Rational Engineering of a Bifunctional Alginate Lyase for Enhancing Thermal Stability and Continuous Preparation of Alginate Oligosaccharides Using an Enzyme–Membrane Coupled Bioreactor

Abstract Alginate oligosaccharides (AOS) are functional marine oligosaccharides with broad application potential, and the enzymatic degradation of alginate is regarded as an efficient and environmentally friendly strategy for their production. However, the practical application of alginate lyases is still limited by poor thermal stability and insufficient process efficiency. In this study, the bifunctional alginate lyase AlgNJU-03 from Vibrio sp. NJU-03 was rationally engineered to improve its thermal stability and applied to continuous AOS production in an enzyme–membrane coupled bioreactor. Two potential stabilizing mutations (A288H and S353R) were identified through rational design and validated via recombinant expression and biochemical characterization. Their specific activities were enhanced 1.09- and 2.74-fold, respectively, compared to the wild-type enzyme. The selected mutant S353R exhibited enhanced thermal stability while retaining desirable catalytic performance toward alginate substrates. Product analysis showed that the enzyme generated oligosaccharides within a relatively defined degree of polymerization range. Furthermore, a membrane-coupled reaction system was established using a 10 kDa ultrafiltration membrane, and the main operating parameters were optimized by single-factor and orthogonal experiments. The optimized system improved AOS production and recovery while also contributing to a certain degree of control over product composition. This study demonstrates that the combination of rational enzyme engineering and enzyme–membrane coupling is an effective approach for continuous AOS production and provides a useful basis for the industrial utilization of marine biomass.