A Novel Alginate Lyase: Identification, Characterization, and Potential Application in Alginate Trisaccharide Preparation

Alginate oligosaccharides (AOS) have many biological activities and significant applications in prebiotics, nutritional supplements, and plant growth development. Alginate lyases have unique advantages in the preparation of AOS. However, only a limited number of alginate lyases have been so far reported to have potentials in the preparation of AOS with specific degrees of polymerization. Here, an alginate-degrading strain Pseudoalteromonas arctica M9 was isolated from Sargassum, and five alginate lyases were predicted in its genome. These putative alginate lyases were expressed and their degradation products towards sodium alginate were analyzed. Among them, AlyM2 mainly generated trisaccharides, which accounted for 79.9% in the products. AlyM2 is a PL6 lyase with low sequence identity (≤28.3%) to the characterized alginate lyases and may adopt a distinct catalytic mechanism from the other PL6 alginate lyases based on sequence alignment. AlyM2 is a bifunctional endotype lyase, exhibiting the highest activity at 30 °C, pH 8.0, and 0.5 M NaCl. AlyM2 predominantly produces trisaccharides from homopolymeric M block (PM), homopolymeric G block (PG), or sodium alginate, with a trisaccharide production of 588.4 mg/g from sodium alginate, indicating its promising potential in preparing trisaccharides from these polysaccharides.


Introduction
Alginate is an acidic linear polysaccharide present in great abundance in the cell wall of brown algae [1], accounting for approximately 30-60% dry weight of brown algae [2]. In addition, alginate also exists in some red algae [3] and can be produced by several bacteria, such as genera Azotobacter and Pseudomonas [4]. Alginate is composed of β-D-mannuronate (M) and its C5 epimer, α-L-guluronate (G), which are linked by 1,4-O-glycoside bonds and arranged in block structures, such as homopolymeric G block (PG), M block (PM), and heteropolymeric MG/GM block [5]. Alginate possesses unique physical properties, such as viscosity, sol/gel transition, and water uptake properties, which contribute to its various applications as stabilizers, emulsifiers, thickeners, and gel-forming agents in the food, cosmetic, and pharmaceutical industries [1].
Pseudoalteromonas contains 48 species of heterotrophic bacteria that are widely distributed in marine environments and are an important group in alginate degradation [35]. However, to date, no alginate-degrading Pseudoalteromonas strains have been isolated from Sargassum. The isolation and identification of strain M9 from Sargassum is helpful to excavate alginate lyases and alginate metabolism related genes.

Bioinformatic Analysis of the Alginate Lyases of Strain M9
To identify the alginate lyases in strain M9, its genome was sequenced and analyzed. In the genome, five genes were predicted to encode alginate lyases. As shown in Table 1, these putative alginate lyases, named AlyM1, AlyM2, AlyM3, AlyM4, and AlyM5, belong to four different PL families. AlyM1 belongs to the PL7 family and has only one domain ( Figure 2). AlyM3 and AlyM5, belonging to the PL17 and PL18 families, respectively, both have two domains, which are conventional in their corresponding families ( Figure 2). AlyM2 and AlyM4, both belonging to the PL6 family, have different modularity. The catalytic PL6 domain of AlyM2 is at the C-terminus and that of AlyM4 To identify strain M9, the 16S rRNA gene (1463 bp) of strain M9 was amplified and sequenced, and a phylogenetic analysis was performed. The result showed that strain M9 was clustered with many Pseudoalteromonas species, most close to Pseudoalteromonas arctica A 37-1-2 T ( Figure 1C). Therefore, strain M9 belongs to the genus Pseudoalteromonas, and was identified as an alginate-degrading strain member of Pseudoalteromonas arctica, named as Pseudoalteromonas arctica strain M9.
Pseudoalteromonas contains 48 species of heterotrophic bacteria that are widely distributed in marine environments and are an important group in alginate degradation [35]. However, to date, no alginate-degrading Pseudoalteromonas strains have been isolated from Sargassum. The isolation and identification of strain M9 from Sargassum is helpful to excavate alginate lyases and alginate metabolism related genes.

