Characterization of a Novel PolyM-Preferred Alginate Lyase from Marine Vibrio splendidus OU02

Alginate lyases are enzymes that degrade alginate into oligosaccharides which possess a variety of biological activities. Discovering and characterizing novel alginate lyases has great significance for industrial and medical applications. In this study, we reported a novel alginate lyase, AlyA-OU02, derived from the marine Vibrio splendidus OU02. The BLASTP searches showed that AlyA-OU02 belonged to polysaccharide lyase family 7 (PL7) and contained two consecutive PL7 domains, which was rare among the alginate lyases in PL7 family. Both the two domains, AlyAa and AlyAb, had lyase activities, while AlyAa exhibited polyM preference, and AlyAb was polyG-preferred. In addition, the enzyme activity of AlyAa was much higher than AlyAb at 25 °C. The full-length enzyme of AlyA-OU02 showed polyM preference, which was the same as AlyAa. AlyAa degraded alginate into di-, tri-, and tetra-alginate oligosaccharides, while AlyAb degraded alginate into tri-, tetra-, and penta-alginate oligosaccharides. The degraded products of AlyA-OU02 were similar to AlyAa. Our work provided a potential candidate in the application of alginate oligosaccharide production and the characterization of the two domains might provide insights into the use of alginate of this organism.


Introduction
Alginate is a linear polysaccharide consisting of β-D-mannuronate (M) and α-L-guluronate (G) residues, arranged in varying sequences of poly β-D-mannuronate (polyM), poly α-L-guluronate (polyG), and the heteropolymer (polyMG) [1]. It has been widely used in biomedical science and engineering, due to its low toxicity, good biocompatibility, low cost, and mild gelation [2]. Alginate now has attracted increasing attention, since it is the most abundant polysaccharide of brown algae, which is considered as the third-generation biofuel [3]. Moreover, studies have showed that alginate oligosaccharides (AOS) possess a variety of biological activities, such as antioxidant, neuroprotective, antibacterial, and antitumor [4][5][6][7]. Therefore, to research the depolymerization of alginate has great importance.

Sequence Analysis of AlyA-OU02
The alginate lyase gene was cloned from marine Vibrio splendidus OU02, and was designated as AlyA-OU02. It consists of 1695 bp encoding 564 amino acids. The calculated molecular mass was

Sequence Analysis of AlyA-OU02
The alginate lyase gene was cloned from marine Vibrio splendidus OU02, and was designated as AlyA-OU02. It consists of 1695 bp encoding 564 amino acids. The calculated molecular mass was Mar. Drugs 2018, 16, 295 3 of 12 64 kDa, and the isoelectric point (PI) was 4.27. The BLASTP searches showed that AlyA-OU02 contained two alginate_lyase2 superfamily modules, and belonged to PL7 family ( Figure 1A), which was similar to AlyA from Vibrio splendidus 12B01 [18]. Figure 1B showed the multiple sequence alignments of AlyA a , AlyA b , and related alginate lyases of PL7 family [19][20][21][22]. Both domains contained the highly conserved regions R(S/T)E(V/L)R, QIH, and YFKAG(I/S)Y, which were necessary for substrate recognition and catalysis [23]. The putative residues involved in the catalytic reaction were pointed out in Figure 1A.

Cloning, Expression, and Purification of Different Versions of AlyA-OU02
To better characterize AlyA-OU02, we cloned, expressed, and purified AlyA-OU02 and its two domains, respectively. The AlyA-OU02, AlyA a , and AlyA b genes were cloned into pET-32a-PreScission vector (modified by our laboratory) respectively, and expressed in the E. coli BL21 (DE3)-pET-32a-PreScission system. The enzymes were then purified by Ni-nitrilotriacetic acid agarose (Ni-NTA) affinity chromatography and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Figure 2 showed that the molecular weights of AlyA-OU02, AlyA a , and AlyA b were approximately 65 kDa, 31 kDa, and 33 kDa, which were consistent with the calculated molecular weights. ~64 kDa, and the isoelectric point (PI) was 4.27. The BLASTP searches showed that AlyA-OU02 contained two alginate_lyase2 superfamily modules, and belonged to PL7 family ( Figure 1A), which was similar to AlyA from Vibrio splendidus 12B01 [18]. Figure 1B showed the multiple sequence alignments of AlyA a , AlyA b , and related alginate lyases of PL7 family [19][20][21][22]. Both domains contained the highly conserved regions R(S/T)E(V/L)R, QIH, and YFKAG(I/S)Y, which were necessary for substrate recognition and catalysis [23]. The putative residues involved in the catalytic reaction were pointed out in Figure 1A.

