A novel nanoprotein particle synthesis: Nanolipase

Article abstract of DOI:10.1016/j.procbio.2011.04.011

Lipases (triacylglycerol acylhydrolases; E.C. 3.1.1.3) constitute a group of enzymes defined as carboxylesterases that catalyse the hydrolysis (and synthesis) of long-chain acylglycerols at the lipid-water interface. In this study, a novel method has been

Full text of DOI:10.1016/j.procbio.2011.04.011

Process Biochemistry 46 (2011) 1688–1692
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Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Short communication
A novel nanoprotein particle synthesis: Nanolipase
Rıdvan Saya,∗, Rüstem Kec¸ ilia, Özlem Bic¸ ena, Filiz Yılmaz S¸ is¸ mana, Deniz Hüra, Adil Denizlib, Arzu Ersöza
a Department of Chemistry, Anadolu University, Eskis¸ehir, Turkey
b Department of Chemistry, Hacettepe University, Ankara, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Lipases (triacylglycerol acylhydrolases; E.C. 3.1.1.3) constitute a group of enzymes defined as car-
boxylesterases that catalyse the hydrolysis (and synthesis) of long-chain acylglycerols at the lipid–water
interface. In this study, a novel method has been developed to prepare nanoprotein particles carrying
lipase using a photosensitive microemulsion polymerization process. The nanostructured lipases with
photosensitive features have been characterized by transmission electron microscopy (TEM) and Zeta
Sizer. The average particle size of nanolipases was found to be about 100 nm. Lipase nanoparticles were
used in hydrolysis of paranitrophenyl palmitate (p-NPP) and the results were compared with free lipase.
The parameters like pH, temperature and hydrolytic activity that affect p-NPP hydrolysis were investi-
gated by using lipase nanoparticles and compared to free lipase. Lastly, reusability of lipase nanoparticles
was investigated and according to our results, this novel lipase nanoparticles showed admirable potential
in reusable catalyst.
Received 4 January 2011
Received in revised form 22 April 2011
Accepted 26 April 2011
Keywords:
Nanolipase
Photosensitive microemulsion
polymerization
Photosensitive cross-linking
Proteinous polymeric particles
© 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Chemical conjugation of enzymes with synthetic polymers
enables their colloidal stability or thermal stability to be enhanced,
Lipases (glycerol ester hydrolase, E.C. 3.1.1.3) have been widely
used for enzymatic conversion in the various biotechnological
applications in dairy industry, manufacturing of specialty chemi-
cals, organic synthesis, and preparation of enantiomerically pure
pharmaceuticals as they combine a broad range of substrate speci-
ficity with a high regio- and enantio-selectivity for many reactions
[1–3]. In most cases, especially in industrial applications, enzymes
are preferably used in their immobilized states owing to many
advantages, such as improved activity and stability of the immobi-
lized enzyme, ready reutilization of the catalyst and possibility of
continuous process operation. To date, numerous efforts have been
towards the development of enzyme immobilization techniques,
e.g. covalent attachment [4,5], physical adsorption [6] and encap-
sulation [7–15]. Adsorption techniques are easy to perform, but
the interactions between the enzyme and the support is relatively
weak. Such biocatalysts are usually not stable enough for a long-
term utilization. Covalent attachment normally leads to improved
enzyme stability. However, partial deactivation is often obtained
due to the conformational restrictions by the covalent bonding of
enzyme moieties to the support [16].
and their enzyme activity to be controlled [17–20]. However,
chemical conjugation has the difficulty that the synthesis is not
always easy, and that conjugation sometimes induces a decrease
in the enzyme activity [20]. Bioconjugation is a useful technique
for modifying the function of enzymes for pharmaceutical and
biotechnological applications. The biconjugation approach entails
the advantages of easy preparation and versatility for various pro-
teins [21–23].
In this study, nanostructured lipase was prepared by the photo-
sensitive cross-linking microemulsion polymerization technique.
