Efficient resolution of (R,S)-1-(1-naphthyl)ethylamine by Candida antarctica lipase B in ionic liquids

Article abstract of DOI:10.1016/j.mcat.2018.01.026

The resolution of (R,S)-1-(1-naphthyl)ethylamine ((R,S)-NEA) by Candida antarctica lipase B (CALB) in ionic liquids (ILs) containing 1-alkyl-3-methylimidazolium cations ([C_nmim]⁺) and [Tf₂N]^?, [BF₄]^?, and [PF₆]^? anions was investigated. When the alkyl chain on the cation contained less than six carbons, the lipase activity corresponded with the hydrophobicity of the ILs, but further increase in the chain length suppressed the enzyme activity. The enzyme activity decreased depending on the anion, where [Tf₂N]^? > [PF₆]^? > [BF₄]^?. The effects of acyl donors, pH, temperature, water activity, and substrate concentration on the resolution were determined. Under the optimal conditions, the conversion of (R,S)-NEA and enantiomer excess of (R)-n-octyl acyl-NEA was 49.3% and 99.2%, respectively. The resolution kinetics of (R,S)-NEA by CALB in [C₆mim][Tf₂N] were studied and a ping-pong mechanism with a two substrate inhibition model was selected. The kinetic parameters of the fitting results were as follows: Michaelis constant of (R,S)-NEA K_ma, 461.8 mmol/L; Michaelis constant of vinyl n-octanoateK_mb, 262.1 mmol/L; inhibition constant of (R,S)-NEA K_ia, 8737.2 mmol/L; inhibition constant of vinyl n-octanoateK_ib, 62336.8 mmol/L; maximum reaction rate r_max, 0.352 mmol/(mg min). Moreover, circular dichroism revealed that incubation of CALB in [C₆mim][Tf₂N] resulted in increased β-sheet content; its secondary structure was stable.

Full text of DOI:10.1016/j.mcat.2018.01.026

MolecularCatalysis448(2018)116–121
Contents lists available at ScienceDirect
Molecular Catalysis
journal homepage: www.elsevier.com/locate/mcat
Efficient resolution of (R,S)-1-(1-naphthyl)ethylamine by Candida antarctica
lipase B in ionic liquids
Bin Wang⁎, Chao Zhang, Qinting He, Hengfei Qin, Guobin Liang, Weiqiao Liu
School of Chemical and Environmental Engineering, Jiangsu University of Technology, Changzhou 213001, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Ionic liquid
Candida antarctica lipase B
Resolution
(R,S)-1-(1-naphthyl)ethylamine
The resolution of (R,S)-1-(1-naphthyl)ethylamine ((R,S)-NEA) by Candida antarctica lipase B (CALB) in ionic
liquids (ILs) containing 1-alkyl-3-methylimidazolium cations ([Cnmim]+) and [Tf2N]−, [BF4]−, and [PF6]−
anions was investigated. When the alkyl chain on the cation contained less than six carbons, the lipase activity
corresponded with the hydrophobicity of the ILs, but further increase in the chain length suppressed the enzyme
activity. The enzyme activity decreased depending on the anion, where [Tf2N]− > [PF6]− > [BF4]−. The
effects of acyl donors, pH, temperature, water activity, and substrate concentration on the resolution were
determined. Under the optimal conditions, the conversion of (R,S)-NEA and enantiomer excess of (R)-n-octyl
acyl-NEA was 49.3% and 99.2%, respectively. The resolution kinetics of (R,S)-NEA by CALB in [C6mim][Tf2N]
were studied and a ping-pong mechanism with a two substrate inhibition model was selected. The kinetic
parameters of the fitting results were as follows: Michaelis constant of (R,S)-NEA Kma, 461.8 mmol/L; Michaelis
constant of vinyl n-octanoateKmb, 262.1 mmol/L; inhibition constant of (R,S)-NEA Kia, 8737.2 mmol/L; inhibi-
tion constant of vinyl n-octanoateKib, 62336.8 mmol/L; maximum reaction rate rmax, 0.352 mmol/(mg min).
