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Bioscience, Biotechnology, and Biochemistry
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20
Enzymatic Properties and Nucleotide and Amino
Acid Sequences of a Thermostable β-Agarase from
the Novel Marine Isolate, JAMB-A94
Yukari OHTA, Yuichi NOGI, Masayuki MIYAZAKI, Zhijun LI, Yuji HATADA,
To cite this article: Yukari OHTA, Yuichi NOGI, Masayuki MIYAZAKI, Zhijun LI, Yuji HATADA,
Susumu ITO & Koki HORIKOSHI (2004) Enzymatic Properties and Nucleotide and Amino Acid
Sequences of a Thermostable β-Agarase from the Novel Marine Isolate, JAMB-A94, Bioscience,
Biotechnology, and Biochemistry, 68:5, 1073-1081, DOI: 10.1271/bbb.68.1073
To link to this article: http://dx.doi.org/10.1271/bbb.68.1073
Published online: 22 May 2014.
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Citing articles: 46 View citing articles
Enzymatic Properties and Nucleotide and Amino Acid Sequences
of a Thermostable -Agarase from the Novel Marine Isolate, JAMB-A94
Yukari OHTA, Yuichi NOGI, Masayuki MIYAZAKI, Zhijun LI, Yuji HATADA,
Susumu ITO,y and Koki HORIKOSHI
Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima, Yokosuka 237-0061, Japan
Received November 6, 2003; Accepted January 29, 2004
A gene, agaA, for a novel -agarase from the marine
bacterium JAMB-A94 was cloned and sequenced. The
16S rDNA of the isolate had the closest match, of only
94.8% homology, with that from Microbulbifer salipaludis JCM11542T. The agaA gene encoded a protein
with a calculated molecular mass of 48,203Da. The
deduced amino acid sequence showed 37–66% identity
to those of known agarases in glycoside hydrolase family
16. A carbohydrate-binding module-like amino acid
sequence was found in the C-terminal region. The
recombinant enzyme was hyper-produced extracellularly when Bacillus subtilis was used as a host. The
purified enzyme was an endo-type -agarase, yielding
neoagarotetraose as the main final product. It was very
thermostable up to 60 C. The optimal pH and temperature for activity were around 7.0 and 55 C respectively. The activity was not inhibited by EDTA (up to
100mM) and sodium dodecyl sulfate (up to 30mM).
Key words: marine bacteria; Microbulbifer; -agarase;
neoagaro-oligosaccharides; cloning
Agar present in the cell walls of some red algae is
composed of agarose and agaropectins. Agarose consists
of a linear chain of alternating residues of 3-O-linked D-galactopyranose and 4-O-linked 3, 6-anhydro--Lgalactose.1) Agaropectins have the same basic disaccharide-repeating units as agarose, although some
hydroxyl groups of 3,6-anhydro--L-galactose residues
can be substituted for sulfoxy, methoxy, or pyruvate
residues.2) Agar-degrading bacteria isolated from marine
and other environments so far have been assigned to the
genera Pseudomonas,3) Pseudoalteromonas,4) Streptomyces,5) Alteromonas,6) Microscilla,7,8) Vibrio,9,10) and
Cytophaga.11) Agarases produced by these bacteria are
classified into two groups based on their mode of action,
namely, -agarase and -agarase, which hydrolyze the
-1,3 linkages and -1,4 linkages in agarose respectively. Agarases have applications in the food, cosmetic, and
medical industries for the production of oligosaccharides
from agar. Neoagaro-oligosaccharides produced by agarase inhibit the growth of bacteria, slow the rate of
degradation of starch, and are used as low-calorie
additives to improve food quality. Moreover, neoagarobiose (NA2) is a rare reagent with both moisturizing
and whitening effects on melanoma cells.12) The
polysaccharide fractions prepared from marine algae
by -agarase also have macrophage-stimulating activity
and are suitable as a source of physiologically functional
foods with protective and immunopotentiating activity.13) Moreover, agarases can be used to degrade the cell
walls of marine algae for extraction of labile substances
with biological activities and for the preparation of
Most agarases that have been purified and characterized belong to the -agarase group except for an agarase from Alteromonas agarlyticus GJ1B.6) Biochemical and genetic studies of -agarases show high
degrees of heterogeneity in terms of their amino acid
(aa) sequences, molecular masses, substrate specificities,
and catalytic properties even though the enzymes are
functionally similar. To understand the catalytic mechanisms and diversity of agarases, we have isolated a
number of deep-sea, agar-degrading microorganisms
and sequenced the genes for agarases and characterized
the enzymes. In this report, we describe the identification of an agar-degrading bacterium designated strain
JAMB-A94 isolated from Suruga Bay, Japan, at a depth
of 2,406m and the sequencing of a gene (agaA) for a
novel agarase (AgaA) that it produced. In addition, we
show the unique catalytic properties of the enzyme
(rAgaA) expressed in Bacillus subtilis cells.
