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Lipase immobilization onto polyethylenimine coated magnetic nanoparticles assisted by divalent metal chelated ions
Accepted Manuscript Title: Lipase immobilization onto polyethylenimine coated magnetic nanoparticles assisted by divalent metal chelated ions Author: Seyed Farshad Motevalizadeh Mehdi Khoobi Armin Sadighi Masoud Khalilvand-Sedagheh Mehrdad Pazhouhandeh Ali Ramazani Mohammad Ali Faramarzi Abbas Shafiee PII: DOI: Reference:
S1381-1177(15)30020-5 http://dx.doi.org/doi:10.1016/j.molcatb.2015.06.013 MOLCAB 3187
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date: Revised date: Accepted date:
24-2-2015 21-6-2015 21-6-2015
Please cite this article as: S.F. Motevalizadeh, M. Khoobi, A. Sadighi, M. KhalilvandSedagheh, M. Pazhouhandeh, A. Ramazani, M.A. Faramarzi, A. Shafiee, Lipase immobilization onto polyethylenimine coated magnetic nanoparticles assisted by divalent metal chelated ions, Journal of Molecular Catalysis B: Enzymatic (2015), http://dx.doi.org/10.1016/j.molcatb.2015.06.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Lipase immobilization onto polyethylenimine coated magnetic nanoparticles assisted by
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divalent metal chelated ions
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Seyed Farshad Motevalizadeh a,b, Mehdi Khoobia, Armin Sadighic, Masoud Khalilvand-Sedagheha,
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Mehrdad Pazhouhandeh e, Ali Ramazani d, Mohammad Ali Faramarzie,* and Abbas Shafieea,*
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a
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Center, Tehran University of Medical Sciences, Tehran 14176, Iran
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b
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Melbourne, VIC 3010, Australia
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Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research
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Particulate Fluids Processing Centre, School of Chemistry, The University of Melbourne,
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c
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Rhode Island, Kingston, Rhode Island, USA
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d
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e
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Center, Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran 14176, Iran
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Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of
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Department of Chemistry, University of Zanjan, P.O. Box 45195-313, Zanjan, Iran
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Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research
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*Corresponding authors: E-mail: [email protected] (A. Shafiee);Tel: +98-21-66406757; Fax: +98-21-
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66461178 and E-mail: [email protected] (M.A. Faramarzi); Tel: +98-21-66954712; Fax: +98-21-
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66954712
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Abstract
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In this study, polyethylenimine coated Fe3O4 magnetic nanoparticles (Fe@PEI) were prepared and
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used for three metal ions (Co2+, Cu2+ and Pd2+ ) chelation. The metal chelated magnetic
4
nanoparticles (Fe@PEI-M) were characterized by using different spectroscopic and analytical
5
techniques. The metal contents were analyzed by inductively coupled plasma atomic emission
6
spectroscopy (ICP) and energy-dispersive X-ray spectroscopy (EDX). Thermomyces lanuginosa
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lipase (TLL) was then immobilized onto the modified magnetic supports by physical adsorption.
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Fe@PEI-Co and Fe@PEI-Cu showed better activity at extreme temperature and pH values than of
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the free enzyme. After 10 cycles of successful biocatalytic activity performed by immobilized lipase
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on Fe@PEI-Co, more than 60% of initial activity was reserved. Furthermore, the immobilized
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lipase onto Fe@PEI-Co retained about 80% of its initial activity after 14 days of storage. The
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immobilized lipase on the selected nanocomposite was then used for synthesis of ethyl valerate.
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After a 24 hr incubation time, the extent of esterification was found to be 70 and 60% in n-hexane
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and DMSO media, respectively.
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Keywords: Lipase immobilization, Adsorption, Metal chelation, Magnetic nanoparticles,
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polyethylenimine, Bioconversion.
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1. Introduction Biological effectiveness of some therapeutical medicines is extensively limited to one
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specific enantiomer of chemical substance that results in high pharmacologic indices and lower side
4
effects. Lipases (triacylglycerol hydrolysis, E.C. 3.1.1.3) are considered as enantioselective and
5
regioselective enzymes, which have been broadly used in pharmaceutical industries [1–3]. Lipases
6
present two physical conformations; an open, active form, and a closed, relatively inactive form.
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The open form is recognized when the α-helix polypeptide chain moves, akin to removing a lid
8
from a container, revealing the active site of molecule [4, 5]. From practical and economical points
9
of view, it is beneficial to utilize immobilized lipases for enantiomeric syntheses [3, 6]. Lipases are
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the most frequently used biocatalyst which can catalyze enantioselective hydrolytic reactions and
11
chemo-, regio- and stereo-selective formation of a diverse range of amide and ester bonds. Lipases
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have gained fame as a best catalyst for resolutions of a wide range of compounds like thioesters,
13
cyclophanes, phosphonates, ferrocenes, calixarenes, and helicenes [7]. Special features of lipases
14
have made them as a best candidate for synthesis of many biologically active compounds and
15
natural products and food processing industries [8].
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Over the past few years, several methods have been developed for lipase immobilization,
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including multipoint covalent attachment, cross-linking, physical entrapment, and adsorption [9].
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The adsorption method, which is simple, cost effective, and preserves catalytic activity of lipase has
19
attracted significant commercial attentions in the recent years [3]. Moreover, there is no need to use
20
cross-linking agents, which reduce the enzyme activity. Some of the adsorbed lipase, however, will
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desorb during washing and operation processes. One of the most critical challenges in enzyme
22
immobilization procedures is preservation of enzyme activity on the support. Poor immobilization
23
technique may change enzyme conformation from the open form to the closed form and therefore
24
reduce enzyme activity. On the other hand, immobilization through non-covalent bonding, such as
25
hydrogen bonding, hydrophobic interaction, and ion- exchange is a gentle enzyme immobilization
26
process. In these methods, lipase was immobilized by physical adsorption, which could be easily
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desorbed from the support. This led to a decrease in immobilized lipase activity. Therefore,
2
selecting an appropriate immobilization process to produce a constant enzyme-support system and
3
open form of enzyme is crucial.
4
Different approaches to stabilize lipase on solid supports have been investigated by various research groups, and a large number of substances have been utilized to make lipase adsorption
6
effective and convenient. For instance, active carbon, porous glass, MCM-41, chitosan
7
microspheres, and magnetic nanoparticles (MNPs) have been successfully used to improve the
8
adsorption process [1, 2, 10]. Two main advantages of magnetic nanoparticles are that they can be
9
readily separated from solution by an external magnet and consequently evaluated for their
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recyclability. A great number of magnetic composite materials have been developed for enzyme
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immobilization during previous years [11–13]. However, naked nanomagnetic particles are not
12
sufficient enough for stabilizing enzymes due to the lack of appropriate functional groups. In order
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to overcome this limitation, magnetic nanoparticles can be decorated with polymers.
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Polyethylenimine (PEI) is an ideal polymer for this role due to its high density of primary,
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secondary, and tertiary amine functional groups across the polymer chain. PEI is also ideal for
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chelating metal ions, which has been successfully applied in biomedicine [14, 15] and
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environmental remediation [16, 17]. The electrostatic repulsive forces and steric hindrance of PEI-
18
coated nanoparticles enhance their dispersion in solution [18]. In our previous study, we have
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demonstrated that modifying the surface of nanoparticles by coating with PEI is inappropriate for
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lipase immobilization via the adsorption method [1].
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Enzyme can be improved and stabilized by chelating metals to the polymer network prior to
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immobilization on the nanoparticle surface [19, 20]. The choice of chelating metal is based on the
23
interaction between chelated metal ion and particular functional groups of the enzyme. Notably, the
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thiol group of cysteine, the indoyl group of tryptophan, or the imidazole group of histidine are the
25
most important responsible functional groups to couple a chelated metal interaction with enzyme.
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In this study, a new approach to avoid the drawbacks of non-stable physical adsorption of enzyme and covalent attachment that restrict the enzyme activity is proposed. The potential of core-
3
shell MNPs-PEI structure with myriad available amine branches to interact with lipase [21] was
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increased by loading divalent metal chelators with the ability to immobilize enzyme [22]. For this
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reason, the surface of MNPs was covalently coated with PEI and three different metal ions (Co2+,
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Cu2+, and Pd2+) were subsequently loaded on the supports to obtain a suitable adsorption capacity.
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Lipase was immobilized onto the Fe@PEI-M via adsorption and then their catalytic activity was
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compared with the free enzyme. Finally, the best lipase immobilized system was utilized in the
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synthesis of ethyl valerate (green apple flavor) in various solvents.
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2. Materials and methods
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2.1. Chemicals and the enzyme
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[3-(2,3-Epoxypropoxy)propyl]trimethoxysilane (EPO, 98% purity), p-nitrophenyl butyrate
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(p-NPB), aqueous ammonia solution (28 wt.%), ferric chloride hexahydrate (FeCl3·6H2O), ferrous
15
chloride tetrahydrate (FeCl2·4H2O) and oleic acid were obtained from Merck (Darmstadt,
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Germany). Hyperbranched polyethylenimine (PEI, MW=60000), palladium chloride (99% purity),
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copper (II) chloride (99% purity), and cobalt (II) chloride (97% purity) were purchased from Merck
18
AG or Aldrich. Lipase derived from Thermomyces lanuginosa (TLL) was generously gifted by
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Novozymes (Bagsvard, Denmark). TLL was refined prior to immobilization on the support via
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adsorbtion on octyl-sepharose beads in 10 mM sodium phosphate, with continuous stirring (pH 7.0).
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The enzyme was then separated by a sintered glass funnel and rinsed three times with the same
22
buffer. Finally, the purified TLL was separated from the supports by immersing the immobilized
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lipase in 25 mM sodium phosphate at pH 7.0 (in a relation of 1/10 w/v) containing 0.6% v/v of
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CTAB during 1 h at 25 °C [23].