Bioinformatic Analysis of the Alginate Lyases of Strain M9
To identify the alginate lyases in strain M9, its genome was sequenced and analyzed. In the genome, five genes were predicted to encode alginate lyases. As shown in Table 1, these putative alginate lyases, named AlyM1, AlyM2, AlyM3, AlyM4, and AlyM5, belong to four different PL families. AlyM1 belongs to the PL7 family and has only one domain ( Figure 2). AlyM3 and AlyM5, belonging to the PL17 and PL18 families, respectively, both have two domains, which are conventional in their corresponding families ( Figure 2). AlyM2 and AlyM4, both belonging to the PL6 family, have different modularity. The catalytic PL6 domain of AlyM2 is at the C-terminus and that of AlyM4 at the N-terminus ( Figure 2). The five alginate lyases all have predicted signal peptides, suggesting that they are likely extracellularly secreted enzymes. at the N-terminus ( Figure 2). The five alginate lyases all have predicted signal peptides, suggesting that they are likely extracellularly secreted enzymes. ACK10595.1 # The sequence identity was obtained by sequence comparison with a characterized alginate lyase whose accession number is listed in the right column.

Expression and Degradation Product Analysis of the Alginate Lyases of Strain M9
To evaluate the application potentials of the alginate lyases of strain M9 in AOS preparation, we expressed all the five putative alginate lyases in Escherichia coli with Histag and purified them by NTA-Ni Sepharose affinity chromatography. Finally, AlyM1, AlyM2, AlyM3, and AlyM5 were successfully purified ( Figure 3A), but AlyM4 failed. Although many attempts were made to improve the expression amount via optimizing the conditions for its expression, the expression amount of AlyM4 was still too low to be purified. To obtain the end products of the four purified enzymes towards sodium alginate, each enzyme was incubated with sodium alginate at 30 °C for 12 h. The degradation products were then determined by gel filtration assay ( Figure 3B). The result showed that AlyM3 is an exotype lyase producing monomer as the sole product, and AlyM1, AlyM2, and AlyM5 are endotype lyases producing different AOS products. AlyM1 produced mainly monosaccharide and disaccharide, with a small amount of trisaccharide. The degradation products of AlyM5 were a mixture of DP3-DP6 saccharides. In contrast, in addition to a slight amount of DP2 and DP4-DP6, the degradation products of AlyM2 are mainly trisaccharides. Trisaccharides accounted for 79.9% of the product amount based on the peak area of the products in the gel filtration chromatogram (total peak area of all products, ~31,380,656; the peak area of trisaccharides, 25,073,144).

Expression and Degradation Product Analysis of the Alginate Lyases of Strain M9
To evaluate the application potentials of the alginate lyases of strain M9 in AOS preparation, we expressed all the five putative alginate lyases in Escherichia coli with His-tag and purified them by NTA-Ni Sepharose affinity chromatography. Finally, AlyM1, AlyM2, AlyM3, and AlyM5 were successfully purified ( Figure 3A), but AlyM4 failed. Although many attempts were made to improve the expression amount via optimizing the conditions for its expression, the expression amount of AlyM4 was still too low to be purified. To obtain the end products of the four purified enzymes towards sodium alginate, each enzyme was incubated with sodium alginate at 30 • C for 12 h. The degradation products were then determined by gel filtration assay ( Figure 3B). The result showed that AlyM3 is an exotype lyase producing monomer as the sole product, and AlyM1, AlyM2, and AlyM5 are endotype lyases producing different AOS products. AlyM1 produced mainly monosaccharide and disaccharide, with a small amount of trisaccharide. The degradation products of AlyM5 were a mixture of DP3-DP6 saccharides. In contrast, in addition to a slight amount of DP2 and DP4-DP6, the degradation products of AlyM2 are mainly trisaccharides. Trisaccharides accounted for 79.9% of the product amount based on the peak area of the products in the gel filtration chromatogram (total peak area of all products, 31  , gel filtration chromatography analysis of the degradation products of the alginate lyases. Gel filtration chromatography analysis was performed using a Superdex peptide 10/300 GL column monitored at a wavelength of 210 nm. The control was treated with pre-heated inactivated lyases. Saturated mannuronate oligosaccharides from DP1 to DP6 were taken as the standards. DP, degree of polymerization.
In previous studies, the products of endotype alginate lyases mainly range from DP2 to DP5, and only a limited number of alginate lyases have been shown to have potentials in the preparation of AOS with specific DPs. While ALFA3, Aly1281, AlySY08, and Val-1 were shown to be suitable for disaccharide production [30,31,36,37], Alg7A, AlyF, and AkAly28 were reported to mainly produce trisaccharides [32,38,39]. The high proportion of trisaccharides in the products of AlyM2 implies that AlyM2 may have potential in trisaccharide production. Thus, we further characterized AlyM2 and evaluated its potential in preparing alginate trisaccharides.