This technique is a new technique which allows the synthesis of
nanoparticles with narrow size distribution [24]. Microemulsion
polymerization is a complex heterogeneous process where trans-
port of monomers, free radicals and other species (such as chain
transfer agent, co-surfactant and inhibitors) between the aqueous
and organic phases, takes place [25]. The polymerization rate is
controlled by monomer partitioning between the phases, parti-
cle nucleation, adsorption and desorption of radicals. The particle
stability is affected by the amount and type of surfactant and
the pH of the dispersing medium [26]. Although microemulsion
polymerization is a complex heterogeneous process, but in this
study, photosensitive cross-linking technique become more easy
and efficient by using the particles which were synthesized accord-
ing to ANADOLUCA (AmiNoAcid (monomer) Decorated and Light
Underpinning Conjugation Approach) method. The proteinous
nanoparticles prepared by photosensitive cross-linking were
∗
Corresponding author at: Anadolu Universitesi, Fen Fakültesi, Kimya Bölümü,
Yunus Emre Kampüsü, 26470 Eskis¸ ehir, Turkey. Tel.: +90 222 3350580x4823;
fax: +90 222 3204910.
E-mail address: rsay@anadolu.edu.tr (R. Say).
1359-5113/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2011.04.011

R. Say et al. / Process Biochemistry 46 (2011) 1688–1692
1689
easily prepared at room temperature, in day light and under nitro-
2.2.3. Synthesis of the photosensitive poly(bis (2-2ꢀ-bipyridyl) MATyr-MATyr-
ruthenium(II))
gen atmosphere. A nano-protein is a new generation polymeric
material prepared in ANADOLUCA concept. This nano-protein
(lipase in current study) can be used directly as a monomer
(enzymes, antibodies or similar proteins) without using any other
platforms. The ANADOLUCA method is used while preparing pho-
tosensitive nanostructured lipase and provides a strategy for
the preparation of photosensitive ruthenium based amino acid
monomers and oligomers, amino acid monomer-protein cross-
linking using photosensitation and conjugation approach on micro
and nanostructures by ruthenium-chelate based monomers [27].
In this study, the nanostructured lipase enzyme with photosen-
sitive features gained lipase was synthesized by the microemulsion
polymerization technique and characterized with fourier transform
infrared spectroscopy (FT-IR), transmission electron microscopy
(TEM) and Zeta-Sizer. After that, the activity of lipase nanoparti-
cles and parameters that affect paranitrophenyl palmitate (p-NPP)
hydrolysis such as pH, temperature and hydrolytic activity by using
lipase nanoparticles were investigated compared with free lipase.
Lastly, the reusability of lipase nanoparticles was determined using
the p-NPP hydrolysis reaction.
The phosensitive polymer was prepared by a conventional polymerization
reaction. The necessary amount of bis (2-2ꢀ-bipyridyl)-MATyr-MATyr ruthe-
nium(II) monomer in dimethyl sulfoxide was mixed to obtain a photosensitive
oligomer. The polymerization reaction was taken place in the presence of 2-2ꢀ-
azobisisobutyronitrile (AIBN) as an initiator. For the MALDI-TOF MS analysis, 2 ␮L of
([Ru(bpy)2MATyr-MATyr]n) solution was mixed with 23 ␮L of a 10 mg mL−1 solution
of ␣-cyano-4-hydroxycinnamic acid (CHCA) in acetonitrile/% 0.3 TFA. The accelera-
tion voltage was set to 20 kV, the delay time is 400 ns, grid voltage 70%, laser intensity
2092 and at reflector mode [27].