Materials and Methods
Bacterial strains and culture conditions. Strain
JAMB-A94 was originally isolated from the sediment
in Suruga Bay at a depth of 2,406m using the unmanned
submersible Kaiko. It was propagated at 25 C in Marine
Broth 2216 (Difco, Detroit, USA). Escherichia coli
y To whom correspondence should be addressed. Tel: +81-46-867-9713; Fax: +81-46-867-9645; E-mail: itos@jamstec.go.jp
Abbreviations: CBM, carbohydrate-binding module; aa, amino acid; nt, nucleotide; CBB, Coomasie Brilliant Blue R-250; NA2, neoagarobiose;
NA4, neoagarotetraose; NA6, neoagarohexaose
Biosci. Biotechnol. Biochem., 68 (5), 1073–1081, 2004
HB101 (F0 supE44 hsdS20 recA13 ara-14 proA2 lacY1
galK2 rpsL20 xyl-5 mtl-1 leuB6 thi-1) was used as the
host for cloning and routinely grown at 37 C in LuriaBertani (LB) broth (Difco) supplemented with ampicillin (100g/ml) or tetracycline (15g/ml) when required. Recombinant B. subtilis ISW1214 (leuA8 metB5
hsrM1) cells were grown at 30 C, with shaking, in a
medium composed of (w/v) 12% corn steep liquor
(Nihon Syokuhin Kako, Shizuoka, Japan), 0.2% LabLemco powder (Oxoid, Hampshire, UK), 0.1% yeast
extract (Difco), 0.1% KH2PO4, 0.02% MgSO47H2O,
0.05% CaCl2, 6% maltose, and tetracycline (15g/ml)
(CLT medium).
Isolation of DNA, transformation, and sequencing.
Genomic DNA and plasmids were prepared as described
by Saito and Miura15) and Birnboim and Doly16)
respectively. Restriction digestion and ligation were
done by the method of Sambrook et al.17) Transformation of E. coli and B. subtilis with plasmids was done by
the methods of Hanahan18) and Chang and Cohen19)
respectively. Double-stranded DNA sequencing was
performed with custom oligonucleotide primers using
an ABI Prism Big Dye Terminator Cycle Sequencing kit
and an ABI 377 sequencer (Applied Biosystems, Foster
City, USA). Computer sequence analysis was carried out
using the GENETYX-MAC program version 10.1 (SDC
Software Development, Tokyo, Japan).
16S rDNA analysis. 16S rDNA analysis was performed by amplifying 16S rRNA genes by PCR using
the eubacterial primers 27f and 1525r, as described by
Weisburg et al.20) PCR was performed using a T
Gradient 96 thermocycler (Whatman Biometra, Goettingen, Germany) programmed as follows: 1min of
denaturation at 96 C, followed by 30 cycles at 96 C for
1min, 63 C for 1min, and 72 C for 1.5min, with a
final extension at 72 C for 2min. The amplified 1.5-kb
PCR product was purified using the High Pure PCR
Product purification kit (Roche Diagnostics, Mannheim,
Germany) according to the manufacturer’s instructions.
The DNA sequence using the primers described by
Weisburg et al.20) was determined directly from the
purified PCR product as described above. The 16S
rDNA sequence of the isolate was analyzed using
the FASTA programs (http://www.ddbj.nig.ac.jp) to
obtain closely matched species.