Moreover, circular dichroism revealed that incubation of CALB in [C6mim][Tf2N] resulted in increased β-sheet
content; its secondary structure was stable.
1. Introduction
antarctica lipase B (CALB) was used for the resolution of (R,S)-NEA
[9,10]. Compared to transaminase, CALB does not require a coenzyme,
(R)-1-(1-naphthyl)ethylamine ((R)-NEA) is an important pharma-
ceutical intermediate for the preparation of calcimimetic Cinacalcet
Hydrochloride [1,2], and is used as a chiral intermediate for the re-
solution of Pregabalin intermediates [3]. It is also an important raw
material for the preparation of heterogeneous chiral hydrogenation
catalysts [4,5]. Therefore, considerable research has been carried out
on preparation methods for chiral NEA, which can be divided into three
categories. The first is chemical resolution methods; for example, (D)-
tartaric acid was used as a chemical separation agent for the resolution
of (R,S)-NEA [6]. The process was long and the optical purity of the
product was low, and a large amount of chemical waste was generated.
The second category is asymmetric synthesis methods; (R,S)-1-(1-
naphthyl)acetophenone was used for the synthesis of (R)-NEA in the
presence of a chiral catalyst [7]. This process was simple, but the chiral
catalyst was structurally complex, expensive, and its large-scale pro-
duction was difficult.The third category is biological methods; an en-
zyme catalyst transaminase was applied for the preparation of chiral
NEA in the presence of 1-(1-naphthyl)ethanone [8] and Candida
is inexpensive, and has other advantages [11].
Notably, organic solvents were used as reaction media for the re-
solution of (R,S)-NEA by CALB; environmental and safety concerns of
the volatile organic solvents directly affected the enzymatic resolution
of (R,S)-NEA. Ionic liquids (ILs), new reaction media with low saturated
vapor pressures, exhibit incombustibility, hydrophobicity, and ex-
cellent thermal stability [12,13]. Recently, ILs have been reported to
show favorable characteristics as reaction media in enzyme catalysis to
improve enzyme enantioselectivity and thermal stability [14,15].
Therefore, in this study, CALB was selected as the catalyst and ILs were
used as the reaction media for the resolution of (R,S)-NEA for the first
time. The effects of the structure of ILs based on 1-alkyl-3-methylimi-
dazolium ([Cnmim]+) cations and ([Tf2N]−), [BF4]−, and [PF6]− an-
ions on the enzymatic catalytic activity were investigated. Moreover,
the effects of the reaction conditions on the enzymatic activity were
also investigated. Circular dichroism (CD) spectroscopy was used to
measure changes in the secondary structure of CALB in different sol-
vents, and the resolution kinetics of (R,S)-NEA by CALB in [C6mim]
Abbreviations: (R,S)-NEA, (R,S)-1-(1-naphthyl)ethylamine; CALB, Candida antarctica lipase B; ILs, ionic liquids; [Tf2N]−, bis[(trifluoromethyl)sulfonyl]imide; [BF4]−, tetrafluoroborate
anion; [PF6]−, hexafluorophosphoric acid anion; [Cnmim]+, 1-alkyl-3-methylimidazolium cations; CD, circular dichroism
⁎
Corresponding author.
E-mail address: wangbin@jsut.edu.cn (B. Wang).
https://doi.org/10.1016/j.mcat.2018.01.026
Received 2 October 2017; Received in revised form 30 December 2017; Accepted 22 January 2018
2468-8231/©2018ElsevierB.V.Allrightsreserved.

B. Wang et al.
MolecularCatalysis448(2018)116–121
Scheme 1. Route of resolution of (R,S)-NEA by CALB.
[Tf2N] was investigated.
expressed in deg·cm2·dmol−1. A buffer sample containing no protein
was subtracted from all spectra to account for any background signal.
Spectra in all the region were averaged after three accumulations.
