Enantioselectivity and catalysis improvements of Pseudomonas cepacia lipase with Tyr and Asp modification

Article abstract of DOI:10.1039/c5cy00110b

A concise strategy to improve the p-NPP (p-nitrophenyl palmitate) catalytic activity and enantioselectivity towards secondary alcohols of Pseudomonas cepacia lipase (PcL) has been described. The PcL was modified by I₃^-, N-acetyl imidazole (NAI), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and ethylenediamine (EDA) in the absence or presence of n-hexane, respectively. After being modified by the four modification reagents, the enantioselectivity (E value) of the PcL towards secondary alcohols was enhanced by 2- to 4-fold. The catalytic activity of EDA-PcL was increased by about 6-fold. The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of modified PcL showed that Tyr⁴, Tyr²⁹, Tyr⁴⁵, Tyr⁹⁵, Asp³⁶ and Asp⁵⁵ were the modified sites. When Tyr²⁹ was modified, the E value of PcL towards secondary alcohols was largely improved. MALDI-TOF-MS characterization and molecular dynamics simulation of the lipase indicated that Tyr²⁹ located inside the catalytic cavity had a significant impact on the E value. The strong steric hindrance of acetyl and iodine ion to the groups on the chiral center of the substrates is responsible for the improvement. In addition, the enhancement of hydrophobicity on the surface of the lipase due to the sidechain replacement of Asp with uncharged hydrophobic groups also improved the E value.

Full text of DOI:10.1039/c5cy00110b

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Enantioselectivity and catalysis improvements of
Pseudomonas cepacia lipase with Tyr and Asp
modification†
Cite this: Catal. Sci. Technol., 2015,
5, 2681
b
b
b
a
Jing Li,ab Lei Yue, Chang Li, Yuanjiang Pan and Lirong Yang
*
*
A concise strategy to improve the p-NPP IJp-nitrophenyl palmitate) catalytic activity and enantioselectivity
towards secondary alcohols of Pseudomonas cepacia lipase (PcL) has been described. The PcL was modi-
fied by I3−, N-acetyl imidazole (NAI), 1-IJ3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and ethyl-
enediamine (EDA) in the absence or presence of n-hexane, respectively. After being modified by the four
modification reagents, the enantioselectivity (E value) of the PcL towards secondary alcohols was enhanced
by 2- to 4-fold. The catalytic activity of EDA-PcL was increased by about 6-fold. The matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of modified PcL showed
that Tyr4, Tyr29, Tyr45, Tyr95, Asp36 and Asp55 were the modified sites. When Tyr29 was modified, the E value
of PcL towards secondary alcohols was largely improved. MALDI-TOF-MS characterization and molecular
dynamics simulation of the lipase indicated that Tyr29 located inside the catalytic cavity had a significant
impact on the E value. The strong steric hindrance of acetyl and iodine ion to the groups on the chiral cen-
ter of the substrates is responsible for the improvement. In addition, the enhancement of hydrophobicity
on the surface of the lipase due to the sidechain replacement of Asp with uncharged hydrophobic groups
also improved the E value.
Received 24th January 2015,
Accepted 17th February 2015
DOI: 10.1039/c5cy00110b
www.rsc.org/catalysis
Chemical modification is an alternative choice besides
DNA-based techniques, which are typically limited to the 20
Introduction
Enantiomerically pure chemicals, such as secondary alcohols,
play significant roles in pharmaceuticals, agrochemicals,
flavors and fragrances.1,2 Biocatalysis is an economic and
green approach for chiral resolution of a racemic mixture,3 in
which lipase (triacylglycerol ester hydrolases, EC 3.1.1.3) is
among the most attractive targets.4–7 Kazlauskas et al. investi-
gated lipase-catalyzed resolution towards 94-pair enantiomers
of secondary alcohols and proposed the well-known empirical
law of lipase's enantiopreference: the enzyme's stereospecific
catalytic cavity contains large and small pockets, and the pre-
ferred enantiomer's large and small groups on the chiral cen-
ter fit better into the corresponding pocket.8 However, when
the substituents on the chiral center are not suitable for the
corresponding pocket, the yields of optically pure compounds
are usually not optimal.4,6
primary proteinogenic amino acids. Chemical modification
of enzymes allows an almost unlimited variety of groups to
be introduced with easily obtained reagents and succinct
operations.9,10 Therefore, chemical modification of proteins
has long been utilized to investigate proteins and is a rapidly
expanding area in chemical biology. Previous studies showed
that chemical modification could change substrate prefer-
ence and specificity,11–13 affect the activity,14 identify key
amino acid residues15–17 and improve stability and resistance
to proteases.18–20 The enantioselectivity (E value) of lipases
from Candida rugosa and Pseudomonas cepacia (CrL and PcL)
increased by 2- and 1.3-fold, respectively, as Kim et al.21 and
Ueji et al.22 described, with no significant improvement or
negative effect on the catalytic activity of lipases. In another
case, Bianchi et al. decreased the E value of PcL by free
amino group acetylation coupled with an enhanced catalytic
activity.23 A recent work improved the E value by 1.9- and 2.7-
fold through immobilization of CrL on two types of costly
nanoparticles with the catalytic activity improved subtly.24
We previously proposed a mechanism of enantioselectivity
towards primary alcohols by lipase from Pseudomonas
cepacia. Chemical modification on the lipase and molecular
dynamics simulations indicated that Tyr29 located within the
catalytic cavity affected the enantioselectivity significantly via
a Institute of Biological Engineering, Department of Chemical & Biological
Engineering, Zhejiang University, Hangzhou 310027, Zhejiang, China.