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2.2. Preparation of metal chelated PEI functionalized MNPs
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Fe3O4 MNPs were easily prepared by a simple co-precipitation method in basic media. In
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order to prepare PEI functionalized magnetic nanoparticles, 1.5 g of PEI was initially added into
3
150 mL dry toluene and stirred for a few minutes. Then, 1 mmol of EPO was added to the mixture
4
and allowed to react at 80 °C for 24 h. After that, 2.5 g of dispersed Fe3O4 MNPs in 25 mL of
5
ethanol were added and the reaction was allowed to proceed for another 24 h at 80 °C. Fe@PEI was
6
easily isolated by applying an external magnet, washed several times with ethanol and dried at 40
7
°C for several days [1]. In order to introduce metals on the surface of Fe@PEI, 300 mg of Fe@PEI
8
were added into a round bottomed flask containing a solution of corresponding chloride salts of the
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single metals (PdCl2, CuCl2 and CoCl2) (0.6 mmol) in acetonitrile (25 mL) and stirred under
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nitrogen atmosphere for 48 h. The resultant residues were separated and washed with
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acetonitrile/acetone, and dried in air for 24 h [24].
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2.3. Immobilization of lipase
A physical adsorption method was used in this study for lipase immobilization. The purified
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TLL solution (1 mL, equal to 5 U) was prepared according to our previous experiments [1,2]. The
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immobilization yield (IY) and efficiency (IE) were calculated based on the following equations: IY
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(%) = [(Yo–Y1) / Yo] × 100 and IE (%) = [E1 / Eo] × 100; where Yo and Y1 indicate the amounts of
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total protein and unbounded protein to the substrates, respectively, whereas Eo and E1 represent the
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total and immobilized enzyme activity, respectively. The essential time for maximum
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immobilization efficiency was determined by immersing the chelated metal support (10 mg) in 10
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mL phosphate buffer (0.1 M, pH 7) containing lipase (1 mL, equal to 5 U), then slowly incubation
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at 25 °C for 15–90 min [25, 26].
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2.4. Characterization
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Thermogravimetric analysis (TGA) was performed by TGA Q50 thermogravimetric
26
analyzer of a TA instrument. A heating rate of 10 °C per min under argon flow was used from room
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temperature to 800 °C. X-ray diffraction (XRD) analysis was executed using a XPert MPD
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advanced diffractometer with a Cu (Kα) radiation source (wavelength: 1.5406 Å) at room
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temperature in the 2θ range of 4° to 120°. Fourier transform infrared (FT-IR) analysis was recorded
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on Nicolet FT-IR Magna 550 spectrographs (KBr disks). The surface morphology of the samples
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was investigated by scanning electron microscopy (SEM, VEGAII TESCAN) with an acceleration
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voltage of 15 kV. In addition, a CM30 Philips Company high-resolution transmission electron
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microscopy (TEM) was utilized to study the final shape of the nanoparticles. Sample preparation
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was done via placing a droplet (1 µL) of sample dispersion latex along with a droplet of water on a
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copper grid covered by Formvar foil (200 mesh). The samples were then dried and analyzed. The
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EDX spectra were acquired using a Philips XL 30 X-Ray spectrometer. The metal content in
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samples was analyzed by ICP-OES (VARIAN-VISTA-PRO). A vibrating sample magnetometer
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(VSM, Meghnatis Kavir Kashan Co., Kashan, Iran) was used at room temperature to investigate
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magnetic properties of the samples. Dynamic light scattering (DLS) was applied to determine the
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particles size (DTS Ver. 4.20). The quantity of ester and residual acid were measured by Digital
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Burette VITLAB continuous RS Titrimeter (made in Germany) and also confirmed by GC mass
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analysis (VARIAN CP-3800, Column properties: CP-WAX 57 CB GLY FS 25X, see
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supplementary data for detailed information).
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2.5. Lipase assay and protein estimation
para-Nitrophenyl butyrate (p-NPB) was applied as substrate to measure the catalytic activity
21
of free and immobilized TLL as described previously [1,2, 10, 27]. Briefly, a mixture of free (0.1
22
ml) or immobilized lipase (10 mg) and 0.9 ml p-NPB solution [0.4 mM in phosphate buffer (100
23
mM, pH 7.4)] was prepared. Then it was incubated at 37 °C and 120 rpm for 30 min followed by
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determining the absorbance at 348 nm using UV-Vis spectrophotometer (UVD 2950, Labomed Inc.,
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Culver City, CA, USA). The quantity of enzyme that release p-nitrophenol at a rate of 1 μmol per
26
min is equal to one unit of enzyme activity [28]. The total amount of free and immobilized protein
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were measured by the Bradford assay at 595 nm based on the bovine serum albumin standard line
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(12.5–400 µg/mL) [1, 2, 29].
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2.6. pH, temperature, and storage stability of immobilized lipases Free and immobilized enzyme were incubated in 100 mM citrate-phosphate buffer (pH 3–7), Tris-HCl buffer (100 mM, pH 8) and 100 mM glycine-NaOH buffer (pH 9–10) at 37 °C for 1h to
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investigate the effect of pH on enzyme consistency by determining the residual enzyme activity as
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previously explained. To study the thermal stability of the free and immobilized lipase, the enzyme
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was placed in a water bath for 1 h at temperatures ranged between 15 to 75 °C and the residual
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lipase activity was measured. The immobilized lipase was placed in the incubator at 25 °C for 14
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days to analyze storage stability by measuring remained activity in taken samples.
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2.7. Reusability of immobilized lipases
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After using immobilized lipase at the first run, the biocatalyst was separated by an external
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magnet and the activity was measured. Then, the immobilized biocatalyst was washed three times
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with phosphate buffer (100 mM, pH 6.5), and enzyme activity was recalculated. The lipase activity
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was determined as 100% at the first run and during the next succeeding runs was defined as relative
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activity.
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2.8. Enzymatic synthesis of ethyl valerate by free and immobilized lipase The immobilized lipase on the Fe@PEI-Co was applied as biocatalyst for preparing ethyl
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valerate in both water and organic solvents. Initially, the optimal temperature was obtained by
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running the reaction at different temperatures (30, 45, and 60 oC). Ethyl alcohol/valeric acid ratio
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was 2:1 and it was carried out for 1, 2, 4, 6, 12, and 24 h under gentle shaking (100 rpm in solvent-
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free medium). With the best temperature in hand, the influence of alcohol to acid molar ratio (i.e.,
26
2:1, 1:1, 1:2, 1:3, 1:4, and 1:5) was studied. To measure the quantity of ester content and residual
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acid, a simple alkalimetric approach was utilized (titration using 0.1 N NaOH and phenolphthalein
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as an indicator). From the consumption of valeric acid after a certain time, the quantity of
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synthesized ester can be determined [30, 31]. Under the conditions without enzyme, the ester yield
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was less than 2%. The optimal condition was applied to investigate the impact of different solvent
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system. A mixture of valeric acid (1 mmol), ethyl alcohols (4 mmol), solvent (15 ml) and
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biocatalyst was prepared and reaction was run at 40 °C and 100 rpm shaking. Inactivated free lipase
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was used as control. Ester formation was also analyzed by GC technique. The crude reaction
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mixture was injected in GC injection port after 12 hours treatment with biocatalyst in n-hexane. The
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results showed that the yield of the esterification was 58% and this result was in good conformity
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with the result obtained by alkalimetric approach (Fig. S4).
3. Results and discussion
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3.1. Preparation of MNPs@PEI
To date several studies have reported PEI grafting to MNPs by electrostatic interaction [32].
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Recently we prepare PEI coated Fe3O4 MNPs via covalent bonds which provide a permanent and
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considered attachment as an advantage compared to coating MNPs with PEI through electrostatic
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interactions (Fig.1) [33, 34]. This PEI coated MNPs were used as a best candidate for different
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metal chelating due to their high accessible amine groups on the surface of MNPs. In this work a
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double capacity substrate was prepared for enzyme immobilization either by divalent metal ions or
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through PEI coated magnetic nanoparticles, which present many available amine groups to interact
21
with hydroxyl and carboxylic groups of amino acids present on the periphery of lipase. TLL, like
22
other lipases, consists of a common α/ß hydrolase fold composed of a nucleophilic serine linked to
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acidic residue (Asp or Glu) and histidine. This catalytic triad is structurally conserved among all
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kinds of lipases and plays the main role in transition of lipase from closed form (inaccessible active
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site) to open form, which facilitate the availability of substrate to catalytic site [35–37]. The affinity
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of three different divalent metal ions (Co, Cu, and Pd) was evaluated for TLL adsorption and the
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performance of the best catalyst was investigated for apple flavor preparation in different media.
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Fig. 1 shows the sequential steps for Fe@PEI-M preparation and lipase immobilization. First, the amine groups in PEI were reacted with the epoxy groups of EPO. Then, MNPs were
5
attached to the silanol groups of the latter. Afterward different metal ions were chelated onto
6
Fe@PEI and finally enzyme was immobilized onto the modified magnetic supports.
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3.2. Characterization of Fe3O4 MNPs and Fe@PEI
FT-IR measurements have been conducted to confirm the coating of Fe3O4 by PEI. As
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shown in Fig. 2, a strong absorption band at around 600 cm-1 of all MNPs could be assigned to Fe–
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O vibrations of the magnetite [38]. Upon EPO coating of Fe3O4, Si–O–H peaks can be seen at
12
1000–1100 cm−1. The Fe–O–Si peak that refers to chemical binding between Fe3O4 and silica
13
cannot be seen in the FT-IR spectrum because it was revealed at around 584 cm−1 and therefore
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overlaps with the Fe–O vibration of MNPs [38]. However, the co-existence of Fe3O4 and silica
15
peaks together indicates the coating of Fe3O4 by silica [39]. The broad OH band at around 3300-
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3400 cm−1 could be attributed to the high amount of amine groups of PEI on the surface of MNPs.
17
The shell/core structure of PEI around MNPs can be also elucidated from the existence of C–N peak
18
at about 1300-1400 cm−1. Moreover, aliphatic C–H stretching vibrations that were appeared at 2924
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and 2831 cm−1 provide further evidence for existence of EPO and polymer on magnetic
20
nanoparticles [39–41].
