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Article

Biochemical Characterization and Elucidation of the Hybrid Action Mode of a New Psychrophilic and Cold-Tolerant Alginate Lyase for Efficient Preparation of Alginate Oligosaccharides

1
College of Food Science and Light Industry, Nanjing Tech University, Nanjing 211816, China
2
Suqian Advanced Materials Industry Technology Innovation Center of Nanjing Tech University, Suqian 223800, China
*
Author to whom correspondence should be addressed.
Mar. Drugs 2022, 20(8), 506; https://doi.org/10.3390/md20080506
Submission received: 13 July 2022 / Revised: 3 August 2022 / Accepted: 3 August 2022 / Published: 5 August 2022

Abstract

:
Alginate lyases with unique biochemical properties have irreplaceable value in food and biotechnology industries. Herein, the first new hybrid action mode Thalassotalea algicola-derived alginate lyase gene (TAPL7A) with both psychrophilic and cold-tolerance was cloned and expressed heterologously in E. coli. With the highest sequence identity (43%) to the exolytic alginate lyase AlyA5 obtained from Zobellia galactanivorans, TAPL7A was identified as a new polysaccharide lyases family 7 (PL7) alginate lyase. TAPL7A has broad substrate tolerance with specific activities of 4186.1 U/mg, 2494.8 U/mg, 2314.9 U/mg for polyM, polyG, and sodium alginate, respectively. Biochemical characterization of TAPL7A showed optimal activity at 15 °C, pH 8.0. Interestingly, TAPL7A exhibits both extreme psychrophilic and cold tolerance, which other cold-adapted alginate lyase do not possess. In a wide range of 5–30 °C, the activity can reach 80–100%, and the residual activity of more than 70% can still be maintained after 1 h of incubation. Product analysis showed that TAPL7A adopts a hybrid endo/exo-mode on all three substrates. FPLC and ESI-MS confirmed that the final products of TAPL7A are oligosaccharides with degrees of polymerization (Dps) of 1–2. This study provides excellent alginate lyase candidates for low-temperature environmental applications in food, agriculture, medicine and other industries.

1. Introduction

Alginate is a structural polysaccharide in the cell wall of Phaeophyta, such as Saccharin japonica and Undaria pinnatifida [1]. Alginate consists of β-ᴅ-mannuronate (M) and its C5 epimer α-ʟ-guluronate (G) randomly combined by 1,4 glycosidic bonds [1]. Alginate has been widely used in food, agriculture, medicine, materials and other industrial fields because of its excellent properties. For example, alginates can be used as food stabilizers and thickeners to improve the stability of ice cream [2]. As the products of alginate lyase degradation of alginate, alginate oligosaccharides (AOS) have the advantages of outstanding solubility and high bio-availability under the same biochemical activity as polysaccharide. Alginate oligosaccharides have probiotic properties and can regulate the intestinal micro-ecological balance by promoting the proliferation of Bifidobacterium [3]. In addition, AOS can promote the growth of crop roots, while polysaccharides did not show a corresponding effect [4].
As one of the modifying enzymes of alginate, alginate is degraded to low molecular oligosaccharides by alginate lyase by a β-elimination mechanism. Alginate lyases are mainly derived from marine bacteria (Pseudomonas, Vibrio), soil bacteria (Klebsiella, Azotobacter), viruses (Chlorella vius), fungi (Corollospora intermedia) and other microorganisms; marine algae (Laminaria japonica); marine echinoderms and mollusks (Haliotis discus hannai) [5]. According to the CAzy database, alginate lyases are classified into 14 polysaccharide lyase (PL) families, including PL5, PL6, PL7, PL8, PL14, PL15, PL17, PL18, PL31, PL32, PL34, PL36, PL39 and PL41 families. In addition, alginate lyases can be classified into polyG-lyase (EC4.2.2.11), polyM-lyase (EC4.2.2.3) and bifunctional lyase because of the difference in substrate specificity [6]. According to the action mode, endo-alginate lyase degrades the substrate to oligosaccharides with a degree of polymerization of 2 or more, while exo-alginate degrades the substrate to monosaccharides. In recent years, alginate lyases with both endo- and exo-activities have been gradually characterized, and their simple and clear product distributions have attracted extensive attention in tailoring special AOS [7]. However, the commercial application of alginate lyases is largely limited by low activity and poor adaptability to industrial environments [8]. Most of the alginate lyases with mixed endo/exo-mode belong to the PL17 family, and only Alg2951 derived from Alteromonas portus HB161718T belongs to the PL7 family. To meet the environmental conditions for industrial applications, the discovery of extreme alginate lyases such as thermophilic and salt-tolerant enzymes have received extensive attention. Among them, cold-adapted enzymes have gradually attracted widespread attention in industrial applications [9]. Biocatalysis at lower temperatures reduce