. This study will benefit futureenzyme applications and oligosaccharide studies.

Agarose is a complex polysaccharide that contains repeated di-saccharide units of 3,6-anhydro-L-galactopyranose-�-1,3-D-

galactose (1). Agarose has been identified to be one of the polysac-charide components of the cell wall and intercellular substances inred algae (Rhodophyta), such as Gracilaria and Porphyra (2). Be-cause of their strong gel-forming ability and high chemical stabil-ity, agarose and its derivatives have been widely used in variousapplications (3–5).

Agarose can be hydrolyzed by �-agarase (EC 3.2.1.158) toproduce agarooligosaccharides (AOs) with 3,6-anhydro-L-galac-topyranose (A) as the reducing end (6) or cleaved by �-agarase(EC 3.2.1.81) to yield neoagarooligosaccharides (NAOs) withD-galactose (G) as the reducing end (7). Agarases have been suc-cessfully applied in many biotechnological applications, such asDNA gel recovery (8) and algal protoplast preparation (9, 10).Agarose-derived oligosaccharides have shown antioxidation andanti-inflammation activities (11, 12) and probiotic and whiteningeffects (13, 14), suggesting their potential usage in the cosmetic,

food, and medical industries. Therefore, agarases are importanttools for oligosaccharide preparation.

During the last decade, numerous agarases have been identi-fied from marine and terrestrial bacteria (2, 15–17). Bacterial aga-

Received 5 February 2016 Accepted 1 June 2016

Accepted manuscript posted online 3 June 2016

Citation Han W, Cheng Y, Wang D, Wang S, Liu H, Gu J, Wu Z, Li F. 2016.Biochemical characteristics and substrate degradation pattern of a novel exo-type�-agarase from the polysaccharide-degrading marine bacterium Flammeovirgasp. strain MY04. Appl Environ Microbiol 82:4944 – 4954.doi:10.1128/AEM.00393-16.

Editor: V. Müller, Goethe University Frankfurt am Main

Address correspondence to Fuchuan Li, fuchuanli@sdu.edu.cn.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.00393-16.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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rases have diverse protein sequences, molecular masses, and aga-rose degradation patterns. Two enzymes have been reported to be�-agarases belonging to glycoside hydrolase (GH) family 96(GH96) (18, 19), while others are �-agarases of the GH16, GH50,GH86, and GH118 families (20, 21). Most agarases are endo-typeenzymes that randomly and internally degrade agarose, producinga series of even-numbered oligosaccharides as products (16), anda few, mainly GH50 �-agarases, are exo-type enzymes that yieldneoagarobiose (NA2) as the sole final agarose degradation prod-uct (22, 23). In bacterial agarose degradation, endo-type agaraseshave an important role in initially depolymerizing agarose intooligomers, while exo-type agarases are essential for releasing thedisaccharide units to facilitate subsequent sugar metabolism inbacteria (24, 25).

Among the exo-type agarases, the structure and enzyme char-acteristics of Aga50D of Saccharophagus degradans strain 2-40have been well characterized (22, 26), which has provided insightsinto the properties of GH50 family enzymes. The Aga50D proteinhas an N-terminal carbohydrate-binding module (CBM) and aC-terminal catalytic module. Crystal structures of the oligosac-charides in complex with Aga50D have shown that two glutamineresidues (Gln534 and Gln695) contributed to the catalytic activesite, which is conserved among GH50 family members. Sugar-binding-site residues are distributed in both the catalytic grooveand the carbohydrate-binding domain, which facilitate activity onthe double helix of agarose (26). However, the biochemical mech-anisms by which agarases degrade substrates and release productsin an exolytic pattern are still unclear.

Bacteria of the Flammeovirga genus have recently been identi-fied from the surface of algae (27), deep-sea and coastal sediments(28–30), and marine animal innards (15). All reported Flammeo-virga strains are efficient in the enzymatic degradation and bacte-rial utilization of multiple polysaccharides, such as agarose,alginate, and starch. Notably, Flammeovirga sp. strain MY04 couldliquefy and grow on agarose as the sole carbon source, indicatingthat MY04 can produce efficient agarose-degrading enzymes (29).To date, one GH86 �-agarase (AgaP4383 of Flammeovirga pacificastrain WPAGA1) (31) and two GH16 �-agarases (AgaG4 of Flam-meovirga sp. strain MY04 and AgaYT of Flammeovirga yaeyamen-sis strain YT) (27, 32) have been identified from Flammeovirgastrains. The AgaYT protein shares a sequence identity of 98.5%with the AgaG4 protein, and Yang et al. determined that neoaga-rotetraose (NA4) and NA2 were the final oligosaccharide productsof recombinant AgaYT (rAgaYT) (27); however, the molecularmasses of the final products were not determined. Moreover, thetwo final oligosaccharide products appeared to be neoagaro-hexaose (NA6) and NA4 rather than NA4 and NA2 because the Rf

value of D-galactose was smaller than that of NA2 but larger thanthat of NA4 under the designated thin-layer chromatography(TLC) system (22, 23, 33, 34). Therefore, all the three studiedagarases of Flammeovirga strains produce NA4 as the smallestoligosaccharide product (31, 32). However, the tetraoligosaccha-ride cannot be utilized directly and efficiently by bacteria until it isfurther enzymatically digested. Thus, there should be essential en-zymes responsible for the generation of disaccharides from aga-rose. Herein, we report the biochemical characteristics and novelenzymatic properties of the AgaO protein from Flammeovirga sp.strain MY04 and identify it as a disaccharide-producing �-agarasefrom polysaccharide-degrading bacteria of the genus Flammeo-virga.

