Agarolytic Pathway in the Newly Isolated Aquimarina sp. Bacterial Strain ERC-38 and Characterization of a Putative β-agarase

Marine microbes, particularly Bacteroidetes, are a rich source of enzymes that can degrade diverse marine polysaccharides. Aquimarina sp. ERC-38, which belongs to the Bacteroidetes phylum, was isolated from seawater in South Korea. It showed agar-degrading activity and required an additional carbon source for growth on marine broth 2216. Here, the genome of the strain was sequenced to understand its agar degradation mechanism, and 3615 protein-coding sequences were predicted, which were assigned putative functions according to their annotated functional feature categories. In silico genome analysis revealed that the ERC-38 strain has several carrageenan-degrading enzymes but could not degrade carrageenan because it lacked genes encoding κ-carrageenanase and S1_19A type sulfatase. Moreover, the strain possesses multiple genes predicted to encode enzymes involved in agarose degradation, which are located in a polysaccharide utilization locus. Among the enzymes, Aq1840, which is closest to ZgAgaC within the glycoside hydrolase 16 family, was characterized using a recombinant enzyme expressed in Escherichia coli BL21 (DE3) cells. An enzyme assay revealed that recombinant Aq1840 mainly converts agarose to NA4. Moreover, recombinant Aq1840 could weakly hydrolyze A5 into A3 and NA2. These results showed that Aq1840 is involved in at least the initial agar degradation step prior to the metabolic pathway that uses agarose as a carbon source for growth of the strain. Thus, this enzyme can be applied to development and manufacturing industry for prebiotic and antioxidant food additive. Furthermore, our genome sequence analysis revealed that the strain is a potential resource for research on marine polysaccharide degradation mechanisms and carbon cycling. Supplementary Information The online version contains supplementary material available at 10.1007/s10126-023-10206-7.

Among them, agar comprises a well-known but complex cell wall polysaccharide family from marine red algae that in general can roughly be divided into the neutral "agarose" fraction and the charged "agaropectin." Agarose is a linear polysaccharide mainly composed of basic alternating units of 3-O-linked-β-D-galactose and 4-O-linked 3,6-anhydro-α-L-galactose, and agaropectin comprises a similar galactosebased backbone but heterogeneously contains acidic side groups, such as sulfate and pyruvate.
Numerous marine microbes have enzymes that degrade polysaccharides, including agar (Arnosti 2011). Bacteroidetes, which is a very diverse bacterial phylum, generally have a greater enzymatic potential for carbohydrate degradation than other bacterial phyla. These carbohydrateactive enzymes often cluster in polysaccharide utilization loci (PULs), which by definition have capacities for sensing, degradation, transport, and metabolism of a given polysaccharide (Lapébie et al. 2019;Grondin et al. 2017). In bacterial cells, polysaccharide-degrading enzymes primarily exist in the periplasmic space and catalyze the breakdown of polysaccharides in the periplasm that are imported by transporters encoded by the susC/D gene pair. The oligomers generated by polysaccharide breakdown can be subsequently degraded by sugar-cleaving enzymes (Lapébie et al. 2019).
Here, we report the complete genome sequence of a marine bacterium, ERC-38, that exhibited a concave surface around colonies on an agar medium. The 16S rRNA gene sequence of the ERC-38 strain indicated that it belongs to the genus Aquimarina and is a potential novel species. To identify possible agarolytic pathways in the ERC-38 strain, the molecular and enzymatic characteristics of its agarolytic enzymes were investigated. This study provides additional information about algal biomass decomposition and recycling by algal polysaccharide-degrading bacteria. Aq1840 agarase reported in this study also has potential for the production of bioactive algal oligosaccharides.