Cloning, Expression, and Purification of Different Versions of AlyA-OU02
To better characterize AlyA-OU02, we cloned, expressed, and purified AlyA-OU02 and its two domains, respectively. The AlyA-OU02, AlyA a , and AlyA b genes were cloned into pET-32a-PreScission vector (modified by our laboratory) respectively, and expressed in the E. coli BL21 (DE3)-pET-32a-PreScission system. The enzymes were then purified by Ni-nitrilotriacetic acid agarose (Ni-NTA) affinity chromatography and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Figure 2 showed that the molecular weights of AlyA-OU02, AlyA a , and AlyA b were approximately 65 kDa, 31 kDa, and 33 kDa, which were consistent with the calculated molecular weights.

Enzymatic Activity Measurements for AlyA-OU02, AlyA a , and AlyA b
Alginate lyases could degrade alginate into unsaturated oligosaccharides with the formation of a double bond between C4 and C5 at the non-reducing terminus. The enzyme activity was measured by monitoring the increased absorbance at 235 nm. Both domains showed alginate lyase activities, and the enzyme activity of AlyA a was much higher than AlyA b at 25 °C ( Figure 3). The enzyme activity of AlyA-OU02 was similar to AlyA a . The km values of AlyA-OU02, AlyA a , and AlyA b toward alginate were 3.55, 4.80, and 0.34 mg/mL respectively. The kcat values of AlyA-OU02, AlyA a , and AlyA b toward alginate were 630, 1052, and 42 min −1 , respectively. The enzymatic kinetics assay was consistent with the enzyme activity assay.
AlyA from Vibrio splendidus 12B01 also contained two PL7 domains, and the sequence similarity between AlyA-OU02 and AlyA was 89%, however, domain 1 of AlyA had no lyase activity [18]. We speculated the potential reason was that AlyA was purified by renaturation from inclusion bodies, and the length of AlyA (53-286) was different from AlyA a [18].  Alginate lyases could degrade alginate into unsaturated oligosaccharides with the formation of a double bond between C 4 and C 5 at the non-reducing terminus. The enzyme activity was measured by monitoring the increased absorbance at 235 nm. Both domains showed alginate lyase activities, and the enzyme activity of AlyA a was much higher than AlyA b at 25 • C ( Figure 3). The enzyme activity of AlyA-OU02 was similar to AlyA a . The k m values of AlyA-OU02, AlyA a , and AlyA b toward alginate were 3.55, 4.80, and 0.34 mg/mL respectively. The k cat values of AlyA-OU02, AlyA a , and AlyA b toward alginate were 630, 1052, and 42 min −1 , respectively. The enzymatic kinetics assay was consistent with the enzyme activity assay.
AlyA from Vibrio splendidus 12B01 also contained two PL7 domains, and the sequence similarity between AlyA-OU02 and AlyA was 89%, however, domain 1 of AlyA had no lyase activity [18]. We speculated the potential reason was that AlyA was purified by renaturation from inclusion bodies, and the length of AlyA (53-286) was different from AlyA a [18]. Crystal structure of FlAlyA showed that His124 and Tyr239 in the highly conserved regions participated in the catalytic reaction [12]. Furthermore, the catalytic activities of the H124N/Y239F mutants were much lower than the wild type. We constructed H139N/Y250F mutants of AlyA a and measured the lyase activities in order to identify the catalytic residues. The purified H139N/Y250F of AlyA a were analyzed by SDS-PAGE ( Figure S1). The alginate lyase activities of the mutants were undetectable, suggesting that His139 and Tyr250 were necessary for catalytic reaction of AlyA a ( Figure S2).
Alginate lyases with different sequences exhibited different optimal temperatures. Most of the PL7 alginate lyases in Table 1 had maximum activity at 30-45 °C . AlyA a had maximum activity at 30 °C , while AlyA b had maximum activity at 40 °C ( Figure 4A). The different optimal temperature of the two domains might help the bacteria to better adapt to the changing environment. The alginate lyases in Table 1 had maximum activity in a pH range of 6.0 to 9.0. Both AlyA a and AlyA b exhibited maximum activity at pH 8.0, which corresponded with the range ( Figure 4B). The optimal temperature/pH for AlyA-OU02 was 30 °C /8.0, the same as for AlyA a . Moreover, the optimal temperature/pH for Algb [20] and ALG-5 [24] in Table 1 was also 30 °C /8.0. Considering that the ion types in the buffer might impact the enzyme activity, we also investigated the pH effect in sodium phosphate buffer (pH 6.0-8.0) and Tris-HCl buffer (pH 7.0-10.0). Our results showed that sodium phosphate buffer decreased enzyme activities of AlyA-OU02, AlyA a , and AlyA b , compared with Tris-HCl buffer ( Figure S3).  Crystal structure of FlAlyA showed that His124 and Tyr239 in the highly conserved regions participated in the catalytic reaction [12]. Furthermore, the catalytic activities of the H124N/Y239F mutants were much lower than the wild type. We constructed H139N/Y250F mutants of AlyA a and measured the lyase activities in order to identify the catalytic residues. The purified H139N/Y250F of AlyA a were analyzed by SDS-PAGE ( Figure S1). The alginate lyase activities of the mutants were undetectable, suggesting that His139 and Tyr250 were necessary for catalytic reaction of AlyA a ( Figure S2).
