APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 2010, p. 6760–6768 Vol. 76, No. 200099-2240/10/$12.00 doi:10.1128/AEM.00758-10Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Discovery and Characterization of a Distinctive Exo-1,3/1,4-�-Glucanasefrom the Marine Bacterium Pseudoalteromonas sp. Strain BB1�†

Yoshio Nakatani, Iain L. Lamont, and John F. Cutfield*Biochemistry Department, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand

Received 25 March 2010/Accepted 12 August 2010

Marine bacteria residing on local red, green, and brown seaweeds were screened for exo-1,3-�-glucanase(ExoP) activity. Of the 90 bacterial species isolated from 32 seaweeds, only one, a Pseudoalteromonas sp., wasfound to display such activity. It was isolated from a Durvillaea sp., a brown kelp known to contain significantamounts of the storage polysaccharide laminarin (1,3-�-D-glucan with some 1,6-� branching). Four chromato-graphic steps were utilized to purify the enzyme (ExoP). Chymotryptic digestion provided peptide sequences forprimer design and subsequent gene cloning. The exoP gene coded for 840 amino acids and was located just 50bp downstream from a putative lichenase (endo-1,3-1,4-�-glucanase) gene, suggesting possible cotranscriptionof these genes. Sequence comparisons revealed ExoP to be clustered within a group of bacterial glycosidaseswith high similarity to a group of glycoside hydrolase (GH3) plant enzymes, of which the barley exo-1,3/1,4-�-glucanase (ExoI) is the best characterized. The major difference between the bacterial and plant proteins isan extra 200- to 220-amino-acid extension at the C terminus of the former. This additional sequence does notcorrelate with any known functional domain, but ExoP was not active against laminarin when this region wasremoved. Production of recombinant ExoP allowed substrate specificity studies to be performed. The enzymewas found to possess similar levels of exoglucanase activity against both 1,4-� linkages and 1,3-� linkages, andso ExoP is designated an exo-1,3/1,4-�-exoglucanase, the first such bacterial enzyme to be characterized. Thisbroader specificity could allow the enzyme to assist in digesting both cell wall cellulose and cytoplasmiclaminarin.

It is well recognized that marine bacteria provide a huge iflargely untapped resource for discovery of potentially usefulcompounds, including biopharmaceuticals and novel enzymes(8, 9, 24, 27, 39). Many marine bacteria reside on seaweeds(multicellular algae) and are able to produce secreted enzymesthat can act upon algal components, in particular, the complexarray of polysaccharides present among the various classes ofseaweed (18, 33, 37). These carbohydrates in general playimportant roles as structural materials and as food reserves.Seaweed cell walls possess a fibrillar component typically com-prising variable amounts of cellulose, xylan, and mannan andan amorphous component which differs in composition amongred, green, and brown algae (34). The galactans agar andcarrageenan are predominantly found in the amorphous com-ponent of red algae, while alginates and fucoidin characterizethe cell walls of brown algae (25). Major storage polysaccha-rides include starch and amylopectin in green algae, florideanstarch in red algae, and laminarin (also known as laminaran) inbrown algae (25, 30). In addition to being a food source,seaweed polysaccharides have a number of commercial usesdue to their thickening and gelling properties, while a range ofbiological activities has been claimed for various preparationsof sulfated polysaccharides (36).

Glycosidases (glycoside hydrolases [GHs]) catalyze the hy-

drolysis of glycosidic linkages between two or more carbohy-drates or between a carbohydrate and a noncarbohydrate com-ponent and have been categorized into over 100 GH familieson the basis of their amino acid sequences and predicted struc-tural similarities (14). These widespread enzymes play impor-tant biological roles in carbohydrate metabolism, morphogen-esis, pathogenesis, and defense mechanisms (7). Some havefound utility in semisynthetic carbohydrate chemistry (5, 32).While many have been characterized biochemically, most gly-coside hydrolases are identified and annotated from gene se-quences; thus, their specific biological function is frequentlynot known or fully understood. A variety of marine bacterialglycoside hydrolases that recognize glucosyl residues in �-linked glycans have been characterized, including endo-1,3-�-glucanases, sometimes reported as laminarinase activities, and�-glucosidases. However, no exclusively exo-1,3-�-glucanase(ExoP) activity has, to our knowledge, been identified in bac-teria, although such enzymes are present in fungi and yeast(22). Using bacteria isolated and cultured from local seaweeds,we adopted a selection/screening process in order to identifyan exo-1,3-�-glucanase. We have isolated a protein displayingthe activity screened for, cloned its gene, and from sequenceand functional analyses established that it is similar to butdistinct from a GH3 subfamily of plant exo-1,3/1,4-�-glu-canases by virtue of a C-terminal extension.

MATERIALS AND METHODS

Chemicals and enzymes. Laminarin, p-nitrophenol (pNP), p-nitrophenyl�-D-glucopyranoside (pNP-Glc), p-nitrophenyl �-D-xylopyranoside (pNPX),4-methylumbelliferone (MU), 4-methylumbelliferyl �-D-glucopyranoside(MU-Glc), and 4-methylumbelliferyl �-D-xylopyranoside (MUX) were fromSigma. Laminaribiose, laminaritriose, laminaritetraose, laminaripentaose,

* Corresponding author. Mailing address: Biochemistry Depart-ment, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand.Phone: 64 3 4797836. Fax: 64 3 4797866. E-mail: john.cutfield@otago.ac.nz.

