Significantly improving the thermal stability of alginate lyase AlyC3 from Psychromonas sp. C-3 by computational redesign

Alginate lyase is a key enzyme in the bioproduction of alginate oligosaccharides. The alginate lyase AlyC3, derived from Psychromonas sp. C-3, can hydrolyze homopolymerized mannuronic acid into oligosaccharides (primarily composed of mannuronate trimer), which possess physiological properties such as bacteriostatic and antioxidant activities and show a better application prospect. However, the poor thermal stability hinders its potential application in industrial production. In this study, AlyC3 mutants A35P, L95F, N138Y and M162I were obtained through the protein repair one-stop shop (PROSS) design combined with protein data bank in Europe (proteins, interfaces, structures and assemblies) (PDBePISA) analysis of the dimer interface. The results demonstrated that the thermodynamic and kinetic stability of these single-point mutants were significantly improved compared to the wild-type (WT), with the melting temperature increased by 4.33, 9.35, 9.74 and 11.24 °C, respectively, while the half-life (t1/2) was 1.66, 1.02, 1.02 and 1.53 times higher than that of WT, respectively. Based on this, the beneficial mutations were further combined to obtain a combinatorial mutant, A35P-L95F–N138Y-M162I, which exhibited the superior thermal stability. Notably, the combinatorial mutant's melting and optimal temperature increased by 17.05 and 10 °C, respectively; moreover, the mutant enzyme's t1/2 and enzyme activity were 8.21 times and 123.93% higher than those of the WT, respectively. Structural analyses indicated that increased hydrophobic interactions and polar bonding between the mutated and nearby residues, with improved rigidity in the flexible loop, might account for the significantly improved thermal stability of the reengineered AlyC3, which makes it a robust candidate for alginate oligosaccharide production.