Phosphotriesterase-Magnetic Nanoparticle Bioconjugates with

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Phosphotriesterase-magnetic nanoparticles bioconjugates with improved enzyme activity in a biocatalytic membrane reactor ABAYNESH YIHDEGO GEBREYOHANNES, Rosalinda Mazzei, Mohamed Yahia Marei Abdelrahim, Giuseppe Vitola, Elena Porzio, Giuseppe Manco, Mihail Barboiu, and Lidietta Giorno Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00214 • Publication Date (Web): 24 May 2018 Downloaded from http://pubs.acs.org on May 29, 2018

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Bioconjugate Chemistry

Graphical abstract

PVDF-EnzSP biocatalytic membrane reactor

Biofunctionalized magnetic nanoparticles

1 ACS Paragon Plus Environment

Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Phosphotriesterase-magnetic nanoparticles bioconjugates with improved enzyme activity in a biocatalytic membrane reactor

Abaynesh Yihdego Gebreyohannes1, Rosalinda Mazzei1*, Mohamed Yahia Marei Abdelrahim1,2,3, Giuseppe Vitola1, Elena Porzio4, Giuseppe Manco4, Mihail Barboiu2, Lidietta Giorno1 1

Institute on Membrane Technology, ITM-CNR, c/o University of Calabria, via P. Bucci, 17/C, 87030 Rende (Cosenza), Italy

2

Institut Européen des Membranes (IEM), Université de Montpellier - Case courrier 047, 2 Place Eugène Bataillon, 34095 Montpellier cedex 5, France

3

Department of Chemistry, Faculty of Science, Helwan University, Ain-Helwan, Cairo 11795, Egypt 4

Institute of Protein Biochemistry, National Research Council, IBP-CNR, via P. Castellino 111, 80131 Naples, Italy

* Corresponding author; tel: +39 0984 492076: fax: +39 0984 49 e-mail: [email protected]


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Abstract The need to find alternative bioremediation solutions for organophosphate degradation

pushed

the

research

to

develop

technologies

based

on

organophosphate degrading enzymes, such as phosphotriesterase. The use of free phosphotriesterase poses limits in terms of enzyme reuse, stability and process development. The heterogenization of enzyme on a support and their use in bioreactors implemented by membrane seems a suitable strategy, thanks to the ability of membranes to compartmentalize, to govern mass transfer and provide microenvironment with tuned physico-chemical and structural properties. Usually, hydrophilic membranes are used since they easily guarantee the presence of water molecules needed for the enzyme catalytic activity. However, hydrophobic materials exhibit a larger shelf life and are preferred for the construction of filters and masks. Therefore, in this work, hydrophobic polyvinylidene fluoride (PVDF) porous membranes were used to develop biocatalytic membrane reactors (BMR). The phosphotriesterase-like lactonase (PLL) enzyme (SsoPox triple mutant from S. solfataricus) endowed with thermostable phosphotriesterase activity was used as model biocatalyst. The enzyme was covalently bound directly to the PVDF hydrophobic membrane or it was bound to magnetic nanoparticles and then positioned on the hydrophobic membrane surface by means of an external magnetic field. Investigation of kinetic properties of the two BMRs and the influence of immobilized enzyme amount revealed that the performance of the BMR was mostly dependent on the amount of enzyme and its distribution on the immobilization support. Magnetic nanocomposite mediated immobilization showed



a much better performance, with an observed specific activity higher than 90% compared to grafting of the enzyme on the membrane. Even though the present work focused on phosphotriesterase, it can be easily translated to other class of enzymes and related application.

Keywords: Nanoparticle bioconjugates; PVDF membrane, biocatalytic membrane reactors; reversible immobilization; organophosphate degradation

Introduction Compartmentalization is one of the fundamental behaviors of living systems, since it enables spatio-temporal control over multi-step biological processes (1). Over the years, many different approaches have been used to mimic natural biocatalytic processes by using synthetic analogues or bioconjugates under confinement. The main purpose is to improve catalytic efficiency, to study and understand processes in living systems and to translate nature design for the production of artificial nano-compartments in different application fields. Specifically to catalytic systems, different approaches were explored in order to create an artificial compartmentalized system that promotes production processes through reactor developments (2-4). Synthetic membranes are widely used to compartmentalize biomolecules in several industrial processes. However, for biotechnological applications, these systems are not yet developed at industrial scale, since several problems related to both biocatalyst activity or membrane transport/fouling need be resolved (5). This is more evident when membrane is used both as support for enzyme immobilization and separation unit to produce biocatalytic membrane reactors (BMR). 4 ACS Paragon Plus Environment

