Overexpression and characterization of a novel thermostable β-agarase YM01-3, from marine bacterium Catenovulum agarivorans YM01T

Article abstract of DOI:10.3390/md12052731

Genome sequencing of Catenovulum agarivorans YM01^T reveals 15 open-reading frames (ORFs) encoding various agarases. In this study, extracellular proteins of YM01^T were precipitated by ammonium sulfate and separated by one-dimensional gel electrophoresis. The results of in-gel agarase activity assay and mass spectrometry analysis revealed that the protein, YM01-3, was an agarase with the most evident agarolytic activity. Agarase YM01-3, encoded by the YM01-3 gene, consisted of 420 amino acids with a calculated molecular mass of 46.9 kDa and contained a glycoside hydrolase family 16 β-agarase module followed by a RICIN superfamily in the C-terminal region. The YM01-3 gene was cloned and expressed in Escherichia coli. The recombinant agarase, YM01-3, showed optimum activity at pH 6.0 and 60°C and had a K_m of 3.78 mg mL^-1 for agarose and a V_max of 1.14 × 10⁴ U mg^-1. YM01-3 hydrolyzed the β-1,4-glycosidic linkages of agarose, yielding neoagarotetraose and neoagarohexaose as the main products. Notably, YM01-3 was stable below 50°C and retained 13% activity after incubation at 80°C for 1 h, characteristics much different from other agarases. The present study highlights a thermostable agarase with great potential application value in industrial production.

Full text of DOI:10.3390/md12052731

Mar. Drugs 2014, 12, 2731-2747; doi:10.3390/md12052731
OPEN ACCESS
marine drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Article
Overexpression and Characterization of a Novel
Thermostable β-Agarase YM01-3, from Marine
Bacterium Catenovulum agarivorans YM01T
Fangyuan Cui 1,†, Sujie Dong 1,†, Xiaochong Shi 1,*, Xia Zhao 2 and Xiao-Hua Zhang 1
1
College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China;
E-Mails: joyceyujian@163.com (F.C.); dongsujie310@163.com (S.D.);
xhzhang@ouc.edu.cn (X.-H.Z.)
2
School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China;
E-Mail: zhaoxia@ouc.edu.cn
†
These authors contributed equally to this work.
* Author to whom correspondence should be addressed; E-Mail: shixiaochong@ouc.edu.cn;
Tel./Fax: +86-532-8203-2767.
Received: 26 November 2013; in revised form: 21 April 2014 / Accepted: 22 April 2014 /
Published: 12 May 2014
Abstract: Genome sequencing of Catenovulum agarivorans YM01T reveals 15 open-reading
frames (ORFs) encoding various agarases. In this study, extracellular proteins of YM01T
were precipitated by ammonium sulfate and separated by one-dimensional gel electrophoresis.
The results of in-gel agarase activity assay and mass spectrometry analysis revealed that
the protein, YM01-3, was an agarase with the most evident agarolytic activity. Agarase
YM01-3, encoded by the YM01-3 gene, consisted of 420 amino acids with a calculated
molecular mass of 46.9 kDa and contained a glycoside hydrolase family 16 β-agarase
module followed by a RICIN superfamily in the C-terminal region. The YM01-3 gene was
cloned and expressed in Escherichia coli. The recombinant agarase, YM01-3, showed
optimum activity at pH 6.0 and 60 °C and had a Km of 3.78 mg mL−1 for agarose and a Vmax
of 1.14 × 104 U mg−1. YM01-3 hydrolyzed the β-1,4-glycosidic linkages of agarose,
yielding neoagarotetraose and neoagarohexaose as the main products. Notably, YM01-3
was stable below 50 °C and retained 13% activity after incubation at 80 °C for 1 h,
characteristics much different from other agarases. The present study highlights a
thermostable agarase with great potential application value in industrial production.

