Microbiological Research 182 (2016) 49–58

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Microbiological Research
journal homepage: www.elsevier.com/locate/micres

Characterization of an extracellular biofunctional alginate lyase from

marine Microbulbifer sp. ALW1 and antioxidant activity of enzymatic
hydrolysates
Yanbing Zhu a,b,c,d,1 , Liyun Wu a,1 , Yanhong Chen a,b,c,d , Hui Ni a,b,c,d , Anfeng Xiao a,b,c,d ,
Huinong Cai a,b,c,d,∗
a
College of Food and Biological Engineering, Jimei University, Xiamen 361021, China
b
Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, Xiamen 361021, China
c
Research Center of Food Biotechnology of Xiamen City, Xiamen 361021, China
d
Key Laboratory of Systemic Utilization and In-depth Processing of Economic Seaweed, Xiamen Southern Ocean Technology Center of China, Xiamen
361021, China

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

Article history: A novel alginate-degrading marine bacterium Microbulbifer sp. ALW1 was isolated from rotten brown alga.
Received 12 May 2015 An extracellular alginate lyase was purified to electrophoretic homogeneity and had a molecular mass
Received in revised form 19 July 2015 of about 26.0 kDa determined by SDS-PAGE and size exclusion chromatography. This enzyme showed
Accepted 20 September 2015
activities towards both polyguluronate and polymannuronate indicating its bifunctionality while with
Available online 25 September 2015
preference for the former substrate. Using sodium alginate as a substrate, strain ALW1 alginate lyase
was optimally active at 45 ◦ C and pH 7.0. It was stable at 25 ◦ C, 30 ◦ C, 35 ◦ C and 40 ◦ C, but not stable at
Keywords:
50 ◦ C. This alginate lyase showed good stability over a broad pH range (5.0–9.0). The enzyme activity
Microbulbifer sp.
Alginate lyase
was increased to 5.1 times by adding NaCl to a final concentration of 0.5 M. Strain ALW1 alginate lyase
Characterization produced disaccharide (majority) and trisaccharide from alginate indicating that this enzyme could be
Alginate oligosaccharides a good tool for preparation of alginate oligosaccharides with low degree of polymerization (DP). The
Antioxidant activity alginate oligosaccharides displayed the scavenging abilities towards radicals (DPPH, ABTS+ and hydroxyl)
and the reducing power. Therefore, the hydrolysates exhibited the antioxidant activity and had potential
as a natural antioxidant.
© 2015 Elsevier GmbH. All rights reserved.

1. Introduction
Alginate is an acidic copolymer consisting of ␤-d-mannuronate
(M) and its C5 epimer ␣-l-guluronate (G) as monomeric units.
These units are linked together with 1,4-O-glycoside bonds to form
a linear polysaccharide arranged as a polyM-block, a polyG-block,
and an alternating or random polyMG-block (Vera et al., 2011).
Alginates are synthesized in great abundance as a major structural
material in cell-wall and intracellular matrix of brown algae, and
also produced by two heterotrophic bacteria Azotobacter vinelandii
(Gorin and Spencer, 1966) and Pseudomonas aeruginosa (Linker and
Jones, 1966). Seaweed Alginate has been widely used in food and
∗ Corresponding author at: College of Food and Biological Engineering, Jimei Uni-
versity, 43 Yindou Rd., Xiamen 361021, China. Fax: +86 592 6181487.
E-mail address: jmswthermo@hotmail.com (H. Cai).
1
These authors contributed equally to this paper.
industrial chemical processes (Tseng 2001; Lee and Mooney, 2012;
Pawar and Edgar, 2012).
Alginate lyase catalyzes the degradation of alginate by
␤-elimination of glycosidic bonds and produces unsaturated
oligosaccharides having 4-deoxy-l-erythro-hex-4-enopyranosy-
luronic acid with double bonds at the non-reducing end (Wong
et al., 2000). According to the substrate specificity, alginate lyase
is generally classified as polyM lyase (EC 4.2.2.3) and polyG lyase
(EC 4.2.2.11), which preferentially break up the polyM and polyG
blocks, respectively (Kim et al., 2011). The degradation products
of alginate exhibit a variety of biological activities such as bac-
teriostasis, antioxidant activity, anti-tumor, immunomodulation,
promotion of plant growth, and prevention of dental caries (Lasky,
1995; Hu et al., 2004; Yokose et al., 2009). They can be exploited
for applications in food, agricultural and medical industries. In addi-
tion to preparation of alginate oligosaccharides, alginate lyase can
also be used as biochemicals in biofilm degradation of mucoid
P. aeruginosa for treating cystic fibrosis patients (Alkawash et al.,