† Supplemental material for this article may be found at http://aem.asm.org/.

� Published ahead of print on 20 August 2010.

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laminarihexaose, cellobiose, cellotriose, cellotetraose, cellopentaose, and cel-lohexaose were from Seikagaku (Tokyo, Japan). Gentiobiose was from Bio-chemika. Curdlan was kindly provided by Takeda. Tryptone, Bacto peptone,casein hydrolysate, yeast extract, and Casamino Acids were from Difco.Platinum Taq DNA polymerase and all restriction enzymes were from In-vitrogen. T4 DNA ligase and Klenow polymerase were from Roche. Glucoseoxidase, horseradish peroxidase, and �-chymotrypsin were from Sigma.

Bacterial culture, growth media, and isolation of bacteria. For isolation andgrowth of marine bacteria, prescreening medium (PSM), a first screening me-dium (1SM), a second screening medium (2SM), and medium B of Alexeeva etal. (2) were used. PSM consisted of a mixture of 50% (vol/vol) Milli-Q water and50% (vol/vol) seawater from the Otago Coast, New Zealand. 1SM contained0.5% (wt/vol) curdlan, 0.1% (wt/vol) Casamino Acids, 0.1% (wt/vol) yeast ex-tract, and 0.05% (wt/vol) potassium phosphate in a mixture of 25% (vol/vol)Milli-Q water and 75% (vol/vol) seawater. 2SM consisted of 0.3% (wt/vol) curd-lan, 0.1% (wt/vol) Casamino Acids, and 0.05% (wt/vol) potassium phosphate ina mixture of 50% (vol/vol) Milli-Q water and 50% (vol/vol) seawater. Unlessstated otherwise, marine bacteria were grown at 28°C on a rotary shaker at 200to 240 rpm. Escherichia coli was grown at 37°C in LB broth supplemented withampicillin (100 �g/ml) and chloramphenicol (34 �g/ml). Thirty-two differentseaweeds were identified and collected locally over the course of a year. Smallpieces of each sample were collected in sterile universal bottles containing 10 mlPSM and incubated for 2 days. Each PSM culture (100 ml) was added to fresh1SM (10 ml) and incubated for 3 days, after which 100 �l was added to fresh 2SM(10 ml) and incubated for a further 3 days. This step was repeated, and then theculture was diluted 1,000-fold in PSM and 100 �l was spread onto a 2% agarplate containing 2SM. The plate was incubated at 28°C until colonies formed.Following enzymatic screening of isolated bacteria (below), genomic DNA(gDNA) was extracted using a PureLink genomic DNA purification kit (Invitro-gen). PCR was carried out following the procedure of Weisburg et al. (38) toamplify the 16S rRNA gene for subsequent bacterial identification. The PCRproduct was purified using a PureLink PCR purification kit (Invitrogen) prior toDNA sequencing.

Enzymatic screening of isolated bacteria. Each culturable strain was grown in10 ml of 2SM at 28°C for 3 days. The culture was frozen, thawed, and vortexedto disrupt the cells, and this procedure was repeated twice. After centrifugationat 10,000 � g for 10 min, cell lysate (0.1 ml) was tested for hydrolysis activityagainst 1,3-�-glucans, either curdlan or laminarin, at 0.5% (wt/vol) in 100 mMsodium phosphate, pH 7.0 (total volume, 0.5 ml). The assay was carried out at37°C for 1 h and stopped by boiling the mixture for 10 min. The sample wascentrifuged, and the supernatant was recovered for use in the assays. Freeglucose was measured using a modification of the glucose oxidase assay of Brussand Black (6). A 50-�l sample was mixed with 200 �l of the glucose oxidasereagent in a microtiter plate. Following incubation at 37°C for 30 min, the A562

was measured in an EL-340 microplate biokinetics reader (Bio-Tec Instruments).Reducing sugar was measured using the modified Somogyi-Nelson assay (29). A200-�l sample was mixed with 200 �l of Somogyi reagent, and the mixture wasboiled for 10 min. After the mixture was cooled to ambient temperature, 200 �lof Nelson reagent was added. This mixture was diluted by addition of 1.4 ml ofwater, and then 200 �l of sample was transferred to a microtiter plate to measurethe A620. To determine �-glucosidase activity, a 100-�l sample of cell lysate wasadded to 400 �l of 100 mM sodium phosphate (pH 7.0) containing 300 �g ofpNP-Glc and the mixture was incubated at 37°C for 1 h. The reaction wasstopped by addition of 0.5 ml of 0.5 M sodium carbonate. A 200-�l volume wastransferred to a microtiter plate to measure the A405. For the curdlan methyl blueplate assay, Pseudoalteromonas sp. strain BB1 was grown in 50 ml of 2SM at 28°Cfor 1 day. The culture was diluted 10- and 100-fold in a mixture of 50% seawaterand 50% water. The undiluted and diluted cultures (10 �l) were spread onto a2% (wt/vol) agar plate containing 2SM and 0.005% (wt/vol) methyl blue as anindicator of 1,3-�-glucan presence (21), and the plate was incubated at 28°C.

