Molecular cloning and functional expression of a new amylosucrase from Alteromonas macleodii

Article abstract of DOI:10.1271/bbb.80891

The presence of amylosucrase in 12 Alteromonas and Pseudoalteromonas strains was examined. Two Alteromonas species (Alteromonas addita KCTC 12195 and Alteromonas macleodii KCTC 2957) possessed genes that had high sequence homology to known amylosucrases. Genomic clones containing the ASase analogs were obtained from A. addita and A. macleodii, and the deduced amino acid sequences of the corresponding genes (aaas and amas, respectively) revealed that they were highly similar to the ASases of Neisseria polysaccharea, Deinococcus radiodurans, and Deinococcus geothermalis. Functional expression of amas in Escherichia coli was successful, and typical ASase activity was detected in purified recombinant AMAS, whereas the purified recombinant AAAS was nonfunctional. Although maximum total activity of AMAS was observed at 45 °C, the ratio of transglycosylation to total activity increased as the temperature decreased from 55 to 25 °C. These results imply that transglycosylation occurs preferentially at lower temperatures while hydrolysis is predominant at higher temperatures.

Full text of DOI:10.1271/bbb.80891

Biosci. Biotechnol. Biochem., 73 (7), 1505–1512, 2009
Molecular Cloning and Functional Expression of a New Amylosucrase
from Alteromonas macleodii
Suk-Jin HA,1 Dong-Ho SEO,1 Jong-Hyun JUNG,1 Jaeho CHA,2 Tae-Jip KIM,3
y
1;
Young-Wan KIM,4 and Cheon-Seok PARK
1Graduate School of Biotechnology and Institute of Life Science and Resources, Kyung Hee University,
Yongin 446-701, Korea
2Department of Microbiology, Pusan National University, Busan 609-735, Korea
3Department of Food Science and Technology, Chungbuk National University, Cheongju 361-763, Korea
4Department of Food and Biotechnology, Korea University, Yeongi 339-700, Korea
Received December 15, 2008; Accepted March 20, 2009; Online Publication, July 7, 2009
[doi:10.1271/bbb.80891]
The presence of amylosucrase in 12 Alteromonas
and Pseudoalteromonas strains was examined. Two
Alteromonas species (Alteromonas addita KCTC 12195
and Alteromonas macleodii KCTC 2957) possessed genes
that had high sequence homology to known amylosu-
crases. Genomic clones containing the ASase analogs
were obtained from A. addita and A. macleodii, and the
deduced amino acid sequences of the corresponding
genes (aaas and amas, respectively) revealed that they
were highly similar to the ASases of Neisseria poly-
saccharea, Deinococcus radiodurans, and Deinococcus
geothermalis. Functional expression of amas in Esche-
richia coli was successful, and typical ASase activity
was detected in purified recombinant AMAS, whereas
the purified recombinant AAAS was nonfunctional.
Although maximum total activity of AMAS was ob-
served at 45 ꢀC, the ratio of transglycosylation to total
activity increased as the temperature decreased from 55
to 25 ꢀC. These results imply that transglycosylation
occurs preferentially at lower temperatures while
hydrolysis is predominant at higher temperatures.
been conducted in Neisseria polysaccharea.3,7,8) In
N. polysaccharea, ASase is secreted and synthesizes
an amylose-like polysaccharide outside the cells. The
N. polysaccharea ASase gene, npas, was cloned and the
functional protein was recombinantly produced in
Escherichia coli. Using the full coding region of NPAS,
including the N-terminal signal peptide, ASase was
isolated from culture supernatants of transformed E. coli
and was able to synthesize linear ꢀ-1,4-glucans from
sucrose.7) Recently, the presence of ASase genes in
various microorganisms other than Neisseria genus has
been described.9,10) These genera include Alteromonas
macleodii, Caulobacter crescentus, Deinococcus radio-
durans, Rhodopirellula baltica SH1, and Xanthomonas
campestris. The putative ASase gene of D. radiodurans
(dras) was actively expressed in Escherichia coli, and
the purified recombinant enzyme catalyzed insoluble
amylose polymer formation together with side-reactions
of sucrose hydrolysis, sucrose isomerization, and soluble
maltooligosaccharide formation.9) Recently, an ASase
gene from the moderately thermostable bacterium
Deinococcus geothermalis (dgas) was also cloned and
functionally expressed, and its recombinant protein was
characterized.10) The recombinant DGAS had excep-
tional thermostable characteristics, as demonstrated by a
half-life of 6.8 h at 55 ꢀC, in addition to typical ASase
enzymatic properties, as found in DRAS.10)
Alteromonas is a Gram-negative, mesophilic, hetero-
trophic bacterium. It belongs to the genus of Proteobac-
teria and is usually found in seawater, either in the open
ocean or on the coast. Alteromonas macleodii is an
obligate marine bacterium isolated from both surface
and deep seawater and is often found with particulate
matter. Alteromonas macleodii secretes a high-molecu-