Alginate lyases with different sequences exhibited different optimal temperatures. Most of the PL7 alginate lyases in Table 1 had maximum activity at 30-45 • C. AlyA a had maximum activity at 30 • C, while AlyA b had maximum activity at 40 • C ( Figure 4A). The different optimal temperature of the two domains might help the bacteria to better adapt to the changing environment. The alginate lyases in Table 1 had maximum activity in a pH range of 6.0 to 9.0. Both AlyA a and AlyA b exhibited maximum activity at pH 8.0, which corresponded with the range ( Figure 4B). The optimal temperature/pH for AlyA-OU02 was 30 • C/8.0, the same as for AlyA a . Moreover, the optimal temperature/pH for Algb [20] and ALG-5 [24] in Table 1 was also 30 • C/8.0. Considering that the ion types in the buffer might impact the enzyme activity, we also investigated the pH effect in sodium phosphate buffer (pH 6.0-8.0) and Tris-HCl buffer (pH 7.0-10.0). Our results showed that sodium phosphate buffer decreased enzyme activities of AlyA-OU02, AlyA a , and AlyA b , compared with Tris-HCl buffer ( Figure S3).  To assess the thermostabilities of AlyA-OU02 and its two domains, the enzymes were incubated in Tris-HCl buffer at different temperatures for 1 h. The enzyme activities were assayed using alginate as the substrate. As shown in Figure 5, the activity of AlyA a remained unchanged, while AlyA b retained 40% activity after incubation at 30 °C for 1 h. The stability of AlyA-OU02 decreased with increasing temperature, and retained 50% activity after incubation at 30 °C for 1 h. All the three enzymes were inactive after incubation at 50 °C . Our findings suggested that AlyA a exhibited more thermostability compared to AlyA b and AlyA-OU02. The activities of alginate lyases might be affected by metal ions, since various organisms survived and evolved in various environments. We first examined the effects of NaCl on the enzymatic activities. As shown in Figure 6A, 200 mM NaCl could effectively enhance the activities of AlyA-OU02 and AlyA a , while moderately affecting AlyA b . Certain alginate lyases could be activated by NaCl, but were not resistant to high concentrations of NaCl, such as AlyH1 [36] and AlgA [39]. AlyA-OU02, AlyA a , and AlyA b all exhibited high salt tolerance, which was analogous to AlgM4 from marine bacterium Vibrio weizhoudaoensis M0101 [32]. The effects of chelating agent and other metal ions on AlyA-OU02, AlyA a , and AlyA b activities were studied in the absence of NaCl. As shown in Figure 6B-D, Ca 2+ showed little effect on the enzymatic activities. Similarly to rAlgSV1-PL7 [35], AlyA-OU02, AlyA a , and AlyA b could be activated by Mg 2+ . Besides, enzymatic activities of AlyA- To assess the thermostabilities of AlyA-OU02 and its two domains, the enzymes were incubated in Tris-HCl buffer at different temperatures for 1 h. The enzyme activities were assayed using alginate as the substrate. As shown in Figure 5, the activity of AlyA a remained unchanged, while AlyA b retained 40% activity after incubation at 30 • C for 1 h. The stability of AlyA-OU02 decreased with increasing temperature, and retained 50% activity after incubation at 30 • C for 1 h. All the three enzymes were inactive after incubation at 50 • C. Our findings suggested that AlyA a exhibited more thermostability compared to AlyA b and AlyA-OU02.  To assess the thermostabilities of AlyA-OU02 and its two domains, the enzymes were incubated in Tris-HCl buffer at different temperatures for 1 h. The enzyme activities were assayed using alginate as the substrate. As shown in Figure 5, the activity of AlyA a remained unchanged, while AlyA b retained 40% activity after incubation at 30 °C for 1 h. The stability of AlyA-OU02 decreased with increasing temperature, and retained 50% activity after incubation at 30 °C for 1 h. All the three enzymes were inactive after incubation at 50 °C . Our findings suggested that AlyA a exhibited more thermostability compared to AlyA b and AlyA-OU02. The activities of alginate lyases might be affected by metal ions, since various organisms survived and evolved in various environments. We first examined the effects of NaCl on the enzymatic activities. As shown in Figure 6A, 200 mM NaCl could effectively enhance the activities of AlyA-OU02 and AlyA a , while moderately affecting AlyA b . Certain alginate lyases could be activated by NaCl, but were not resistant to high concentrations of NaCl, such as AlyH1 [36] and AlgA [39]. AlyA-OU02, AlyA a , and AlyA b all exhibited high salt tolerance, which was analogous to AlgM4 from marine bacterium Vibrio weizhoudaoensis M0101 [32]. The effects of chelating agent and other metal ions on AlyA-OU02, AlyA a , and AlyA b activities were studied in the absence of NaCl. As shown in Figure 6B-D, Ca 2+ showed little effect on the enzymatic