Process Biochemistry 46 (2011) 328–334

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Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Augmented production of poly-␤-d-mannuronate and its acetylated forms

by Pseudomonas
Yaligara Veeranagouda a , Chitragara Basavaraja b , Hyun-Sook Bae c , Kwang-Hyeon Liu d , Kyoung Lee a,∗
a
Department of Microbiology, Changwon National University, Changwon, Kyongnam 641-773, Republic of Korea
b
Department of Chemistry, Institute of Functional Materials, Inje University, Kimhae, Kyungnam 621-749, Republic of Korea
c
Department of Clothing & Textiles, Changwon National University, Changwon, Kyongnam 641-773, Republic of Korea
d
Department of Pharmacology, Inje University, College of Medicine, Busan 614-736, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: Poly-␤-d-mannuronate (PM) and its derivatives have potential to be used in pharmaceutical applications.
Received 16 February 2010 A spontaneous mutant (E1) of Pseudomonas alkylphenolia KL28 produced large amounts of a highly viscous
Received in revised form 25 August 2010 polymer consisting of repetitive units of ␤-d-mannuronic acid with acetylation at the 2nd and/or 3rd
Accepted 14 September 2010
carbon (AcPM). The alg gene cluster (18,275 bp with 12 ORFs) is essential for the production of AcPM and
has been sequenced. Mutations in the E1 strain were found in both the mucA (resulting in a truncated
Keywords:
protein) and the algG (non-synonymous amino acid substitution in the conserved epimerase domain
Poly-␤-d-mannuronate
of mannuronan C-5-epimerase) genes. Disruption of algI (acetylase) resulted in the production of a de-
Polymannuronate
Alginate
acetylated polymer, PM. The molecular weight (viscosity) and the AcPM production level were influenced
Pseudomonas by the addition of NaCl into the culture media. Under optimized flask culture conditions, 8–14 g/L of
Polymannan AcPM with different viscosities could be produced. In addition, some rheological properties of the high
alg gene molecular weight AcPM produced by the E1 mutant were better suited for pharmaceutical use than
commercial alginate. In conclusion, P. alkylphenolia E1 and its algI mutant may be suitable candidates for
mass production of AcPM and PM, respectively.
© 2010 Elsevier Ltd. All rights reserved.

1. Introduction of novel uses for PM in various medical and pharmaceutical fields

Biopolymers with defined chemical compositions are of spe-
cial interest in biomedical research. Poly-␤-d-mannuronate (PM)
is a component of alginate and is relevant to many medical and
pharmaceutical applications. For example, PM and its degrada-
tion products activate immune cells, resulting in the secretion
of cytokines such as tumor necrosis factor ␣, interleukin-1 and
interleukin-6, and thus PM can be used to increase immune pro-
tection against various infections [1–3]. Sulfated PM has been
shown to significantly inhibit HIV-1 replication both in vitro and
in vivo [4]. Recent studies demonstrate that oligomannuronates
are highly effective tumor angiogenesis and metastasis inhibitors
and are promising candidates for cancer therapy [5]. Matrix
tablets prepared from highly viscous alginate (rich in mannuronic
acid) demonstrated an enhanced drug release rate in their acidic
phase [6]. It has been recently demonstrated the application
of PM in nanocomposite synthesis, in which newly synthesized
polyaniline–PM nanocomposites had enhanced conductive proper-
ties compared to polyaniline nanocomposites [7,8]. The exploration
∗ Corresponding author. Tel.: +82 55 213 3486; fax: +82 55 213 3480.
E-mail address: kyounglee@changwon.ac.kr (K. Lee).
will likely lead to an increased demand for its production.
The PMs in the aforementioned applications are mainly sup-
plied from seaweed algae alginate consisting of ␤-d-mannuronic
acid and ␣-l-guluronic acid, which are linked by a ␤-1,4 glycosidic
bond with varying compositions and sequential structures [9,10].
However, concerns have been raised regarding the use of PMs from
seaweed in biomedical research because algal alginate is suscep-
tible to composition variation due to uncontrolled environmental
factors [11]. Moreover, extraction of PM from algal alginate can
have low purity and high production costs. Thus, there is a grow-
ing interest in the production of bacterial PM where qualities such
as monomer composition, polymer chain length and the degree of
acetylation can be manipulated [12]. Thus far, two bacterial gen-
era, Azotobacter and Pseudomonas, have been shown to produce PM
as a component of alginate. Alginate produced by Azotobacter and
brown algae contains a higher proportion of l-guluronic acid than
that of Pseudomonas. Although a few Pseudomonas strains, including
Pseudomonas aeruginosa, produce acetylated PM (AcPM) with little
or no guluronic acid content [9,13,14], studies on the efficient pro-
duction of PM and AcPM in bacterial hosts are lacking. In addition,
culture conditions that affect the molecular sizes of PM and AcPM
have not been thoroughly investigated. In this study, the effect of
culture conditions on AcPM productivity and polymer properties

1359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2010.09.009
 Y. Veeranagouda et al. / Process Biochemistry 46 (2011) 328–334 329