Mar. Drugs 2014, 12
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Keywords: Catenovulum agarivorans YM01T; expression; characterization; YM01-3;
β-agarase; thermostable
1. Introduction
Agar, as one of the marine polysaccharides used widely at present, is a component found in the cell
walls of some marine red algae, Gracilariales and Gelidiales, and consists of two different components,
agarose and agaropectin [1]. Agarose is a neutral linear polysaccharide composed of alternating
residues of 3-O-linked β-D-galactopyranose and 4-O-linked 3,6-anhydro-α-L-galactopyranose [2].
Agaropectin has the same basic disaccharide-repeating units as agarose, although some hydroxyl groups
of 3,6-anhydro-α-L-galactose residues are substituted by sulfoxy or methoxy and pyruvate residues.
Agarases are a group of glycoside hydrolases (GH) that degrade agar into oligosaccharides.
According to the cleaving mechanisms, agarases could be classified into two groups, i.e., α-agarases
(EC 3.2.1.158), which cleave α-1,3 linkages to produce agaro-oligosaccharides [3], and β-agarases
(EC 3.2.1.81), which cleave β-1,4 linkages to produce neoagarooligosaccharides [4]. According to the
CAZy database [5,6], β-agarases are classified into four families of GH16, GH50, GH86 and GH118,
based on the amino acid sequence similarity [7]. To date, many bacterial genera with agarolytic
activity have been identified, which were mainly isolated from seawater and marine sediment [8],
including the genera, Agarivorans [9], Alteromonas [10], Microscilla [11], Pseudoalteromonas [12],
Pseudomonas [13], Pseudozobellia [14], Saccharophagus [15], Vibrio [16], etc. In addition, there are a
few agarolytic bacterial genera from freshwater and terrestrial soils, such as Acinetobacter [17],
Bacillus [18], Cellvibrio [8] and Cytophaga [19]. Agarases produced by the above bacteria are
potential biocatalysts to modify agars and alter their properties, producing new, specific algal
biomolecules for foods, cosmetics and pharmaceuticals [20]. Catenovulum agarivorans YM01T, an
agar-hydrolyzing marine bacterium, recently isolated from seawater of the Yellow Sea of China, was
identified as a novel genus and species [21]. It has been shown that the extracellular proteins of
YM01T have high agarolytic activity and thermostability. Moreover, 15 complete coding sequences of
agarases (including two α-agarases and 13 β-agarases) from the C. agarivorans YM01T genome were
identified by the whole-genome sequencing [22]. In this study, to explore the characteristics and
commercial significance of C. agarivorans agarase, the β-agarase gene, YM01-3, with the most
evident agarolytic activity according to the in-gel agarase activity assay and mass spectrometry
analysis was cloned and overexpressed. The purified recombinant agarase was characterized, and its
enzymatic products were analyzed. On account of its noticeable high agarolytic activity and
thermostability, this enzyme has potential industrial applications.
2. Results and Discussion
2.1. Agarolytic Activity of the Extracellular Proteins of YM01T
The result of SDS-PAGE and in-gel detection of agarase activity (Figure 1) showed that the
extracellular proteins of YM01T had agarolytic activity, and the protein band of approximately 40 kDa

Mar. Drugs 2014, 12
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on SDS-PAGE showed the most evident agarase activity. The result of mass spectrometry analysis
revealed that the 40-kDa protein band contained the YM01-3 gene encoded agarase along with several
other proteins, e.g., lipoprotein, flagellin and aspartate-semialdehyde dehydrogenase. The YM01-3
gene (GenBank accession No. KF413621) was therefore chosen for further study.
Figure 1. SDS-PAGE and in situ detection of agarolytic activity. Lane M, molecular mass
marker (Fermentas SW0431); Lane 1, the crude extracellular proteins of YM01T; Lane 2,
in situ detection of the crude agarase, and the arrow indicates the band cut for MS analysis.
SDS-PAGE was performed with 10% (w/v) polyacrylamide gel. The gel with Lane M and
Lane 1 was stained with Coomassie brilliant blue R-250; the agar plate used for in-gel
detection (Lane 2) was stained by Lugol’s iodine solution.
2.2. Cloning and Sequence Analysis of the YM01-3 Gene
The YM01-3 gene, a 1263-bp open reading frame (ORF), was amplified from the genomic
DNA of C. agarivorans YM01T using the primer sets. The ORF encoded a deduced protein of 420 amino
acids, with an estimated molecular mass of 46.9 kDa and a putative isoelectric point of 5.66. No
putative signal peptide was detected with the SignalP 4.0 server [23]. Sequence analysis using BLAST
search in the database, NCBI, showed that the deduced protein was mainly comprised of a 269 amino
acid β-agarase domain that was homologous to the catalytic module of family 16 glycoside hydrolase
(GH16) and a 134 amino acid sequence belonging to the RICIN superfamily (ricin-type beta-trefoil;
carbohydrate-binding domain formed from presumed gene triplication). The protein displayed the
catalytic residues (Glu130-Asp132-Glu135) and the catalytic motif (E[ILV]D[IVAF][VILMF][0,1]E), which
are prevalent in the agarases belonging to the GH16 family. Glu130 and Glu135 act as nucleophile and
an acid/base, respectively, while Asp132 is probably important in maintaining the charges in the
environment of catalytic amino acids [24]. Moreover, the encoded protein showed high identity to
other β-agarases of the GH16 family: 76% to the β-agarase AgaB34 from Glaciecola agarilytica NO2
(WP_008302724.1), 76% to the β-agarase I from Pseudoalteromonas atlantica (AAA91888), 74% to
the β-agarase from Aeromonas sp. (AAF03246), 56% to the β-agarase A from Zobellia galactanivorans

