Figure 4
3.3 Determination of organic solvent resistance against different DMSO concentrations
The organic solvent resistance of WT and the “best” mutant M3 against different DMSO concentrations was performed. The purified enzymes of WT and M3 were incubated in 25%, 35%, and 45% DMSO. The samples were incubated for 1~500 min and collected for activity measurement. Under 25% DMSO, the half-life (t 1/2) of M3 was 114.4 min, which was 61.6 min longer than WT (52.8 min). As the DMSO concentration increased to 35% and 45%, the t 1/2 of WT decreased to 48.7 and 13.5 min, respectively, whereas M3 was decreased to 75.6 and 19.4 min, respectively (Table 1). The t 1/2 values of WT and M3 suggested that the substitutions of T23I, T200K, and P260S were beneficial for At ATA overall structure against DMSO concentration. The specific activities of M3 in 25%, 35%, and 45% of DMSO were higher than WT. The phenomenon of activity-stability trade-off was off-target, whereas variant M3 displayed co-evolution toward activity and DMSO tolerance. In general, the residues affecting stability often appear in the loop of protein surface, and the residues affecting activity are often located near the active center. However, in many other cases of trade-off escape between activity and stability, the location of beneficial substitution is far from enzyme active site.[31-33] The beneficial residue position 23, 200, and 260 in At ATA with improving the organic solvents stability, locate at loop region of surface that are far from active center, which may explain why M3 enhanced organic solvent stability without sacrificing activity.