activities. Similarly to rAlgSV1-PL7 [35], AlyA-OU02, AlyA a , and AlyA b could be activated by Mg 2+ . Besides, enzymatic activities of AlyA- The activities of alginate lyases might be affected by metal ions, since various organisms survived and evolved in various environments. We first examined the effects of NaCl on the enzymatic activities. As shown in Figure 6A, 200 mM NaCl could effectively enhance the activities of AlyA-OU02 and AlyA a , while moderately affecting AlyA b . Certain alginate lyases could be activated by NaCl, but were not resistant to high concentrations of NaCl, such as AlyH1 [36] and AlgA [39]. AlyA-OU02, AlyA a , and AlyA b all exhibited high salt tolerance, which was analogous to AlgM4 from marine bacterium Vibrio weizhoudaoensis M0101 [32]. The effects of chelating agent and other metal ions on AlyA-OU02, AlyA a , and AlyA b activities were studied in the absence of NaCl. As shown in Figure 6B-D, Ca 2+ showed little effect on the enzymatic activities. Similarly to rAlgSV1-PL7 [35], AlyA-OU02, AlyA a , and AlyA b could be activated by Mg 2+ . Besides, enzymatic activities of AlyA-OU02 and AlyA a were inhibited by the chelating agent ethylenediaminetetraacetic acid (EDTA), indicating that AlyA-OU02 and AlyA a might be ion-dependent alginate lyases.
Mar. Drugs 2018, 16, x FOR PEER REVIEW 6 of 12 OU02 and AlyA a were inhibited by the chelating agent ethylenediaminetetraacetic acid (EDTA), indicating that AlyA-OU02 and AlyA a might be ion-dependent alginate lyases. To determine the substrate specificity, 0.2% alginate, polyG, and polyM were used as the substrates to study the enzyme activities. The relative activities of AlyA a toward alginate, polyG, and polyM were 48.1% ± 2.2%, 20.0% ± 2.2%, and 100.0% ± 6.2%, respectively, indicating that AlyA a was a polyM-preferred alginate lyase ( Figure 7A). AlyA b showed slight polyG preference ( Figure 7B). The relative activities of AlyA-OU02 toward alginate, polyG, and polyM, were 60.0% ± 8.7%, 20.7% ± 0.4%, and 100.0% ± 9.1%, respectively ( Figure 7C), which was similar to AlyA a . The specific activities of AlyA-OU02, AlyA a , and AlyA b toward alginate, polyG, and polyM are shown in Table 2. The substrate specificities of PL7 alginate lyases were related with the protein sequences in the conserved regions [17]. Crystal structures and sequence analyses showed that the three conserved regions in PL7 family formed the cavity composed of a jelly roll β-sandwich structure, which was assumed to bind to a suitable substrate [24]. The conserved amino acids are thought to be pivotal in catalytic activity or folding of the structure. Recent studies have revealed that the polyM-specific, polyGspecific, and polyMG alginate lyases contained QVH, QIH, and QIH in the conserved regions, respectively [17]. As shown in Table 1, the alginate lyases that showed activities toward polyG and polyM contained QIH in the conserved region, with the exception of rAlgSV1-PL7 [35]. A9mT and PyAly contained QVH and were polyM-specific [24,38]. The polyG-specific enzymes, such as Alg2A, ALG-5, and Aly2, all contained QIH sequence [26,30,37]. Contrary to this accepted rule, AlyA a was active toward polyM, containing QIH sequence which was similar to FlAlyA and AlyDW11 [27,31]. To determine the substrate specificity, 0.2% alginate, polyG, and polyM were used as the substrates to study the enzyme activities. The relative activities of AlyA a toward alginate, polyG, and polyM were 48.1% ± 2.2%, 20.0% ± 2.2%, and 100.0% ± 6.2%, respectively, indicating that AlyA a was a polyM-preferred alginate lyase ( Figure 7A). AlyA b showed slight polyG preference ( Figure 7B). The relative activities of AlyA-OU02 toward alginate, polyG, and polyM, were 60.0% ± 8.7%, 20.7% ± 0.4%, and 100.0% ± 9.1%, respectively ( Figure 7C), which was similar to AlyA a . The specific activities of AlyA-OU02, AlyA a , and AlyA b toward alginate, polyG, and polyM are shown in Table 2. The substrate specificities of PL7 alginate lyases were related with the protein sequences in the conserved regions [17]. Crystal structures and sequence analyses showed that the three conserved regions in PL7 family formed the cavity composed of a jelly roll β-sandwich structure, which was assumed to bind to a suitable substrate [24]. The conserved amino acids are thought to be pivotal in catalytic activity or folding of the structure. Recent studies have revealed that the polyM-specific, polyG-specific, and polyMG alginate lyases contained QVH, QIH, and QIH in the conserved regions, respectively [17]. As shown in Table 1, the alginate lyases that showed activities toward polyG and polyM contained QIH in the conserved region, with the exception of rAlgSV1-PL7 [35]. A9mT and PyAly contained QVH and were polyM-specific [24,38]. The polyG-specific enzymes, such as Alg2A, ALG-5, and Aly2, all contained QIH sequence [26,30,37]. Contrary to this accepted rule, AlyA a was active toward polyM, containing QIH sequence which was similar to FlAlyA and AlyDW11 [27,31].