Optimal growth conditions. A freshly grown single colony of Pseudoalteromo-nas sp. strain BB1 on a medium B plate was inoculated in 50 ml of medium B (pH6.0) and grown at 28°C to an optical density at 600 nm (OD600) of �0.2. A 2-mlsample of this culture was diluted in 18 ml of PSM. For determination of optimalgrowth pH, 100 �l diluted culture was added to 50 ml of fresh medium B (pH 5.5to 9.0) and incubated at 28°C. To determine the optimal growth temperature, 100�l diluted culture was added to 50 ml fresh medium B (pH 6.0) and incubated at4, 14, 22, 28, and 37°C. The OD600 was monitored for bacterial growth.

Purification of native ExoP. Pseudoalteromonas sp. strain BB1 was grown at28°C in 1.6 liters medium B (OD600, 1.9 to 2.2, stationary phase). Preparation ofExoP was carried out at 4°C. Buffer exchange and concentration of the sampleused Vivaspin 20-ml concentrators (30-kDa cutoff; Vivascience). Chromato-graphic procedures were performed at a flow rate of 1.0 ml min�1, except where

stated otherwise. Cells were harvested by centrifugation, and the pellet wasresuspended in 100 ml of 20 mM Tris-HCl (pH 7.0) containing 300 mM NaCl.Cells were broken by ultrasonic treatment. The cell lysate supernatant wasextensively dialyzed against 50 mM Tris-HCl, pH 7.0, and then 160 ml was loadedonto a DEAE Sepharose CL6B column (2 � 10 cm; Pharmacia) equilibratedwith 50 mM Tris-HCl, pH 7.0. Elution was performed with a linear gradient of0 to 100% 1.0 M NaCl in 50 mM Tris-HCl (pH 7.0) for 3 h. Fractions (5 ml) werecollected, and those that showed ExoP activity by laminarin/glucose oxidase(GO) assay were pooled and concentrated 2- to 3-fold, prior to gel permeationchromatography. A 3- to 4-ml volume of the concentrated sample was loadedonto a Superdex 200 HR16/60 column (Pharmacia) equilibrated with 50 mMTris-HCl (pH 7.0) and 150 mM NaCl, and elution was performed using the samebuffer. Fractions (3 ml) were collected, and those with activity were pooled andammonium sulfate (final concentration, 1.5 M) was added, prior to hydrophobicinteraction chromatography. A sample (5 ml) was loaded onto a Phenyl SuperoseHR5/5 column (Pharmacia) equilibrated with 20 mM sodium phosphate (pH 7.0)containing 1.5 M ammonium sulfate. Elution was performed using a reverselinear gradient of 1.5 to 0 M ammonium sulfate in 20 mM sodium phosphate (pH7.0) over 30 min at a flow rate of 0.5 ml min�1. Active fractions (1 ml) werepooled, and buffer exchange was performed with 50 mM Tris-HCl (pH 7.0), priorto Mono Q chromatography. The sample was then loaded onto a Mono Q HR5/5column (Pharmacia) equilibrated with 20 mM Tris-HCl (pH 7.0). Elution wasperformed using a two-step linear gradient of NaCl. The first gradient wasapplied using 0 to 0.5 M NaCl over 20 min, and the second was applied imme-diately after the first gradient using 0.5 to 1 M NaCl for 5 min. Purified ExoP waspooled, buffer exchange was performed with 50 mM sodium phosphate (pH 7.0)and 100 mM NaCl, and ExoP was stored at 4°C.

Identification and cloning of exoP. Peptide sequences on which to base PCRexperiments were derived from isolated native ExoP following in-gel digestion(26), as described in the supplemental material. For exoP gene identification, thefirst PCR step was performed using degenerate primers based on the N-terminalsequence of the intact protein (4.2 �M, 5�-ACIGARCAYGARCARGTIAAYTGGCCITA-3�) and the internal sequence TDAMHGHSN (4.2 �M, 5�-TTISWRTGICCRTGCATIGCRTCIGT-3�) together with 100 ng of Pseudoalteromonassp. strain BB1 genomic DNA in a 20-�l reaction mixture containing 20 mMTris-HCl (pH 8.4), 1.5 mM MgCl2, 0.2 mM deoxynucleoside triphosphates, and2.5 U of Platinum Taq polymerase (Invitrogen). The initial denaturation step wasfor 2 min at 94°C, followed by 30 cycles of amplification (30 s at 94°C, 30 s at56°C, and 1.5 min at 72°C). A final extension step was carried out at 72°C for 5min. The second PCR experiment was performed in the same way as the first onebut used the exoP gene-specific forward primer (0.5 �M, 5�-CAGCAATGGACTCAACCCTT-3�) and the reverse degenerate primer based on the conservedC-terminal region (AFVAAWLPG) of GH3 family members (3.5 �M, 5�-CCIGGIARCCAIGCIGCIACRAAIGC-3�). Southern hybridization, colony hybrid-ization, and DNA cloning were carried out as described by Sambrook et al. (28).