E-mail: lryang@zju.edu.cn; Fax: +86 57187952363; Tel: +86 57187952363
b Department of Chemistry, Zhejiang University, Hangzhou, 310027, Zhejiang,
China. E-mail: panyuanjiang@zju.edu.cn; Fax: +86 57187951629;
Tel: +86 57187951264
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c5cy00110b
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the hydrogen bond with the Onon-α of the substrate.25 In the
present study, a combination of chemical modification,
molecular dynamics simulation and MALDI-TOF-MS was
employed to shed light on effects of chemical modification
on the p-NPP catalytic activity and enantioselectivity towards
secondary alcohols of PcL. Enantioselectivity catalyzed by
modified PcL towards 5 pairs of different chiral secondary
alcohol esters was improved by 2- to 4-fold and the catalytic
activity of EDA-PcL increased by about 6-fold. Since lipases
are widely known to be activated at the aqueous–hydrophobic
interface, n-hexane was employed as the activator of PcL in
this study.26,27
nitrophenol (p-NP) released as a result of enzymatic hydroly-
sis of p-NPP at 410 nm. The enzyme activity was defined
as the amount of protein liberating 1 μmol of p-nitrophenol
per minute.
Hydrolysis of racemic esters
A mixed solution of PcL (10 mg mL−1), substrates (esters of
secondary alcohols, 100 mM) and PBS (100 mM, pH 7.0,
including 10% V/V isopropanol as co-solvent) was incubated
at 27 °C under 200 rpm stirring. All the conversion ratios
were controlled between 10% and 20%.31 The substrate and
the product were extracted from the reaction mixture with
equal volume of n-hexane and dried with sodium sulfate
IJNa2SO4), then analyzed by gas chromatography (GC).
Experimental
Materials
Gas chromatography
Butan-2-ol, hexan-2-ol, octan-2-ol, NAI, NaI, I2, EDA and EDC
(Fig. 1) were purchased from Tokyo Chemical Industry Co.
Ltd. Trypsin and PcL were obtained from Amano Enzyme Inc.
Other chemical reagents were of analytical grade.
All the samples extracted from the hydrolysis reaction were
injected into an Agilent 6890® GC instrument (Agilent Inc.)
equipped with a CP-cyclodextrin-β-2,3,6-M-19, FS 50 m ×
0.25 mm column with a film thickness of 0.25 μm (Varian
Inc.) and a flame-ionization detector for analysis. Nitrogen
was utilized as the carrier gas. Both of the injection and
detector temperatures were 260 °C.
Synthesis of substrates
Esters of secondary alcohols were synthesized from their cor-
responding alcohol in our lab following the procedure
given.25 Alcohol and triethylamine (molar ratio = 1 : 1.5) were
mixed in an ice bath. Acyl chloride was slowly added next.
The final products were washed with saturated sodium bicar-
bonate solution several times. The pure products were
obtained by reduced pressure distillation.
One-dimension SDS-PAGE and lipase digestion with trypsin
in a gel
SDS-PAGE was performed according to the method of Das.32
After recovery and decolorization of the lipase, 40 ng of trypsin
was added. Digestion was performed at 37 °C overnight. The
peptides were extracted by water with 5% TFA, then lyophilized
using a Labconco FreeZone 18 freeze drier (Labconco Corpora-
tion, Kansas City, MO, USA) vacuum concentrator centrifuge.