Sequence Analysis and Structural Prediction of the Alginate Lyase AlyM2
AlyM2 is a PL6 alginate lyase containing 910 amino acid residues (Table 1). Alginate lyases in the PL6 family are divided into three subfamilies, that is, PL6_1, PL6_2, and PL6_3. Based on phylogenetic analysis, AlyM2 is a PL6_3 lyase, clustered with the only characterized PL6_3 alginate lyase MASE_04180 from Alteromonas macleodii ATCC 27126 [40] and many predicted PL6_3 alginate lyases in the CAZy database ( Figure 4A). AlyM2 exhibited a quite low sequence identity (28.3%) with MASE_04180 (Table 1). , gel filtration chromatography analysis of the degradation products of the alginate lyases. Gel filtration chromatography analysis was performed using a Superdex peptide 10/300 GL column monitored at a wavelength of 210 nm. The control was treated with pre-heated inactivated lyases. Saturated mannuronate oligosaccharides from DP1 to DP6 were taken as the standards. DP, degree of polymerization.
In previous studies, the products of endotype alginate lyases mainly range from DP2 to DP5, and only a limited number of alginate lyases have been shown to have potentials in the preparation of AOS with specific DPs. While ALFA3, Aly1281, AlySY08, and Val-1 were shown to be suitable for disaccharide production [30,31,36,37], Alg7A, AlyF, and AkAly28 were reported to mainly produce trisaccharides [32,38,39]. The high proportion of trisaccharides in the products of AlyM2 implies that AlyM2 may have potential in trisaccharide production. Thus, we further characterized AlyM2 and evaluated its potential in preparing alginate trisaccharides.