A gene probe for exoP was made using 60 ng of the second-round PCR productas a template. A total of 750 ng of 3- to 5-kbp genomic DNA fragments wascloned into 200 ng of pUC18 (Stratagene). The colonies were then transferred toHybond-N� membranes (Amersham). Plasmids were purified using a PureLinkQuick plasmid miniprep kit (Invitrogen). To produce recombinant ExoP, theexoP gene was amplified from genomic DNA of Pseudoalteromonas sp. strainBB1 using primers 5�-CATGCCATGGCAGAGCATGAGCAAGTCAATTGG-3� and 5�-CCGCTCGAGCTTGGCACACGTTAATGATATTGA-3�. Theinitial denaturation step was 4 min at 94°C, followed by 30 cycles of amplification(1 min at 94°C, 1 min at 56°C, and 2.5 min at 72°C). A final extension step wascarried out at 72°C for 5 min. The PCR product was digested using NcoI andXhoI (the underlined sites) and cloned into pET21d (Novagen). The expressionconstruct (plasmid) was transformed into E. coli BL21(DE3)/pLysS.

Purification of recombinant ExoP. Transformed E. coli was inoculated, induplicate, in 500 ml of LB broth containing appropriate antibiotics and grown at37°C to reach an OD600 of approximately 0.3. After the cell culture was cooledat 4°C for 10 min, isopropyl-�-D-thiogalactopyranoside (final concentration, 0.5mM) was added to the culture medium and incubation was carried out at 18°C.Approximately 1 day later, the OD600 of the cell cultures reached 1.6 to 1.8. Cellswere harvested by centrifugation and resuspended in 100 ml of 50 mM Tris-HClbuffer, pH 7.0, containing 300 mM NaCl. Cell lysate was prepared as describedabove for native ExoP. Immobilized metal ion affinity chromatography (IMAC)was performed using 3 ml Talon resin (BD Biosciences) packed and equilibratedwith a 10 times volume of 50 mM Tris-HCl (pH 7.0) containing 300 mM NaCl.Cell lysate was loaded and thoroughly washed with 30 ml of 50 mM Tris-HCl (pH7.0) containing 1 M NaCl. Recombinant ExoP was eluted using 30 ml of 50 mMTris-HCl (pH 7.0) containing 300 mM NaCl and 50 mM imidazole. After puri-

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fication by IMAC, recombinant ExoP was purified as described for native ExoP,except without the initial DEAE purification step.

Enzyme assays. To detect exo-1,3-�-glucanase activity in chromatography frac-tions, the hydrolysis assay employed 0.5% (wt/vol) laminarin as substrate in atotal volume of 120 �l containing 100 mM sodium phosphate (pH 7.0) and 100mM NaCl. The reaction was stopped by boiling for 10 min. Free glucose wasmeasured as described above. For determination of the optimal pH, the lami-narin assay was carried out using 0.04 �g of recombinant ExoP in a total volumeof 120 �l using various 100 mM buffers at 30°C for 3 to 4 h. For determinationof the optimal temperature, the laminarin assay was carried out using 0.04 �g ofrecombinant ExoP in a total volume of 120 �l containing 100 mM sodiumphosphate (pH 7.0) at various temperatures for 2 h. Free glucose was measuredas described above using glucose oxidase. Kinetic parameters for various oligo-saccharide substrates were determined from rate data measured at 30°C using 0.7to 16.6 pmol of recombinant ExoP in a total volume of 120 �l containing 100 mMsodium phosphate (pH 7.0) and 100 mM NaCl. Reactions were stopped byboiling for 10 min, and the glucose released was measured as described. ForpNP-Glc and MU-Glc, reactions were stopped by adding 10 �l of 0.5 M sodiumcarbonate or 10 �l of 0.5 M glycine-NaOH, pH 10.3, to a 50-�l aliquot. Thereleased pNP was measured at A405, while the fluorescence of MU was measuredat 384 nm (excitation) and 450 nm (emission). All kinetic data were analyzed byPrism (version 5) software (GraphPad Software, Inc.). The hydrolysis reactionsof several polysaccharides were also tested. A total of 120 �l reaction mixture,which contained 5 mg ml�1 substrate and 0.0625 �g of ExoP in 100 mM sodiumphosphate buffer, pH 7.0, and 100 mM NaCl, was incubated at 30°C for 30 min.Subsite affinities were calculated according to published procedures (15, 31).

Comparison of bacterial and plant GH3 sequences. Protein sequences (e-value cutoff, 1e�54) were aligned using the ClustalW program (20) with defaultsettings. To create an unrooted tree, the Clustal output was read into thesoftware package Geneious, and a neighbor-joining tree was constructed usingthe Geneious default settings.

Nucleotide sequence accession number. The 7,162 bp of DNA comprising theexoP gene sequence and two partial gene sequences was submitted to GenBankand is found under accession number DQ361032.

Protein Data Bank accession number. The structure of ExoP may be found inthe Protein Data Bank under accession code 3F93.