2. Experimental
2.5. Water activity
2.1. Materials
The substrates and ILs were separately pre-equilibrated at different
water activities (aw) prior to the resolution of (R,S)-NEA. All samples
were placed in a closed vessel above saturated salts, and equilibrium
was reached overnight at room temperature. The salts used in this test
included Na2HPO4·2H2O/Na2HPO4, aw = 0.17; NaAc·3H2O/NaAc,
aw = 0.28; CuSO4·5H2O/CuSO4·3H2O, aw = 0.42; Na2HPO4·7H2O/
Na2HPO4·2H2O, aw = 0.63; and Na2HPO4·12H2O/Na2HPO4·7H2O,
aw = 0.78.
(R,S)-NEA (98%), ionic liquids[C4mim][BF4], [C4mim][PF6],
[Cnmim][Tf2N] (n = 2,4,6,8,10) were purchased from Alfa Aesar che-
mical Co. Ltd (Shanghai, China). The purity of IL was 99%.
Recombinant lipase B from Candida antarctica from Aspergillus oryzae
(9 U/mg) was obtained from Sigma-Aldrich(Shanghai, China). All other
reagents were of analytical grade, and were obtained from Sinopharm
Chemical Reagent Co. Ltd (Shanghai, China).
2.2. (R,S)-NEA kinetic resolution study
2.6. Dynamic parameter fitting
Unless otherwise indicated, in a typical resolution of (R,S)-NEA by
CALB (Scheme 1), reactions were carried out in a 10 mL flask with a
stopper; 200 mmol/L(R,S)-NEA and 120 mmol/Lvinyl acetate were
added to 5 mL IL (or 5 mL toluene). The reaction was catalyzed in the
presence of 100 mg CALB at 40 °C with shaking at 180 rpm. The water
activities of substrates, solvents and enzyme were not specially con-
trolled. The pH of CALB was also not specially controlled.
The initial enzymatic reaction rates were determined at different
substrate concentrations with certain amounts of (R,S)-NEA
(50 ∼ 400 mmol/L) and vinyl n-octanoate (50 ∼ 240 mmol/L) respec-
tively, according to the method described in Section 2.2. The dynamic
parameters were fitted according to the dynamic model, substrate
concentration, and initial reaction rate using Origin 11.0 software.
3. Results and discussion
2.3. Analysis conditions
3.1. Effect of solvent on resolution of (R,S)-NEA catalyzed by CALB
Aliquots were withdrawn from the reaction mixture at various in-
tervals and the samples were analyzed using HPLC equipped with a
Chiralcel® OJ-H chiral column (250 mm × 4.6 mm, 5 μm) and detected
using UV at 222 nm at 25 °C. The mobile phase was composed of a
mixture of hexane, isopropanol, and ethanol (300:50:0.8, V/V) with a
flow rate of 1 mL/min. The retention times of (R)-NEA and (S)-NEA
were 6.5 and 7.7 min, respectively; the retention time of (R)-n-octyl
acyl-NEA and its enantiomer were 11.4 (R) and 14.6 (S) min, respec-
tively.
As (R,S)-NEA has poor solubility in hydrophilic ILs, several hydro-
phobic ILs with various anions and cations were used. The initial re-
action rates of the resolution of (R,S)-NEA catalyzed by CALB in ILs and
toluene are shown in Table 1. The initial reaction rates of the resolution
by CALB in ILs were lower than that in toluene. Organic solvents, like
toluene, have lower viscosities than ILs [12], thus the enzyme and
substrates have higher mass transfer coefficients in toluene than in ILs.
However, the conversion of (R,S)-NEA was not dominant in toluene
with prolonged reaction times, and the conversion of (R,S)-NEA in
[C6mim][Tf2N] gradually caught up and exceeded that in toluene
(Fig. 1). The ee value of R-n-octyl acyl-NEA remained relatively stable
(95.1%–99.5%) in [C6mim][Tf2N]; however, the ee value of R-n-octyl
acyl-NEA rapidly decreased as the resolution time increased in toluene.
