Cloning and Characterization of a Novel Endo-Type Metal-Independent Alginate Lyase from the Marine Bacteria Vibrio sp. Ni1

The applications of alginate lyase are diverse, but efficient commercial enzymes are still unavailable. In this study, a novel alginate lyase with high activity was obtained from the marine bacteria Vibrio sp. Ni1. The ORF of the algB gene has 1824 bp, encoding 607 amino acids. Homology analysis shows that AlgB belongs to the PL7 family. There are two catalytic domains with the typical region of QIH found in AlgB. The purified recombinant enzyme of AlgB shows highest activity at 35 °C, pH 8.0, and 50 mmol/L Tris-HCl without any metal ions. Only K+ slightly enhances the activity, while Fe2+ and Cu2+ strongly inhibit the activity. The AlgB preferred polyM as substrate. The end products of enzymatic mixture are DP2 and DP3, without any metal ion to assist them. This enzyme has good industrial application prospects.

In the present study, a novel alginate gene was cloned from Vibrio sp. Ni1 and overexpressed in Escherichia coli. The purified recombinant enzyme has high activity toward alginate, poly M and poly G at high temperature and pH, independent of ions. The properties of the recombinant enzyme and its products were analyzed.

The Analysis of Alginate-Degrading Activity of Vibrio sp. Ni1
After growth on the alginate plate for 48 h, three colonies of Vibrio sp. Ni1 and their alginate-degraded circles were observed and measured ( Figure 1 and Table 1). The results of plate assay showed that the central bright yellow zones were formed by iodate was reduced to I2. It suggested that Vibrio sp. Ni1 can produce alginate lyase to degrade algin and yield the unsaturated uronic acid with reducibility. The diameter of alginate-degraded zones were more than four times greater than that of the colonies. The result showed that Vibrio sp. Ni1 has good ability to degrade alginate.

The LC-MS/MS Analysis of Crude Alginate Lyase
The crude alginate lyase solution was purified by 70% (NH4)2SO4 precipitation. The majority of the extracellular proteins were exhibited in the range of 20-75 kDa in SDS-PAGE gel ( Figure 2). The protein bands in lane 1 were cut-out to extract the proteins, then the proteins were analyzed by LC-MS/MS. There were 147 peptides detected and 6 peptides (Table 2) were discovered to match with one candidate alginate lyase gene. According to the homology of AIY22661.1 and WP_118120558.1, and peptide locations, No. 3 and 4 peptide sequences were chosen to design a pair of degenerate primers named algB-F0/R0 to amplify the partial algB gene.

The LC-MS/MS Analysis of Crude Alginate Lyase
The crude alginate lyase solution was purified by 70% (NH 4 ) 2 SO 4 precipitation. The majority of the extracellular proteins were exhibited in the range of 20-75 kDa in SDS-PAGE gel ( Figure 2). The protein bands in lane 1 were cut-out to extract the proteins, then the proteins were analyzed by LC-MS/MS. There were 147 peptides detected and 6 peptides ( Table 2) were discovered to match with one candidate alginate lyase gene. According to the homology of AIY22661.1 and WP_118120558.1, and peptide locations, No. 3 and

The Characterization of the Full Length algB Gene
Based on the sequencing result of the partial algB gene, using SiteFinding-PCR technology, a series of primers were designed to obtain the full length algB gene. Finally, a pair of primers named algB-QF/QR were designed to amplify the full length of algB gene ( Figure 3). It consisted of 1824 bp and encodes 607 amino acids with a sequence of 18 amino acids at the N-terminus that was predicted to be signal peptide by online software Signal P5.0. The AlgB protein had a calculated molecular weight 67.7 kDa and pI 4.34. It was subjected to Blast analysis in the NCBI database. The DNA sequence displays high similarity with only one partial genome of Vibrio penaeicida (AP025146.1, 93.81% identity of 86% length). The amino acid sequence displays high similarity of 93.87% identity with three alginate lyases (WP_148112135.1 from Vibrio penaeicida, WP_224056095.1 from Vibrio penaeicida, WP_224915578.1 from Vibrio alginolyticus). These three alginate lyases have not been characterized. Other alginate lyases have about 70-80% identities, such as AIY22661.1 from Vibrio sp. W13, 81.13%; and WP_118120558.1 from Vibrio sp. Dhg, 77.86% (Figure 4a). The algB sequence has been submitted to NCBI (GenBank accession No. MZ680650).

