Efficient Degradation of Alginate and Preparation of Alginate Oligosaccharides by a Novel Biofunctional Alginate Lyase with High Activity and Excellent Thermophilic Features

Abstract

The enzymatic degradation of seaweed polysaccharides is gaining interest for its potential in the production of functional oligosaccharides and fermentable sugars. Herein, a novel alginate lyase, AlyRm3, was cloned from a marine strain, Rhodothermus marinus DSM 4252. The AlyRm3 showed optimal activity (37,315.08 U/mg) at 70 °C and pH 8.0, with the sodium alginate used as a substrate. Noticeably, AlyRm3 was stable at 65 °C and also exhibited 30% of maximal activity at 90 °C. These results indicated that AlyRm3 is a thermophilic alginate lyase that efficiently degrades alginate at high industrial temperatures (>60 °C). The FPLC and ESI−MS analyses suggested that AlyRm3 primarily released disaccharides and trisaccharides from the alginate, polyM, and polyG in an endolytic manner. In the saccharification process of sodium alginate (0.5%, w/v), the AlyRm3 yielded numerous reducing sugars (1.73 g/L) after 2 h of reaction. These results indicated that AlyRm3 has a high enzymatic capacity for saccharifying the alginate, and could be used to saccharify the alginate biomass before the main fermentation process for biofuels. These properties make AlyRm3 a valuable candidate for both fundamental research and industrial applications.

A Novel Bifunctional Alginate Lyase and Antioxidant Activity of the Enzymatic Hydrolysates

“…It has been reported that the active site of FsAlgB is located within two flexible loops, and that both polyG and polyM in the substrate molecules could bind to the active site . Furthermore, structural analysis of the bifunctional alginate lyase, AlyRm3, revealed that the complex substrate ΔMG can bind to a long cleft formed between the N-terminal domain and central domain . To the best of our knowledge, this study is the first to explore different binding sites in a bifunctional alginate lyase for different substrates.…”

“…Understanding the detailed mechanisms of bifunctional enzymes will help to optimize protein engineering applications and elucidate how enzymes might have evolved . In recent years, the structure and substrate-binding sites of several bifunctional alginate lyases have been predicted, such as AlyPL17, AlyRm3, and FsAlgB . However, there has been no mutation verification work so far, and this is the first time mutation verification was conducted based on docking results.…”

“…25 Furthermore, structural analysis of the bifunctional alginate lyase, AlyRm3, revealed that the complex substrate ΔMG can bind to a long cleft formed between the N-terminal domain and central domain. 54 To the best of our knowledge, this study is the first to explore different binding sites in a bifunctional alginate lyase for different substrates. The results of this study provide further insights into the substrate recognition and degradation pattern of alginate lyases with broad substrate specificity, as well as offer a theoretical basis for the development of alginate lyases with exceptional properties through molecular modification technology.…”