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XRD patterns of Fe3O4 MNPs and Fe@PEI are shown in Fig. S1. All peaks were well
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demonstrated as spinel magnetite which are in good agreement with the standard literature data
23
(JCPDF card number: 86-1354) [42].These sharp diffraction peaks revealed that the spinel
24
magnetite product has well-defined crystallinity. In addition, no impurity diffraction peaks were
25
detected, suggesting a pure phase of the synthesized Fe3O4 MNPs. The XRD data for the
26
synthesized MNPs showed the diffraction peaks at 2θ=30.23ο, 35.16 ο, 43.22 ο, 53.64 ο, 57.54 ο, and
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62.38ο, which can be related to the 220, 311, 400, 422, 511, and 440 planes of Fe3O4. After
2
functionalization of magnetic nanoparticles, the XRD patterns exhibited similar diffraction peaks
3
that do not undergo any notable change, suggesting that the magnetite structure was retained. It can
4
be concluded that the structure of Fe3O4 MNPs was preserved after loading the PEI and
5
subsequently, it was demonstrated that modification of the Fe3O4 nanoparticles do not lead to any
6
change in crystal phase. Crystallite size of Fe@PEI was also measured using Scherrer's equation
7
(17.2 nm).
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TGA curves of PEI, pure Fe3O4 MNPs and Fe@PEI (Fig. S2a) were used for a quantitative
9
comparison of degradation behavior of the prepared samples. The weight loss of Fe3O4 MNPs that
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was detected at 100–280 °C can arise from removal of the hydroxyl group adsorbed water and/or on
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the surface of Fe3O4 MNPs. The same results were obtained by Cao et al. [43] following similar
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preparation methods. Fig. S2a also shows that the weight-loss of Fe3O4 MNPs in the range of 280°C
13
to 800°C is around 2.5 wt%, resulting from the oxidation of Fe3O4 MNPs. TGA analysis of the sole
14
PEI also shows that the polymer decomposition occurs in the range of 100 and 517 °C. As PEI is in
15
the form of 50 wt.% aqueous solution, the first weight loss of PEI at 100–200 °C can arise from
16
water evaporation and the next weight loss in the range of 200°C to 517 °C can be ascribed to the
17
polymer decomposition. In the same region, the weight-loss of Fe@PEI increases to 30 wt%. This
18
could be due to chemical binding of EPO and PEI groups. By subtracting the weight-loss of
19
Fe@PEI and Fe3O4 MNPs, the weight of PEI modified on the surface of MNPs can be estimated as
20
27 wt%.
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Surface coating with various phases, morphology, physical characteristics and size can
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affect on the magnetic properties of MNPs. To investigate magnetization (M) and the impact of
23
coating on paramagnetic properties of MNPs, an external magnetic field (H) ranging from -8 KOe
24
to +8 KOe were applied using VSM. Fig. S2b show the M–H curves of Fe3O4 MNPs and Fe@PEI.
25
No hysteresis in the magnetization was observed and both remanence and coercivity were zero in
26
magnetization versus applied magnetic field (M–H loop) curves. It suggests that both unmodified
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and PEI-modified MNPs show super paramagnetic character. The coercive field (Hc) and the
2
particular saturation magnetization (σs) for Fe3O4 MNPs are 32 Oe and 46.4 emu g-1, respectively.
3
But these values changed as 30.2 Oe and 33.8 emu g-1 for the Fe@PEI, respectively. Although,
4
coatings affected the magnetic power, it still could be collected by permanent magnets (200 mT)
5
within 20 s. It can be concluded that anchoring of the polymers on the surface of MNPs may alter
6
the surface magnetic anisotropy. This can result in the increasing of the surface spins disorientation
7
which leads to the reduced saturated magnetization [44].
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SEM and TEM were used to verify the fine morphology and structure of MNPs. Fig. 3a and b represent the SEM and TEM images of Fe@PEI-Co as best support. The diameter of the pristine
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Fe3O4 nanoparticles is less than 70 nm (Fig S3a) and the PEI grafting onto the MNPs exhibits a
11
morphology resembling that of the original material, but the size of the Fe@PEI expands (Fig. S3b
12
and S3c). The diameter and morphology of Fe@PEI-Co was also similar to Fe@PEI. The samples
13
show polydispersity and aggregation because of the high surface/volume ratio and subsequent
14
enhancement of magnetic dipole–dipole interactions.
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ICP results showed that the incorporated Co, Cu, and Pd percentage in Fe@PEI-M
16
substrates were 6.76, 8.87, and 5.03%, respectively. The EDX spectra of Fe@PEI-M are shown in
17
Fig 4. Atomic weight ratio of C: N: Fe: Metal were 20.40: 12.78: 11.72: 4.36, 19.90: 13.52: 18.08:
18
3.13 and 15.35: 13.75: 11.50: 3.06 for Fe@PEI-Co, Fe@PEI-Cu and Fe@PEI-Pd, respectively,
19
which demonstrate that the MNPs are coated with PEI, and that the metals were chelated
20
successfully.
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3.3. Selection of chelated metal ions Chemical bonds between atoms with similar rating are elucidated by hard and soft acids and
24
bases (HSAB theory or Pearson acid base concept) e.g. hard base bonded with a hard acid are the
25
strongest. Based on this concept, metal ions such as Fe3+, Mg2+, Ca2+ and K+ are categorized as hard
26
Lewis acids, while metal ions such as Cu+, Ag+ and Pd2+ are classified as soft Lewis acids. The
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Page 12 of 29
transition metal ions, such as Co2+, Cu2+, and Zn2+ are defined as ‘‘borderline acids”. Ligands
2
including oxygen (carboxylate) and aliphatic nitrogen (asparagine) are classified as hard Lewis
3
bases. Ligands containing sulfur (cysteine) and aromatic nitrogen (e.g., histidine and tryptophan)
4
are categorized as soft bases and borderline bases, respectively. The borderline acids, comprising
5
Co2+, Cu2+ and Zn2+ coordinate favorably with borderline bases (e.g., aromatic nitrogen atoms) and
6
also soft base like sulphur atoms [45, 46].
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1
In this special approach, strong interaction between metal ions and enzyme is crucial.
Tryptophan, cysteine, and histidine are promising residues on the surface of enzyme to provide
9
strong interaction with metal ions, while histidine has the strongest affinity for metal ions. In
10
interactions between immobilized Co2+, Pd2+, Cu2+, Zn2+ and amino acid residues on protein
11
surfaces, amine groups are the main targets for the metal ions [46, 47].
12
Three metal ions, Co2+, Pd2+ and Cu2+, were designated as the chelated ions, which can easily form
13
coordination bonds with the residual groups of lipase.
M
an
us
cr
8
3.4. Characterization of immobilized lipase
Ac ce pt e
15
d
14
16
The amount of immobilized lipase and the relative activity of lipase versus immobilization
17
time are shown in Fig 5a. The lowest time (15 min) for maximum lipase loading (95%) was found
18
for Fe@PEI-Co. In the case of Fe@PEI-Cu, 90% of lipase was loaded after 15 min incubation
19
while only 30% of TLL was loaded on the Fe@PEI-Pd at the same time. For Fe@PEI-Co and
20
Fe@PEI-Cu, the immobilization process was complete after 30 min incubation in the presence of
21
TLL, whilst Fe@PEI-Pd required 90 min for complete immobilization. Maximum immobilization
22
yield (88±1.1%) and immobilization efficiency (82±2.2%) was obtained for Fe@PEI-Co followed
23
by Fe@PEI-Cu (IY%, 73±0.9% and IE%, 68±1.8%) and Fe@PEI-Pd (IY% of 66±2.6% and IE% of
24
58±1.4%).
25 26
Türkmen and co-workers reported the possibility of cytochrome c adsorption to the PEI modified poly (2-hydroxyethylmethacrylate) (mPHEMA) magnetic beads via chelated Cu2+ ions.
13
Page 13 of 29
The authors mentioned that Cu+2 chelation through three amine groups of PEI significantly
2
increased the loading efficiency of protein onto magnetic beads compare to the absence of metal
3
chelation [48]. The presence of Ser-His-Asp catalytic triad has been revealed in the three
4
dimensional crystallographic structure of lipases [49,50], which could be the main player in metal
5
ions chelation and subsequent immobilization on the surface of Fe@PEI particles. In our study TLL
6
was immobilized on the chelated support via two possible interactions. First, charge of TLL is
7
negative (isoelectric point of 4.4) [4] and this may lead to interaction between TLL and positive
8
charge of metal chelated in the constructed support. Second, immobilization could have achieved
9
via coordination bond between metal ion groups and residual moieties in TLL. It is worth to
us
cr
ip t
1
mention that, if the net charge of support and enzyme were opposite, the enzyme would be more
11
stable on the support [6]. It was also established that multipoint interaction might aid to increase the
12
structural constancy of enzymes [51, 52]. Along with the possible interactions between chelated
13
metal ions with the TLL that are responsible for enzyme immobilization, the potential of PEI
14
available amine groups to establish a dipole-dipole interaction with the carboxylic groups of acidic
15
amino acids present on the enzyme surface could be considered as the reason of TLL activity in the
16
absence of metal ions. Indeed, metal chelated ions increase the capacity of enzyme immobilization.