The Characterization of the Full Length algB Gene
Based on the sequencing result of the partial algB gene, using SiteFinding-PCR technology, a series of primers were designed to obtain the full length algB gene. Finally, a pair of primers named algB-QF/QR were designed to amplify the full length of algB gene ( Figure 3). It consisted of 1824 bp and encodes 607 amino acids with a sequence of 18 amino acids at the N-terminus that was predicted to be signal peptide by online software Signal P5.0. The AlgB protein had a calculated molecular weight 67.7 kDa and pI 4.34. It was subjected to Blast analysis in the NCBI database. The DNA sequence displays high similarity with only one partial genome of Vibrio penaeicida (AP025146.1, 93.81% identity of 86% length). The amino acid sequence displays high similarity of 93.87% identity with three alginate lyases (WP_148112135.1 from Vibrio penaeicida, WP_224056095.1 from Vibrio penaeicida, WP_224915578.1 from Vibrio alginolyticus). These three alginate lyases have not been characterized. Other alginate lyases have about 70-80% identities, such as AIY22661.1 from Vibrio sp. W13, 81.13%; and WP_118120558.1 from Vibrio sp. Dhg, 77.86% (Figure 4a). The algB sequence has been submitted to NCBI (GenBank accession No. MZ680650).

The Characterization of the Full Length algB Gene
Based on the sequencing result of the partial algB gene, using SiteFinding-PCR technology, a series of primers were designed to obtain the full length algB gene. Finally, a pair of primers named algB-QF/QR were designed to amplify the full length of algB gene ( Figure 3). It consisted of 1824 bp and encodes 607 amino acids with a sequence of 18 amino acids at the N-terminus that was predicted to be signal peptide by online software Signal P5.0. The AlgB protein had a calculated molecular weight 67.7 kDa and pI 4.34. It was subjected to Blast analysis in the NCBI database. The DNA sequence displays high similarity with only one partial genome of Vibrio penaeicida (AP025146.1, 93.81% identity of 86% length). The amino acid sequence displays high similarity of 93.87% identity with three alginate lyases (WP_148112135.1 from Vibrio penaeicida, WP_224056095.1 from Vibrio penaeicida, WP_224915578.1 from Vibrio alginolyticus). These three alginate lyases have not been characterized. Other alginate lyases have about 70-80% identities, such as AIY22661.1 from Vibrio sp. W13, 81.13%; and WP_118120558.1 from Vibrio sp. Dhg, 77.86% (Figure 4a). The algB sequence has been submitted to NCBI (GenBank accession No. MZ680650).  substrate binding and catalysis [23,24]. These conserved motifs also exist in each of the two structural domains in AlgB (RSEVR, QIH, YFKAGVYNQ; RSELR, QIH, YFKAGIYPH). The predicted three-dimensional structure of AlgB was generated by LOMETS ( Figure 4c). The typical protein structure of PL7 is a single domain β-jelly roll [3,23]. The first structural domain is residues 78-310, the second structural domain is residues 319-606. These two structural domains connect closely with an octapeptide linking region. It is rare that PL7 alginate lyases have two catalytic domains [25,26].  AlgB is classified in the polysaccharide lyase family 7 (PL7). Conserved domain analysis revealed two structural domains in AlgB (Figure 4b). Each structural domain has a predicted catalytic domain. Alginate lyases in PL7 contain three highly conserved motifs, R*E*R, Q(I/V)H, Y*KAG*Y*Q, which form the active site and play an important role in substrate binding and catalysis [23,24]. These conserved motifs also exist in each of the two structural domains in AlgB (RSEVR, QIH, YFKAGVYNQ; RSELR, QIH, YFKAGIYPH). The predicted three-dimensional structure of AlgB was generated by LOMETS ( Figure 4c). The typical protein structure of PL7 is a single domain βjelly roll [3,23]. The first structural domain is residues 78-310, the second structural domain is residues 319-606. These two structural domains connect closely with an octapeptide linking region. It is rare that PL7 alginate lyases have two catalytic domains [25,26].

