Figure 1
3.2 Identification of At ATA key amino acid sites for improving
organic solvents stability via regional epPCR
The crystal structure of At ATA-WT has been reported (PDB ID:
4CE5) and it is a homodimer with a substrate-binding pocket located in
the dimer interface.[22] In each monomer, there
are three main loop regions on the surface and one of the three loop
regions is located at the homodimer interface. Many literatures suggest
that multimer interfaces of enzymes are essential for their stability
since the interfaces are more hydrophobic and form a number of
inter-H-bonds and hydrophobic interactions between subunits for
affecting the overall function of protein.[29]Besides, loop regions in the surface-exposed position of enzyme
represent a potential targeting hot location for improving organic
solvents tolerance, since enzyme requires some essential water to bound
to the surface of enzyme for exhibiting conformational flexibility and
maintaining the enzyme structure intact via
H-bonds.[28,30] Subsequently, regional epPCR
covering amino acid residues at the three main loop regions was designed
and conducted (Figure 2). The residue methionine (Met) at position 1 is
not considered since it is translated from the initiation codon. Three
epPCR libraries were generated by adding 0.1 mM MnCl2 on
the regional At ATA gene (from the 2nd to
30th containing a segment of α-helix,
75th to 105th,
197th to 261st bp) usingAt ATA-WT as template. High-throughput screening was performed
using 4-nitrophenylethylamine and 1-acetylnaphthalene as substrates. As
shown in Figure 3, about 9600 clones (96-well per plate × 100 plates)
were constructed to assay the residual activity, which 95.8% (9196
clones) lost or not detected their activity, and 404 clones remained
activity. Notably, four mutants containing A13V/T200K, L91P/T200K, T23I,
and P260S exhibited improved residual activity against high
concentration DMSO (incubated in
80% DMSO, v/v ), which were 60.7%, 59.2%, 55.3%, and 64.5%
higher than WT, respectively. Furthermore, the mutants obtained from
epPCR were re-screened at the optimal DMSO concentration (25% DMSO,v/v , Figure 3B), all four mutants were acquired with enhanced
activity and stability against DMSO, and the single mutant T23I obtained
the highest activity enhancement. Four mutants contained 5 amino acid
residues (A13, T200, L91, P260, and T23). Combinatorial mutations were
performed to obtain a “best” mutant against high concentration DMSO.
As shown in Figure 4, the residual activities of single-, double- and
triple-mutant incubated in different concentrations of DMSO were
assayed. With the DMSO concentration increasing, the residual activity
of WT and mutants decreased gradually. Compared with WT, double-mutant
T200K/P260S and triple-mutant L91P/T200K/P260S, T23I/T200K/P260S
displayed higher activities under different DMSO concentrations. And
under 15~45% DMSO concentration, the “best”
triple-mutant T23I/T200K/P260S exhibited the highest activity against
DMSO. T23I/T200K/P260S was set as the research object in the following
work.