Cloning, Expression, and Characterization of a New PL25 Family Ulvan Lyase from Marine Bacterium Alteromonas sp. A321

Ulvan lyases can degrade ulvan to oligosaccharides with potent biological activity. A new ulvan lyase gene, ALT3695, was identified in Alteromonas sp. A321. Soluble expression of ALT3695 was achieved in Escherichia coli BL21 (DE3). The 1314-bp gene encoded a protein with 437 amino acid residues. The amino acid sequence of ALT3695 exhibited low sequence identity with polysaccharide lyase family 25 (PL25) ulvan lyases from Pseudoalteromonas sp. PLSV (64.14% identity), Alteromonas sp. LOR (62.68% identity), and Nonlabens ulvanivorans PLR (57.37% identity). Recombinant ALT3695 was purified and the apparent molecular weight was about 53 kDa, which is different from that of other polysaccharide-degrading enzymes identified in Alteromonas sp. A321. ALT3695 exhibited maximal activity in 50 mM Tris-HCl buffer at pH 8.0 and 50 °C. ALT3695 was relatively thermostable, as 90% activity was observed after incubation at 40 °C for 3 h. The Km and Vmax values of ALT3695 towards ulvan were 0.43 mg·mL−1 and 0.11 μmol·min−1·mL−1, respectively. ESI-MS analysis showed that enzymatic products were mainly disaccharides and tetrasaccharides. This study reports a new PL25 family ulvan lyase, ALT3695, with properties that suggest its great potential for the preparation of ulvan oligosaccharides.

Several methods for depolymerizing polysaccharides have been studied. Qi et al. prepared different molecular weight ulvans by treatment with H 2 O 2 and by changing the depolymerization conditions [7]. Yu et al. degraded ulvan by microwaving under different pressures [8]. Mild acid hydrolysis has also been used to degrade ulvan [1]. However, such chemical and physical methods

Influence of Temperature on the Activity of Recombinant ALT3695
The influence of temperature on enzyme stability and the optimum temperature for ALT3695 activity were investigated. Ulvan lyases from different families exhibit different optimum temperatures. The optimum temperature for ALT3695 activity was 50 • C (Figure 4a), which is higher than that of another ulvan lyase from the PL25 family (45 • C) [16]. Ulvan lyases from other families, such as FaPL28 (GenBank accession no. WP_038530530.1, PL28) [23] and PsPL (GenBank accession no. AMA19992.1, PL24) [22], showed maximum activity at lower temperatures of 29.5 • C and 35 • C, respectively. Little has been reported concerning the thermal stability of PL25 family members. FaPL28 had poor thermal stability, and less than 10% activity was observed after incubation for 3 h at or above 30.9 • C [23]. In contrast, ALT3695 was relatively thermostable and 90% activity was observed even after incubation for 3 h at 40 • C (Figure 4b). However, the stability of ALT3695 decreased as the temperature was increased above 40 • C (Figure 4b), 49% residual activity was observed after incubation for 3 h at 45 • C, and no activity was observed after incubation at 55 • C for 1 h.
Mar. Drugs 2019, 17, x FOR 5 of 11 than that of another ulvan lyase from the PL25 family (45 °C) [16]. Ulvan lyases from other families, such as FaPL28 (GenBank accession no. WP_038530530.1, PL28) [23] and PsPL (GenBank accession no. AMA19992.1, PL24) [22], showed maximum activity at lower temperatures of 29.5 °C and 35 °C, respectively. Little has been reported concerning the thermal stability of PL25 family members. FaPL28 had poor thermal stability, and less than 10% activity was observed after incubation for 3 h at or above 30.9 °C [23]. In contrast, ALT3695 was relatively thermostable and 90% activity was observed even after incubation for 3 h at 40 °C ( Figure 4b). However, the stability of ALT3695 decreased as the temperature was increased above 40 °C (Figure 4b), 49% residual activity was observed after incubation for 3 h at 45 °C, and no activity was observed after incubation at 55 °C for 1 h.