Cloning of the agaA gene. The genomic DNA of
strain JAMB-A94 was digested with PstI or EcoRI. The
digests were purified using a High Pure PCR Product
Purification kit (Roche Diagnostics) and ligated into
pUC18, which had been digested with PstI or EcoRI and
then treated with a shrimp alkaline phosphatase (Roche
Diagnostics), using a DNA ligation kit version 2
(TaKaRa Bio, Kyoto, Japan). The ligation mixture was
transformed into competent E. coli HB101 cells, and
transformants were selected on LB agar supplemented
with ampicillin (100g/ml). A positive clone expressing agarolytic activity was detected as a colony, forming
a shallow crater around it on the agar. To confirm the
agarolytic activity expressed in E. coli HB101 cells, a
2% (w/v) iodine solution (Wako Pure Chemical, Kyoto,
Japan) was poured onto the agar. The positive clones
were visualized as clear zones around the colonies with
a dark brown background. The recombinant plasmids
prepared from the 3 positive clones obtained were
digested with PstI or EcoRI. All the plasmids had the
same insert of a 3.9-kb PstI fragment, and one of them
was designated pUAB1.
Expression and purification of rAgaA. To produce
rAgaA extracellularly, the agaA gene was amplified
from genomic DNA of strain JAMB-A94 by PCR using
LA Taq DNA polymerase (TaKaRa Bio). The primers
GGC-30, where SalI and BamHI restriction sites were
incorporated (underlined) into the 50 and 30 ends of the
agaA gene respectively. The amplification product was
digested with SalI and BamHI, and the digest was
ligated into the SalI-BamHI site of the expression vector,
pHSP64, constructed by Sumitomo et al.21) as a shuttle
vector between E. coli and B. subtilis. This vector
contains an upstream region of the gene of an alkaline
endoglucanase from Bacillus sp. KSM-64 and has been
examined for high-level expression of foreign genes in
B. subtilis.22–24) pHSP64 carrying the SalI-BamHI gene
fragment for the agarase activity was designated
pBAG1. It was then introduced into both E. coli
HB101 and B. subtilis ISW1214 cells. E. coli HB101
cells harboring pBAG1 were grown for 16 h on LB agar
supplemented with ampicillin (100g/ml). The agarolytic activities of E. coli HB101 cells harboring
pBAG1 were confirmed by pit formation in the agar.
B. subtilis ISW1214 cells harboring the plasmid were
grown for 72 h in the CLT medium.
All procedures for enzyme purification were done at
temperatures below 4 C. Cells in the spent CLT
medium were removed by centrifugation at 12,000 
g for 10min. The centrifugal supernatant was used for
enzyme purification. The supernatant (35ml) was
brought to 80% saturation with solid ammonium sulfate.
The precipitates formed were collected by centrifugation
(8,000  g, 25min) and resolved in a small volume of
20mM Tris–HCl buffer (pH 7.0). The solution was
dialyzed against the same buffer overnight. After
removal of insoluble materials by centrifugation at
8,000  g for 15min, the retentate was applied to a
DEAE-Toyopearl 650M column (2:5 15 cm; Tosoh,
Tokyo, Japan) that had been equilibrated with 20mM
Tris-HCl buffer (pH 7.0). After washing the column
with 200ml of the equilibration buffer, the enzyme was
eluted over 500ml with a linear 0–100mM NaCl
gradient in the same buffer. The active fractions were
combined and concentrated on a PM-10 membrane
1074 Y. OHTA et al.
(10,000-Mr cutoff; Millipore, Bedford, USA) to 1ml.
The concentrate was then applied to a Hi prep 26/60
Sephacryl S-100 HR column (2:6 60 cm; Pharmacia
Biosciences, Piscataway, USA) equilibrated with 20mM
Tris–HCl buffer (pH 7.0) plus 0.5 M NaCl, and the
proteins were eluted with 320ml of the equilibration
buffer at a flow rate of 0.5ml/min. The fractions
containing agarase activity were combined and concentrated on a PM-10 membrane and dialyzed overnight
against 20mM Tris–HCl buffer (pH 7.0). The retentate
(0.6ml) was used as the final preparation of purified
enzyme throughout the experiments.