Purification of the Recombinant Enzyme AlgB
To characterize the recombinant enzyme, the algB gene was cloned into the expression vector pET32a (+) and transformed to E. coli BL21. The fusion proteins of AlgB were soluble when expressed in host cells and purified by nickel affinity chromatography. Since the mass of the fusion tag protein (TrxA) is approximately 20 kDa, the recombinant AlgB is about 88 kDa. The SDS-PAGE analysis is consistent with the prediction ( Figure 5). The activity of purified AlgB was measured ( Figure 6). Along with the increase in time, the absorption at λ235 was enhanced rapidly within 1 min and, subsequently, increased slowly. It suggested that AlgB shows the activity of alginate lyase.

Purification of the Recombinant Enzyme AlgB
To characterize the recombinant enzyme, the algB gene was cloned into the expression vector pET32a (+) and transformed to E. coli BL21. The fusion proteins of AlgB were soluble when expressed in host cells and purified by nickel affinity chromatography. Since the mass of the fusion tag protein (TrxA) is approximately 20 kDa, the recombinant AlgB is about 88 kDa. The SDS-PAGE analysis is consistent with the prediction ( Figure 5). The activity of purified AlgB was measured ( Figure 6). Along with the increase in time, the absorption at λ235 was enhanced rapidly within 1 min and, subsequently, increased slowly. It suggested that AlgB shows the activity of alginate lyase.

Characteristics of the Recombinant Enzyme AlgB
The optimal pH for the recombinant enzyme AlgB activity was 8.0 and the highest activity was 979 U/mL ( Figure 7). More than 50% of the maximum activity was measured at a wide pH range 5.0-10.0 and the activity was stable at pH range of 6.0-10.0. The activity of AlgB sharply decreased when pH lower than 5.0. Beyond Tris-HCl buffer capacity, the activity was not measured on the condition of pH higher than 10.0. The results suggest that the AlgB has good activity at basic pH conditions. The characteristic of some alginate

Purification of the Recombinant Enzyme AlgB
To characterize the recombinant enzyme, the algB gene was cloned into the expression vector pET32a (+) and transformed to E. coli BL21. The fusion proteins of AlgB were soluble when expressed in host cells and purified by nickel affinity chromatography. Since the mass of the fusion tag protein (TrxA) is approximately 20 kDa, the recombinant AlgB is about 88 kDa. The SDS-PAGE analysis is consistent with the prediction ( Figure 5). The activity of purified AlgB was measured ( Figure 6). Along with the increase in time, the absorption at λ235 was enhanced rapidly within 1 min and, subsequently, increased slowly. It suggested that AlgB shows the activity of alginate lyase.

Characteristics of the Recombinant Enzyme AlgB
The optimal pH for the recombinant enzyme AlgB activity was 8.0 and the highest activity was 979 U/mL (Figure 7). More than 50% of the maximum activity was measured at a wide pH range 5.0-10.0 and the activity was stable at pH range of 6.0-10.0. The activity of AlgB sharply decreased when pH lower than 5.0. Beyond Tris-HCl buffer capacity, the

