Alginate lyases can fully degrade alginate into various size-defined unsaturated oligosaccharide products by β-elimination. Here, we identified the bifunctional endolytic alginate lyase Aly35 from the marine bacterium Vibrio sp. Strain H204. The enzyme Aly35 is classified into the polysaccharide lyase 7 superfamily and contains two alginate lyase catalytic domains. The relationship and function of the two lyase domains are not well known. Thus, the full-length recombinant enzyme and its truncated proteins Aly35-CD1 (catalytic domain 1), Aly35-CD2 (catalytic domain 2 domain) were constructed. The three enzymes showed similar biochemical characteristics and exhibited temperature and pH stability. Further research showed that Aly35 and Aly35-CD2 can efficiently degrade alginate, polymannuronate (PM) and polyguluronate (PG) into a series of unsaturated oligosaccharides, while Aly35-CD1 exhibits greater PM-degrading activity than that of Aly35-CD2 but can not degraded PG efficiently. The results suggest that the domain (Trp²⁹⁵-His⁵⁸²) is critical for PG-degrading activity, the domain has (Leu⁵³-Lys²⁸⁶) higher PM-degrading activity, both catalytic domains together confer increased alginate (including M-blocks and G blocks)-degrading activity. The enzyme Aly35 and its truncations Aly35-CD1 and Aly35-CD2 will be useful tools for structural analyses and for preparing bioactive oligosaccharides, especially Aly35-CD1 can be used to prepare G unit–rich oligosaccharides from alginate.

In the context of peptide drug development, glycosylation plays a pivotal role. Accordingly, L-type peptides were synthesized predicated upon the PD-1/PD-L1 blocker ^DPPA-1. Subsequent glycosylation resulted in the production of two distinct glycopeptides, D-glu-^LPPA-1 and D-gal-^LPPA-1, by using D-glucose (D-glu) and D-galactose (D-gal), respectively, during glycosylation. Both glycopeptides significantly inhibited the interaction between PD-1 and PD-L1, and the measured half maximal inhibitory concentrations (IC_50s) were 75.5 μM and 101.9 μM for D-glu-LPPA-1 and D-gal-LPPA-1, respectively. Furthermore, D-gal-LPPA-1 displayed a pronounced ability to restore T-cell functionality. In an MC38 tumor-bearing mouse model, D-gal-LPPA-1 demonstrated a significant inhibitory effect. Notably, D-gal-LPPA-1 substantially augmented the abundance and functionality of CD8⁺ T cells in the tumor microenvironment. Additionally, in the lymph nodes and spleens, D-gal-LPPA-1 significantly increased the proportion of CD8⁺ T cells secreting interferon-gamma (IFN-γ). These strong findings position D-gal-LPPA-1 as a potent enhancer of the antitumor immune response in MC38 tumor-bearing mice, underscoring its potential as a formidable PD-1/PD-L1 blocking agent.