Mar. Drugs 2014, 12
2734
(1O4Y) and 52% to the β-agarase from Coraliomargarita akajimensis DSM 45221 (YP_003547559.1)
(Figure 2). The GH16 family, consisting of more than 2700 members, is classified into ten specific
subfamilies, including β-agarase (EC 3.2.1.81), licheninase (EC 3.2.1.73), κ-carrageenase
(EC 3.2.1.83), xyloglucanase (EC 3.2.1.151), endo-β-1,3-galactanase (EC 3.2.1.181), β-porphyranase
(EC 3.2.1.178), etc. [6,25]. To determine the relationship between the YM01-3 protein and other
known glycoside hydrolases from GH16, a neighbor-joining tree (Figure 3) based on amino acid
sequences was constructed, which revealed the evolutionary relationship between protein YM01-3 and
other known glycoside hydrolases and showed that YM01-3 formed a tight phylogenetic cluster in the
β-agarase clade.
2.3. Expression and Purification of the Recombinant YM01-3
The recombinant plasmid pET-24a (+)/YM01-3 was conditionally expressed in the E. coli BL21 (DE3)
as a C-terminally His-tagged recombinant protein. After IPTG (isopropyl-β-D-thiogalactopyranoside)
induction, the agarolytic activity of the recombinant protein was detected in the supernatant of the cell
lysate. To maintain its bioactivity, the recombinant protein was purified from the supernatant under
native conditions. The purified enzyme (including the 6-His tag) gave a single band with a molecular
weight of about 46 kDa on SDS-PAGE (Figure 4). Further analysis by mass spectroscopy showed that
the accurate molecular mass of YM01-3 (including the histidine tag and 16 amino acids from pET 24a
(+)) was 49.528 kDa [26]. To find out whether agarase YM01-3 was an endo- or exo-glycoside
hydrolase, as well as to determine its end hydrolysis products, a time course hydrolysis analysis was
carried out with the purified recombinant enzyme. As shown in Figure 5, four major spots were clearly
detected. Ion trap mass spectra analysis of the Spot 1 revealed that it has a molecular ion at m/z of
629.19 [M − H]− and 651.17 [M + Na − 2H]−, and it was determined to be neoagarotetraose (DP4).
Meanwhile, Spot 2, Spot 3 and Spot 4 were assigned as neoagarohexaose (DP6) (m/z = 935.29 [M − H]−,
997.29 [M + HCO3]−), neoagarooctaose (DP8; m/z = 1241.37 [M − H]−, 1303.37 [M + HCO3]−,
1325.35 [M + HCO3 + Na − 2H]−, 620.18 [M − 2H]2−) and neoagarodecaose (DP10; m/z = 1547.47
[M − H]−, 1609.47 [M + HCO3]−, 1631.45 [M + HCO3 + Na − 2H]−, 773.23 [M − 2H]2−), respectively.
According to the results of thin-layer chromatography (TLC) and MS analysis, agarose incubation with
YM01-3 generated DP10 and DP8 firstly, followed by DP6 and DP4, and the amount of DP6 and DP4
increased in a time-dependent manner. This hydrolysis pattern indicated that β-agarase YM01-3 was
an endo-type β-agarase. In addition, after incubation for 24 h, the main products were DP6 and DP4,
which is a characteristic of the GH16 family agarases. Therefore, with the evidence obtained above,
we conclude that the β-agarase YM01-3 should be cataloged into family 16 of glycoside hydrolases
and proceeded with a common catalytic mechanism of GH16 family, which led to the overall retention
of the anomeric configuration and to trans-glycosylating activity [25].