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Chemical binding of enzymes to a membrane provides improvement in their stability and reactor performance, since it avoids enzyme leakage. In addition, when multi-point covalent attachment is involved, enzyme stability is also improved in the presence of high temperature, high shear stress, solvents, pH changes and impurities (6). However, when covalent attachment of the biomolecule is carried out on a hydrophobic membrane, additional problems related to both non-specific adsorption of the proteins and poor interaction with hydrophilic substrate are possible. In addition, a water activity is needed for almost all biocatalysts, which in the above mentioned conditions could be limited by the hydrophobic support. Biomolecule-loaded

hydrophilic

nanoparticles

represent

innovative

tailored

materials, which integrates specific properties of nanoelements (e.g. optical, electrical) with the catalytic or sensing function of biomacromolecules (7). Nowadays, emerging alternative approaches to overcome limits related to BMR is to build biohybrid multilevel systems in which bionanoconjugates are associated with membranes (8-13). Biomolecules can be loaded on superparamagnetic nanoparticles (MNP) and reversibly deposited on the membrane using an external magnetic force (13) or covalently bound to the membrane by means of nanoparticles with surface functional groups (14). In the first system, the enzyme is first immobilized on the MNP to form a stimuliresponsive bio-nanocomposite. Then an external magnetic field is used to guide and trigger the reversible immobilization of the bionanocomposites on the membrane. The use of MNPs ensures easy biocatalyst recovery for membrane cleaning and enzyme reuse. Thanks to the special alignment of the MNPs in response to the external magnetic field, MNP mediated enzyme immobilization



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allows to pack multi-levels of enzyme over a given membrane surface without possible molecular crowding phenomena (15) . MNP mediated reversible enzyme immobilization is particularly suitable to protect the membrane from direct contact with contaminants, to preserve enzyme water activity,

while

avoiding

alterations

of

the

desired

membrane

surface

hydrophobicity. These properties are particularly important when the significant effect of the microenvironment on the immobilized enzyme activity is taken into consideration (2). However, although high hydrophobicity can negatively affect enzymes activity, it is a valuable property for membranes employed in some applications like filtration of air and aerosol suspensions (e.g. for filters and mask production). In these applications, pore wetting must be prevented, since it can cause pore blockage, due to water absorption, which is prevented by hydrophobic membrane. In the present work, a multilevel bioconjugate membrane system in which hydrophilic

MNPs

were

phosphotriesterase-like

used

lactonase

as

carriers

(PLL)

for

enzyme

the

immobilization

SsoPox

triple

of

mutant

(C258L/I261F/W263A (Sso-3M)(16)) was studied. To reach this aim, the enzyme was covalently immobilized on commercial MNP, which were further dispersed on the surface of hydrophobic PVFD membranes, using an external magnetic field. The enzyme was also immobilized on PVDF membrane by covalent bond. Both enzyme-loaded

supports

were

used

for

the

hydrolysis

of

a

model

organophosphate (paraoxon-ethyl) in a BMR. Such hydrophobic bio-hybrid membrane can in the future be employed in the development of bio functionalized systems for the bioremediation of organophosphates contained in air or in suspension.


