Alginate lyases are important biocatalysts for the preparation of bioactive alginate oligosaccharides from brown algae. However, the application of currently available alginate lyases is often constrained by insufficient thermostability and catalytic efficiency. In this study, a PL39 family alginate lyase, designated Algtm3, was identified from the thermophilic deep-sea bacterium Rhodothermus profundi (type strain PRI 2902ᵀ = DSM 22212ᵀ = JCM 15944ᵀ) through genome mining, heterologously expressed in Escherichia coli, and systematically characterized. Algtm3 exhibited optimal activity at 70 °C and pH 8.0, and retained more than 90% of its initial activity after incubation at 65 °C for 60 min, indicating excellent thermal stability. Domain truncation analysis revealed that the Chondroitin AC/alginate lyase superfamily domain and the Hepar_II_III catalytic domain are essential for maintaining proper protein folding, soluble production, and catalytic activity. In contrast, deletion of the C-terminal T9SS sorting domain improved enzyme performance in the heterologous expression system, with the truncated variant Algtm3dc exhibiting a 1.98-fold higher activity than the wild-type enzyme. Structural modeling and molecular docking analyses suggested that C-terminal truncation preserved the catalytic core while inducing local changes in the substrate-binding region, which may contribute to enhanced catalytic efficiency. TLC and ESI-MS analyses demonstrated that Algtm3 could degrade sodium alginate, polyM, and polyG, producing alginate oligosaccharides mainly with degrees of polymerization ranging from 1 to 4, and exhibited both endolytic and exolytic degradation characteristics. Collectively, these findings provide insights into the domain organization and structure-function relationship of PL39 family alginate lyases and highlight the potential of Algtm3 as a thermostable biocatalyst for the efficient production of functional alginate oligosaccharides.
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