18
M
d
Ac ce pt e
17
an
10
3.5.1. Effect of changing pH and temperature on free and immobilized lipase
19
The pH value of the catalysis environment has a great effect on the biotransformation
20
efficiency. This effect is more pronounced when the free and immobilized enzymes are compared in
21
terms of activity. The effect of pH on the activity of the immobilized and free lipase was
22
investigated at the pH range between 3 to 10 (Fig 5b). For the free lipase, the maximum activity was
23
observed at a pH of 7, while higher relative activity and excellent adaptability was observed at a
24
wider pH range for immobilized lipase. For example, immobilized lipase on Fe@PEI-Cu preserved
25
more than 80% of its initial activity at the pH range from 3 to 10. According to the Fig 5b, higher
26
pH stability was observed for immobilized lipase on the metal chelated MNPs compared to the
14
Page 14 of 29
1
absence of metal. It could be assigned to the immobilization method, carrier charge and structure [6,
2
52]. High resistance of immobilized enzyme in the wide range of pH is associated with the ionic
3
interactions between the negative residual groups on the enzyme and the positive charge of the
4
metal ion on the surface, as well as changes in the enzyme oligomerization [53]. Similar behavior
5
has also been observed for immobilized invertase and polyphenol oxidase [54, 55]. One of the most critical properties for industrial application of lipase is thermal stability.
7
The thermal stability of the free and immobilized lipases was studied in the temperature range of
8
15–75 oC and results are shown in Fig 5c. The optimum temperature for activity of free lipase is 25
9
o
cr
ip t
6
us
C, whereas relative activity for immobilized lipase on Fe@PEI-Co was more than 80% within 15–
65 oC. Higher thermal stability and relative activity for immobilized enzyme onto Fe@PEI in the
11
presence and absence of metal in comparison to free lipase is probably due to the increase of the
12
rate of a coordination reaction, which prevents lipase from conformational changes and
13
denaturation at high temperature. However, temperatures above 65 °C can result in a clear decrease
14
in activity of immobilized lipases due to the enzyme denaturation.
d
M
an
10
16
Ac ce pt e
15
3.5.2 The operational and storage stability of free and immobilized lipase Recycling of enzyme after multiple rounds, which results in decreasing the unit cost of
17 18
enzymatic process is one of the most important features of immobilization [56]. Fig. 6a shows the
19
number of successful catalytic cycles accomplished by the immobilized lipase against their relative
20
activity. Fe@PEI-Co maintained more than 80% of its initial activity after 8 times running in the
21
process, while more than 70 and 60% of initial activity for Fe@PEI-Cu and Fe@PEI-Pd were
22
maintained, respectively, during the same period. When the operation time was increased,
23
immobilized lipase activity decreased gradually. The denaturation of the lipase and the leakage of
24
enzyme from the support could be an explanation for the inactivation of the enzyme after each reuse
25
cycle.
15
Page 15 of 29
1
Generally, enzymes in solution are not constant and their activities decline with time. It makes the storage of free enzymes particularly difficult. One of the best methods to address this
3
issue is immobilization. This prevents the gradual reduction of enzyme activity in solutions,
4
maintaining their practical application in biochemical studies. Free and immobilized lipase were
5
stored at 25°C, and activity measurements were carried out over a period of 14 days. Fig. 6b
6
illustrates that immobilization increased storage stability of free lipase. The free enzyme lost 90%
7
of its activity within 14 days, whereas the immobilized lipase onto Fe@PEI-Co, Fe@PEI, Fe@PEI-
8
Cu, and Fe@PEI-Pd retained more than 80, 65, 60, and 55% of their initial activity during the same
9
period time, respectively. According to the depicted results, it can be concluded that lipase
us
cr
ip t
2
immobilization improves storage stability and Co chelated substrates exhibited the higher stability
11
along with the other supports.
an
10
14
3.6. Esterification studies
Biotechnological approaches are predominantly used in bioprocess of flavor compounds in
d
13
M
12
modern food industries. Some of the esters, which are known as flavors, have been prepared by
16
esterification reactions via enzymes. In this study, the potential of free and the immobilized lipases
17
were compared in esterification of valeric acid with ethanol to form ethyl valerate in various media
18
(Fig. 6c). Ethyl valerate was synthesized with a 4:1 molar ratio of valeric acid to alcohol at 45 oC at
19
optimized condition and only 23% conversion was observed for ethyl valerate (data not shown).
20
The immobilized lipase on Fe@PEI-Co showed about 40% bioconversion of valeric acid into ethyl
21
valerate in 24 h under similar conditions.
22
Ac ce pt e
15
The choice of organic solvent has a huge impact on the yield of the final product (Fig. 5c).
23
This is due to esters transferring into the organic phase, and this may lead to shifting equilibrium
24
towards the ester synthesis. These results are entirely consistent with other studies performed by
25
other groups [57, 58].
16
Page 16 of 29
1
We also applied leaching test to investigate the amount of the metal desorption from the solid catalyst. In order to determine metal species leached from the solid catalyst, Fe@PEI-Co was
3
stirred in aqueous media for one day. MNPs were magnetically separated from the reaction mixture.
4
The solution was concentrated and checked for determination of the leached metal ion by ICP
5
analysis and isolated MNPs were also used for the next three runs under the above mentioned
6
condition. ICP analysis was shown that amount of the leached cobalt from the catalyst is less than
7
0.1 ppm for the first run and less than 0.015 ppm for the next runs. The European Food Safety
8
Authority [59], the United Kingdom Expert Group on Vitamin and Minerals [60], and Finley et al
9
[61] have proposed that 600, 1400, and 2100 μg Co/day should be considered safe [62]. These
us
cr
ip t
2
results revealed that amount of the metal leached from the biocatalyst is negligible and the catalyst
11
could be used in the food industry [63]. It could be attributed to the presence of high amount of
12
hydroxyl and carboxyl groups on the surface of enzyme with a good affinity for chelating to the
13
divalent metal ions.
16
4. Conclusion
M
Ac ce pt e
15
d
14
an
10
Different approaches to improve the conformational stability of enzymes have been studied.
17
Enzyme type, immobilization route and structure of support play a significant role in the extent of
18
stabilization. In this work a double capacity substrate was used for enzyme immobilization either by
19
divalent metal ions (e.g., Co, Cu, and Pd) or through PEI coated magnetic nanoparticles, which
20
present numerous available amine groups to interact with hydroxyl and carboxylic groups of amino
21
acids present on the periphery of lipase. Herein, we evaluate the affinity of three different divalent
22
metal ions (Co, Cu, and Pd) for TLL adsorption on the MNPs and performance of the catalyst for
23
apple flavor preparation. The results show that metal chelate ions increase the capacity of enzyme
24
immobilization and cobalt could be a good candidate for TLL immobilization rather than other
25
metal. The storage ability of immobilized lipases was improved and they retained their relative
26
activity in the wide range of temperature and pH compared with free lipase, due to the chelation.
17
Page 17 of 29
1
This biocatalyst can be easily separated from mixture of the reaction by using an external magnet
2
without need of filtration or decanting.
3
5 6
Acknowledgements This work was supported financially by the grants from Tehran University of Medical Sciences, and Iran National Science Foundation (INSF) Tehran, Iran.
Ac ce pt e
d
M
an
us
cr
7
ip t
4
18
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Page 22 of 29
Figure Captions
2
Fig.1: A schematic illustration of synthetic pathway for preparation of Fe@PEI-M and enzyme
3
immobilization via adsorption.
4
Fig.2: FT-IR spectra of the prepared (a) Fe3O4 MNPs, (b) Fe@PEI, (c) Fe@PEI-Co, (d) Fe@PEI-
5
Cu, (e) Fe@PEI-Pd.
6
Fig.3: The SEM micrograph and TEM images for Fe@PEI-Co.
7
Fig.4: The EDX spectra of (a) Fe@PEI-Co (b) Fe@PEI-Cu (c) Fe@PEI-Pd.
8
Fig.5: a) Time, b) temperature, and c) storage stability of free and immobilized lipase on supports.
9
Fig.6: (a) reusability, (b) storage profile of lipase immobilized on prepared supports. (c)
us
cr
ip t
1
Conversion of valeric acid in the presence of ethyl alcohol using free and immobilized lipase onto
11
the Fe@PEI-Co in aqueous and non-aqueous media.
M
12
an
10
Graphical abstract
14
Lipase immobilization onto polyethylenimine coated magnetic nanoparticles assisted by
15
divalent metal chelated ions
16
Seyed Farshad Motevalizadeh, Mehdi Khoobi, Armin Sadighi, Masoud Khalilvand-Sedagheh, Mehrdad
17
Pazhouhandeh, Ali Ramazani, Mohammad Ali Faramarzi,* and Abbas Shafiee*
Ac ce pt e
18
d
13
Lipase Immobilization
Fe@PEI-M
O OH
Fe@PEI-M-Lipase HN
N n
O
EtOH
H N
N
H2N HN
N
M 2+
O Apple flavour
19 20
Highlights 23
Page 23 of 29
•
Synthesis of metal chelated magnetic nanoparticles.
2
•
Lipase immobilized on nanoparticles by adsorption and metal chelation.
3
•
Good reusability, storage time, optimal pH, and temperature of immobilized lipase.