lar-weight polysaccharide in culture.11) This exopoly-
saccharide has similarities to xanthan, a bacterial
polysaccharide used as a food additive and a rheology
modifier in industry. The backbone of xanthan consists
of two ꢁ-D-glucose units linked through the 1 and 4
positions.12–14) The side chain consists of two mannoses
and one glucuronic acid. Thus the chain consists of
repeating modules of 5 sugar units. The genome of
A. macleodii Deep ecotype, an obligate marine bacte-
Key words: Alteromonas macleodii; amylosucrase;
transglycosylation
Amylosucrase (ASase, EC 2.4.1.4) is a glycosyltrans-
ferase that belongs to family 13 of the glycoside
hydrolases, which contains a variety of known polysac-
charide-degrading enzymes (e.g., ꢀ-amylase).1) This
enzyme transfers a D-glucopyranosyl moiety from
sucrose to maltooligosaccharides, producing an insolu-
ble ꢀ-1,4-linked glucan polymer.2,3) The most interesting
aspect of synthesizing this amylose-like polymer is the
use of sucrose as the sole substrate, unlike starch or
glycogen synthases, which normally require an expen-
sive nucleotide-activated sugar (e.g., ADP- or UDP-
glucose) as a donor.3–5) It is known that ASase catalyzes
sucrose hydrolysis, sucrose isomerization, and polymer
synthesis (soluble malto-oligosaccharides and insoluble
ꢀ-1,4-linked glucan).1,3)
Although the presence of ASase in microorganisms
was first found in cultures of Neisseria perflava,6) most
enzymatic and biochemical studies of ASase have
y
To whom correspondence should be addressed. Tel: +82-31-201-2631; Fax: +82-31-204-8116; E-mail: cspark@khu.ac.kr

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S.-J. HA et al.
template. The PCR cycling conditions were as follows: initial
denaturation at 94 ꢀC for 5 min, followed by 30 cycles of denaturation
at 94 ꢀC for 30 s, annealing at 50 ꢀC for 30 s, and an extension at 72 ꢀC
for 1 min, with an additional extension at 72 ꢀC for 5 min during the
final cycle. After amplification, the PCR products in each reaction were
analyzed by agarose gel electrophoresis. The amplified PCR products
were then purified using the QIAquick gel extraction kit and were
cloned into the pGEM-T Easy vector. The nucleotide sequence of the
subcloned PCR products was determined using the BigDye Terminator
Cycle Sequencing Kit for an ABI377 PRISM (PE Applied Biosystems,
Boston, MA). The resulting DNA sequences were analyzed using the
Basic Local Alignment Search Tool (BLAST) server available at NCBI
(Bethesda, MD, http://blast.ncbi.nlm.nih.gov/Blast.cgi).
rium isolated at a depth of 3,500 meters in the Urania
Basin, has been sequenced and made available at
the National Center for Biotechnology Information
(NCBI).15) A putative protein tagged MADE 00676,
which is significantly homologous to known ASases, is
located in the genome of the A. macleodii Deep ecotype.
Based on the fact that A. macleodii produces a xanthan-
like exopolysaccharide and ASase forms an ꢀ-1,4-linked
glucan polysaccharide, which has a similar backbone to
xanthan polymers, we hypothesized that the putative
ASase homolog of A. macleodii is a functional protein
with a role in the biosynthesis of exopolysaccharides.
In this study, we sought homologs of Neisseria and
Deinococcus ASase in various Alteromonas strains and
cloned the potential ASase gene from A. macleodii
(amas). We further expressed amas in E. coli and
characterized the enzymatic properties of the recombi-
nant protein.
Cloning and analysis of the amas gene. Southern blot analysis was
performed with A. macleodii KCTC 2957 genomic DNA. Genomic
DNA was isolated from A. macleodii KCTC 2957 and digested with
BamHI, EcoRI, HindIII, SphI, XbaI, or XhoI. The DNA digests were
separated on 0.6% agarose gels and then transferred by capillary-
blotting onto Hybond-Nþ nylon membranes (GE Healthcare, Buck-
inghamshire, UK). Membranes were hybridized with digoxigenin
(DIG)-labeled probe, which was generated by PCR with the AS-N1
and AS-C1 primer set. Detection was performed with a chemilumi-
nescent detection system according to the manufacturer’s protocols
(Roche Applied Science, Indianapolis, IN). Restriction endonuclease
mapping localized the amas gene within a 7.5 kb fragment spanning
two SphI sites. Colony hybridization was used to screen a mini-library
made from 7–8 kb SphI digests of A. macleodii KCTC 2957 genomic
DNA. A positive clone was identified with the DIG-labeled probe used
in Southern blot analysis. An isolated positive clone was designated
pGEM-Sph7.5. The insert was fully sequenced by the dideoxy chain
termination method, using T7 or SP6 sequencing primers and the
BigDye Terminator Cycle Sequencing Kit for the ABI 377 PRISM
(PE Applied Biosystems). The DNA and amino acid sequences were
subsequently analyzed with DNASIS version 2.1 (Hitachi Software,
Yokohama, Japan) and the BLAST server at NCBI.