Chemical modification of Tyr and Asp/Glu
−
Modification of Tyr was carried out using NAI and I3 in the
procedure described by Cacace et al.28 Asp/Glu modification
was performed by employing EDA and EDC following the
strategies of Barbosa.29 According to the standard reaction
conditions of the chemical modification methods, 10% IJV/V)
of n-hexane was added to the reaction mixture as the activator.
MALDI-TOF-MS analysis of modified PcL
All MALDI-TOF-MS analyses were performed using a Bruker
ultrafleXtreme™ TOF/TOF system mass spectrometer
equipped with a modified Nd:YAG laser in positive ion mode,
with data acquisition using the Bruker flexControl 3.4
(Germany). Saturated α-cyano-4-hydroxycinnamic acid (HCCA)
was chosen as the matrix. The samples were dissolved in
40 μL of water with 0.1% TFA. A portion (1.5 μL) of the sample
and matrix (1.5 μL) were spotted on a MALDI target plate
sequentially and dried at room temperature prior to MALDI-
TOF-MS analysis. Ions were extracted into the mass spectrome-
ter in reflection mode using an extraction potential of 20 kV
with a low-mass detection method.
Lipase assay in aqueous solution
The activity of native and modified PcL was assayed by
employing the well-known p-nitrophenyl palmitate (p-NPP).30
The basis of this assay protocol was the estimation of para-
Docking of the substrate and binding free energy calculation
The crystal structure of PcL was obtained from the work of
Luis (PDB entry 2NW6).33 The secondary alcohol esters'
enantiomers were docked into the catalytic cavity of PcL
using the CDOCKER protocol in Discovery Studio 3.0 software
and the complex conformation with the highest score was
recorded. The hydroxyl group of Ser87 was dehydrogenated
Fig. 1 Structures of modification reagents and substrates.
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manually in the Discovery Studio molecule window. The car-
boxyl C of the docked molecule was changed from the sp2 to
sp3 hybridization state and covalently bonded to OG of Ser87.
The CO bond was changed to a C–O single bond, and the
carbonyl O was charged with −1. All other parameters were
set to the standard values.
All the binding free energies were obtained from the
results of molecular dynamic calculations (MD) which were
carried out using the Discovery Studio 3.0 program and the
CHARMM force field parameters. The simulation was
performed over 5 ns at constant temperature and pressure
using the Berendsen algorithm with a coupling constant of
2 ps for both parameters. Electrostatic interaction was calcu-
lated using the particle mesh Ewald (PME) method with a
non-bonded cutoff of 12 Å. The conformations of the system
were recorded every 10 ps for further analysis.
is not convenient.34,35 As a powerful tool, matrix-assisted
laser desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF-MS) can be used to solve the problem. MALDI-
TOF-MS has become a popular method to study peptides,
proteins and other organic molecules because of its high
mass accuracy, fast analysis speed and simplicity of
operation.36–39 Recently, MALDI-TOF-MS was applied to
determine the modification sites of congII and the affinity
binding sites of boronic acid-decorated lectins.40,41
Here MALDI-TOF-MS was used to verify that Tyr and Asp
−
residues were reactive towards NAI, I3 , EDC and EDA. Native
and modified PcL samples were treated with trypsin before
undergoing peptide mass fingerprinting. The theoretical pep-
tide masses taken from native PcL should appear at m/z
881.411, 1020.572, 1047.507, 1211.638, 1424.760, 1519.746,
1527.858, 1706.897, 2124.994, 2138.160 and 2231.077
(Table S4†). The peptide fragments of residues 1–8, 23–40,
41–61 and 95–115, which contained Tyr4 (ADNYAATR), Tyr29
(YAGVLEYWYGIQEDLQQR), Tyr45 (GATVYVANLSGFQSDDGPNGR)
and Tyr95 (YVAAVAPDLVASVTTIGTPHR), were detected at m/z
881.416, 2231.074, 2124.995 and 2138.162, in line with the
theoretical peptide masses of native PcL (Fig. S2†). The analy-
sis of the solvent accessible surface (ASA) showed that the
potential modification sites were Tyr4, Tyr23, Tyr29, Tyr31,
Tyr45, Tyr95, Asp36, Asp55, Asp102, Asp303, Glu63 and Asp302
(Fig. S1†). As expected, a mass shift of 42.02 Da (correspond-
ing to the weight of the fragment of NAI covalently bonded to
Tyr) for peptide fragments of modified PcL with n-hexane was
found at m/z 923.436 (Tyr4), 2273.094 (Tyr29), 2167.015 (Tyr45)
and 2180.212 (Tyr95) (Fig. 2). When n-hexane was absent, only
Tyr4, Tyr45 and Tyr95 were modified and the peptide frag-
ments were detected at m/z 923.429, 2167.027 and 2180.181
Results and discussion
Chemical modification of PcL
Scheme 1 shows the modification strategies of PcL at Tyr,
Asp or Glu residues. NAI reacted with the hydroxyl group of
Tyr selectively. The hydrogen atom of the hydroxyl group was
substituted by the acetyl and a mass shift of 42.01 Da was
−
detected. When I3 was used as the modification reagent, the
hydrogen atom at the ortho site of the hydroxyl group was
replaced by the iodine ion. The mass shift was identified by
125.90 Da. The carboxyl group reacted with EDA to form the
respective amide. The mass shift was 42.06 Da. When car-
boxyl group was reacted with EDC, the hydrogen atom was
replaced by CH3–NIJCH3)–CH2–CH2–NH–CHNH–CH2–CH3
and the mass shift was 142.13 Da.
