Improving the thermostability of alginate lyase FlAlyA with high expression by computer-aided rational design for industrial preparation of alginate oligosaccharides

FlAlyA, a PL7 alginate lyase with industrial potential, is widely applied in the preparation the alginate oligosaccharide because of its high activity of degradation the alginate. However, heat inactivation still limits the industrial application of FlAlyA. To further enhance its thermostability, a group of mutants were designed, according to evaluating the B-factor value and free energy change via computer-aided calculation. 25 single-point mutants and one double-points mutant were carried out by site-directed mutagenesis. The optimal two single-point mutants H176D and H71K showed 1.20 and 0.3? increases in the values of T (m), while 7.58 and 1.73 min increases in the values of half-life (t (1/2)) at 50?, respectively, compared with that of the wild-type enzyme. Interestingly, H71K exhibits the comprehensive improvement than WT, including expression level, thermal stability and specific activity. In addition, the mechanism of these two mutants is speculated by multiple sequence alignment, structural basis and molecular dynamics simulation, which is likely to be involved in the formation of new hydrogen bonds and decrease the SASA of the mutants. These results indicate that B-factor is an efficient approach to improves the thermostability of alginate lyase composed of beta-sheet unit. Furthermore, the highest yield of the mutant reached about 650 mg/L, which was nearly 36 times that of previous studies. The high expression, excellent activity and good thermal stability make FlAlyA a potential candidate for the industrial production of alginate oligosaccharides.

Automated summary

This study reports the enhancement of thermostability of FlAlyA, an alginate lyase with industrial potential. By computer-aided calculation and site-directed mutagenesis, two single-point mutants with better thermostability were optimized and the mechanisms of these mutants were investigated. The expression level of the mutants was greatly increased, making them potential candidates for the industrial production of alginate oligosaccharides.