Thin-Layer Chromatography Analysis of the Degradation Products
To investigate the action modes of AlyA-OU02, AlyA a , and AlyA b , the degradation products of alginate, polyG, and polyM after 1 h were analyzed by thin-layer chromatography (TLC). As shown in Figure 8, AlyA a degraded alginate and polyM into di-, tri-, and tetra-oligosaccharides, and trisaccharide was the main product. The products patterns of AlyA-OU02 were similar to AlyA a . For AlyA b , tri-, tetra-, and penta-oligosaccharides were the main hydrolysis products, and AlyA b had limited activity toward polyM. The results were consistent with the enzymatic assays. We also analyzed the overnight products of alginate, polyG, and polyM, and the product patterns remained the same ( Figure S4). This indicated that AlyA-OU02 and AlyA a might be potential tools for preparation of lower molecular weight polyM products which have wide pharmaceutical applications.

Thin-Layer Chromatography Analysis of the Degradation Products
To investigate the action modes of AlyA-OU02, AlyA a , and AlyA b , the degradation products of alginate, polyG, and polyM after 1 h were analyzed by thin-layer chromatography (TLC). As shown in Figure 8, AlyA a degraded alginate and polyM into di-, tri-, and tetra-oligosaccharides, and trisaccharide was the main product. The products patterns of AlyA-OU02 were similar to AlyA a . For AlyA b , tri-, tetra-, and penta-oligosaccharides were the main hydrolysis products, and AlyA b had limited activity toward polyM. The results were consistent with the enzymatic assays. We also analyzed the overnight products of alginate, polyG, and polyM, and the product patterns remained the same ( Figure S4). This indicated that AlyA-OU02 and AlyA a might be potential tools for preparation of lower molecular weight polyM products which have wide pharmaceutical applications.

