Studies toward stereoselective bionanocatalysis on gold nanoparticles

Article abstract of DOI:10.1016/j.molcatb.2013.01.008

As yet, different enzymes were immobilized on gold nanoparticles both through adsorption and covalent binding. However, there is only one evaluation if such immobilization influenced enzyme enantioselectivity, which is an essential parameter in biocatalys

Full text of DOI:10.1016/j.molcatb.2013.01.008

Journal of Molecular Catalysis B: Enzymatic 90 (2013) 12–16
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Studies toward stereoselective bionanocatalysis on gold nanoparticles
Hanna Je˛drzejewska, Ryszard Ostaszewski∗
Institute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
As yet, different enzymes were immobilized on gold nanoparticles both through adsorption and covalent
binding. However, there is only one evaluation if such immobilization influenced enzyme enantiose-
lectivity, which is an essential parameter in biocatalysis. Therefore systematic studies with enzymes
immobilized on gold nanoparticles through covalent binding and embedded through adsorption were
performed. Adsorption was not efficient method and it significantly lowered enantioselectivity of
enzymes. In turn, covalent binding was in most cases very good method of immobilization, especially
for Pseudomonas cepacia lipase, where conversion and enantioselectivity were even slightly better than
for native enzyme. It was also evaluated that in case of adsorption size of nanoparticles did not influence
enantioselectivity, but in case of covalent binding small nanoparticles gave much better results than big
ones.
Received 31 October 2012
Received in revised form
21 December 2012
Accepted 10 January 2013
Available online 20 January 2013
Keywords:
Biocatalysis
Enantioselectivity
Gold nanoparticles
Enzyme immobilization
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
had three times lower Km parameter (Michaelis constant) in reac-
tion of hydrolysis of p-nitrophenyl palmitate when enzyme was
Stereoselectivity is one of a key parameter in biocatalysis and
in biosensors based on enzymatic reactions. Such biosensors have
been improved by adding enzymes immobilized on gold nanopar-
ticles on an electrode surface. Sensors of glucose, fundamental in
diagnostics and food testing, had lower detection limit and were
more selective, if enzymes on AuNPs in silicate [1], mesoporous
carbon [2] or composite [3] were placed on electrodes. Sensor of
hydrogen peroxide were more stable when horseradish peroxidase
was immobilized on AuNPs, instead of direct immobilization on
gold electrode [4]. l-lactate concentration is measured indirectly
by measuring concentration of NADH. NADH is reduced during oxi-
dation of lactate to pyruvate catalyzed by lactate dehydrogenase.
Gold electrode is not sensitive and selective for NADH, but gold
electrode covered with silicate gel and AuNP with dehydrogenase
was more sensitive [5].
immobilized on AuNPs. Maximal velocity of reaction was not
changed. Smaller AuNPs (13 nm diameter) gave better results than
big ones [7]. Aspartyl protease [8] and pepsin [9] after immobiliza-
tion on gold nanoparticles had similar activity in reaction of protein
hydrolysis as native enzymes. In addition, enzymes with AuNPs
embedded on zeolite were active in wider temperature and pH
range. In these experiments stereochemical aspect of bionanocatal-
ysis was not investigated.
Aside from non-thiolated AuNPs, also nanoparticles capped with
thiols were used in biocatalysis. Lysine-tagged aminopeptidase
II from Bacillus stearothermophilus was covalently bound to gold
nanoparticles thiolated with 16-mercaptohexadecanoic acid [10].
Amount of immobilized enzyme was 50 and 12.5 times bigger for
enzyme tagged with respectively 9 and 3 lysine residues than for
native enzyme. In case of enzyme modified with 3 Lys, 64% of immo-
bilized enzyme was active and its pH and temperature stability
was improved. Enzyme was reused a few times and was active in 5
catalytic cycle (24% of the initial activity in 5th cycle).
