*2.4. Repertoire of CAZymes*

We focused on investigation and comparison of the CAZymes genes in the *Zobellia* genomes in order to speculate about their bacterial lifestyles, as well as to identify relevant CAZymes for potential application in medicine and biotechnology.

CAZymes are a class of enzymes that synthesize, modify, or break down saccharides, and their classification comprises the following modules: Glycoside hydrolase families (GHs), polysaccharide lyase families (PLs), carbohydrate esterase families (CEs), glycosyltransferase families (GTs), auxiliary activity families (AAs), and carbohydrate-binding module (CBM) families [13].

A genomic approach was used to explore all CAZymes of a genome (CAZome) more profoundly. Identification of CAZymes across *Zobellia* genomes was carried out using the dbCAN2 meta server (http://cys.bios.niu.edu/dbCAN2). The server allows us to make a more accurate prediction of the CAZome because it integrates three annotation tools: HMMER, DIAMOND, and Hotpep searches [43]. The proportions of CAZymes predicted in the genomes of *Zobellia* are shown in Table 5. Calculations were based on the data obtained by RAST gene prediction and dbCAN2 CAZyme annotation.


**Table 5.** Proportions of predicted carbohydrate-active enzymes (CAZymes) in the genomes of *Zobellia* strains.

As discussed by Barbeyron et al. [30] and Boncan et al. [14], a CAZome is characteristic of species, which gives insights into bacterial behavior, lifestyle, and ecological niche. Therefore, for free-living species the proportion of CAZymes in their genomes typically corresponds to 1%–5% of all predicted coding sequences. In the five *Zobellia* strains studied, the proportion of CAZymes in the genomes ranged from 5.93% in *Z. laminariae* KMM 3676<sup>Т</sup> to 6.74% in *Z. galactanivorans* DsiJT, indicating the ability to consume various polysaccharides. Other *Zobellia* had slightly lower proportion of CAZymes than *Z. galactanivorans* DsiJT, these values being sufficient to argue that a broad biodegradation potential is conserved at the genus level. Total statistics of CAZymes' classes predicted across the genomes are in Figure 4.

**Figure 4.** Carbohydrate-active enzymes in *Zobellia* species. GH, glycoside hydrolase; GT, glycosyltransferase; PL, polysaccharide lyase; CE, carbohydrate esterases; AA, auxiliary activities; CBM, carbohydrate-binding module.

The determination of core and pan CAZomes for the *Zobellia* genus is of particular interest and importance. Obviously, the core CAZomes are composed of genus-specific enzymes, while the enzymes identified in singletons and inparalogs are species-specific. In terms of lifestyle peculiarities, the most interesting are the core multigenic CAZyme families. According to this idea, the core and pan CAZomes of the *Zobellia* genus were determined, and the repertoire of CAZymes is summarized in Figure 5 and Table S1.

GHs are enzymes that catalyze the hydrolytic cleavage of the glycosidic bond between two or more carbohydrates or between a carbohydrate and a non-carbohydrate moiety. These enzymes are involved in the degradation of the majority of biomass, including seaweeds [44]. In the present study, a total of 775 GHs were classified into 62 families in five *Zobellia* genomes. Among the identified core glycoside hydrolases, the most dominant were the GH29, GH109, GH2, GH13, and GH117 families in order of abundance. It is worth noting that seven particular GH13 subfamilies—GH13\_11, GH13\_19, GH13\_3, GH13\_31, GH13\_38, GH13\_7, and GH13\_9—were predicted. Based on the CAZy database (http://www.cazy.org/) definitions, enzymes of predicted families might act as broad spectrum α-fucosidases, α-N-acetylgalactosaminidase, β-glycosidases with Koshland double-displacement mechanism, as well as glycosidases acting on substrates with α-glucoside linkages, and α-1,3-L-(3,6-anhydro)-galactosidases.

GTs are principal enzymes that catalyze oligosaccharide, polysaccharide, and glycoconjugate synthesis. They also assist in glycosyl group transfer to specific acceptor molecules and utilize various sugar-1-phosphate derivatives [45]. A total of 16 GT families including 297 GTs were identified for the strains. The GT2 and GT4 families were ranked as key glycosyltransferases for the genus, which are polyspecific enzymes.

PLs are a group of enzymes that cleave uronic acid-containing polysaccharides via a β-elimination mechanism [46]. In the *Zobellia* genomes, a total of 100 PLs were classified in 13 families, among which PL14 lyases, possessing alginate, exo-oligoalginate, and β-1,4-glucuronan lytic activities, were the most abundant.

CEs are a class of esterases that catalyze the de-O or de-N-acylation of substituted saccharides [47]. There are two core multigenic families, namely CE1 and CE10, with wide substrate specificities, which generally help to degrade substrates leading to saccharification [48].

CBMs are non-catalytic proteins with carbohydrate-binding activity, capable of binding carbohydrate ligands and enhancing the catalytic efficiency of other CAZymes [49]. In the present study, a total of 186 CBMs were classified into 18 families, among which three multigenic families (CBM6, CBM47, CBM50) were identified in *Zobellia* genus.

AAs are the last class created in the CAZy classification, comprising enzymes that break glycosidic bonds via an oxidation mechanism [50]. Today, CAZy lists 16 AA families of enzymes playing a significant role in the degradation of biopolymers (CAZy database; http://www.cazy.org/). CAZyme annotation revealed that there are six different AA families in *Zobellia* strains: AA1, AA2, AA3, AA5, AA7, and AA12. The majority of AAs are AA3 with up to five AA3 family members in individual genomes. Moreover, this enzyme group was observed in all studied *Zobellia* strains, while other families were less populated (from zero to two AAs per genome).

**Figure 5.** Number of CAZymes in the *Zobellia* species. Number of (**A**) GH families; (**B**) PL families; (**C**) CE families; (**D**) AA families; (**E**) CBM families; (**F**) GT families; GHs, glycoside hydrolases; PLs, polysaccharide lyases; AAs, Auxiliary Activities; CBMs, carbohydrate-binding modules; GTs, glycosyltransferases.