Materials and Methods
Bacterial strains, media, and plasmids. Twelve strains belonging to
the genera Alteromonas and Pseudoalteromonas were obtained from
the Korean Collection for Type Cultures (KCTC). These strains were
Alteromonas addita KCTC 12195, Alteromonas macleodii KCTC
2957, Pseudoalteromonas byunsanensis KCTC 12274, Pseudoalter-
omonas issachenkonii KCTC 12958, Pseudoalteromonas maricaloris
KCTC 12960, Pseudoalteromonas marina KCTC 12241, Pseudoalter-
omonas phenolica KCTC 12086, Pseudoalteromonas ruthenica KCTC
12959, Pseudoalteromonas sp. KCTC 12273, Pseudoalteromonas sp.
KCTC 12275, Pseudoalteromonas ulvae KCTC 12940, and Pseudoal-
teromonas undina KCTC 12423. They were grown in Marine broth or
on Marine agar (Difco Laboratories, Detroit, MI) at 25–30 ꢀC for 48 h
and used as the source of chromosomal DNA. E. coli DH5ꢀ
þ
[Fꢁꢀ80lacZ ꢁM15 ꢁ(lacZYA-argF)U169 recA1 endA1 hsdR17(rk
,
Phylogenetic analysis. Known ASase sequences of N. polysaccharea,
D. geothermalis, and D. radiodurans and the complete amino acid
sequences of AAAS and AMAS were aligned with the default
parameters of the Clustal X (version 1.81) multiple sequence alignment
algorithm.16) A phylogenetic tree was constructed using the Neighbor-
joining (NJ) method, version 3.6b of PHYLIP software. The phyloge-
netic tree was evaluated by a bootstrap test on 1,000 replicates.17)
mkþ) phoA supE44 thi-1 gyrA96 relA1ꢂꢁ] and E. coli BL21 [FꢁompT
hsdSB (rBꢁ, mBꢁ) gal dcm ꢂ(DE3) pLysS T1R] were used as hosts in
DNA manipulation and for gene expression respectively. The selection
of recombinant E. coli clones was performed on LB agar plates
supplemented with 100 mg/ml ampicillin, 0.5 mM of isopropyl-ꢁ-D-
thiogalactopyranoside (IPTG), and 40 mg/ml of 5-bromo-4-chloro-
3-indolyl-ꢁ-D-galactopyranoside (Xgal). pGEM-T Easy plasmid
(Promega, Madison, WI) and the pGEX-4T-1 vector (Amersham
Biosciences, Buckinghamshire, UK) were utilized in the cloning
of PCR products and the construction of the expression vector
respectively.
Nucleotide sequence accession numbers. The AAAS and AMAS
nucleotide sequences were submitted to GenBank (accession
nos. AB469415 and AB469558 respectively).
Expression and purification of AMAS. The putative AMAS coding
region was amplified by PCR from pGEM-Sph7.5 with primers
AMASNBamHI (50-GGA TCC ATG AGC TAT GCT GCT GAC-30)
and AMASCXhoI (50-CTC GAG TTA ATC AGC AAG AAG CCA-
30). The introduced BamHI and XhoI restriction sites of each primer are
underlined. The amplified products were cloned into pGEX-4T-1
vector (Amersham Biosciences, Piscataway, NJ, USA) to create
pGEX-AMAS, in which the glutathione S-transferase (GST) was fused
to AMAS. Cultures of recombinant E. coli BL21(DE3)/pGEX-AMAS
were grown in 1 liter of LB medium supplemented with 0.1 mg/ml of
ampicillin at 37 ꢀC with agitation. When the optical density at 600 nm
reached 0.5 to 0.6, IPTG was added to a final concentration of 1 mM,
and the cells were grown for 3 h to induce expression of the amas gene.
After induction, the cells were harvested by centrifugation at 5;000 ꢂ g
for 20 min at 4 ꢀC. The pellet was resuspended in 5 ml of phosphate-
buffered saline (PBS) buffer (140 mM NaCl, 2.7 mM KCl, 10 mM
Na2HPO4, and 1.8 mM KH2PO4, pH 7.3) per g (wet weight) of cells.
