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affect the overall secondary structure of an enzyme (Figure S5).
It was expected that the incorporation of (4R)-FP into w-TA
would increase the stability of the enzyme. To validate the hy-
pothesis, melting point (Tm) of enzyme and its variants were
examined through differential scanning fluorimetry (Figure S6).
The w-TA and its NCAA variants showed two Tm values; here
we depicted it as Tm1 and Tm2. In the case of the wild-type w-
TA and w-TAdopa, both exhibit similar Tm values whereas the
(4R)-FP incorporated variants share similar Tm values. The Tm1
and Tm2 of the wild type w-TA and w-TAdopa are ꢀ65 and
ꢀ748C, respectively. Similarly, Tm1 and Tm2 of w-TA[(4R)-FP]
and w-TAdp[(4R)-FP] are ꢀ77 and ꢀ898C, respectively
(Table S5). Earlier, it was reported that the fluorophenylalanine
incorporated phosphotriesterase showed an slight increase in
melting temperature (ꢀ28C) when compared to wild-type.[12]
In this study, w-TA[(4R)-FP] and w-TAdp[(4R)-FP] showed en-
hanced Tm greatly compared with the wild-type w-TA and w-
TAdopa. Furthermore, the thermal stability of the enzyme was
examined based on enzyme activity under different tempera-
tures. The residual activities of w-TA and its variants were ex-
amined after 1 h incubation in 100 mm Tris/HCl buffer (pH 8.0)
containing 100 mm PLP at different temperatures. w-TA[(4R)-FP]
and w-TAdp[(4R)-FP] showed higher residual activity than w-TA
and w-TAdopa (Figure S7). The half-lives of the enzymes were
then measured in the presence of 100 mm PLP at 50, 60, and
708C (Table 1 and Figure S8). At all the temperatures indicated
Scheme 1. Engineered transaminase (w-TAdp[(4R)-FP]) containing multiple
noncanonical amino acids such as fluoroproline (red star) and DOPA. Fluoro
moiety of fluoroproline enhances the stability of protein and catechol
moiety of DOPA facilitates the site specific immobilization onto chitosan or
polystyrene beads (blue sphere).
have been developed and extensively studied.[10] Furthermore,
considerable effort has been spent to develop a tool for the
immobilization of biocatalyst onto a solid support system, be-
cause this would offer many advantages, such as biocatalyst
reusability, easy separation of the product and stabilization of
the enzymes.[11] Recently, to improve the biocatalytic synthesis
of chiral amines, (R)- and (S)-w-TA were immobilized onto chi-
tosan beads through glutaraldehyde treatment that showed
improved thermal stability and higher conversion.[11d,h] Howev-
er, by using this approach, the enzyme cannot be immobilized
onto a solid support in a site-specific manner, which is desira-
ble for enzyme immobilization. So here we aimed to utilize site
specific immobilization of w-TA through site-specific incorpora-
tion of DOPA.
Table 1. Half-lives of w-TA and its variants at different temperature.
Enzyme
508C [h]
608C [h]
708C [h]
To achieve this, w-TA from Sphaerobacter thermophilus (YP_
0033191071) was chosen for the incorporation of FP and
DOPA. Our first method involved the incorporation of both
(4S)-FP and (4R)-FP into w-TA by the residue-specific incorpora-
tion method. The whole cell protein expression profile clearly
showed that the w-TA expressed with (4S)-FP and (4R)-FP was
comparable with w-TA expressed with canonical amino acids.
However, the most of the w-TA were in insoluble form when
incorporated with (4S)-FP. Conversely, the incorporation of
(4R)-FP into w-TA favored to form soluble protein, comparable
with the parent w-TA (Figure S1). Therefore, (4R)-FP was
chosen for the residue-specific incorporation into w-TA. For the
site-specific incorporation of DOPA into w-TA, an amber codon
(TAG) was introduced at the surface exposed N-terminal resi-
due (Arg23). Under optimized conditions, (4R)-FP and DOPA
were incorporated into w-TA by coupling method.[3] Electro-
spray ionization-tandem mass spectrometry (ESI-MS/MS) analy-
sis of the tryptic digested fragments confirmed the presence
of (4R)-FP in w-TA[(4R)-FP] and w-TAdp[(4R)-FP] (Figure S2,
Table S1 and S2). In addition, redox cycling staining with nitro-
blue tetrazolium confirmed the presence of DOPA in w-TAdopa
and w-TAdp[(4R)-FP] (Figure S3, Table S3). Based on the gel per-
meation chromatography analysis, it is clear that w-TA and w-
TAdp[(4R)-FP] exist as dimeric structure (Figure S4). CD profiles
of w-TA and its NCAA variants showed similar kind of sharp
negative major deflection pattern at 210–220 nm. Based on
these result, it is clear that the incorporation of NCAA does not
w-TA
19.5
31.0
19.4
29.5
8.1
11.5
7.5
2.2
4.5
2.1
4.4
w-TA[(4R)-FP]
w-TAdopa
w-TAdp[(4R)-FP]
11.0
Half-lives of w-TA and its variants were measured in 100 mm Tris/HCl
buffer (pH 8.0) containing 100 mm PLP at 50, 60 and 708C.
above, w-TA[(4R)-FP] and w-TAdp[(4R)-FP] showed enhanced
thermal stability. For example, the half-lives of w-TA, w-TA[(4R)-
FP], w-TAdopa and w-TAdp[(4R)-FP] at 708C were 2.2, 4.5, 2.1,
and 4.4 h, respectively (Table 1). These results clearly showed
that incorporation of (4R)-FP enhanced the thermal stability of
w-TA.
As co-solvent is often added to the w-TA reaction mixture to
dissolve the substrate,[10] the effects of different solvents on
enzyme activity were examined. The relative activity of the en-
zymes was analyzed in the presence of different water miscible
solvents [ethyl acetate, acetonitrile, and dimethyl sulfoxide
(DMSO)] and water immiscible solvents (toluene, benzene, and
hexane). In all the cases, w-TA[(4R)-FP] and w-TAdp[(4R)-FP]
showed better activity than w-TA and w-TAdopa (Figure 1).
Overall, the replacement of Pro residues with (4R)-FP in w-TA,
conferred enhanced thermal stability and higher activity in or-
ganic solvents. Similarly, recent reports demonstrated that the
incorporation of NCAA enhances the activity of lipase even in
ChemCatChem 2015, 7, 417 – 421
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