Screening of Strain HB226069
The screening results by agar plate method showed that strain HB226069 from Sargassum sp. exhibited significant alginate lyase and fucoidanase activities. Under the role of 1 M CaCl2, a white ring produced by gelation reaction was observed on the plate, indicating that alginate lyase was secreted. Under the action of 0.5% CTAB, a transparent halo of gelation reaction was observed, meaning that fucoidanase was secreted (Fig. S1).
Genome Specifics
The complete genome of strain HB226069 was determined and one circular chromosome was obtained, with the NCBI GenBank accession number CP130317. A total of 33,734 clean reads were analyzed, with an average read length of 7997 bp, possessing a total of 269.8 Mb and the coverage depth of 67×. Strain HB226069 presents a genome of 4,021,337 bp with chromosomal G + C content of 56.5%. A total of 3482 genes were predicted, including 3393 protein-coding genes, 50 tRNA and 9 rRNA sequences. The general features of HB226069 genome are shown in Fig. 1 and Table S1.
The gene functions were classified with eggNOG and KEEG databases, and it was shown that 1800 proteins had KEGG homologous genes and 2946 proteins had eggNOG classification. The most common genes in eggNOG annotation were related to the general functions of bacterial cells (7.7%). In addition, the higher proportion (> 5%) included genes related to amino acid transport and metabolism (6.6%), cell wall/membrane/envelope biogenesis (6.1%), energy production and conversion (5.9%), replication, recombination and repair (5.8%), transcription (5.6%), and carbohydrate transport and metabolism (5.1%). Bioinformatics analysis by antiSMASH predicted four secondary metabolite gene clusters in strain HB226069, including RiPP-like, ectoine, NRP-metallophore, and non-ribosomal peptide synthase (NPRS).
Identification of Strain HB226069
A nearly complete 16S rRNA gene sequence (1385 bp) by PCR amplification of strain HB226069 was obtained, and complete sequence (1535 bp) from genome with GenBank No. CP130317. Phylogenetic analysis of the complete 16S rRNA gene sequences indicated that strain HB226069 belonged to the genus Microbulbifer. The closest phylogenetically related species was M. thermotolerans JAMB A94T (100% similarity), and the other 16S rRNA gene sequence similarities were less than 97.0%, which was consistent with the result of 16S rRNA gene obtained by amplification. The phylogenetic tree reconstructed by the neighbour-joining method showed that the strain clustered together with M. thermotolerans JAMB A94T and M. thermotolerans DAU221 (Fig. 2), significantly supported by the high bootstrap value of 100%. Phylogenetic analysis indicated that strain HB226069 had the closest phylogenetic affinity with the type strain of M. thermotolerans.
At the genomic level, strain HB226069 exhibited ANI value of 99.3% and DDH value (the recommended results from formula 2) of 93.9% with the closest relative of M. thermotolerans JAMB A94T (from GenBank No. FOKT00000000.1), which were higher than the threshold values of the species boundary (ANI 94–96% and DDH 70%) [Meier-Kolthoff et al. 2013; Yoon et al. 2017]. Hence clearly, the results of the phylogenetic and genotypic analysis clearly indicate that the isolate HB226069 should be classified as Microbulbifer thermotolerans.
Genetic Basis of Polysaccharide Degradation
To clarify the genes related to carbohydrate-active enzyme in the genome, the relevant proteins were predicted on the basis of the CAZyme database. M. thermotolerans HB226069 has 161 CAZymes, including 67 glycoside hydrolases (GHs), 23 glycosyl transferases (GTs), 11 carbohydrate esterases (CEs), 9 auxiliary activities (AAs), 7 polysaccharide lyases (PLs) and 40 carbohydrate-binding modules (CBMs). The capability of degrading various polysaccharides was evidently proved according to the annotation of its genome. Many polysaccharide-degrading enzymes were predicted, such as alginate lyase, pectate lyase, fucosidase, agarase, xylanase, cellulase, amylase and chitinase (Table 1). All of the enzymes belong to GHs and PLs, 24 of them contain carbohydrate-binding modules (CBM) defined as contiguous amino acid sequences having carbohydrate-binding activity. The PLs are classified into five families: 1, 6, 7, 17 and 31, which are predicted as alginate lyases and pectate lyases. Bacteria depolymerize polysaccharides by a series of lyases or glycosidases and produce oligosaccharides or monosaccharides that can be utilized by cells. These masses of carbohydrate-active proteins comprise a complex system for carbohydrate catabolism in M. thermotolerans HB226069.