Thin-Layer Chromatography Analysis of the Degradation Products
To investigate the action modes of AlyA-OU02, AlyA a , and AlyA b , the degradation products of alginate, polyG, and polyM after 1 h were analyzed by thin-layer chromatography (TLC). As shown in Figure 8, AlyA a degraded alginate and polyM into di-, tri-, and tetra-oligosaccharides, and trisaccharide was the main product. The products patterns of AlyA-OU02 were similar to AlyA a . For AlyA b , tri-, tetra-, and penta-oligosaccharides were the main hydrolysis products, and AlyA b had limited activity toward polyM. The results were consistent with the enzymatic assays. We also analyzed the overnight products of alginate, polyG, and polyM, and the product patterns remained the same ( Figure S4). This indicated that AlyA-OU02 and AlyA a might be potential tools for preparation of lower molecular weight polyM products which have wide pharmaceutical applications.

Materials
Alginate was purchased from Shanghai Yuanye co., Ltd. The alginate viscosity is 4500 cP at 2% (w/v) concentration at 25 • C. The M/G ratio of alginate is 0.6 as determined by NMR analysis. PolyG and polyM were prepared according to the reference [10]. Other chemicals and reagents used in this study were of analytical grade.

Sequence Analysis of AlyA-OU02
For functional annotation, the BLAST algorithm on the National Center for Biotechnology Information server (http://www.ncbi.nlm.nih.gov) was used to analyze the similarity of the amino acid sequences. Molecular weights of the enzymes were estimated by the peptide mass tool on the ExPASY server of the Swiss Institute of Bioinformatics (http://swissmodel.expasy.org/). Multiple sequence alignments were performed by MEGA version 5.05.

Expression and
Purification of AlyA-OU02, AlyA a and AlyA b E. coli BL21 (DE3) cells harboring the plasmid were initially cultured in LB broth containing ampicillin at 37 • C. When the cell density reached an A 600 value of 0.6, 0.1 mM (final concentration) isopropyl β-D-1-thiogalactopyranoside was used to induce enzyme expression for 8 h at 16 • C. The cells were harvested by centrifugation (1500× g, 30 min, 4 • C) and sonicated in 20 mM Tris, 500 mM NaCl (pH 7.5). The lysates were clarified by centrifugation (20,000× g, 30 min, 4 • C). The supernatant containing soluble proteins was loaded onto a Ni-NTA column. The samples were eluted with a linear gradient of 10-500 mM imidazole. The molecular mass and the purities of the enzymes were analyzed by 12% SDS-PAGE.

