Engineered N57P Variant of Ulvan Lyase with Improvement of Catalytic Efficiency and Thermostability via Reducing Loop Flexibility and Anchoring Substrate

Ulvan lyase degrades the ulvan from green macroalgae, which is a very attractive biocatalyst in algae biomass degradation. Here, a novel N57P mutant from Nonlabens ulvanivorans ulvan lyase (NuPL28) with improved catalytic efficiency and thermostability was rationally designed. Compared with the wild type, the kinetic parameters (k(cat) and k(cat)/K-m) of N57P were 2.46 and 3.20 times higher than that of WT, respectively. The N57P variant cooperating with commercial cellulase showed 1.33-fold higher activity for degradation of ulva powder to unsaturated sugar than that of WT. Molecular dynamics (MD) simulations were performed to reveal that the N57P mutant anchored the substrate in the active site through reducing the flexibility of the two loops T102-R117 and G209-R216. Moreover, it is also found that, compared with WT, the catalytic residues of N57P possessed a suitable nucleophilic attack distance and more hydrogen bonds with the tetrasaccharide. These findings would facilitate the discovery of the improved catalytic behavior of ulvan lyase and reduce the cost of algal biomass conversion.