Mar. Drugs 2014, 12
Figure 2. Multiple amino acid sequence alignment of the YM01-3 agarase of Catenovulum agarivorans YM01T with known GH16 family
2735
β-agarases. Identical residues in all sequences are indicated by asterisks under the column; conserved substitutions are indicated by colons,
and semi-conserved substitutions are indicated by dots; pound signs highlight the catalytic residues, and black rectangles highlight the
catalytic motif. Deletions are indicated by dashes.

Mar. Drugs 2014, 12
Figure 3. Neighbor-joining tree based on amino acid sequences of YM01-3 and other
2736
GH16 family members showing the relationship among protein YM01-3 and other known
glycoside hydrolase from GH16. Numbers at nodes are the levels of bootstrap support (%).
Scale bar, 0.2 substitutions per amino acid position.
Figure 4. SDS-PAGE of purified recombinant agarase YM01-3. Lane M, molecular mass
marker (Fermentas SW0431); Lane 1, purified agarase YM01-3. SDS-PAGE was
performed with 12% (w/v) polyacrylamide gel. Gels were stained with coomassie brilliant
blue R-250.

Mar. Drugs 2014, 12
Figure 5. TLC chromatogram depending on the reaction time. The hydrolysis reactions
2737
were carried out at 50 °C for 24 h in 20 mM Tris-HCl buffer (pH 8.0) containing
0.25% agarose and then separated on a Silica Gel 60 TLC plate at appropriate
time intervals.
2.4. Biochemical Characterization of YM01-3
Both the effects of temperature and pH on the activity of the recombinant agarase were evaluated.
The results showed that the optimal temperature for β-agarase YM01-3 was 60 °C (Figure 6a), and
YM01-3 retained more than 80% agarolytic activity after being kept in 50 °C for 1 h, 13% residual
activity after incubation in 80 °C for 1 h (Figure 6a) and 12% residual activity, even if being boiled
for 5 min [26]. The enzyme exhibited maximum agarase activity at pH 6.0 and retained more than 80%
of activity after incubation at a wide range of 4.0–9.0 for 12 h at 4 °C (Figure 6b).
The results of the effect of various metal ions and agents on the activity of the purified agarase
(Table 1) showed that some metal ions, such as Cu2+, Mn2+, Fe3+ and Ni2+, had a highly significant
negative effect on the activity of the agarase, YM01-3, while Na+, K+ and Ca2+ increased the agarase
activity significantly. In addition, Mg2+, urea, EDTA and SDS did not have obvious effects on
the activity.
Based on the results obtained above, all enzymatic reactions were performed in citric
acid-sodium citrate buffer (pH 6.0) at 60 °C, the Km and Vmax for agarose were 3.78 mg mL−1
and 1.14 × 104 U mg−1 of protein, respectively. Compared the kinetic parameters of YM01-3 to those
of other known β-agarases, the Vmax value (1.14 × 104 U mg−1) was remarkably higher than that of
agarase AgaA (909.1 U mg−1) [27], which is the highest Vmax value reported (Table 2).

Mar. Drugs 2014, 12
Figure 6. Effect of temperature (a) and pH (b) on the agarolytic activity and stability of the
2738
recombinant β-agarase YM01-3. Temperature profiles were measured at different temperatures
(0–100 °C) in 20 mM Tris-HCl buffer (pH 8.0). pH profiles were measured at 50 °C in
different buffers. All data shown are the mean values from at least three replicate experiments.
Table 1. Effects of various reagents on the activity of the recombinant β-agarase YM01-3.
Reagent
None
SDS
Urea
EDTA
Na+
Concentration (mM) a
Relative Activity (%) b
100
1
1
109 ± 0.02
101 ± 0.056
81 ± 0.016
1
1
122 ± 0.13 **
122 ± 0.12 **
113 ± 0.13 *
88 ± 0.07
K+
1
Ca2+
1
Mg2+
Mn2+
Fe3+
1
1
28 ± 0.04 **
14 ± 0.03 **
34 ± 0.08 **
1 ± 0.01 **
1
Cu2+
Ni2+
1
10
The activity was analyzed with 0.25% (w/v) agarose in 20 mM Tris-HCl buffer (pH 8.0) at 50 °C for 10 min.
a
Results are shown as the mean ± SEM from three independent experiments. The concentration is the final
b
concentration of the reagents in the activity detection system; the activity measured under the condition
without being treated with various ions and reagents was defined as 100%; * p < 0.05; ** p < 0.01.