The resuspended cells were incubated on ice for 30 min after adding
1 mg/ml lysozyme and were disrupted by sonication (Sonifier 450,
Branson, Danbury, CT; output 4, 6 times for 10 s, constant duty) in an
ice bath. The cell lysate was centrifuged at 10;000 ꢂ g for 10 min at
4 ꢀC. The clarified supernatant was applied directly onto a Glutathione-
Sepharoseꢀ high performance affinity column (Amersham Bioscien-
ces) that was pre-equilibrated with PBS. The column was washed with
Enzymes and chemicals. Restriction endonucleases and other DNA
modifying enzymes, such as T4 DNA ligase and Pfu DNA polymerase,
were purchased from New England Biolabs (Beverly, MA) and
Stratagene (La Jolla, CA). The genomic DNAs of Alteromonas and
Pseudoalteromonas strains was isolated and purified with the Genomic
DNA Prep Kit for Bacteria (Solgent, Seoul, Korea). The purification of
PCR products and DNA restriction fragments was performed using the
QIAquick gel extraction kit from Qiagen (Valencia, CA). All other
chemicals used were of reagent-grade quality and were obtained from
Sigma Chemical (St. Louis, MO).
Design of degenerate primers specific to the ASase gene and
detection of it. To detect potential ASase genes from the Alteromonas
and Pseudoalteromonas strains, a degenerate ASase primer set was
developed (Fig. 1). The primer set AS-N1 and AS-C1, specific to the
ASase gene, was designed based on conserved regions of known
ASases of N. polysaccharea, D. radiodurans, and D. geothermalis
(Fig. 2). The presence of an ASase was detected by PCR amplification
using Taq polymerase (Promega). Standard PCR reactions were carried
out in a 20 ml volume at the following final concentrations: 50 mM KCl,
10 mM Tris–HCl (pH 9.0 at 25 ꢀC), 0.1% Triton X-100, 1.5 mM MgCl2,
100 mM of each dNTP, 0.5 mM of each primer, and 0.5 U Taq DNA
polymerase. Thirty nanograms of genomic DNA was used as a

A New Amylosucrase from Alteromonas macleodii
1507
Fig. 1. The Degenerate Primer Set Specific to ASase Genes.
The degenerate primer set was designed from two highly conserved regions from the N. polysaccharea, D. geothermalis, and D. radiodurans
ASase genes.
2 volumes of PBS. Bound recombinant AMAS was eluted with 50 mM
Tris–HCl (pH 8.0) containing 30 mM of reduced glutathione. The size
which are two highly conserved sequences among the
ASase genes from various microbial sources (Figs. 1
and purity of isolated recombinant AMAS were determined by sodium
and 2). When PCR was performed with genomic DNA
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and
from 12 Alteromonas and Pseudoalteromonas strains
using AS-N1 and AS-C1, two Alteromonas strains
Coomassie brilliant blue staining (Bio-Rad, Hercules, CA). The fused
GST protein was removed from the purified recombinant AMAS by
(A. addita KCTC 12195 and A. macleodii KCTC
2957) displayed distinct bands around 450 bp (Fig. 3),
although a few non-specific minor bands were noticed in
some of the Pseudoalteromonas strains. Based on a
preliminary BLAST sequence alignment, both cloned
PCR products had strong homology with known ASases,
NPAS, DRAS, and DGAS. The deduced amino acid
sequence of the A. addita KCTC 12195 PCR product
was 64% identical (97 amino acids out of 150 amino
acids) to that of NPAS, 47% (74/155) to that of DGAS,
and 47% (73/155) to that of DRAS, whereas the amino
acid sequence of A. macleodii KCTC 2957 was 66%
identical (101 amino acids out of 153 amino acids)
to NPAS, 49% (77/157) identical to DGAS, and 49%
(77/157) identical to DRAS. This suggests that the
cloned PCR products were newly-described ASase
genes in A. addita KCTC 12195 and A. macleodii
KCTC 2957.
treatment with thrombin as suggested by the manufacturer (Amersham
Biosciences).
Enzymatic assay of AMAS. The enzymatic activity of recombinant
AMAS was assayed at 45 ꢀC with 50 g/l sucrose in 50 mM Tris–HCl
(pH 8.0) for 10 min. Since the formation of fructose reflects total
consumption of sucrose, total activity was defined as the amount of
enzyme needed to catalyze the production of 1 mmole of fructose per
min under the assay conditions. Hydrolysis activity was defined as the
amount of enzyme required to catalyze the production of 1 mmole of
glucose per min. The release of glucose results from sucrose hydrolysis
when water is used as an acceptor. Transglycosylation activity was
calculated by subtracting the amount of glucose from the amount of
fructose. The effect of pH on enzymatic activity was investigated within
a range of pH 5.0 to pH 10.0 (0.1 M sodium citrate for pH 5.0, 6.0, and
7.0, 0.1 M Tris–HCl for pH 7.0, 8.0, and 9.0, and 0.1 M glycine-NaOH
for pH 9.0 and 10.0) at 45 ꢀC. The effect of temperature on the activity
was studied between 25 ꢀC and 55 ꢀC at pH 8.0. The thermostability of
purified AMAS was observed by pre-incubating the enzyme without
substrate at 35 ꢀC, 40 ꢀC, 45 ꢀC, and 50 ꢀC. After various durations, the
residual activity was measured under standard assay conditions.