−
(Fig. S7†). Tyr45 and Tyr95 were modified by I3 without
Characterization of native and modified PcL by MALDI-TOF-MS
n-hexane as the activator and a mass shift of 125.90 Da (cor-
responding to the weight of the iodine atom covalently
bonded to Tyr) for the peptide fragments of modified PcL
was found at m/z 2250.897 and 2264.051 (Fig. S3†). After
addition of n-hexane, one more peptide fragment of modified
These reactions of chemical modification are typically non-
specific in nature and direct identification of modified sites
−
PcL by I3 showed up at m/z 2356.964 (Fig. S4†), which indi-
cated that Tyr29 was modified. In Fig. S5,† Asp36
(YAGVLEYWYGIQEDLQQR) of PcL was identified as the site
modified by EDA. A mass shift of 41.93 Da for the peptide
fragment of modified PcL was detected at m/z 2273.009. When
EDC was the modifier, Asp55 (GATVYVANLSGFQSDDGPNGR)
was modified and a mass shift of 142.10 Da appeared at m/z
−
2267.097 (Fig. S6†). These data confirmed that NAI, I3 , EDA
and EDC were covalently bonded to Tyr and Asp residues.
Effect of chemical modification on p-NPP catalytic activity
of PcL
The hydrolysis of p-NPP was examined to assess the effect of
chemical modification on the catalytic activity of PcL. By vary-
ing the molar ratio of the reagent over the lipase, the maxi-
mum concentration of the modifier was determined. All the
degrees of modification were obtained under the maximal
Scheme 1 Modification strategies of PcL at Tyr, Asp or Glu.
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Fig. 2 Expanded MALDI-TOF mass spectra by tryptic digestion of NAI-PcL in the presence of n-hexane. (A) Tyr4 modification, ADNYAATR, 1–8,
m/z change from 881.416 to 923.436. (B) Tyr45 modification, GATVYVANLSGFQSDDGPNGR, 41–61, m/z change from 2124.995 to 2167.015. (C)
Tyr29 modification, YAGVLEYWGIQEDLQQR, 23–40, m/z change from 2231.074 to 2273.094. (D) Tyr95 modification, YVAAVAPDLVASVTTIGTPHR,
95–115, m/z change from 2138.162 to 2180.212.
concentration of the reagents and were calculated according
to the strategy described by Pelton et al.42 For all modifica-
tion reagents, the maximal degree of modification was
reached at 50–70% (Table S3†). Fig. 3 shows that PcL is quite
sensitive to the four modifiers and the reagents have no
effect on the hydrolysis of the substrate when PcL was absent.
For NAI-PcL in the absence of n-hexane, the relative activity
was reduced to about half, whereas for modification in the
presence of n-hexane, the relative activity of NAI-PcL was
almost lost (Fig. 3A). The modification of PcL with I3− (Fig. 3B)
reduced both of the relative activities in the presence and
absence of n-hexane. The EDC modification (Fig. 3C) also
decreased the relative activity. By contrast, PcL modified by
EDA enhanced the relative activity by about 6-fold (Fig. 3D).