Characteristics of the Recombinant Enzyme AlgB
The optimal pH for the recombinant enzyme AlgB activity was 8.0 and the highest activity was 979 U/mL (Figure 7). More than 50% of the maximum activity was measured at a wide pH range 5.0-10.0 and the activity was stable at pH range of 6.0-10.0. The activity of AlgB sharply decreased when pH lower than 5.0. Beyond Tris-HCl buffer capacity, the activity was not measured on the condition of pH higher than 10.0. The results suggest that the AlgB has good activity at basic pH conditions. The characteristic of some alginate lyases with similar molecular mass from Vibrio strains are summarized (Table 3). These alginate lyase showed optimal pH ≥ 7.0, the highest pH 8.9 [25][26][27][28][29][30][31][32].
Mar. Drugs 2022, 20, 479 6 of 15 lyases with similar molecular mass from Vibrio strains are summarized (Table 3). These alginate lyase showed optimal pH ≥ 7.0, the highest pH 8.9 [25,[26][27][28][29][30][31][32].  The optimal temperature for AlgB was 35 °C and the highest activity was 1059 U/mL (Figure 8a). More than 50% of the maximum activity was measured at a wide temperature range 20-60 °C. The activity of AlgB has sharply decreased over 60 °C. Even at 4 °C, the activity of AlgB was measured 35% maximum. The enzyme retained more than 50% of the highest activity after being incubated at 30 °C for 36 h, and the enzyme retained more than 40% of the highest activity after being incubated at 40 °C for 24 h (Figure 8b). The results show that the activity of AlgB is stable at 30-40 °C. The optimal temperature of AlgB was similar to Aly-Ⅳ from Vibrio sp. QD-5 [31].  The optimal temperature for AlgB was 35 • C and the highest activity was 1059 U/mL (Figure 8a). More than 50% of the maximum activity was measured at a wide temperature range 20-60 • C. The activity of AlgB has sharply decreased over 60 • C. Even at 4 • C, the activity of AlgB was measured 35% maximum. The enzyme retained more than 50% of the highest activity after being incubated at 30 • C for 36 h, and the enzyme retained more than 40% of the highest activity after being incubated at 40 • C for 24 h (Figure 8b). The results show that the activity of AlgB is stable at 30-40 • C. The optimal temperature of AlgB was similar to Aly-IV from Vibrio sp. QD-5 [31]. The activity of AlgB was inhibited by 10 mmol/L Mg 2+ , Ca 2+ , Mn 2+ , Co 2+ , Ni 2+ , Zn 2+ ; furthermore, strongly inhibited by Fe 2+ and Cu 2+ . Among the metal ions, only K + displayed slight activation of enzyme activity (Figure 9a). The enzyme retained more than 50% of the highest activity in 1-2 mol/L NaCl (Figure 9b). Enhancing the concentration of NaCl to more than 3 mol/L, the activity sharply decreases. These results suggested that AlgB has a good tolerance to NaCl. Previous investigations report that many metal ions can increase the enzyme activity, such as Mg 2+ and Ca 2+ ( Table 3). The effect of metal ion on AlgB was similar to Aly-IV except for Mg 2+ (Table 3). With the same concentration, Mg 2+ strongly activates Aly-IV but slightly inhibits AlgB. The kinetic parameters of AlgB were determined at the optimal reaction conditions. The specific activity of AlgB towards sodium alginate was 2118 U/mg, the Km value of The activity of AlgB was inhibited by 10 mmol/L Mg 2+ , Ca 2+ , Mn 2+ , Co 2+ , Ni 2+ , Zn 2+ ; furthermore, strongly inhibited by Fe 2+ and Cu 2+ . Among the metal ions, only K + displayed slight activation of enzyme activity (Figure 9a). The enzyme retained more than 50% of the highest activity in 1-2 mol/L NaCl (Figure 9b). Enhancing the concentration of NaCl to more than 3 mol/L, the activity sharply decreases. These results suggested that AlgB has a good tolerance to NaCl. Previous investigations report that many metal ions can increase the enzyme activity, such as Mg 2+ and Ca 2+ ( Table 3). The effect of metal ion on AlgB was similar to Aly-IV except for Mg 2+ (Table 3). With the same concentration, Mg 2+ strongly activates Aly-IV but slightly inhibits AlgB. The activity of AlgB was inhibited by 10 mmol/L Mg 2+ , Ca 2+ , Mn 2+ , Co 2+ , Ni 2+ , Zn 2+ ; furthermore, strongly inhibited by Fe 2+ and Cu 2+ . Among the metal ions, only K + displayed slight activation of enzyme activity (Figure 9a). The enzyme retained more than 50% of the highest activity in 1-2 mol/L NaCl (Figure 9b). Enhancing the concentration of NaCl to more than 3 mol/L, the activity sharply decreases. These results suggested that AlgB has a good tolerance to NaCl. Previous investigations report that many metal ions can increase the enzyme activity, such as Mg 2+ and Ca 2+ ( Table 3). The effect of metal ion on AlgB was similar to Aly-IV except for Mg 2+ (Table 3). With the same concentration, Mg 2+ strongly activates Aly-IV but slightly inhibits AlgB. The kinetic parameters of AlgB were determined at the optimal reaction conditions. The specific activity of AlgB towards sodium alginate was 2118 U/mg, the Km value of  The kinetic parameters of AlgB were determined at the optimal reaction conditions. The specific activity of AlgB towards sodium alginate was 2118 U/mg, the K m value of AlgB was 8.22 mg/mL, the value of V max was 2.6 mg/min, and the value of K cat was 26 min −1 at pH 8.0, 35 • C ( Figure 10).