2.2.4. Synthesis of lipase proteinous nanoparticles
Lipase nanoparticles were synthesized according to microemulsion polymeriza-
tion technique. Microemulsion system was prepared by dispersing 0.5 g of poly(vinyl
alcohol) (PVA) in 45 mL of deionized water. On the other hand, 0.5 g of MATyr was
dissolved in 5 mL of dimethylsulfoxide and added into the lipase solution prepared
by dissolving 10 mg of lipase in 5 mL of deionized water. Then, 0.5 mL of rutenium
based polymer p(MATyr-Ru (bipyr)2-MATyr) was added into the mixture and mixed
for 1 h. This mixture was added into 25 mL of PVA dispersing medium. Beside this,
20 mL of initiator solution prepared by dissolving 0.02 g of APS in 45 mL of deion-
ized water was added into reaction mixture. Finally, 0.3 mL of EDMA was added
and mixed for 48 h under nitrogen atmosphere at room temperature, at daylight.
Lipase nanoparticles were separated from the reaction solution by centrifugation
at 6000 rpm for 20 min and washed with deionized water to remove unreacted
substances.
2. Experimental
2.3. Characterization of lipase proteinous nanoparticles
2.1. Chemicals
The average particle size and size distribution lipase nanoparticles were deter-
mined by Zeta Sizer (Malvern Instruments, Model 3000 HSA, England) and a FEI
120 kV transmission electron microscope (TEM).
Lipase (E.C. 3.1.1.3 from Candida rugosa), p-nitrophenyl palmitate, isopropanol,
p-nitrophenol, Triton X-100 were purchased from Sigma (St. Louis, MO, USA).
Ethyleneglycol dimethacrylate (EDMA) was purchased from Fluka A.G. (Buchs,
Switzerland), distilled under reduced pressure in the presence of hydroquinone
inhibitor and stored at 4 ◦C until use. 2,2-Dimethoxy-2-phenyl acetophenone was
supplied from Aldrich Chem. Co. (Milwaukee, WI, USA). Poly(vinyl alcohol) (Mw
27,000) was purchased from Fluka Chemica, Dimethylsulfoxide was purchased from
Sigma. Ammonium persulfate (APS) was purchased from local sources. All glassware
was extensively washed with dilute HNO3 before use. All other chemicals were of
analytical grade purity and purchased from Merck AG (Darmstadt, Germany). All
water used in the experiments was purified using a Barnstead (Dubuque, IA) ROpure
LP® reverse osmosis unit with a high flow cellulose acetate membrane (Barnstead
D2731) followed by a Barnstead D3804 NANO pure® organic/colloid removal and
ion exchange packed-bed system.
2.4. Activity of lipase proteinous nanoparticles
The activity of lipase nanoparticles was measured by the spectrophotometric
method using paranitrophenyl palmitate (p-NPP) as a substrate. The activity was
assayed by measuring the absorbance of released p-nitrophenol at 405 nm. For this
purpose, firstly stock solution of substrate containing 20 mM p-NPP in isopropanol
was prepared. Then, working substrate was prepared by diluting the p-NPP stock
solution (1:20) using 20 mM Tris HCl buffer (pH 8) and synthetic lipase activity
was measured by mixing 0.9 mL of working substrate and 0.25 mg mL−1 of lipase
proteinous nanoparticles.
3. Results and discussion
2.2. ANADOLUCA method for the preparation of nanoenzyme
3.1. Properties of lipase proteinous nanoparticles
2.2.1. Synthesis of N-methacryloyl-(l)-tyrosine
The N-Methacryloyl-(l)-Tyrosine (MATyr) was selected as the functional
monomer for lipase proteinous nanoparticles. 3-(4-hydroxyphenyl)-2-[(2-
methylacryloyl)amino]propanoic acid (methacryloyl tyrosine, (MATyr)), was
prepared according to the previously published method [28]: 5.0 g of l-tyrosine
methylester and 0.2 g of hydroquinone were dissolved in 100 mL of dichloromethane
solution. This solution was cooled down to 0 ◦C and 12.7 g triethylamine was added
into the solution. 5.0 mL of methacryloyl chloride was poured slowly into this
solution and the solution was stirred magnetically at room temperature for
2 h. At the end of the chemical reaction period, hydroquinone and unreacted
methacryloyl chloride were extracted with 10% NaOH solution. Aqueous phase was
evaporated in a rotary evaporator. The residue (i.e., MATyr) was crystallized in an
ether–cyclohexane mixture and then dissolved in ethyl alcohol.