Sequence Analysis and Structural Prediction of the Alginate Lyase AlyM2
AlyM2 is a PL6 alginate lyase containing 910 amino acid residues (Table 1). Alginate lyases in the PL6 family are divided into three subfamilies, that is, PL6_1, PL6_2, and PL6_3. Based on phylogenetic analysis, AlyM2 is a PL6_3 lyase, clustered with the only characterized PL6_3 alginate lyase MASE_04180 from Alteromonas macleodii ATCC 27126 [40] and many predicted PL6_3 alginate lyases in the CAZy database ( Figure 4A). AlyM2 exhibited a quite low sequence identity (28.3%) with MASE_04180 ( Table 1).
The catalytic mechanisms of some PL6 alginate lyases have been revealed [40]. In these PL6 enzymes, a conserved lysine and a conserved arginine usually act as the two catalytic residues, and several conserved residues interact with the water molecule or calcium ion to neutralize the negative charge of the carboxyl group [40]. However, multiple sequence alignment of AlyM2 with alginate lyases from three PL6 subfamilies showed that the amino acid of PL6_3 alginate lyases corresponding to the reported catalytic lysine of other enzymes is glutamine, and the amino acid of most PL6_3 alginate lyases corresponding to the catalytic arginine is aspartic acid ( Figure 4B). Moreover, the amino acids coordinated with water or Ca 2+ in PL6_3 alginate lyases are likely different from those in other PL6 enzymes ( Figure 4B). These data suggest that AlyM2 and other PL6_3 alginate lyases may have a potentially distinct catalytic mechanism from those of PL6_1 and PL6_2, which awaits further confirmation.   Among PL6 alginate lyases, the structures of seven enzymes have been reported, including five from PL6_1 and two from PL6_2 ( Figure 4A). These structures can be divided into two kinds. Some, represented by AlyF (PDB code: 6ITG), contain only a catalytic domain exhibiting the β-helix fold [38]. The others, represented by AlyGC (PDB code: 5GKQ), contain two domains both exhibiting the β-helix fold, and their N-terminal domain is the catalytic domain [41] ( Figure 5A). The overall structure of AlyM2 was predicted by Alphafold2 [42], which suggests that AlyM2 consists of five domains, including domain 1 (D1), domain 2 (D2), domain 3 (D3), domain 4 (D4), and domain 5 (D5) from the N-terminus to the C-terminus ( Figure 5A). The C-terminal D5 exhibits the typical β-helix fold of the catalytic domains of the reported PL6 alginate lyases [38,41], suggesting that D5 is likely the catalytic domain of AlyM2. Domains D1, D2, D3, and D4 all consist of five or six β strands. Thus, the overall structure of AlyM2 may be quite different from the reported PL6 alginate lyase structures, which awaits further investigation. Furthermore, an alignment of the predicted AlyM2 structure with seven reported PL6 structures showed that a glutamine and an aspartic acid of AlyM2 spatially correspond to the known catalytic lysine and arginine of these alginate lyases, respectively ( Figure 5B). This is consistent with the above sequence alignment result ( Figure 4B), further suggesting that AlyM2 and other PL6_3 alginate lyases may have a different catalytic mechanism from those of PL6_1 and PL6_2.
lytic lysine of other enzymes is glutamine, and the amino acid of most PL6_3 alginate lyases corresponding to the catalytic arginine is aspartic acid ( Figure 4B). Moreover, the amino acids coordinated with water or Ca 2+ in PL6_3 alginate lyases are likely different from those in other PL6 enzymes ( Figure 4B). These data suggest that AlyM2 and other PL6_3 alginate lyases may have a potentially distinct catalytic mechanism from those of PL6_1 and PL6_2, which awaits further confirmation.
Among PL6 alginate lyases, the structures of seven enzymes have been reported, including five from PL6_1 and two from PL6_2 ( Figure 4A). These structures can be divided into two kinds. Some, represented by AlyF (PDB code: 6ITG), contain only a catalytic domain exhibiting the β-helix fold [38]. The others, represented by AlyGC (PDB code: 5GKQ), contain two domains both exhibiting the β-helix fold, and their Nterminal domain is the catalytic domain [41] ( Figure 5A). The overall structure of AlyM2 was predicted by Alphafold2 [42], which suggests that AlyM2 consists of five domains, including domain 1 (D1), domain 2 (D2), domain 3 (D3), domain 4 (D4), and domain 5 (D5) from the N-terminus to the C-terminus ( Figure 5A). The C-terminal D5 exhibits the typical β-helix fold of the catalytic domains of the reported PL6 alginate lyases [38,41], suggesting that D5 is likely the catalytic domain of AlyM2. Domains D1, D2, D3, and D4 all consist of five or six β strands. Thus, the overall structure of AlyM2 may be quite different from the reported PL6 alginate lyase structures, which awaits further investigation. Furthermore, an alignment of the predicted AlyM2 structure with seven reported PL6 structures showed that a glutamine and an aspartic acid of AlyM2 spatially correspond to the known catalytic lysine and arginine of these alginate lyases, respectively ( Figure 5B). This is consistent with the above sequence alignment result ( Figure 4B), further suggesting that AlyM2 and other PL6_3 alginate lyases may have a different catalytic mechanism from those of PL6_1 and PL6_2.