Bacterial Strains and Culture Conditions
A seawater sample collected from the Yellow Sea coast (Buan-gun), South Korea, was diluted with sterilized seawater and spread onto marine broth 2216 (Difco) containing 2% agar (MA) plates. The strain was routinely cultured on MA plates and maintained at − 80 °C with 20% (v/v) glycerol. To examine carbon source utilization, the strain was pregrown in marine broth containing 0.5% glucose, inoculated into marine medium containing 0.1-1% glucose, galactose, agarose (low melting agarose, Duchefa), κ-carrageenan (Sigma) or ι-carrageenan (Sigma), and incubated at 25 °C for 3 days. To determine polysaccharide degradation ability using a plate assay, the pre-grown cells were inoculated on a plate containing marine broth diluted 10-time supplemented with 3.6% sea salt (Sigma), and 2% agar (Bacto™ Agar, Difco), κ-carrageenan or ι-carrageenan as the carbon source and incubated at 25 °C for 4 days. The degradation ability was determined by colony growth and iodine staining.

Phylogenetic Analysis and Whole-Genome Sequencing
The 16S rRNA gene was amplified from purified genomic DNA using universal primers (27F and 1492R). The pairwise gene similarity of 16S rRNA was determined using the EzBioCloud database (https:// www. ezbio cloud. net/, (Yoon et al. 2017)) and the BLAST program. Phylogenetic tree was conducted using MEGA X after performing multiple alignments of the sequences and constructed with neighbor-joining (NJ) method (Kumar et al. 2018;Saitou and Nei 1987).
For whole-genome sequencing of the ERC-38 strain, genomic DNA was extracted from cells using a genomic DNA purification kit (Invitrogen), according to the method provided by the manufacturer. Sequencing and assembly method is presented in Table 1. Circular genome was drawn using the CGView program (Grant and Stothard 2008). DNA-DNA hybridization (DDH) and the average amino acid identities (AAIs) were calculated using a method described in previous studies (Kang et al. 2022) and the color-coded heatmap was generated using CIMminer software (http:// disco ver. nci. nih. gov/ cimmi ner).

Sequence Analysis of the Agar-degrading Enzyme Genes
Genes involved in polysaccharide metabolism were identified based on annotations in the Swiss-Prot and Carbohydrate-Active Enzyme (CAZy) databases. The molecular weight and predicted structure of the proteins were estimated using the pI/ Mw or modeling tools on the ExPASy server of the Swiss Institute of Bioinformatics (Gasteiger et al. 2005). The presence of a signal sequence was predicted using the SignalP 5.0 server (Almagro Armenteros et al. 2019), and the conserved domain was detected using the NCBI's conserved domain database (CDD). The conserved domain was reconfirmed by comparison with characterized GH16 family members in the CAZy database. Multiple alignment of the amino acid sequences of agarases was constructed using the Clustal_W algorithm in MEGA X as described above.

Cloning and Expression of the β-Agarase Gene
The mature sequences of agarase genes were amplified from the genomic DNA of the ERC-38 strain using primer sets (Table S1). Each of the amplified fragments were inserted into the pET21a vector using the AccuRapid Cloning kit (Bioneer Inc.) to generate expression vector, pET21a-Aq1828, -Aq1829, -Aq1830, -Aq1832, -Aq1839, or -Aq1840. After confirmation of the accuracy of the sequences, each expression vector was transformed into Escherichia coli BL21 (DE3) codon + competent cells.

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The transformed E. coli BL21 (DE3) codon + cells were cultured in LB medium containing ampicillin (100 μg/mL) (LA). To induce gene expression, the transformed cells were pre-grown at 37 °C for 14 h and transferred to new LA medium containing 0.5 mM IPTG, and incubated for 6 h at 37 °C. Crude enzymes were obtained from the gene induced cells by disruption using sonication under a resuspension buffer (40-mM Tris-HCl buffer (pH 8.0), supplemented with 50 mM NaCl and 1 mM dithiothreitol (DTT)). Protein expression was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the gels were stained with Coomassie Brilliant Blue R-250 (Bio-Rad).

Purification of Recombinant Aq1840 β-Agarase
To purify the recombinant enzyme, the crude enzyme solution was applied to a HisTrap HP column (GE Healthcare) using an FPLC system (ÄKTA Pure 25, GE Healthcare). The enzyme sample was washed with three volumes of washing buffer (40 mM Tris-HCl, 500 mM NaCl, and 20 mM imidazole, pH 8.0) and eluted with an elution buffer (40 mM Tris-HCl, 500 mM NaCl, and 500 mM imidazole, pH 8.0). The eluted enzyme was concentrated using an Amicon Ultra-15 Centrifugal Filter Unit with an Ultracel-3 membrane (EMD Millipore) for desalting and resuspended in a 40-mM Tris-HCl buffer (pH 8.0) supplemented with 1 mM DTT. The enzyme solution was used to measure agarase activity.