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Results and Discussion Activity of immobilized enzyme in batch The amount of Sso-3M enzyme immobilized on the PVDF membrane via direct grafting was 1 mg corresponding to 50 µg/cm2. The amount of Sso-3M immobilized on MNP, measured by directly injecting the EnzSP into the BCA reagent was 44.5 (±4) µgEnz/mgMNP. This result was similar to the amount of immobilized enzyme obtained by mass balance (Eq. 1) (48.9 ±2.8 µgEnz/mgMNP). Taking the 15 m2/g available surface area of the MNP, the enzyme loading capacity was 0.31± 0.003 mgEnz/cm2. The SEM micrographs (Fig. 2) of free nanoparticles before (Fig. 2a) and after enzyme immobilization (Fig. 2b) revealed a monodispersed particle size, that range from 200 to 220 nm, respectively. The slightly higher particle size on Fig 2b, is due to enzyme immobilization as previously observed in Gebreyohannes et al. (11) by using a different enzyme. Fig. 2c shows SEM cross-section of a PVDF membrane that contained EnzSP, which were magnetically deposited on the surface of the PVDF membrane to form the PVDF-EnzSP biocatalytic membrane.The activity of Sso-3M enzyme chemically grafted on the surface of modified PVDF membrane (PVDF-DAMP-GA-Enz) or on the MNP (EnzSP) was determined by measuring the hydrolysis product formation as a function of time. The specific activity of enzyme immobilized on the surface of the PVDF membrane (PVDF-DAMP-GA-Enz) and the EnzSP were 0.06 µmol/minmg and 0.91 µmol/minmg respectively. Reactions with both types of immobilized enzyme were carried out in static conditions and the reported specific activities were calculated from the product diffused in the bulk. Therefore, they are "observed" rather than "intrinsic" values.



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Nevertheless, they clearly show that the enzyme on the nanoparticles showed a much better performance, with a 93% higher observed specific activity. a

c

b

Fig. 2: SEM micrographs of MNP: a) before enzyme immobilization, b) after enzyme immobilization and c) after magnetically depositing EnzSP on a PVDF membrane (cross section).

Immobilized enzyme performance in biocatalytic membrane reactor (BMR)

The performance of enzyme in both immobilized forms (i.e. covalently linked to the PVDF membrane, PVDF-DAMP-GA-Enz, or first covalently bound to the nanoparticles and then deposited on the membrane surface, PVDF-ENZSP) was studied using the BMR set-up illustrated in Fig. 1, where the reagent transport through the reaction environment was obtained by convective flow by applying transmembrane

pressure (TMP). In this system, the TMP is an important

parameter to be tuned, as it influences the flux through the membrane and therefore the residence time of the reagent in contact with the biocatalyst. Fig. 3a shows that for both BMRs, the flux increased with increase in TMP with slightly higher steady-state values for PVDF-DAMP-GA-Enz BMR compared to PVDF-ENZSP BMR.In the entire TMP and related flux range, the reaction rate for the PVDF-DAMP-GA-Enz BMR was stable around 2.310-3 µM/min (Fig. 3b). 8 ACS Paragon Plus Environment

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Whereas for the PVDF-EnzSP BMR, the reaction rate decreased from 810-3 µM/min to 510-3 µM/min with flux increase from 1.22 L/hm2 to 1.8 L/hm2, respectively and then it remained constant for further increase of flux. Since the flux influenced trasport and residence time, results showed that, in the studied range, the reaction rate of PVDF-DAMP-GA-Enz BMR was not influenced by these parameters. This might be due to the fact that, despite the high amount of immobilized enzyme, only a minor quantity is available to catalyze the reaction. This quantity resulted saturated at all flux values tested. Whereas, for the PVDF-ENZSP BMR, the reaction rate was higher at the highest residence time tested, (i.e. lower flux). In this system, the decrease of reaction rate with flux increase was mainly due to the decrease of residence time, which negatively influenced the enzyme saturation. This behaviour was previously observed in other BMR systems (2). On the basis of these results, 0.5 bar was selected as suitable driving force to guarantee appropriate residence time for both BMRs. It is worth nothing that, the amount of immobilized enzyme utilized in the PVDFEnzSP BMR was 0.30 µg/cm2 while for the PVDF-DAMP-GA-Enz BMR it was 50 µg/cm2. However, the reaction rate for the PVDF-EnzSP BMR was higher compared to the PVDF-DAMP-GA-Enz BMR, in particular, at 0.5 bar was more than 70%. In agreement with the results obtained under static conditions, the PVDF-EnzSP BMR gave better performance also under convective flow. This better performance is mainly due to the better spatial orientation of the enzyme on the spherical nanoparticles (that avoids intermolecular crowding) and to the better magnetic dispersion of enzyme on the surface of the membrane, i.e. the possibility to form a highly porous biofunctionalized layer using magnetic nanoparticles that