Cloning and analysis of two Alteromonas ASase
genes
Analysis of reaction products. The quantification of the released
fructose and glucose was performed using a D-glucose/D-fructose kit
(Roche Diagnostics) and high performance anion exchange chroma-
tography (HPAEC) analysis. HPAEC analysis was performed with an
analytical column for carbohydrate detection (CarboPac PA100,
Dionex, Sunnyvale, CA) and an electrochemical detector (ED40,
Dionex). Filtered samples were eluted with a linear gradient from
100% buffer A (100 mM NaOH in water) to 60% buffer B (500 mM of
sodium acetate in buffer A) over 70 min. The flow rate of the mobile
phase was maintained at 1.0 ml/min. Soluble maltooligosaccharides
were quantified using the linear relationship existing between the
detector response per mole ꢀ-1,4-glucan chains and the degree of
polymerization, as described by Pizzut-Serin.14)
The A. addita KCTC 12195 and A. macleodii KCTC
2957 ASase genes were cloned from genomic DNA of
the two strains. The A. macleodii KCTC 2957 ASase
gene (amas) was located in a 7.5 kb SphI fragment, and
that of A. addita KCTC 12195 (aaas) was located in a
6.5 kb EcoRI fragment. DNA sequence analysis of aaas
identified an ORF of 1,950 nucleotides that encoded a
protein of 649 amino acids with a calculated molecular
mass of 73,735 Da and an isoelectric point (pI) of 5.3.
Similarly, amas sequence analysis revealed an ORF of
1,950 nucleotides that translated to a protein of 649
amino acids with an estimated molecular mass of
73,863 Da and an isoelectric point (pI) of 6.1. The
deduced amino acid sequences of aaas and amas had
relatively strong homology to ASases from various
microorganisms (Fig. 2). The predicted amino acid
sequences of aaas and amas were 48% (301/623) and
49% (311/629) similar to NPAS, 40% (260/644) and
41% (263/628) similar to DRAS, and 41% (261/627)
Results
Alteromonas sp. ASase
The existence of the ASase gene in the Alteromonas
and Pseudoalteromonas strains was investigated by
PCR using a degenerate primer set designed from
253WVWTTFN259 (AS-N1) and 400HDDIGW405 (AS-
C1) (numbers are from the NPAS amino acid sequence),

1508
S.-J. HA et al.
Fig. 2. Amino Acid Sequence Alignment of Alteromonas sp. ASases with Known ASases.
Alteromonas sp. ASases are AAAS (accession no. BAG82877) and AMAS (accession no. BAG82876) and known ASases from various
microorganisms include NPAS (accession no. AAM51153), DRAS (accession no. NP 294657), DGAS (accession no. ABF44874), and AMAS
(Deep ecotype) (accession no. YP 002124988). Amino acid sequences from the indicated sources were aligned by CLUSTALW. The numbers
on the right refer to the amino acid sequence position. Black boxes highlight residues identical in all sequences, whereas gray boxes indicate
regions where a majority of the residues matched. Open triangles indicate amino acid residues required for catalytic activity. The two arrows are
the regions against which the degenerate primer sets were designed.
Fig. 3. Agarose Gel Electrophoresis of PCR-Amplified Products with ASases-Specific Degenerate Primers.
M, 100 bp plus marker (Solgent): 1, Alteromonas addita KCTC 12195; 2, Alteromonas macleodii KCTC 2957; 3, Pseudoalteromonas
byunsanensis KCTC 12274; 4, Pseudoalteromonas issachenkonii KCTC 12958; 5, Pseudoalteromonas maricaloris KCTC 12960;
6, Pseudoalteromonas marina KCTC 12241; 7, Pseudoalteromonas phenolica KCTC 12086; 8, Pseudoalteromonas ruthenica KCTC 12959;
9, Pseudoalteromonas sp. KCTC 12273; 10, Pseudoalteromonas sp. KCTC 12275; 11, Pseudoalteromonas ulvae KCTC 12940;
12, Pseudoalteromonas vndina KCTC 12423.
and 41% (259/631) similar to DGAS respectively. Both
ASases (AAAS and AMAS) were highly homologous
[76% (497/648) and 87% (563/646)] to a putative ASase
from A. macleodii Deep ecotype. AAAS and AMAS
displayed 77% (501/647) homology to each other.
Phylogenetic analysis demonstrated that the ASases of

A New Amylosucrase from Alteromonas macleodii
1509
Fig. 4. Phylogenetic Tree of Six ASases.
Phylogenetic tree of six ASases (NPAS, DGAS, DRAS, AMAS-Deep ecotype, AMAS, and AAAS) was constructed by the neighbor-joining in
PHYLIP based on a CLUSTALW version 1.81 alignment with default parameters. The scale bar represents an amount of evolutionary change
corresponding to an expected 0.05 changes per site.
the Alteromonas strains were more closely related to
those of Neisseria than to those of Deinococcus (Fig. 4).