Enzyme Activity Assay and Biochemical Characterization
For the enzymatic reaction, 100 μL of the purified enzyme solution was mixed with an equal volume of 1.5% (w/v) agarose dissolved in a 40-mM Tris-HCl buffer containing 1 mM DTT (pH 8.0). Temperature tests were performed with the mixtures in a temperature range of 30-50 °C (5 °C intervals) after incubation for 3 h. pH tests were performed in a range of 5.5-9.0 (0.5 unit intervals) using a McIlvaine buffer (pH 5.5-7) or a Tris-HCl buffer (pH 7.5-9.0). Thermostability of the enzyme was investigated by pre-incubating the enzyme at 30-60 °C for 3 h and then analyzing the residual activity at 40 °C for 3 h with equal volume of 1.5% (w/v) agarose. The effects of metal ions on enzyme activity were assayed using a buffer supplemented with various ions (10 mM). Quantification of agarase activity using the previously described enzyme reaction conditions was performed using the 3,5-dinitrosalicylic acid method, which detects reducing sugars released from agarose.

Identification of Enzyme Reaction Products by Thin-layer Chromatography
Hydrolysis reactions using purified recombinant Aq1840 were performed at 40 °C with 0.75% (w/v) agarose in a 40 mM-Tris-HCl buffer containing 30 mM NaCl and 1 mM DTT (pH 8.0), and the reaction time was varied from 15 min to 72 h. To identify the minimum hydrolysis substrate, 1% AOSs and NAOSs (Biosynth, Ltd.) were included as the only substrate using the method mentioned above. TLC was performed using silica gel 60 aluminum plates (F254 Merck) to detect the hydrolytic products, which were developed using n-butanol-acetic acid-water (2:1:1, v/v). Saccharides were detected using a visualization solution containing 10% (v/v) sulfuric acid in ethanol, followed by heating at 100 °C.

Isolation and Identification of the Agar-degrading Bacterium ERC-38
To understand the diverse degradation mechanisms of marine polysaccharides by marine bacteria, various bacteria were isolated from seawater. Among these, we found that one bacterium generated a concave surface on the agar medium (Fig. S1a). Because this phenotype indicated that the strain could degrade agar, the isolate was selected to examine the diversity of hydrolytic enzymes present in the strain.
To assess the agar-decomposing ability of the ERC-38 strain, we performed a plate assay that visualized this ability using iodine staining. In agar-containing plates, the strain grew with a concave surface and transparent zone around the colonies (Fig. S1a). Interestingly, the isolate grew well in agar-containing marine medium but did not grow in a standard marine broth. The isolate grew in marine broth supplemented with carbon sources such as glucose, galactose, or agarose (Fig. S1b), which implies that the isolate requires a sufficient carbon source for growth.
To identify the taxonomic location of the strain, the 16S rRNA gene sequence was amplified from the genome of the isolate and sequenced. A comparative analysis of the 16S rRNA gene using the EzBioCloud database showed that a 1451-bp continuous sequence of the ERC-38 strain was most similar to that of Aquimarina celericrescens NS08 T , with a sequence similarity of 95.2%. The ERC-38 strain also showed 92.6-94.9% sequence similarity with other members of the Aquimarina genus, containing 29 recognized species (LPSN, List of Prokaryotic names with Standing in Nomenclature), including the recently published species A. acroporae D1M1 T (Sun et al. 2022). In phylogenetic analysis based on the 16S rRNA gene, ERC-38 comprised a clade with A. celericrescens NS08 T within the genus Aquimarina, belonging to the family Flavobacteriaceae (Fig. 1).

