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

Abstract

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°C 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°C, 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 β-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.

Keywords: alginate lyase; alginate oligosaccharide; high-level expression; molecular dynamics simulation; rational design; thermostability.

FIGURE 1

Flexible residues are determined based on the structure. (A) The evolutionary conservation is determined based on the structure and multiple sequence alignment. The amino acid residues are colored in gradient representing the conservation grades from the most variable (turquoise) to the most conserved (maroon) obtained with ConSurf program. (B) Identification of the flexible residue according to the B-factor values of the WT FlAlyA determined from the crystal structure (PDB: 5y33). The top ten sites with B-factor value were selected as mutation sites and marked with red dots. (C) Spatial location of all mutation sites in WT FlAlyA. Mutation amino acid residues are shown as stick models and marked in magenta.

FIGURE 2

Protein thermal shift analysis of Wild-type and mutants. The positions of the black, blue, red and green dashed lines represent the T _m values of WT, H71K, H176D, and Var, respectively.

FIGURE 3

CD characterization of the WT and mutants between 190 and 260 nm. The dotted line indicates the position of the characteristic peaks (195 and 216 nm) of β-sheet.

FIGURE 4

Enzymatic properties of WT and mutants. Effects of temperature on the activity (A) and stability (B) of WT and mutants. Error bars are standard deviation of three replicates.

FIGURE 5

Structural analysis for mutational effects on the thermostability of mutants. Potential hydrogen bonding interactions caused by mutants H71K (A) and H176D (B) are indicated by yellow dashed lines. (C) The effect of mutation on protein B-factor. (D) Changes in surface electrostatic charge distribution caused by mutations. The structures shown are H71K, WT, and H176D from left to right.

FIGURE 6

MD simulations for the wild-type enzyme and mutants. RMSF computed from MD simulations for the wild-type enzyme and mutant H71K (A) and H176D (B) at 300 K for 100 ns. (C) SASA of the WT and mutants H71K and H176D changed with simulation time over 50 ns at 323K.