Enzymatic Activity Assay
To determine the activities of the alginate lyases, 20 µL diluted enzyme (5 µM) was added to 180 µL substrate solution containing 0.2% (w/v) substrate, 50 mM Tris-HCl, and 200 mM NaCl (pH 7.5). After incubation at 25 • C for 10 min, alginate lyase activity was determined by measuring the increased absorbance at 235 nm of the reaction buffer. One unit was defined as the amount of enzyme required to increase the absorbance at 235 nm by 0.1 per min [40].
For kinetic analysis, the substrate concentrations varied from 0.33 mg/mL to 4 mg/mL. The reactions (200 µL for each assay) were carried out in 50 mM Tris-HCl (pH 7.5) with 200 mM NaCl at 25 • C for 10 min. After incubation, 200 µL of dinitrosalicylic acid was added to the reaction mixture, followed by heating at 100 • C for 5 min and centrifugation. The absorbance at 520 nm of the resulting supernatant was measured.
To determine the effects of temperature on enzyme activities, the reactions (200 µL for each assay) were carried out in 50 mM Tris-HCl (pH 7.5) with 200 mM NaCl at different temperatures (10-60 • C) for 10 min, with alginate as substrate. After incubation, 200 µL of dinitrosalicylic acid was added to the reaction mixture, followed by heating at 100 • C for 5 min and centrifugation. The absorbance at 520 nm of the resulting supernatant was measured. To determine the effects of pH on enzyme activities, the reactions (200 µL for each assay) were carried out in buffers with different pH values, including NaAC-HAC buffer (pH 5.0-6.0) and Tris-HCl buffer (pH 7.0-10.0) at 25 • C for 10 min, with alginate as substrate. The increased absorbance at 520 nm was measured.
To determine the thermostabilities, the enzyme solutions were incubated at various temperatures for 1 h. The reactions (200 µL for each assay) were carried out in 50 mM Tris-HCl (pH 7.5) with 200 mM NaCl at 25 • C for 10 min with 0.2% (w/v) alginate as substrate. After incubation, 200 µL of dinitrosalicylic acid was added to the reaction mixture, followed by heating at 100 • C for 5 min and centrifugation. The absorbance at 520 nm of the resulting supernatant was measured.
The effects of metal ions on enzyme activities were carried out by incubating enzymes at 4 • C for 1 h in the presence of various metal ions (1 mM) or EDTA (5 mM) in 50 mM Tris-HCl buffer (pH 7.5). The reactions (200 µL for each assay) were carried out in 50 mM Tris-HCl (pH 7.5) at 25 • C for 10 min with alginate as substrate. The increased absorbance at 235 nm was measured.
To study the substrate specificities, the reactions (200 µL for each assay) were carried out in 50 mM Tris-HCl (pH 7.5) with 200 mM NaCl at 25 • C for 10 min, using 0.2% (w/v) alginate, polyM, and polyG separately as substrates. The increased absorbance at 235 nm was measured.

TLC Analysis of the Degradation Products
To analyze the oligosaccharides produced by the enzymes, 20 µL diluted enzyme (5 µM) was added to 180 µL substrate solution containing 0.2% (w/v) substrate, 50 mM Tris-HCl, and 200 mM NaCl (pH 7.5). After incubation at 25 • C for 1 h or 12 h, the reaction buffer was boiled for 5 min, and analyzed by TLC with the solvent system (1-butanol/formic acid/water 4:6:1). The TLC plate was sprayed using 10% (v/v) sulfuric acid in ethanol, and then heated at 130 • C for 5 min.

Conclusions
In this study, we reported a new alginate lyase derived from the marine Vibrio splendidus OU02. AlyA-OU02 was characterized as a novel PL7 alginate lyase containing two PL7 domains. Surprisingly, both of the two domains showed alginate lyase activities. Although both domains contained QIH sequence in the conserved regions, which was thought to be polyG-specific, AlyA-OU02 turned out to be polyM-preferred. The two domains exhibited different enzymatic properties. The optimal temperature, pH, metal ions effects, and degradation patterns of AlyA-OU02 were similar to AlyA a . The putative reason was that the enzyme activity of AlyA a was much higher than AlyA b at 25 • C. Further work will be focused on obtaining crystals of the enzymes to elucidate the catalytic mechanism of AlyA-OU02.