Genomic Sequence Analysis of Bacterium ERC-38
Genome sequence data of the ERC-38 strain have been submitted to the GenBank database under accession number CP098511. Whole-genome sequencing showed that the ERC-38 strain has a circular chromosome consisting of 4,431,522 bp with a GC content of 34.8 mol% and no plasmid (Fig. 2a). Genome annotation using the PGAP revealed that the genome of strain ERC-38 was predicted to encode 3615 coding sequences (CDSs), 40 tRNAs, and 9 rRNAs ( Table 1). The majority of the CDSs (3,381/3,685; 91.8%) could be assigned a putative function according to their annotated functional feature (Clusters of Orthologous Groups, COG) categories (Fig. 2a).
The heat map results for the percentage of genome and protein similarities between ERC-38 and other species belonging to the Aquimarina genus are presented in Fig. 3. The in silico DDH and AAI values ranged from 19.0 to 21.0 and from 59.0 to 65.5, respectively. Although the DDH and AAI values of ERC-38 were somewhat lower than those of other strains, they were within the range of values of all strains of the Aquimarina genus (17.2-47.7 and 58.5-93.7, respectively; Fig. 3).

Expression and Activity Test of Recombinant 1840 Agarases
To examine the properties of the six putative β-agarases, the genes were cloned and expressed in E. coli BL21 (DE3) codon + strains. However, only recombinant Aq1840 was presented in the soluble fraction together with agar-degrading activity, and other recombinant enzymes were present in the cell lysate precipitate as inclusion bodies (Fig. S5).
Aq1840, the only successfully expressed β-agarase in this study, includes an open reading frame consisting of 1263 nucleotides, encoding a 421-amino acid protein, and its predicted molecular mass is 46.3 kDa. Aq1840 contains a signal peptide (1-20 amino acids), a bacterial immunoglobulin (Ig) domain (31-118 amino acids) belonging to the Big_9 superfamily, and a GH16 catalytic module (160-416 amino acids) (Fig. 5a). In the amino acid sequence alignment of relative β-agarases, Fig. 2 Circular genome diagram and carbohydrateactive enzymes predicted to be encoded in the Aquimarina sp. ERC-38 genome. a Schematic representation of the genome. Labeling, from the center to outside, is as follows: circle 1, scale line in Mbps; circle 2, regions with a GC percentage higher or lower than the average value are shown outside or inside the center; circle 3, GC skew index according to the formula, (G − C)/(G + C), showing a higher G content (in green) and a higher C content (purple peaks); circles 4 and 5, CDS/tRNA/rRNA genes; circles 6 and 7, CDSs colored according to COG function categories.

Biochemical Characterization and Enzyme Activity of Aq1840 Agarase
To characterize recombinant Aq1840, protein purification was performed using Ni-NTA resin. Purified recombinant Aq1840 with His-tag showed a single band with a molecular weight of approximately 46.2 kDa on SDS-PAGE analysis (Fig. 6a). To characterize the enzymatic activity of recombinant Aq1840, we investigated various enzymatic factors such as temperature, pH, effects of metal ions, and temperature stability. Purified recombinant Aq1840 was examined at various temperatures (30-50 °C, 5 °C intervals), and the highest enzymatic activity was found at 40 °C. The optimal pH was 8 at 40 °C. More than 80% of the maximum activity was retained throughout the temperature range of 20-40 °C. When the pH was above 9 or below 7, recombinant Aq1840 showed less than 80% of its maximum activity (Fig. 6b-d). The presence of Na + and Ca 2+ ions enhanced the activity of recombinant Aq1840 by 1.56fold and 1.31-fold, respectively, compared with that in the absence of ions (Fig. 6e). This result indicates that the ion-binding site regulates the conformational equilibrium and catalytic activity of the Aq1840 β-agarase by binding with Na + or Ca 2+ ions.