The enantiomeric excess (ee) formulas for (R)c-n-octyl acyl-NEA and
c
− c
+ c
− c
+ c
PR
PS
S
c
R
(S)-NEA were eeP
=
× 100% and eeS =
× 100%, respec-
c
tively. The conversionPoRf cPSwas calculated accordSingRto the changes in
the number of substrates. CPR and cPS were the contents of R and Sn-
octyl acyl-NEA, and cR and cS were the contents of (R)- and (S)-NEA,
respectively. The enantioselectivity was determined using the following
ln[1-c (1+ee )]
p
equation: E =
.
ln[1-c (1-ee )]
p
Table 1
Effect of solvents on resolution of (R,S)-NEA catalyzed by CALB.
2.4. CD spectroscopy
Entry Solvent
Conversion/%
eep/%
E
v0/mmol mg−1 min−1
CALB powder was incubated in [C6mim][Tf2N] (or toluene) at 4 °C
for 2 h, washed with cooled ethyl acetate to remove the IL, and sub-
sequently washed with cooled ether to remove ethyl acetate; the ether
was volatilized at room temperature. The treated CALB powders were
dissolved in phosphate buffered solution (50 mM) to a concentration of
0.1 mmol/L, and the enzyme solution was used for CD measurements.
Spectra were recorded at 4 °C from 260 to 190 nm (0.5 nm increments)
with a 0.1 cm path-length cell, 20 nm/min scan rate, 4 s response time,
1
2
3
4
5
6
7
8
[C4mim][BF4]
0.347
0.377
0.426
0.455
0.494
0.439
78.1
79.8
92.0
88.6
95.1
84.1
86.5
71.2
12
14
49
37
0.025
0.027
0.034
0.045
[C4mim][PF6]
[C2mim][Tf2N]
[C4mim][Tf2N]
[C6mim][Tf2N]
[C8mim][Tf2N]
137 0.052
23
24
11
0.038
0.031
0.056
[C10mim][Tf2N] 0.401
Toluene 0.465
and 2 nm bandwidth. The mean residue ellipticity, [Θ]MR
,
was
E = Enantiomeric ratio, v0 = Initial reaction rate, reaction time 6 h.
117

B. Wang et al.
MolecularCatalysis448(2018)116–121
reaction media (Table 1, entry 5). The reasons for the improvement of
enantioselectivity of enzymes in ionic liquids are multiaspect and
complex. The evaluation of enzyme enantioselectivity in ionic liquids
was carried out with alcohols as the substrates, and the enantioselec-
tivities of lipases, in general, were higher in hydrophobic ionic liquids
than in hydrophilic ionic liquids [25]. The enzyme enantioselectivity
was affected by the hydrophobicity of ILs. However, the lipase-cata-
lyzed enantioselective acylation of 1-phenylethylamine and 2-phenyl-1-
propylamine were investigated in ionic liquids, the enantioselectivity of
the lipase in acylation of 1-phenylethylamine was almost perfect in all
ionic liquids, and the enantioselectivity for acylation of 2-phenyl-1-
propylamine was very low and did not vary much with the nature of the
ionic liquids [26]. The structural difference of substrates accounts for
the enantioselectivity of their enzymatic acylation in ionic liquids. The
resolution of (R,S)-1-phenylethanol catalyzed by a lipase from Pseudo-
monas sp. in [BMIm][Tf2N] remained highly enantioselective with the
E-value of 200 at 55 °C; while in MTBE, the E-value dropped dramati-
cally to 4 [27]. The improvement of enantioselectivity in IL was due to
the high enzyme thermostability in IL. Now the question is why enzyme
shows such high enantioselectivity in ionic liquids. At this time, no
satisfactory answers are available since the enzyme enantioselectivity
could be affected by several factors, including the nature of medium,
the structure of substrate, the enzyme thermostability and so on.
