Discovery and characterization of a novel mannanase targeting Porphyra insoluble mannan and its application in the liquefaction of Porphyra

Porphyra is rich in various bioactive polysaccharides and holds great potential for high-value applications. Nevertheless, the insoluble β-1,4-mannan in its cell wall constitutes a structural barrier that limits the efficient extraction of these polysaccharides. This study aims to discover and characterize a Porphyra mannanase to improve the enzymatic saccharification of Porphyra, thereby providing a tool enzyme for its high-value extraction and industrial application. Through database mining and sequence analysis, a potential Porphyra mannanase was obtained, followed by heterologous expression and purification. The enzyme was systematically characterized for its optimal temperature, optimal pH, stability, metal ion tolerance, and kinetic parameters. The hydrolysis products were analyzed by high-performance liquid chromatography to determine the hydrolysis pattern and product profile. The effectiveness of the enzyme in enhancing the liquefaction of Porphyra biomass was evaluated in a multi-enzyme system. The multidomain mannanase Man5_8cb from Clostridium bornimense exhibited obvious degrading activity towards Porphyra mannan. Its optimal reaction conditions were 45  ℃ and pH 7.5, and it showed good stability below 40  ℃ and at pH 5~10. The enzyme acted as a random endo-mannanase, producing mainly di- and trisaccharides. In a multi-enzyme system for Porphyra saccharification, the addition of Man5_8cb increased the liquefaction rate by approximately 8 %, confirming that it effectively cooperates with other enzymes to degrade the cell wall polysaccharides. The presence of a carbohydrate-binding module significantly improved the thermal stability, substrate affinity, catalytic efficiency, and tolerance to some inhibitory ions. Man5_8cb exhibited efficient and stable degradation activity towards Porphyra mannan and significantly improved the liquefaction rate of Porphyra. This enzyme provides a powerful tool for the efficient bioprocessing of Porphyra and related algal resources.