Substrate Specificity of AlgB
To investigate their substrate specificity, sodium alginate, polyM and polyG were used as substrate for the recombinant enzyme AlgB. Compared to sodium alginate, the relative activity of AlgB to polyM was 16% higher, while polyG was 14% lower at the same reaction conditions (Figure 11). This illustrates that AlgB degrades M-M links more efficiently. Both AlyA and AlyA-OU2 which were favorably to degrade polyM in activity assays ( Table 3). The structure of these two alginate lyases are similar to that of AlgB. There is a double region of QIH within them which determines the enzyme has polyM specificity. Some alginate lyases from Vibrio strains were reported sodium alginate-preferred, such as the alginate lyase from Vibrio sp. YWA, Aly-IV from Vibrio sp. QD-5 and Alg7A from Vibrio sp. W13. A few polyG-preferred alginate lyases were discovered, such as AlyB and AlgNJ04. It can be inferred that the QIH region is not absolutely determined by the substrate specificity. Figure 11. Substrate specificity of AlgB. The concentration of substrate was 1 mg/mL. The activity measured in 35 °C and 50 mmol/L Tris-HCl buffer (pH 8.0) using sodium alginate as substrate was taken as 100%. The data represent the mean of three experimental repeats with SD ≤ 5%.

Analysis of AlgB Degradation Products
Degradation products of AlgB activity were analyzed by TLC and ESI-Q-TOF. The result of TLC showed that the distribution of the end products from the sodium alginate

Substrate Specificity of AlgB
To investigate their substrate specificity, sodium alginate, polyM and polyG were used as substrate for the recombinant enzyme AlgB. Compared to sodium alginate, the relative activity of AlgB to polyM was 16% higher, while polyG was 14% lower at the same reaction conditions ( Figure 11). This illustrates that AlgB degrades M-M links more efficiently. Both AlyA and AlyA-OU2 which were favorably to degrade polyM in activity assays ( Table 3). The structure of these two alginate lyases are similar to that of AlgB. There is a double region of QIH within them which determines the enzyme has polyM specificity. Some alginate lyases from Vibrio strains were reported sodium alginate-preferred, such as the alginate lyase from Vibrio sp. YWA, Aly-IV from Vibrio sp. QD-5 and Alg7A from Vibrio sp. W13. A few polyG-preferred alginate lyases were discovered, such as AlyB and AlgNJ04. It can be inferred that the QIH region is not absolutely determined by the substrate specificity.

Substrate Specificity of AlgB
To investigate their substrate specificity, sodium alginate, polyM and polyG were used as substrate for the recombinant enzyme AlgB. Compared to sodium alginate, the relative activity of AlgB to polyM was 16% higher, while polyG was 14% lower at the same reaction conditions (Figure 11). This illustrates that AlgB degrades M-M links more efficiently. Both AlyA and AlyA-OU2 which were favorably to degrade polyM in activity assays ( Table 3). The structure of these two alginate lyases are similar to that of AlgB. There is a double region of QIH within them which determines the enzyme has polyM specificity. Some alginate lyases from Vibrio strains were reported sodium alginate-preferred, such as the alginate lyase from Vibrio sp. YWA, Aly-IV from Vibrio sp. QD-5 and Alg7A from Vibrio sp. W13. A few polyG-preferred alginate lyases were discovered, such as AlyB and AlgNJ04. It can be inferred that the QIH region is not absolutely determined by the substrate specificity. Figure 11. Substrate specificity of AlgB. The concentration of substrate was 1 mg/mL. The activity measured in 35 °C and 50 mmol/L Tris-HCl buffer (pH 8.0) using sodium alginate as substrate was taken as 100%. The data represent the mean of three experimental repeats with SD ≤ 5%.

Analysis of AlgB Degradation Products
Degradation products of AlgB activity were analyzed by TLC and ESI-Q-TOF. The result of TLC showed that the distribution of the end products from the sodium alginate  Figure 11. Substrate specificity of AlgB. The concentration of substrate was 1 mg/mL. The activity measured in 35 • C and 50 mmol/L Tris-HCl buffer (pH 8.0) using sodium alginate as substrate was taken as 100%. The data represent the mean of three experimental repeats with SD ≤ 5%.