from a hyperproducer of Pseudomonas alkylphenolia was examined, 2.4. Generation of an algI non-polar mutant
and genes related to AcPM production were also characterized.
A tetracycline-resistant (TcR ) cassette without a transcriptional-terminator was
constructed as follows. First, the TcR cassette was amplified from p34S-Tc [22]
2. Materials and methods
with primers Tc-F1 (5 -CCGAGCTCATGTTTGACAGCTTATC-3 , SacI restriction site
underlined) and Tc-R1 (5 -TGCCGCCGAGCTCCATTCAGGTCG-3 , SacI restriction site
2.1. Bacterial strains, media and culture conditions
underlined). The amplified fragment was cloned into pGEM® -T Easy vector by select-
ing tetracycline-resistant E. coli DH5␣ recombinants. The intact nucleotide sequence
P. alkylphenolia KL28 was previously described as an n-alkylphenol-degrading
was also confirmed by DNA sequencing. From the resulting vector pGEM-Teasy–Tc2,
bacterium [15,16]. A spontaneous mutant of strain KL28 (E1) that formed a mucoid
the new TcR cassette (Tc2) from the SacI digestion was cloned into p34S-Tc where
colony under conditions used for the induction of aerial structures by p-cresol [16]
the original TcR gene was removed by SacI digestion. The resulting plasmid was
was used in this study and maintained on LB medium. Recombinant Escherichia
named p34S-Tc2 (Fig. S1 in Supporting Information).
coli and Pseudomonas strains were treated with antibiotics when necessary using
A non-polar E1 algI knockout mutant was generated by insertional muta-
concentrations as described previously [17]. Modified King’s B medium (PG) con-
genesis with the Tc2 cassette, followed by allelic replacement as described by
sisting of 20 g/L of peptone, 10 g/L of glycerol and 1.5 g/L of MgSO4 ·7H2 O in 50 mM
Schafer et al. [23]. Briefly, a 1.1-kb fragment including parts of the algI and
potassium phosphate buffer (pH 6.0) was used for the production of AcPM or PM by
algJ genes was PCR-amplified using the P. alkylphenolia KL28 chromosome as a
Pseudomonas. To assess the effect of pH on AcPM production, the pH of the medium
template. The primers AlgIF1 (5 -CCGACCATTGCTTCGCCCTACAGA-3 ) and AlgJR1
was adjusted with 1 M NaOH or 1 M HCl. The medium was sterilized by autoclav-
(5 -GCATGCTCGTCGTGAACAGCCACT-3 ) were used. The amplified product was puri-
ing at 15 lb for 20 min. Seed cultures were prepared by scraping E1 cells from PG
fied, ligated into pGEM® -T Easy vector, and named pT-algI-J. The presence of
plates grown overnight and suspending them in saline. The absorbance of the cell
an intact amplified gene sequence in pT-algI-J was confirmed by DNA sequenc-
suspension was set to 6.0 at 660 nm. Then, 50 ␮L of the cell suspension was added to
ing. The EcoRI–SphI fragment from pT-algI-J was cloned into pK18mobsacB [23]
250-mL Erlenmeyer flasks containing 50 mL of medium. The flasks were incubated
to yield pK18mobsacB-algI-J. The Tc2 cassette from p34S-Tc2 was cloned into the
at 30 ◦ C on a rotary shaker (160 rpm), and 1 mL of the sample was withdrawn at the
unique SacI site of algI in pK18mobsacB-algI-J to disrupt the gene. The resulting
specified time intervals. Growth was estimated by measuring the absorbance of the
plasmid was called pK18mobsacB-algI-J-Tc2 (Fig. S1 in Supporting Information)
cell suspension at 660 nm with a spectrophotometer (Model 2130, Sinco Co., Korea).
and used to disrupt the algI gene in E1 via a triple mating as described previ-
Cells were removed by centrifugation and culture supernatants were appropriately
ously [17]. The E1(algI::Tc2) mutant was selected as described previously, except
diluted with distilled water to measure the AcPM production, and the AcPM content
that the LB agar was supplemented with tetracycline [23]. algI disruption in
was estimated as detailed below.
E1(algI::Tc2) was further confirmed by the sequencing of the PCR product obtained
using the primers AlgIFa (5 -CGACCTGTGCATCCTCGGCTATTTC-3 ) and TcIR (5 -
2.2. Sequencing of the alg gene cluster and mucA gene TGCGACTCCTGCATTAGGAAGC-3 ) with template DNA from E1(algI::Tc2). The two
primers bind upstream of the AlgIF1-binding sequence and the TcR gene, respec-
The alg gene cluster of the KL28 strain was probed by homologous recombi- tively.
nation using a suicide vector. The alg genes retrieved from the junction plasmids
were cloned and sequenced. To construct a suicide vector that cannot replicate
in Pseudomonas, pRL27 [18] was first digested with EcoRI and the transposon-
2.5. Construction of an algG expression vector and complementation in E1
free fragment was self-ligated. The resulting plasmid was named pRL27Tnp. The
DNA sequence (1558 bp) upstream of the alginate gene cluster and a small por-
The algG expression vector, pAlgG was constructed as follows. The algG gene
tion of the 5 -terminus of algD were PCR-amplified using the P. alkylphenolia KL28
was PCR-amplified with primers AlgGF (5 -CCGGATCCTTGAGCCAGTCGGTCGA-3 ,
chromosome as a template. Primers E1-algDpF (5 -CGACGACCTGCTGCTCAACC-3 )
BamHI site underlined) and AlgGR (5 -AAGGCAGAGCTCAGGCCCAGCAGTT-3 , SacI
and E1-algDpR (5 -ATCGGTGGCTGGTCGTAA-3 ) were used. These primers were
site underlined) with the KL28 chromosome as a template. The 1.8-kb product
designed based on conserved sequences from Pseudomonas alg gene clusters. The