Fig. 1. Time-course curves of resolution of (R,S)-NEA catalyzed by CALB in [C6mim]
[Tf2N] and toluene.
[C6mim][Tf2N] may provide a more moderate catalytic environment
for CALB. [C6mim][Tf2N] is a polar substance; it enhanced the elec-
trostatic interactions between the protein molecules, and thus the
protein was more flexible [16], which was conducive to the catalytic
reaction. However, organic solvents could form hydrogen bonds with
the protein and facilitate weak electrostatic interactions between them;
thus, the protein structure was more rigid [17,18], which explained
why the late conversion in [C6mim][Tf2N] was higher than that in to-
luene.
3.2. Effect of acyl donors on the enzymatic resolution of (R,S)-NEA
The (R,S)-NEA can catalyzed itself amidation reaction with acyl
donor. The self-catalyzed reaction is non-selective, and non-enzymatic
reactions could lead to a decrease in the eep. However, an acyl donor
with a specific structure might suppress the self-catalyzed reaction [28].
As such, acyl donors with varying levels of structural complexity and
steric hindrance were tested, as shown in Table 2. Accordingly, the
conversions of the self-catalyzed amination reaction was reduced from
6.5% to 0%.
The above acyl donors were used for the resolution of (R,S)-NEA,
and the results are shown in Table 3. Higher conversion of the self-
catalyzed amination reaction led to lower eep values of R-n-octyl acyl-
NEA. When vinyl n-octanoate was the acyl donor, the conversion was
49.3%, and the eep was 99.2%. This result suggested that the non-en-
zymatic amidation reaction was one of main factors affecting the re-
solution; structurally complex acyl donors led to lower conversions of
the self-catalyzed amidation reaction.
The effects of alkyl chain length on the cation of the IL on the con-
version of (R,S)-NEA are illustrated in Table 1 (entries 3–7). As the number
of carbon atoms on the cation alkyl chain increased, the hydrophobicity of
the IL increased accordingly. However, the conversion decreased in the
order
of
[C6mim]+ > [C4mim]+ > [C8mim]+ > [C2mim]+
>
[C10mim]+, which was not consistent with the hydrophobicity of the ILs.
This phenomenon might be explained as follows: (1) In the micro-
environment of hydrophobic IL, a small amount of water was needed to
maintain the conformation of the enzyme by hydrogen bonds [19]. The
layer of water around the enzyme had a higher dielectric constant than
that of the ILs and could not be easily separated from the enzyme. This, in
turn, will stabilize the hydrogen bond responsible for the ordered struc-
tures of CALB to display its biological function in ILs [20,21]. (2) The IL
with shorter alkyl chain on the cation has a lower viscosity and the slight
increase of alkyl chain length might raises the viscosity largely [22], and
the reaction rates may be affected by the significant viscosity change. In
general, the less viscous ILs lead to more active enzymes, and this trend is
different from the alkyl chain length in the cation.
Table 2
As shown in Table 1 (entries 1–2, 4), the anions of the ILs also in-
fluenced the CALB activity，and the activity decreased in the order of
[Tf2N]− > [PF6]− > [BF4]−. Bearing in mind the nucleophilicity
order is [Tf2N]− > [BF4]− > [PF6]−, the increase in activity was not
in agreement with the decrease in this parameter. The nucleophilicity of
anions on the activity of CALB for acylation of amines was investigated,
the reaction rate order was found to be following the order of the nu-
cleophilicity of anions [23]. The reason might be the anion with strong
nucleophilicity can interact with the amino acid residues and steady the
structure of enzymes. However, the half-life times of the stability of
CALB in different ILs showed that the half-life times were not only in-
fluenced by the nucleophilicity of anions, but also influenced by the
hydrophobicity of cation [24]. The activity of CALB might be influ-
enced by multiple factors of ILs, including nucleophilicity, hydro-
phobicity, viscosity and so forth.
Non-enzymatic amidation reaction of acyl donors in [C6mim][Tf2N].