Fig. 3 Effect of chemical modification on relative activity of PcL in the absence or presence of n-hexane (100 μL). (A) NAI for (■), NAI + PcL for
−
−
−
(▲) and NAI + PcL + n-hexane for (♦); (B) I3 for (▲), I3 + PcL for (♦) and I3 + PcL + n-hexane for (■); (C) EDC for (■) and EDC + PcL for (♦); (D)
EDA for (■) and EDA + PcL for (♦).
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Table 1 The initial rate, enantioselectivity, and the binding free energy for the hydrolysis of sec-butyl acetate catalyzed by the native and modified PcL
Binding free energy
(kJ mol−1
)
Entry
Modification reagent
Initial rate (μmol min−1)νR
E value
Enantiopreference
RIJΔG)
SIJΔG)
|ΔIJΔG)| (kJ mol−1
)
1
2
3
4
5
6
7
Native PcL
NAI + n-hexane
NAI alone
14.0
12.0
13.0
8.0
9.0
16.0
5.0
1.2
3.2
1.2
2.0
1.8
2.3
3.1
R
R
R
R
R
R
R
−107.37
−113.91
−22.27
−106.74
−80.04
−21.74
−93.95
−97.39
−102.26
−65.13
0.6
33.9
0.5
17.0
5.2
8.0
I3− + n-hexane
−110.91
−102.57
−110.27
−87.55
I3− alone
EDA alone
EDC alone
22.4
All initial rates of native and modified PcL were given
towards the five substrates (Table 1 and S1†). Table 1 and
S1† show the initial rate of EDA-PcL, which is higher than
the native one.
respectively. It demonstrated that the E value increased with
an increase in the modification degree. The enhancement of
the E value catalyzed by EDC- and EDA-PcL is attributed to
the replacement of the charged amino acid (aspartic acid res-
idue) on the lipase surface with the uncharged hydrophobic
group.22
As shown in Fig. 3A and B, the reduction tendency of the
−
relative activity of NAI- and I3 -PcL in the absence and pres-
ence of n-hexane is diverse which indicated that PcL was acti-
vated by n-hexane and the amino acid residue in the cavity of
the catalytic center was modified by NAI and I3−. Preliminary
studies showed that introduction of more hydrophobic modi-
fiers or the change in conformation was the major cause of
hydrolytic activity diversity.43,44 The latter factor might result
in the different catalytic activities of modified PcL in our
experiments, especially for the hydrolytic activity increase of
EDA-PcL.
Molecular dynamics study
The complex of PcL and sec-butyl acetate was simulated by
using molecular dynamics (MD) computation. For native and
modified PcL, all binding free energies of the (R)-configura-
tion were lower than the (S)-configuration (Table 1). After
modification, |ΔIJΔG)| between (R)- and (S)-configurations was
much higher than that of the native one. That is to say sub-
strates of the (R)-configuration were liable to be hydrolyzed.
The correlation between |ΔIJΔG)| and enantioselectivity corre-
sponding to native and modified PcL at Tyr was given in
Fig. 4. The tendency of |ΔIJΔG)| was consistent with the
enantioselectivity. The |ΔIJΔG)| and enantioselectivity were
almost a linear relation (R2 = 0.9566). It indicated that the
enantioselectivity was highly correlated with |ΔIJΔG)|. Towards
the other four substrates, sec-butyl butyrate, sec-butyl
hexanoate, hexan-2-yl acetate and octan-2-yl acetate, the laws
Enantioselectivity improvement towards secondary alcohol by
chemical modification
PcL-catalyzed resolution of five pairs of different chiral sec-
ondary alcohols via hydrolysis of their corresponding esters
was investigated. Hydrolysis of sec-butyl acetate was chosen
as a model (Table 1). Both native and modified PcL's enantio-
preference was the (R)-configuration. Enantioselectivity of the
native PcL was poor which was around one (entry 1). When
modified by NAI in the absence of n-hexane, the E value was
not improved (entry 3). After addition of n-hexane, the modi-
fication of PcL with NAI enhanced the E value by 3-fold
(entry 2). The E value was enhanced from 1.2 to 1.8 in the
absence of n-hexane (entry 5), and 2.0 in the presence of
−
n-hexane by I3 modification (entry 4). The modification with
EDA and EDC also favored an improvement in the E value by
about 2- and 3-fold (entries 6 and 7), respectively.