Identification of Hydrolytic Products and Minimum Hydrolysis Substrate of Aq1840
To identify the final hydrolyzed agarose products generated by the Aq1840 enzyme, we performed TLC analysis of these hydrolysis products after time-kinetic treatment of agarose with purified recombinant Aq1840. Because Aq1840 showed homology with GH16 family proteins, it is speculated to be an endo-type enzyme and degrade agarose into NAOS with 4-10° of polymerizations (DPs). As a result of   Fig. 4 Genes of the PUL-a and tentative pathway involved in the agarose metabolism in the ERC-38 genome. a Each arrow indicates a separate gene, and the size is proportional to the gene length. Predicted functions are color-coded and presented above the arrows. b Enzymes are indicated at each reaction step along with the CAZy family or EC number. Solid arrows represent the proposed pathway for ERC-38 based on functional and bioinformatic analyses and dotted arrow represents an absent pathway. NAOSs, neoagarooligosaccharides; AOSs, agaro-oligosaccharides; Gal, D-galactose; L-AHG, 3,6-anhydrogalactose; L-AHGA, 3,6-anhydro-L-galactonate; L-KDGal, 2-keto-3-deoxy-L-galactonate; L-DDGal, 2,5-diketo-3-deoxy-L-galactonate; KDG, 2-keto-3-deoxy-D-gluconate; KDPG, 2-keto-3-deoxy-6-phospho-D-gluconate 1 3 TLC analysis, the degradation model of Aq1840 is shown in Fig. 7a. Similar to other GH16 family β-agarases, Aq1840 showed endo-typic hydrolysis of agarose from the initiation of the reaction, and the final hydrolyzed product mainly consisted of NA4 (Fig. 7b). When NA6 and NA4 were used as substrates to investigate the minimum hydrolysis substrates of recombinant Aq1840, the recombinant enzyme eventually hydrolyzed NA6 to yield NA4 and NA2, but NA4 was not hydrolyzed (Fig. 7c). When the agaropentaose (A5) or agarotriose (A3) was used as the substrate, recombinant Aq1840 weakly hydrolyzed A5 to yield A3 and NA2, but it did not hydrolyze A3 (Fig. 7d). These results show that recombinant Aq1840 cleaves the β-1,4-linkage between A3 and NA2, which consistent with the mechanism of substrate recognition of ZgAgaC (Fig. 7a) (Naretto et al. 2019).