MATERIALS AND METHODSBacterial strains and growth conditions. Unless otherwise noted, Esche-richia coli strains were cultured at 37°C in Luria-Bertani (LB) medium,supplemented with antibiotics, e.g., ampicillin (100 �g/ml), if necessary.The marine bacterium Flammeovirga sp. strain MY04 (CGMCC 2777)was cultured at 30°C in medium (pH 7.0) composed of 0.40% (wt/vol)tryptone, 0.25% yeast extract, and 3.0% NaCl. Agar powder was added ata concentration of 1.5% (wt/vol) to prepare solid media. Agar, agarose,alginate, carrageenan, �-carrageenan, �-carrageenan, cellulose, and xylanwere purchased from Sigma-Aldrich (USA).

Sequence analysis of the genes and proteins. DNA sequences weretranslated into protein sequences using the software BioEdit, version 7.2.1(35), and the percent GC contents were calculated. For functional predic-tion, searches of the similarity of the protein sequences to those of knownsequences were performed using the BLAST algorithm from the NationalCenter for Biotechnology Information (NCBI) server (http://www.ncbi.nlm.nih.gov). Signal peptides and their types were analyzed using theSignalP (version 4.1) and the LipoP (version 1.0) online servers (http://www.cbs.dtu.dk/services/), respectively. The molecular weights of theproteins were estimated using the peptide mass tool on the ExPASy serverof the Swiss Institute of Bioinformatics (http://swissmodel.expasy.org/). The modules and domains of the proteins were identified usingthe Simple Modular Architecture Research Tool (https://en.wikipedia.org/wiki/Simple_Modular_Architecture_Research_Tool), the Pfam da-tabase (http://pfam.xfam.org), and the Carbohydrate-Active Enzymedatabase (http://www.cazy.org). Multiple-sequence alignments andphylogenetic analysis were performed using MEGA software, version6.06 (36).

Construction of expression vectors. Total genomic DNA of Flam-meovirga sp. strain MY04 was extracted using a universal DNA purifica-tion kit (TianGen Co., Ltd., Beijing, China). To express the AgaO protein,the full-length gene was amplified using the primers BAgaO-F andBAgaO-R (Table 1) and the high-fidelity DNA polymerase PrimeSTARHS(TaKaRa, Dalian, China). Primer pairs with restriction enzyme sites forXhoI and XbaI (underlined in Table 1) were designed to generate a 6�Histag at the C terminus of the recombinant protein (rAgaO). The DNAproducts were gel purified and then cloned into the expression vectorpBAD/gIII A (Invitrogen, USA). The plasmid carrying the recombinant(pBAgaO) was transformed into Escherichia coli TOP10 cells. The integ-rity of the nucleotide sequences was confirmed by sequencing the expres-sion plasmids thrice.

Moreover, the full length of the agaO gene was amplified using theprimer pair BAgaO-F and TF-AgaO-R (Table 1) with restriction enzymesites for XhoI and XbaI, to generate a recombinant protein (rTFAgaO)fused with a cold shock protein (TF) at the N terminus and a 6�His tag atthe C terminus. The DNA products were finally cloned into the expressionvector pCold TF (TaKaRa, Dalian, China). The resulting plasmid, pCTF-AgaO, was transformed into E. coli BL21(DE3) cells.

Heterologous expression and purification of the recombinant pro-teins. Unless otherwise noted, protein analyses were performed at 4°C(32). Briefly, E. coli TOP10 cells harboring the plasmid pBAgaO wereinitially cultured in LB broth. When the cell density reached an A600 of 0.6to 0.8, L-arabinose was added at final concentrations ranging from 0.001to 1.0 mM. After continued cultivation for an additional 24 h at 16°C, thecells were harvested by centrifugation, washed twice with ice-cold buffer A(50 mM Tris, 150 mM NaCl, pH 8.0), resuspended, and disrupted bysonication. After centrifugation, the soluble fraction was collected andloaded onto a Ni-nitrilotriacetic acid (Ni-NTA) agarose column (Nova-gen, USA), and the column was then eluted using imidazole at increasinggradient concentrations (0, 10, 50, 100, and 250 mM). Purified proteinsamples were diluted and dialyzed against buffer B (25 mM Tris, 5%[vol/vol] glycerol, pH 8.0) (1:50, vol/vol). SDS-PAGE was performedusing 12% (wt/vol) polyacrylamide gels as described by Sambrook andRussell (37). Proteins were detected by staining the gel with Coomassiebrilliant blue R-250. Protein concentrations were determined by the

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Folin-Lowry method using the Folin-Ciocalteu phenol reagent (Sigma-Aldrich, USA) and bovine serum albumin as a standard.

E. coli BL21(DE3) cells harboring the plasmid pCTF-AgaO were usedto express the recombinant fusion protein rTFAgaO, and IPTG (isopro-pyl-�-D-thiogalactopyranoside) was added at final concentrations rang-ing from 0.001 to 1.0 mM. The recombinant protein rTFAgaO was ex-pressed and purified using a strategy similar to that described above forrAgaO.