As seen in Fig. 1A, the average particle size and size distribution
of the nanolipase were measured by Zeta Sizer (Malvern Instru-
ments, Model 3000 HSA, England) and about 100 nm. The average
particle size was an average of minimum 30 measurements, and
the size distribution (Fig. 1A) was of these repeated measurements
recorded automatically by the software. The average particle size of
the lipase nanoparticles was measured as about 100 nm. The aver-
age particle size was an average of minimum 4 measurements, and
the size distribution (Fig. 1A) was of these repeated measurements
recorded automatically by the software. Also, lipase nanoparti-
cles were investigated by TEM analysis (Fig. 1B). As seen in Fig. 1,
nanoparticles have regular distribution.
2.2.2. Chemical synthesis of photosensitive ruthenium based
aminoacid-monomer bis (2-2ꢀ-bipyridyl) MATyr-MATyr-ruthenium (II)
A
dichlorobis(2-2ꢀ-bipyridyl)ruthenium(II) (RuCl2(bipyr)2) was synthesized
according to previously published method [29]: RuCl2(bipyr)2 (0.1 g, 1 eq.) was dis-
solved in water and the solution was cooled to 0 ◦C. Then, triethylamine and the
aqueous solution of MATyr (0.1 g, 2 eq.) were added dropwise into that solution and
the solution stirred at room temperature for 30 min. The mixture was heated to
80 ◦C for refluxing ca. 24 h. The brown complex was filtered off, washed with ether
and dried under vacuum. M.p.: >200 ◦C.
3.2. Effect of pH on paranitrophenyl palmitate (p-NPP) hydrolysis
The pH is one of the important parameters that has the ability
of changing enzymatic activities in aqueous solutions [30]. In this
study, the optimum pH was determined applying different pH val-
ues in the range of 6.0–10.0 using 20 mM p-NPP. The effect of pH
on the time course of p-NPP hydrolysis is shown in Fig. 2. As seen
from the figure, the maximum hydrolytic activity was observed at
pH 8.0. All fat-digesting enzymes act in an alkaline media and the
optimal pH varies slightly from species to species. This situation
can be explained as proteinous nanoparticles are mimic the natural
lipase.
Anal. for C46H44N6O8Ru: found: C 61.54%, H 4.63%, N 10.56%, calcd.: C 60.72%, H
4.87%, N 9.24%.
1H NMR (500 MHz, CD3OD), ppm: 9.78 (1H, s), 7.8 (1H, d, J = 8.15 Hz), 7.73 (t,
1H, J = 4.1 Hz), 7.64 (t, 2H, J= 4.11 Hz), 7.47–7.41 (q, 2H), 7.3–7.11 (m, 11H), 7.05 (t,
3H, J = 8.18 Hz), 6.95 (d, 2H, J = 8.18 Hz), 6.78 (s, 9H), 6.69 (d, 1H, J = 12.75 Hz), 6.34
(s, 1H), 5.84 (s, 1H), 4.5 (s, 1H), 4.25 (s, 2H), 2.06 (s, 3H), 1.91 (s, 3H).
MALDI-TOF-MS: the ion peaks at 79, 128 and 155 m/z relating to bipyridyl. m/z
101, 413 peaks show Ru and Ru(bpy)2, respectively. m/z 250, 599 and 755 data show
MATyr monomer, Ru-(MATyr)2 and -Ru(bpy)- MATyr complex.