Mar. Drugs 2014, 12
2739
Table 2. The partial characterization of agarases from different families.
Optimal
Vmax
(μmol min−1mg−1)
1.14 × 104
909.1
22.5
Family
Bacterial Species and Protein
Accession No.
Thermostability
Reference
Temperature
60 °C
40 °C
40 °C
40 °C
40 °C
50 °C
50 °C
55 °C
55 °C
55 °C
40 °C
40 °C
40 °C
40 °C
30 °C
40 °C
40 °C
40 °C
40 °C
45 °C
42.5 °C
45 °C
35 °C
40 °C
52 °C
Catenovulum agarivorans YM01T (YM01-3)
Agarivorans sp. LQ48 (AgaA)
AGU13985
ACM50513
BAF62129
BAF34350
AAN39119
BAC99022
None a
Stable below 50 °C for 1 h.
Stable below 40 °C for 1 h.
Stable under 37 °C for 10 min.
Stable under 45 °C for 10 min.
Stable below 30 °C for 1 h.
Stable up to 50 °C.
This study
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[16]
[12]
[34]
[35]
[36]
[37]
[38]
[4]
Vibrio sp. strain PO-303 (AgaA)
Vibrio sp. strain PO-303 (AgaD)
101
GH16
Pseudoalteromonas sp. CY24 (AgaA)
Microbulbifer elongatus JAMB-A7 (RagaA7)
Microbulbifer sp. Strain CMC-5 ( β-agarase)
Microbulbifer thermotolerans JAMB-A94 (AgaA)
Vibrio sp. F-6 (AG-b)
482
(β-agarase)
398
62% activity remained at 50 °C for 30 min.
Stable up to 60 °C.
0.133
517
BAK08910
None a
Stable below 60 °C for 30 min.
Stable below 55 °C for 1 h.
Stable up to 60 °C.
307.4
ND b
ND b
Pseudoalteromonas sp. AG4 (AgaP)
Agarivorans sp. JA-1 (β-agarase)
Vibrio sp. Strain CN41 (AgaACN41)
Agarivorans sp. JAMB-AII (AgaAII)
Streptomyces coelicolor A3(2) (Sco3487)
Saccharophagus degradans 2-40 (Aga50D)
Alteromonas sp. E-l (β-agarase)
ADD60418
ABK97391
ADM25828
BAD99519
CAB61811
ABD81904
BAE97587
P85974
Stable below 40 °C.
3
Stable up to 40 °C.
371
Stable below 50 °C for 1h.
Stable up to 40 °C.
10.75
GH50
17.9
ND b
(β-agarase)
Stable up to 40 °C for 30 min.
Stable up to 50 °C for 1 h.
Stable below 50 °C for 30 min.
ND b
Agarivorans albusYKW-34 (AgaA34)
Vibrio sp. F-6 (AG-a)
529
[39]
[16]
[40]
[41]
[3]
None a
230
Agarivorans sp. HZ105 (HZ2)
ADY17919
BAK08903
AAF26838
BAF44076
BAF03590
AAQ56237
GU975829
235
ND b
ND b
GH86 (β-agarase)
GH96 (α-agarase)
Microbulbifer sp. JAMB-A94 (rAgaO)
Alteromonas agarilytica GJB (AgaA)
Thalassomonas sp. JAMB-A33 (AgaA33)
Vibrio sp. Strain PO-303 (Agarase C)
Pseudoalteromonas sp. CY24 (AgaB)
Catenovulum sp. X3 (AgaXa)
Stable up to 40 °C .
Stable up to 30 °C during for several days.
Stable up to 40 °C for 30 min.
Stable under 37 °C.
40.7
[42]
[43]
[44]
[45]
329
ND b
GH118 (β-agarase)
Stable under 35 °C for 1 h.
Stable below 42 °C.
588.2
a The accession No. is not available in the reference; b the characterization is not done.