Analysis of AlgB Degradation Products
Degradation products of AlgB activity were analyzed by TLC and ESI-Q-TOF. The result of TLC showed that the distribution of the end products from the sodium alginate were separated to two different regions with different colors (Figure 12). The negative ESI-Q-TOF results were similar to that of TLC ( Figure 12). The main products of degrading sodium alginate, polyM, and polyG were similar and consist of DP2 and DP3. It suggests that the AlgB belongs to endo-alginate lyase family. These results suggest that dimers and trimers are the primary end products of AlgB activity.
Mar. Drugs 2022, 20, 479 9 of 15 were separated to two different regions with different colors (Figure 12). The negative ESI-Q-TOF results were similar to that of TLC ( Figure 13). The main products of degrading sodium alginate, polyM, and polyG were similar and consist of DP2 and DP3. It suggests that the AlgB belongs to endo-alginate lyase family. These results suggest that dimers and trimers are the primary end products of AlgB activity. were separated to two different regions with different colors (Figure 12). The negative ES Q-TOF results were similar to that of TLC ( Figure 13). The main products of degradin sodium alginate, polyM, and polyG were similar and consist of DP2 and DP3. It sugges that the AlgB belongs to endo-alginate lyase family. These results suggest that dime and trimers are the primary end products of AlgB activity. The end products from AlgB degradation are similar to Aly-IV (Table 3). It is differe from most other alginate lyases currently reported. The degradation mode of AlyA mainly DP1-4; that of AlyA-OU02 is mainly DP2-4; Algb and AlgNJ04 are mainly DP 5; and Alg7A is mainly DP2-6 ( Table 3).
The process of the recombinant enzyme AlgB was monitored in a viscosity ass ( Figure 14). The viscosity of the sodium alginate solution rapidly decreased in the firs min, then the viscosity slowly decreased in the following 25 min. The variation trend AlgB degradation resembled to Aly-IV [31]. Figure 14. Viscosity variation during enzymatic degradation of alginate. The viscosity was me ured in a 100 mL reaction system with 1% (w/v) sodium alginate, 50 mmol/L Tris-HCl buffer (p 8.0) and RT. The data represents the mean of three repeats with SD ≤ 5%.

Bacteria and Substrate
The strain of Vibrio sp. Ni1 was isolated from the sludge of the Minjiang river estuar located in Fujian province, China. This strain was kept in our lab and China Center f Type Culture Collection (CCTCC No. M2018916). E. coli BL21(DE3) was used as an e pression host. The sodium alginate donated by Haizhilin Biotechnology Development C Ltd. (Qingdao, China) was used as the substrate to test alginate lyase activity. The visco ity of this sodium alginate sample was 1250 mPa.s. The sodium alginate used to prepa medium was from Solarbio (Beijing, China). The end products from AlgB degradation are similar to Aly-IV (Table 3). It is different from most other alginate lyases currently reported. The degradation mode of AlyA is mainly DP1-4; that of AlyA-OU02 is mainly DP2-4; Algb and AlgNJ04 are mainly DP2-5; and Alg7A is mainly DP2-6 ( Table 3).
The process of the recombinant enzyme AlgB was monitored in a viscosity assay ( Figure 14). The viscosity of the sodium alginate solution rapidly decreased in the first 5 min, then the viscosity slowly decreased in the following 25 min. The variation trend of AlgB degradation resembled to Aly-IV [31]. The end products from AlgB degradation are similar to Aly-IV (Table 3). It is different from most other alginate lyases currently reported. The degradation mode of AlyA is mainly DP1-4; that of AlyA-OU02 is mainly DP2-4; Algb and AlgNJ04 are mainly DP2-5; and Alg7A is mainly DP2-6 ( Table 3).
The process of the recombinant enzyme AlgB was monitored in a viscosity assay ( Figure 14). The viscosity of the sodium alginate solution rapidly decreased in the first 5 min, then the viscosity slowly decreased in the following 25 min. The variation trend of AlgB degradation resembled to Aly-IV [31]. Figure 14. Viscosity variation during enzymatic degradation of alginate. The viscosity was measured in a 100 mL reaction system with 1% (w/v) sodium alginate, 50 mmol/L Tris-HCl buffer (pH 8.0) and RT. The data represents the mean of three repeats with SD ≤ 5%.

