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.