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.