was purified and ligated into pGEM® -T Easy (Promega Co.), yielding pT-AlgG. The
amplified PCR product was purified and ligated into pGEM® -T Easy vector, yield-
presence of an intact algG gene in pT-AlgG was confirmed by DNA sequencing.
ing pT-algP. The presence of the intact, amplified, alg sequence in pT-algP was
The amplified BamHI–SacI fragment from pT-AlgG was ligated into the same sites
confirmed by DNA sequencing. The EcoRI fragment from pT-algP containing the
of pBBR1MCS-5, which has a broad host range [24]. The resulting pAlgG plasmid
amplified gene was cloned into the suicide vector pRL27Tnp. The resulting plas-
(Fig. S1 in Supporting Information) was transformed into strain E1 by tri-parental
mid was named pRL27Tnp-algP (Fig. S1 in Supporting Information) and was used
mating to yield the E1(pAlgG) strain.
in a homologous recombination reaction with the alg cluster of KL28. A single
crossover mutant of the alg gene was created through a triple mating between P.
alkylphenolia KL28, E. coli DH5␣ (pRL27Tnp-algP), and E. coli DH5␣ (pRK2013)
[19]. Mutants were selected on LB agar containing ampicillin and kanamicin and 2.6. Preparation and quantification of polymers for analysis
confirmed by PCR. Chromosomal DNA from the mutants was digested with BamHI
and self-ligated to recover the whole alg gene cluster containing the suicide plas- The AcPM and PM contents in the culture supernatants were measured by the
mid. A 22-kb junction plasmid obtained from the above procedure was sequenced m-hydroxybiphenyl method as previously described with purified AcPM and PM as
at SolGent Co. Ltd. (Taejeon, Korea) using an automated sequencing apparatus standards [25]. To obtain the large amount of AcPM and PM required for various
(ABI PRISM 377, PE Biosystems Inc.). ORFs were identified using the GETORF pro- measurements, culture broths were centrifuged at 12,000 rpm for 1 h at 4 ◦ C. When
gram (http://bioweb.pasteur.fr/seqanal/interfaces/getorf.html, Pasteur Institute). the culture broths were too viscous, they were diluted with saline and centrifuged.
The deduced amino acid sequences were compared with the protein sequence AcPM and PM were precipitated by adding equal volumes of isopropyl alcohol and
database (GenBank) using the BLASTX algorithm (http://www.ncbi.nlm.nih.gov). removed with a glass rod. The precipitate was dissolved in distilled water and pre-
The nucleotide sequence of the alg gene cluster was deposited in the NCBI nucleotide cipitated with three volumes of ethanol. The resulting PM was washed several times
sequence database under the accession number GU597845. with 100% ethanol and dried at 80 ◦ C to a constant weight.
The mucA gene was PCR-amplified with primers MucAF (5 -GAGTTT-
TACGACGGCGATCATGG-3 ) and MucAR (5 -GCTCACACCAGACACCAAGGCC-3 ) with
KL28 and E1 chromosomes. The amplified PCR product (1.1 kb) was purified and
2.7. HPLC analyses to determine the sugar acid composition and molecular weight
subjected to nucleotide sequence analysis. The nucleotide sequence of mucA and its
of the polymer
flanking genes was deposited in the NCBI nucleotide sequence database under the
accession number HM172485.
The sugar acid compositions of AcPM and PM were analyzed with trifluo-
roacetic acid hydrolysates by a high-performance anion-exchange chromatography
2.3. Measurement of PalgD -gfp gene expression with pulsed amperometric detection system (Dionex, Sunnyvale, CA, USA) using
a CarboPacTM PA-1 column as previously described [26]. The molecular weight of
The amplified EcoRI fragment from the pT-algP vector was cloned into the same AcPM was measured by a Waters GPC system equipped with a PL Aquagel-OH mixed
restriction site of the GFP reporter vector pPROBE-GT to assess alg promoter activ- column (Polymer Laboratories Ltd., UK, 8 ␮m, 7.8 mm × 300 mm) and a Waters 515
ity [20]. The resulting plasmid with the correct promoter orientation was called HPLC pump. Samples eluted from the column were detected with a Waters 2410 dif-
pPROBE-GT-algP (Fig. S1 in Supporting Information) and complemented the KL28 ferential refractometer. AcPM samples were filtered through a 0.22-␮M Millipore
and E1 strains. To measure alg promoter activity (PalgD ), strains containing pPROBE- membrane to remove microgels and a 100-␮L sample (3 mg/mL) was subsequently
GT-algP were grown in PG liquid medium with gentamicin under the conditions injected into the GPC system. The AcPM was eluted using 0.2 M NaNO3 and 10 mM
described above. At specified time points, 1-mL aliquots were harvested by cen- NaH2 PO4 (pH 7.0) at 35 ◦ C with a flow rate of 1 mL/min. Pullulans of 11,800, 22,800,
trifugation, washed twice with saline, and resuspended in saline at an OD660 of ∼0.4. 47,300, 112,000, 212,000, 404,000 and 788,000 Da were used as standards. The
The GFP-expression level was measured using a spectrofluorophotometer (Model molecular weight of AcPM was calculated based on the retention times of the stan-
RF-5391PC, Shimadza Co.) as previously described with excitation and emission dards and AcPM. This experiment was carried out by the Korea Polymer Testing &
wavelengths of 450 and 509, respectively, with 3.0 nm wavelength splits [21]. Research Institute, Seoul.
330 Y. Veeranagouda et al. / Process Biochemistry 46 (2011) 328–334