Enzyme assay. A suitably diluted solution of enzyme
preparation was incubated in 1ml of 20mM Tris–HCl
buffer (pH 7.0) containing 0.2% (w/v) of agar (Nacalai
Tesque, Kyoto, Japan) for 15min at 50 C. Activity was
determined by measuring the release of reducing sugars
using the 3,5-dinitrosalicylic acid procedure25) with Dgalactose as standard. One unit (U) of enzymatic activity
was defined as the amount of protein that produced
1mol of reducing sugar as D-galactose per min under
the conditions of the assay. The kinetic parameters of Km
and kcat for agar and neoagarohexaose (NA6; Dextra
Laboratories, Reading, UK) were determined at 55 C in
20mM Tris–HCl buffer (pH 7.0). The initial rates of
hydrolysis of agar were determined at 7 different
substrate concentrations, ranging from 0.15 to 2.5 times
the estimated Km value. The hydrolysis of NA6 was
monitored quantitatively by gel filtration chromatography on an Asahipak GS220 G7 column (6:7 500mm;
Asahi Kasei, Tokyo, Japan) using an LC-10Avp with the
CLASS-VP HPLC system (Shimadzu, Kyoto, Japan)
equipped with a refractive index detector (RID-10A,
Shimadzu). Reaction conditions were chosen so that
<10% of the substrate was hydrolyzed, and the initial
rates were determined based on the rates of substrate
disappearance. Protein was determined using a protein
assay kit (Bio-Rad, Hercules, USA) with bovine serum
albumin as the standard protein.
Electrophoresis and mass spectrometry. Sodium
dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) was done essentially as described by
Laemmli26) on 12.5% (w/v) acrylamide slab gels
(70 50mm, 2.0-mm thick), and the gels were stained
for protein with Coomasie Brilliant Blue R-250 (CBB).
The molecular mass of rAgaA was estimated by SDSPAGE using 12.5% (w/v) acrylamide slab gels with low
range molecular mass standards (Bio-Rad), which
included phosphorylase b (97.4 kDa), serum albumin
(66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase
(31 kDa), trypsin inhibitor (21.5 kDa), and lysozyme
(14.4 kDa). It was also determined by a Thermo
Finnigan LCQ Deca-plus ion-trap mass spectrometer
(Thermo Electron, San Jose, USA) equipped with a
nanospray ion source, using borosilicate PicoTips
(Econo 10; New Objective, Woburn, USA). The purified
rAgaA was infused into 0.1% trifluoroacetic acid in 2%
acetonitrile (v/v). One l of the sample (500 fmol/l)
was loaded and sprayed for about 7min. The multiplecharged mass spectral data of rAgaA were acquired over
a range of 800–2000 m/z and analyzed using Biomass
Deconvolution software (Thermo Electron).
Activity staining. After SDS-PAGE of the enzyme,
SDS in the gels was removed by soaking the gels in
20mM Tris–HCl buffer (pH 7.0) three times for a total of
30min. The gels were then overlaid onto the agar sheets
containing 1.5% (w/v) agar and 20mM Tris–HCl buffer
(pH 7.0) and incubated at 25 C for 1 h. The agar sheets
were then flooded with 2% (w/v) iodine solution.
Agarase activities were visualized as clear zones on a
brown background.
Sequencing of amino-terminal regions of protein.
Each enzyme sample was blotted on a polyvinylidene
difluoride membrane (Applied Biosystems) that had
been wetted with methanol. The N-terminal aa sequence
of the protein was determined on a protein sequencer
(model 476A, Applied Biosystems).
Chromatographic analysis of the products of agarose
and neoagaro-oligosaccharides hydrolysis. Thin-layer
chromatography (TLC) was used to identify products.
Enzymatic hydrolysis of agarose (TaKaRa Bio), NA6
Dextra Laboratories, Reading, UK, or neoagarotetraose
(NA4; Dextra Laboratories) was carried out at 40 C in
20mM Tris–HCl buffer (pH 7.0) containing 1.0% (w/v)
of each substrate. The reaction mixtures were applied to
silica gel 60 TLC plates (Merck AG, Darmstadt,
Germany) according to the method of Aoki et al.27)
The plates were developed using a solvent system
composed of n-butanol–acetic acid–H2O (2:1:1, v/v).
The spots, which were oligosaccharides resulting from
the hydrolysis of the substrates, were visualized by
spraying with 10% (v/v) H2SO4 and heating. Dgalactose (Sigma Chemical, St. Louis, USA), NA4,
and NA6 were used as standards. The quantification of
reaction products was done by gel filtration chromatography using the HPLC system as described above.
Chemicals. Unless otherwise stated, all chemicals
used were from Wako Pure Chemical. p-Chloromercuribenzoate and 2-mercaptoethanol were from Nacalai
Tesque. SDS was from Bio-Rad. Iodoacetamide was
from Kanto Chemical (Tokyo, Japan). Dithiothreitol was
from Pharmacia Biosciences.