Gold nanoparticles have influence on activity of enzymes used
as biocatalysts. Wu et al. [6] found out that Candida rugosa lipase
Interactions of gold nanoparticles with proteins change confor-
mation and structure of proteins, e.g. hemoglobin [11] and albumin
[12]. Amount of ␣-helices decrease whereas ␤-sheets and ␤-turns
increase. In case of enzymes such interactions, mainly near active
site, should cause changes in substrate specificity or stereoselec-
tivity of biocatalysts. All cited examples of enzymes immobilized
on gold nanoparticles were kinetic studies. But there is only one
case of studies where stereoselectivity of such enzyme was men-
tioned, and it turned out that immobilization on gold nanoparticles
Abbreviations: AuNPs, gold nanoparticles; c, conversion; C. antarctica, Candida
Antarctica; C. rugosa, Candida rugosa; CDI, carbonyldiimidazole; DLAP, duck liver
acetone powder; E, enantioselectivity; ees, enantiomeric excess of substrate; NADH,
nicotinamide adenine dinucleotide; M. javanicus, Mucor javanicus; PBS, phosphate
buffer saline; PLE, pig liver esterase; PPL, porcine pancreatic lipase; P. cepacia, Pseu-
domonas cepacia; TLAP, turkey liver acetone powder.
∗
Corresponding author. Tel.: +48 22 3432120; fax: +48 22 6326681.
E-mail addresses: ryszard.ostaszewski@icho.edu.pl,
rysza@icho.edu.pl (R. Ostaszewski).
1381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.01.008

H. Je˛drzejewska, R. Ostaszewski / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 12–16
13
Fig. 1. Covalent immobilization of enzymes (CDI – carbonyldiimidazole, Enz-NH2 – enzyme, PBS – phosphate buffer saline).
did not change enantioselectivity of alcohol dehydrogenase [13].
We decided to investigate in systematic way how gold nanoparti-
cles of different kinds and sizes affect stereoselectivity of selected
hydrolytic enzymes.
the mixture was centrifuged (20 min, 13,000 rpm), the pellet was
rinsed 3 times with buffer and re-dispersed in 0.3 mL of PBS.
2.3. Ethyl 3-hydroxy-3-phenylpropanoate
2. Experimental
Synthesis of racemic ethyl 3-hydroxy-3-phenylpropanoate was
performed according to known procedure [17]. HPLC method for
resolution of ethyl 3-hydroxy-3-phenylpropanoate enantiomers:
Chiralcel-OB column, eluent was hexan/isopropanol 96:4; flow
0.8 mL/min. Retention times: 19.08 min (R-enantiomer), 21.81 min
(S-enantiomer).
Chemicals (except solvents) and enzymes were purchased from
Sigma–Aldrich and used without further purification. Solvents
were purchased from POCh and purified with distillation. Liver ace-
tone powders were prepared [14] and enzymes were purified [15]
in our laboratory by standard protocols. Thin layer chromatogra-
phy was performed on TLC Plates 60 F-254 from Merck. Silica gel 60
230–400 Mesh was from Merck. UV–vis spectra were recorded on
Hitachi U-1900 Spectrophotometer. High performance liquid chro-
matography was performed with Varian ProStar (with UV Varian
ProStar 330 detector and Chromatopac C-R6A analyzer). Centrifu-
gation: Eppendorf Centrifuge 5804 R.
2.4. Kinetic resolution
10 mg of selected enzyme was incubated 45 min at 23 ◦C with
0.2 mL solution of 3.5 nm AuNPs, 0.1 mL solution of 20 nm AuNPs
or 0.2 mL of water. Enzyme prepared in this way was added to
1 mL of PBS with 0.1 mmol of 3-hydroxy-3-phenylpropanoate. The
mixture was shaking at 23 ◦C until conversion was about 50% (mon-
itored with TLC). The mixture was extracted with diethyl ether
and washed with saturated solution of sodium bicarbonate. Extract
was dried with anhydrous magnesium sulfate, filtered and solvent
was evaporated under reduced pressure. Recovered substrate was
purified with column chromatography.
Protein was measured with the Bradford dye calibrated against
BSA [16], according to procedure attached to the Bradford reagent.
2.1. Nanoparticles
Non-thiolated nanoparticles were prepared according to known
procedure: 3.5 0.7 nm AuNPs were obtained using sodium boro-
hydride [8], 13 1.5 nm and 20 4 nm AuNPs were obtained using
sodium citrate [6,10]. Concentrations of nanoparticles solutions
were 200 nM, 12 nM and 18 nM respectively. Observed absorption
maxima were 514 nm, 520 nm and 522 nm, which was in agree-
ment with literature data.
In case of immobilized enzymes 0.3 mL of enzyme on AuNPs in
PBS was added instead of 10 mg of enzyme incubated with AuNPs.
Selected experiments were performed 5 times. Differences in
conversion and enantiomeric excess were less than 5%.
Thiolated AuNPs: To 70 mL 10.3 mM methanolic solution of
HAuCl4·H2O, 1 mL 0.24 M methanolic solution of ␣-lipoic acid was
added. Next, 35 mL 32 mM water solution of NaBH4 was added.