Biochemical Characterization of AlyM2
The recombinant AlyM2 was further biochemically characterized. As shown in Figure 6A, AlyM2 exhibited the maximum activity at 30 • C. Its activity was more than 60% of the maximum activity at 20 • C, and less than 50% at 50 • C. The activity of AlyM2 was the highest at pH 8.0 and maintained higher than 50% at a range of pH 7.0-9.0. At pH 10.0, the enzyme activity dropped to 18.2% ( Figure 6B). Low NaCl concentrations less than 1 M significantly promoted AlyM2 activity. In particular, 0.5 M NaCl increased the activity of AlyM2 by nearly 4 times compared with that without NaCl ( Figure 6C), indicating that AlyM2 has a salt-activated characteristic. These characteristics reflect the adaptation of AlyM2 to the seawater environment.
To investigate the substrate specificity of AlyM2, we measured the activity of AlyM2 towards four alginate substrates, PM, PG, PMG, and sodium alginate. AlyM2 had activity towards all these substrates (11.7-29.7 U/mg). Among them, its activity towards PMG was the highest (29.7 U/mg), and that towards PG was the lowest (11.7 U/mg) ( Figure 6D). This result suggested that AlyM2 is capable of cleaving the M-M bond, the G-G bond, and the M-G bond in the substrates. To date, only one PL6_3 alginate lyase, MASE_04180, has been characterized, which can cleave only the M-G bond [40]. Thus, AlyM2 is distinct from MASE_04180 in substrate specificity.

Biochemical Characterization of AlyM2
The recombinant AlyM2 was further biochemically characterized. As shown in Figure 6A, AlyM2 exhibited the maximum activity at 30 °C. Its activity was more than 60% of the maximum activity at 20 °C, and less than 50% at 50 °C. The activity of AlyM2 was the highest at pH 8.0 and maintained higher than 50% at a range of pH 7.0-9.0. At pH 10.0, the enzyme activity dropped to 18.2% ( Figure 6B). Low NaCl concentrations less than 1 M significantly promoted AlyM2 activity. In particular, 0.5 M NaCl increased the activity of AlyM2 by nearly 4 times compared with that without NaCl ( Figure 6C), indicating that AlyM2 has a salt-activated characteristic. These characteristics reflect the adaptation of AlyM2 to the seawater environment. To investigate the substrate specificity of AlyM2, we measured the activity of AlyM2 towards four alginate substrates, PM, PG, PMG, and sodium alginate. AlyM2 had activity towards all these substrates (11.7-29.7 U/mg). Among them, its activity towards PMG was the highest (29.7 U/mg), and that towards PG was the lowest (11.7 U/mg) ( Figure 6D). This result suggested that AlyM2 is capable of cleaving the M-M bond, the G-G bond, and the M-G bond in the substrates. To date, only one PL6_3 algi-

