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

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--5-hexoseulose uronate (DEH), a promising material for bioethanol production and biorefinery systems.In this study, we cloned and characterized an exo-type polysaccharide lyase family 7 (PL7) alginate lyase VxAly7D from the marine bacterium 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 Dsij as the template. The action mode and the end products of the W295A mutant revealed that Trp is a key amino acid residue responsible for the exolytic action mode of VxAly7D.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.