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could avoid diffusional limitations. Besides, during chemical grafting of an enzyme to the membrane surface, the enzyme solution may intrude to the membrane matrix, resulting in complex substrate transport and reaction kinetics. The presence of the enzyme within the PVDF-DAMP-GA-Enz membrane thickness was previously demonstrated in Vitola et al. (17) by the enzyme immuno in-situ detection and TEM microscopy. This could partially explain, the lower reaction rate observed in PVDF-DAMP-GA-Enz BMR even though the mass of immobilized enzyme was significantly higher than the PVDF-EnzSP BMR. The comparison of the results of the present with a previous work (17) also confirmed this behavior. In fact (Table 1), for the mutant phosphotriesterase SsoF immobilized on hydrophobic PVDF membrane, the reactor performance (specific activity) decreases with the increase of the immobilized enzyme amount. b

a

Fig. 3: a) Water flux in both BMR configurations as a function of transmembrane pressure increase and b) Reaction rate of 0.21 mg and 1 mg of immobilized Sso3M enzyme in the PVDF-EnzSP and PVDF-DAMP-GA-Enz BMR system, as a function of flux incres, respectively (paraoxon-ethyl concentration: 1mM)


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Table 1 Immobilized enzyme amount and specific activity of phosphotriesterase SsoF immobilized on PVDF hydrophobic membrane Biocatalyst

Membrane

Immobilized

Specific

enzyme

(µmol/minmgENZ)

activity Ref

amount (µg/cm2) Phosphotriesterase Hydrophobic

12

0.0035

(SsoF)

16

0.0030

23

0.0022

PVDF

(17)

Apparent kinetics of immobilized enzyme in biocatalytic membrane reactor

The apparent kinetic parameters were studied using a substrate concentration range from 0.0625 mM to 1mM. As mentioned before, a TMP of 0.5 bar was used to supply substrate through the catalytic microenvironment. Starting from same initial enzyme concentration solution (1 mg/ml), the amount of immobilized enzyme in the PVDF-EnzSP BMR and PVDF-DAMP-GA-Enz BMR were 0.21 mg and 1 mg, respectively. Most probably, the higher enzyme amount immobilized in the PVDF membrane was due to not only to the covalent bond with the GA bifunctional agent but also to non-specific hydrophobic interactions of the enzyme with the membrane. Fig. 4 illustrates the graphs from which kinetic parameters were evaluated for both BMRs. The observed reaction rates were evaluated as the slope of the product concentration vs time. Fig. 4a and 4b show a very linear increase of reaction product as a function of time for PVDF-EnzSP and PVDF-DAMP-GA-Enz BMR, respectively. This means that the system works as a batch reactor with time variant conditions. This is due to the reaction occurrence not only in the membrane or MNP thickness/void volume, but also on the surface facing the bulk, where the



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reaction product is accumulated. The permeation through the membrane under convective flow permits to collect samples as a function of time from the batch reactor. The slope of each straight line gives the observed reaction rate, which is then plotted as a function of substrate concentration to build the apparent MichaelisMenten plot (Fig. 4c). The kinetic behaviour seems to be linear reflecting low affinity for the substrate as it was also observed for the free enzyme (KM 1.5 mM) (16). Higher substrate concentration was not used due to safety in handling the substrate. Although a plateau was not observed for the product concentration vs time plot, assuming that we are observing the linear part of a Michaelis-Menten (M-M) curve, it is possible to calculate the enzyme specificity (kcat/KM) from the slopes of the two straight lines. In fact, when the catalyst affinity for the substrate is low, many enzymes catalyse reactions of second-order kinetics. In the mentioned conditions, the denominator in Michaelis-Menten equation becomes Km, the enzyme concentration [E] is the total enzyme concentration [ETOT] and Vmax is given by kcat [ETOT], so the MichaelisMenten equation is given by (18): =

(3)

where, v is reaction rate (µM/min), kcat is the catalytic constant or turnover number (s-1), Km is the Michaelis-Menten constant (µM), [Etot] is the total enzyme concentration (M) and [S] is the substrate concentration (µM). Using equation 3 and the data in Fig. 3c, the slopes were 210-6 and 410-6 for PVDF-DAMP-GA-Enz BMR and PVDF-EnzSP BMR, respectively. From these values we can conclude that the PVDF-EnzSP BMR gave a significantly high 12 ACS Paragon Plus Environment