Evaluation of the Potential of AlyM2 in Alginate Trisaccharides Preparation
We further analyzed the end degradation products of AlyM2 towards PM, PG, and PMG. Similar to the result of AlyM2 on sodium alginate (79.9% trisaccharides in the products), trisaccharides were also the main component of the degradation products ( Figure 7A), accounting for 78.2% in those of PM, 79.6% in those of PG, and 76.75% in those of PMG. This result suggested that AlyM2 can be used to produce different alginate trisaccharides, including trimannuronate from PM, triguluronate from PG, and a mixture of trisaccharides from PMG and sodium alginate.
To investigate the effect of degradation time on the products, the four alginate substrates were degraded with AlyM2 for different times from 0 to 12 h, and the degradation products were detected by thin layer chromatography (TLC). As shown in Figure 7B, after oligosaccharides with DP3-DP6; after 6 h degradation or more time, trimer was the main product, with only a small amount of tetramer and a negligible amount of dimer. Therefore, the degradation time can be shortened to 6 h. By using commercial saturated mannuronate trisaccharides as standards, we further calculated the production of trisaccharides when sodium alginate was degraded by AlyM2, which was determined to be 588.4 mg/g sodium alginate ( Figure 7C).   Figure 7A for 0, 3, 6, or 12 h. The degradation products were analyzed by TLC. Lane M, saturated oligosaccharides standards including monomer, dimer, trimer, tetramer, and pentamer. (C), determination of the production of trisaccharides from sodium alginate degradation by AlyM2. The degradation reaction was performed in a 500 mL reaction mixture containing 1 g sodium alginate and 40 mg AlyM2 in the buffer containing 50 mM Tris-HCl (pH 8.0) and 0.5 M NaCl at 30 • C for 6 h. The degradation products were separated by gel filtration and monitored using a refractive index detector. A standard curve of trisaccharide concentration versus peak area was drawn by using commercial saturated mannuronate trisaccharide at the concentrations of 0-6 mg/mL as standards. The trisaccharide amount released from sodium alginate by AlyM2 was determined based on the standard curve.
By using commercial saturated mannuronate trisaccharides as standards, we further calculated the production of trisaccharides when sodium alginate was degraded by AlyM2, which was determined to be 588.4 mg/g sodium alginate ( Figure 7C).
The biological activity of AOS is closely related to its DP [43]. It has been reported that AOS with an average DP of 3, including ∆GG and a mixture of ∆MM and ∆MM' (the β anomer of M), have conspicuous root growth-promoting activities in barley seedlings [16], and treatment with sodium AOS of DP3 (3 α-L-guluronate and/or β-D-mannuronate) can attenuate spontaneous hypertension [17]. Till now, according to the summary from Cheng et al., 3 alginate lyases have been shown to mainly produce trisaccharides [2], that is, AlyF from Vibrio splendidus OU02 [38], Alg7A from Vibrio sp. W13 [32] and AkAly28 from Aplysia kurodai [39] (Table 2). AlyF is a PG specific lyase, and AkAly28 is a PM lyase, both of which are distinct from AlyM2 in substrate specificity. Alg7A is similar to AlyM2 in substrate specificity, having activity towards PM, PG, PMG, and sodium alginate [32]. While trimers are the predominant products of Alg7A towards all the four substrates, dimers and tetramers are also the main products [32] (Table 2). However, in the degradation products of AlyM2 towards the four substrates, trimers are the main products, with only a small amount of DP4 and a negligible amount of DP2 ( Figure 7B). Therefore, AlyM2 is promising for the preparation of different alginate trisaccharides with high purity. Since AOS of DP3 have been reported to have many bioactivities [16][17][18], AlyM2 may have application potential in the production of trisaccharides with healthcare potential and other industrial applications, such as treatment of hypertension, acting as plant growth regulators and healthcare supplements, which awaits further investigation.  [38,44] -, no data available.

Materials and Strains
Sodium alginate derived from brown seaweed was purchased from Sangon (Shanghai, China). PM, PG (6-8 kDa), and alginate oligosaccharides (purity ≥ 97%) were purchased from Zzstandard (Qingdao, China). PMG was prepared as previously described [45]. Other chemicals and reagents used in this study are of analytical grade. E. coli strains were from TransGen (Beijing, China) and cultured in Lysogeny broth (LB) medium at 37 • C.

Determination of Extracellular Alginate Lyase Activity
Extracellular alginolytic activity of strain M9 was quantitatively determined by measuring the amount of reducing sugars released from sodium alginate by using the DNS method [46,47]. The fermentation broth of strain M9 cultured at 25 • C with stir (180 rpm) for 24 h was centrifuged at 15,871× g for 5 min to obtain the supernatant as crude enzyme.
Then, 50 µL crude enzyme was mixed with 50 µL substrate solution composed of 1% (w/v) sodium alginate dissolved in 50 mM Tris-HCl (pH 8.0), and the mixture was incubated at 30 • C for 60 min. Afterwards, the reaction mixture was terminated by the addition of 100 µL DNS and then boiled at 100 • C for 10 min. A blank control was set by mixing DNS solution and the substrate solution followed by an addition of crude enzyme to inactivate the alginate lyase activity before incubation at 30 • C. The absorbance values of the mixtures were determined at 540 nm. The amount of reducing sugars released into the mixture was determined with glucose as the standard. One unit of enzyme activity is defined as the amount of enzyme required to release 1 µg reducing sugars from sodium alginate per min.

