*Article* **Characterization of an Alkaline Alginate Lyase with pH-Stable and Thermo-Tolerance Property**

**Yanan Wang 1,**†**, Xuehong Chen 1,**†**, Xiaolin Bi 2, Yining Ren 1, Qi Han 1, Yu Zhou 1, Yantao Han 1,\*, Ruyong Yao <sup>3</sup> and Shangyong Li 1,\***


Received: 23 April 2019; Accepted: 14 May 2019; Published: 24 May 2019

**Abstract:** Alginate oligosaccharides (AOS) show versatile bioactivities. Although various alginate lyases have been characterized, enzymes with special characteristics are still rare. In this study, a polysaccharide lyase family 7 (PL7) alginate lyase-encoding gene, *aly08*, was cloned from the marine bacterium *Vibrio* sp. SY01 and expressed in *Escherichia coli.* The purified alginate lyase Aly08, with a molecular weight of 35 kDa, showed a specific activity of 841 U/mg at its optimal pH (pH 8.35) and temperature (45 ◦C). Aly08 showed good pH-stability, as it remained more than 80% of its initial activity in a wide pH range (4.0–10.0). Aly08 was also a thermo-tolerant enzyme that recovered 70.8% of its initial activity following heat shock treatment for 5 min. This study also demonstrated that Aly08 is a polyG-preferred enzyme. Furthermore, Aly08 degraded alginates into disaccharides and trisaccharides in an endo-manner. Its thermo-tolerance and pH-stable properties make Aly08 a good candidate for further applications.

**Keywords:** Alginate lyase; Thermo-tolerant; pH-stability; Endo-manner; *Vibrio* sp. SY01

#### **1. Introduction**

Alginate is an acidic hetero-polysaccharide extracted from brown algae, which accounting for 22–44% of its dry weight [1–3]. Alginate mainly contains two different uronic acids, including α-l-guluronic acid (G) and β-d-mannuronic acid (M). They are arranged into three different kinds of blocks by (1→4)-linked monosaccharides: homopolymeric G blocks, polyguluronate (PolyG); homopolymeric M blocks, polymannuronate (PolyM); and random or heteropolymeric blocks of alternating M and G units (PolyMG) [4,5].

Alginate lyase (E.C. 4.2.2.3 and E.C. 4.2.2.11) is a kind of polysaccharide lyase that degrades alginate by β-eliminating the glycoside 1-4 O-bonds between C4 and C5 at the non-reducing end, thus producing unsaturated alginate oligosaccharides (UAOS) as main products [6,7]. Due to its high efficiency, specificity and mild degradation function, alginate lyases have attracted widespread attention in industrial applications, especially in the preparation of alginate oligosaccharides [8,9].

According to the Carbohydrate-Active enZYmes (CAZy) databases, alginate lyases belong to PL families 5, 6, 7, 14, 15, 17, and 18 based on the analysis of their amino acid sequences [10–12]. Based on the substrate specificity, alginate lyases can be further classified into two types, one type is the G block-specific lyases (polyG lyases, EC 4.2.2.11), and the other type is the M block-specific lyases (polyM lyases, EC 4.2.2.3) [13,14]. In PL families 5, 7, 14, 17, and 18, most of the reported alginate lyases are polyM lyases. Only the alginate lyase reported in PL family 6 is mainly comprised of polyG

lyases. Thus far, hundreds of alginate lyases have been purified, cloned, and characterized from marine microorganisms, brown seaweeds, and mollusks [15–18]. However, these reported enzymes with characteristics specific for commercial use are rare. Cold-adapted alginate lyases can run biocatalytic processes at low temperature and reduce the danger of contamination. Thermo-tolerant enzymes persist at high temperatures, thereby not only improving degradation efficiency but also reducing production costs. Meanwhile, high proportion product in a mixture of products will be propitious to the purification of oligosaccharide. Therefore, there is an urgency to obtain an alginate lyase with the optimal characteristics (e.g., pH-stability, thermo-tolerance, and single product distribution) needed for industrial applications.

In this study, a new alginate lyase-encoding gene, *aly08*, was cloned from *Vibrio* sp. SY01, and expressed in *Escherichia coli* BL21 (DE3). The recombinant enzyme Aly08 degraded alginate, yielding alginate disaccharides and trisaccharides as main products. This study also revealed that Aly08 was a polyG-preferred enzyme with special characteristics, such as wide pH-stability, thermo-tolerance, and single product distribution. These special features suggest that Aly08 may play essential roles in saccharification processes of alginate and carbon cycling.