Entry
1
Acyl donor
eep/%
0
Conversion/%
6.5
2
3
0
0
4.1
1.5
4
5
6
0
0
0
2.7
1.2
0
The enantioselectivity of CALB in acylation of (R,S)-NEA was also
shown in Table 1. The value for enantioselectivity in ionic liquids was
actually higher than that in toluene, and [C6mim] [Tf2N] was the best
Reaction condition: 5 mL of [C6mim][Tf2N], 200 mmol/L(R,S)-NEA, 120 mmol/Lacyl
donor, reaction time 6 h.
118

B. Wang et al.
MolecularCatalysis448(2018)116–121
Table 4
Effect of temperature on resolution of (R,S)-NEA catalyzed by CALB in [C6mim][Tf2N].
Table 3
Effect of acyl donors on resolution of (R,S)-NEA catalyzed by CALB in [C6mim][Tf2N].
Entry
1
Acyl donor
Conversion/%
49.4
eep/%
95.1
E
T/°C
v0/mmol mg−1 min−1
Conversion/%
eep/%
E
137
30
35
40
45
50
0.046
0.054
0.068
0.074
0.078
32.5
44.0
49.3
46.2
42.1
99.3
99.1
99.2
97.0
95.7
> 400
> 400
> 400
170
2
3
49.0
49.1
96.3
98.4
180
95
> 400
Reaction condition:5 mL of [C6mim][Tf2N], 200 mmol/L(R,S)-NEA, 120 mmol/Lvinyl n-
octanoate, reation time 5 h.
4
5
6
49.4
48.5
49.3
97.2
98.4
99.2
259
temperatures (30–40 °C). At higher temperatures, the enzyme was de-
activated, resulting decreased activity and enantioselectivity. The self-
catalyzed reaction rate of (R,S)-NEA with vinyl n-octanoate was also
accelerated at higher temperatures, which led to decreased enantios-
electivity. At a temperature above 60 °C, the difference between IL and
n-hexane could be explained by the strong protective effect of ionic
liquid against enzyme thermal deactivation described already in lit-
erature [27].
> 400
> 400
Reaction condition: 5 mL of [C6mim][Tf2N], 200 mmol/L(R,S)-NEA, 120 mmol/L acyl
donor, reaction time 6 h.
3.3. Effect of pH on the enzymatic resolution of (R,S)-NEA in [C6mim]
[Tf2N]
3.5. Effect of water activity (aw) on CALB activity in [C6mim][Tf2N]
The water activities of[C6mim][Tf2N], (R,S)-NEA, and vinyl n-oc-
tanoate were controlled by the salts showed in Section 2.5; the effects of
water activity are shown in Fig. 3. Under nearly anhydrous conditions
(aw = 0.17), the initial activity was lower in [C6mim][Tf2N], and the
enzyme activity was the highest when aw = 0.28; the enzyme activity
decreased with further increase in the water activity. Catalytic activity
is related to the water molecules adsorbed on the surface of the protein;
however, under high water activities, the enzyme is easy to gather and
the hydrolysis of vinyl n-octanoate affected the catalytic activity of the
enzyme [10].
In non-aqueous enzymatic catalysis, pH plays an important role on
lipase activity by regulating the amino acid residues in the active site of
the enzyme. The effect of pH on the catalytic activity of the lipase in
[C6mim][Tf2N] was evaluated by varying the pH of the enzymatic so-
lution before lyophilization (Fig. 2). At pH 7, the conversion of (R,S)-
NEA was the highest. Rios et al. [29] found that the synthesis of
ethylene glycol esters by CALB was maximized when the pH was 7.0 in
ILs. Salis et al. [30] found that decreased pH values led to the proto-
nation of Glu341 and His449 residues of Candida rugosa lipase, which did
not facilitate the nucleophilic attack on carbon in the substrate by
oxygen in Ser209, thereby reducing the enzymatic activity.