Influence of pH on the Activity of Recombinant ALT3695
To investigate the effect of pH on ALT3695 activity, different buffers (pH 4-9.5) were used. The optimum pH of other ulvan lyases ranged from 7.5 to 9.0 [16,22,23], which corresponds to the pH of slightly alkaline seawater [24]. Similarly, ALT3695 showed maximal activity at pH 8.0 (Figure 4c). In addition, activity in Tris-HCl buffer was higher than that in Na2HPO4-citric acid buffer at pH 8.0, suggesting that the catalytic activity of ALT3695 was also affected by the buffer salt. ALT3695 retained 80% activity after pre-incubation at pH 4.0-9.0 for 2 h at 4°C, indicating that ALT3695 has excellent pH stability (Figure 4d). Table 2a shows the influence of various metal ions on ALT3695 activity. K + had no significant influence on ALT3695 activity. Additionally, Fe 2+ and Cu 2+ decreased enzyme activity, and this effect was also found in the ulvan lyases, PsPL [22] and FaPL28 [23]. Furthermore, ulvan lyases from

Influence of pH on the Activity of Recombinant ALT3695
To investigate the effect of pH on ALT3695 activity, different buffers (pH 4-9.5) were used. The optimum pH of other ulvan lyases ranged from 7.5 to 9.0 [16,22,23], which corresponds to the pH of slightly alkaline seawater [24]. Similarly, ALT3695 showed maximal activity at pH 8.0 (Figure 4c). In addition, activity in Tris-HCl buffer was higher than that in Na 2 HPO 4 -citric acid buffer at pH 8.0, suggesting that the catalytic activity of ALT3695 was also affected by the buffer salt. ALT3695 retained 80% activity after pre-incubation at pH 4.0-9.0 for 2 h at 4 • C, indicating that ALT3695 has excellent pH stability (Figure 4d). was also found in the ulvan lyases, PsPL [22] and FaPL28 [23]. Furthermore, ulvan lyases from different families exhibit different responses to Co 2+ , the activity of PsPL was increased by Co 2+ [22], while the activity of FaPL28 [23] and ALT3695 was decreased by Co 2+ . This may be attributed to structural differences among these enzyme families. Moreover, Cd 2+ , Hg 2+ , and Fe 3+ deactivated ALT3695 completely, which may be caused by the binding of these ions to the SH, CO, and NH moieties of amino acids, resulting in structural changes and inactivation [21]. It is worth mentioning that although ALT3695 has Zn 2+ -containing ligands, Zn 2+ strongly inhibited enzymatic activity. Thus, Zn 2+ may play a dual role, as inherent Zn 2+ is essential for maintaining enzyme structure, but exogenous Zn 2+ may bind to other amino acid residues and inhibit activity [25]. The enzyme activity was increased by Ca 2+ , Mg 2+ , and Ba 2+ . The addition of Ca 2+ would not only increase the activity of PL25 ulvan lyase, but also stimulate the activity of PsPL [22] and FaPL28 [23]. With more Ca 2+ added, the activity of ALT3695 was also increased. In addition, ALT3695 activity decreased by 68% after pre-incubation in 5 mM ethylenediaminetetraacetic acid (EDTA), similar phenomenon was also found in PsPL and FaPL28, this may due to the removal of divalent cations bound to enzymes. Additionally, structure analysis suggested that divalent metal ions play a structural role in all three ulvan lyase families.

Influences of Surfactants, Metal Ions, and Metal Chelators on the Activity of Recombinant ALT3695
The effect of surfactants and metal chelators on ALT3695 activity was also investigated (Table 2b). It has been reported that non-ionic surfactants could increase the activity of some enzymes [26,27]. However, surfactants had little effect on the activity of ALT3695.

Kinetic Parameters of Recombinant ALT3695
V max is the maximum initial rate of an enzymatic reaction. Each enzyme has a specific K m for each substrate, which is inversely related to the enzyme's affinity for the substrate [28]. The kinetic parameters of other ulvan lyases from the PL25 family have not yet been reported. The K m and V max of recombinant ALT3695 toward ulvan were 0.43 mg·mL −1 and 0.11 µmol·min −1 ·mL −1 , respectively, as determined by the Lineweaver-Burk plot. ALT3695 showed a higher affinity for ulvan than PsPL and FaPL28, which had a K m of 2.10 mg·mL −1 [22] and 0.75 mg·mL −1 [23], respectively, suggesting the potential of ALT3695 for application.

