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.