Improving the thermal stability and catalytic activity of ulvan lyase by the combination of FoldX and KnowVolution campaign

Thermal stability is one of the most important properties of ulvan lyases for their application in algae biomass degradation. The Knowledge gaining directed eVolution (KnowVolution) protein engineering strategy could be employed to improve thermostability of ulvan lyase with less screening effort. Herein, the unfolding free energies (ΔΔG) of the loop region were calculated using FoldX and four sites (D103, G104, T113, Q229) were selected for saturation mutagenesis, resulting in the identification of a favorable single-site mutant Q229M. Subsequently, iteration mutation was carried out with the mutant N57P (previously obtained by our group) to further enhance the performance of ulvan lyase. The results showed that the most beneficial variant N57P/Q229M exhibited a 1.67-fold and 2-fold increase in residual activity compared to the wild type after incubation at 40 °C and 50 °C for 1 h, respectively. In addition, the variant produced 1.06 mg/mL of reducing sugar in 2 h, which was almost four times as much as the wild type. Molecular dynamics simulations revealed that N57P/Q229M mutant enhanced the structural rigidity by augmenting intramolecular hydrogen bonds. Meanwhile, the shorter proton transmission distance between the general base of the enzyme and the substrate contributed to the glycosidic bond breakage. Our research showed that in silico saturation mutagenesis using position scan module in FoldX allowed for faster screening of mutants with improved thermal stability, and combining it with KnowVolution enabled a balanced effect of thermal stability and enzyme activity in protein engineering.