These results indicate that the newly-discovered ASases
of Alteromonas strains must be characterized to define
their activities and function.
Expression and characterization of AMAS
Both ASase genes were expressed in E. coli using the
pGEX expression system, in which the recombinant
protein is fused to glutathione-S-transferase (GST). The
vectors for aaas and amas expression were designated
pGEX-AAAS and pGEX-AMAS respectively. The
recombinant proteins were purified with Glutathione-
Sepharoseꢀ high performance affinity columns and the
GST-tags were removed, as shown in Fig. 5. The
apparent molecular mass of the recombinant proteins
was calculated to be about 70 kDa by SDS–PAGE, close
to the estimated molecular masses of AAAS and AMAS
(73,735 Da and 73,863 Da respectively).
Enzymatic activity was determined by incubating a
sucrose solution with a crude cell extract prepared from
recombinant E. coli strains harboring pGEX-AAAS or
pGEX-AMAS. Cleavage of sucrose to both glucose and
fructose was detected with the AMAS crude cell extract
but not with that of AAAS. These results did not change
even when purified AAAS was used in lieu of the crude
cell extract. It will be very interesting to investigate how
sequence differences in AAAS render it non-functional
as compared to NPAS, DGAS, and DRAS. However, in
this study, we focused on detailed enzymatic character-
ization of AMAS.
The optimum temperature for hydrolysis and trans-
glycosylation by recombinant AMAS was found to be
between 25 and 55 ꢀC (Fig. 6). Hydrolysis and trans-
glycosylation activities were highest at 45 ꢀC (432 U/mg
of protein and 304 U/mg of protein respectively), and
decreased drastically as the temperature increased to
55 ꢀC. As the temperature decreased, from 45 ꢀC to
25 ꢀC, hydrolysis also decreased, from 432 to 138 U/mg
of protein, whereas transglycosylation was not signifi-
cantly affected (254–304 U/mg of protein). The ratio of
transglycosylation/hydrolysis (T/H) increased as the
reaction temperature decreased, and was highest (1.86) at
25 ꢀC. In contrast, hydrolysis correlated directly with
temperature, and as a result, the lowest ratio of T/H
(0.79) was observed at 55 ꢀC. These data imply that there
are two different catalytic activities of AMAS that can be
modulated by regulating the reaction temperature.
Fig. 5. SDS–PAGE Analysis of Recombinant AMAS and AAAS
Expressed in E. coli BL21.
M, standard size markers; lane 1, cell extract of E. coli BL21
(control); lanes 2 and 7, cell extract of E. coli BL21 harboring
pGEX-AMAS and pGEX-AAAS, respectively; lanes 3 and 8, cell
extract of E. coli BL21 harboring pGEX-AMAS and pGEX-AAAS
respectively, after adsorption to a Glutathione-Sepharoseꢀ high
performance affinity column; lanes 4 and 9, purified recombinant
AMAS and AAAS tagged with GST respectively; lanes 5 and 10,
purified recombinant AMAS and AAAS after GST-tags were
removed by thrombin treatment.
The optimum pH of recombinant AMAS for hydrol-
ysis and transglycosylation was determined using various
buffers, including sodium citrate (pH 5.0, 6.0, and 7.0),
Tris–HCl (pH 7.0, 8.0, and 9.0), and glycine-NaOH
(pH 9.0 and 10.0) (Fig. 6). Hydrolysis and transglyco-
sylation were highest at pH 7.0 (481 U/mg protein) and
pH 8.0 (399 U/mg protein) respectively. As the pH
decreased from pH 8.0 to pH 5.0, hydrolysis changed
little and ranged from 425–481 U/mg protein; however,
transglycosylation declined to below 116 U/mg of protein.
In all pH ranges except pH 8.0 and 9.0 (Tris–HCl), the
T/H ratio was below 0.25, implying that hydrolysis is
dominant over transglycosylation. However, transglyco-
sylation is relatively preferred at pH 8.0 and 9.0 (Tris–
HCl) with T/H ratios of 0.89 and 1.27 respectively. The
maximum hydrolysis and transglycosylation activities of
recombinant AMAS were observed at pH 7.0 (sodium
citrate) and pH 8.0 (Tris–HCl) respectively.
Thermostability and polymerization activity
The thermal stability of recombinant AMAS was
investigated by incubating the enzyme in 50 mM Tris–
HCl (pH 8.0) at various temperatures (35, 40, 45,

1510
S.-J. HA et al.
Fig. 6. Effects of Temperature and pH on Hydrolysis Activity, Transglycosylation Activity, and the Ratio of Transglycosylation/Hydrolysis (T/H)
of Recombinant AMAS.