Fig. 2. HPLC analysis of sugar acid content of the acid-hydrolyzed polymers.

Fig. 1. Polymer produced by the E1 strain. Polymer produced from 500 mL of E1
culture supernatant was recovered using a glass stirring rod following isopropyl
alcohol precipitation. Petri dish size: Ø 100× h 20 mm.
1
2.8. H NMR determinations
The AcPM and PM obtained from the procedure described above were partially
acid hydrolyzed to avoid line broadening caused by polymer viscosity. In brief, the
AcPM and PM were dissolved in water and dialyzed against triple distilled water for
48 h, and the PM was recovered by isopropyl alcohol precipitation. About 400 mg
of this sample was dissolved in 20 mL of distilled water and the pH of the solution
was adjusted to 3.0 using 1 M HCl. The solution was then boiled for 1 h and neutral-
ized to pH 7.0 with 1 M NaOH. The resulting solution was dialyzed against 10 L of
distilled water for 48 h and concentrated by freeze-drying. Then, 20 mg of the acid-
hydrolyzed polymers were dissolved in 1 mL of D2 O and the spectra were recorded
at 82 ◦ C. All of the NMR experiments were performed on a Bruker spectrometer at
400.13 M Hz for 1 H nuclei. The following acquisition parameters were used: number
of scans = 32; pulse width = 9.3 ␮s; number of points in time domain = 32; inter-pulse
delay = 1 s; spectral width = 6793 Hz; acquisition time = 2.41 s; and line broadening
for exponential window function = 0.3 Hz. The reference peak was DHO at 4.71 ppm
at 82 ◦ C. The degree of O-acetylation in AcPM ((number of acetyl groups/uronic
acid) × 100) was deduced from the 1 H NMR spectra by comparing the intensities
of acetyl proton signals (Ia , at 2.6–2.8) with those of the anomeric proton signal (Ib ,
5.1–5.9) of mannuronic acid with a calculation of Ia /(Ib − Ia /3) as previously described
[13].
gle peak with a similar retention time to the second peak formed
by acid-hydrolyzed commercial alginate (Fig. 2). Because commer-
cial uronic acid standards for alginate are not currently available,
the structure of the E1 polymer was determined using 1 H NMR
spectroscopy. The 1 H NMR spectrum of partially acid-hydrolyzed
E1-polymers is shown in Fig. 3A. The chemical shift assignment of
the E1 polymers was achieved by comparing previously reported
chemical shifts of AcPM [9]. The acetylation and H1–H5 pro-
tons have been assigned as shown in Fig. 3A. No resonance was
detected in the spectrum for guluronic acid. H2 (5.58 ppm) and
H3 (5.96 ppm) in the anomeric region were assigned to the H2
and H3 protons, respectively, in the acetylated mannuronic acid
components [9]. From these results, it was concluded that the E1
polymers were mono-acetylated at the O-2/O-3 position or di-
acetylated at both the O-2 and O-3 positions to a degree of 37.4%
acetylation.
These results were confirmed with de-acetylated PM produced
by the engineered acetylase-negative mutant E1(algI::Tc2). The PM
produced by the mutant E1 did not show chemical shifts for acetyl

2.9. Measurement of PM viscosity

The viscosity of culture supernatants and purified AcPM or PM was measured

with a Vibro Viscometer (SV-10, A & D Company, Japan) at 25 ◦ C with a vibration
frequency of 30 Hz. The influence of temperature on viscosity of 0.25% (w/v) poly-
mer solutions was determined at a fixed temperature and after thermal treatment.
To measure the influence of pH on viscosity, the pH of aqueous polymer solutions
(0.25%, w/v) was adjusted with either 1 M HCl or 1 M NaOH. The effect of salt on
the viscosity was determined by preparing polymer solutions with different salt
concentrations. The control sodium alginate from brown algae was purchased from
Aldrich Chemical Co. (No. 180947).