3.6. Substrate concentration on the enzymatic resolution of (R,S)-NEA in
[C6mim][Tf2N]
3.4. Effect of temperature on the enzymatic resolution of (R,S)-NEA in
[C6mim][Tf2N]
Next, the effect of substrate concentration on the enzymatic re-
solution of (R,S)-NEA was studied (Fig. 4) and the formation rate of (R)-
n-octyl acyl-NEA was calculated (Fig. 4, inset). As the substrate con-
centration increased, the time at which the reaction reached the end
point was prolonged. When the substrate concentration was 400 mmol/
L, the reaction time was about 12 h and the conversion was 49.1%.
However, when the substrate concentration was 300 mmol/L, the for-
mation rate reached the highest 24.7 mmol/(L h).
Next, the effect of temperature on the resolution of (R,S)-NEA in
[C6mim][Tf2N] with CALB was evaluated (Table 4). The initial reaction
rate (v0)increased significantly with increased temperatures and was
the highest at 50 °C. However, the conversion was the highest at 40 °C,
and higher enantioselectivity (> 400) could be achieved at lower
Mutti [8] applied four (R)-ω-transaminases for the asymmetric
Fig. 2. Effect of pH on resolution of (R,S)-NEA catalyzed by CALB in [C6mim][Tf2N].
Reaction condition: 5 mL of [C6mim][Tf2N], 200 mmol/L(R,S)-NEA, 120 mmol/Lvinyl n-
octanoate, reaction time 5 h.
Fig. 3. Initial activities at different water activity in [C6mim][Tf2N]. Reaction condition:
5 mL of [C6mim][Tf2N], 200 mmol/L(R,S)-NEA, 120 mmol/Lvinyl n-octanoate.
119

B. Wang et al.
MolecularCatalysis448(2018)116–121
Table 5
Percentages of secondary structural elements in CALB at solvents calculated from CD
spectra.
Solvent
Secondary structure
α-helix/%
β-sheet/%
β-Turn/%
Random coil/%
None
[C6mim][Tf2N]
Toluene
39.6
38.2
32.7
9.4
10.1
15.2
17.7
17.2
18.1
33.3
34.5
34.0
that of the β-sheet structure increased compared to the native enzyme,
while the other structures remained unchanged. Therefore, it could be
concluded that certain solvents induced the formation of β-sheets from
α-helices.The increase in β-sheet content could be attributed to the
more rigid structure of the treated enzyme [33], which was caused by
the loss of water molecules on the surface of the protein in the solvents.
In other words, enzymes with high α-helical contents might have a
softer structure, which would be responsible for the high enzyme ac-
tivity [34]. Lipase araujiain hI exhibited a significant decrease in en-
zyme activity in organic media, which was accompanied by a sig-
nificant decrease in α-helix content [35]. The deactivation of
Burkholderia cepacia lipase in methanol was also reported to be related
to conformational changes [36].
Fig. 4. Effect of substrate concentration on resolution of (R,S)-NEA catalyzed by CALB in
[C6mim][Tf2N]. Experimental results of conversion at: (■) = 100 mmol/L;
(●) = 200 mmol/L; (▲) = 300mmol/L; (▼) = 400 mmol/L, experimental results of eep
at: (□) = 100 mmol/L−1; (○) = 200 mmol/L; (△) = 300 mmol/L; (▽) = 400 mmol/L.
amination of ketones leading to optically pure (R)-amines, including the
(R) -NEA. However, the concentration substrate was 1 mmol/L. Ditrich
[9] applied lipase B from Candida antarctica for the resolution of NEA,
and the concentration substrate was 100 mmol/L. In this paper, the
resolution of NEA was carried out in 300 mmol/L with eep of 49.3% and
yield of 99.2%.