After 2 h of mixing at ambient temperature, the mixture was cen-
trifuged (20 min, 13,000 rpm), the pellet was washed 4 times with
methanol and dried. 120 mg of gold nanoparticles of 5 1 nm diam-
eter were obtained.
3. Results and discussion
3.1. Kinds of nanoparticles
Nanoparticles have dimensions similar to proteins, therefore
their size should play crucial role in interactions with enzymes. This
problem was examined only for C. rugosa lipase [7]. For our stud-
ies a few selected kinds of AuNPs were generated. Non-thiolated
AuNPs reduced with sodium citrate [6,10] or sodium borohydride
[8] were obtained as water solutions. Reaction progress and grow-
ing of nanoparticles was followed up by spectroscopic methods [8].
Absorption maxima indicated that nanoparticles of relevant sizes
were formed. To obtain 5 nm thiolated nanoparticles procedure
known for 11-mercaptoundecanoic acid was used [18] with ␣-
lipoic acid as a thiol. Reduction was performed in water–methanol
solution and reducing agent was sodium borohydride. Some mod-
ifications of procedure, including changing of thiol amount, were
brought in to form larger nanoparticles, but effective formation of
nanoparticles was observed only for original conditions. Obtained
nanoparticles, were next used as a base to immobilization of
selected enzymes.
2.2. Immobilization
Adsorption: solution of non-thiolated nanoparticles (0.2 mL,
initial concentration) was centrifuged (20 min, 13,000 rpm) and re-
dispersed in 1.1 mL of distilled water. 0.1 mL enzyme solution (10 U)
was added. After 30 min incubation at 23 ◦C, the probe was cen-
trifuged and the pellet was rinsed with buffer (0.01 M PBS pH 7.4)
until the supernatant had no catalytic activity, and re-dispersed in
0.3 mL of PBS.
Covalent binding: 8 mg AuNPs thiolated with ␣-lipoic acid were
dissolved in 1.4 mL 0.02 mM CDI in PBS and shaking 2 h at 23 ◦C.
Concentration of ␣-lipoic acid was 1.4 mM. Next, 0.1 mL enzyme
solution (10 U) was added and shaking another 2 h. After this time

14
H. Je˛drzejewska, R. Ostaszewski / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 12–16
Scheme 1. Kinetic resolution of ethyl 3-hydroxy-3-phenylpropanoate.
3.2. Immobilization
was found [19,20]. In our laboratory, this class of compounds is
also well-known [21]. The hydrolysis reaction was chosen because
non-thiolated nanoparticles are stable in water solutions. Ester
was obtained by reduction of ethyl benzoylacetate with procedure
reported by Hattori et al. [17].
Non-thiolated AuNPs of various sizes were used to embedding
selected enzymes by adsorption [7]. Due to the great affinity of
thiol and amino groups, which are present in proteins, to gold
such procedure was reasonable. Water solutions of enzymes were
added to water solutions of nanoparticles, mixed, centrifuged
and re-dispersed in PBS (0.01 M phosphate buffer saline pH 7.4).
Absorption maximum showed small red shift related to bigger size
of conjugates than nanoparticles alone. Aggregation of AuNPs was
not observed.
Covalent immobilization (Fig. 1) was formation of amide bond
between carboxylated gold nanoparticles and amino groups of
enzymes using carbonyldiimidazole as a coupling agent. AuNPs
capped with ␣-lipoic acid were added to solution of CDI in PBS. After
shaking, enzyme solution was added and the mixture was shaking
2 h. Next, immobilized enzyme was centrifuged, washed with PBS
and re-dispersed in PBS. Deep gray-black color of solution indicated
that good dispersion was obtained. For nanoparticles bigger than
5 nm immobilization was less effective.
Firstly, kinetic resolution reactions were catalyzed by native
enzymes and by mixtures of enzymes and nanoparticles
(Scheme 1). Catalytic effect was a sum of adsorbed and free enzyme
in solution. Results are presented in Table 1. Among over twenty
hydrolases used, nine were good catalysts of the model reaction.
Nanoparticles did not affect conversion of the reaction. In case
of pig liver esterase (PLE), porcine pancreatic lipase (PPL) and Can-
dida antarctica lipase adding of AuNPs changed enantioselectivity of
the reaction. For PLE and PPL enantioselectivity was improved and
effect depended on size of nanoparticles. For majority of enzymes
influence of nanoparticles was non-significant. In case of Pseu-
domonas cepacia lipase, which is the best biocatalyst of examined
reaction, enantioselectivitydidnotdecreasewhichwasveryimpor-
tant and promising result (entries 13–15, Table 1).