Bacteria and Substrate
The strain of Vibrio sp. Ni1 was isolated from the sludge of the Minjiang river estuary, located in Fujian province, China. This strain was kept in our lab and China Center for Type Culture Collection (CCTCC No. M2018916). E. coli BL21(DE3) was used as an expression host. The sodium alginate donated by Haizhilin Biotechnology Development Co. Ltd. (Qingdao, China) was used as the substrate to test alginate lyase activity. The viscosity of this sodium alginate sample was 1250 mPa.s. The sodium alginate used to prepare medium was from Solarbio (Beijing, China). Figure 14. Viscosity variation during enzymatic degradation of alginate. The viscosity was measured in a 100 mL reaction system with 1% (w/v) sodium alginate, 50 mmol/L Tris-HCl buffer (pH 8.0) and RT. The data represents the mean of three repeats with SD ≤ 5%.

Bacteria and Substrate
The strain of Vibrio sp. Ni1 was isolated from the sludge of the Minjiang river estuary, located in Fujian province, China. This strain was kept in our lab and China Center for Type Culture Collection (CCTCC No. M2018916). E. coli BL21(DE3) was used as an expression host. The sodium alginate donated by Haizhilin Biotechnology Development Co. Ltd. (Qingdao, China) was used as the substrate to test alginate lyase activity. The viscosity of this sodium alginate sample was 1250 mPa.s. The sodium alginate used to prepare medium was from Solarbio (Beijing, China).

Identification of Alginate Lyase Fragments
The crude alginate lyase was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The 10% acrylamide gel was stained with Coomassie blue and detained with 30% acetonitrile and 0.1 mol/L NH 4 HCO 3 . The gel slice was dried in a vacuum centrifuge. The proteins were reduced in-gel with dithiothreitol (10 mmol/L DTT and 100 mmol/L NH 4 HCO 3 ) for 30 min at 56 • C, then alkylated with iodoacetamide (20 mmol/L IAA and 100 mmol/L NH 4 HCO 3 ) in the dark at 25 • C for 30 min. The gel slice was briefly rinsed with 100 mmol/L NH 4 HCO 3 and acetonitrile, followed by digestion with 12.5 ng/µL trypsin in 25 mmol/L NH 4 HCO 3 overnight. The digested peptides were extracted three times with 60% (v/v) acetonitrile and 0.1% (v/v) trifluoroacetic acid. The extracts were pooled and dried completely in a vacuum centrifuge. The peptide mass and sequence were determined by Liquid Chromatography (LC)-Electrospray Ionization (ESI) Tandem mass spectrometry (MS/MS) in a Q Extractive mass spectrometer which was coupled to Easy nLC (Proxeon Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). The MS data were analyzed using Max Quant (Version 1.6.4.0) by searching the data against the amino acid sequence of alginate lyases from Vibrio, and the intensity of sequenced peptide in the target protein was calculated.

Cloning and Sequence Analysis of the Alginate Lyase Gene
Based on the LC-MS/MS results, a pair of degenerate primers (algB-F0/R0) was designed to amplify fragments of the algB gene. According to the sequencing result of the partial algB gene, a series of primers were designed to apply the SiteFinding-PCR method to extend the target gene [34,35]. The details of primers used in this experiment are listed in Table 4. Chromosome Walking Kit and Pfu DNA polymerase (TaKaRa, Osaka, Japan) were used in this study. Finally, a pair of primers named algB-QF/R was designed to amplify the full-length gene. The expression recombinant plasmid was constructed with pET32a on sites of BamH I and Xhol I and transformed into E. coli BL21(DE3).

Purification of the Alginate Lyase
The E. coli cells harboring the recombinant plasmid pET32a-algB were cultured at 37 • C in liquid LB medium with 100 µg/mL ampicillin until OD 600 reached about 0.6. Gene expression was induced with the addition of isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.8 mmol/L and then grown at 20 • C for 24 h. The cells were harvested by centrifugation 8000 r/min, 5 min, at 4 • C, then suspended in 0.05 mol/L Tris-HCl buffer (pH 7.4) to concentrate 1/10 volume. The cells were disrupted by ultrasonication. The lysate was harvested by centrifugation 10,000× g, 30 min, at 4 • C. The supernatant was filtered with 0.22 micrometer cellulose acetate membrane before passage through a Ni-NTA Sepharose column (GE, Boston, MA, USA). The column was washed with washing buffer (0.05 mol/L Tris-HCl buffer (pH 7.5), 0.5 mol/L NaCl, 20 mmol/L imidazole), and the recombinant alginate lyase AlgB was eluted with elution buffer (0.05 mol/L Tris-HCl buffer (pH 7.5), 0.5 mol/L NaCl, 0.3 mol/L imidazole). The eluate was dialyzed in 0.05 mol/L Tris-HCl buffer (pH 7.5) overnight and analyzed by 12% SDS-PAGE. The protein concentration was measured by the Bradford method.