Sequencing of the 16S rRNA Gene and the Genomic DNA of Strain M9
Genomic DNA of strain M9 was extracted using a BioTekeDNA extraction kit (Beijing, China). The 16S rRNA gene was amplified from the genome DNA via PCR using the primers 1492R and 27F [26] ( Table 3). The PCR product was ligated to the pMD19-T vector (TaKaRa, kusatsu, Japen) and sequenced on an Applied Biosystems DNA sequencer (model 3730XL). The sequence of the 16S rRNA gene was compared with those in the GenBank (https://blast.ncbi.nlm.nih.gov/Blast.cgi; accessed on 17 December 2021) and Ezbiocloud (http://www.ezbiocloud.net; accessed on 17 December 2021) databases using BLASTN.
The similarity values of paired sequences were obtained by the EzBioCloud server. The genomic DNA of strain M9 was sequenced on the Illumina Hiseq sequencing platform (Majorbio, Shanghai, China). The genome assembly was performed using the ABySS 2.1.5 analysis process implemented in SMRT Link (V6.0.0.47841) to get the draft genome [48]. The genome data of strain M9 was uploaded to the Genbank database under the accession number JAEKKD000000000.1. Table 3. Primers used in this study a .

Primer
Sequence a Restriction enzyme sites used for cloning are underlined.

Gene Cloning, Protein Expression and Purification
Genes of alginate lyases without the signal peptides were amplified from the genomic DNA of strain M9 via PCR using the primer pairs with the restriction enzyme sites of NdeI and XhoI (Table 3) and cloned into the expression vector pET-22b between these restriction sites along with a C-terminal His tag. The constructed expression vectors were transferred into E. coli BL21 (DE3). The recombinant E. coli BL21 (DE3) strains were cultured in LB medium and induced by adding 0.3 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) at 15 • C for 16 h. After cultivation, cells were collected and disrupted by sonication in the buffer containing 50 mM Tris-HCl buffer (pH 8.0) and 100 mM NaCl. The recombinant proteins in the cell extract were purified by nickel-nitrilotriacetic acid resin (Qiagen, Germantown, Germany). The purified proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Biochemical Characterization of AlyM2
The concentration of AlyM2 was determined by the bicinchoninic acid (BCA) protein assay kit (Thermo, Waltham, MA, USA), with bovine serum albumin (BSA) as the standard. The activities of AlyM2 on PM, PG, PMG, and sodium alginate were measured by the ultraviolet absorption spectrometry method [54]. Briefly, a 200 µL mixture containing 60 µg/mL enzyme and 2 mg/mL substrate in 50 mM Tris-HCl (pH 8.0) and 0.5 M NaCl was incubated at 30 • C for 10 min. The reaction was then terminated by boiling for 10 min. The increase in the absorbance at 235 nm (A 235 ), resulting from the release of unsaturated uronic in the mixture, was monitored. One unit (U) of enzyme activity was defined as the amount of enzyme required to cause an increase of 0.1 at 235 nm per minute.
The optimum temperature for AlyM2 activity was determined at a range of 20 to 50 • C at pH 8.0. The optimum pH for AlyM2 activity was determined at 30 • C in the Britton-Robinson (B-R) buffer ranging from pH 5.0 to 10.0. B-R buffer was prepared with boric acid, acetic acid, and phosphoric acid, all with final concentration of 0.04 M in an aqueous solution, and was adjusted to different pH with 0.2 M NaOH. The effect of NaCl on AlyM2 activity was determined at NaCl concentrations ranging from 0 to 2.0 M. The substrate specificity of AlyM2 was determined with PM, PG, PMG, and alginate sodium at 30 • C in 50 mM Tris-HCl (pH 8.0) and 0.5 M NaCl.