Analysis of Enzymatic Products
The main repetitive disaccharide units in ulvan are Rha3S-GlcA, Rha3S-IduA, and Rha3S-Xyl. ALT3695 could cleave the bond on both Rha3S-GlcA and Rha3S-IdoA, while PL24 family ulvan lyase specifically cleaves the bond between Rha3S-GlcA. The products of ulvan degradation by ALT3695 were analyzed by negative electrospray ionisation mass spectrometry (ESI-MS). Mass spectroscopic analysis of products showed two major types of oligosaccharides. The peak at m/z 401 was equivalent to the disaccharide ∆GlcA-Rha3S, with a molecular weight of 402 Da. There was an unsaturated double bond between C4 and C5 [18]. The peak at m/z 379 was equivalent to double-charged molecular ions of the tetrasaccharide ∆GlcA-Rha3S-Xyl-Rha3S, with a molecular weight of 760 Da [14]. A low abundance of single-charged molecular ions of this tetrasaccharide was also identified as the peak at m/z 759. However, the products of ulvan degradation by PL24 ulvan lyase were different. The products included the two most abundant oligosaccharides which comprised of disaccharides (∆GlcA-Rha3S) and tetrasaccharides (∆GlcA-Rha3S-IdoA-Rha3S), with minor tetrasaccharides (∆GlcA-Rha3S-Xyl-Rha3S) [15].
Moreover, the change in the full wavelength during enzymatic degradation was studied ( Figure 5b). As the enzymatic reaction continued, the absorption peak at 235 nm gradually increased, which may be due to the generation of ∆GlcA-Rha3S and ∆GlcA-Rha3S-Xyl-Rha3S, as C-4=C-5 double bonds have a high absorbance at 235 nm [29].

Analysis of Enzymatic Products
The main repetitive disaccharide units in ulvan are Rha3S-GlcA, Rha3S-IduA, and Rha3S-Xyl. ALT3695 could cleave the bond on both Rha3S-GlcA and Rha3S-IdoA, while PL24 family ulvan lyase specifically cleaves the bond between Rha3S-GlcA. The products of ulvan degradation by ALT3695 were analyzed by negative electrospray ionisation mass spectrometry (ESI-MS). Mass spectroscopic analysis of products showed two major types of oligosaccharides. The peak at m/z 401 was equivalent to the disaccharide ∆GlcA-Rha3S, with a molecular weight of 402 Da. There was an unsaturated double bond between C4 and C5 [18]. The peak at m/z 379 was equivalent to double-charged molecular ions of the tetrasaccharide ∆GlcA-Rha3S-Xyl-Rha3S, with a molecular weight of 760 Da [14]. A low abundance of single-charged molecular ions of this tetrasaccharide was also identified as the peak at m/z 759. However, the products of ulvan degradation by PL24 ulvan lyase were different. The products included the two most abundant oligosaccharides which comprised of disaccharides (∆GlcA-Rha3S) and tetrasaccharides (∆GlcA-Rha3S-IdoA-Rha3S), with minor tetrasaccharides (∆GlcA-Rha3S-Xyl-Rha3S) [15].
Moreover, the change in the full wavelength during enzymatic degradation was studied ( Figure  5b). As the enzymatic reaction continued, the absorption peak at 235 nm gradually increased, which may be due to the generation of ∆GlcA-Rha3S and ∆GlcA-Rha3S-Xyl-Rha3S, as C-4=C-5 double bonds have a high absorbance at 235 nm [29].

Strains, Plasmids, and Medium
The ulvan-degrading strain Alteromonas sp. A321 was provided by Peng Wang (Ocean University of China, Qingdao, China). Genomic DNA was extracted, and the whole genome was sequenced. E. coli strains DH5α and BL21 (DE3) were purchased from TIANGEN Biotech Co. (Beijing, China). The pProEX-HTa vector was used to clone and express the ulvan lyase gene. The E. coli strains were cultured in Luria-Bertani medium.

Sequence Analysis
An ulvan lyase gene, named ALT3695, was identified in the genome, and its sequence was deposited into the NCBI database (GenBank accession no. MN347032). DNAMAN 9 (Lynnon Biosoft, San Ramon, CA, USA) software was used to analyze the sequence of ALT3695. The homology was analyzed using BLAST at the NCBI website. A phylogenetic tree based on the homology of various ulvan lyases was constructed using MEGA 5.0 (Koichiro, Tokyo, Japan).

Construction of the Recombinant ALT3695-Expressing Strain
The ALT3695 gene was amplified by PCR using Phanta ® Turbo Super-Fidelity DNA Polymerase (Vazyme Biotech, Nanjing, China).