3. Results

3.1. Isolation and identification of the polymer produced by the

E1 strain

A spontaneous mutant of KL28, designated E1, which produce a

large amount of polymer when grown in PG medium was isolated
as described in Materials and methods. The mucoid phenotype
associated with E1 was stable for more than 100 generations. The
polymers produced by this strain could be readily recovered by
isopropyl alcohol precipitation as described in Section 2 (Fig. 1).
Chemical analysis revealed that the polymers were mainly com-
posed of uronic acid. In contrast, the wild-type KL28 strain was not
Fig. 3. 1 H NMR spectra of the polymers formed by the E1 strain (A) and the algI
mucoid and did not produce any detectable amount of uronic acid. mutant of E1 (B). H2 and H3 are the corresponding H2 and H3 protons in the
HPLC analysis of the acid-hydrolyzed E1 polymers revealed a sin- acetylated components [9].
 Y. Veeranagouda et al. / Process Biochemistry 46 (2011) 328–334
A
P(alg) algD alg8 alg44 algK
Number of aa 438 496 388 557
Highest aa 92 91 78 77
Identity (%)
Fig. 4. Organization of the alg gene cluster of P. alkylphenolia KL28. (A) Arrangement of alg genes. (B) Alignment of a portion of the AlgG amino acid sequence. The AlgG
amino acid sequences were from the following sources. KL28, P. alkylphenolia; PAO1, P. aeruginosa PAO1; PSY, P. syringae pv. syringae B728a; PfO1, P. fluorescens PfO1; PEL48,
P. entomophila L48; PPF1, P. putida F1; KT2440, P. putida KT2440; and E1, spontaneous mutant of KL28. The amino acids identical to those of strain KL28 were indicated with
dots. The amino acids shown to be essential for the epimerase function of AlgG were boxed.
groups but did display five major signals whose chemical shifts
were identical to those of previously reported 1 H chemicals shifts
of PM (Fig. 3B) [9]. In addition, the acid-hydrolyzed polymer formed
by E1(algI::Tc2) produced a single peak corresponding to the sec-
ond peak of alginate in the HPLC analysis (Fig. 2). Therefore, based
on the HPLC and 1 H NMR analyses, it was confirmed that the poly-
mers produced by the E1 and E1(algI::Tc2) strains are AcPM and PM,
respectively. In addition, the monomers, mannuronic acid, mono-
acetyl mannuronic acid and di-acetyl mannuronic acid obtained
by the acid hydrolysis of those polymers were identified using
electrospray tandem spectrometry as shown in Fig. S2 (see Sup-
porting Information).
3.2. Nucleotide sequence and level of alg gene expression in
strains KL28 and E1
The alg gene cluster necessary for alginate production was
retrieved from P. alkylphenolia KL28 and sequenced as described
in Section 2. The alginate biosynthesis genes of KL28 were
organized in a manner similar to other alginate-producing Pseu-
domonas species [12,27]. Based on sequence homology, the 12
ORFs (18,275 bp) were arranged in the same transcriptional ori-
entation (Fig. 4A). The proteins from the deduced amino acid
sequences had identities of 77–93% to known alginate biosyn-
thesis proteins of other Pseudomonas species. Expression level of
the promoter upstream of algD was investigated using a GFP-
based reporter assay. The specific GFP expression found in the
strains KL28(pPROBE-GT-algP) and E1(pPROBE-GT-algP) after 48 h
of culture was 2.5 ± 0.25 and 68 ± 3, respectively. These results
indicated that the alg cluster was strongly expressed only in the
E1 strain and were consistent with the observation that AcPM
was not detected from the culture supernatant of wild-type strain
KL28.
The mucA gene product inhibits the function of AlgU, a sigma
factor that induces the expression of algD [12]. In many cases, over-
expression of alginate coincides with mutations in mucA, and for
this reason, mucA was sequenced in the E1 and KL28 strains. Inter-
estingly, the mucA gene in E1 contained a point mutation (C169T)
resulting in a stop codon (Fig. S3 in Supporting Information). This
shortened MucA (56 amino acid residues of 196 residues) likely
did not inhibit AlgU well, and thus, this mutation led to the mucoid
phenotype in E1.
331
algE algG algX algL algI algJ algF algA
493 521 466 434 485 385 215 483
87 90 87 79 92 78 79 93
3.3. Identification of a point mutation in the E1 algG gene
Although strain E1 contains an algG gene that encodes the
enzyme required for catalysis of the epimerization of C-5 in man-
nuronate to form guluronic acid residues in AcPM, it produced
polymers without guluronic acid residues (Figs. 2 and 3). Point
mutations in P. aeruginosa algG have been shown to affect epimer-
ization, leading to the production of alginate without l-guluronic
acid residues [14]. Because of this, we compared the E1 algG
nucleotide sequence to the wild-type strain KL28. E1 contained
a point mutation (G1264C) resulting in an amino acid substitu-
tion (G422R) of the well-conserved AlgG sequence in Pseudomonas
species (Fig. 4B) (Fig. S4 in Supporting Information). The mutated
amino acid residue has been shown to be essential for the epimer-
ization function of AlgG [28]. To confirm the proposed function of
the mutated amino acid residue, a pAlgG vector expressing wild-
type KL28 algG from KL28 was constructed and introduced into E1
as described in Section 2. When the polymer formed by E1(pAlgG)
was subjected to acid hydrolysis and sugar composition analysis, it
yielded l-guluronic acid and d-mannuronic acid at a ratio of 33–67
(Fig. 2). The presence of l-guluronic acid residues in the polymer
was also confirmed by 1 H NMR analysis (data not shown). This
further defined the role of the G422 residue in the epimerization
function of AlgG.
3.4. Primary production of AcPM by E1
Various culture parameters for maximal AcPM production by
the E1 strain were examined. King’s B [29] Medium with 1% (w/v)
of various carbon sources was used for the initial carbon screening.
Cultures were incubated at 30 ◦ C for 72 h and the AcPM content
