Improving thermostability of a PL 5 family alginate lyase with combination of rational design strategies.

Alginate lyases with strict substrate specificity possess potential in directed production of alginate oligosaccharides with specific composition. However, their poor thermostability hampered their applications in industry. In this study, an efficient comprehensive strategy including sequence-based analysis, structure-based analysis, and computer-aid ΔΔG _fold value calculation was proposed. It was successfully performed on alginate lyase (PMD) with strict poly-β-D-mannuronic acid substrate specificity. Four single-point variants A74V, G75V, A240V, and D250G with increased T _m of 3.94 °C, 5.21 °C, 2.56 °C, and 4.80 °C, respectively, were selected out. After ordered combined mutations, a four-point mutant (M4) was finally generated which displayed remarkable increase on thermostability. The T _m of M4 increased from 42.25 °C to 51.59 °C and its half-life at 50 °C was about 58.9-fold of PMD. Meanwhile, there was no obvious loss of enzyme activity (more than 90% retained). Molecular dynamics simulation analysis insisted that the improvement of thermostability might be attribute to the rigidified region A which might be caused by the newly formed hydrogen bonds and salt bridges introduced by mutations, the lower distance of original hydrogen bonds, and the more compact overall structures.
Competing Interests: Declaration of competing interest The authors declare that they have no competing interests.
(Copyright © 2023. Published by Elsevier B.V.)