The reactions were performed at various temperatures and pH levels under standard assay conditions. Total activity ( ), hydrolysis activity
(
), transglycosylation activity ( ), the ratio of hydrolysis/total activity ( ), and the ratio of transglycosylation/total activity ( ) are described
in the figure. Sodium-citrate (pH 5.0, 6.0, and 7.0), Tris–HCl (pH 7.0, 8.0, and 9.0), and glycine-NaOH (pH 9.0 and 10.0) buffers were used in
the experiment.
and 50 ꢀC). As shown in Fig. 7, hydrolysis and trans-
glycosylation were relatively stable below 40 ꢀC, but
decreased significantly above 45 ꢀC. The half-life of the
transglycosylation activity was 20.2 min and 9.1 min at
45 ꢀC and 50 ꢀC respectively. However, the half-life of
the hydrolysis activity was 33.3 min and 14.3 min at
45 ꢀC and 50 ꢀC respectively, implying that transglyco-
sylation is more sensitive to temperature than hydrolysis.
Glucan polysaccharide synthesis was analyzed by
HPAEC to determine the dependence of the trans-
glycosylation activity of AMAS on temperature. As
shown in Fig. 8, recombinant AMAS exhibited strong
glucan polysaccharide synthesis activity at 35 ꢀC as
compared to 30 ꢀC and 40 ꢀC. HPAEC analysis revealed
that the reaction mixture included various maltooligo-
saccharides, from maltotriose (G3) to glucan polymers
longer than G35 at 35 ꢀC. More glucan polymers were
formed at 30 ꢀC than at 40 ꢀC. The yields of soluble
maltooligosaccharides and insoluble glucan were 16.9%
and 40.7% (w/w%) respectively. But they were much
lower at 30 ꢀC and 40 ꢀC.
A
B
C
Discussion
Until now, only three ASases from two microbial
genera (Neisseria and Deinococcus) have been reported,
although the presence of ASase homologs is expected
based on whole microbial genome sequence pro-
jects.1,3,9) The function of the ASase secreted from
N. polysaccharea may be to produce insoluble amylose-
like polymers, whereas the function of the ASases of
Deinococcus is not understood.18,19) Recently, the whole
genome of A. macleodii Deep ecotype was sequenced,
revealing a putative ASase homolog.15) The putative
protein has 49% identity (307/622) with NPAS, 40%
identity (255/627) with DGAS, and 40% identity (253/
627) with DRAS. Our investigation of the 12 different
Alteromonas and related Pseudoalteromonas strains
Fig. 7. Thermostability of the Total (A), Hydrolysis (B), and Trans-
glycosylation (C) Activity of Recombinant AMAS.
The enzyme in 50 mM Tris–HCl buffer (pH 8.0) was incubated at
35 ꢀC ( ), 40 ꢀC ( ), 45 ꢀC ( ), and 50 ꢀC ( ).
available in our laboratory demonstrated that two
Alteromonas strains (A. addita and A. macleodii) had
genes highly homologous to the putative ASase gene of
the A. macleodii Deep ecotype [76% (497/648) se-

A New Amylosucrase from Alteromonas macleodii
1511
strains. We analyzed the locations of ASases of Alter-
omonas species within the genome to identify possible
involvement in the synthesis of exopolysaccharides. In
the A. macleodii Deep ecotype, the ASase gene is not
located in any exopolysaccharide biosynthetic gene
cluster, based on the fact that there are no putative
homologs to the genes involved in exopolysaccharide
biosynthesis near the ASase gene (Fig. 9). This is also
true of A. addita KCTC 12195 and A. macleodii KCTC
2957. The deduced amino acids of the genes near aaas
and amas did not align significantly with any exopoly-
saccharide-related proteins, indicating that the ASases of
Alteromonas species are not involved in the biosynthesis
of exopolysaccharides.
The hydrolysis and transglycosylation activities of
AMAS were substantially dependent on temperature.
The highest activities for both hydrolysis and trans-
glycosylation were observed at 45 ꢀC. However, as
reaction temperature increased from 25 to 55 ꢀC,
hydrolysis was favored over transglycosylation. The
ratio of T/H was 1.86 at 25 ꢀC, whereas it was 0.79 at
55 ꢀC. Temperature dependence of hydrolysis and trans-
glycosylation reactions has been reported for other
enzymes. A trans-sialidase expressed by Trypanosoma
cruzi, the causative agent of Chagas disease, exhibits
maximal transglycosylation at 13 ꢀC, while the hydrol-
ysis rate increases for temperatures up to 35 ꢀC.24) We
noted that as the rate of hydrolysis decreased, the rate of
transglycosylation increased. This result was explained
by differences in the optimal temperatures of hydrolysis
and transglycosylation resulting from the differential
binding of the acceptor to the enzyme. With increased
reaction temperatures the affinity for the acceptor
decreases, resulting in a simultaneous increase in the
rate of transfer to water, which in turn is suppressed by
increasing the acceptor concentration. In a thermostable
ꢁ-glycosidase of Thermus thermophilus, however, both
hydrolysis and transglycosylation increase as temper-
ature is elevated.25) A cyclodextrin glucanotransferase
from Bacillus macerans synthesized cyclic ꢀ-1,4-glucan
products (transglycosylation) more rapidly at 60 ꢀC
compared to 40 ꢀC, whereas the opposite was found
for the coupling reaction, resulting in hydrolysis mainly
of the larger cyclic ꢀ-1,4-glucans.13) These results imply
that the effect of temperature on hydrolysis and trans-
glycosylation cannot be generalized, and that it is an
intrinsic characteristic of each enzyme.