Enzyme activity assay. The polysaccharide-degrading activity ofrAgaO was determined by quantifying reducing sugars using the 3,5-dini-trosalicylic acid (DNS) assay (38). The absorbance of the reducing sugarproduct was measured at 540 nm, with D-galactose used as a standard.One unit of enzyme was defined as the amount of enzyme that produced1 �mol reducing sugars per minute.

To determine the optimal substrate of rAgaO, various polysaccharides(e.g., agar, agarose, alginate, carrageenan, �-carrageenan, �-carrageenan,cellulose, and xylan) were individually dissolved in deionized water toprepare stock solutions (1.0 mg/ml). Each stock solution (500 �l) wasmixed with the appropriately diluted enzyme in buffer B at an equal vol-ume and then incubated for 12 h at different temperatures ranging from 0to 70°C. Enzyme-treated samples were initially heated in boiling water for10 min and then cooled on ice. After centrifugation at 15,000 � g for 15min, the supernatant was collected and analyzed for the enzymatic prod-ucts.

Biochemical characterization of rAgaO. To determine the optimalconditions for rAgaO activity, stock solutions of agarose (1.0 mg/ml) wereprepared using buffers with different pH values, including acetate buffer(50 mM, pH 4.0 to 6.5), NaH2PO4-Na2HPO4 buffer (50 mM, pH 6.0 to8.0), and Tris-HCl buffer (50 mM, pH 7.0 to 10). The optimal tempera-ture of rAgaO was assayed by monitoring the agarase activity at tempera-tures ranging from 0 to 70°C at pH 7.0 for 1 h. The pH dependence ofrAgaO was tested at 45°C for 1 h. The thermostability of rAgaO was eval-uated by measuring the residual activity after incubating the enzyme atvarious temperatures for 2 h. The effects of pH on rAgaO stability weredetermined by measuring the residual activities after incubating the en-zyme in various pH environments (4.0 to 10) at 4°C for 2 h. The effects ofmetal ions and chelating agents on the activity of rAgaO were examined by

determining its activity in the presence of 1 mM and 10 mM variouschemicals, respectively.

Analysis of the agarose-degrading pattern of rAgaO. Agarose (1.0mg/ml) was digested by rAgaO (0.01 U/ml) at 45°C for over 72 h. Aliquotsof the digestions were collected for time course analysis by thin-layerchromatography (TLC) using silica gel 60 plates (F254; Merck, Germany)(32). The Rf value of each oligosaccharide fraction was calculated accord-ing to the spots visualized on the TLC plates. The concentrations ofoligosaccharides from the enzymatic digestions were determined usingthe DNS reducing sugar assay (38).

Characterization of the final oligosaccharide products. To obtainsize-defined oligosaccharides, 100 mg agarose was degraded by excessrAgaO at 45°C for 72 h. The final enzymatic digests were loaded onto aSuperdex peptide 10/300 GL column (GE Healthcare, USA) and moni-tored with a refractive index detector (Shimadzu, Kyoto, Japan). The mo-bile phase was a NH4HCO3 solution (0.20 M), which was used at a flowrate of 0.4 ml/min. Data analysis was performed using the software LC-solution, version 1.25. The size-defined oligosaccharide fractions werecollected and repeatedly freeze-dried to remove the NH4HCO3. The rateof agarose conversion was calculated as the mass ratio of the obtainedoligosaccharide products to the initial polysaccharide substrates.

To determine the molecular masses, approximately 0.1 to 1.0 �g pu-rified oligosaccharide products was assayed using matrix-assisted laserdesorption ionization–time of flight mass spectrometry (MS) (Axima-CFR plus; Shimadzu, Japan). For nuclear magnetic resonance (NMR)spectroscopy, purified oligosaccharide samples (�2 mg) were dissolved in0.5 ml of D2O in 5-mm NMR tubes and repeatedly freeze-dried. Thespectra were recorded on a JNM-ECP600 (JEOL, Japan) apparatus set at600 MHz. Data analyses were performed using MestReNova (version6.1.0-6224) software.

Analysis of the oligosaccharide degradation pattern of rAgaO. Toassay the smallest substrate, various oligosaccharides were reacted withthe enzyme rAgaO, and the digests were analyzed by TLC as describedabove. D-Glucose and D-galactose (1.0 mg/ml) were used as standardmarkers to assay the lactose-degrading activity as described previously(39).

To determine the molar ratios of the final disaccharide products to the

TABLE 1 Bacterial strains, plasmids, and primers used in the present study

Strain, plasmid, or primer Descriptiona or sequenceb Source or reference

StrainsFlammeovirga sp. strain MY04 A polysaccharide-degrading marine bacterium (patented as CGMCC 2777) 29E. coli BL21(DE3) F ompT hsdSB (rB

mB) dcm gal �(DE3)pLysS Cmr Novagen

E. coli TOP10 F mcrA (mrr-hsdRMS-mcrBC) �80dlacZM15 lacX74 deoR recA1 araD139 (araA-leu)7697galU galK rpsL endA1 nupG

Invitrogen

PlasmidspBAD/gIII A Expression vector; Apr InvitrogenpCold TF Expression vector; Apr TaKaRapBAgaO pBAD/gIII A carrying an amplified NcoI-XbaI fragment encoding the recombinant protein

rAgaOThis study

pCTF-AgaO pCold TF carrying an amplified NcoI-XhoI fragment encoding the recombinant proteinrTFAgaO