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performance than the PVDF-DAMP-GA-Enz BMR although it was not possible to provide a reliable estimate of the apparent kcat and/or Km values. It is worth nothing that in PVDF-DAMP-GA-Enz BMR system the saturation trend was much lower compared to the PVDF-EnzSP BMR, despite the higher amount of immobilized enzyme. This means that in the PVDF-DAMP-GA-Enz BMR, most of the immobilized enzyme sites were not engaged in the biocatalysis, probably due to crowding phenomena, limited mass transfer, repulsion of the hydrophobic membrane character towards hydrophilic substrate, etc. On the contrary, in the PVDF-EnzSP BMR, the enzyme was able to efficiently interact with the hydrophilic substrate, thanks to the hydrophilic MNP character and to the radial distribution in the space of the enzyme immobilized on the spherical nanoparticle.



1.2

a

1

y = 0.0085x R² = 0.9957

0.3

y = 0.0067x R² = 0.9901

0.25

b

0.6

y = 0.0058x R² = 0.9911

0.4

y = 0.0055x R² = 0.9961

0.2

PNP (µM)

0.8

0 0

20

40

60

80

y = 0.0029x R² = 0.9855

b

y = 0.0024x R² = 0.9979

0.2

y = 0.002x R² = 0.9993

0.15

y = 0.0016x R² = 0.9971

0.1 0.05

y = 0.0050x R² = 1.0000

y = 0.0014x R² = 0.9986

0

100 120 140

0

20

40

Time (min) 0.0625

Reaction rate (µM/min)

PNP (µM)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.125 mM

60

80

100

120

140

Time (min)

0.25 mM

0.5 mM

1mM

0.0625 mM

0.125 mM

0.25 mM

0.5 mM

1 mM

PVDF-EnzSP BMR

0.01

c PVDF-DAMP-GA-Enz BMR

0.008 0.006 0.004 0.002 0 0

200

400

600

800

1000

Substrate concentration (µM)

Fig. 4: Effect of paraoxon-ethyl concentration on reaction rate on PVDF-EnzSP BMR and PVDF-DAMP-GA-Enz BMR systems containing 0.21 mg and 1 mg of immobilized Sso-3M, respectively at 0.5 bar and 30 mL feed volume: a) Product concentration vs time for PVDF-EnzSP BMR; b) Product concentration vs time for PVDF-DAMP-GA-Enz BMR; c) Michaelis-Menten plot.


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Effect of amount of immobilized enzyme

Fig. 5 shows the reaction rates for paraoxon-ethyl (1 mM) degradation in the BMR at 0.5 bar, when the amount of EnzSP on the membrane was varied from 0.160 mg of MNP (0.01 mg enzyme) to 27 mg MNP (1.2 mg enzyme). The reaction rate linearly increased until it leveled-off after 0.41 mg of immobilized enzyme. This behavior is congruent with the fact that increasing the enzyme amount, the reaction rate increased until the substrate availability did not become limiting; in other words, the reaction rate increased until the increased amount of enzyme was saturated by the substrate. Afterwards, the slope of the curve changed. The performance of the PVDF-DAMP-GA-ENZ BMR as a function of immobilized enzyme amount was not carried out given its much lower efficiency compared to PVDF-ENZSP. In fact, when the same immobilized enzyme amount and substrate concentration (1 mg and 1mM) for the two BMRs were used, the reaction rate for the PVDF-DAMP-GA-ENZ BMR (2.310-3 µM/min) was an order of magnitude lower than the one observed in the PVDF-ENZSP BMR (1.010-2 µM/min). Fig. 5 shows the flux behavior through the same membrane as a function of immobilized EnzSP. Since EnzSP is mainly constrained on the surface of the membrane (Fig. 2c) due to the presence of an external magnetic field at 2 cm below the membrane, the increment in the amount of EnzSP over the membrane surface created a slightly thicker layer of biofunctionalized bed which led to the flux decline observed in Fig. 6.



Fig. 5: Reaction rate of native PVDF membrane loaded with various amounts of EnzSP layer when the BMR was fed-batch with 30 mL, 1 mM paraoxon-ethyl at 0.5 bar. 1.6

1.2

Flux (L/m2 h)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.8

0.4

0 0

400

800

1200

1600

Immobilized enzyme (µg)

Fig. 6: Flux through PVDF EnzSP membrane as a function of various amounts of EnzSP in the BMR fed-batch with 30 mL, 1 mM paraoxon-ethyl at 0.5 bar.