Nucleotide sequence accession numbers. The 16S
rDNA sequence of JAMB-A94 and the nucleotide (nt)
sequence data for agaA have been submitted to the
DDBJ, EMBL, and GenBank databases under accession
numbers AB124836 and AB124837 respectively.
Thermostable -Agarase from the Marine Isolate, JAMB-A94 1075
Phylogenetic analysis of the isolate
Strain JAMB-A94 occurs as Gram-negative rods 3.5–
4.5m in length and 0.4–0.8m in width and is motile
by means of peritrichous flagella. The temperature range
for growth is 20–52 C, and optimal growth occurs at
around 43 C. The bacterium is positive for catalase,
oxidase, gelatinase, and amylase, and negative for the
producton of indole and H2S, and reduction of nitrite. It
produces acid from L-arabinose, cellobiose, D-galactose,
and D-glucose. To determine the phylogenetic position
of strain JAMB-A94, its 16S rDNA sequence was
determined and analyzed using comparative sequence
analysis against known 16S rDNA sequences. The
phylogenetic tree was constructed using the neighborjoining method (Fig. 1). The sequence of the isolate had
a closest match of only 94.8% homology with that from
Microbulbifer salipaludis JCM11542T. The next highest
similarity was with Microbulbifer hydrolyticus
DSM11525T (93.6% homology). Because strains of the
genus Microbulbifer are non-motile and those of the
genus Pseudomonas are motile by polar flagella, our
isolate was affiliated into neither of these genera and
may be of a new genus. Further taxonomic approaches
for classification of our isolate are now in progress.
Cloning of the agarase gene
The genomic DNA of strain JAMB-A94 was digested
with PstI or EcoRI. The digests were inserted into
pUC18 that had been digested with PstI or EcoRI. The
ligated plasmids were introduced into competent E. coli
HB101 cells, and positive clones were selected on LB
agar plus ampicillin (100g/ml). The clones with
agarolytic activity were detected as colonies pitting or
depressing the agar surrounding them. To confirm
agarolytic activity further, the positive clones were
visualized as clear zones around colonies by flooding
2% (w/v) iodine solution onto the agar, and a plasmid
prepared from one of the positive clones was designated
The insert (3.9-kb PstI fragment) in pUAB1 was
sequenced in both directions. The insert contained
3,901 bp with a G + C content of 51 mol%, and a
single open reading frame (ORF) was found in the
sequence, which begins with an ATG at nt 91 and ends
with a TAA at nt 1,392. This agaA gene encodes a
protein of 433 aa with a calculated molecular mass of
48,203Da, as shown in Fig. 2. A putative ribosome
binding site of 50-AAGGAG-30 is present 9 bp upstream
from the initiation codon ATG. A putative sigma70-type
promoter sequence, 50-TTGTAG-30 for the 35 region
and 50-TATGGT-30 for the 10 region, are located 42 bp
upstream from the initiation codon with 17 bp spacing.
An inverted-repeat sequence was found 48 bp downstream from the TAA stop codon. The free energy value
of this sequence for a stem-loop structure was calculated
to be 167:2 kJ/mol, which would be sufficient for the
termination of transcription. The deduced aa sequence
was analyzed using a PSORT program (http://psort.
nibb.ac.jp). A possible signal sequence of 20 aa is
present with a potential cleavage site between Ala20 and
Ala21. AgaA was suggested to localize on the outer
membrane or in the periplasmic space in the case of
Gram-negative bacteria. The calculated molecular mass
and isoelectric point of the mature enzyme deduced
from its aa sequence were 46,045Da (413 aa) and pH
5.12 respectively.