Discussion
The previous result of statistical and clustering analyses of the marine heterotrophic bacteria shows that their carbohydrate catabolism correlates with both taxonomy and habitat (Barbeyron et al. 2016b). The genomes of marine bacteria reportedly contain genes encoding CAZymes for the degradation of marine polysaccharides (Hehemann et al. 2012;Boncan et al. 2018;Schultz-Johansen et al. 2018;Valdehuesa et al. 2018). Studies of the polysaccharide-degrading abilities and polysaccharide hydrolase characteristics of bacteria revealed their polysaccharide-metabolizing pathway and suggested that that pathway may be involved in the carbon cycle and biomass utilization in marine ecosystems (Imran et al. 2017;Valdehuesa et al. 2018;Gao et al. 2019). The marine bacterium ERC-38 strain grew well in marine broth supplemented with carbon sources representing abundant sugars of seaweed, such as glucose, galactose, or agarose (Fig. S1b). However, the isolate did not grow in medium containing another marine polysaccharide, carrageenan, as the carbon source (data not shown). This result implied that the isolate could not hydrolyze carrageenan and could not utilize it as a carbon source. Interestingly, the isolate could not grow on medium containing carrageenan as the carbon source but had several genes encoding enzymes involved in carrageenan degradation in a PUL (PUL-c; locus tag, NBT05_06965 ~ 07,125), which is including two putative GH150 family proteins (Aq1437 and Aq1450), and two GH82 family proteins (Aq1435 and Aq1440) (Fig. S2). According to the tentative carrageenan metabolic pathway, putative genes encoding enzymes such as α-1,3-(3,6)-anhydro-D-galactosidase (GH127, Aq0699), galactose mutarotase (Aq1864, in the PUL-a), galactokinase (Aq1452), galactose-1-phosphate uridylyltransferase (Aq1451), and UDP-galactose epimerase (Aq0608) were also found in the PUL-c or other loci of the genome (Fig. S3). Moreover, 17 putative S1-type sulfatases were also identified by a blast search on the sulfatase database server SulfAtlas (https:// sulfa . c In the 3D structure, blue, red, and white represent basic, acidic, and other amino acid residues, respectively. Catalytic or metal ion-binding residues are indicated in the structure. d Predicted residues involved in substrate recognition are indicated in the structure as colored in the 3D structure. The amino acids involved in substrate recognition are colored as follows: subsite − 2 or − 3, orange; subsite − 1, yellow; subsite + 1, blue; subsite + 2, green; catalytic residues, red 1 3 tlas. sb-rosco ff. fr/) (Stam et al. 2023), and these enzymes have been reported to be important for marine microbes to utilize highly sulfated algal polysaccharides (Barbeyron et al. 2016a;Hettle et al. 2018). Of these sulfatases, Aq2073 (locus tag, NBT05_10190), identified as S1_19, was classified as an S1_19B-type sulfatase and had the highest homology (57.6%) with PsS1_19B (6PRM_A) of Pseudoalteromonas fuliginea when the proteins were compared against Protein Data Bank proteins (PDB) using NCBI BLAST. S1_19B is an exo-G4S κ-carrageenan sulfatase that has κ-NC2 and κ-NC4 activity but does not have ι-NC2 or ι-NC4 activity (Hettle et al. 2019). The other 14 putative sulfatase genes, except for Aq2073 (S1_19), Aq3091 (S1_16), and Aq3095 (S1_16), were concentrated in a sizeable PUL containing the PUL-c (Fig. S2). These in silico data explain why the isolate could not grow when carrageenan was the carbon source. Although the isolate had many genes involved in carrageenan degradation and metabolism, it is assumed that carrageenan could not be completely hydrolyzed to neocarrabiose; ι-carrageenan oligosaccharides were produced from ι-carrageenan by Aq1435, Aq1440, or Aq1863, but desulfation could not occur. Additionally, exo-G4S κ-carrageenan sulfatase was present, but κ-carrageenase, which is used to generate κ-carrageenan oligosaccharides, was absent in the genome (Fig. S3). Therefore, further study is needed to verify this pathway.
Unlike its inability to degrade carrageenan, the isolate could degrade agarose. Therefore, we also investigated the agarose degradation process of the isolate at the genomic and enzymatic levels. Several species of the genus Aquimarina, as shown by their species name, including A. agarivorans HQM9 T , A. agarilytica ZC1 T , and A. aggregata RZW4-3-2 T , exhibit agar-degrading activity (Zhou et al. 2015;Lin et al. 2012;Wang et al. 2016). Among 29 species of the Aquimarina genus, five species including A. latercula DSM2041 T and A. algiphila 9Alg 151 T as well as the above-mentioned three species have demonstrated agardecomposing ability. Sixteen species do not have this ability, and another eight species have not been evaluated at this time ( Fig. 1) (Nedashkovskaya et al. 2018;Li et al. 2014;Han et al. 2017;Park et al. 2013;Chen et al. 2012;Sun et al. 2021;Zhou et al. 2015;Wang et al. 2016;Lin et al. 2012). Although several species belonging to the Aquimarina genus have agar-degrading ability, only enzymatic characteristics of Aga672 and Aga575 of A. agarilytica ZC1 T have currently been reported. Aga672 is a GH16 β-agarase, and its final hydrolyzed products are NA4, NA6, and NA8, and Aga575 is an exo-type agarase and generates only NA2 as a final product (Dong et al. 2021;Lin et al. 2017). Besides, the production of neoagarooligosaccharides using a β-agarase of A. agarilytica NI125 has been