4
•
The immobilized lipase was used as biocatalyst for the synthesis of green apple flavor. H2N
+
Fe (III)
+ NH4OH
O
H2 N NH
O
O
H N
NH N
N H
N
N
HPEI
NH
n
+ MeO
O MeO
Si
O
OH H O N
Si
H2N
NH N
N
N H
MeO
n
MeO
NH
EPO
NH
us
NH2
N n
HPEI
MeO MeO
N H
OH
Si
Fe3O 4
H2N
O
cr
H
Fe (II)
ip t
1
H 2N
Fe@PEI
Metal Chelating
an
Lipase Immobilization
Fe@PEI-M-Lipase N
H2N HN N n
7
M2+
Fig. 1
Ac ce pt e
6
N
d
5
H N
M
Fe@PEI-M HN
24
Page 24 of 29
ip t cr us
1 Fig. 2
an
2
Ac ce pt e
d
M
3
25
Page 25 of 29
Fig. 3
cr
2
ip t
1
Ac ce pt e
d
M
an
us
3
26
Page 26 of 29
ip t cr us
1
M d
4
Fig. 4
Ac ce pt e
3
an
2
27
Page 27 of 29
ip t cr us an M d Ac ce pt e 1 2
Fig.5
28
Page 28 of 29
ip t cr us an M d Ac ce pt e 1 2
Fig.6
3 29
Page 29 of 29
S1381-1177(15)30020-5 http://dx.doi.org/doi:10.1016/j.molcatb.2015.06.013 MOLCAB 3187
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date: Revised date: Accepted date:
24-2-2015 21-6-2015 21-6-2015
Please cite this article as: S.F. Motevalizadeh, M. Khoobi, A. Sadighi, M. KhalilvandSedagheh, M. Pazhouhandeh, A. Ramazani, M.A. Faramarzi, A. Shafiee, Lipase immobilization onto polyethylenimine coated magnetic nanoparticles assisted by divalent metal chelated ions, Journal of Molecular Catalysis B: Enzymatic (2015), http://dx.doi.org/10.1016/j.molcatb.2015.06.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Lipase immobilization onto polyethylenimine coated magnetic nanoparticles assisted by
2
divalent metal chelated ions
3
Seyed Farshad Motevalizadeh a,b, Mehdi Khoobia, Armin Sadighic, Masoud Khalilvand-Sedagheha,
4
Mehrdad Pazhouhandeh e, Ali Ramazani d, Mohammad Ali Faramarzie,* and Abbas Shafieea,*
5 6
a
7
Center, Tehran University of Medical Sciences, Tehran 14176, Iran
8
b
9
Melbourne, VIC 3010, Australia
cr
ip t
Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research
us
Particulate Fluids Processing Centre, School of Chemistry, The University of Melbourne,
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c
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Rhode Island, Kingston, Rhode Island, USA
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e
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Center, Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran 14176, Iran
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Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of
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Department of Chemistry, University of Zanjan, P.O. Box 45195-313, Zanjan, Iran
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Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research
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*Corresponding authors: E-mail: [email protected] (A. Shafiee);Tel: +98-21-66406757; Fax: +98-21-
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66461178 and E-mail: [email protected] (M.A. Faramarzi); Tel: +98-21-66954712; Fax: +98-21-
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66954712
27 1
Page 1 of 29
Abstract
2
In this study, polyethylenimine coated Fe3O4 magnetic nanoparticles (Fe@PEI) were prepared and
3
used for three metal ions (Co2+, Cu2+ and Pd2+ ) chelation. The metal chelated magnetic
4
nanoparticles (Fe@PEI-M) were characterized by using different spectroscopic and analytical
5
techniques. The metal contents were analyzed by inductively coupled plasma atomic emission
6
spectroscopy (ICP) and energy-dispersive X-ray spectroscopy (EDX). Thermomyces lanuginosa
7
lipase (TLL) was then immobilized onto the modified magnetic supports by physical adsorption.
8
Fe@PEI-Co and Fe@PEI-Cu showed better activity at extreme temperature and pH values than of
9
the free enzyme. After 10 cycles of successful biocatalytic activity performed by immobilized lipase
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on Fe@PEI-Co, more than 60% of initial activity was reserved. Furthermore, the immobilized
11
lipase onto Fe@PEI-Co retained about 80% of its initial activity after 14 days of storage. The
12
immobilized lipase on the selected nanocomposite was then used for synthesis of ethyl valerate.
13
After a 24 hr incubation time, the extent of esterification was found to be 70 and 60% in n-hexane
14
and DMSO media, respectively.
16
Keywords: Lipase immobilization, Adsorption, Metal chelation, Magnetic nanoparticles,
17
polyethylenimine, Bioconversion.
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Page 2 of 29
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1. Introduction Biological effectiveness of some therapeutical medicines is extensively limited to one
3
specific enantiomer of chemical substance that results in high pharmacologic indices and lower side
4
effects. Lipases (triacylglycerol hydrolysis, E.C. 3.1.1.3) are considered as enantioselective and
5
regioselective enzymes, which have been broadly used in pharmaceutical industries [1–3]. Lipases
6
present two physical conformations; an open, active form, and a closed, relatively inactive form.
7
The open form is recognized when the α-helix polypeptide chain moves, akin to removing a lid
8
from a container, revealing the active site of molecule [4, 5]. From practical and economical points
9
of view, it is beneficial to utilize immobilized lipases for enantiomeric syntheses [3, 6]. Lipases are
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the most frequently used biocatalyst which can catalyze enantioselective hydrolytic reactions and
11
chemo-, regio- and stereo-selective formation of a diverse range of amide and ester bonds. Lipases
12
have gained fame as a best catalyst for resolutions of a wide range of compounds like thioesters,
13
cyclophanes, phosphonates, ferrocenes, calixarenes, and helicenes [7]. Special features of lipases
14
have made them as a best candidate for synthesis of many biologically active compounds and
15
natural products and food processing industries [8].
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Over the past few years, several methods have been developed for lipase immobilization,
17
including multipoint covalent attachment, cross-linking, physical entrapment, and adsorption [9].
18
The adsorption method, which is simple, cost effective, and preserves catalytic activity of lipase has
19
attracted significant commercial attentions in the recent years [3]. Moreover, there is no need to use
20
cross-linking agents, which reduce the enzyme activity. Some of the adsorbed lipase, however, will
21
desorb during washing and operation processes. One of the most critical challenges in enzyme
22
immobilization procedures is preservation of enzyme activity on the support. Poor immobilization
23
technique may change enzyme conformation from the open form to the closed form and therefore
24
reduce enzyme activity. On the other hand, immobilization through non-covalent bonding, such as
25
hydrogen bonding, hydrophobic interaction, and ion- exchange is a gentle enzyme immobilization
26
process. In these methods, lipase was immobilized by physical adsorption, which could be easily
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Page 3 of 29
1
desorbed from the support. This led to a decrease in immobilized lipase activity. Therefore,
2
selecting an appropriate immobilization process to produce a constant enzyme-support system and
3
open form of enzyme is crucial.
4
Different approaches to stabilize lipase on solid supports have been investigated by various research groups, and a large number of substances have been utilized to make lipase adsorption
6
effective and convenient. For instance, active carbon, porous glass, MCM-41, chitosan
7
microspheres, and magnetic nanoparticles (MNPs) have been successfully used to improve the
8
adsorption process [1, 2, 10]. Two main advantages of magnetic nanoparticles are that they can be
9
readily separated from solution by an external magnet and consequently evaluated for their
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recyclability. A great number of magnetic composite materials have been developed for enzyme
11
immobilization during previous years [11–13]. However, naked nanomagnetic particles are not
12
sufficient enough for stabilizing enzymes due to the lack of appropriate functional groups. In order
13
to overcome this limitation, magnetic nanoparticles can be decorated with polymers.
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Polyethylenimine (PEI) is an ideal polymer for this role due to its high density of primary,
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secondary, and tertiary amine functional groups across the polymer chain. PEI is also ideal for
16
chelating metal ions, which has been successfully applied in biomedicine [14, 15] and
17
environmental remediation [16, 17]. The electrostatic repulsive forces and steric hindrance of PEI-
18
coated nanoparticles enhance their dispersion in solution [18]. In our previous study, we have
19
demonstrated that modifying the surface of nanoparticles by coating with PEI is inappropriate for
20
lipase immobilization via the adsorption method [1].
21
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Enzyme can be improved and stabilized by chelating metals to the polymer network prior to
22
immobilization on the nanoparticle surface [19, 20]. The choice of chelating metal is based on the
23
interaction between chelated metal ion and particular functional groups of the enzyme. Notably, the
24
thiol group of cysteine, the indoyl group of tryptophan, or the imidazole group of histidine are the
25
most important responsible functional groups to couple a chelated metal interaction with enzyme.
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Page 4 of 29
1
In this study, a new approach to avoid the drawbacks of non-stable physical adsorption of enzyme and covalent attachment that restrict the enzyme activity is proposed. The potential of core-
3
shell MNPs-PEI structure with myriad available amine branches to interact with lipase [21] was
4
increased by loading divalent metal chelators with the ability to immobilize enzyme [22]. For this
5
reason, the surface of MNPs was covalently coated with PEI and three different metal ions (Co2+,
6
Cu2+, and Pd2+) were subsequently loaded on the supports to obtain a suitable adsorption capacity.
7
Lipase was immobilized onto the Fe@PEI-M via adsorption and then their catalytic activity was
8
compared with the free enzyme. Finally, the best lipase immobilized system was utilized in the
9
synthesis of ethyl valerate (green apple flavor) in various solvents.
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2. Materials and methods
12
2.1. Chemicals and the enzyme
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[3-(2,3-Epoxypropoxy)propyl]trimethoxysilane (EPO, 98% purity), p-nitrophenyl butyrate
14
(p-NPB), aqueous ammonia solution (28 wt.%), ferric chloride hexahydrate (FeCl3·6H2O), ferrous
15
chloride tetrahydrate (FeCl2·4H2O) and oleic acid were obtained from Merck (Darmstadt,
16
Germany). Hyperbranched polyethylenimine (PEI, MW=60000), palladium chloride (99% purity),
17
copper (II) chloride (99% purity), and cobalt (II) chloride (97% purity) were purchased from Merck
18
AG or Aldrich. Lipase derived from Thermomyces lanuginosa (TLL) was generously gifted by
19
Novozymes (Bagsvard, Denmark). TLL was refined prior to immobilization on the support via
20
adsorbtion on octyl-sepharose beads in 10 mM sodium phosphate, with continuous stirring (pH 7.0).
21
The enzyme was then separated by a sintered glass funnel and rinsed three times with the same
22
buffer. Finally, the purified TLL was separated from the supports by immersing the immobilized
23
lipase in 25 mM sodium phosphate at pH 7.0 (in a relation of 1/10 w/v) containing 0.6% v/v of
24
CTAB during 1 h at 25 °C [23].