in the culture supernatants was measured as described in Section
2. The levels of AcPM production by E1 were 0.4, 2.3, 0.2, 0.5, 0.8,
0.4, 3.8 and 0.3 g/L with glucose, fructose, lactose, pyruvate, citrate,
acetate, glycerol and succinate, respectively, as carbon sources. The
results indicated that glycerol was the best carbon source for the
optimum production of AcPM. In P. aeruginosa M-BR, glycerol was
also shown to be the best carbon source for maximal alginate pro-
duction [13]. AcPM production was associated with an increase in
broth viscosity (1.5–42.3 ± 5.6 cps) and a decrease in pH (7–5.8) of
the culture media. A decrease in the pH of the media was prevented
by increasing the buffer strength of the King’s B Medium from 10
332 Y. Veeranagouda et al. / Process Biochemistry 46 (2011) 328–334
Fig. 5. The effect of different concentrations of glycerol and NaCl on production of
AcPM (A) and viscosity (B) by the E1 strain. Culture conditions were described in
Section 2. The PM content and viscosity of culture broth were measured from culture
supernatants following 72 h of incubation. Values represent the averages for three
independent replicates. The standard deviation was less than 15%.
to 50 mM potassium phosphate. It was found that initial pH values
of 5, 7, and 8 were inferior to pH 6.0 for maximal AcPM production
(data not shown). Based on the above results, a new production
medium was designed in which the pH and buffer strength were
maintained at 6.0 and 50 mM, respectively. In this modified King’s
Medium (PG), E1 produced 4.5 ± 0.5 g/L of high viscosity AcPM,
and hence PG medium was used for polymer production in our
subsequent studies.
In Pseudomonas species, increased osmolarity has been shown
to stimulate alginate biosynthesis [30,31]. Thus, it was attempted to
increase the AcPM yield by incorporating NaCl as an osmotic stress
inducer in PG medium with varying concentrations of glycerol. An
increase of glycerol concentration in the media from 1% to 4% (v/v)
had no significant effect on growth, AcPM production or AcPM vis-
cosity, but 5% glycerol drastically reduced viscosity and increased
AcPM production. A large effect in the viscosity and AcPM produc-
tion was also seen with the addition of NaCl to the PG medium
(Fig. 5A and B). Adding 1% NaCl to the PG medium linearly increased
AcPM production to almost double. Interestingly, the viscosity was
maximal at 0.25% NaCl, but a dramatic reduction was observed
above 0.75%, with a viscosity nearly equal to that of water. This
indicated that at high NaCl concentrations, the E1 strain produced
lower molecular weight AcPM at an increased yield. In this study,
almost 8 g/L of AcPM was obtained with the highest viscosity being
160 cps. In addition, 14 g/L of AcPM was obtained with the lowest
viscosity being 1.2 cps.
3.5. Analysis of the molecular weight of AcPM
The molecular weight of AcPM produced at different viscosities
was examined by gel permeation chromatography. AcPM at the
Fig. 6. Distribution of AcPM molecular weights obtained from the high (black line)
and low (red line) viscosity-culture media. (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of the article.)
highest viscosity (160 cps) showed three broad peaks with median
molecular weights of 415, 3.4 and 0.95 kDa at a ratio of 4:3:3 (Fig. 6).
This demonstrated that the high broth viscosity was mainly due to
the production of high molecular weight AcPM, though the reason
for the distribution of these molecular weights was unclear. Fur-
thermore, AcPM obtained at the lowest viscosity (1.2 cps) showed
homogeneous, lower molecular weight polymers with a median
molecular mass of 35 kDa (Fig. 6).
3.6. Rheological properties of AcPM
The viscosity of an aqueous AcPM solution under different con-
ditions has not been previously reported. Because mannuronic acid
is also a component of alginate, the viscosity of AcPM was compared
to that of commercial alginate. As the polymer content increased,
so did the viscosity broth of the AcPM and alginate (Fig. 7A). At a
concentration of 1.5% polymer, the viscosity of AcPM was three-fold
higher compared to commercial alginate. The viscosities of AcPM
and alginate aqueous solutions (0.25%, w/v) were determined at
different temperatures ranging from 25 to 80 ◦ C (Fig. 7B). Both poly-
mers had decreased viscosities at higher temperatures but retained
their original viscosity when temperatures were dropped to room
temperature. The viscosity of the AcPM solution was stable when
shifting the pH to either highly basic or moderately acidic values
(Fig. 7C). At pH 1.0, the viscosity of AcPM decreased to about 50%; in
contrast, commercial alginate lost its viscosity and precipitated. The
effects of some representative monovalent and divalent cations on
the viscosity of polymer solutions were also investigated (Fig. 7D).
A slight decrease in the viscosity values for both polymer solutions
was detected up to 10% (w/v) of NaCl and MgCl2 . The determi-
nation of the viscosity of commercial alginate in the presence of
CaCl2 was not possible because it formed a gel even at low CaCl2
concentrations. In contrast, the viscosity of the AcPM solution was
not affected even after the addition of 10% CaCl2 . This difference
was most likely due to the presence and absence of guluronic acid
residues in polymers [11]. In fact, the commercial alginate con-
tained l-guluronic acid and d-mannuronic acid residues at a ratio
of 31:69 (Fig. 2).
4. Discussion
The potential applications of PM and its derivatives in mate-
rial science and pharmaceutical research led to develop a microbial
system that efficiently produces PM. The results presented in this
study clearly indicate that the E1 and E1(algI::Tc2) strains have the
capacity to produce large quantities of AcPM and PM, respectively.
 Y. Veeranagouda et al. / Process Biochemistry 46 (2011) 328–334 333