RESULTS

Identification of a marine bacterial isolate possessing puta-tive exo-1,3-�-glucanase activity. A total of 32 seaweed sam-ples representing green, red, and brown algae were collectedfrom shallow seawater (8 to 14°C) at a local beach under bothsummer and winter conditions. Following an initial wash step,the associated bacteria were incubated under progressivelymore defined conditions (see Materials and Methods) andspread on plates containing curdlan (1,3-�-glucan) as the ma-jor carbon source. Up to three colonies from each plate werethen selected on the basis of fast growth. Enzymatic screeningof cell lysates from a total of 90 marine bacterial isolates usingthree different glycosidase assays showed that only one of thestrains displayed putative exo-1,3-�-glucanase activity, by vir-tue of a positive glucose oxidase assay result following incuba-tion with laminarin, but negligible glucosidase activity. Thepositive result from the Somogyi-Nelson assay did not distin-guish exo-1,3-�-glucanase from endo-1,3-�-glucanase activityfor this isolate. Interestingly, many other species displayedglucosidase activity but not exo-1,3-�-glucanase activity. Thecandidate bacterial species was isolated from the summer sea-weed collection and specifically from the brown alga Durvillaeasp. However, eight other types of brown seaweed did not har-bor bacteria with exoglucanase activity. The ability of this ma-rine bacterium to hydrolyze 1,3-�-glucan in the form of insol-uble curdlan was visualized on methyl blue-impregnated plates(Fig. 1), and is likely to be the result of a combination of endo-and exo-acting enzymes. The marine bacterial isolate was iden-

tified as a Pseudoalteromonas species using 16S rRNA geneanalysis. Pseudoalteromonas was officially classified as a newgenus in 1995, on the basis of phylogenetic comparisons (12).

Purification and identification of the ExoP enzyme. Opti-mized growth conditions for the bacterium were established inorder to improve the yield of the exo-1,3-�-glucanase (ExoP).Figure 2A and B shows that the optimal growth temperaturewas close to 28°C, with no growth being observed at 4°C or at37°C over a period of 3 days. The optimum pH was in the rangeof 6.0 to 8.0 but favored slightly acidic conditions rather thanthe slightly alkaline pH of seawater. Relatively low exoglu-canase activity was detected in culture supernatant, but celllysate supernatant possessed significantly higher activity. Acombination of anion-exchange, gel permeation, and hydro-phobic interaction chromatographies (see Materials and Meth-ods) allowed purification of the enzyme to homogeneity, albeitat a low yield. The gel permeation step that followed the initialDEAE Sepharose chromatography was needed to separate outa major protein contaminant, subsequently identified by N-terminal sequence analysis to be an arylsulfatase. The finalyield of ExoP was only about 50 �g from a 10-liter culture.SDS-PAGE indicated that ExoP migrated at about 80 kDa (seethe data in the supplemental material). Modified Rosenfeldin-gel digestion allowed two pure peptides (GTDAMHGHSNVY and ESYSEDP) to be sequenced. The protein wasidentified as a likely member of the GH3 family of glycosidehydrolases following a BLASTP search (3), as these sequencesfortuitously corresponded to conserved regions in this family.

Gene identification. Primers based on the N-terminalsequence of the intact protein (AEHEQVNWPYVNTK . . .),together with an internal sequence, derived as describedabove, and inosine at sites of 4-fold base redundancy, wereused in a PCR to obtain a 350-bp product. With an N-terminalsequence of 118 amino acids now determined, the proteincould be assigned to the GH3 family with reasonable certainty.On the basis of this assumption, the most C-terminal conservedsequence region in this family was identified for utilization inanother PCR, utilizing a new 3� primer, to produce a 1.3-kbproduct and a deduced protein sequence of 417 amino acids.As this represented only about half of the predicted length ofabout 800 amino acids, Southern hybridization was employed

FIG. 1. Curdlan hydrolysis by Pseudoalteromonas sp. strain BB1 inthe presence of methyl blue. Sectors: 1, no bacteria; 2, 1/100 dilution;3, 1/10 dilution; 4, undiluted sample (OD600 1.0).

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to identify gDNA fragments containing the rest of the gene.Two clones provided not only the missing exoP gene sequencebut also considerable sequence data on both the 5� and 3�flanks. Altogether, 7,162 bp of DNA sequence (GenBank ac-cession no. DQ361032) was obtained, revealing four completegenes, including exoP and two partial genes, as schematized inFig. 3A. The putative lichenase gene (licA) was of interest, asit also codes for a glycoside hydrolase and is located only 50 bpupstream of the exoP gene. This LicA enzyme, comprising 258

amino acids, is a member of the GH16 family and is presumedto hydrolyze linear 1,3-1,4-�-glucan specifically at 1,4 linkagesin an endo manner.

The exoP gene consists of 2,523 nucleotides which translateinto an 840-amino-acid sequence, including a 27-amino-acidsignal sequence (Fig. 3B), as determined by the programSignalP (4) and confirmed by N-terminal sequencing of theisolated mature protein. The mature protein (813 amino acids)has a calculated molecular mass of 89,320 Da. A BLASTP

FIG. 2. Comparison of optimal pH and temperature for growth of Pseudoalteromonas sp. strain BB1 and for ExoP enzyme activity. (A and B)Bacterial growth curves over pH and temperature, respectively, using medium B; (C and D) enzyme activity profiles for purified recombinant ExoPagainst pH and temperature, respectively. The assay system used glucose oxidase to detect the glucose released from the nonreducing end oflaminarin. The buffer employed for the temperature study was 0.1 M sodium phosphate, pH 7. Note that Tris and HEPES were inhibitory.