Conclusion

An important challenge in enzyme immobilization is to provide proper spatial distribution

in

order

to

avoid

crowding

phenomena

and

microenvironment to retain biocatalytic activity of the bioconjugate.


hydrophilic

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This work demonstrated that immobilization of Sso-3M on MNP and their subsequent deposition on the PVDF membrane increased enzyme performance in terms of reaction rate by almost 80% compared to the immobilization of Sso-3M directly on PVDF membrane. This can be attributed to the hydrophilic property of MNPs that provided a suitable micro-environment for the enzyme as well as the fact that the enzyme distribution on these spherical MNPs limited the effect of molecular crowding. In addition, being the substrate hydrophilic, the presence of the hydrophilic MNPs promoted a higher interaction between the enzyme and the substrate under a hydrophobic microenvironment imparted by the membrane. Considering that organophosphate compounds include harmful gases and that hydrophobic membranes, such as the PVDF membrane used here are appropriate to construct systems (such as masks or filters) to treat gas streams, the proposed strategy represents a solid base for future development of decontamination of gaseous systems. Furthermore the strategy of immobilizing enzyme on hydrophobic membrane through hydrophilic nanoparticles opens for breakthrough of heterogenized biocatalytic systems. To our best knowledge, the dynamic bioconjugated catalytic membrane process can be regarded as pioneer work.

Materials and Methods Materials

Phosphotriesterase enzyme

(Sso-3M)

was

provided

by

IBP-CNR

(Italy),

glutaraldehyde (GA) and 1,5-diamino-2-methyl pentane (DAMP) were purchased from Sigma Aldrich, Italy. Flat sheet polyvinylidene fluoride (PVDF) membrane



Page 18 of 26

with an average pore size of 0.2 µm, 47 mm diameter and 200 µm thickness, which were supplied by GVS Spa (Italy), was used as support for biomolecule immobilization.

Magnetic

nanoparticles

(MNPs)

(Amino-Adembeads)

were

purchased from Ademtech, France. They are monodispersed and superparamagnetic beads composed of iron encapsulated by a hydrophilic polymer shell. The surface was activated with amine functionality. EGDE (Ethylene Glycol Diglycidyl Ether) and EDC (1-ethyl-3-(3-dimethylamino propyl) carbodiimide hydrochloride), supplied together with the MNPs, were used as activators for the MNPs (Fig. S1). In order to test the efficiency of the system, paraoxon-ethyl (from Sigma Aldrich) was used as substrate.

Eterologous expression and protein purification

The C258L/I261F/W263A (Sso-3M) gene was expressed and the protein purified as reported in (16). In short, expression was under the direct control of the IPTG (isopropyl-β-D-thiogalactopyranoside)-inducible T7 promoter. 7 liters of LB medium containing 100 µg/ml ampicillin were inoculated at 0.05 O.D.600 with E. coli BL21 (DE3) trasformed cells with vigorous bubbling of sterile air at 37°C. After overnight growth, the protein expression was induced by adding 0.5 mM IPTG and 0.5 mM CoCl2, at 37 ° C for further 4 hours; bacterial culture was centrifuged (3000 g, 4°C, 10 minutes) and harvested cells were stored at -80°C. Wet thawed cells (≈ 20 g) were dissolved in 60 ml of 20 mM Hepes pH 8.5, 0.5 mM CoCl2 and lysed by French pressure cell disruption (Aminco Co., Silver Spring, MD, USA) at a pressure setting of 2,000 lb/in2 (1.38 MPa). Cells debris were removed by centrifugation (80000 g, 20 minutes, 4°C). E. coli proteins were partially fractionated by incubating the crude extract for 20 minutes at 60 and 70°C, under


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gentle stirring and with clarification, by centrifugation (80000 g, 20 minutes, 4°C). Such enzyme solution was loaded onto Q Sepharose Fast Flow FPLC column (Pharmacia) connected to an AKTA Explorer system (GE Healthcare). After washing, a linear gradient of NaCl (0-0.5 M) was applied. The fractions with Sso3M activity were collected, analysed by SDS (sodium dodecyl sulphate)-PAGE, pooled, concentrated by ultrafiltration, dialyzed against Hepes 20 mM pH 8.5, 0.2 M NaCl and loaded onto a Superdex 75 column 16 cm x 60 cm (GE Healthcare). The fractions containing the active protein were pooled, concentrated by ultrafiltration and stored at 4°C until use.

