Expression and characterization of a porphyranase from marine bacterium Wenyingzhuangia fucanilytica and its application in compound enzymolysis of Pyropia

Pyropia is one of the most commercially important marine algae. Pyropia cultivation of China ranks first in the world, but the production value remains low. Porphyran is the major polysaccharide from Pyropia. It consists of alternating 4-linked α-L-galactopyranose-6-sulfate (L6S) residues and 3-linked β-D-galactopyranose (G) residues. Porphyran and its degradation products were confirmed to have various bioactivities, which is considered as an important way to realize the high-value application of Pyropia. In order to explore the glycoside hydrolase for degrading porphyran and verify the feasibility of its application, here, a porphyranase coding gene por16B was cloned from the marine bacterium Wenyingzhuangia fucanilytica and heterologously expressed in Escherichia coli. The biochemical properties and hydrolysis patterns of the recombinant protein Por16B_Wf were characterized. Por16B_Wf exhibited maximum activity at 40 °C and pH 7.0, and it possessed stable activity under different pH conditions. Por16B_Wf hydrolyzed porphyran by an endo-acting manner. The end products of Por16B_Wf were mainly composed of porphyran disaccharides L6S-G. The subsite specificity of Por16B_Wf was clarified by glycomics strategy. It specifically hydrolyzed the β-1,4 glycosidic linkage of G-L6S, whereas it tolerated 3,6-anhydro-α-L-galactopyranose and methyl-D-galactose in subsites –2 and +2, respectively. Furthermore, Por16B_Wf was applied in the compound enzymolysis of Pyropia. Compared with the classical enzymolysis technique of Pyropia using neutral protease, the compound enzymatic hydrolysate was characterized with low viscosity (8.5 Pa·s) and high conversion rate (79.4%±0.3%) of Pyropia. Por16B_Wf could be utilized as a promising tool for the degradation of porphyran. In this study, the compound enzymolysis of Pyropia was realized for the first time, which was beneficial to the full development of Pyropia, and provided new insights into the deep-processing and high-value utilization of Pyropia.