http://dx.doi.org/10.1016/j.micres.2015.09.004
0944-5013/© 2015 Elsevier GmbH. All rights reserved.
50 Y. Zhu et al. / Microbiological Research 182 (2016) 49–58
2006), and be employed in preparation of algal protoplasts (Inoue
et al., 2011), extraction of intracellular bioactive substances or DNA
from brown algae, and analysis of the alginate structure (Currie and
Turvey, 1982).
Alginate lyases are present in a large diversity of organisms
distributing over bacteria, fungi, brown algae, herbivorous marine
mollusks, and Chlorella virus (Madgwick et al., 1973; Suda et al.,
1999; Rahman et al., 2010; Singh et al., 2011; Wang et al., 2013).
In the past several years, a number of alginate lyases from bac-
teria have been characterized. These include alginate lyases from
Agarivorans sp. (Kobayashi et al., 2009), Alteromonas sp. (Iwamoto
et al., 2001), Corynebacterium sp. (Matsubara et al., 1998), Flavobac-
terium sp. (Huang et al., 2013; Inoue et al., 2014), Microbulbifer sp.
(Swift et al., 2014), Pseudoalteromonas elyakovii (Ma et al., 2008),
Pseudomonas aeruginosa (Farrell and Tipton, 2012), Pseudomonas
alginovora (Lundqvist et al., 2012), Pseudomonas fluorescens (Li et al.,
2011b), Pseudomonas syringae (Preston et al., 2000), Saccharophagus
degradans (Kim et al., 2012), Sphingomonas sp. (Ryu and Lee 2011;
Park et al., 2012), Stenotrophomas maltophilia (Lee et al., 2012),
Streptomyces sp. (Cao et al., 2007; Kim et al., 2009), and Vibrio sp.
(Wang et al., 2013).
In this study, we report the purification and biochemical
characterization of a new alginate lyase from marine bacterium
Microbulbifer sp. ALW1, which was isolated from rotted brown sea-
weed.
2. Materials and methods
2.1. Screening of alginate-degrading bacteria
The rotten brown alga sample was collected from a market
in Xiamen, China. It was incubated in 100 ml of medium A (3%
(w/v) NaCl, 0.5% (w/v) (NH4 )2 SO4 , 0.2% (w/v) K2 HPO4 , 0.1% (w/v)
MgSO4 ·7H2 O and 0.01% (w/v) FeSO4 ·7H2 O, pH 7.5) with shaking
(180 rpm) at 25 ◦ C for 15 days. Then the suspension was inoculated
into 50 ml of the selection medium (medium A containing 0.5%
(w/v) sodium alginate) and the sample was incubated under the
same conditions as described above for 48 h. The culture was plated
on the selection medium agar plates and incubated at 25 ◦ C for 72 h.
The obtained 26 single strains were individually cultivated in 10 ml
of the selection medium at 25 ◦ C with shaking (180 rpm) for 60 h.
For each strain, 5 ml of the culture was centrifuged at 10,000 × g for
10 min, and the supernatant was collected and used as the extracel-
lular alginate lyase preparation. The cell pellet was resuspended in
5 ml of 100 mM sodium phosphate buffer (pH 7.0) and then broken
by ultrasonication on ice. After removal of cell debris by centrifu-
gation at 10,000 × g for 20 min, the supernatant was used as the
intracellular alginate lyase preparation. Determination of alginate
lyase activity was performed by the method described below. The
results showed that all the selected strains had low intracellu-
lar alginate lyase activity and high extracellular enzyme activity.
Among them, strain ALW1 displayed the highest extracellular algi-
nate lyase activity and was chosen for further study.
2.2. Identification of bacterial strain
The genomic DNA of strain ALW1 was extracted using the
bacterial genomic DNA extraction kit (Guangzhou Dongsheng
Biotech Co., Ltd., China) according to the manufacturer’s instruc-
tion. The 16S rRNA gene was amplified from the genomic DNA by
PCR using the primers 27F (5 -AGAGTTTGATCCTGGCTCAG-3 ) and
1492R (5 -GGCTACCTTGTTACGACTT-3 ) (Moreno et al., 2002). The
PCR product was sequenced. Homology search of the 16S rRNA gene
sequence was performed using the BLAST program (Altschul et al.,
1997) against GenBank database. A bootstrapped phylogenetic tree
was built using MEGA 6.0 software (Tamura et al., 2013) with the
neighbor-joining method (Saitou and Nei, 1987).
2.3. Production and purification of alginate lyase
Strain ALW1 was cultivated aerobically with shaking (180 rpm)
at 25 ◦ C in 10 flasks (250 ml) containing 50 ml of the selection
medium. After incubation for 60 h, the supernatant of the culture
was obtained by centrifugation at 15,000 × g, 4 ◦ C for 20 min. All the
following purification steps were carried out at 4 ◦ C. Solid ammo-
nium sulfate was slowly added to the supernatant with constant
stirring to 30% saturation. After 2 h at 4 ◦ C, the sample was cen-
trifuged at 15,000 × g for 20 min, and then ammonium sulfate was
added to the supernatant to 75% saturation. After 12 h at 4 ◦ C, the
precipitated proteins from the centrifugation (15,000 × g, 20 min)
was dissolved in 30 ml of 50 mM Tris–HCl buffer (pH 7.0) and
dialyzed against the same buffer. The sample was loaded on a
DEAE-Sepharose FF (GE Healthcare, Sweden) that had been equili-
brated with 50 mM Tris–HCl buffer (pH 7.0). Elution was performed
with a linear gradient of 0–1.0 M NaCl in the same buffer at a
flow rate of 1.0 ml/min, and fractions of 3.0 ml each were col-
lected. The fractions with alginate lyase activity were pooled and
then concentrated by a Millipore centrifugal filter 5 K device (5000
nominal molecular weight limit) (Millipore, USA). The sample was
applied to a HiPrep 16/60 Sephacryl S-100HR column (GE Health-
care, Sweden) equilibrated with 50 mM Tris–HCl buffer (pH 7.0).
The fractions were eluted with the same buffer at a flow rate
of 0.3 ml/min, and 0.6 ml fractions were collected each 2.0 min.
Fractions showing alginate lyase activity were pooled for further
enzyme characterization. The protein concentration was deter-
mined by Bradford method (Bradford, 1976) with bovine serum
albumin as the standard. The homogeneity and molecular mass of
the purified enzyme was analyzed by 12% sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) gel. The proteins
were developed by silver staining. Size exclusion chromatogra-
phy was used to determine the native molecular mass of the
enzyme. Experiments were performed on a HiPrep 16/60 Sephacryl
S-200HR column (GE Healthcare) developed in 50 mM Tris–HCl (pH
7.0). The column was calibrated by chromatographic protein stan-
dards (aldolase, 158 kDa; conalbumin, 75 kDa; carbonic anhydrase,
29 kDa; ribonuclease A, 13.7 kDa; aprotinin, 6.5 kDa).
2.4. Enzyme activity assay
Unless otherwise noted, the standard assay of alginate lyase
activity was determined by measuring the amount of released
reducing sugar equivalent using the 3,5-dinitrosalicylic acid (DNS)
method (Miller, 1959). The reaction was initiated by adding 10 ␮l
of enzyme solution (0.3 mg/ml) to 500 ␮l of 100 mM sodium phos-
phate buffer (pH 7.0) containing 0.5% (w/v) sodium alginate. After
incubation at 45 ◦ C for 30 min, it was stopped by adding 500 ␮l
of DNS reagent and heating at 100 ◦ C for 10 min. After cooling to
room temperature, the release of reducing sugar was monitored
at 540 nm using a Cary 50 spectrophotometer (Varian, USA). One
unit of alginate lyase activity was defined as the amount of enzyme
that released 1 ␮mol of the reducing sugar (glucose equivalent) per
minute under the assay conditions.
2.5. Substrate specificity
To investigate the substrate specificity, 0.5% (w/v) of sodium
alginate, poly-mannuronate (polyM) and poly-guluronate (polyG)
were used as the substrates to determine the enzyme activity.
Activity of the alginate lyase was determined by the increase in
absorbance at 235 nm due to the formation of a carbon–carbon dou-
ble bond at the end of the product generated from lyase-mediated
 Y. Zhu et al. / Microbiological Research 182 (2016) 49–58 51