Fig. 7. Rheological properties of AcPM and alginate. The viscosity of aqueous solutions of AcPM produced by strain E1 (open symbols) and commercial alginate (filled symbols)
was recorded under different conditions: (A) effect of polymer concentration on viscosity; (B) effect of temperature (the viscosity of polymer solutions was measured at a
fixed temperature (triangle) and after reaching room temperature (square)); (C) effect of pH; (D) effect of NaCl (square), MgCl2 (triangle), and CaCl2 (circle). Conditions used
to measure viscosity were described in the Materials and methods. Values represent the averages for three independent replicates.

Interestingly, the salt concentration of the production medium
influenced the molecular weight of AcPM. Under optimal culture
conditions, the E1 strain produced a relatively good yield (8 g/L)
of highly viscous AcPM and an enhanced yield (14 g/L) of lower
viscosity AcPM at a 1% NaCl concentration. From a technological
perspective, this finding will aid in the development of a process
where one can control polymer size by manipulating the salt con-
centration of the production medium. The molecular mechanism
resulting from the salt concentration is currently under investiga-
tion.
The highest reported volume of alginate produced by Pseu-
domonas species is approximately 20 g/L [32] in Pseudomonas
mendocina under nitrogen-limiting continuous culture conditions
in a minimal glucose media. In this case, the fermentation yielded a
low-molecular weight polymer with low viscosity due to secreted
alginate lyase. Recently, one patent reported that more than 10 g/L
of AcPM and PM were produced by Pseudomonas fluorescens and
variants [33]. In this experiment, protease was added to the
medium to slow the degradation of the produced polymers by
extracellular alginate lyase. Studies on the control of polymer size
have not been reported in the study.
Controlling the molecular weight of a polymer requires com-
plex procedures in most polymer-producing bacteria. For instance,
in Azotobacter vinelandii, increased dissolved oxygen tension led
to the formation of alginate with a high molecular weight and
greater l-guluronic acid content [34,35]. In addition, P. aeruginosa
was reported to produce high mass alginate at higher oxygen con-
tents [36] and low mass alginate at an Mg2+ concentration of less
than 3 mM [37].
The applications for PM mainly depend on its rheological behav-
ior. Compared to high molecular weight PM, low molecular weight
PM has a lower viscosity and higher solubility, with properties sim-
ilar to printing grade alginate. In addition, low molecular weight
PM has increased functional activities, such as lowering the choles-
terol level in experimental animals. Supplementing the diet of male
Sprague Dawley rats with low molecular weight PM (30–50 kDa)
prevents obesity, controls serum lipids, enhances liver function,
and removes heavy metals from the body [38].
High molecular weight polymers have better rheological prop-
erties and may eventually be useful in pharmaceuticals [39].
For instance, high molecular weight PMs have potential to be
used as matrix material in tablet preparations and nanocom-
posite syntheses [6–8]. Exploitation of high molecular weight
polymers for pharmaceutical purposes mainly depends on their
physicochemical characteristic, rheological behavior and stability
in the presence of acid, alkalinity, salt, and temperature. When
the rheological behavior of AcPM was compared to commercial
alginate, AcPM had exceptional viscosity in aqueous solutions.
The difference in viscosity between the polymers is also due
to different molecular weights, different composition of uronic
acid or the acetylation content of the polymer. In fact, several
researchers have attempted to increase the viscosity of algal algi-
nate by acetylation using chemical or microbiological methods
[40,41]. In this regard, it is worth emphasizing the remarkable
rheological behavior of AcPM produced by the E1 strain at high
electrolyte concentrations and over a wide pH and temperature
range.
Recently, ␤-d-mannuronic acid (M2000) has been described as
a potential treatment for inflammatory and autoimmune diseases
[3]. Because ␤-d-mannuronic acid is not currently commercially
available, the authors attempted to produce M2000 by conven-
tional methods, including alginate production from P. fluorescens,
polymer recovery by alcohol precipitation followed by centrifu-
gation, de-acetylation using alkaline conditions, and polymer acid
hydrolysis [42]. In contrast, the use of E1(algI::Tc2) described in this
work has several advantages. A large quantity of pure PM can easily
be recovered using a glass rod following isopropyl alcohol precip-
itation and the de-acetylation step is not necessary. Hence, E1 algI
mutant can be exploited for the production of a large quantity of
highly pure ␤-d-mannuronic acid.
334 Y. Veeranagouda et al. / Process Biochemistry 46 (2011) 328–334
5. Conclusion
The present results identified the genetic backgrounds of the
polymer-producing P. alkylphenolia mutant E1 and screened the
culture conditions for maximal production of the PM and its deriva-
tives. The identified genes such as algG, algI and mucA could be
further exploited to modify polymers and strain improvement. The
intriguing findings were in this study that the use of P. alkylphenolia
KL28 derivatives was much beneficial to make tailor-made poly-
mers because changes of glycerol and salt concentrations in culture
media resulted in the dramatic effects on the polymer size and
productivity. The volumetric polymer production by strain E1 and
its derivatives is comparable to the maximal production by other
Pseudomonas. Additional optimization of the production conditions
with stirred fermenters and controlling the metabolic fluxes in P.
alkylphenolia cells by genetic engineering could further increase the
production of PM and AcPM adopting certain material properties.
Acknowledgements
This research was supported by Basic Science Research Program
through the National Research Foundation of Korea (NRF) grant
funded by the Ministry of Education, Science and Technology (nos.
2009-0073913 and 2007-0055799).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.procbio.2010.09.009.
References
[1] Otterlei M, Ostgaard K, Skjåk-Bræk G, Smidsrod O, Soon-Shiong P, Espevik
T. Induction of cytokine production from human monocytes stimulated with
alginate. J Immunother 1991;10:286–91.
[2] Jahr TG, Ryan L, Sundan A, Lichenstein HS, Skjåk-Bræk G, Espevik T. Induction of
tumor necrosis factor production from monocytes stimulated with mannuronic
acid polymers and involvement of lipopolysaccharide-binding protein, CD14,
and bactericidal/permeability-increasing factor. Infect Immun 1997;65:89–94.
[3] Mirshafiey A, Rehm BHA. Alginate and its comonomer mannuronic acid: med-
ical relevance as drugs alginates. In: Rehm BHA, editor. Alginates: biology and
applications. Berlin Heidelberg: Springer-Verlag; 2009. p. 229–60.
[4] Meiyu G, Fuchuan L, Xianliang X, Jing L, Zuowei Y, Huashi G. The potential
molecular targets of marine sulfated polymannuroguluronate interfering with
HIV-1 entry. Interaction between SPMG and HIV-1 rgp120 and CD4 molecule.