Mar. Drugs 2014, 12
2740
Bacterial agarases are used in various preparative processes, namely, for liberating DNA
and other embedded molecules from agarose gels, isolating protoplasts from red algae and generating
neo-oligosaccharides, which exhibit important physiological and biological activities [46]. The industrial
oligosaccharide production from agar or marine algae requires the agarases to have high activity and
thermostability at temperatures higher than the gelling temperature of agar (around 40 °C) [41]. The
use of thermostable agarase makes it possible to conduct the process at an elevated temperature,
thereby considerably decreasing the risk of contamination, the cost of external cooling and enzyme
dosage [47]. The elevated temperature also provides a better solubility of substrates and a lower
viscosity, allowing accelerated mixing and pumping [48]. However, the optimal temperatures for most
of the known agarases in the GH50 family were between 30 °C and 40 °C (Table 2). The β-agarase
members of the GH86 and GH118 families exhibited the greatest activity at a temperature range of
35–52 °C, and the optimal temperatures of the two α-agarases were a little higher than 40 °C. Most of
the agarases in the GH16 family had an optimal temperature around 40–50 °C. Although the agarase,
YM01-3, showed similarity to other known agarases belonging to the GH16 family, whose highest
optimal temperature for the agarolytic activity of the known agarases was 55 °C [12,16,33], the
optimal temperature of the agarase, YM01-3, was 60 °C. Moreover, most of the characterized agarases
were stable below 42 °C after incubation for 10 min to 1 h, while YM01-3, which was stable after
incubation at 50 °C for 1 h, showed a higher thermostability than most of the other agarases, except
AgaP from Pseudoalteromonas sp. AG4, AG-b from Vibrio sp. F-6, β-agarase from Agarivorans sp.
JA-1 and AgaA from Microbulbifer thermotolerans JAMB-A94, which are stable up to
55–60 °C [12,16,33,34]. Some of the agarases showed the same thermostability with YM01-3, but lost
most of the activity after incubation at 70 °C for 1 h [37,39]. However, YM01-3 maintained about 12%
agarolytic activity, even if the recombinant agarase was boiled for 5 min. These characterizations
revealed that the agarase, YM01-3, is thermostable, significantly different from the other reported
agarases of GH16. Recently, another β-agarase gene, agaXa, was cloned from Catenovulum sp. X3,
which is in the same genus as YM01T [45]. AgaXa is also a thermostable agarase, but there are several
differences between YM01-3 and AgaXa. YM01-3 showed very low identity to AgaXa (14.5%), and
YM01-3 had maximum activity at 60 °C , while AgaXa was at 52 °C. In addition, YM01-3 was stable
below 50 °C , while AgaXa was below 40 °C. Therefore, YM01-3 is more thermostable than AgaXa,
and YM01-3 would provide more information for the thermostable mechanism and the
structure-function relationship.
The thermostable mechanism of YM01-3 is not clear at present. It was reported that the fold state of
the thermophilic protein showed a higher structural flexibility than that of the mesophilic homologue [49].
Compared with other agarases of GH16, YM01-3 owns less predicted parallel β-helixes (five helixes).
Thus, it is assumed that a mechanism characterized by entropic stabilization could be responsible for
the high thermostability of the enzyme. The three-dimensional structure would provide more information
for the thermostable mechanism and the structure-function relationship; however, only four structural
data of agarases are available to date, i.e., three wild type β-agarases, AgaA, AgaB and AgaD, and a
mutant, AgaB-E189D [24]. Further studies on the three-dimensional structure of β-agarase YM01-3
would provide not only a clearer understanding of its thermostable mechanism, but also additional data
to other agarases from the GH16 family.