Expression of Recombinant ALT3695
E. coli BL21 (DE3) cells containing pProEX-HTa-ALT3695 were cultivated in Luria-Bertani medium supplemented with ampicillin (100 µg/mL). The culture conditions were 37 • C and 200 rpm. When the strain entered logarithmic growth phase (OD600~0.7), IPTG was added at a final concentration of 1 mM to induce the expression of ALT3695. Then, the culture temperature was decreased to 28 • C, but 200 rpm was retained with further cultivation for 18 h. Finally, cells were collected by centrifugation under freezing conditions (4 • C) and resuspended in 20 mM Tris-HCl buffer (pH 7.5).

Enzyme Purification
After homogenization by sonication, the supernatant containing crude enzymes was obtained by centrifugation under freezing conditions. The His-tagged ulvan lyase was purified using BeaverBeads™ IDA-Nickel (Beaverbio, Suzhou, China) and eluted using 20 mM Tris-HCl buffer (pH 7.5) supplemented with 0.5 M NaCl and 300 mM imidazole. The concentration of soluble protein was determined by the Folin-Ciocalteu method [30]. Purified enzyme was then subjected to SDS-PAGE to analyze the purity and molecular mass [31].

Enzyme Activity Assay
Ulvan was extracted from Ulva clathrate (Fujian, China) according to the method of Qi et al. [32]. The ulvan substrate was added at 0.1% (w/v) in 50 mM Tris-HCl buffer (pH 8.0) and the reaction was conducted at 35 • C. The activity of ulvan lyase was examined by measuring the change in absorbance at 235 nm [14]. One unit (U) of ulvan lyase activity was defined as the amount of protein needed to generate 1 µmol of unsaturated glucuronyl residue (extinction coefficient, 4800 M −1 ·cm −1 ) per minute [23]. All assays were repeated three times.

Characterization of Recombinant ALT3695
To examine the optimum temperature of recombinant ALT3695, the reaction was conducted at 35-60 • C under standard conditions. The change in enzyme stability at different temperatures was analyzed by conducting enzyme activity assays after pre-incubating the enzyme for various times (5,15,30,60,90,120, and 180 min) at 35-60 • C.
The optimal pH of recombinant ALT3695 was investigated by examining its activity in 50 mM Na 2 HPO 4 -citric acid buffer (for pH 4.0-8.0) and 50 mM Tris-HCl buffer (for pH 7.5-9.5). To investigate the change of enzyme stability by pH, purified enzymes were stored for 2 h at 4 • C in buffers of different pH levels, and then assayed for residual enzyme activity.
To examine the influence of different metal ions on recombinant ALT3695 activity, the enzyme was incubated with various metal ions (at 10 mM and 20 mM) for 2 h at 4 • C. The following metal ions were tested: K + , Ca 2+ , Mg 2+ , Zn 2+ , Ba 2+ , Cu 2+ , Fe 2+ , Hg 2+ , Co 2+ , Cd 2+ , and Fe 3+ . The effect of the following surfactants and metal chelators on recombinant ALT3695 activity was also determined: Tween-20, Tween-80, Triton X-100, EDTA, and 1,10-phenanthroline. The enzyme solution was stored with each surfactant or potential inhibitor (at 5 mM and 10 mM) for 2 h at 4 • C. Then, enzyme activity was assayed and compared with the activity in the absence of additive.

Kinetic Measurements
To determine the kinetic parameters of recombinant ALT3695 using ulvan as a substrate, reactions were performed with various concentrations (0.03125-1.0 mg·mL −1 ) of substrate under standard conditions for 5 min. Then, the K m and V max values were calculated by constructing a Lineweaver-Burk double reciprocal plot.

Conclusions
ALT3695 is a new ulvan lyase identified from Alteromonas sp. A321. The gene was cloned, and the recombinant ALT3695 was expressed in soluble fraction. The enzymatic properties were also investigated. The enzyme exhibited excellent stability and substrate affinity. Maximum activity was observed at 50 • C, and 90% activity remained after incubation at 40 • C for 3 h. ALT3695 catalyzes β-elimination at the internal bonds between uronic acids and Rha3S, leading to increased absorbance at 235 nm. ESI-MS results showed that disaccharides and tetrasaccharides were the major enzymatic products. In conclusion, ALT3695 shows great potential for the preparation of ulvan oligosaccharides. Further research on this recombinant strain and a structural analysis of ulvan lyase ALT3695 are warranted based on results of this study.