Enhancing the alkaline tolerance of Pseudoalteromonas porphyrae κ-carrageenase through surface charge engineering

κ-Carrageenases with good alkaline tolerance are highly desirable for the green resource utilization of κ-carrageenan industrial waste residues. In this study, three mutants D49I, Q100R, and D49I-Q100R with improved alkaline tolerance were identified by optimizing the surface charge distribution of Pseudoalteromonas porphyrae κ-carrageenase KCgCD (WT). After 90 min of incubation at pH 10.0, the residual enzyme activities of D49I, Q100R, and D49I-Q100R increased by 28.4 %, 30.5 %, and 39.2 %, respectively, compared to the wild-type (WT) enzyme. Following 7 days of treatment at pH 10.0, WT lost all activity, whereas D49I, Q100R, and D49I-Q100R retained 63.3 %, 29.8 %, and 91.0 % of their initial activities, respectively. The double mutant D49I-Q100R exhibited an additive beneficial effect from the single mutations. The enhanced alkaline resistance could be attributed to the reduced surface hydrophobicity, increased van der Waals interactions, and a decrease in ΔG_elec. Molecular dynamics simulations further showed that at pH 10.0, the RMSD values of the mutant-κ-neocarratetraose complexes were significantly lower than that of the wild-type complex. Moreover, the binding energies of key residue R150 and the catalytic triad E162-D164-E167 were reduced in the mutant complexes. These changes might contribute to the enhanced binding stability between the mutants and κ-neocarratetraose. These findings provided molecular-level insights into the κ-carrageenase alkaline resistance mechanism. This knowledge would facilitate the resource-efficient application of this enzyme in treating κ-carrageenan industrial waste residues.