Mar. Drugs 2014, 12
2741
Since the superior limit of the stable range and optimal temperatures of YM01-3 are higher than the
gelling temperature of agar (40 °C) and the Vmax value (1.14 × 104 U mg−1) is remarkably higher than
any other agarases, the recombinant YM01-3 appears to be a powerful enzyme for use at low cost in
industrial application with comparatively high agarolytic activity and thermostable characterization.
3. Experimental Section
3.1. Bacterial Strains and Growth Conditions
The marine bacterium, Catenovulum agarivorans gen. nov. sp. nov. YM01T (=CGMCC 1.10245T =
DSM 23111T = JCM 16580T) [21] was recently isolated from seawater of the Yellow Sea in the coastal
region of Qingdao, China, and it was cultured at 28 °C on marine agar 2216 (MA) (Difco, Franklin
lakes, NJ, USA). The pUCm-T (TaKaRa, Otsu, Japan) and plasmid pET24a (+) (Novagen, Madison,
WI, USA) were used as the vector of cloning and expression, respectively. Escherichia coli JM109
(New England Biolabs, Beverly, MA, USA) and E. coli BL21 (DE3) (Novagen, Madison, WI, USA),
which were used as the host for cloning and expression, were routinely grown at 37 °C in
Luria-Bertani (Difco, Franklin lakes, NJ, USA) broth supplemented with ampicillin or kanamycin
(100 μg mL−1) when required.
3.2. Analysis of the Agarolytic Activity of the Extracellular Proteins of YM01T
The broth culture of YM01T was centrifuged at 12,000 rpm for 15 min at 4 °C to remove the cells.
Solid ammonium sulfate was added to the supernatant to 60% saturation at 4 °C. Precipitated proteins
were pelleted by centrifugation (12,000 rpm for 15 min, 4 °C), resuspended in PBS (0.01 M, pH 7.2)
and then dialyzed in the same buffer for 24 h at 4 °C. The SDS-PAGE was performed, and the gel was
overlaid onto a plate sheet containing 2% agarose and incubated at 37 °C for 3 h. Following incubation,
the agarose sheets were stained by 2% (w/v) iodine solution, and then, agarase activity was visualized
as clear zones on a brown background; meanwhile, protein bands were visualized by staining with
Coomassie Brilliant Blue R-250 [42]. Finally, by comparing the zymogram and the gel, the gel band
that contained the protein with the highest agarase activity was sliced, washed and then analyzed by
mass spectrometry (Bo-Yuan Biological Technology Co., LTD, Shanghai, China).
3.3. Cloning and Sequence Analysis of the YM01-3 Gene
The YM01-3 gene (GenBank accession No. KF413621) was amplified by polymerase chain
reaction (PCR) using the primer sets, YMa (5′-CCGGAATTCATGTATGCAGCAGACTGGGAT-3′)
and YMb (5′-CCGCTCGAGTTGGAACTTCCATTGCTGG-3′), which contained EcoRI and XholI
sites (underline), respectively. The termination codon (TAA) was removed in the reverse primer in
order to add His-tag to the C-terminal of the recombinant agarase. The PCR products with an
appropriate size were gel purified and ligated into the pUCm-T vector. The plasmid was transformed
into E. coli JM109 competent cells, and the cloned gene was confirmed by sequencing. The
recombinant plasmid, pUCm-T/YM01-3, was digested by EcoRI and XholI restriction endonucleases,
and the product was purified and inserted into the linearized plasmid, pET-24a (+).

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The prediction of the amino acid sequence was performed with the software, BioEdit (Ibis
Biosciences, Carlsbad, CA, USA). The signal peptide was predicted using the SignalP 4.0 server [23].
Homology searches were performed using BLASTP [50,51]. Multiple alignments of protein sequences
from different species were performed by ClustalW [52,53]. The phylogenetic tree based on the amino
acid sequence was constructed by MEGA5.02 [54].
3.4. Overexpression and Purification of the Recombinant YM01-3 Protein
The expression vector, pET-24a (+)/YM01-3, was transformed into E. coli JM109 competent cells,
and correct recombinants were selected after sequencing confirmation. The sequence confirmed that
pET-24a (+)/YM01-3 was transformed into E. coli BL21 (DE3) cells and grown at 37 °C in LB broth
supplemented with kanamycin (100 μg mL−1). At the mid-exponential growth phase, IPTG was added
at a final concentration of 0.1 mM, and the cultivation was continued further at 16 °C, 150 rpm,
for 10–12 h.
The induced E. coli cells were collected by centrifugation, and the His-tagged agarase was purified
using the His Bind Kit, according to the recommendations of the manual. The purified agarase
YM01-3 was dialyzed and freeze-dried for determination of molecular mass by mass spectroscopy
(Thermo Scientific LTQ Orbitrap XL, Waltham, MA, USA).
3.5. Agarolytic Activity Assay of the Recombinant YM01-3
The agarolytic activity of purified recombinant YM01-3 was assayed by the modified
3,5-dinitrosalicylic acid method [27]. Briefly, the standard reaction contained 100 μL of diluted enzyme
solution and 900 μL of 0.1 M citric acid-sodium citrate buffer (pH 6.0) containing 0.25% (w/v) agarose
as the substrate. After incubation at 60 °C for 10 min, 1 mL of 3,5-dinitrosalicylic acid was added to
the mixture and boiled for 5 min until the color developed. The reaction was terminated by cooling the
test tube in a cold water bath. The absorbance of reducing sugar was measured at 540 nm and
compared to the standard curve of D-galactose [45]. Enzyme activity (U) was defined as the amount of
enzyme that liberated 1 μmol of D-galactose per minute.
3.6. Biochemical Characterization of the Recombinant YM01-3
The optimal temperature of the recombinant agarase was determined by monitoring the relative
activity at temperatures ranging from 40 to 100 °C (at 10 °C intervals) in 20 mM Tris-HCl buffer
(pH 8.0) for 10 min. The thermostability of the protein was determined by measuring the residual activity
of the enzyme after incubation at temperatures ranging from 0 to 100 °C for 1 h. The relative activity
was defined as the percentage of activity determined with respect to the maximum agarolytic activity.
The optimal pH of the agarase was assayed with a pH range of 4.0–11.0 (at 1.0 intervals) at 50 °C.
The buffers used were 0.1 M citric acid/sodium citrate buffer (pH 4.0–6.0), 20 mM Tris-HCl buffer
(pH 7.0–8.0), 50 mM Tris-glycine buffer (pH 9.0–10.0) and 50 mM Na2HPO4-NaOH buffer
(pH 11.0). The pH stability of the agarase was determined by pre-incubating the enzyme in buffers
with a pH range of 4.0–11.0 at 4 °C for 12 h, and the residual enzymatic activity was measured.

