**1. Introduction**

Alginate is derived from the cell wall and intercellular substance of brown seaweed, accounting for approximately 22–44% (*w*/*w*) of seaweed biomass [1]. It is a kind of linear, water-soluble, and acidic polysaccharide composed of one or both of *β*-D-mannuronic acid (M) and its C-5 epimer *α*-L-guluronic acid (G) [2]. The M and G subunits form polyguluronic acid (poly-*α*-L-guluronate, polyG), polymannuronic acid (poly-*β*-D-mannuronate, polyM), and mixed random polymer (polyGM) in three types of block structures by α/β-1,4 glycosidic bonds [3]. Alginate is widely used in the field of food and pharmaceutical industry owing to its high viscosity and gelling properties. Further, it has attracted attention as a promising marine biomass for the production of biofuels and chemicals in biorefinery applications [4].

Alginate oligosaccharide (AOS) is an oligomer of alginate, which has the characteristics of low relative molecular weight, good solubility, high safety, and high stability. It is

**Citation:** Huang, H.; Li, S.; Bao, S.; Mo, K.; Sun, D.; Hu, Y. Expression and Characterization of a Cold-Adapted Alginate Lyase with Exo/Endo-Type Activity from a Novel Marine Bacterium *Alteromonas portus* HB161718T. *Mar. Drugs* **2021**, *19*, 155. https://doi.org/10.3390/ md19030155

Academic Editor: Irina M. Yermak

Received: 20 February 2021 Accepted: 12 March 2021 Published: 17 March 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

reported that AOS has potential applications in agricultural, health product, and medical industries, since it exhibits significant pharmacological, physiological, and biological activities, including antioxidant, immunomodulation, antibacterial, antitumor, anticoagulation, and plant growth-promoting activities [5–7].

Alginate lyase is adopted to catalytically degrade alginate into AOS by *β*-elimination under relatively mild and controllable conditions. According to the substrate specificity, alginate lyases can be classified into three groups: polyM-specific lyase (EC 4.2.2.3), polyGspecific lyases (EC 4.2.2.11), and polyMG-specific lyases (EC 4.2.2) that can degrade polyG, polyM, and polyMG blocks of alginate, respectively [8]. Alginate lyases can be classified into endo- and exo-lytic fashions, according to the mode of degradation of alginate. Endolytic enzymes can cleave the glycosidic bonds inside the alginate with unsaturated oligosaccharides as the main products, while exotype ones can degrade oligomeric alginates and alginate polymers into monomers [9,10]. Furthermore, on the basis of their amino acid sequence similarities, the enzymes are generally grouped into seven polysaccharide lyases (PL) families in the Carbohydrate-Active Enzymes (CAZy) database—that is, PL-5, -6, -7, -14, -15, -17, and -18 [11,12]. Alginate lyases from the PL7 family have been widely studied, e.g., NitAly from *Nitratiruptor* sp. SB155-2 [13], lyA from *Isoptericola halotolerans* NJ-05 [14], Aly1281 from *Pseudoalteromonas carrageenovora* ASY5 [15], and MtAl138 from *Microbulbifer thermotolerans* DAU221 [16]. Alginate lyases are widely derived from marine algae; mollusks; and microorganisms (including bacteria, fungi, and viruses), among which marine bacteria are the most common resources. Many kinds of alginate lyase-secreting bacteria have been reported from marine bacteria, such as *Pseudoalteromona*, *Alteromonas*, *Microbulbifer*, *Zobellia*, *Vibrio*, and *Bacillus* [9,17–19].

In our previous study, a novel alginate lyase-producing strain, *Alteromonas portus*, was obtained from marine sand sample and taxonomically studied [17]. In this work, a gene *alg2951*, encoding alginate lyase Alg2951, was studied for a cloning, expression, and degradation product analysis. The results showed that Alg2951 was a cold-adapted PL7 alginate lyase with significant preference toward polyG. Moreover, Alg2951 was shown to be a NaCl- and KCl-activated enzyme with exolytic and endolytic activity, which released 4- deoxy-L-erythro-5-hexoseulose uronic acid (DEH) and trisaccharide from sodium alginate. In addition, the antioxidant capacity of the obtained AOS was investigated.