Fig. 1. Unrooted neighbor-joining phylogenetic tree derived from 16S rRNA gene sequence of strain ALW1. Sequence alignment was performed using ClustalW and the
tree was built by MEGA 6.0 software. Numbers in parentheses represent the sequence accession numbers in GenBank. The number at each branch point is the percentage
supported by bootstrap. The scale indicates the number of amino acid substitutions per site.

cleavage of alginate (Linker et al., 1956). The reaction was initi-
ated by adding 5 ␮l of enzyme solution (0.3 mg/ml) to 200 ␮l of
the corresponding substrate solution and carried out at 45 ◦ C for
20 min. The amount of new unsaturated uronic acid yielded was
monitored by recording the absorbance of the reaction mixture
at 235 nm using a UV-5200 spectrophotometer (Shanghai Metash
Instruments Co., Ltd., China).
2.6. Effect of temperature on enzyme activity
The optimum temperature of alginate lyase was determined
at temperatures ranging from 25 ◦ C to 60 ◦ C. The thermal stabil-
ity of alginate lyase was investigated by incubating the enzyme at
25 ◦ C, 30 ◦ C, 35 ◦ C, 40 ◦ C, 45 ◦ C and 50 ◦ C for 0.5, 1–4 h, individually.
The residual activity of the enzyme was measured by the standard
method as described above. The activity of the enzyme without the
treatment was defined as 100%.
2.7. Effect of pH on enzyme activity
The optimal pH of alginate lyase was examined by assaying
enzyme activity over the pH ranges of 3.0–11.0. The buffer sys-
tems used were 100 mM acetate buffer (pH 3.0–6.0), 100 mM
sodium phosphate buffer (pH 6.0–8.0), 100 mM Tris–HCl buffer (pH
8.0–9.0), and 100 mM glycine–NaOH buffer (pH 9.0–11.0). Reac-
tions were carried out using 0.5% (w/v) sodium alginate as the
substrate in the appropriate buffer at 45 ◦ C for 30 min. The pH sta-
bility assay was performed by measuring the residual activity after
keeping the enzyme in buffers with different pH values (ranging
from 3.0 to 11.0) at 25 ◦ C for 30 min. The activity of the enzyme
without the treatment was defined as 100%.
2.8. Effects of metal ions on enzyme activity
The effects of metal ions on enzyme activity were determined
by adding various metal ions (LiCl, KCl, MgCl2 , CaCl2 , BaCl2 , NiCl2 ,
ZnCl2 , CoCl2 , CuCl2 , FeCl3 , and AlCl3 ) at final concentration of 1 mM
and NaCl at different concentrations (1, 10, 50, 100, 300, 500, 700,
900 and 1000 mM) directly to the substrate in 50 mM Tris–HCl
buffer (pH 7.0), individually. The reactions were initiated by adding
the enzyme solution to the substrate mixtures and carried out
at 45 ◦ C for 30 min. And then the residual activities were deter-
mined. The activity of the enzyme without addition of metal ion
was defined as 100%.
2.9. Effects of inhibitors and detergents on enzyme activity
The effects of inhibitors on enzyme activity were investigated by
using ethylenediaminetetraacetic acid (EDTA), phenylmethylsul-
fonyl fluoride (PMSF), ␤-mercaptoethanol (␤-ME), and dithiothre-
itol (DTT) at final concentrations of 1 mM or 10 mM. The enzyme
was incubated with each inhibitor at 25 ◦ C for 30 min, individually.
Residual activity was measured by the standard assay as described
above. The effects of detergents on enzyme activity were deter-
mined by using sodium dodecyl sulfate (SDS), Triton X-100, Tween
20, Tween 80, and 3-[(3-cholamidopropyl)dimethylammonio]-1-
propane sulfonate (Chaps) at final concentrations of 0.1% (w/v or
v/v) or 1% (w/v or v/v). They were also monitored by the same
procedure as described above. The activity of the enzyme without
additives was defined as 100%.
2.10. Determination of enzyme kinetic parameters
The kinetic properties of the enzyme were determined using
sodium alginate at different concentrations (0.05, 0.15, 0.25,
52 Y. Zhu et al. / Microbiological Research 182 (2016) 49–58
Fig. 2. SDS-PAGE of Microbulbifer sp. ALW1 alginate lyase. Lane 1: molecular weight
markers; lane 2: purified Microbulbifer sp. ALW1 alginate lyase. The arrow indicates
the position of alginate lyase.
0.35, 0.45 and 0.50 mg/ml) by the DNS method (Miller, 1959).
Michaelis–Menten substrate affinity constant (Km ), the turnover
number (kcat ), and catalytic efficiency (kcat /Km ) for sodium alginate
were calculated.
2.11. Analysis of enzymatic hydrolysates
After 0.5 U of alginate lyase was added to 100 ml of 50 mM
Tris–HCl buffer (pH 7.0) containing 1% (w/v) sodium alginate, the
enzymatic degradation was maintained at 37 ◦ C. Samples were