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R. Say et al. / Process Biochemistry 46 (2011) 1688–1692
Fig. 1. (A) Size distribution of the lipase nanoparticles distrubution result (mean/area) = 100.2 d nm/100.0% Zeta potential = −31 mV, conductivity: 0.0344 mS cm−1. (B) TEM
image of the lipase nanoparticles (the average particle size of nanolipases was about 94 9 nm).
3.3. Effect of temperature on paranitrophenyl palmitate (p-NPP)
hydrolysis
perature values ranging from 20 ◦C to 50 ◦C. As can be seen from
Fig. 3, the optimum temperature is approximately 40 ◦C. This sit-
uation can be explained as free lipase and lipase nanoparticles
have shown similar active site and this is an indication of the
Fig. 3 shows the effect of temperature on p-NPP hydrolysis.
The optimum temperature was determined applying different tem-
Fig. 3. The effect of temperature on p-NPP hydrolysis using free lipase and lipase
nanoparticles.
Fig. 2. The effect of pH on p-NPP hydrolysis using free lipase and lipase nanoparti-
cles.

R. Say et al. / Process Biochemistry 46 (2011) 1688–1692
1691
Fig. 4. Lineweaver–Burk Plot of kinetics data of hydrolysis of paranitrophenyl
palmitate.
Fig. 5. Reusabilities of lipase nanoparticles (0.25 mg mL−1).
lipase enzyme was represented in the structure of the polymeric
matrix.
4. Conclusion
A novel photosensitive nanolipase selective for p-NPP palmitate
was prepared by a microemulsion polymerization technique. The
microemulsion polymerization by using photosensitive crosslink-
ing conjugation of the protein on nano-structure without affecting
conformation and function of protein through the aminoacid-
monomer and/or ruthenium-chelate based aminoacid monomers
is not reported in the literature. Prepared lipase nanoparticles
showed high level of activity over a wider range of pH between
6.0 and 10.0, especially at the optimum pH 8.0 than that of
free form and could be removed from a reaction mixture eas-
ily and used repeatedly. The nanoparticles are very stable even
after 10 cycle usage. Thermal stability was considerably enhanced
by the photosensitive crosslinking polymerization. Since, the pro-
teinous nanoparticle preserves high enzyme loading, activity
with enhanced stability and reusability, this simple and effective
nanoparticle synthesis method is useful in enzyme engineering or
biotechnology.
3.4. Hydrolytic activity of lipase nanoparticles
The kinetic constants of p-NPP hydrolysis were obtained fitting
the data in Eq. (1).
Vm · S
V =
(1)
Km + S
where V is the initial rate, Vm is the maximum rate, S is the
concentration of substrate, Km is the Michealis–Menten constant.
Hydrolytic activity of free lipase and nanolipase proteinous parti-
cles was evaluated in the framework of Michealis–Menten kinetics.
For this purpose, p-NPP substrate concentrations in the range of
2.0–20 mM were used in each case and initial reaction rates (IRR)
of hydrolysis were determined. Then, 1/IRR versus reciprocal of the
substrate concentration (1/S) for nanoparticles was plotted that is
called Lineweaver–Burk plot (Fig. 4) and Vm and Km values were
found to be 0.72 mM min−1 and 9.3 mM, respectively.
In Michealis–Menten kinetics, Km reflects the affinity of an
enzyme for a particular substrate (the lower value of Km the higher
affinity). In the present case, Km represents the affinity of functional
groups of lipase proteinous nanoparticles for substrate p-NPP. In
studying the enzyme kinetics of substrate hydrolysis, the Km value
determined for the nanolipase is lower than free enzyme (10.5 mM)
indicating that the affinity of nanolipase towards the substrate is
higher than that of free lipase. On the other hand, the Vmax of the
free enzyme (1.7 mM min−1) was higher than that of the nanolipase,
thus suggesting that the because of the crosslinking of nanolipase,
the number of active site that the substrate binds decreases. At
the same time, the regeneration of active sites of the nanolipase
is possible without loosing the affinity towards the substrate. This
situtation brings with the reusability advantage of nanolipase.
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