Comparison of the deduced amino acid sequence of
the agaA gene product with those of other agarases
Database searches using the BLAST algorithm
(http://www.ddbj.nig.ac.jp/) with the full-length deduced aa sequence of AgaA showed substantial homology with other known agarases, which are members of
the glycoside hydrolases family 16 (http://afmb.cnrsmrs.fr/CAZY/). The sequence identities of AgaA and
the other agarases are as follows: 66% with AagA
(AB063259–1) from Pseudomonas sp. ND137, 54%
with AgaB (AF098955–1) from Zobellia galactanivorans Dsij, 54% with AagA (U61972–1) from Aeromonas sp. B9, 47% with AgaA (AF098954–1) from Z.
galactaninovorans Dsij, 52% with -agarase I
(M73783–1) from Pseudoalteromons atlantica
ATCC19262, 47% each with -agarase (AguB:
AY212801–21, AguD: AY212800–13, AguH:
AY236225–2, AguK: AY236223–3) from uncultured
bacteria, 40% with AgaA (AY150179–1) from Pseudomonas sp. CY24, and 37% with DagA (AL133236–6)
Fig. 1. Phylogenetic Tree Based on 16S rDNA Sequence Analysis
Showing the Position of Strain JAMB-A94 in the Microbulbifer
Bootstrap values expressed as percentages of 1000 replications
are shown at branch points. The bar indicates the distance
corresponding to one nucleotide substitution per 100 nucleotides.
Sequence sources are M. salipaludis JCM11542T (AF479688), M.
hydrolyticus DSM11525T (U58338), M. elongatus DSM6810T
(AF500006), Pseudomonas aeruginosa LMG1242T and E. coli
ATCC11775T (used as the outer groups, Z76651 and X80725,
1076 Y. OHTA et al.
from Streptomyces coelicolor A3(2). The multiple
alignments showed that the homologous regions in
these agarases were located in the N-terminal halves
Ala21–Val293 (AgaA numbering). A conserved domain
search using the NCBI Conserved Domain Search
(http://www.ncbi.nlm.nih.gov) with the full-length
deduced aa sequence as a query suggested the existence
of the putative carbohydrate-binding module 6 (CBM 6)
at Gly338–Leu433 (Fig. 2).
Extracellular expression and purification of rAgaA
High-level exoproduction of rAgaA was achieved
using B. subtilis ISW1214 and pBAG1. Supernatant
from 35ml of culture broth was collected after 72 h of
culture. The total activity in the supernatant of the
culture broth was 45,000U/l. The rAgaA was purified
220-fold after anion-exchange chromatography and gel
filtration chromatography, with a high specific activity
of 517U/mg and a final yield of 9.0%, as summarized in
Table 1. The SDS-PAGE and activity staining of the
purified enzyme gave a single band with an apparent
molecular mass of 32 kDa, as shown in Fig. 3. This
value is smaller than that estimated from the agaA gene
sequence. The calculated molecular mass of the recombinant mature enzyme is 46,045Da. rAgaA may be
produced as a result of proteolytic digestion during
excretion. The N-terminal aa sequence of rAgaA was
determined to be Tyr-Ala-Ala-Asp-Trp-Asp-Gly-ValPro-Val from aa 19 to 28, where the unexpected Tyr19
instead of Ala21 occurs at the N-terminus. To determine
the aa of the N- and C-termini, the correct molecular
mass of rAgaA was determined with an ion-trap mass
Fig. 2. Complete Nucleotide Sequence of the agaA Gene and Its Deduced Amino Acid Sequence.
In the 3,901 bp determined, the nt sequence of the agaA gene and its flanking regions are shown. Sequences similar to the 35 and 10
consensus promoters of E. coli are underlined. A putative ribosome-binding site is double underlined. The deduced aa sequence of the gene
product is indicated by the single-letter codes under the nt sequence determined. The putative signal peptide is shown by the dotted line. Thick
bars under the nucleotide sequence show 50 and 30-terminal sequences inserted into the expression vector. The putative CBM is boxed. Inverted
repeats downstream from the stop codons TAA (*) of the ORF are designated by convergent arrows.
Thermostable -Agarase from the Marine Isolate, JAMB-A94 1077
spectrometer. As shown in Fig. 4, the molecular mass of
rAgaA excreted was 31; 998 5Da, indicating that the
N- and C-terminal aa residues are Tyr19 and Thr300,
respectively, in rAgaA. The homologous region among
the related agarases (Ala21–Val293 in AgaA numbering) was kept intact in rAgaA. Thus, the CBM-like
domain (Gly338–Leu433 in AgaA numbering) was not
essential for agarolytic activity and may facilitate the
reaction by binding to substrates. We are now trying to
purify the agarase from strain JAMB-A94 to confirm its
actual cleavage site, signal peptide, and molecular mass.