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The sensitivity of YM01-3 to various metal ions, denaturants and reducing agents was determined
by the measurement of activity after incubation in 1% agarose solution supplemented with different
ions (1 mM) and agents (10 mM) at 45 °C for 30 min. Km and Vmax values for agarase acting on
agarose (final concentration of 1–5 mg mL−1) were calculated by linear regression analysis of
Lineweaver-Burk double reciprocal plots of initial velocity data obtained under the standard condition
described above.
3.7. Identification of Hydrolysis Products by Purified YM01-3
The hydrolysis reactions were conducted at 50 °C in 20 mM Tris-HCl buffer (pH 8.0) containing
the purified YM01-3 agarase and 0.25% agarose. Subsequently, 1 mL of the reaction solution was
spotted onto Silica Gel 60 TLC plates (Merck, Germany), which were developed using a solvent
system composed of n-butanol–acetic acid–water (2:1:1, v:v:v). The oligosaccharides spots that
resulted from hydrolysis of substrates were visualized after the plate was sprayed with a modified
diphenylamine-aniline reagent and heated at 100 °C for 10 min [30].
To identify the agarose degradation products by YM01-3 agarase, the reaction mixtures after
incubation for 1 h were applied to Silica Gel 60 TLC plates. Three quarters of the plate was covered,
and the remainder was sprayed and heated, as mentioned above, for visualization. According to the
position of the spots in the visualized part, the spots corresponding to the hydrolyzed products were
scraped out with a razor from the TLC plate without diphenylamine-aniline reagent treatment and
dissolved in the mixture of hexanenitrile and 1 mM NH4HCO3 (1:1, v:v). The molecular mass
distribution of the products was determined by ion trap mass spectrometer (Thermo Scientific LTQ
Orbitrap XL, Waltham, MA, USA).
3.8. Statistical Analysis
Multiple comparisons were performed by the one-way analysis of variance (ANOVA) using the
Tukey test (SPSS statistics 19.0 software, IBM, Armonk, NY, USA). Results were expressed as the
mean ± SEM from three independent experiments, with p < 0.05 and p < 0.01 taken to show statistical
significance and distinct significance, respectively.
4. Conclusions
The present study represents cloning and overproducing of YM01-3, which showed the most
evident activity on agar degradation among the 15 putative agarases from Catenovulum agarivorans
YM01T. The recombinant YM01-3 reveals high agar degrading activity and produces neoagarotetraose
and neoagarohexaose as the main products. Notably, YM01-3 shows higher optimal temperature and
thermostability than most of the other agarases, which reveals its potentially high effectiveness for
application in industrial oligosaccharide production.

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Acknowledgments
This work was supported by projects from the National Natural Science Foundation of China
(NSFC; No. 41206117), the National High Technology Research and Development Program of China
(863 program; No. 2007AA09Z434) and the NSFC for Creative Research Groups (No. 41221004).
Author Contributions
Xiaochong Shi and Xiao-Hua Zhang designed the experiment and contributed to writing
manuscript. Fangyuan Cui performed the mass spectrometry analysis of agarolytic activity of the
extracellular proteins of YM01T, cloning and expression of the YM01-3 agarase, and contributed to
writing manuscript. Sujie Dong performed the purification and characterization of the YM01-3
agarase, analyzed the data and wrote the manuscript. Xia Zhao contributed to the determination of
hydrolysis products by purified YM01-3.
Conflicts of Interest
The authors declare no conflict of interest.
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