**1. Introduction**

Alginate is the major component of cell wall of brown algae [1]. It is a linear anionic polysaccharide and consists of α-L-guluronate (G) and its C5 epimer β-D-mannuronate (M), which are linked by α-1, 4-glycosidic bonds [2]. The two monomeric units are arranged into three groups: poly-α-L-guluronate (polyG), poly-β-D-mannuronate (polyM), and the heteropolymer (polyMG) [3]. Due to its high viscosity, gelling properties, and versatile activities, alginate has been widely applied in food, chemical, and pharmaceutical industries [4–6]. However, the applications of this polysaccharide are still limited by its high molecular weight and poor solubility [7]. The alginate oligosaccharide, as the degradation product of alginate, retains various specific physiological functions and activities of polysaccharide but possesses good bioavailability [8]. For instance, Pack et al. found that alginate oligosaccharide (AOS) can reduce plasma LDL-cholesterol levels by regulating the expression of LDLR [9]. Iwamoto et al. studied the effect of AOS with different structures on the induction of cytokine production from RAW264.7 cells and found that G8 and M7 showed the most potent activity [10]. Yamamoto et al. reported that mannuronate oligomers (M3–M7) could induce the

production and secretion of multiple cytokines, such as tumor necrosis factor- α (TNF-α), granulocyte colony-stimulating factor (GCSF), and monocyte chemoattractant protein-1 (MCP-1) [11].

Alginate lyase, a member of polysaccharide lyase, can catalyze the alginate by the β-elimination, producing unsaturated oligosaccharides with double bonds between C4 and C5 [12]. Until now, a number of alginate lyases have been identified, gene-cloned, purified, and characterized from various sources, such as marine and terrestrial bacteria, marine mollusks, and algae [13–18]. According to the substrate specificities, alginate lyases can be classified into three types: polyM-specific lyases (EC 4.2.2.3), polyG-specific lyases (EC 4.2.2.11), and bifunctional lyases (EC 4.2.2.-) [19]. Additionally, the alginate lyases are generally organized into seven polysaccharide lyase (PL) families according to the sequence similarity, namely PL-5, -6, -7, -14, -15, -17, and -18 families [20]. Moreover, in terms of the mode of action, alginate lyases can be grouped into endolytic and exolytic alginate lyases [21]. Endolytic enzymes can cleave glycosidic bonds inside alginate polymer and release unsaturated oligosaccharides as main products [22], while exolytic ones can further degrade oligosaccharides into monomers [23]. Now alginate lyases, especially endolytic enzymes, have been widely used to produce alginate oligosaccharides for food and nutraceutical industries [24,25]. Moreover, the enzymes can also be used to elucidate the fine structures of alginate and prepare protoplast of brown algae [26–28]. Furthermore, alginate lyases also show great potential in the treatment of cystic fibrosis by degrading the polysaccharide biofilm of pathogen bacterium [29]. So far, many alginate lyases originating from marine microorganisms have been well characterized. However, few of these enzymes have been commercially used in the food and nutraceutical industries due to the poor substrate specificity and low activity [30–36]. Thus, to explore novel enzymes with high activity and high substrate specificity will be of great importance for both research and commercial purposes.

In this work, a new alginate lyase with high substrate specificity and pH stability has been identified and characterized from *Serratia marcescens* NJ-07. To evaluate the enzyme for potential use in the food and nutraceutical industries, the kinetics and analysis of degrading products has also been characterized, which suggests that it would be a potential candidate for expanding applications of alginate lyases.

#### **2. Results and Discussions**

#### *2.1. Screening and Identification of Strain NJ-07*

The strain was isolated from rotten red algae from the Yellow Sea. The 16S rRNA sequence of the strain was sequenced and submitted to GeneBank (accession number MH119760). According to the phylogenetic analysis of 16S rRNA sequence (Figure 1), the strain was assigned to the genus Serratia and named *Serratia marcescens* NJ-07.

**Figure 1.** The phylogenetic analysis of strain NJ-07 and other similar strains. The phylogenetic tree was constructed by MEGA 6.0 on the basis of the 16S rRNA gene sequences of strain AlgNJ-07 and other known Serratia species.

#### *2.2. Purification of Alginate Lyase*

The strain NJ-07 was cultured in optimized liquid medium for 40 h until alginate lyase reached the highest activity. The supernatant containing alginate lyase was subjected to further purification by anion exchange chromatography with Source 15Q. After purification, the alginate lyase was purified 7.43-fold with a yield of 68.1%. The final specific activity of the purified alginate lyase was 2742.5 U/mg towards sodium alginate. The result of SDS-PAGE showed a single protein band with a molecular weight of 25 kDa (Figure 2), which was designated as AlgNJ-07. The alginate lyases are grouped into three types based on their molecular weights: small alginate lyases (25–30 kDa), medium-sized alginate lyases (around 40 kDa), and large alginate lyases (>60 kDa). As a result, the AlgNJ-07 belongs to the small ones. Similarly, the AlyA from *Azotobacter chroococcum* 4A1M has a small molecular weight of 24 kDa [31]. While the AlyA from *Pseudomonas* sp. E03, AlyA from *Pseudomonas aeruginosa*, and AlyA from *Pseudomonas* sp. strain KS-408 possess the medium-sized molecular weights of 40.4 kDa, 43 kDa, and 44.5 kDa, respectively [33,34,36]. The ALYII from *Pseudomonas* sp. OS-ALG-9 has a large molecular weight of 79 kDa [32].