Degradation Product Analysis
The end degradation products of alginate lyases on sodium alginate were analyzed by using 2 mg/mL substrate and excess enzyme (AlyM1, 132 µg/mL; AlyM2, 80 µg/mL; AlyM2, 144 µg/mL; AlyM5, 58 µg/mL). The degradation reaction was carried out in a buffer containing 50 mM Tris-HCl (pH 8.0) and 0.5 M NaCl at 30 • C for 12 h, after which the kind and amount of AOS in the products were no longer changed even if the reaction time was further extended. The end degradation products of AlyM2 on PM, PG, and PMG were obtained with the same method. The resultant degradation products were analyzed by gel filtration chromatography on a Superdex Peptide 10/300 GL column (GE Healthcare, Pittsburgh, PA, USA) using high performance liquid chromatography (HPLC). The flow rate was 0.3 mL/min with 0.2 M ammonium hydrogen carbonate as the running buffer. Elution was monitored at 210 nm using a UV detector. LabSolutions 6.108 (Shanghai, China) software was used for online monitoring and data analysis. The proportion of trisaccharides in the product of AlyM2 was calculated based on the peak area of the trisaccharides divided by that of all the products in the gel filtration chromatogram. The effect of degradation time on the products of AlyM2 were analyzed by the TLC method. A 200 µL mixture containing 80 µg/mL AlyM2 and 2 mg/mL substrate (PM, PG, PMG, or sodium alginate) in the buffer containing 50 mM Tris-HCl (pH 8.0) and 0.5 M NaCl was incubated at 30 • C for 0, 3, 6, or 12 h. Then, the degradation products and saturated oligosaccharides standards at DP1-DP5 were separated using a solvent system of 1-butanol/acetic acid/water (4:6:1, v/v) and visualized by heating the TLC plates at 90 • C for 15 min after spraying with 10% (v/v) sulfuric acid in ethanol.
To determine the production of trisaccharides from sodium alginate degradation by AlyM2, a 500 mL reaction mixture containing 1 g sodium alginate and 40 mg AlyM2 in the buffer containing 50 mM Tris-HCl (pH 8.0) and 0.5 M NaCl was incubated at 30 • C for 6 h. The production of trisaccharides was determined by gel filtration on a Superdex Peptide 10/300 GL column at a flow rate of 0.3 mL/min using ultrapure water as the running buffer. Elution was monitored using a refractive index detector (RID). LabSolutions software was used for online monitoring and data analysis. A standard curve of trisaccharide concentration versus peak area was drawn by using different concentrations of commercial saturated mannuronate trisaccharide as the standard. The trisaccharide amount released from sodium alginate by AlyM2 was determined based on the standard curve.

Conclusions
Although many alginate lyases have been studied, only a few lyases have been shown to have potential in the preparation of AOS with specific DPs. In this study, we identified and characterized an alginate lyase, AlyM2, that has potential in the preparation of different alginate trisaccharides. AlyM2 was identified from an alginate-degrading strain Pseudoalteromonas arctica M9 that was screened out from Sargassum. AlyM2 is a PL6_3 alginate lyase with low sequence identity to the characterized lyases and does not possess the conserved catalytic amino acids reported in the other PL6 lyases. AlyM2 is an endotype lyase, exhibiting the highest activity at 30 • C, pH 8.0, and 0.5 M NaCl. It mainly produces trisaccharides from PM, PG, PMG, or sodium alginate, and trisaccharides account for approximately 80% of the products. The production of trisaccharides from sodium alginate degradation is 588.4 mg/g. These results demonstrated that AlyM2 is a novel alginate lyase that likely has good potential application in the production of different alginate trisaccharides such as trimannuronate from PM, triguluronate from PG, and a mixture of trisaccharides from PMG and sodium alginate. The structures of these trisaccharides and their potential bioactivities await further investigation.