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2.2. Preparation of metal chelated PEI functionalized MNPs
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Page 5 of 29
Fe3O4 MNPs were easily prepared by a simple co-precipitation method in basic media. In
2
order to prepare PEI functionalized magnetic nanoparticles, 1.5 g of PEI was initially added into
3
150 mL dry toluene and stirred for a few minutes. Then, 1 mmol of EPO was added to the mixture
4
and allowed to react at 80 °C for 24 h. After that, 2.5 g of dispersed Fe3O4 MNPs in 25 mL of
5
ethanol were added and the reaction was allowed to proceed for another 24 h at 80 °C. Fe@PEI was
6
easily isolated by applying an external magnet, washed several times with ethanol and dried at 40
7
°C for several days [1]. In order to introduce metals on the surface of Fe@PEI, 300 mg of Fe@PEI
8
were added into a round bottomed flask containing a solution of corresponding chloride salts of the
9
single metals (PdCl2, CuCl2 and CoCl2) (0.6 mmol) in acetonitrile (25 mL) and stirred under
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nitrogen atmosphere for 48 h. The resultant residues were separated and washed with
11
acetonitrile/acetone, and dried in air for 24 h [24].
14
2.3. Immobilization of lipase
A physical adsorption method was used in this study for lipase immobilization. The purified
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TLL solution (1 mL, equal to 5 U) was prepared according to our previous experiments [1,2]. The
16
immobilization yield (IY) and efficiency (IE) were calculated based on the following equations: IY
17
(%) = [(Yo–Y1) / Yo] × 100 and IE (%) = [E1 / Eo] × 100; where Yo and Y1 indicate the amounts of
18
total protein and unbounded protein to the substrates, respectively, whereas Eo and E1 represent the
19
total and immobilized enzyme activity, respectively. The essential time for maximum
20
immobilization efficiency was determined by immersing the chelated metal support (10 mg) in 10
21
mL phosphate buffer (0.1 M, pH 7) containing lipase (1 mL, equal to 5 U), then slowly incubation
22
at 25 °C for 15–90 min [25, 26].
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2.4. Characterization
25
Thermogravimetric analysis (TGA) was performed by TGA Q50 thermogravimetric
26
analyzer of a TA instrument. A heating rate of 10 °C per min under argon flow was used from room
6
Page 6 of 29
temperature to 800 °C. X-ray diffraction (XRD) analysis was executed using a XPert MPD
2
advanced diffractometer with a Cu (Kα) radiation source (wavelength: 1.5406 Å) at room
3
temperature in the 2θ range of 4° to 120°. Fourier transform infrared (FT-IR) analysis was recorded
4
on Nicolet FT-IR Magna 550 spectrographs (KBr disks). The surface morphology of the samples
5
was investigated by scanning electron microscopy (SEM, VEGAII TESCAN) with an acceleration
6
voltage of 15 kV. In addition, a CM30 Philips Company high-resolution transmission electron
7
microscopy (TEM) was utilized to study the final shape of the nanoparticles. Sample preparation
8
was done via placing a droplet (1 µL) of sample dispersion latex along with a droplet of water on a
9
copper grid covered by Formvar foil (200 mesh). The samples were then dried and analyzed. The
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EDX spectra were acquired using a Philips XL 30 X-Ray spectrometer. The metal content in
11
samples was analyzed by ICP-OES (VARIAN-VISTA-PRO). A vibrating sample magnetometer
12
(VSM, Meghnatis Kavir Kashan Co., Kashan, Iran) was used at room temperature to investigate
13
magnetic properties of the samples. Dynamic light scattering (DLS) was applied to determine the
14
particles size (DTS Ver. 4.20). The quantity of ester and residual acid were measured by Digital
15
Burette VITLAB continuous RS Titrimeter (made in Germany) and also confirmed by GC mass
16
analysis (VARIAN CP-3800, Column properties: CP-WAX 57 CB GLY FS 25X, see
17
supplementary data for detailed information).
19 20
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2.5. Lipase assay and protein estimation
para-Nitrophenyl butyrate (p-NPB) was applied as substrate to measure the catalytic activity
21
of free and immobilized TLL as described previously [1,2, 10, 27]. Briefly, a mixture of free (0.1
22
ml) or immobilized lipase (10 mg) and 0.9 ml p-NPB solution [0.4 mM in phosphate buffer (100
23
mM, pH 7.4)] was prepared. Then it was incubated at 37 °C and 120 rpm for 30 min followed by
24
determining the absorbance at 348 nm using UV-Vis spectrophotometer (UVD 2950, Labomed Inc.,
25
Culver City, CA, USA). The quantity of enzyme that release p-nitrophenol at a rate of 1 μmol per
26
min is equal to one unit of enzyme activity [28]. The total amount of free and immobilized protein
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Page 7 of 29
1
were measured by the Bradford assay at 595 nm based on the bovine serum albumin standard line
2
(12.5–400 µg/mL) [1, 2, 29].
3 4 5
2.6. pH, temperature, and storage stability of immobilized lipases Free and immobilized enzyme were incubated in 100 mM citrate-phosphate buffer (pH 3–7), Tris-HCl buffer (100 mM, pH 8) and 100 mM glycine-NaOH buffer (pH 9–10) at 37 °C for 1h to
7
investigate the effect of pH on enzyme consistency by determining the residual enzyme activity as
8
previously explained. To study the thermal stability of the free and immobilized lipase, the enzyme
9
was placed in a water bath for 1 h at temperatures ranged between 15 to 75 °C and the residual
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lipase activity was measured. The immobilized lipase was placed in the incubator at 25 °C for 14
11
days to analyze storage stability by measuring remained activity in taken samples.
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2.7. Reusability of immobilized lipases
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After using immobilized lipase at the first run, the biocatalyst was separated by an external
15
magnet and the activity was measured. Then, the immobilized biocatalyst was washed three times
16
with phosphate buffer (100 mM, pH 6.5), and enzyme activity was recalculated. The lipase activity
17
was determined as 100% at the first run and during the next succeeding runs was defined as relative
18
activity.
20 21
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2.8. Enzymatic synthesis of ethyl valerate by free and immobilized lipase The immobilized lipase on the Fe@PEI-Co was applied as biocatalyst for preparing ethyl
22
valerate in both water and organic solvents. Initially, the optimal temperature was obtained by
23
running the reaction at different temperatures (30, 45, and 60 oC). Ethyl alcohol/valeric acid ratio
24
was 2:1 and it was carried out for 1, 2, 4, 6, 12, and 24 h under gentle shaking (100 rpm in solvent-
25
free medium). With the best temperature in hand, the influence of alcohol to acid molar ratio (i.e.,
26
2:1, 1:1, 1:2, 1:3, 1:4, and 1:5) was studied. To measure the quantity of ester content and residual
8
Page 8 of 29
acid, a simple alkalimetric approach was utilized (titration using 0.1 N NaOH and phenolphthalein
2
as an indicator). From the consumption of valeric acid after a certain time, the quantity of
3
synthesized ester can be determined [30, 31]. Under the conditions without enzyme, the ester yield
4
was less than 2%. The optimal condition was applied to investigate the impact of different solvent
5
system. A mixture of valeric acid (1 mmol), ethyl alcohols (4 mmol), solvent (15 ml) and
6
biocatalyst was prepared and reaction was run at 40 °C and 100 rpm shaking. Inactivated free lipase
7
was used as control. Ester formation was also analyzed by GC technique. The crude reaction
8
mixture was injected in GC injection port after 12 hours treatment with biocatalyst in n-hexane. The
9
results showed that the yield of the esterification was 58% and this result was in good conformity
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with the result obtained by alkalimetric approach (Fig. S4).
3. Results and discussion
13
3.1. Preparation of MNPs@PEI
To date several studies have reported PEI grafting to MNPs by electrostatic interaction [32].
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Recently we prepare PEI coated Fe3O4 MNPs via covalent bonds which provide a permanent and
16
considered attachment as an advantage compared to coating MNPs with PEI through electrostatic
17
interactions (Fig.1) [33, 34]. This PEI coated MNPs were used as a best candidate for different
18
metal chelating due to their high accessible amine groups on the surface of MNPs. In this work a
19
double capacity substrate was prepared for enzyme immobilization either by divalent metal ions or
20
through PEI coated magnetic nanoparticles, which present many available amine groups to interact
21
with hydroxyl and carboxylic groups of amino acids present on the periphery of lipase. TLL, like
22
other lipases, consists of a common α/ß hydrolase fold composed of a nucleophilic serine linked to
23
acidic residue (Asp or Glu) and histidine. This catalytic triad is structurally conserved among all
24
kinds of lipases and plays the main role in transition of lipase from closed form (inaccessible active
25
site) to open form, which facilitate the availability of substrate to catalytic site [35–37]. The affinity
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Page 9 of 29
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of three different divalent metal ions (Co, Cu, and Pd) was evaluated for TLL adsorption and the
2
performance of the best catalyst was investigated for apple flavor preparation in different media.
3
Fig. 1 shows the sequential steps for Fe@PEI-M preparation and lipase immobilization. First, the amine groups in PEI were reacted with the epoxy groups of EPO. Then, MNPs were
5
attached to the silanol groups of the latter. Afterward different metal ions were chelated onto
6
Fe@PEI and finally enzyme was immobilized onto the modified magnetic supports.
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3.2. Characterization of Fe3O4 MNPs and Fe@PEI
FT-IR measurements have been conducted to confirm the coating of Fe3O4 by PEI. As
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7
shown in Fig. 2, a strong absorption band at around 600 cm-1 of all MNPs could be assigned to Fe–
11
O vibrations of the magnetite [38]. Upon EPO coating of Fe3O4, Si–O–H peaks can be seen at
12
1000–1100 cm−1. The Fe–O–Si peak that refers to chemical binding between Fe3O4 and silica
13
cannot be seen in the FT-IR spectrum because it was revealed at around 584 cm−1 and therefore
14
overlaps with the Fe–O vibration of MNPs [38]. However, the co-existence of Fe3O4 and silica
15
peaks together indicates the coating of Fe3O4 by silica [39]. The broad OH band at around 3300-
16
3400 cm−1 could be attributed to the high amount of amine groups of PEI on the surface of MNPs.
17
The shell/core structure of PEI around MNPs can be also elucidated from the existence of C–N peak
18
at about 1300-1400 cm−1. Moreover, aliphatic C–H stretching vibrations that were appeared at 2924
19
and 2831 cm−1 provide further evidence for existence of EPO and polymer on magnetic
20
nanoparticles [39–41].
