Genome Analysis of a Potential Novel Vibrio Species Secreting pH- and Thermo-Stable Alginate Lyase and Its Application in Producing Alginate Oligosaccharides

Abstract

Alginate lyase is an attractive biocatalyst that can specifically degrade alginate to produce oligosaccharides, showing great potential for industrial and medicinal applications. Herein, an alginate-degrading strain HB236076 was isolated from Sargassum sp. in Qionghai, Hainan, China. The low 16S rRNA gene sequence identity (<98.4%), ANI value (<71.9%), and dDDH value (<23.9%) clearly indicated that the isolate represented a potential novel species of the genus Vibrio. The genome contained two chromosomes with lengths of 3,007,948 bp and 874,895 bp, respectively, totaling 3,882,843 bp with a G+C content of 46.5%. Among 3482 genes, 3332 protein-coding genes, 116 tRNA, and 34 rRNA sequences were predicted. Analysis of the amino acid sequences showed that the strain encoded 73 carbohydrate-active enzymes (CAZymes), predicting seven PL7 (Alg1-7) and two PL17 family (Alg8, 9) alginate lyases. The extracellular alginate lyase from strain HB236076 showed the maximum activity at 50 °C and pH 7.0, with over 90% activity measured in the range of 30-60 °C and pH 6.0-10.0, exhibiting a wide range of temperature and pH activities. The enzyme also remained at more than 90% of the original activity at a wide pH range (3.0-9.0) and temperature below 50 °C for more than 2 h, demonstrating significant thermal and pH stabilities. Fe²⁺ had a good promoting effect on the alginate lyase activity at 10 mM, increasing by 3.5 times. Thin layer chromatography (TLC) and electrospray ionization mass spectrometry (ESI-MS) analyses suggested that alginate lyase in fermentation broth could catalyze sodium alginate to produce disaccharides and trisaccharides, which showed antimicrobial activity against Shigella dysenteriae, Aeromonas hydrophila, Staphylococcus aureus, Streptococcus agalactiae, and Escherichia coli. This research provided extended insights into the production mechanism of alginate lyase from Vibrio sp. HB236076, which was beneficial for further application in the preparation of pH-stable and thermo-stable alginate lyase and alginate oligosaccharides.

Keywords: Vibrio sp. HB236076; alginate lyase; antimicrobial activity; genome; oligosaccharide.

Figure 1

Circos maps of the two chromosome genomes of strain HB236076. (a) Chromosome 1; (b) chromosome 2. The maps were divided into 7 circles from outside to inside, namely, markers of genome size (5 kb per scale), genes on the positive strand, genes on the negative strand, repetitive sequences, genes of tRNA (blue) and rRNA (purple), GC content (buff: above mean; blue: below mean), and GC-skew (dark gray: G higher than the C; red: G lower than the C).

Figure 2

Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences between strain HB236076 and related species of the genus Vibrio by comparison of 1478 nucleotides. Bootstrap values (1000 replicates) are shown as percentages at each node for values; only values > 50% are shown. Grimontia hollisae ATCC 33564^T was used as an outgroup. The scale bar represents 0.01 nucleotide substitutions per position.

Figure 3

(a) Domain architectures of the nine alginate lyases of strain HB236076. (b) Neighbor-joining phylogenetic tree of alginate lyases based on the predicted amino acid sequences. Bootstrap values (1000 replicates) are shown as percentages at each node for values. The scale bar represents 0.1 nucleotide substitutions per position. Putative alginate lyases of strain HB236076 are highlighted in bold. Poly (beta-D-mannuronate) lyase (ABG42142.1) from the PL6 family was used as an outgroup.

Figure 4

Multiple sequence alignments of Alg1–7 and nine well-characterized alginate lyases of the PL7 family. The conserved amino acid regions are marked in the black boxes.

Figure 5

The biochemical characteristics of the extracellular alginate lyases produced by strain HB236076. (a) Effect of different temperatures on the alginate lyase activity assayed at 4–80 °C. (b) Thermal stability analysis of alginate lyase assayed at 4–80 °C for 2 h. (c) Effect of different pH values on the alginate lyase activity assayed at pH 3–10. (d) pH stability analysis of alginate lyase assayed at pH 3–10 for 24 h. (e) Effects of metal ions, EDTA, and SDS on the alginate lyase activity. * p < 0.05, *** p < 0.001. The highest activity was taken as 100% in Figure 5a–d, and the initial activity without additional substance was taken as 100% in Figure 5f. Data are shown as the means ± standard deviation, n = 3.

Figure 6

Degradation product analysis of alginate performed by alginate lyase from strain HB236076. (a) Degradation product analysis with TLC. Lane M, the guluronic acid sodium salt monomers, dimers, and trimers. Lane 3, 6, 24, and 36, the degradation products performed for 3, 6, 24, and 36 h, respectively. (b) Degradation product analysis with ESI-MS. The DP2 and DP3 peaks represent disaccharide and trisaccharide, respectively.