taken every one hour to determine the amount of released reducing
sugars by the DNS method (Miller, 1959). After 8 h, 0.1 U of alginate
lyase was added to the reaction mixture and the reaction was car-
ried out at the same temperature for 9 h. At this time, the amount
of released reducing sugars had no more change. The hydrolysates
were loaded on a Millipore centrifugal filter 10 K device (10,000
nominal molecular weight limit) (Millipore, USA). The filtrate was
collected and concentrated into powder by Labconco FreeZone 6
plus (ThermoFisher, USA). The composition of the hydrolysates was
profiled by electrospray ionization mass spectrometry (ESI–MS)
(Bruker Esquire HCT, USA) in a negative-ion mode with the follow-
ing conditions: capillary exit voltage, −260.0 V; Skimmer voltage,
−40.0 V; accumulation time, 100,000 ␮s; scan range, 100–1200
m/z.
2.12. Antioxidant activity assay of the enzymatic hydrolysates
2.12.1. DPPH radical scavenging assay
The above prepared hydrolysate powder was dissolved in dis-
tilled water, yielding a series of sample solutions with different
concentrations for the following antioxidant activity assays. The
scavenging activity of DPPH radical was assayed according to the
reported method (Kong et al., 2012) with some modifications. In
brief, 0.4 ml of the hydrolysate sample solution was mixed with
0.4 ml of 1 mM DPPH dissolved in ethanol. After the mixture was
shaken and incubated at room temperature for 30 min in the dark,
the absorbance of the resulting solution was measured at 517 nm.
The DPPH radical scavenging effect of the sample was calculated as
follows:
 A1

Scavenging effect(%) = 1− × 100
A0
where A0 is the absorbance of the control without sample, A1 is the
absorbance in the presence of the sample. The sample concentra-
Fig. 3. Substrate specificity of Microbulbifer sp. ALW1 alginate lyase. Enzyme activity
against the substrates at 45 ◦ C and pH 7.0 was determined spectrophotometrically at
235 nm. Values are mean ± standard deviation (SD) from three independent exper-
iments.
tion at which the inhibition percentage reaches 50% is defined as
the IC50 value. It can be used to assess the scavenging effect.
2.12.2. ABTS radical cation scavenging assay
The scavenging of ABTS radical cation was determined according
to the reported method of Alashi et al. (2014) with some modifi-
cations. ABTS radical cation (ABTS•+ ) stock solution was prepared
by dissolving 7 mM ABTS and 2.4 mM K2 S2 O8 in 50 mM sodium
phosphate buffer (pH 7.0) and the mixture was incubated at room
•
temperature for 12–16 h in the dark to generate ABTS + . The work-
•+
ing solution was prepared by diluting the ABTS stock solution
with 50 mM sodium phosphate buffer (pH 7.0) until the absorbance
was 0.70 ± 0.02 at 734 nm. Reaction solution consisted of 0.1 ml of
the hydrolysate sample solution and 1 ml of the working solution.
After the mixture was incubated at 30 ◦ C for 30 min, its absorbance
•
was determined at 734 nm. The ABTS + scavenging effect of the
sample was calculated by the formula above.
2.12.3. Hydroxyl radical scavenging assay
The scavenging of hydroxyl radical was determined according
to the reported method (Wu et al., 2012) with some modifications.
Reaction solution consisted of 0.2 ml of the hydrolysate sample
solution, 0.1 ml of 9 mM FeSO4 , 0.1 ml of 9 mM salicylic acid dis-
solved in ethanol, 0.1 ml of 8.8 mM H2 O2 , and 0.6 ml H2 O. After
the mixture was incubated at 37 ◦ C for 10 min, the absorbance was
detected at 510 nm. The hydroxyl radical scavenging effect of the
sample was calculated by the formula above.
2.12.4. Reducing power assay
The reducing power was investigated by the reported method
(Chang et al., 2007) with some modifications. Reaction solution
contained of 0.3 ml of the hydrolysate sample solution, 0.35 ml of
0.2 M sodium phosphate buffer (pH 6.6), and 0.35 ml of 1% (w/v)
potassium ferricyanide. The mixture was incubated at 50 ◦ C for
20 min, and then 0.35 ml of 10% (w/v) trichloroacetic acid (TCA)
and 0.15 ml of 0.1% (w/v) ferric chloride were added to the reaction
solution. The absorbance was determined at 700 nm. The increased
absorbance indicated the increased reducing power of the sample.
3. Results
3.1. Identification of strain ALW1
By utilizing the alginate as the sole carbon source, twenty-
six alginate metabolizing strains were obtained. Among them,
 Y. Zhu et al. / Microbiological Research 182 (2016) 49–58 53