This study

PrimersBAgaO-F 5=-CTCGAGTTTTTTTTAGCATTCAATCCGCC-3= This studyBAgaO-R 5=-TCTAGAAAGTTATTCACATTACCCAAACGG-3= This studyTC-D344-R 5=-TCTAGAAAATCAAATTGTTGATAGAATCCTAATGGCTTCC-3= This studyTC-A530-R 5=-TCTAGAAAAGCATATACATCTTCATTGGCACCATCACC-3= This studyTC-D626-R 5=-TCTAGAAAGTCTTTAGCTACTTTTACCCCTGGGTGG-3= This studyTN-F164-F 5=-CTCGAGTTTGGGATGTTGAAACGTGATGTGACC-3= This studyTN-N350-F 5=-CTCGAGAATTTCTATCAAGCCAACTTATATCG-3= This studyTF-AgaO-R 5=-TCTAGATTAGTTATTCACATTACCCAAACGG-3= This study

a Cmr, chloramphenicol resistant; Apr, ampicillin resistant.b XhoI and XbaI restriction enzyme sites are underlined.

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starting oligosaccharide substrate, each substrate (�1 �g) and its enzy-matic digests were individually labeled using 2-aminobenzamide (2-AB)(40) and were then analyzed using the Superdex 10/300 GL column, mon-itored with excitation and emission wavelengths of 330 and 420 nm, re-spectively. A mixture composed of 2-AB-labeled oligosaccharides, e.g.,D-galactose, NA2, NA4, NA6, and neoagarooctaose (NA8), was used as astandard marker. The molar ratios were calculated according to the cor-responding peak area shown in high-performance liquid chromatography(HPLC) analysis.

To further examine the oligosaccharide degradation pattern of rAgaO,each size-defined oligosaccharide fraction (�1 �g) was labeled with ex-cess 2-AB, purified with gel filtration using the Superdex 10/300 GL col-umn, and repeatedly freeze-dried to eliminate NH4HCO3. Each 2-AB-labeled oligosaccharide substrate (�0.1 �g each) was mixed with rAgaO(0.01 U) in a 30-�l volume, incubated at 45°C, and traced over 12 h. Thefinal enzymatic digests were analyzed using the HPLC system with fluo-rescence detection.

Saccharide sequencing of oligosaccharides. To determine the se-quences of the oligosaccharides produced by rAgaO, the enzymatic digests(�1 �g) of the oligosaccharides or agarose were fluorescently labeledusing excess 2-AB. Oligosaccharide samples and 2-AB-labeled oligosac-charide mixtures were initially analyzed by mass spectrometry (MS) andsubsequently by two-stage MS (MS/MS) analyses. Data analysis was per-formed using the software LCMSsolution, version 3.80.410.

Gene truncation, protein expression, and enzyme characterization.The full-length gene of AgaO was PCR amplified using the primers listedin Table 1 to generate truncated gene fragments, as indicated in Fig. 1A.

The DNA products were individually gel purified and cloned into theplasmid pBAD/gIII A. The resulting plasmids were each expressed usingthe same method described for pBAgaO. Soluble bacterial fractions con-taining the truncated proteins were each used as an enzyme preparationfor further activity tests and product analyses.

Accession number(s). The nucleotide sequence of agaO in strainMY04 has been submitted to the GenBank database under accession num-ber KU524066.

RESULTSAgaO gene and protein sequences. In the genomic sequence ofFlammeovirga sp. strain MY04, the agaO open reading frame is2,118 bp in length (GenBank accession no. KU524066) and has aGC content of 35.5%. The predicted protein, AgaO, has a molec-ular mass of �81.3 kDa, and its calculated isoelectric point is 7.07.SignalP (version 4.1) and LipoP (version 1.0) analyses indicatedthat the type II signal peptide of AgaO was composed of 25 aminoacid residues (Met1 to Phy25) (Fig. 1A).

AgaO, which has no homologous gene in the MY04 genome, isa unique protein. According to BLASTp searches, the mature pro-tein of AgaO (Asn26 to Asn705) shared sequence identities of lessthan 40% with characterized �-agarases of the GH50 family. Themain fragment of AgaO (Met163 to Asn702) showed 35% identitywith the exo-type �-agarase Aga50D (Met155 to Ser749; PDB acces-sion no. 4BQ4, chain A) of S. degradans strain 2-40, a type member

FIG 1 Sequence properties of the agarase AgaO from Flammeovirga sp. strain MY04. (A) The gene encoding AgaO and the module organization of AgaO. Thenumbers indicate nucleotides. The protein contains an N-terminal signal peptide (Met1 to Phy25, in black on the left) and a hypothetical catalytic module (Ser395

to Ile602, in black on the right). The primer pairs indicated at the top and bottom were used to generate the full-length gene and various truncated fragments. (B)Phylogenetic analysis based on the protein sequence alignment. The neighbor-joining tree was obtained using MEGA (version 5.05) software. The numbers onthe branches indicate the bootstrap confidence values from 1,000 replicates. The bar is equal to the distance corresponding to 1 amino acid substitution per 10amino acid residues.