In the second step, enzymes embedded on gold nanoparticles
were used to the kinetic resolution reaction. Two enzymes were
active after adsorption, and only P. cepacia lipase catalyzed the
reaction stereoselectively (Table 2). Enantioselectivity was lower
because of long reaction time and increasing participation of auto-
hydrolysis.
3.3. Kinetic resolution
Aim of our research was to investigate various immobilization
procedures and their influence on enantioselectivity of reaction,
and not to work out a new optically pure compound. There-
fore racemic ethyl 3-hydroxy-3-phenylpropanoate was picked out
which is well-examined compound. Reaction conditions of its
kinetic resolution were optimized and the best native biocatalyst
Size of nanoparticles had rather small influence on enantiose-
lectivity of the reaction. Also time of incubation of enzyme with
nanoparticles (results not showed) was not essential. The process
of adsorption was very fast (it took a few minutes).
Table 1
Kinetic resolution: native enzymes with or without nanoparticles.
Entry
Enzyme
Time [h]
Conversion [%]
ees [%]
Ea
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
Turkey liver acetone powder
TLAP + 3.5 nm AuNPs
TLAP + 20 nm AuNPs
5
5
5
3
3
3
8
8
8
2
2
2
5
5
5
1.5
1.5
1.5
16
16
16
2.5
2.5
2.5
3
40
40
40
40
40
40
45
45
45
50
50
50
55
55
55
55
55
55
40
40
40
40
40
40
40
40
40
9
8
10
1
3
4
38
50
44
29
37
43
1.4
1.4
1.6
–
–
–
3.9
6.7
5.0
2.3
3.1
3.8
Wheat germ lipase
Wheat germ lipase + 3.5 nm AuNPs
Wheat germ lipase + 20 nm AuNPs
Pig liver esterase
PLE + 3.5 nm AuNPs
PLE + 20 nm AuNPs
Porcine pancreatic lipase
PPL + 3.5 nm AuNPs
PPL + 20 nm AuNPs
P. cepacia lipase
>99
72
P. cepacia lipase + 3.5 nm AuNPs
P. cepacia lipase + 20 nm AuNPs
C. antarctica lipase
C. antarctica lipase + 3.5 nm AuNPs
C. antarctica lipase + 20 nm AuNPs
Duck liver acetone powder
DLAP + 3.5 nm AuNPs
>99
>99
28
29
18
10
7
10
12
11
10
13
12
12
117
117
2
2.1
1.6
1.5
1.3
1.5
1.6
1.6
1.5
1.6
1.6
1.6
DLAP + 20 nm AuNPs
Rhizopus arrhizus lipase
Rhizopus arrhizus lipase + 3.5nmAuNPs
Rhizopus arrhizus lipase + 20nmAuNPs
Rhizopus niveus lipase
Rhizopus niveus lipase + 3.5 nm AuNPs
Rhizopus niveus lipase + 20 nm AuNPs
3
3
a
Calculated from E = [ln((1 − c) × (1 − ees))]/[ln((1 − c) × (1 + ees))].

H. Je˛drzejewska, R. Ostaszewski / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 12–16
15
Table 2
Kinetic resolution catalyzed by enzymes adsorbed on gold nanoparticles.
Entry
Enzyme
Time [d]
Conversion [%]
ees [%]
Ea
1
2
3
4
5
6
P. cepacia lipase on 3.5 nm AuNPs
P. cepacia lipase on 13 nm AuNPs
P. cepacia lipase on 20 nm AuNPs
PLE on 3.5 nm AuNPs
PLE on 13 nm AuNPs
PLE on 20 nm AuNPs
2
2
2
2
2
2
20
20
30
25
25
25
8
2.1
2.7
2.2
–
–
–
10
14
rac
rac
rac
a
Calculated from E = [ln((1 − c) × (1 − ees))]/[ln((1 − c) × (1 + ees))].
Table 3
Kinetic resolution catalyzed by enzymes immobilized covalently.