Table 1
Purification of the alginate lyase from Microbulbifer sp. ALW1.

Purification step Total protein (mg) Total activity (U) Specific activity (U/mg) Purification fold Yield (%)

Culture supernatant 615.00 88.89 0.14 1.0 100.0

(NH4 )2 SO4 precipitation 92.70 15.42 0.17 1.2 17.3
DEAE-Sepharose FF 11.80 3.21 0.27 1.9 3.6
Sephacryl S-100HR 0.99 1.48 1.49 10.6 1.7

one strain designated as ALW1 showed the highest extracellu-
lar alginate lyase activity. Moreover, the lyase activity was not
detected in the non-sodium alginate medium under the same
conditions, which indicated that the enzyme was inducible. The
16S rRNA gene of strain ALW1 (GenBank accession number
KJ719305) was compared with available 16S rRNA gene sequences
from Nucleotide collection database of NCBI. It shared 100%,
98.3%, 97.4%, 97.1%, 96.8% and 96.3% identities with that from
Microbulbifer sp. MG10 (GenBank accession number JN791316),
Microbulbifer arenaceous RSBr-1 (GenBank accession number
NR114885), Microbulbifer maritimus TF-17 (GenBank accession
number NR025772), Microbulbifer elongatus DSM6810 (GenBank
accession number NR025246), Microbulbifer donghaiensis CN85
(GenBank accession number NR044478), and Microbulbifer sali-
paludis SM-1 (GenBank accession number NR025232), respectively.
In the phylogenetic tree, strain ALW1 16S rRNA gene formed a
distinct group with that from Microbulbifer sp. MG10 (Fig. 1).
indicated that the enzyme had a relatively narrow range of thermal
stability.
3.5. Effect of pH on alginate lyase activity
The optimal pH of Microbulbifer sp. ALW1 alginate lyase was
investigated at various pH values at 45 ◦ C. As shown in Fig. 5A,
the enzyme was most active at pH 7.0, and presented over 30%
of its maximal activity in the pH range of 5.5–9.0. There was no
detectable enzyme activity at pH 3.5. The enzyme still had 11% of
relative activity at pH 11.0. The pH stability of alginate lyase was
determined by measuring the residual activity after incubating the
enzyme in a series of buffers with different pH values (3.0–11.0)
at 25 ◦ C for 30 min (Fig. 5B). The results showed that strain ALW1
alginate lyase was stable over a broad pH range of 5.0–9.0, retaining
over 60% of its original activity. It also could maintain 32% and 42%
of residual activities at pH 4.0 and 11.0, respectively.

