The catalytic efficiency of Bapul-T

The catalytic efficiency of Bapul-TS showed a decrease in kcat/Km
from 1473.2mL/mgs of the WT to 1333.8mL/mgs.
E518I is surrounded by a hydrophobic core that consists of V491,
F494, V516, A520, and A522 (Fig. 6A). Replacing of the hydrophilic
E518 with the hydrophobic I518 might stabilize the hydrophobic
interaction, which is one of the most powerful interactions for protein
stabilization [28,29]. S662R, which was selected because of
the high B-factor, can be attributed to the involvement of the R662
residue in hydrogen bond formation. Hydrogen bonding is a critical
factor that contributes to enzymatic thermostability [30]. In addition
to the hydrogen bonding interaction, electrostatic interaction
among charged residues is considered as another factor that contributes
to the thermostability of S662R. A comparative study of
homolog proteins showed that the percentage of charged amino
acids in thermophilic proteins was larger than that in mesophilic
proteins [31]. Several reports have revealed that the rational optimization
of charge–charge interactions on the protein surface can
be a viable protein stability enhancement strategy [32–34]. However,
electrostatic stabilization is not achieved uniquely, but rather
varies with the protein family [35]. The stabilization of Q706P may
be caused by the reduction in the entropy of the unfolded state
because of the following reasons: (a) proline has a distinctive cyclic
side chain structure that locks its backbone, thereby resulting in
an exceptional conformational rigidity in the turns and loops [36]
and (b) replacing the polar Gln706 with a non-polar proline within
the vicinity of five other hydrophobic residues (F699, P704, V707,
V709, and I710; Fig. 6A) increases the hydrophobic interactions in
this region.