Improving the thermostability of a novel PL-6 family alginate lyase by rational design engineering for industrial preparation of alginate oligosaccharides

Alginate is degraded into alginate oligosaccharides with various biological activities by enzymes. However, the thermostability of the enzyme limits its industrial application. In this study, a novel PL-6 alginate lyase, AlyRm6A from Rhodothermus marinus 4252 was expressed and characterized. In addition, an efficient comprehensive strategy was proposed, including automatic design of heat-resistant mutants, multiple computer-aided AAGfold value calculation, and conservative analysis of mutation sites. AlyRm6A has naturally high thermostability. Compared with the WT, T43I and Q216I kept their original activities, and their half-lives were increased from 3.68 h to 4.29 h and 4.54 h, melting point temperatures increased from 61.5 degrees C to 62.9 degrees C and 63.5 degrees C, respectively. The results of circular dichroism showed that both the mutants and the wild type had the characteristic peaks of j3-sheet at 195 nm and 216 nm, which indicated that there was no significant effect on the secondary structure of the protein. Molecular dynamics simulation (MD) analyses suggest that the enhancement of the hydrophobic interaction network, improvement of molecular rigidity, and denser structure could improve the stability of AlyRm6A. To the best of our knowledge, our findings indicate that AlyRm6A mutants exhibit the highest thermostability among the characterized PL-6 alginate lyases, making them potential candidates for industrial production of alginate oligosaccharides.