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XRD patterns of Fe3O4 MNPs and Fe@PEI are shown in Fig. S1. All peaks were well
22
demonstrated as spinel magnetite which are in good agreement with the standard literature data
23
(JCPDF card number: 86-1354) [42].These sharp diffraction peaks revealed that the spinel
24
magnetite product has well-defined crystallinity. In addition, no impurity diffraction peaks were
25
detected, suggesting a pure phase of the synthesized Fe3O4 MNPs. The XRD data for the
26
synthesized MNPs showed the diffraction peaks at 2θ=30.23ο, 35.16 ο, 43.22 ο, 53.64 ο, 57.54 ο, and
10
Page 10 of 29
62.38ο, which can be related to the 220, 311, 400, 422, 511, and 440 planes of Fe3O4. After
2
functionalization of magnetic nanoparticles, the XRD patterns exhibited similar diffraction peaks
3
that do not undergo any notable change, suggesting that the magnetite structure was retained. It can
4
be concluded that the structure of Fe3O4 MNPs was preserved after loading the PEI and
5
subsequently, it was demonstrated that modification of the Fe3O4 nanoparticles do not lead to any
6
change in crystal phase. Crystallite size of Fe@PEI was also measured using Scherrer's equation
7
(17.2 nm).
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TGA curves of PEI, pure Fe3O4 MNPs and Fe@PEI (Fig. S2a) were used for a quantitative
9
comparison of degradation behavior of the prepared samples. The weight loss of Fe3O4 MNPs that
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was detected at 100–280 °C can arise from removal of the hydroxyl group adsorbed water and/or on
11
the surface of Fe3O4 MNPs. The same results were obtained by Cao et al. [43] following similar
12
preparation methods. Fig. S2a also shows that the weight-loss of Fe3O4 MNPs in the range of 280°C
13
to 800°C is around 2.5 wt%, resulting from the oxidation of Fe3O4 MNPs. TGA analysis of the sole
14
PEI also shows that the polymer decomposition occurs in the range of 100 and 517 °C. As PEI is in
15
the form of 50 wt.% aqueous solution, the first weight loss of PEI at 100–200 °C can arise from
16
water evaporation and the next weight loss in the range of 200°C to 517 °C can be ascribed to the
17
polymer decomposition. In the same region, the weight-loss of Fe@PEI increases to 30 wt%. This
18
could be due to chemical binding of EPO and PEI groups. By subtracting the weight-loss of
19
Fe@PEI and Fe3O4 MNPs, the weight of PEI modified on the surface of MNPs can be estimated as
20
27 wt%.
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Surface coating with various phases, morphology, physical characteristics and size can
22
affect on the magnetic properties of MNPs. To investigate magnetization (M) and the impact of
23
coating on paramagnetic properties of MNPs, an external magnetic field (H) ranging from -8 KOe
24
to +8 KOe were applied using VSM. Fig. S2b show the M–H curves of Fe3O4 MNPs and Fe@PEI.
25
No hysteresis in the magnetization was observed and both remanence and coercivity were zero in
26
magnetization versus applied magnetic field (M–H loop) curves. It suggests that both unmodified
11
Page 11 of 29
and PEI-modified MNPs show super paramagnetic character. The coercive field (Hc) and the
2
particular saturation magnetization (σs) for Fe3O4 MNPs are 32 Oe and 46.4 emu g-1, respectively.
3
But these values changed as 30.2 Oe and 33.8 emu g-1 for the Fe@PEI, respectively. Although,
4
coatings affected the magnetic power, it still could be collected by permanent magnets (200 mT)
5
within 20 s. It can be concluded that anchoring of the polymers on the surface of MNPs may alter
6
the surface magnetic anisotropy. This can result in the increasing of the surface spins disorientation
7
which leads to the reduced saturated magnetization [44].
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SEM and TEM were used to verify the fine morphology and structure of MNPs. Fig. 3a and b represent the SEM and TEM images of Fe@PEI-Co as best support. The diameter of the pristine
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Fe3O4 nanoparticles is less than 70 nm (Fig S3a) and the PEI grafting onto the MNPs exhibits a
11
morphology resembling that of the original material, but the size of the Fe@PEI expands (Fig. S3b
12
and S3c). The diameter and morphology of Fe@PEI-Co was also similar to Fe@PEI. The samples
13
show polydispersity and aggregation because of the high surface/volume ratio and subsequent
14
enhancement of magnetic dipole–dipole interactions.
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ICP results showed that the incorporated Co, Cu, and Pd percentage in Fe@PEI-M
16
substrates were 6.76, 8.87, and 5.03%, respectively. The EDX spectra of Fe@PEI-M are shown in
17
Fig 4. Atomic weight ratio of C: N: Fe: Metal were 20.40: 12.78: 11.72: 4.36, 19.90: 13.52: 18.08:
18
3.13 and 15.35: 13.75: 11.50: 3.06 for Fe@PEI-Co, Fe@PEI-Cu and Fe@PEI-Pd, respectively,
19
which demonstrate that the MNPs are coated with PEI, and that the metals were chelated
20
successfully.
21 22 23
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3.3. Selection of chelated metal ions Chemical bonds between atoms with similar rating are elucidated by hard and soft acids and
24
bases (HSAB theory or Pearson acid base concept) e.g. hard base bonded with a hard acid are the
25
strongest. Based on this concept, metal ions such as Fe3+, Mg2+, Ca2+ and K+ are categorized as hard
26
Lewis acids, while metal ions such as Cu+, Ag+ and Pd2+ are classified as soft Lewis acids. The
12
Page 12 of 29
transition metal ions, such as Co2+, Cu2+, and Zn2+ are defined as ‘‘borderline acids”. Ligands
2
including oxygen (carboxylate) and aliphatic nitrogen (asparagine) are classified as hard Lewis
3
bases. Ligands containing sulfur (cysteine) and aromatic nitrogen (e.g., histidine and tryptophan)
4
are categorized as soft bases and borderline bases, respectively. The borderline acids, comprising
5
Co2+, Cu2+ and Zn2+ coordinate favorably with borderline bases (e.g., aromatic nitrogen atoms) and
6
also soft base like sulphur atoms [45, 46].
7
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In this special approach, strong interaction between metal ions and enzyme is crucial.
Tryptophan, cysteine, and histidine are promising residues on the surface of enzyme to provide
9
strong interaction with metal ions, while histidine has the strongest affinity for metal ions. In
10
interactions between immobilized Co2+, Pd2+, Cu2+, Zn2+ and amino acid residues on protein
11
surfaces, amine groups are the main targets for the metal ions [46, 47].
12
Three metal ions, Co2+, Pd2+ and Cu2+, were designated as the chelated ions, which can easily form
13
coordination bonds with the residual groups of lipase.
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3.4. Characterization of immobilized lipase
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The amount of immobilized lipase and the relative activity of lipase versus immobilization
17
time are shown in Fig 5a. The lowest time (15 min) for maximum lipase loading (95%) was found
18
for Fe@PEI-Co. In the case of Fe@PEI-Cu, 90% of lipase was loaded after 15 min incubation
19
while only 30% of TLL was loaded on the Fe@PEI-Pd at the same time. For Fe@PEI-Co and
20
Fe@PEI-Cu, the immobilization process was complete after 30 min incubation in the presence of
21
TLL, whilst Fe@PEI-Pd required 90 min for complete immobilization. Maximum immobilization
22
yield (88±1.1%) and immobilization efficiency (82±2.2%) was obtained for Fe@PEI-Co followed
23
by Fe@PEI-Cu (IY%, 73±0.9% and IE%, 68±1.8%) and Fe@PEI-Pd (IY% of 66±2.6% and IE% of
24
58±1.4%).
25 26
Türkmen and co-workers reported the possibility of cytochrome c adsorption to the PEI modified poly (2-hydroxyethylmethacrylate) (mPHEMA) magnetic beads via chelated Cu2+ ions.
13
Page 13 of 29
The authors mentioned that Cu+2 chelation through three amine groups of PEI significantly
2
increased the loading efficiency of protein onto magnetic beads compare to the absence of metal
3
chelation [48]. The presence of Ser-His-Asp catalytic triad has been revealed in the three
4
dimensional crystallographic structure of lipases [49,50], which could be the main player in metal
5
ions chelation and subsequent immobilization on the surface of Fe@PEI particles. In our study TLL
6
was immobilized on the chelated support via two possible interactions. First, charge of TLL is
7
negative (isoelectric point of 4.4) [4] and this may lead to interaction between TLL and positive
8
charge of metal chelated in the constructed support. Second, immobilization could have achieved
9
via coordination bond between metal ion groups and residual moieties in TLL. It is worth to
us
cr
ip t
1
mention that, if the net charge of support and enzyme were opposite, the enzyme would be more
11
stable on the support [6]. It was also established that multipoint interaction might aid to increase the
12
structural constancy of enzymes [51, 52]. Along with the possible interactions between chelated
13
metal ions with the TLL that are responsible for enzyme immobilization, the potential of PEI
14
available amine groups to establish a dipole-dipole interaction with the carboxylic groups of acidic
15
amino acids present on the enzyme surface could be considered as the reason of TLL activity in the
16
absence of metal ions. Indeed, metal chelated ions increase the capacity of enzyme immobilization.