reported, but the classification . a Recombinant Aq1840 was purified using a His-Trap column, and its purity was verified by SDS-PAGE analysis. Control or Crude Aq1840 indicates the soluble cell lysate obtained from an E. coli transformant containing an empty or a pET21a-Aq1840 vector, respectively. Purified Aq1840 indicates recombinant His-tagged Aq1840 purified from soluble cell lysate using a HisTrap column. b and c The effects of temperature and pH on the enzyme activity were measured using 0.75% agarose and the purified recombinant enzyme. d Temperature stability was investigated following a 3-h incubation at the indicated temperature. e The effects on the enzyme activity of 10-mM metal ion or EDTA (chelating agent) supplementation were compared with those in a non-treated control 1 3 of the enzyme and the characteristics of the hydrolyzed products have not been specified (Farahat 2019).
For characterization of the β-agarases of the isolate, the six putative genes were expressed together with the addition of a His 6 tag in E. coli BL21 (DE3) codon + strains. In a plate assay performed to detect agar-degrading activity, lysates of cells expressing recombinant Aq1829 and Aq1840 showed activity after iodine staining, but other putative enzymes did not show activity (Fig. S5a). SDS-PAGE showed that all expressed enzymes except recombinant Aq1840 were present in the cell lysate precipitate as inclusion bodies (Fig. S5b). Because the recombinant Aq1829 lysates exhibited activity in the plate assay, we attempted to obtain recombinant Aq1829 protein in a soluble state. However, it was insufficiently expressed as a soluble protein, and the precipitated protein lost activity after renaturation (data not shown).
In the amino acid sequence alignment, the Ig domain was presented in Aq1840 but not in other relative β-agarases. Iglike domains, which have been previously shown to be present in bacteriophage and cell-cell adhesion proteins, are also distributed in a variety of GHs. However, the function of Ig domains is still not well understood, but it is speculated that these domains facilitate enzymatic function (Xing et al. 2022).
In TLC analysis, Aq1840 showed that hydrolysis of NA6 or A5 but not NA4. This is probably because at least fivesugar units are essential for substrate recognition like ZgA-gaC. Although the recombinant Aq1840 could cleave the β-1,4-linkage between A3 and NA2 of A5, it showed greater efficiency on the longer substrates such as NA6 or agarose. This result shows that the degradation by Aq1840 can be influenced by presence of extra sugars in addition to the five-sugar units.
Meanwhile, during the hydrolysis of NA6 by recombinant Aq1840, the substrate seemed to polymerize into larger oligosaccharides, but this polymerization was not observed in other reactions using smaller substrates. Even in several repeated tests, this type of polymerization still occurred (Fig. S6). Similarly, a previous study reported that a GH16 family β-agarase derived from the Microbulbifer sp. BN3 presented polymerization of oligosaccharides at the initial stage of NA8 hydrolysis (Li et al. 2020). Similar to this result, recombinant Aq1840 also exhibited polymerization properties, and the end-products of agarose were larger than NA4 until 72 h. Just as genomic studies showed that marine Bacteroidetes may adapt to their habitat, analyses of the human gut metagenome and gut bacteria genomes revealed the presence of genes encoding porphyranases and agarases. These genes may have been laterally acquired from marine bacteria associated with seaweeds, and their products may function to decompose agarose or porphyran to oligo-or mono-saccharides (Pluvinage et al. 2018;Hehemann et al. 2010;Yun et al. 2021;Robb et al. 2022). The oligosaccharides could stimulate the growth of some probiotic bacteria, such as bifidobacteria and lactobacilli; thus, hydrolysates are considered to have prebiotic effects (Hu et al. 2006). As prebiotics, NAOSs (DPs of 4-6) increase the lactobacilli population, but larger NAOSs (DPs of 8-12) have better effects (Hu et al. 2006). Additionally, NAOS mixtures produced by treatment with β-agarase derived from Vibrio natriegens CICC 23,820 have the potential to be utilized as antioxidant and prebiotic additives (Zhang et al. 2019). The enzymatic characterization showed that recombinant Aq1840 could be used to produce the variable sizes of NAOS or AOS, and also the oligosaccharides can be utilized as prebiotic and antioxidant additives because of their large sizes.
In summary, we showed through whole-genome sequencing analysis that the genome of the ERC-38 strain contains all genes required for agarose degradation and metabolism and it can perform degradation at least via a functional β-agarase Aq1840. The result of minimum hydrolysis substrate test has shown that Aq1840 cleaves the β-1,4-linkage between A3 and NA2, which implies that the substrate recognition way of Aq1840 is identical to that of ZgAgaC. Characterization of Aq1840 supports the understanding of the action of relatively unique ZaAgaC-like enzymes and may facilitate industrial application of this recombinant enzyme for the production of oligosaccharides for use as food additives such as prebiotics or antioxidants. Moreover, the here presented in silico genomic analysis provides the gene basis for understanding the mechanism of marine polysaccharide degradation by Aquimarina sp. ERC-38.