Engineering a Novel Alginate Lyase for Improved Thermostability and Evaluation of Promoting Effects of Its Degradation Products on Brassica rapa var. chinensis under Salt Stress Environments

Alginate lyases catalyze depolymerization of alginate, a major polysaccharide in brown algae, into bioactive oligosaccharides. Herein, the gene (CelPL7A) encoding a PL7 family alginate lyase from Cellulophaga lytica was cloned, heterologously expressed in Escherichia coli, and characterized. CelPL7A exhibited maximum activity at 30 °C and pH 8.0 but limited thermostability. To enhance its thermal stability, rational design was employed and yielded three single mutants (N99G, T331D, and G346S). All three mutants exhibited significantly enhanced thermal stability. Their specific activities were enhanced to 2.7-, 2.3-, and 3.2-fold, respectively, compared to the wild-type enzyme. Both CelPL7A and the G346S mutant efficiently degraded pretreated kelp powder, primarily yielding trisaccharides. Application tests demonstrated that alginate oligosaccharides (AOS) produced by CelPL7A significantly promoted the seed germination and seedling vigor of pak choi (Brassica rapa var. chinensis) under salt stress. These results suggest that engineered variants are promising biocatalysts for AOS production and agricultural biostimulant development.