FIG. 3. (A) Schematic diagram of the gene arrangement in the 7,162 bp of DNA sequence obtained following PCR amplification and Southernhybridization. The putative lichenase gene (licA) is located only 50 bp upstream of the exoglucanase gene (exoP). The exoP gene contained 2,523nucleotides, which translates into a protein of 840 amino acids. (B) Schematic representation of the ExoP protein structure showing signal peptide(S), N-terminal domain (A), central domain (B), and C-terminal domain (X). Barley ExoI is shown for comparison.

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search confirmed that ExoP is indeed a member of the GH3family, which includes subfamilies of plant and bacterial exo-�-glucanases and �-glucosidases, as shown by sequence com-parison analysis (Fig. 4).

Recombinant ExoP. Given the low yields of native ExoP andthe desire to determine the biochemical and structural prop-erties of the enzyme, the gene was expressed as a His-taggedrecombinant protein. The optimum temperature for obtaining

the best yield of soluble protein was found to be 18°C. Recom-binant protein was deemed pure by SDS-PAGE and nativePAGE analysis and was essentially equivalent to native ExoP,allowing for the slight increase in molecular mass due to theaddition of 9 extra amino acids (N-terminal Met, C-terminalLeu-Glu-His6). Further comparisons of native and recombi-nant ExoP confirmed that they were virtually identical, as far asbiochemical properties (pH optimum, temperature optimum,

FIG. 4. Sequence comparison of GH3 plant and bacterial exo-�-glucanases (subgroups 1 and 2, respectively [13]). Domains A and B were usedin a BLASTP search, and matched sequences were extracted and aligned using the ClustalW program. The unrooted tree was made using theneighbor-joining method. Plant enzymes are circled; ExoP is highlighted. Some of these enzymes have been assigned EC numbers 3.2.1.x, wherex is 21 (�-glucosidase), 58 (exo-1,3-�-glucanase), 74 (exo-1,4-�-glucanase), or hyphen (exo-1,3/1,4-�-glucanase).

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and Km and kcat values) are concerned, when laminarin wasused as the substrate (data not shown). With this assurance,more detailed kinetic and specificity studies were carried outusing the recombinant protein, as it could be prepared in largerquantities.

Activity and specificity studies of ExoP. Profiles of enzymeactivity, using laminarin as the substrate, over a range of pHsand temperatures are shown in Fig. 2C and D. With an opti-mum pH of 7.0 and an optimum temperature of 30°C, theenzyme was observed to be compatible with the optimal growthconditions of Pseudoalteromonas sp. strain BB1 (Fig. 2A andB). Table 1 summarizes the kinetic data for a variety of lami-nari-oligosaccharide (1,3-�-linked) and cello-oligosaccharide(1,4-�-linked) substrates and two aryl �-glucosides. No hydro-lysis with two aryl �-xylopyranosides was seen. Gentiobiose(1,6-� linked) was also tested, but no activity was detected. Theenzyme showed greater hydrolytic efficiency against the longeroligosaccharides, with the highest kcat/Km value being for cel-lohexaose. These kinetic data were used to map enzyme sub-sites that bind to the individual glucosyl residues, as seen inFig. 5. Three main subsites for the binding and subsequenthydrolysis of both 1,3-�- and 1,4-�-oligosaccharides are evi-dent, with the highest subsite binding affinity being at subsite�1; i.e., the binding of the second sugar from the nonreducingend of the polysaccharide contributes the most to the stabili-zation of the transition state complex. Several polysaccharideswere also tested as substrates for hydrolysis. Compared tolaminarin as the substrate, the specific activity of ExoP towardbarley 1,3-1,4-�-glucan was about 10-fold less. It was 25-foldless toward carboxymethyl cellulose and 36-fold less towardcarboxymethyl curdlan, indicating that the presence of a bulkycarboxymethyl group on a glucosyl residue is not favored by theenzyme. However, all three of these substrates form viscoussolutions, which may slow enzyme activity.

A significant finding was that a sample of ExoP that hadbeen left at room temperature for several weeks was partlycleaved, presumably by a protease contaminant, to form a65-kDa truncated protein, as shown by SDS-PAGE (data notshown). Treatment of the native protein with trypsin replicatedthis finding over a much shorter time span. Mass spectrometry

analysis showed that the cleavage site was directly after thedibasic motif K627-K628, suggesting that these residues arelocated in an exposed region. In both cases of truncation,almost complete loss of hydrolytic activity against laminarinwas observed.