Antiviral Res 2003;59:127–35.
[5] Zhao H, Liu H, Chen Y, Xin X, Li J, Hou Y, et al. Oligomannurarate sulfate, a novel
heparanase inhibitor simultaneously targeting basic fibroblast growth factor,
combats tumor angiogenesis and metastasis. Cancer Res 2006;66:8779–87.
[6] Liew CV, Chan LW, Ching AL, Heng PW. Evaluation of sodium alginate as drug
release modifier in matrix tablets. Int J Pharm 2006;309:25–37.
[7] Basavaraja C, Veeranagouda Y, Lee K, Pierson R, Huh DS. Surface characteriza-
tion and electrical behavior of polyaniline–polymannuronate nanocomposites.
J App Poly Sci 2008;47:36–45.
[8] Basavaraja C, Veeranagouda Y, Lee K, Pierson R, Huh DS. Surface characteriza-
tion and electrical behavior of polyaniline-polymannuronate nanocomposites.
J Polym Sci Part B: Polym Phys 2009;47:36–45.
[9] Skjåk-Bræk G, Grasdalen H, Larsen B. Monomer sequence and acetylation pat-
tern in some bacterial alginates. Carbohydr Res 1986;154:239–50.
[10] Sabra W, Zeng AP, Deckwer WD. Bacterial alginate: physiology, product quality
and process aspects. Appl Microbiol Biotechnol 2001;56:315–25.
[11] Skjåk-Bræk G. Alginates: biosyntheses and some structure–function relation-
ships relevant to biomedical and biotechnological applications. Biochem Soc
Trans 1992;20:27–33.
[12] Remminghorst U, Rehm BH. Bacterial alginates: from biosynthesis to applica-
tions. Biotechnol Lett 2006;28:1701–12.
[13] Marty N, Dournes JL, Chabanon G, Montrozier H. Influence of nutrient media
on the chemical composition of the exopolysaccharide from mucoid and non-
mucoid Pseudomonas aeruginosa. FEMS Microbiol Lett 1992;77:35–44.
[14] Franklin MJ, Chitnis CE, Gacesa P, Sonesson A, White DC, Ohman DE.
Pseudomonas aeruginosa AlgG is a polymer level alginate C5-mannuronan
epimerase. J Bacteriol 1994;176:1821–30.
[15] Jeong JJ, Kim JH, Kim CK, Hwang I, Lee K. 3- and 4-Alkylphenol degradation path-
way in Pseudomonas sp. strain KL28: genetic organization of the lap gene cluster
and substrate specificities of phenol hydroxylase and catechol 2,3-dioxygenase.
Microbiology 2003;149:3265–77.
[16] Lee K, Veeranagouda Y. Ultramicrocells form by reductive division in macro-
scopic Pseudomonas aerial structures. Environ Microbiol 2009;11:1117–25.
[17] Yun JI, Cho KM, Kim JK, Lee SO, Cho K, Lee K. Mutation of rpoS enhances Pseu-
domonas sp. KL28 growth at higher concentrations of m-cresol and changes its
surface-related phenotypes. FEMS Microbiol Lett 2007;269:97–103.
[18] Larsen RA, Wilson MM, Guss AM, Metcalf WW. Genetic analysis of pigment
biosynthesis in Xanthobacter autotrophicus Py2 using a new, highly efficient
transposon mutagenesis system that is functional in a wide variety of bacteria.
Arch Microbiol 2002;178:193–201.
[19] Figurski DH, Helinski DR. Replication of an origin-containing derivative of plas-
mid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci
U S A 1979;76:1648–52.
[20] Miller WG, Leveau JH, Lindow SE. Improved gfp and inaZ broad-host-range
promoter-probe vectors. Mol Plant Microbe Interact 2000;13:1243–50.
[21] Choi EN, Cho MC, Kim Y, Kim CK, Lee K. Expansion of growth substrate range
in Pseudomonas putida F1 by mutations in both cymR and todS, which recruit a
ring-fission hydrolase CmtE and induce the tod catabolic operon, respectively.
Microbiology 2003:149.
[22] Dennis JJ, Zylstra GJ. Plasposons: modular self-cloning minitransposon deriva-
tives for rapid genetic analysis of gram-negative bacterial genomes. Appl
Environ Microbiol 1998;64:2710–5.
[23] Schafer A, Tauch A, Jager W, Kalinowski J, Thierbach G, Puhler A. Small mobiliz-
able multi-purpose cloning vectors derived from the Escherichia coli plasmids
pK18 and pK19: selection of defined deletions in the chromosome of Corynebac-
terium glutamicum. Gene 1994;145:69–73.
[24] Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop 2nd RM, Peterson
KM. Four new derivatives of the broad-host-range cloning vector pBBR1MCS,
carrying different antibiotic-resistance cassettes. Gene 1995;166:175–6.
[25] Vargas-Garcia MC, Lopez MJ, Elorrieta MA, Suarez F, Moreno J. Prop-
erties of polysaccharide produced by Azotobacter vinelandii cultured on
4-hydroxybenzoic acid. J Appl Microbiol 2003;94:388–95.
[26] Veeranagouda Y, Lim EJ, Kim DW, Kim JK, Cho K, Heipieper HJ, et al. Formation of
specialized aerial architectures by Rhodococcus during utilization of vaporized
p-cresol. Microbiology 2009;155:3788–96.
[27] Muhammadi, Ahmed N. Genetics of bacterial alginate: alginate genes
distribution, organization and biosynthesis in bacteria. Curr Genomics
2007;8:191–202.
[28] Gimmestad M, Sletta H, Ertesvåg H, Bakkevig K, Jain S, Suh SJ, et al.
The Pseudomonas fluorescens AlgG protein, but not its mannuronan C-5-
epimerase activity, is needed for alginate polymer formation. J Bacteriol
2003;185:3515–23.
[29] King EO, Ward MK, Raney DE. Two simple media for the demonstration of
pyocyanin and fluorescin. J Lab Clin Med 1954;44:301–7.
[30] Singh S, Koehler B, Fett WF. Effect of osmolarity and dehydration on alginate
production by fluorescent pseudomonads. Curr Microbiol 1992;25:335–9.
[31] Penaloza-Vazquez A, Kidambi SP, Chakrabarty AM, Bender CL. Characterization
of the alginate biosynthetic gene cluster in Pseudomonas syringae pv. syringae.
J Bacteriol 1997;179:4464–72.
[32] Hacking AJ, Taylor IWF, Jarman TR, Govan JRW. Alginate biosynthesis by Pseu-
domonas mendocina. J Gen Micorbiol 1983;129:3473–80.
[33] Gimmestad M, Sletta H, Karunakaran KP, Bakkevig K, Ertesvåg H, Ellingsen T, et
al. Mutant stains of Pseudomonas fluorescens and variants thereof, methods for
their production, and uses thereof in alginate production. US patent application
publication 2009; US 2009/0226977 A1.
[34] Sabra W, Zeng AP, Lunsdorf H, Deckwer WD. Effect of oxygen on formation and
structure of Azotobacter vinelandii alginate and its role in protecting nitroge-
nase. Appl Environ Microbiol 2000;66:4037–44.
[35] Galindo E, Pena C, Nunez C, Segura D, Espin G. Molecular and bioengineer-
ing strategies to improve alginate and polyhydroxyalkanoate production by
Azotobacter vinelandii. Microb Cell Fact 2007;6:7.
[36] Leitao JH, Sa-Correia I. Oxygen-dependent upregulation of transcription of
alginate genes algA, algC and algD in Pseudomonas aeruginosa. Res Microbiol
1997;148:37–43.
[37] Dunne WM, Buckmire FLA. Effect of divalent cations on the synthesis of alginic
acid-like exopolysaccharide from mucoid Pseudomonas aeruginosa. Microbios
1985;43:193–216.
[38] Byon JH, Lee JW, Lee DS, Nam TJ. Low molecular weight polymannuronate.
World intellectual property organization WO/2001/056404 2001.
[39] d’Ayala GG, Malinconico M, Laurienzo P. Marine derived polysaccharides
for biomedical applications: chemical modification approaches. Molecules
2008;13:2069–106.
[40] Lee JW, Day DF. Bioacetylation of seaweed alginate. Appl Environ Microbiol
1995;61:650–5.
[41] Skjåk-Bræk G, Zanetti FH, Paoletti S. Effect of acetylation on some solution and
gelling properties of alginates. Carbohydr Res 1989;185:131–8.
[42] Mirshafiey A, Rehm B, Abhari RS, Borzooy Z, Sotoude M, Razavi A. Production
of M2000 (␤-d-mannuronic acid) and its therapeutic effect on experimental
nephritis. Environ Toxicol Pharmacol 2007;24:60–6.