Biochemical characteristics and molecular mechanism of an exo-type alginate lyase VxAly7D and its use for the preparation of unsaturated monosaccharides

Background: As the most abundant polysaccharide in brown algae, alginate has become a promising economical material for bioethanol production. Recently, exo-type alginate lyases have received extensive attention because the unsaturated monosaccharides produced by their degradation of alginate can be easily converted into 4-deoxy-l-erythro-5-hexoseulose uronate (DEH), a promising material for bioethanol production and biorefinery systems.

Results: In this study, we cloned and characterized an exo-type polysaccharide lyase family 7 (PL7) alginate lyase VxAly7D from the marine bacterium Vibrio xiamenensis QY104. Recombinant VxAly7D was most active at 30 °C and exhibited 21%, 46% and 90% of its highest activity at 0, 10 and 20 °C, respectively. Compared with other exo-type alginate lyases, recombinant VxAly7D was shown to be a bifunctional alginate lyase with higher specific activity towards sodium alginate, polyG and polyM (462.4 ± 0.64, 357.37 ± 0.53 and 441.94 ± 2.46 U/mg, respectively). A total of 13 μg recombinant VxAly7D could convert 3 mg sodium alginate to unsaturated monosaccharides in 1 min with a yield of 37.6%, and the yield reached 95% in 1 h. In addition, the three-dimensional structure of VxAly7D was modelled using the crystal structure of AlyA5 from Zobellia galactanivorans Dsij^T as the template. The action mode and the end products of the W295A mutant revealed that Trp²⁹⁵ is a key amino acid residue responsible for the exolytic action mode of VxAly7D.

Conclusion: Overall, our results show that VxAly7D is a PL7 exo-type alginate lyase with high activity and a high conversion rate at low/moderate temperatures, which provides a useful enzymatic tool for the development of biofuel production from brown algae and enriches the understanding of the structure and functional relationships of polysaccharide lyases.