DISCUSSION

Despite the many reports of hydrolytic activity by marinebacteria, there have been very few studies that have properlycharacterized the enzyme systems involved in the degradationof substrates that are relevant in marine systems (1). Thehypothesis underlying our work was that marine bacteria re-siding on seaweeds that contain laminarin as a storage poly-saccharide might secrete an exo-1,3-�-glucanase, in addition toendo-acting enzymes, in order to fully digest the 1,3-�-glucan.Such synergistic action has been reported for the hyperther-mophilic archaeon Pyrococcus furiosus with respect to diges-tion of laminarin and barley glucan substrates by a mixture oftwo endoglucanases and a �-glucosidase (10). As red and greenseaweeds do not contain significant amounts of 1,3-�-glucan, itwas reassuring, in terms of the screen employed, to discoverthat the only marine bacterium in this study that exhibited thedesired enzyme activity was associated specifically with a spe-cies of brown algae. The enzyme isolated (ExoP) displayed avery low level of activity against aryl �-glucosides, suggesting

FIG. 5. Subsite mapping of ExoP on the basis of hydrolysis of (A)1,3-�-oligosaccharides and (B) 1,4-�-oligosaccharides. Subsites are la-beled from �1 to �5, where the �1 subsite binds to the nonreducingend of the substrate. Subsite affinities (Ai) were calculated using the Kmand kcat values from Table 1. The intrinsic rate of hydrolysis (kint) wascalculated as described by Tanaka et al. (31).

TABLE 1. Kinetic constants for ExoP hydrolysisof oligosaccharidesa

Oligosaccharide Km (mM) kcat (s�1) kcat/Km (s�1 mM�1)

Laminaribiose 2.55 0.19 237.8 6.9 93.2Laminaritriose 0.94 0.08 298.4 11.0 318.2Laminaritetraose 0.73 0.06 272.9 10.1 375.9Laminaripentaose 0.72 0.05 264.6 9.3 369.0Laminarihexaose 0.69 0.05 261.0 8.9 380.7Cellobiose 1.77 0.24 24.7 1.8 13.9Cellotriose 0.99 0.14 242.4 14.9 243.7Cellotetraose 0.69 0.05 246.4 8.2 357.8Cellopentaose 0.45 0.04 216.3 7.0 483.4Cellohexaose 0.33 0.02 208.8 4.8 624.6pNP-Glc 7.71 0.37 6.7 0.2 0.9MU-Glc 0.95 0.08 6.2 0.2 6.5Laminarinb 0.70 0.03 204.9 2.5 290.9

a Laminari-oligosaccharides are 1,3-� linked; cello-oligosaccharides are 1,4-�linked; pNP-Glc and MU-Glc are aryl �-glucosides.

b Laminarin is assumed to have an average degree of polymerization of 28(molecular mass, 4,500 Da).

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that it was not likely a �-glucosidase per se. It was of interestthat the bacterium was found to produce relatively largeamounts of an arylsulfatase, indicative of the need to removesulfate groups prevalent in seaweed polysaccharides.

Pseudoalteromonas species are frequently found in associa-tion with eukaryotic hosts in the marine environment, includ-ing certain fish, shellfish, tunicates, sponges, and marine plants(16). These bacteria have been shown to display antibioticeffects: bacteriolytic, algicidal, and agarolytic activities. Whilethe production of extracellular glycoside hydrolases, includinglaminarinase, has been reported for a species of Pseudoaltero-monas, no individual enzymes were fully characterized (2).Genomic sequencing of several Pseudoalteromonas species hasturned up one clear homologue of ExoP (45% identity), whichwas from Pseudoalteromonas tunicata D2.

What is the natural substrate for ExoP? When the exoPgene was sequenced and its relationship to a group of plantexo-1,3/1,4-�-glucanases of the GH3 family was established,the specificity of the enzyme toward a variety of oligosaccha-rides was tested, revealing an almost equal catalytic efficiencyfor ExoP in hydrolyzing a glucose residue from the nonreduc-ing ends of both 1,3-�- and 1,4-�-linked glucans. This resultbegged the question: could a natural substrate for ExoP actu-ally be 1,3-1,4-�-D-glucan, or does the enzyme preferentiallytarget both 1,3-�-glucan (laminarin) and 1,4-�-glucan (cellu-lose) substrates? The 1,3-1,4-�-glucan polymer is found in thecell walls of some lichens and of the Poaceae family of higherplants, being particularly abundant in the endosperm of thedeveloping seedling. Both endo- and exohydrolases capable ofdepolymerizing this polysaccharide have been well character-ized in barley (17). However, such a polysaccharide has notbeen reported to be present either in the cell walls of marinealgae or inside the cells. ExoP was shown to be 10-fold lessactive against barley 1,3-1,4-�-glucan than against laminarin.The barley homologue, ExoI, also showed a similar difference,probably due to the different solubilities of the two substrates,so this result does not really help identify the physiologicalsubstrate of ExoP. It should be noted that natural laminarinalso contains 1,6-� branches in the 1,3-�-glucan chain; how-ever, ExoP did not show activity toward gentiobiose, whichcontains a 1,6-� linkage. While cellulose is present in the cellwall and laminarin is present in the cytoplasm of brown sea-weeds, the homology between ExoP and barley ExoI sequencesand their shared enzyme specificities together suggest thatExoP may act on a similar mixed-linkage substrate(s). ExoP,with its 1,3-� or 1,4-� specificity, may in fact be a more general-purpose enzyme not restricted to assisting with digestion ofbrown seaweed carbohydrate but part of the adaptive strategyof Pseudoalteromonas species to allow their survival in a varietyof marine habitats.