Analysis of the role of the CBM-like domain will be
necessary using full-length protein encoded by the agaA
Effects of metal salts and chemical reagents
The activity of rAgaA was examined after the enzyme
had been incubated with various cations and chemicals
in 20mM Tris-HCl buffer (pH 7.0) at 25 C for 30min. It
was not essentially affected by NaCl up to at least 1.0 M.
The other metal ions found in seawater, such as Mg2þ,
Kþ, and Ca2þ ions (each at 5 or 100mM), also did not
affect the activity to any practical extent. The activity
was abolished by Hg2þ, Cu2þ, Pb2þ, and Zn2þ ions
(each at 1mM). It was not affected by iodoacetamide,
p-chloromercuribenzoate, dithiothreitol, or 2-mercaptoFig. 3. SDS-PAGE and Activity Staining of Purified rAgaA.
(A) SDS-PAGE of the purified enzymes (0.5g) on a 12.5% (w/
v) polyacrylamide gel. The proteins were stained with CBB (lane P).
Protein mass markers (in kDa) are indicated on the left (lane M). (B)
Activity staining of the purified enzyme. After SDS–PAGE, the slab
gel was overlaid onto the agar sheet containing 1.5% (w/v) agar,
20mM Tris–HCl buffer (pH 7.0), followed by incubation at 25 C for
1 h. The activity was visualized as a clear zone by flooding with an
iodine solution on the agar sheet.
Table 1. Typical Summary of Purification of rAgaA
Total Total Specific
Yield Purification
Purification step Protein activity activity
(mg) (U) (U/mg)
(%) (-fold)
Culture supernatant 668.6 1572 2.4 100.0 1.0
80% Ammonium sulfate 211.8 1398 6.6 88.9 2.8
DEAE-Toyopearl 650M 12.1 774 64.0 49.2 27.2
Hi prep 26/60 Sephacryl S-100 HR 0.3 142 517.3 9.0 220.0
Fig. 4. Determination of Molecular Mass of rAgaA by Mass Spectrometry.
The full scan mass spectrum by electrospray ionization of rAgaA and the charge states of peaks recorded with more than 50% relative
abundance are shown. The inset shows the deconvolution of the electrospray ionization mass spectrum. The calculated average molecular mass
of the enzyme is indicated by the arrow.
1078 Y. OHTA et al.
ethanol (each at 1mM), but was slightly inhibited by
diethyl pyrocarbonate and 1-ethyl-3-(3-dimethyl-aminopropyl)carbonate (each at 1mM) by 10–20%. It was
abolished by N-bromosuccinimide (0.1mM), suggesting
that a tryptophan residue(s) are important for catalysis.
Characteristically, rAgaA showed strong resistance to
EDTA up to 100mM and to SDS up to 30mM.
Effects of pH on activity and stability
The pH optimum for activity of rAgaA (0.02U/ml)
was examined at 50 C in 50mM Britton-Robinson
universal buffers (pH 3–12). The enzyme showed an
optimal pH of about 7.0. To assess the pH stability of
rAgaA, the enzyme (4.0U/ml) was preincubated for
30min at 25 C at various pHs in 50mM BrittonRobinson buffers. After 1:20 dilution, the samples
(0.1ml) were used for measurements of the residual
activity under the standard conditions of enzyme assay.
rAgaA was very stable between pH 6 and pH 9, having
95% of the original activity. It also retained approximately 75% of the original activity after treatment at pH
4–5 (data not shown).
Effects of temperature on activity and stability
The optimal temperature for the activity of rAgaA
was around 55 C (Fig. 5A), which is the highest among
those reported so far for agarases, which have ranged
from 30 C to 40 C.28) The thermostability curve for
agalolytic activity of rAgaA was not essentially altered
regardless of whether NaCl (up to 0.5 M) or CaCl2 (up to
10mM) was present. To assess the thermostability of
rAgaA, the enzyme was heated at various temperatures
for 15min in 20mM Tris–HCl buffer (pH 7.0). As shown
in Fig. 5B, rAgaA was stable to incubation up to 60 C
and retained more than 80% of the original activity even
after heating at 65 C for 15min.
Analysis of hydrolysis products and substrate specificity
The time course of hydrolysis products from agarose
was examined with 0.04U/ml of rAgaA incubated at
40 C for up to 24 h, as shown in Fig. 6A. In the initial
stage, the enzyme hydrolyzed agarose to generate many
oligosaccharides. After incubation for 24 h, the main
product by the enzyme was NA4 with a small amount of
NA6 and NA2, as judged by the Rf values on TLC.