3.2. Purification of alginate lyase from Microbulbifer sp. ALW1 3.6. Effect of metal ions on alginate lyase activity

As summarizes in Table 1, alginate lyase from Microbulbifer sp. The effects of metal ions on the alginate lyase activity were
ALW1 was purified through the steps of centrifugation, ammo- investigated by using various metal ions (Na+ , Li+ , K+ , Mg2+ , Ca2+ ,
nium sulfate fractionation, anion-exchange chromatography and Ba2+ , Ni2+ , Zn2+ , Co2+ , Cu2+ , Fe3+ and Al3+ ) at a final concentration of
size exclusion chromatography. The enzyme was purified 10.6 fold. 1 mM (Table 2). The enzyme was inhibited by Ba2+ , Ni2+ , Zn2+ , Co2+ ,
Using 0.5% sodium alginate as the substrate, the specific activity of Cu2+ , Fe3+ and Al3+ , and the effect of Al3+ was the most prominent.
the purified alginate lyase was 1.49 U/mg. A single band with the With Li+ , K+ , Mg2+ and Ca2+ , the enzyme activity was not affected.
molecular mass of 26.2 kDa was detected by SDS-PAGE (Fig. 2, lane Na+ at 1 mM stimulated the alginate lyase activity. Effects of dif-
2). The molecular weight of the native protein was determined to ferent concentrations of Na+ were further investigated (Fig. 6). The
be 25.9 kDa by size exclusion chromatography using a Sephacryl results showed that the highest activity (511% of relative activity)
S-200 column (data not shown). These results suggested that the was observed in the presence of 0.5 M NaCl. These indicated that
purified alginate lyase from Microbulbifer sp. ALW1 was a monomer NaCl is essential for strain ALW1 alginate lyase activity.
with the molecular mass of about 26.0 kDa.
3.7. Effect of inhibitors and detergents on alginate lyase activity
3.3. Substrate specificity
The effects of some inhibitors on alginate lyase activity were
The substrate specificity was investigated by measuring the examined by using EDTA, PMSF, ␤-ME and DTT (Table 3). The
OD235 value of the unsaturated uronic acids that were generated enzyme was strongly inhibited by EDTA at 1 mM and 10 mM. When
from the oligomers via a ␤-elimination reaction using alginate, PMSF was added, very slight inhibition on the enzymatic activity
polyG, and polyM as the substrates. As shown in Fig. 3, strain ALW1 was observed at both concentrations tested. DTT at 1 mM had no
alginate lyase showed activity towards alginate, polyG and polyM. influence on alginate lyase activity, whereas at 10 mM, its effect
It degraded polyG more efficiently than polyM. became positive. With ␤-ME, the enzyme activity was not affected
at both concentrations tested. The effects of some detergents on
3.4. Effect of temperature on alginate lyase activity
Table 2
The optimum temperature of Microbulbifer sp. ALW1 alginate Effects of metal ions on Microbulbifer sp. ALW1 alginate lyase activity. The enzyme
lyase was determined over the temperature range of 25–60 ◦ C. As was incubated with each metal ion (final concentration of 1 mM) in 50 mM Tris–HCl
buffer (pH 7.0) containing 0.5% sodium alginate at 45 ◦ C for 30 min, individually.
shown in Fig. 4A, the enzyme had an optimum temperature of 45 ◦ C,
The activity was determined by using the standard assay described in Section 2. The
and presented over 35% of relative activity in temperatures ranging alginate lyase activity of the enzyme without addition of metal ion was defined as
from 30 to 50 ◦ C. It had nearly no activity at 60 ◦ C. The thermostabil- 100%. Values are mean ± SD from three independent experiments.
ity of strain ALW1 alginate lyase was investigated by measuring the
Metal ion Relative activity (%) Metal ion Relative activity (%)
residual activity after incubating the enzyme at 25 ◦ C, 30 ◦ C, 35 ◦ C,
40 ◦ C, 45 ◦ C and 50 ◦ C for different times, respectively (Fig. 4B). The Control 100.0 ± 0.7 Ni2+ 41.9 ± 1.9
Na+ 115.1 ± 0.9 Zn2+ 31.6 ± 2.9
alginate lyase was relatively stable at 25 ◦ C, 30 ◦ C, 35 ◦ C and 40 ◦ C Li+ 102.9 ± 2.9 Co2+ 10.9 ± 0.9
after incubation for 4 h. Incubation at higher temperatures resulted K+ 102.0 ± 3.5 Cu2+ 10.1 ± 1.2
in enzyme activity loss. The enzyme maintained 68% and 21% of Mg2+ 102.6 ± 4.3 Fe3+ 11.3 ± 4.2
residual activities after incubation at 45 ◦ C for 1 h and 4 h, respec- Ca2+ 104.1 ± 2.4 Al3+ 5.7 ± 0.9
Ba2+ 72.3 ± 1.2
tively. It had nearly no activity at 50 ◦ C for 30 min. These results
54 Y. Zhu et al. / Microbiological Research 182 (2016) 49–58

Fig. 4. Effect of temperature on Microbulbifer sp. ALW1 alginate lyase activity. (A) Optimum reaction temperature of the alginate lyase. The enzymatic activity at different
temperatures was determined. Activity at 45 ◦ C was taken as 100%. (B) Thermostability of the alginate lyase. The enzyme was incubated at 25 ◦ C, 30 ◦ C, 35 ◦ C, 40 ◦ C, 45 ◦ C
and 50 ◦ C for different times, individually. Residual activity was measured by using the standard assay described in Section 2. The initial activity was taken as 100%. Data are
presented as mean ± SD from three independent experiments.

Table 3 ual activity was maintained after treatment with these detergents
Effects of inhibitors and detergents on Microbulbifer sp. ALW1 alginate lyase activ-
at both concentrations tested.
ity. The enzyme was incubated with each inhibitor and detergent with different
final concentrations at 25 ◦ C for 30 min, individually. Residual activity was mea-
sured by using the standard assay described in Section 2. Reaction mixture without
inhibitors and detergents was used as a reference. Values are mean ± SD from three 3.8. Enzyme kinetic parameters
independent experiments.

