Rational design for thermostability improvement of a novel PL-31 family from Paenibacillus YN15

Currently, alginate oligosaccharides (AOS) become attractive due to their excellent physiological effects. AOS has been widely used in food, pharmaceutical, and cosmetic industries. Generally, AOS can be produced from alginate using alginate lyase (ALyase) as the biocatalyst. However, most ALyase display poor thermostability. In this study, a thermostable ALyase from Paenibacillus sp. YN15 (Payn ALyase) was characterized. It belonged to the polysaccharide lyase (PL) 31 family and displayed poly beta-D-mannuronate (Poly M) preference. Under the optimum condition (pH 8.0, 55 degrees C, 50 mM NaCl), it exhibited maximum activity of 90.3 U/mg and efficiently degraded alginate into monosaccharides and AOS with polymerization (DP) of 2-4. Payn ALyase was relatively stable at 55 degrees C, but the thermostability dropped rapidly at higher temperatures. To further improve its thermostability, rational design mutagenesis was carried out based on a combination of FireProt, Consensus Finder, and PROSS analysis. Finally, a triple-point mutant K71P/Y129G/S213G was constructed. The optimum temperature was increased from 55 to 70 degrees C, and the Tm was increased from 62.7 to 64.1 degrees C. The residual activity after 30 min incubation at 65 degrees C was enhanced from 36.0 % to 83.3 %. This study provided a promising ALyase mutant for AOS industrial production.

Automated summary

In this study, a thermally stable alginate lyase (ALyase) from Paenibacillus sp. YN15 (Payn ALyase) was characterized. Rational design mutagenesis was carried out to improve its thermostability, resulting in a triple-point mutant with increased optimum temperature and residual activity. This study provides a promising ALyase mutant for the industrial production of alginate oligosaccharides (AOS).