Table 1
Diverse genes related to polysaccharide degradation identified in the genome of M. thermotolerans HB226069
Catabolic Enzymes | Enzyme Family | No. of Enzymes |
Alginate lyase | CBM16|CBM16|CBM32|PL7 | 1 |
CBM16|CBM32|PL7 | 1 |
PL17 | 1 |
Pectate lyase | PL1 | 3 |
Chondroitinase | PL6 | 2 |
Chitinase | CMB5|GH18 | 1 |
CMB73|GH18 | 3 |
β-Agarase | GH16|CBM6|CBM6 | 1 |
GH16|CBM6 | 1 |
α-l-fucosidase | GH95 | 1 |
α-Amylase | GH13|CBM20 | 1 |
GH13 | 7 |
Cellulase | CMB2|GH6 | 1 |
CMB2|GH5 | 1 |
Endo-1,3(4)-β-glucanase | GH81|CBM6 | 1 |
α-Glucuronidase | GH67 | 1 |
β-Xylosidase | GH43|CBM91 | 1 |
β-Hexosaminidase | GH20 | 1 |
α-Glucuronidase | GH67 | 1 |
α-1,6-Glucosidase | GH13 | 1 |
Peptidoglycan β-N-acetylmuramidase | GH171 | 1 |
Further analysis based on the dbCAN database identified three putative PLs, namely GE000077 (PL7, accession number: WP 302736807.1), GE000078 (PL7, accession number: WP 302736809.1), and GE000084 (PL17, accession number: WP 266073123.1), which were associated with alginate degradation. The coding sequences (CDS) of the ge000077, ge000078, and ge000084 genes are composed of 2253, 1794 and 2262 bp; encode 750, 597 and 753 amino acids, respectively. The predicted molecular weights of the three alginate lyases are 80.2, 63.1 and 83.6 kDa, belonging to the PL7, PL7 and PL17 families, respectively (Table S2).
To confirm the attribution of the alginate lyases, a phylogenetic tree was constructed based on the amino acid sequences of the three alginate lyases and other related enzymes. As shown in Fig. 3, the phylogenetic analysis clearly indicates that GE000077 and GE000078 fall within the PL7 family branch, while GE000084 is situated within the PL17 family branch. These results are consistent with the predicted classifications from the CAZy database.
In addition, the results of dbCAN revealed that one α-l-fucosidase belonging to GH95 family and two chondroitinase B belonging to PL6 family were predicted. Fucosidase in GH95 family is an exo-type fucoidan-degrading enzyme that hydrolyzes from the non-reducing end of polysaccharides α-l-fucoside bond and release fucose. Microorganisms can produce a range of α-l-fucosidases of diverse substrate specificity cleaving the nonreducing terminal α-l-fucose from these glycoconjugates [Wu et al. 2023]. Three exo-type fucoidases were isolated from the marine bacterium Vibrio sp. N-5, and the main enzymatic transformation of fucoidan were sulfated fucose and sulfated fucobioses [Furukawa et al. 1992]. Chondroitinase B has the ability to cleave glycosaminoglycan and can enhance the antioxidant activity of chondroitin sulfate B [Kevin et al. 2002].
Four chitinases belonging to the GH18 family were predicted, each with additional carbohydrate-binding modules CBM5 or CBM73. Chitin, a critical component of extracellular matrices such as fungal cell walls and insect exoskeletons, can be efficiently degraded by chitinases. This ability has important applications in agriculture, as it can control chitin-containing pests, fungal pathogens, and nematodes [Chen et al. 2023]. Two cellulases were also predicted, both of which possessed additional carbohydrate-binding modules CBM2.
Furthermore, two β-agarases were predicted in strain HB226069. The enzyme cleaves β-(1,4) linkages of agarose to produce the neo-agarotetraose and neo-agarohexaose at the end of hydrolysis [Kikuchi et al. 2020]. Strain HB226069 is also predicted to possess other CAZymes such as endo-1,3(4)-β-glucanase, α-glucuronidase, β-xylosidase, β-hexosaminidase, α-1,6-glucosidase, and peptidoglycan β-N-acetylmuramidase, et al. The presence of complex carbohydrate catabolic systems enables strain HB226069 to effectively degrade and utilize various carbohydrates present in its surrounding.