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of the GH50 family (22, 26). Protein sequence alignment showedthat AgaO shares homologous regions with GH50 �-agarases,mostly in the C-terminal region (see Fig. S1 in the supplementalmaterial), which was identified to be the catalytic module inAga50D (26). Furthermore, within these partially conserved re-gions, AgaO contained two glutamine residues (Gln458 andGln606) that were strictly conserved in the catalytic motif of GH50�-agarases (see Fig. S1 in the supplemental material), Gln534 andGln695 in Aga50D, respectively, while it did not contain the sameN-terminal CBM-like module, as previously reported for Aga50D(26). Analysis using the Carbohydrate-Active Enzyme databaseand the Simple Modular Architecture Research Tool suggestedthat AgaO contained only one putative catalytic module (Ser395 toIle602). Phylogenetic analysis showed that AgaO is far distant fromother characterized GH50 �-agarases (Fig. 1B). The results indi-cate that AgaO is a novel agarase obtained from bacteria of thegenus Flammeovirga and that it belongs to a novel branch of theGH50 family to be defined.

Heterologous expression of AgaO in E. coli. The full-lengthagaO gene was amplified from the genomic DNA of Flammeovirgasp. strain MY04. The 2.1-kb DNA product was gel purified andcloned into the vector pBAD/gIII A downstream of a PBAD pro-moter. A secretion peptide from the gIII phage and a 6�His tag,respectively, were individually added to the N and C termini ofthe protein product (rAgaO) in the expression vector (pBAgaO).SDS-PAGE analysis indicated that TOP10 cells harboring the plas-mid pBAgaO could produce soluble recombinant proteins (�120mg/liter) with an appropriate molecular mass (i.e., 85 kDa) (seeFig. S2 in the supplemental material). After sonication and cen-trifugation, soluble crude enzyme was extracted from the E. colicultures. Protein fractions of rAgaO could be eluted from the Ni-NTA column using imidazole at concentrations higher than 50mM. The rAgaO protein was further purified through gel filtra-tion chromatography, if needed. SDS-PAGE analysis indicatedthat the purified soluble protein rAgaO had a purity of �99%, agel recovery of �60%, and an initial concentration of �5 mg/ml(see Fig. S2 in the supplemental material).

To obtain high protein yields, the full-length agaO gene wasalso cloned into the vector pCold TF. BL21(DE3) cells harboringthe resulting plasmid, pCTF-AgaO, could produce solublerTFAgaO at a yield of �1.4 g/liter, nearly 12-fold greater than thatof rAgaO.

Enzymatic characteristics of AgaO. The recombinant pro-teins rAgaO and rTFAgaO did not digest alginate, carrageenan,�-carrageenan, �-carrageenan, cellulose, or xylan but could effi-ciently hydrolyze agar and agarose to produce oligosaccharideproducts. The enzymatic digests of agar and agarose exhibited astrong absorbance at 540 nm in the DNS assay, suggesting thatAgaO is a hydrolase of agarose.

The full-length enzyme rAgaO demonstrated its highest activ-ity at 45°C when agar or agarose was used as the substrate (Fig.2A). The rAgaO enzyme retained more than 80% of its residualactivity after incubation for 2 h at temperatures ranging from 0 to40°C (Fig. 2A). The optimal pH, determined at 45°C in variousbuffers, was 7.0 (Fig. 2B). The enzyme retained more than 80% ofits residual activity after incubation at 4°C for 2 h in environmentswith pHs ranging from 6 to 8 (Fig. 2B).

The enzyme activity of rAgaO was strongly inhibited by mostof the tested chemicals, such as the metal ions Ag , Co2 , andFe3 , at 10 mM or even 1.0 mM (Fig. 2C). The enzyme activity was

inhibited by sodium dodecyl sulfate, phenylmethylsulfonyl fluo-ride, imidazole, and disodium EDTA. In contrast, the enzymeactivity was increased to 124% by MgSO4 (10 mM) and to 140%by glycerol at 5% (vol/vol) (Fig. 2C). Notably, sodium chloride atconcentrations under 0.9 M increased the activity, with optimalactivity (134%) being at �0.2 M (Fig. 2D). The results indicatethat, consistent with its origin from a marine bacterium, the aga-rase AgaO can be activated by Mg2 and Na , common metal ioncomponents in the sea.

Under optimal conditions (45°C, 50 mM NaH2PO4-Na2HPO4,200 mM NaCl, pH 7.0), the specific activity of rAgaO for agarosewas measured as described in Materials and Methods and was�185 U/mg of protein.

Agarose degradation pattern of AgaO. To determine the po-lysaccharide-degrading pattern, the digestion of agarose by rAgaOwas monitored at 45°C. The reaction time varied from 0 to 72 h.The digests (�1 �g) were loaded onto a silica gel plate and subse-quently developed. TLC analysis indicated that rAgaO initiallyproduced a series of even-numbered oligosaccharide productswhich had Rf values similar to those of standard NAO markers(Fig. 3A). Remarkably, rAgaO rapidly converted them into thefinal product, which had the same Rf value as NA2 (Fig. 3A). Theresults suggest that AgaO is a neoagarobiose-producing �-agarase.

Characterization of the disaccharide product. To identify thepredominant oligosaccharide, 100 mg agarose was degraded usingexcess rAgaO, and the digests were fractionated by gel filtrationchromatography. The main fraction, which had a peak retentiontime of 44.6 min in the designated HPLC system (Fig. 3B), wasrecovered as the final oligosaccharide product. A total of �54 mgpure oligosaccharides was obtained from the final digests, indicat-ing that AgaO could convert agarose into oligosaccharides at aratio of nearly 50% (wt/wt).

