**1. Introduction**

Alginic acid has the ability to form viscous solutions and gels in aqueous media and is nontoxic to living organisms. Therefore, it has been widely used in the pharmaceutical, cosmetic, food, and biotech industries [1]. In addition, the degradation products of alginic acid—alginate oligosaccharides—have a wide range of biological activities, such as the promotion of growth and the alleviation of abiotic stress in plants; antitumour, antibacterial, anti-inflammatory, anticoagulant, antioxidative, and immunomodulatory activities; and the reduction of free radicals and blood glucose and lipids. Alginate oligosaccharides have broad application prospects in the green agriculture, medical, food, and household chemical industries, among others [2].

Alginic acid, also known as algin or alginate, is a straight-chain polysaccharide composed of β-D-mannuronic acid (M) and its C5 stereoisomer α-L-guluronic acid (G), which are randomly linked via α-1,4-glycosidic bonds. Alginate molecules can have the sugar monomers M and G arranged in three ways: M monomers can be linked in succession, forming poly-mannuronic acid (polyM); G monomers can be linked in succession, forming poly-guluronic acid (polyG); and M and G monomers can be randomly and alternately linked, forming poly-guluronic acid -mannuronic acid (polyMG) [3]. The relative proportions of M and G vary among the alginic acids derived from different organisms [4,5].

Alginate lyases catalyze the cleavage of the 1,4-glycosidic bonds between the uronic acid monomers in alginate, resulting in the production of oligouronic acids or uronic acid monomers. Alginate lyases are present in a wide range of organisms and can be isolated from marine algae, molluscs, and microorganisms, as well as soil microorganisms. Currently, the primary sources of alginate lyases are marine bacteria, including members of the genera *Pseudomonas* and *Vibrio* [6].

The catalytic mechanisms of alginate lyases are well understood. Alginate lyases catalyze the cleavage of 1,4-glycosidic bonds in alginic acid via a β-elimination reaction. In addition, a double bond is formed between C4 and C5 of the saccharide ring containing the 4-*O* glycosidic bond, generating an oligomer with 4-deoxy-L-erythro-hex-4-enepyranosyluronate at the nonreducing end [7]. Based on their catalytic characteristics, alginate lyases are divided into endolytic and exolytic alginate lyases [8,9]. According to the substrate specificity, endolytic alginate lyases can be divided into mannuronate lyases (polyM lyase, EC 4.2.2.3) and guluronate lyases (polyG lyase, EC 4.2.2.11). Bifunctional enzymes that exhibit activities towards both polyG and polyM have also been identified [10]. While numerous alginate lyases have been discovered, few studies have focused on the enzymatic properties of alginate lyases. In recent years, a number of alginate lyases have been discovered and reported, including the cold-adapted alginate lyases [11–13], thermostable alginate lyases [14], high-alkaline alginate lyases [15], and salt-activated alginate lyases [12,15–17]. According to the evolution and homology of amino acid sequences, most alginate lyases belong to seven families of polysaccharide lyases (PL-5, PL-7, PL-14, PL-15, PL-17, and PL-18) [18]. Most of the reported alginate lyases have endolytic activity [15,19–21] and hydrolyze sodium alginate (SA) to produce oligosaccharides. Alginate lyase A1-IV, produced by the bacterial strain *Sphingomonas* sp. A1 [22], and alginate lyase Atu3025, produced by *Agrobacterium tumefaciens* C58 [23], possess exolytic activity and hydrolyze SA into mannuronate or guluronate. Alginate lyases that degrade alginate into monosaccharides are part of the PL15 family [22,23], whereas alginate lyases with endolytic activity belong to the PL-5, 6, 7, 14, 16, 17, and 18 families [24].

The marine bacterium *Vibrio weizhoudaoensis* M0101 harbors the *algM4* gene, which encodes an alginate lyase belonging to polysaccharide lyase family 7 (PL7). In this study, we purified exogenously expressed AlgM4 and observed it to exhibit high salt tolerance, as AlgM4 activity increased more than sevenfold in the presence of 1 mol/L NaCl. The result is different from those for other salt-activated alginate lyases for which enzyme activity is decreased at 1 mol/L NaCl [15,25]. In the depolymerization of a high content of sodium alginate, Alg2A generated equal total molar amounts of oligosaccharides, but the amounts of oligosaccharides with DP of 5–10 were higher than those for both of the two commercial enzymes [26]. AlgM4 showed activities toward both polyM and polyG, which may degrade alginate more effectively. Moreover, AlgM4 catalyzing polyM released oligosaccharides with DP 7–9 from the polyM, which was different from the previously reported endolytic alginate lyases, despite their diverse substrate specificities [27,28]. Therefore, the unique endolytic reaction mode of AlgM4 gives it a distinct advantage in facilitating uronic acid oligosaccharides with high DPs. AlgM4 could be a good tool for the preparation of alginate oligosaccharides. AlgM4 not only functions as a key enzyme in the preparation and functional study of oligosaccharides but also plays an important role in utilization of alginate for ethanol fermentation.