18
M
d
Ac ce pt e
17
an
10
3.5.1. Effect of changing pH and temperature on free and immobilized lipase
19
The pH value of the catalysis environment has a great effect on the biotransformation
20
efficiency. This effect is more pronounced when the free and immobilized enzymes are compared in
21
terms of activity. The effect of pH on the activity of the immobilized and free lipase was
22
investigated at the pH range between 3 to 10 (Fig 5b). For the free lipase, the maximum activity was
23
observed at a pH of 7, while higher relative activity and excellent adaptability was observed at a
24
wider pH range for immobilized lipase. For example, immobilized lipase on Fe@PEI-Cu preserved
25
more than 80% of its initial activity at the pH range from 3 to 10. According to the Fig 5b, higher
26
pH stability was observed for immobilized lipase on the metal chelated MNPs compared to the
14
Page 14 of 29
1
absence of metal. It could be assigned to the immobilization method, carrier charge and structure [6,
2
52]. High resistance of immobilized enzyme in the wide range of pH is associated with the ionic
3
interactions between the negative residual groups on the enzyme and the positive charge of the
4
metal ion on the surface, as well as changes in the enzyme oligomerization [53]. Similar behavior
5
has also been observed for immobilized invertase and polyphenol oxidase [54, 55]. One of the most critical properties for industrial application of lipase is thermal stability.
7
The thermal stability of the free and immobilized lipases was studied in the temperature range of
8
15–75 oC and results are shown in Fig 5c. The optimum temperature for activity of free lipase is 25
9
o
cr
ip t
6
us
C, whereas relative activity for immobilized lipase on Fe@PEI-Co was more than 80% within 15–
65 oC. Higher thermal stability and relative activity for immobilized enzyme onto Fe@PEI in the
11
presence and absence of metal in comparison to free lipase is probably due to the increase of the
12
rate of a coordination reaction, which prevents lipase from conformational changes and
13
denaturation at high temperature. However, temperatures above 65 °C can result in a clear decrease
14
in activity of immobilized lipases due to the enzyme denaturation.
d
M
an
10
16
Ac ce pt e
15
3.5.2 The operational and storage stability of free and immobilized lipase Recycling of enzyme after multiple rounds, which results in decreasing the unit cost of
17 18
enzymatic process is one of the most important features of immobilization [56]. Fig. 6a shows the
19
number of successful catalytic cycles accomplished by the immobilized lipase against their relative
20
activity. Fe@PEI-Co maintained more than 80% of its initial activity after 8 times running in the
21
process, while more than 70 and 60% of initial activity for Fe@PEI-Cu and Fe@PEI-Pd were
22
maintained, respectively, during the same period. When the operation time was increased,
23
immobilized lipase activity decreased gradually. The denaturation of the lipase and the leakage of
24
enzyme from the support could be an explanation for the inactivation of the enzyme after each reuse
25
cycle.
15
Page 15 of 29
1
Generally, enzymes in solution are not constant and their activities decline with time. It makes the storage of free enzymes particularly difficult. One of the best methods to address this
3
issue is immobilization. This prevents the gradual reduction of enzyme activity in solutions,
4
maintaining their practical application in biochemical studies. Free and immobilized lipase were
5
stored at 25°C, and activity measurements were carried out over a period of 14 days. Fig. 6b
6
illustrates that immobilization increased storage stability of free lipase. The free enzyme lost 90%
7
of its activity within 14 days, whereas the immobilized lipase onto Fe@PEI-Co, Fe@PEI, Fe@PEI-
8
Cu, and Fe@PEI-Pd retained more than 80, 65, 60, and 55% of their initial activity during the same
9
period time, respectively. According to the depicted results, it can be concluded that lipase
us
cr
ip t
2
immobilization improves storage stability and Co chelated substrates exhibited the higher stability
11
along with the other supports.
an
10
14
3.6. Esterification studies
Biotechnological approaches are predominantly used in bioprocess of flavor compounds in
d
13
M
12
modern food industries. Some of the esters, which are known as flavors, have been prepared by
16
esterification reactions via enzymes. In this study, the potential of free and the immobilized lipases
17
were compared in esterification of valeric acid with ethanol to form ethyl valerate in various media
18
(Fig. 6c). Ethyl valerate was synthesized with a 4:1 molar ratio of valeric acid to alcohol at 45 oC at
19
optimized condition and only 23% conversion was observed for ethyl valerate (data not shown).
20
The immobilized lipase on Fe@PEI-Co showed about 40% bioconversion of valeric acid into ethyl
21
valerate in 24 h under similar conditions.
22
Ac ce pt e
15
The choice of organic solvent has a huge impact on the yield of the final product (Fig. 5c).
23
This is due to esters transferring into the organic phase, and this may lead to shifting equilibrium
24
towards the ester synthesis. These results are entirely consistent with other studies performed by
25
other groups [57, 58].
16
Page 16 of 29
1
We also applied leaching test to investigate the amount of the metal desorption from the solid catalyst. In order to determine metal species leached from the solid catalyst, Fe@PEI-Co was
3
stirred in aqueous media for one day. MNPs were magnetically separated from the reaction mixture.
4
The solution was concentrated and checked for determination of the leached metal ion by ICP
5
analysis and isolated MNPs were also used for the next three runs under the above mentioned
6
condition. ICP analysis was shown that amount of the leached cobalt from the catalyst is less than
7
0.1 ppm for the first run and less than 0.015 ppm for the next runs. The European Food Safety
8
Authority [59], the United Kingdom Expert Group on Vitamin and Minerals [60], and Finley et al
9
[61] have proposed that 600, 1400, and 2100 μg Co/day should be considered safe [62]. These
us
cr
ip t
2
results revealed that amount of the metal leached from the biocatalyst is negligible and the catalyst
11
could be used in the food industry [63]. It could be attributed to the presence of high amount of
12
hydroxyl and carboxyl groups on the surface of enzyme with a good affinity for chelating to the
13
divalent metal ions.
16
4. Conclusion
M
Ac ce pt e
15
d
14
an
10
Different approaches to improve the conformational stability of enzymes have been studied.
17
Enzyme type, immobilization route and structure of support play a significant role in the extent of
18
stabilization. In this work a double capacity substrate was used for enzyme immobilization either by
19
divalent metal ions (e.g., Co, Cu, and Pd) or through PEI coated magnetic nanoparticles, which
20
present numerous available amine groups to interact with hydroxyl and carboxylic groups of amino
21
acids present on the periphery of lipase. Herein, we evaluate the affinity of three different divalent
22
metal ions (Co, Cu, and Pd) for TLL adsorption on the MNPs and performance of the catalyst for
23
apple flavor preparation. The results show that metal chelate ions increase the capacity of enzyme
24
immobilization and cobalt could be a good candidate for TLL immobilization rather than other
25
metal. The storage ability of immobilized lipases was improved and they retained their relative
26
activity in the wide range of temperature and pH compared with free lipase, due to the chelation.
17
Page 17 of 29
1
This biocatalyst can be easily separated from mixture of the reaction by using an external magnet
2
without need of filtration or decanting.
3
5 6
Acknowledgements This work was supported financially by the grants from Tehran University of Medical Sciences, and Iran National Science Foundation (INSF) Tehran, Iran.
Ac ce pt e
d
M
an
us
cr
7
ip t
4
18
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Page 22 of 29
Figure Captions
2
Fig.1: A schematic illustration of synthetic pathway for preparation of Fe@PEI-M and enzyme
3
immobilization via adsorption.
4
Fig.2: FT-IR spectra of the prepared (a) Fe3O4 MNPs, (b) Fe@PEI, (c) Fe@PEI-Co, (d) Fe@PEI-
5
Cu, (e) Fe@PEI-Pd.
6
Fig.3: The SEM micrograph and TEM images for Fe@PEI-Co.
7
Fig.4: The EDX spectra of (a) Fe@PEI-Co (b) Fe@PEI-Cu (c) Fe@PEI-Pd.
8
Fig.5: a) Time, b) temperature, and c) storage stability of free and immobilized lipase on supports.
9
Fig.6: (a) reusability, (b) storage profile of lipase immobilized on prepared supports. (c)
us
cr
ip t
1
Conversion of valeric acid in the presence of ethyl alcohol using free and immobilized lipase onto
11
the Fe@PEI-Co in aqueous and non-aqueous media.
M
12
an
10
Graphical abstract
14
Lipase immobilization onto polyethylenimine coated magnetic nanoparticles assisted by
15
divalent metal chelated ions
16
Seyed Farshad Motevalizadeh, Mehdi Khoobi, Armin Sadighi, Masoud Khalilvand-Sedagheh, Mehrdad
17
Pazhouhandeh, Ali Ramazani, Mohammad Ali Faramarzi,* and Abbas Shafiee*
Ac ce pt e
18
d
13
Lipase Immobilization
Fe@PEI-M
O OH
Fe@PEI-M-Lipase HN
N n
O
EtOH
H N
N
H2N HN
N
M 2+
O Apple flavour
19 20
Highlights 23
Page 23 of 29
•
Synthesis of metal chelated magnetic nanoparticles.
2
•
Lipase immobilized on nanoparticles by adsorption and metal chelation.
3
•
Good reusability, storage time, optimal pH, and temperature of immobilized lipase.
4
•
The immobilized lipase was used as biocatalyst for the synthesis of green apple flavor. H2N
+
Fe (III)
+ NH4OH
O
H2 N NH
O
O
H N
NH N
N H
N
N
HPEI
NH
n
+ MeO
O MeO
Si
O
OH H O N
Si
H2N
NH N
N
N H
MeO
n
MeO
NH
EPO
NH
us
NH2
N n
HPEI
MeO MeO
N H
OH
Si
Fe3O 4
H2N
O
cr
H
Fe (II)
ip t
1
H 2N
Fe@PEI
Metal Chelating
an
Lipase Immobilization
Fe@PEI-M-Lipase N
H2N HN N n
7
M2+
Fig. 1
Ac ce pt e
6
N
d
5
H N
M
Fe@PEI-M HN
24
Page 24 of 29
ip t cr us
1 Fig. 2
an
2
Ac ce pt e
d
M
3
25
Page 25 of 29
Fig. 3
cr
2
ip t
1
Ac ce pt e
d
M
an
us
3
26
Page 26 of 29
ip t cr us
1
M d
4
Fig. 4
Ac ce pt e
3
an
2
27
Page 27 of 29
ip t cr us an M d Ac ce pt e 1 2
Fig.5
28
Page 28 of 29
ip t cr us an M d Ac ce pt e 1 2
Fig.6
3 29
Page 29 of 29