Approximately 100 ng pure oligosaccharides was used in eachMS analysis. The results indicated that the purified oligosaccha-ride product had a molecular mass of 324. Further MS/MS analy-sis showed a strong signal of the pseudo ion [G Na] at m/z 203and a weak signal of the pseudo ion [A Na] at m/z 185 (Fig. 4A).Thus, the predominant oligosaccharide product yielded by AgaOwas identified to be a disaccharide of agarose composed of A and Gunits. However, direct MS analyses could not determine whetherthe disaccharide product is an agarobiose or a neoagarobiose.

To sequence the disaccharide product, i.e., to determine whichsugar unit, A or G, comprises the reducing end, we fluorescentlylabeled the reducing ends of the purified disaccharide productusing excess 2-AB and then tested the resulting derivative (�50ng) by MS analyses. Initial MS analysis indicated that the signal ofthe 2-AB-labeled disaccharide was m/z 444. Further MS/MS anal-ysis showed strong signals from the 2-AB-labeled G units (m/z323) and associated 2-AB-labeled fragments (m/z 201 and 231)but not from the 2-AB-labeled A unit (Fig. 4B). Thus, the finaldisaccharide product of AgaO was identified to be a neoagarobiosethat had a G unit as the reducing end.

To confirm the results of the above-described fluorescence as-says, �2 mg of the purified disaccharide products was analyzed toobtain a 13C NMR spectrum. The result showed two resonancetypes at �92 and �97 ppm, which indicated the existence of a freeanomeric carbon in the G unit at the reducing end (Fig. 4C) (7).Moreover, only signals from the A units at the nonreducing end(�98 ppm) were found, with no signals from G units at the nonre-ducing end or A units at the reducing end being found (Fig. 4C).

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The results demonstrated that AgaO yields neoagarobiose insteadof agarobiose as the disaccharide product when degrading aga-rose. Thus, AgaO is identified to be a neoagarobiose-producing�-agarase from Flammeovirga sp. strain MY04. Moreover, the 13CNMR spectrum analysis confirmed that the fluorescence assaysare useful in determining the glycoside-type selectivity of a newagarase.

Candidate by-products in the agarose digestions of rAgaO.TLC analysis suggested that during the agarose digestion, rAgaOappeared to have yielded a low proportion of by-products, withthe Rf values being quite different from those of even-numberedNAOs, e.g., NA2, NA4, and NA6 (Fig. 3A). To identify the pro-portion of these abnormal by-products, the final agarose digestsby rAgaO (�1.0 �g) were fluorescently labeled using excess 2-ABand subsequently gel filtered using an HPLC system with fluores-cence detection. The 2-AB-labeled products showed retentiontimes different from those of the 2-AB-labeled NAO markers

(Fig. 3C). According to the peak area, the candidate by-productseach accounted for �0.01 to 0.10% (molar ratio) of the predom-inant product, NA2 (Fig. 3C). The results confirmed that AgaOproduces NA2 as the sole final product in agarose degradation.

Oligosaccharide degradation patterns of rAgaO. To assay thesmallest substrate, oligosaccharides with different chain lengths(e.g., NA2, NA4, NA6, NA8, and NA10) were individually dis-solved in water and then reacted with excess rAgaO at pH 7.0 and45°C for 24 h. TLC analysis indicated that rAgaO could degradeoligosaccharides larger than NA2 in size and produced NA2 as thefinal product (Fig. 5A). Moreover, rAgaO could not further de-grade NA2 to produce any detectable monosaccharide units (Fig.5B). Therefore, the smallest neoagarooligosaccharide substrate ofAgaO is NA4, and the smallest oligosaccharide product is NA2.

To further determine the molar ratios of the NA2 products toeach oligosaccharide substrate, the substrates and the products oftheir final digestions by rAgaO were initially labeled using excess

FIG 2 Biochemical characteristics of the agarase rAgaO. (A) Thermostability of rAgaO and effects of temperature on its enzyme activities. (B) The pH stabilityof rAgaO and effects of pH on its enzyme activities. (C) Effects of various compounds on enzyme activities. PMSF, phenylmethylsulfonyl fluoride; 2-ME,2-mercaptoethanol; DTT, dithiothreitol. (D) Effects of NaCl on enzyme activities.

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2-AB and then analyzed using an HPLC system with fluorescencedetection. According to the peak area, the molar ratios of theNA10, NA8, NA6, NA4, and NA2 substrates to their NA2 prod-ucts were determined to be approximately 5:1, 4:1, 3:1, 2:1, and1:1, respectively (Fig. 5C). The results demonstrate that AgaO can

degrade agarose and associated oligosaccharides completely,yielding neoagarobiose as the final disaccharide product.