A
B
C
Fig. 8. HPAEC Analysis of Maltooligosaccharides Synthesized by
AMAS.
Reactions were performed for 45 h at 30 ꢀC (A), 35 ꢀC (B), and
40 ꢀC (C) by recombinant AMAS. Samples were diluted 10-fold and
filtered before injection.
quence identity to A. addita KCTC 12195 and 87%
(563/646) sequence identity to A. macleodii KCTC
2957]. Expression of the putative ASase of A. macleodii
KCTC 2957 indicated that this gene encodes an ASase;
the recombinant gene product had all the activities
(hydrolysis, isomerization, and polymerization) typical
of ASases. Curiously, however, the gene product
obtained from E. coli expressing a putative ASase of
A. addita KCTC 12195 did not display ASase activity,
although it was successfully purified from the cells. All
amino acids known to be important are well conserved
in the aaas gene. However, there is still a possibility of a
point mutation located in an amino acid, not proved but
critical to the enzymatic activity in the aaas gene
product. In-depth studies are needed to determine the
relationship between critical amino acid residues and the
enzymatic activities of AAAS to determine why AAAS
is non-functional.
HPAEC analysis revealed that significantly more
transglycosylation products were produced at 35 ꢀC than
at 30 and 40 ꢀC, although maximum transglycosylation
activity was observed at 45 ꢀC and the highest T/H at
25 ꢀC. At 45 ꢀC, no noticeable transglycosylation prod-
ucts were detected, most likely due to its low thermo-
stability. In fact, AMAS was extremely unstable at
45 ꢀC, with a half-life (t1=2) of 30 min. At 40 ꢀC, some
G3 was produced, but there was markedly little of the
longer maltooligosacchrides; this may be because the
reaction proceeded for 48 h and the enzyme activity
might have been limited as a result of relatively low
thermostability (t1=2 at 40 ꢀC ¼ 11:8 h). In contrast,
considerably more maltooligosaccharides were detected
at 30 ꢀC than at 40 ꢀC, consistently with the observation
that AMAS transglycosylation was more active at 30 ꢀC
than at 40 ꢀC. These data indicate that there are two
There are few publications describing the exopoly-
saccharides produced by Alteromonas species.20–22)
A
subspecies of Alteromonas macleodii is reported to
secrete an extremely viscous polysaccharide in cul-
ture,11,23) with rheological and chemical properties
similar to xanthan, a commercial polysaccharide. The
backbone of xanthan consists of two ꢁ-D-glucose units
linked through the 1 and 4 positions, and the side chain
is composed of two mannoses and one glucuronic
acid.12,14) Xanthan biosynthetic genes are clustered in
the genomes of Xanthomonas campestris pv. campestris

1512
S.-J. HA et al.
A
B
C
Fig. 9. Schematic Gene Organization of the Genomic Regions Containing the ASases.
A, Gene organization of Alteromonas macleodii (Deep ecotype); B, gene organization of Alteromonas macleodii KCTC 2957; and C, gene
organization of Alteromonas addita KCTC 12195. Arrows indicate transcriptional orientation. Potential functions based on homologies are
indicated.
cation,’’ ed. Park KH, CRC press, Boca Raton, pp. 125–140
(2008).
catalytic activities of AMAS that can be modulated by
adjusting the reaction temperature.
11) Raguenes G, Cambon-Bonavita MA, Lohier JF, Boisset C, and
Guezennec J, Curr. Microbiol., 46, 448–452 (2003).
Acknowledgment
12) Garcia-Ochoa F, Santos VE, Casas JA, and Gomez E,
Biotechnol. Adv., 18, 549–579 (2000).
This work was supported by the Korea Research
Foundation Grant funded by the Korean Government
(MOEHRD, Basic Research Promotion Fund) (KRF-
2008-313-F00145).
13) Qi Q, She X, Endo T, and Zimmermann W, Tetrahedron, 60,
799–806 (2004).
14) Becker A, Katzen F, Puhler A, and Ielpi L, Appl. Microbiol.
Biotechnol., 50, 145–152 (1998).
15) Ivars-Martinez E, Martin-Cuadrado AB, D’Auria G, Mira A,
Ferriera S, Johnson J, Friedman R, and Rodriguez-Valera F,
ISME J., 2, 1194–1212 (2008).
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