Assay of Carbohydrate Utilization
After incubating at 37°C and 180 rpm for 48 h, the capacity of strain HB226069 to utilize different carbohydrates was evaluated by measuring the optical density OD600 of the fermentation broth. Cell growth indicates that the tested carbohydrates can be used as carbon sources by the strain. As shown in Table S3, the fermentation broth had the higher OD600 values (1.462 and 1.253, respectively) when pectin and sodium alginate were used as the sole carbon sources, suggesting that strain HB226069 has strong capacity to utilize pectin and sodium alginate. Furthermore, the strain demonstrated the ability to utilize various other carbohydrates for its growth, including alginate, fucoidan, agar, starch, cellulose, chitin, xylan, pectin, sucrose, d-glucose; except for mannitol and xylitol. The extensive utilization of carbohydrates indicates that the strain can effectively obtain energy and nutrients from various carbohydrates, enhancing its versatility and adaptability in different environments.
Effects of Temperature and pH on Alginate Lyase Activity and Stability
The extracellular alginate lyase was prepared by using ammonium sulfate saturated solution (80%) to precipitate the culture supernatant. The specific activity was increased from the initial 7.5 U/mL to 117.4 U/mL, with a yield of 15.7 times. In order to identify the optimal temperature and pH, the alginate lyase activity of strain HB226069 was determined under different temperatures and pH values by ultraviolet absorption method. As shown in the Fig. 4a, the optimal temperature and thermostability of alginate lyase were determined within the temperature range of 20–90°C. The alginate lyase of strain HB226069 exhibited the maximum activity at 50°C. Above 60% of the maximum activity was maintained at the range of 20–55°C, meanwhile, the enzymatic activity still kept above 40% at the range of 60–90°C. The alginate lyase remained relatively stable at temperatures lower than 30°C; approximately 100% of the activity was maintained after incubation at less than 30°C for 1 h. As the temperature rose above 30°C, the activity declined dramatically; meanwhile, the enzymatic activity still kept above 40% at 90°C. The results indicate that the alginate lyase of strain HB226069 is a hot-adapted enzyme, exhibiting good activity under high temperature.
As shown in Fig. 4b, the alginate lyase had the best activity at pH 7.0, and above 60% of the maximum activity when the pH value was between 5.5 and 7.5. The activity was the most stable at pH 7.0, above 80% of the activity was retained at pH 5.0–7.0 and about 60% of the activity at pH 4.5 and pH 7.5. The results indicate that the alginate lyases have good pH stability.
Effects of Ions on Alginate Lyase Activity
As shown in Fig. 4c, the results indicated that 0.05 M Fe3+ and Cu2+ had significant promoting impact on the enzyme activity, with activity increased by 2.5 and 2.1 times compared to the absence of ions, respectively. However, at 0.05 M, NH4+, K+ and Na+ had no effect on enzyme activity; Ca2+ displayed a slightly promoted effect on the enzyme activity; Ni2+ and Zn2+ showed some inhibitory effects on enzyme activity. As shown in Fig. 4d, the enzyme activity was significantly increased when 0.01–0.06 M FeCl3 was added. The maximum enzyme activity reached 117.4 U/mL at 0.05 M, 2.5 times higher than that without FeCl3.
Analysis of Degradation Products
The sodium alginate oligosaccharides were analyzed by TLC and ESI-MS. As shown in Fig. 5a, monosaccharides and oligosaccharides with different degrees of polymerization (DP) were produced in the process of sodium alginate degradation. Oligosaccharides of DP1–DP6 were displayed while reacting for 12 h. As the reaction time prolonged, the content of DP5 and DP6 gradually decreased, and DP1–DP5 were shown while reacting for 36 h. The alginate products degraded for 36 h were also analyzed by ESI-MS in negative ion mode (Fig. 5b). The main products of the enzymatic hydrolysates were clearly distributed in monosaccharides ([ΔDP1–H]– =175.02), disaccharides ([ΔDP2–H]– =351.05), trisaccharides ([ΔDP3–H]– =527.32), tetrasaccharide ([ΔDP4–H2O–H]– =685.44), and pentasaccharide ([ΔDP5–H]–=877.24, not shown). The results showed that exolytic and endolytic activity existed in the partially purified alginate lyases.