Lichenase involvement. The discovery of a putative lichen-ase (endo-1,3-1,4-�-glucanase) gene located closely upstreamof ExoP raises the possibility of coexpression of these genes.Sequences controlling transcription in Pseudoalteromonas spe-cies are not well-defined, although a consensus promoter se-quence based on analysis of a limited number of promoters hasbeen proposed for P. haloplanktis (11). The 50 bp of DNA thatseparate the licA and exoP genes are extremely AT rich (82%AT) and do not contain any sequences that resemble thisconsensus. There is also no evidence of potential GC-rich

stem-loop structures that may form a transcriptional termina-tor. It is therefore likely that exoP is cotranscribed with theupstream licA gene, although we cannot exclude the possibilitythat the intergenic region contains an exoP promoter. It ispossible, then, that ExoP and the lichenase may act together todigest a 1,3-1,4-�-glucan, although, as mentioned above, such astructure is evidently not present in seaweed. Attempts toproduce recombinant LicA resulted in insoluble protein, sofunctional studies have not been able to be carried out.

Some lichenases, designated 1,3(4)-�-glucanases, have beenshown to be capable of depolymerizing 1,3-�-glucans as well as1,3-1,4-�-glucans; thus, laminarin is a substrate for these en-zymes (23). However, these enzymes show limited sequenceidentity to the archetypal Bacillus lichenases and contain ad-ditional amino acids in the region of the active site. These arenot characteristics of the LicA that we have identified, whichhas 30% identity to the Bacillus enzymes. A BLASTP searchrevealed that LicA is most closely related (63% identity) to alichenase (called GluC) from another marine bacterial species,Pseudomonas sp. strain PE2 (19). Of interest was the discoverythat this bacterium is effective at degrading the cell wall of theoomycete Pythium porphyrae, the causative agent of red rotdisease in marine red algae. The hyphal cell walls of the genusPythium contain 1,3-�-, 1,4-�-, and 1,6-�-linked glucans whichcan be hydrolyzed by a group of glycosidases with overlappingspecificity.

The C-terminal extension. ExoP belongs to the GH3 familyof glycoside hydrolases, which comprises a variety of enzymesfrom bacteria, fungi, and plants. These include �-glucosidases,1,4-�-xylosidases, 1,3-�-glucosidases, 1,4-�-glucosidases, N-acetyl�-hexosaminidases, �-L-arabinofuranosidases, and exo-1,3-1,4-�-glucanases. Harvey et al. (13) subdivided this family into sixsubfamilies, two of which contain plant and bacterial exo-�-glucanases or �-glucosidases, respectively. Interestingly, theseenzymes have been annotated both 1,3-� and 1,4-� largely onthe basis of their sequence similarities and not substrate spec-ificity studies. The main difference between the plant and bac-terial enzymes is the additional length of about 200 aminoacids associated with the bacterial enzymes. The first 600 to650 amino acids of both subgroups show significant homology(43% identity with barley ExoI, for example) and comprise twodomains (domains A and B), both of which contribute to thearchitecture of the active site (35).

The C-terminal extension (domain X) is a particularly inter-esting feature of the ExoP sequence and also of some othermarine bacterial GH3 members present in the sequence data-base. Sequence alignment of these proteins indicates relativelyfew conserved residues, however, and these are mainly in thecarboxy-terminal region. Notable among these are four cys-teines which may well be involved in disulfide linkages, giventhat the enzyme is extracellular (Fig. 6). There is, however,little homology with any known protein or domain which mightgive a clue to its biological function, such as a carbohydratebinding module. The observation that truncated ExoP (628amino acids) retained very little enzyme activity points to anessential role for the additional C-terminal sequence, possiblya domain that influences substrate binding or that serves amolecular anchor role. Certainly, by analogy to barley ExoI,the 628-residue polypeptide sequence ought to contain all thecontributing residues of the active site and at least two sugar-

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binding sites, so it was somewhat surprising to note the loss ofhydrolytic activity observed. Interestingly, recently depositedgenomic sequence data for the marine bacterial speciesPseudoalteromonas atlantica T6c and Saccharophagus degrad-ans 2-40 contain an individual hypothetical protein which hassignificant similarity to the last 80 or so amino acids of ExoP(Fig. 6), suggesting a particular biological role for this part ofthe protein.

In conclusion, the selection and screening strategy employedwas designed to identify exo-1,3-�-glucanase activity with nopreconceived notions of sequence similarity to known �-glu-canases. A metagenomics approach might not have identifiedExoP specifically. Only 1 bacterial isolate out of 90 displayedsuch activity, but as it transpired, the enzyme specificity wasbroader than this and included 1,4-� linkages as well. Thediscovery and characterization of a marine bacterial exo-1,3/1,4-�-glucanase belonging to the GH3 family and related to agroup of plant enzymes but containing an extra domain nec-essary for activity but of unknown function suggest an inter-esting evolutionary relationship. We have now successfullycrystallized ExoP and solved its structure (PDB accession code3F93), revealing that the C-terminal extension is a separatefolded domain (unpublished data).

ACKNOWLEDGMENTS

We thank Sue Cutfield, Judy Broom, and Alan Carne for helpfuladvice and Diana Carne for peptide sequencing. We are grateful toTakeda Chemical Industries Ltd. (Osaka) for a gift of curdlan.

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