To determine the oligosaccharide-degrading pattern of AgaO,the oligosaccharide NA6 was initially labeled using excess 2-AB,subsequently reacted with rAgaO, and monitored over time usingHPLC analysis with fluorescence detection. The agarase rAgaOinitially degraded 2-AB-labeled NA6 into 2-AB–NA4 and unla-beled NA2 and finally converted 2-AB–NA4 into 2-AB–NA2 andunlabeled NA2, thus yielding NA2 and 2-AB–NA2 as the finalproducts (Fig. 5D). Notably, almost 1% (molar ratio) of the sub-strate 2-AB–NA4 could not be hydrolyzed completely to releasethe NA2 and 2-AB–NA2 products, even if the enzyme amount orthe reaction time was doubled (Fig. 5D). This suggested that the2-AB label at the reducing end of NA4 caused a stereospecificblockade and thus weakly inhibited the enzymatic degradation byrAgaO. The results demonstrate an exo-type disaccharide-yield-ing property of the �-agarase AgaO, which releases neoagarobioseby gradually cleaving the units from the nonreducing end of 2-AB-labeled oligosaccharides.

Because AgaO shares low homology with the �-galactosidaseVadG925 of Victivallis vadensis strain ATCC BAA-548, two differ-ent disaccharides, lactose and neoagarobiose, were used as thesubstrates to assay the galactosidase activities. The former disac-charide contained a G unit at its nonreducing end, while the lattercontained the G unit as its reducing end. TLC (Fig. 5B) and HPLCanalyses with fluorescence detection (Fig. 5C) indicated that, un-like the �-galactosidase VadG925 (39), rAgaO could not degradethe disaccharide lactose to release any monosaccharide units ofD-galactose or D-glucose. Thus, AgaO is a neoagarobiose-produc-ing �-agarase without detectable galactosidase activities.

Agarose bioconversion properties of the truncated proteins.To identify functional modules within the protein AgaO, variousgene fragments were generated via PCR, using the full-lengthAgaO gene as the DNA template. Five truncated proteins weresuccessfully generated, i.e., three proteins (TC-D344, TC-A50,and TC-626) with deletion of the N-terminal noncatalytic regionfrom AgaO and two recombinant proteins (TN-F164 and TN-N350) containing the full-length putative catalytic module (Fig.1A and Table 1). The crude enzyme preparations containing eachtruncated protein were initially reacted with agarose at 30°C to45°C for 12 h, and the digestions were assayed. DNS assays showedthat three of the truncated proteins, i.e., TC-626, TN-F164, andTN-N350, weakly produced reducing sugars. However, furtherTLC analysis indicated that all five protein preparations lost theability to produce NA2 as products (negative results; data notshown). Therefore, the putative catalytic module is not the solefunctional element required for the full-length enzyme AgaO toproduce disaccharide products. Moreover, it is suggested that theinteraction between the putative modules is essential for the�-agarase AgaO to act in an exo-type pattern, which requires strictstructure-function coordination.

DISCUSSION

Because of the excellent agarose degradation and utilization capa-bilities of Flammeovirga strains, various agarase preparations,such as crude extracellular enzymes (29) and pure recombinantagarases (27, 31, 32), have been investigated. And all of these en-zyme preparations yielded NA4 rather than NA2 as the smallestoligosaccharide product. Recently, we have sequenced and anno-tated the genome of a polysaccharide-degrading marine bacte-

FIG 3 Polysaccharide degradation pattern of the agarase rAgaO. (A) TLCanalysis of the degradation products of agarose (1.0 mg/ml) by rAgaO (0.1U/ml) at 45°C. Lane M, standard oligosaccharide markers; lane (), negativecontrol. (B) HPLC analysis of the final agarose digests by rAgaO at 45°C for 72h. The oligosaccharide products were purified with a Superdex peptide 10/300GL column monitored by use of a parallax detector. (C) HPLC analysis withfluorescence detection of the final agarose digests by rAgaO at 45°C for 72 h.The final products were 2-AB labeled, purified by gel filtration, and monitoredat an excitation wavelength of 330 nm and a monitoring wavelength of 420 nm.M1, M2, M4, M6, and M8, 2-AB-labeled oligosaccharide markers of D-galac-tose, NA2, NA4, NA6, and NA8, respectively. The oligosaccharide by-productsin the final agarose digestions are indicated by asterisks.

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rium, Flammeovirga sp. strain MY04, to identify the genes in-volved in diverse polysaccharide metabolic pathways (41). Inaddition to a previously reported GH16 �-agarase, AgaG4, whichyielded NA4 and NA6 as the final oligosaccharide products (32),

we have identified another candidate gene (agaO) in the MY04genome. Interestingly, due to its novel sequence properties andlow homologies to previously identified enzymes, AgaO appearedto be an exo-type GH50 �-agarase, while it contains a putative

FIG 4 Identification of the disaccharide product yielded by rAgaO. (A) Direct MS/MS analysis of the purified disaccharide product (m/z 324). (B) MS/MS analysis of2-AB-labeled disaccharide product (m/z 444). (C) 13C NMR spectrum of the purified neoagarobiose product. Inten, intensity; A, the 4-O-linked 3,6-anhydro-�-L-galactopyranose; G, the 3-O-linked �-D-galactopyranose; r and nr, residues at the reducing and nonreducing ends, respectively; �/�, the respective anomer.

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catalytic module (Fig. 1A and B). In this study, we expressed theAgaO protein in E. coli cells to obtain soluble proteins (see Fig. S2in the supplemental material) for enzyme characterization. Therecombinant enzyme rAgaO was identified as a disaccharide-producing �-agarase but not a galactosidase of the Flammeo-virga genus.