Entry
Enzyme
Time [d]
Conversion [%]
ees [%]
Ea
1
2
3
4
5
6
7
8
9
P. cepacia lipase – native enzyme
P. cepacia lipase on thiol-AuNPs
PPL – native enzyme
PPL on thiol-AuNPs
PLE – native enzyme
2
2
3
3
2
2
5
5
2
2
3
3
47
48
28
23
19
10
23
19
22
28
16
11
68
72
13
9
2
rac
2
4
rac
7
rac
rac
15.6
16.7
2.3
2.0
–
–
–
1.5
–
1.5
–
PLE on thiol-AuNPs
Wheat germ lipase – native enzyme
Wheat germ lipase on thiol-AuNPs
C. antarctica lipase – native enzyme
C. antarctica lipase on thiol-AuNPs
M. javanicus lipase – native enzyme
M. javanicus lipase on thiol-AuNPs
10
11
12
–
a
Calculated from E = [ln((1 − c) × (1 − ees))]/[ln((1 − c) × (1 + ees))].
Table 4
P. cepacia lipase – 5 catalytic cycles.
Entry
Enzyme
Time [d]
Conversion [%]
ees [%]
Ea
1
2
3
4
5
6
Native enzyme
2
2
2
2
2
2
47
48
42
37
25
22
68
72
53
40
16
7
15.6
16.7
11.2
8.0
3.3
1.9
Enzyme on thiol-AuNPs – 1st cycle
Enzyme on thiol-AuNPs – 2nd cycle
Enzyme on thiol-AuNPs – 3rd cycle
Enzyme on thiol-AuNPs – 4th cycle
Enzyme on thiol-AuNPs – 5th cycle
a
Calculated from E = [ln((1 − c) × (1 − ees))]/[ln((1 − c) × (1 + ees))].
Adsorption as a method of immobilization was inefficient and
enantioselectivity was very low. Therefore we decided to use cova-
lently immobilized enzymes (Table 3). Amount of immobilized
enzyme was measured as a difference between concentration of
enzyme in the probe and in supernatant after centrifugation. The
same amount of native enzyme was used in reference reaction.
Reactions were performed with nanoparticles of various sizes, but
only for small 5 nm AuNPs effective biocatalysts were obtained.
It turned out that in most cases conversion and enantioselec-
tivity of the reaction with immobilized enzymes were similar to
results obtained for the same amount of native enzymes. Lipases
loose at most 30% of initial activity, only esterase loose 50%.
Results obtained for P. cepacia lipase were significantly better
than for any other enzyme. We checked structure of this enzyme
in Protein Data Bank (Fig. 2) [22]. There are seven lysine residues
near the protein surface. Therefore immobilization through the
amide bond was effective for this enzyme. The structure of enzyme
is an explanation of the fact that only small nanoparticles were
good base for immobilization. Small AuNPs had size similar to the
enzyme and therefore they could connect through one or two lysine
residues, which did not cause significant deformation of the lipase.
Bigger nanoparticles could bind more lysine residues of one enzyme
molecule and it could deactivate the lipase.
3.4. Reusability
Fig. 2. Structure of P. cepacia lipase (red – cysteine, green – lysine). (For interpreta-
tion of the references to color in this figure legend, the reader is referred to the web
version of the article.)
The main advantage of immobilized enzymes is possibility
to recycle them. Enzymes embedded on nanoparticles through

16
H. Je˛drzejewska, R. Ostaszewski / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 12–16
adsorption were difficult to recover and were inactive in sec-
ond cycle. For P. cepacia lipase, which is the best catalyst of the
model reaction, five catalytic cycles with the same portion of the
enzyme immobilized covalently on gold nanoparticles were per-
formed (Table 4).
abilities were greatly improved. This immobilized enzyme was also
used in five catalytic cycles and retained 50% of its activity.
Acknowledgments
Conversion and enantioselectivity were gradually lower
because of a slight release of the enzyme from AuNPs. This effect is
especially important in biosensors, where enzyme must be per-
manently bound with electrode to ensure stable measurement
conditions. Lipases are not typical enzymes for biosensors but cases
of their usage are known [23,24]. Gold nanoparticles have not been
used in biosensors with lipases yet, but having in mind that amide
bond is not very stable in water conditions, usage of other bonds
should be considered in the future.
This work was supported by project “Biotransformations for
pharmaceutical and cosmetics industry” No. POIG.01.03.01-00-
158/09-01 part-financed by the European Union within the
European Regional Development Fund.
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4. Conclusion
This work describes systematic studies on influence of
gold nanoparticles on enzyme enantioselectivity. Two different
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unfortunately were unsuccessful. Size of nanoparticles had negli-
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