Inhibitors and detergents Concentration Relative activity (%) Using sodium alginate as the substrate, the Km and Vmax values of
strain ALW1 alginate lyase evaluated from Lineweaver–Burk dou-
Control – 100.0 ± 1.6
EDTA 1 mM 49.7 ± 2.8 ble reciprocal plot were 1.03 mg/ml and 4.63 U/mg, respectively.
10 mM 10.6 ± 2.1 The turnover number kcat and the catalytic efficiency kcat /Km values
PMSF 1 mM 94.0 ± 4.0 were 69.38 s−1 and 67.36 s−1 mg−1 ml, respectively.
10 mM 95.3 ± 1.2
␤-ME 1 mM 101.7 ± 3.4
10 mM 102.7 ± 4.9
DTT 1 mM 100.9 ± 2.8 3.9. Analysis of the hydrolysates by ESI-MS
10 mM 128.8 ± 1.0
SDS 0.1% 98.5 ± 1.1 The sodium alginate (1 g) was degraded with strain ALW1 algi-
1% 88.1 ± 2.4
nate lyase until the released reducing sugars stabilized (data not
Triton X-100 0.1% 104.6 ± 2.5
1% 106.9 ± 1.3
shown). The amount of the hydrolysates was 630 mg. These prod-
Tween 20 0.1% 100.3 ± 1.1 ucts were then determined by ESI-MS. Under the negative mode,
1% 107.1 ± 5.0 the degrees of polymerization (DPs) with mass-to-charge ratios
Tween 80 0.1% 99.3 ± 1.1 (m/z) of the products were mainly presumed to be 2 (352 m/z) and
1% 109.8 ± 4.6
3 (527 m/z) from ion peaks of [DPx – H]− (x = 2, 3) species (Fig. 7).
Chaps 0.1% 96.9 ± 0.9
1% 92.4 ± 1.0 For peaks of [DPx – 2H + Na]− (x = 2, 3) species, DPs of the products
were 2 (374 m/z) and 3 (549 m/z) (Fig. 7). These results revealed
that prolonged strain ALW1 alginate lyase-mediated degradation
alginate lyase activity were examined by using SDS, Triton X-100, of alginate produced disaccharides and trisaccharides, and disac-
Tween 20, Tween 80, and Chaps (Table 3). More than 85% of resid- charides were the most abundant products.
 Y. Zhu et al. / Microbiological Research 182 (2016) 49–58 55

Fig. 5. Effect of pH on Microbulbifer sp. ALW1 alginate lyase activity. (A) Optimum reaction pH of the alginate lyase. The enzyme activity was determined at 45 ◦ C in 100 mM
acetate buffer (filled rhombus), 100 mM sodium phosphate buffer (filled square), 100 mM Tris–HCl buffer (filled triangle), and 100 mM glycine-NaOH buffer (open triangle).
Activity at pH 7.0 was taken as 100%. (B) pH stability of the alginate lyase. The enzyme was incubated in buffers with different pH values at 25 ◦ C for 30 min. Residual activity
was measured by using the standard assay described in Section 2. Activity before treatment was defined as 100%. Each value represents the mean of three replicates ± SD.

3.10. Antioxidant activity of the hydrolysates
Assays of reducing power, scavenging DPPH, ABTS and hydroxyl
radicals were used to assess the antioxidant activity (Fig. 8). The
enzymatic hydrolysates had the inhibitory effect on the DPPH rad-
icals and the scavenging activity improved with the increasing
of the concentration (Fig. 8A). At the concentration of 18 mg/ml,
the inhibitory effect was 81.0%. The IC50 value of the hydrolysates
was 10.42 mg/ml. As shown in Fig. 8B, the ABTS radical scaveng-
ing activity of the hydrolysates was in a dose-dependent manner.
When the hydrolysate concentration was 3.0 mg/ml, the scaveng-
ing rate reached 100%. The IC50 value of the hydrolysates was
1.23 mg/ml. As shown in Fig. 8C, the hydrolysates exhibited evident
hydroxyl radical scavenging activity in a concentration-dependent
manner. The activity increased dramatically from 8.9% to 52.1% at
the hydrolysate concentration of 1.0–3.0 mg/ml. The IC50 value of
the hydrolysates was 3.35 mg/ml. In the reducing power assay, the
reducing power improved with the increasing of the concentration,
displaying a linear correlation (Fig. 8D).
4. Discussion