3.8. Dynamic parameter fitting
The resolution reaction kinetics of (R,S)-NEA by CALB in [C6mim]
[Tf2N] were evaluated. Due to the transesterification reaction catalyzed
by lipase with a ping-pong mechanism, a two substrate inhibition
model was selected and the kinetic parameters were fitted; the kinetic
model was as follows [37]:
3.7. CD spectroscopy
CD spectroscopy is one of the most convenient and efficient means
of detecting the secondary structure of a protein, and is a convenient,
sensitive, and time-saving method to explore the conformation stability
of a protein in various solvents [31]. As shown in Fig. 5, all permuted
proteins maintained a mixed α-helix/β-sheet spectral signature, and the
native enzyme showed two negative peaks at 208 and 222 nm, and a
positive peak at 192 nm. Upon treatment with [C6mim][Tf2N] and to-
luene, the mean residue ellipticity (Θ) of CALB at 192 nm was con-
sistently lower, and the intensity of the minima at 208 and 222 nm
decreased as compared to that of the native enzyme. The interpretation
of the data was confirmed using secondary-structure models of the MRE
curves byDICHROWEB (Table 5).
The minima at 208 and 222 nm and maximum at 192 nm were
thought to correspond to helical structures, and the absolute values of
these points were closely related to the percentage of helical residues in
the protein [32]. As shown in Table 4, upon warming the CLAB solution
in [C6min][Tf2N] and toluene, the percentage of α-helix decreased and
rmax cVIccNEA
r =
c
VI
cVIcNEA + KmbcNEA (1 + NEA ) + Kma cVI (1 +
)
K
ib
K
ia
where r is the reaction rate, rmax is the maximum reaction rate; Kma and
Kmb are Michaelis constants for (R,S)-NEA and vinyl n-octanoate, re-
spectively; Kia and Kib are inhibition constants of (R,S)-NEA and vinyl n-
octanoate respectively; and cNEA and cVI are the substrate concentra-
tions of (R,S)-NEA and vinyl n-octanoate, respectively.
The kinetic parameters of the fitting results were as follows:
Kma = 461.8 mmol/L, Kmb = 262.1 mmol/L, Kia = 8737.2 mmol/L,
Kib = 62336.8 mmol/L, and rmax = 0.352 mmol/(mg min). These re-
sults indicated that the enzymatic reaction in [C6mim][Tf2N] was
mainly inhibited by the substrate (R,S)-HMPC and substrate inhibition
by vinyl n-octanoate was relatively small, suggesting that increasing the
concentration of vinyl n-octanoate could promote the lipase-catalyzed
reaction rate.
4. Conclusion
The effect of ILs on the activity of CALB in the resolution of (R,S)-
NEA was investigated. The alkyl chain length on the cation and type of
anion influenced the activity of CALB. The enzyme activity as a function
of cation decreased as follows: [C6MIM]+ > [C4MIM]+ > [C8MIM]+
> [C2MIM]+ > [C10MIM]+, which was not completely in accord
with the hydrophobicity of the ILs, and the best anion was [Tf2N]−. The
resolution of NEA by CALB in [C6mim][Tf2N] was also influenced by
the acyl donor, pH, temperature, water activity, and substrate con-
centration; the optimal conditions were as follows: vinyl n-octanoate,
pH 7.0, 40 °C, water activity = 0.28, and substrate concentra-
tion = 300 mmol/L. Under the optimal conditions, the conversion was
49.3% and ee value of R-n-octyl acyl-NEA was 99.2%. The resolution
reaction kinetics of (R,S)-NEA by CALB in [C6mim][Tf2N] were de-
termined using a ping-pong mechanism with a two substrate inhibition
model. The kinetic parameters of the fitting results were
Fig. 5. Far-UV circular dichroism spectra of CALB in different solvents.
120

B. Wang et al.
MolecularCatalysis448(2018)116–121
Kma = 461.8 mmol/L, Kmb = 262.1 mmol/L, Kia = 8737.2 mmol/
L, Kib = 62336.8 mmol/L, and rmax = 0.352 mmol/(mg·min). CD spec-
troscopy revealed that the secondary structure of CALB in [C6mim]
[Tf2N] was slightly different than that of native lipase.
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