Background

Alginate Structure and Composition

Alginate is an acidic linear polysaccharide widely found in brown algae, composed of β-D-mannuronic acid (M) and α-L-guluronic acid (G) linked by 1,4-glycosidic bonds in alternating or consecutive arrangements to form polymers.

Depending on the arrangement of M and G residues, alginate can form polyG (poly-guluronic acid), polyM (poly-mannuronic acid), or alternating M/G sequences (alternating M/G sequences).

Alginate Lyase

Alginate oligosaccharides (AOSs), obtained by the cleavage of alginate with alginate lyase, possess diverse biological activities, including antioxidant, anti-inflammatory, antitumor, and plant growth-promoting effects4. Their activity is closely related to degree of polymerization (DP), with low-molecular-weight AOSs (DP 2~4) showing particularly strong physiological effects.

Therefore, increasing the proportion of tetrasaccharides (DP4) in alginate lyase products is valuable for understanding substrate recognition and for the targeted preparation of high-value oligosaccharides.

Alginate lyases depolymerize alginate via a β-elimination mechanism, producing unsaturated AOSs. They are classified as endo-type, cleaving randomly within the chain, or exo-type, degrading from the ends. PL7 family alginate lyases have been extensively studied, with well-characterized structures and catalytic mechanisms.

Alginate Lyases Structure

In endolytic PL7 enzymes, loop1 and loop2 in the substrate-binding pocket significantly influence the product distribution. Longer loop1 generally favors disaccharides and trisaccharides, while shorter loops favor higher DP products. Importantly, structural features of PL7 family alginate lyases have been shown to increase substrate affinity at the -3 and -4 subsites, which correlates with stable accumulation of DP4, while the number and position of hydrogen bonds formed in the binding pocket also influence DP4 production, indicating that tetrasaccharide yield can serve as a direct indicator of enzyme engineering outcomes. PyAly, a PL7 alginate lyase from Pyropia yezoensis, is polyM-specific and primarily produces tetrasaccharides, making it an ideal candidate for rational engineering.

The Aim of Our Project

In this study, we aimed to improve tetrasaccharide yield of PyAly via single- and multiple-site directed evolution combined with machine learning. Based on structural analysis of the substrate-binding pocket and loop2, four residues (R143, R159, F170, K172) were selected for saturation mutagenesis, with its tetrasaccharide content of the products was measured. Guided by single-site mutagenesis results and computational predictions, four multiple-site mutants were designed and verified to enhance tetrasaccharide production.