Alginate lyase can be used to degrade alginate for preparation

Fig. 6. Effect of NaCl on Microbulbifer sp. ALW1 alginate lyase activity. Different con-
centrations of NaCl were directly added to the reaction mixtures in 50 mM Tris–HCl
buffer (pH 7.0), individually. The enzyme activity without addition of NaCl was
defined as 100%. Values are mean ± SD from three independent experiments.
of alginate oligosaccharides having many interesting biologi-
cal activities. In addition, it also plays an important role in
medical application, seaweed engineering, and study of alginate
metabolism. In this study, an alginate lyase-producing bacterium
was isolated which was identified as Microbulbifer sp. ALW1. An
alginate lyase was successfully purified from strain ALW1 with a
molecular mass of approximately 26.0 kDa.
Strain ALW1 alginate lyase exhibited activity towards both
polyG and polyM substrates. These excellent characteristics sug-
gested this alginate lyase had potential applications in production
of alginate oligosaccharides with low DPs. Similar results have been
observed in alginate lyases from Isoptericola halotolerans CGMCC
5336 (Dou et al., 2013), Microbulbifer sp. 6532A (Swift et al., 2014), P.
elyakovii (Ma et al., 2008), Streptomyces sp. ALG-5 (Kim et al., 2009)
and Vibrio sp. QY105 (Wang et al., 2013). Some alginate lyases are
polyG or polyM-specific (Cao et al., 2007; Kam et al., 2011; Huang
et al., 2013).
Microbulbifer sp. ALW1 alginate lyase exhibited the maximal
activity at 45 ◦ C, which is exactly the same as that for alginate lyase
56 Y. Zhu et al. / Microbiological Research 182 (2016) 49–58
Fig. 7. ESI-MS analysis of the hydrolysis products by Microbulbifer sp. ALW1 alginate lyase.
from Flavobacterium sp. S20 (Huang et al., 2013). Good enzyme ther-
mostability may create favorable condition for its applications. In
this study, strain ALW1 alginate lyase retained 91% residual activ-
ity after incubation at 40 ◦ C for 4 h. It also maintained 68% and 21%
residual activities after incubation at 45 ◦ C for 1 h and 4 h, respec-
tively. Its thermal stability is comparable to alginate lyase from
I. halotolerans CGMCC 5336 (Dou et al., 2013) and alginate lyase
Alg2A from Flavobacterium sp. S20 (Huang et al., 2013). Microbulb-
ifer sp. ALW1 alginate lyase is less stable than its counterparts from
Streptomyces sp. A5 (Cao et al., 2007) and Vibrio sp. QY105 (Wang
et al., 2013). However, it is substantially more stable than alginate
lyase from P. elyakovii (83.5% residual activity after 10 min at 30 ◦ C)
(Ma et al., 2008), alginate lyase from Pseudoalteromonas sp. SM0524
(half-life period of 41 min at 40 ◦ C) (Li et al., 2011a), alginate lyases
B and C from P. fluorescens HZJ216 (about 30% and 40% residual
activities of 30 min at 40 ◦ C, individually) (Li et al., 2011b), and algi-
nate lyase from Vibrio sp. YWA (about 30% residual activity after
60 min at 40 ◦ C) (Wang et al., 2006). The optimum pH of Microb-
ulbifer sp. ALW1 alginate lyase was 7.0, which was the same as
that for the alginate lyases from I. halotolerans CGMCC 5336 (Dou
et al., 2013), P. fluorescens HZJ216 (Li et al., 2011b), S. degradans
(Kim et al., 2012), and Vibrio sp. QY105 (Wang et al., 2013). In this
study, strain ALW1 alginate lyase showed an excellent stability in
pH range of 5.0–9.0, maintaining more than 60% of residual activity
after incubation at 25 ◦ C for 30 min. These properties could convert
Microbulbifer sp. ALW1 alginate lyase into an attractive enzyme for
its biotechnological applications.
The effects of some metal ions, inhibitors or detergents on
alginate lyase activity were determined in this study. NaCl could
strongly activate the enzyme activity of Microbulbifer sp. ALW1
alginate lyase, which is similar to alginate lyases from Agarivorans
sp. JAM-A1m (Kobayashi et al., 2009), I. halotolerans (Dou et al.,
2013), Microbulbifer sp. 6532A (Swift et al., 2014), and Vibrio sp.
QY105 (Wang et al., 2013). Strain ALW1 alginate lyase was greatly
inhibited by chelating agent EDTA, indicating that it might be a
metalloenzyme having some requirement for divalent cations. For
alginate lyase from Microbulbifer sp. 6532A, EDTA does not alter
the enzyme activity (Swift et al., 2014). Except EDTA, Microbulbifer
 Y. Zhu et al. / Microbiological Research 182 (2016) 49–58
Fig. 8. Antioxidant activity of the hydrolysates produced by Microbulbifer sp. ALW1 alginate lyase. (A) Scavenging effect on DPPH radical. (B) Scavenging effect on ABTS
radical cation. (C) Scavenging effect on hydroxyl radical. (D) Reducing power of the hydrolysates. Values are presented as mean ± SD from three independent experiments.
sp. ALW1 alginate lyase exhibited good resistance towards other
inhibitors and detergents tested, which reinforces its potential
applications in related industrial process.
Enzymatic hydrolysates analysis by MS revealed that the prod-
ucts contained disaccharides and trisaccharides, which indicated
that the smallest substrate of strain ALW1 alginate lyase is pen-
tasaccharides. This feature is similar to that of the alginate lyase
from Pseudoalteromonas sp. SM0524 (Li et al., 2011a). The alginate
lyase-mediated degradation products by Microbulbifer sp. ALW1 is
different from that by Microbulbifer sp. 6532A (Swift et al., 2014).
Oligosaccharides possess many biological activities, which are
affected by many factors, including monosaccharide composition,
glycosyl residues, and chain conformation (Bao et al., 2001). In
this study, in vitro antioxidant activity of alginate oligosaccharides
yielded by Microbulbifer sp. ALW1 alginate lyase was investigated.
Judged by the scavenging abilities towards radicals (DPPH, ABTS+
and hydroxyl) and the reducing power, the hydrolysates showed
the effective antioxidant activity. Unlike the chemical compounds,
the hydrolysates in this study did not show poisonous effects at
the high concentrations, and they might be exploited as available
antioxidant products in food and pharmaceutical industries.
Acknowledgements
This work was financially supported by National Natural Sci-
ence Foundation of China (No. 31401632), Projects of Xiamen
Southern Ocean Technology Center of China (Nos. 13PZP002NF03
and 13GZP004NF10), and the Open Research Foundation of Fujian
57
Provincial Key Laboratory of Food Microbiology and Enzyme Engi-
neering, China (No. M20130905).
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