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Mar Drugs
2023 Oct 27;2111:. doi: 10.3390/md21110564.
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Genome Sequencing-Based Mining and Characterization of a Novel Alginate Lyase from Vibrio alginolyticus S10 for Specific Production of Disaccharides.
Shu Z
,
Wang G
,
Liu F
,
Xu Y
,
Sun J
,
Hu Y
,
Dong H
,
Zhang J
.
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Alginate oligosaccharides prepared by alginate lyases attracted great attention because of their desirable biological activities. However, the hydrolysis products are always a mixture of oligosaccharides with different degrees of polymerization, which increases the production cost because of the following purification procedures. In this study, an alginate lyase, Alg4755, with high product specificity was identified, heterologously expressed, and characterized from Vibrio alginolyticus S10, which was isolated from the intestine of sea cucumber. Alg4755 belonged to the PL7 family with two catalytic domains, which was composed of 583 amino acids. Enzymatic characterization results show that the optimal reaction temperature and pH of Alg4755 were 35 °C and 8.0, respectively. Furthermore, Alg4755 was identified to have high thermal and pH stability. Moreover, the final hydrolysis products of sodium alginate catalyzed by Alg4755 were mainly alginate disaccharides with a small amount of alginate trisaccharides. The results demonstrate that alginate lyase Alg4755 could have a broad application prospect because of its high product specificity and desirable catalytic properties.
Figure 1. Screening and identification of strain S10. (A) Transparent circle of solid culture medium (S1, S2, S10, and S11 are four strains that can degrade sodium alginate screened from the intestinal microorganisms of sea cucumber). (B) SEM results of strain S10. (C) The neighbor-joining phylogenetic tree of strain S10 based on the 16S rRNA gene sequences. The bootstrap values of each branch were tested by 1000 repetitions.
Figure 2. Sequence analysis of Alg4755. (A) Schematic diagram of the structural domains of alginate lyase Alg4755. The signal peptide (SP) was predicted using the SignalP 5.0 Server, while the conserved domains were analyzed using the Conserved Domain Database. (B) Phylogenetic analysis of Alg4755 with other alginate lyases from the PL7 family using MEGA 11. The bootstrap values of each branch were tested by 1000 repetitions. Alg4755 is marked with a dot. The characterized alginate lyases are marked with triangles. Structure-solved alginate lyases are marked with a five-pointed star. (C) Multiple sequence alignment of Alg4755 with related alginate lyases (MBY7662542.1, alginate lyase from V. atlanticus; UUW33102.1, alginate lyase from Vibrio sp.; AIY22661.1, alginate lyase from Vibrio sp. W13; WP_118120558.1, and alginate lyase from Vibrio sp. dhg). The conserved sequences of the PL7 family catalytic domains are marked with black boxes (N-terminal, Domain1) and green boxes (C-terminal, Domain2). Black triangles indicate neutralizing residues. Black stars indicate catalytic acids/bases. Amino acids below the black circles indicate residues associated with catalytic substrates.
Figure 3. SDS-PAGE analysis of Alg4755. Lane M, molecular weight marker (Solarbio, Beijing, China); Lane 1, uninduced cell lysate; Lane 2, induced cell lysate; Lane 3, supernatant of induced cell lysate; Lane 4, pellet of induced cell lysate; and Lane 5, purified Alg4755 from the pellet of induced cell lysate.
Figure 4. Effect of temperature on Alg4755 activity. (A) The optimal temperature of Alg4755. Reactions were conducted in 50 mM Tris–HCl buffer (pH 8.0) at different temperatures for 30 min. (B) Thermal stability of Alg4755. The residual activity of Alg4755 was measured after preincubation at different temperatures for 2 h in 50 mM Tris-HCl buffer (pH 8.0). The highest activity was set to 100%. Each value represents the mean of three replicates ± standard deviations.
Figure 5. Effect of pH on the activity of Alg4755. (A) The optimal pH of Alg4755. The reactions were carried out at 35 °C for 30 min in different buffers of pH 6 to 10.5. (B) The pH stability of Alg4755. After incubation of Alg4755 in buffers of different pH (6–10.5) for 2 h at 35 °C, residual enzyme activity was measured. The highest activity was set to 100%. Each value represents the mean of three replicates ± standard deviations.
Figure 6. (A) Effect of different metal ions on the activity of Alg4755. (B) Effect of different concentrations of inhibitors and detergents on the activity of Alg4755. The enzymatic activity without other reagents served as the control, and the enzymatic activity was designated as 100%. (C) Substrate specificity of Alg4755. The activity towards sodium alginate was determined as the 100% relative activity. Each value represents the mean of three replicates ± standard deviations.
Figure 7. HPLC analysis of 0–24 h products of Alg4755 with (A) sodium alginate, (B) polyM, and (C) polyG. The degradation products were analyzed by monitoring at 210 nm using a UV detector. Saturated mannuronate oligosaccharides from DP2 to DP6 were taken as the standards. Analysis of the 24 h product composition of Alg4755 by ESI-MS with (D) sodium alginate, (E) polyM, and (F) polyG as substrates.
Figure 8. Three-dimensional structure analysis of Alg4755. (A) Predicted three-dimensional structure of Alg4755 using AlphaFold2. The predicted Alg4755 has two structural domains. The red region (left) is at the N-terminal (residues 57 to 286) and the green region (right) is at the C-terminal (residues 295 to 582). The cyan area is an octapeptide linker. (B) Electrostatic surface analysis of the entire structure of Alg4755. The residues in both structural domains that potentially play a key role in the catalytic process are located within the positively charged gaps, marked by yellow circles. The blue color in the diagram indicates a positive charge and the red color indicates a negative point.
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