Assay of the Recombinant Alginate Lyase Activity
The activity of the alginate lyase AlgB was measured in a mixture solution containing 1% (w/v) sodium alginate dissolved in 0.05 mol/L Tris-HCl buffer (pH 8.0) (0.9 mL) and enzyme solution (0.1 mL) at 40 • C for 30 min. Enzyme activity was calculated by constructing a standard curve with uronic acid under the same reaction conditions with DNS solution [36]. One unit (U) activity was defined as the amount of enzyme that generated 1 µg of uronic acid per min under standard assay conditions. In order to distinguish reaction products, the enzyme activity was also determined as the increase in the absorbance 235 nm to measure the amount of unsaturated uronic acid [15].

Characterization of the Recombinant Alginate Lyase Activity
The activity of AlgB at a specific pH range (3-10) and temperature range (4-90 • C) was determined by the enzyme assay described above using 1% alginate as substrate. The thermostability was evaluated by measuring the residual activity after different treatment time at different temperatures. Kinetic parameters of the enzyme towards alginate were calculated by the substrate at different concentrations (1-5 mg/mL). The enzyme activity was measured as described previously.
The effect of metal ions on the activity of AlgB was investigated. The reaction mixtures were added with different metal ions (KCl, MgSO 4 , ZnSO 4 , FeSO 4 , CuSO 4 , MnSO 4 , CaCl 2 , CoCl 2 and NiCl 2 ) to a final concentration of 10 mmol/L. The parallel reaction without metal ions served as the control. The activity on different concentrations of NaCl (1-5 mol/L) was also measured.

Substrate Specificity of the Recombinant Alginate Lyase Activity
The purified AlgB was added to the reaction mixture containing 1 mg/mL alginate, polyG and polyM, respectively. The average molecular weights of both polyG and polyM (Shanghai Yuanye Bio-Technology Co. Ltd., Shanghai, China) were 6-8 kDa.

Analysis of Reaction Mode and Products
The mixture of enzymatic products was purified by centrifugation (10,000 r/min, 30 min, 4 • C) after react 12 h at optimal conditions. The degraded products of AlgB were analyzed by thin-layer chromatography (TLC) (TLC Silica gel 60 F254, Merck KGaA, Darmstadt, Germany) [37]. The enzymatic products on silica gel plate were separated with a solvent solution of n-butyl alcohol/acetic acid/H 2 O (3:2:3) and visualized by heating 80 • C for 15 min after spraying a diphenylamine/aniline/phosphate (1:1:5) reagent. To further investigate the enzymatic products, ESI-QqQ-MS/MS was conducted. Five microliter samples were injected into a triple quadrupole tandem mass spectrometry (QqQ-MS/MS) (Aligent Technologies Inc., Palo Alto, CA, USA) after filtration with 0.22 µm cellulose acetate membrane. The products were detected in a negative ion mode using the following settings: pressure of the atomizer (N 2 ), 50 psi; flow rate of the dryer (N 2 ), 10 L/min; temperature of the dryer (N 2 ), 350 • C; capillary voltage, 4000 V; scanning the mass range, 100-1000 m/z [25]. The viscosity of substrate was intermittently determined by viscometer (Shanghai Hengping No. NDJ-1, Shanghai, China) during reaction.

Conclusions
In this study, a novel alginate lyase of AlgB with high activity was obtained from Vibrio sp. Ni1. The purified recombinant enzyme of AlgB showed highest activity at 35 • C, pH 8.0, 50 mmol/L Tris-HCl without metal ions. The AlgB preferred polyM as substrate. The predominant end products of the enzymatic reaction are DP2 and DP3.

Data Availability Statement:
The data presented in this study are available on reasonable request from the corresponding author.