−
NAI- and I3 -PcL in the presence of n-hexane had more
influence on the E value towards sec-butyl acetate because of
the modification of the Tyr29 in the catalytic cavity. From the
data listed in Table S3,† the E value of PcL depended not only
on the nature of the modification group but also on the
degree of the modification. The modification degrees of Tyr29
Fig. 4 Correlation of |ΔIJΔG)| and enantioselectivity corresponding to
native and modified PcL at Tyr (substrate: sec-butyl acetate).
−
of NAI- and I3 -PcL were 19.2 and 12.3 (Table S3†),
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Fig. 5 Conformation of a sec-butyl acetate enzyme complex resulting from MD simulation. In the figure, all non-polar hydrogen atoms have been
hidden to avoid visual disturbance. (A) (R)-sec-Butyl acetate + native PcL (purple for unmodified Tyr29). (B) (S)-sec-Butyl acetate + native PcL (pur-
ple for unmodified Tyr29). (C) (R)-sec-Butyl acetate + modified PcL (purple for Tyr29-O-acetylated by NAI). (D) (S)-sec-Butyl acetate + modified PcL
(purple for Tyr29-O-acetylated by NAI).
of E values (Fig. S8†) and binding free energies were similar
to that of the model one (Fig. 4). The experimental results
were approved by the MD data.
at the hydrophobic side chain of Tyr4, Tyr45 and Tyr95 was
replaced by a larger hydrophobic group, acetyl, on the surface
of PcL in the absence of n-hexane, the E value was not
improved. The reason was that the hydrophobic side chains
of Tyr were folded back on to the modified PcL's surface in
aqueous conditions and could not extend fully. The confor-
mation or flexibility of modified PcL slightly changed so that
the E value was not enhanced. On the other hand, the E value
can be improved by the replacement of the charged amino
acid on the lipase surface with the uncharged hydrophobic
group in most cases. However, Tyr was the uncharged amino
acid. Instead, in the presence of n-hexane, Tyr29 in the
catalytic cavity was acetylated which led to an increase in the
Mechanism discussion of enantioselectivity improvement
Our experimental results are well in line with Kazlauskas's
law and indicated that chemical modification was an effec-
tive method to improve the enantioselectivity of PcL.
According to Kazlauskas's work, enantioselectivity was related
to the size difference between groups directly connected to
the chiral center of the substrate.8
Before modification, the size difference between methyl
and ethyl was not so big that (R)-configuration enantio-
selectivity was poor (Table 1). The (R)-sec-butyl acetate
enantiopreference was confirmed by the lower binding free
energy of the (R)-sec-butyl acetate (Table 1). MD simulation
revealed that the small substituent on the chiral center,
methyl, and the large substituent, ethyl of the (R)-sec-butyl
acetate, fit better into the corresponding pocket
(Fig. 5A and B). Because the groups on the chiral center were
the same with sec-butyl acetate, the results above can be
applied to interpret the phenomena of the other two sub-
strates, sec-butyl butyrate and sec-butyl hexanoate catalyzed
by native PcL (Tables S1 and S2†). When the substrates were
hexan-2-yl acetate and octan-2-yl acetate, (R)-configuration
enantioselectivity was enhanced with the size difference of
the groups on the chiral center increase (Tables S1 and S2†).
The enantioselectivity of modified lipase depended on the
nature of the modification group. When the hydrogen atom
E
value. The modification group, acetyl, facilitated a
much stronger steric hindrance to methyl than ethyl
(Fig. 5C and D). It made the (R)-sec-butyl acetate combination
to the enzyme more convenient and hydrolysis reaction much
faster. Because iodide ion (diameter: 440 pm) was smaller
than acetyl (diameter: 893 pm), its steric hindrance to the
methyl group was not as strong as that to the acetyl group.
Thus, whenever the modification process was in the presence
or absence of n-hexane, the E value changed slightly (Table 1
and Table S1†).
Conclusions
In conclusion, this study demonstrated that PcL was modi-
fied through a one-step reaction by NAI, I3−, EDA and EDC.
This method successfully improved the p-NPP catalytic
activity and enantioselectivity towards secondary alcohols of
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PcL. The modified sites were identified by MALDI-TOF-MS
and it was indicated that Tyr29 was critical for the improve-
ment of enantioselectivity catalyzed by PcL. Chemical modi-
fication in vitro is an effective method to modify a protein
and further to modulate its function. In addition, the investi-
gation confirms the crucial roles of a steric hindrance.
This cost-effective and environmentally friendly method is
promising for the resolution of chiral compounds in many
industrial fields.
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