Figure 8
In addition to the increase of interactions within or between proteins for improving the stability of proteins, the interaction between solvent molecules and proteins in the reaction system is also a key factor. Organic solvents can interact with residues on the surface of enzyme and affect the structure of the enzyme.[8,28] Water molecules attached to the enzyme surface would make the enzyme and the substrate bind better, and maintain enzyme activity and stability via noncovalent interactions.[9,34] Organic solvent will compete with water molecules for enzymes, reducing the surface water layer of enzyme, thereby changing the structure of the protein and changing its stability.[9,38,39] Herein, three mutation sites T23, T200, and P260 obtained by regional epPCR were located in the loop on the surface of the At ATA. The mutated amino acids K200 and S260 are polar amino acids, respectively, and their residues are more easily combined with water molecules to increase the hydration shell on the protein surface and maintain enzyme activity and organic solvent resistance. However, site 23 of polar amino acid threonine on WT was substituted with nonpolar amino acid isoleucine, and the organic solvents stability was not decreased. This might be site 23 is located at the N-terminal of At ATA, the hydrophobic side chains prefer to be buried within the core of the protein rather than been exposed to organic solvents to make the overall structure in a more favorable state.[40]
3.8 Asymmetric synthesis of (R )-(+)-1-(1-naphthyl) ethylamine by recombinant E. coli whole cells expressing WT and M3
Compared with purified enzyme, whole cells as bio-catalyst can reduce the time and cost of purification, simplify the cofactor regeneration system and require no additional expensive external cofactors.[41] Furthermore, whole cells can protect and stabilize the enzymes for resisting severe external environments.[42] Moreover, whole cells as bio-catalyst can simplify the separation and purification of downstream processing for acquiring target products.[43] In order to optimize the amplification experiment using whole cells, the substrate concentration was set as 3~20 mM in 20-mL scale. The substrates of 3~20 mM 1-acetylnaphthalene and 1-(R )-PEA were dissolved in 25% DMSO due to the activity ofAt ATA in 25% DMSO was considerably higher than that in higher concentration DMSO, and the time course of the asymmetric synthesis of (R )-NEA in the presence of 25% DMSO was monitored and compared using 6 g dcw/L recombinant E. coli expressing WT and M3. As shown in Figure 9A, the substrate concentration was 3 mM, the yield of (R )-NEA using WT reached the maximum value of 88.3% within 5 h, while the yield of (R )-NEA using M3 reached the maximum yield of 94.2% after 7 h. With the substrate concentration increased to 5, 10, 15, and 20 mM, the yield of (R )-NEA catalyzed by M3 was higher than that of WT, and the yield catalyzed by both WT and M3 was decreased. All the reactions reached equilibrium within 10 h and the optical purity of (R )-NEA was > 99.5% (Table 5). Overall, improving the tolerance of organic solvents for M3 plays a vital role in asymmetric synthesis (R )-NEA.