The quantifications of reaction products after 48 h of
incubation were performed by gel filtration chromatography. The composition (mol%) of the products was
2.8% of NA6, 78.0% of NA4, 17.1% of NA2, and 2.2%
of unidentified saccharides. rAgaA acted on NA6 to
yield NA4 and NA2, as shown in Fig. 6B. No product
was generated from NA4. If rAgaA cleaved the -1,3
linkage in NA6, the products must be mono- and/or triand/or pentameric sugars. These results imply that
rAgaA is an endo--agarase and hydrolyzes agarose,
neoagaro-oligosacchrides larger than NA6, and NA6 to
yield NA4 as the main product. The Km values for agar
and NA6 were 4.8 and 87.6mg/ml respectively. The
kcat=Km values for them were 3:9 102 and 0.35
(mg/ml)1 sec1 respectively. The catalytic efficiency
(kcat=Km) for agar is 3 orders of magnitude greater than
that for NA6.
The agarases reported to date have not been used
widely for industrial applications due to their low
activity, low stability, and low productivity. In this
study, we cloned and sequenced the agaA gene from the
marine bacterium JAMB-A94, isolated from a sample of
deep-sea sediments at a depth of 2,406m. Judging by the
16S rDNA sequence and other taxonomic analyses, our
isolate may be placed in a new genus. The aa sequence
of AgaA showed homology with those of other known
Fig. 5. Effects of Temperature on Activity and Stability of rAgaA.
(A) The temperature-activity curve of rAgaA (0.02U/ml) is
shown. The activities were determined at the temperatures indicated
for 15min and at pH 7.0 in 20mM Tris–HCl buffer. Values are
expressed as percentages of the optimal temperature of about 55 C,
which are taken to be 100%. (B) To assess the thermostabilty of
rAgaA, the enzyme (0.2U/ml) was preincubated at the indicated
temperatures for 15min in 20mM Tris–HCl buffer (pH 7.0). The
samples (0.1ml) were used for measurement of residual activities
under standard conditions of enzyme assay. The values are shown as
percentages of the original activity without heating, which is taken
to be 100%.
Thermostable -Agarase from the Marine Isolate, JAMB-A94 1079
agarases of the glycoside hydrolases family 16. In the
C-terminal half, there was a region that showed
homology with a noncatalytic module that resembles
in part CBM family 6. Polysaccharide-degrading enzymes are known in general to have modular structures
in which the catalytic modules are attached via linker
sequences to noncatalytic modules that bind to substrates.29) The binding ligands and their specificities are
dominated by hydrophobic interactions between the
sugar rings and hydrophobic and aromatic aa residues on
the surface of binding sites.30) At present, we do not
know how the CBM-like sequence contributes to the
catalysis of AgaA.
The recombinant enzyme rAgaA had a molecular
mass of 32 kDa and a high specific activity of 517U/mg
with optimal pH and temperature of about 7.0 and 55 C
respectively. rAgaA is an endo-type -agarase, and the
final main product is NA4. -Agarase I from P. atlantica ATCC19262 with a molecular mass of 32 kDa
shows maximal activity in the pH region between 6 and
7 and hydrolyzes agarose to yield NA4 as the main
product.31,32) Agarase 0107 from Vibrio sp. JT0107 with
a high molecular mass of 105 kDa shows maximal
activity at pH 8 and hydrolyzes not only agarose but also
NA4 to yield NA2.33) rAgaA (AgaA) is much more
beneficial than the reported enzymes for use in industrial
applications. The high activity and thermostability at
temperatures higher than the gelling temperature of agar
(around 40 C) represent advantages for industrial
oligosaccharide production from agar or marine algae.
Moreover, the strong resistance to EDTA, SDS, and salts
at high concentrations shows that crude marine algae in
high-salt seawater can be hydrolyzed by AgaA to
produce oligosaccharides. The properties of AgaA can
also be used for molecular biology applications such as
extraction of DNA fragments from agarose gel after
electrophoresis, because most buffers for electrophoresis
of DNA contain EDTA.
We are grateful to Professor Y. Sakano of the Tokyo
University of Agriculture and Technology, Japan, for
stimulating discussions.
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Thermostable -Agarase from the Marine Isolate, JAMB-A94 1081

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