During the process of agarose degradation by rAgaO, we alsofound a series of candidate by-products in the final oligosaccha-ride products by TLC and HPLC analyses with fluorescence detec-tion (Fig. 3A and C). These products had very low molar propor-tions in the final agarose degradation products (Fig. 3C), even ifwe increased the amount of enzyme by 2-fold or doubled thereaction time. However, similar products were not found in thefinal digests of various size-defined NAO substrates (Fig. 5A andC). Because AgaO is an agarase without galactosidase activities, itis suggested that the candidate by-products are not oligosaccha-rides produced by rAgaO via any glycosyl transferase activity butthat they are complex components of agarose, e.g., sulfated oligo-saccharide units, and indigestible by rAgaO.

The 13C NMR spectrum analysis of oligosaccharide productscan easily distinguish AOs from NAOs by analyzing the 13C NMRsignals of the anomeric carbon, which belongs to the A or G unit atthe reducing end and thus determines the � or � hydrolysis pat-tern of agarases (6, 7). However, the NMR analysis requires oligo-saccharide products with a purity of �99% and is efficient at amass grade of a milligram, which is time-consuming and expen-sive. MS/MS analysis can easily distinguish unit A from unit G byassaying the molecular masses (Fig. 4A). Moreover, it does notrequire purification of the target oligosaccharide products, whileit is efficient at a lower mass grade of a microgram or even ananogram. However, direct MS and subsequent MS/MS analyseswere insufficient to determine the reducing end of the final disac-charide product (Fig. 4A and B). To solve the problem, we initiallylabeled the reducing end of the disaccharide product using 2-AB,followed by MS/MS analysis. In the MS/MS spectrum, a mainpeak of 2-AB–G (m/z 323) without any 2-AB–A peaks was de-tected (Fig. 4B). Thus, although it has a low molecular mass, thefinal disaccharide product produced by rAgaO was easily demon-strated to be NA2 with a G unit as the reducing end, which isconsistent with the result of the 13C NMR spectrum analysis (Fig.4C). This strategy can also be used to identify larger oligosaccha-ride products (42) and therefore is very useful for rapidly deter-mining the glycoside cleavage patterns of the corresponding en-zymes.

Agarase can degrade agarose by either an endolytic or an exo-lytic pattern. Numerous disaccharide-producing agarases havebeen well characterized with regard to their biochemical charac-teristics and agarose degradation products (22, 23, 26, 33). How-ever, only one NA2-producing �-agarase, Aga50D of S. degradansstrain 2-40, which has the exolytic pattern, determined by crystalstructure analysis of the protein-oligosaccharide complex, hasbeen reported (26). Therefore, biochemical demonstration ofthe exolytic pattern of agarases is still needed. In the presentstudy, through the use of fluorescence labeling and furtherHPLC analyses, we demonstrated that AgaO is an NA2-pro-ducing exolytic agarase. First, when degrading agarose or associ-ated oligosaccharides, rAgaO always produces NA2 as the solefinal product (Fig. 3C and 5C). Second, when degrading 2-AB-labeled oligosaccharides, rAgaO releases the NA2 product bygradually cleaving the unit from the nonreducing end of oligosac-

FIG 5 Pattern of oligosaccharide degradation by the agarase rAgaO. (A) TLCanalysis of the final digests of NA2, NA4, NA6, NA8, and NA10. Lane M,standard NAO markers; lanes 1, 3, 5, 7, and 9, control groups; lanes 2, 4, 6, 8,and 10, groups reacting with rAgaO. (B) TLC analysis of the reaction productsof lactose with rAgaO. Lane 1, D-galactose; lane 2, D-glucose; lane 3, controlgroup for lactose; lane 4, lactose degradation by rAgaO. (C) HPLC analysiswith fluorescence detection of the final digests of lactose and NAOs. Each finalproduct was 2-AB labeled and analyzed by HPLC. (D) HPLC analysis withfluorescence detection of the exo-type degradation pattern of rAgaO. Theoligosaccharide NA6 was initially 2-AB labeled and then reacted with rAgaO.

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charide chains (Fig. 5D). To the best of our knowledge, the secondfinding represents novel biochemical visualization data on theexolytic pattern of an agarase.

Conclusion. AgaO is a disaccharide-producing �-agarase ob-tained from agarolytic bacteria of the genus Flammeovirga. It be-longs to a novel branch of the GH50 family to be defined. Theenzyme can efficiently degrade agarose and associated oligosac-charides into neoagarobiose by sequential digestion from thenonreducing end of sugar chains. Moreover, this study has pro-vided a novel biochemical visualization of the exolytic pattern ofan agarase. The fluorescence assays will be helpful for demonstrat-ing the substrate-degrading and product-yielding patterns of var-ious agarases.

ACKNOWLEDGMENTS

We declare that we have no competing interests.W.H. designed the study under the guidance of F.L. F.L. and W.H.

drafted and corrected the manuscript. W.H., Y.C., D.W., S.W., H.L., J.G.,and Z.W. carried out the experiments. All authors approved the finalmanuscript.

FUNDING INFORMATIONThis work was financially supported by the Major State Basic ResearchDevelopment Program of China (grant no. 2012CB822102), the NationalNatural Science Foundation of China (grant no. 31300664 and31570071), and the State Key Laboratory of Microbial Technology ofShandong University (grant no. M2013-11).

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