Enzyme immobilization

Chemical grafting of enzyme on PVDF membrane The PVDF membrane functionalizations based on the formation of (–CH=CF) groups when (–CH2CF2–) groups are treated with DAMP in alkaline condition and subsequent addition of 1,5-diamino-2-methyl pentane (DAMP) on double bond (14, 19). A flat sheet PVDF membrane with 47 mm diameter was soaked for 6 h, in a 20 mL DAMP solution containing 1 M carbonate buffer at pH 11 and 50ºC in order to graft the DAMP onto the membrane surface. Subsequently, the membrane was washed with ultrapure water. The PVDF–DAMP membrane was then treated with (20% v/v) glutaraldehyde (GA) solution for 2 h at 25ºC to introduce an aldehyde terminal group to the membrane (PVDF–DAMP–GA). The membrane was thoroughly washed with distilled water until all residual GA was removed. The PVDF–DAMP–GA membrane were then immersed in 25 mL Sso3M enzyme solution of 1 g/L enzyme in 50 mM HEPES buffer at pH 8.5) at 25ºC for 24 h under gentle stirring (50 rpm) (Fig. S2). The membrane was washed



Page 20 of 26

thoroughly with 50 mM HEPES buffer in order to remove biomolecules which were not stably linked to the membrane. To quantify the amount of immobilized enzyme, on the PVDF-DAMP-GA-Enz, the concentration of protein in the immobilization solution before (Massinitialsol), after (Massfinalsol) immobilization and washing buffer (Masswashing) was measured by the BCA assay. The amount of immobilized enzyme (Mimmob) was calculated based on equation 1: = − −

(1)

Each functionalization step caused a slight decrease of flux (Table S1). The overall flux decline accounts for partial penetration of enzyme into the membrane matrix, which was also previously observed by Vitola et al. (17) using the enzyme immuno in-situ detection and TEM microscopy.

Enzyme immobilization on magnetic nanoparticles (MNPs)

Activation of magnetic nanoparticles In this work, we used a 1% aqueous suspension with monodispersed superparamagnetic nanoparticles (11). These particles have magnetic properties only when they are subjected to an external magnetic field and exhibiting no residual magnetism when the external magnetic field is removed. They have a diameter of 200 nm, a specific surface area of 15 m2/g, and carrying positive charges in the pH range 2 to 10. They contain 70% iron oxide and have a magnetic susceptibility of about 40 emu/g. The covalent immobilization of Sso-3M on MNP occurs by the functionalization of the polymer coated MNP with carboxyl groups through which the links with the


Page 21 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


amino groups of the enzyme are established. Prior to enzyme immobilization, the MNP were activated with EGDE (Ethylene glycol diglycidyl ether MW= 174.2) and an activation buffer (patented ADEMTECH, Li StarFish s.r.l., Milan, Italy) to form a MNP containing epoxy intermediate (Fig. S1).

Enzyme immobilization For the covalent immobilization of Sso-3M on the PVDF membrane, 1 mg of enzyme dissolved in 1 mL HEPES buffer (20 mM, pH 8.5, 25ºC) was mixed with 10 mg of EDGE activated MNPs. Amino groups present within the enzyme will then react with the free endo of the epoxy rings of the EGDE molecule, resulting in covalent

immobilization

of

the

enzyme

to

form

a

superparamagnetic

biofunctionalized particle (EnzSP). The mixture was kept in suspension by using mechanical stirring at 200 rpm for 2 h, at ambient temperature. Then, the enzyme-loaded MNPs (EnzSP) were separated from the reaction mixture by using an external magnet. The magnetically isolated supernatant was kept at 4oC for indirect quantification of the amount of immobilized enzyme (equation 1). Subsequently, the particles were washed trice with buffer. These solutions were spectrophotometrically analyzed at a wavelength of 280 nm to assess the possible presence of protein leached during the washing. Alternatively, a fixed amount of EnzSP was directly put in contact with the BCA for direct quantification of the amount of enzyme immobilized on the surface of the MNP. Flux behavior for PVDF membrane alone and with MNP and ENZSP is reported in Table S1.



The morphology of the MNP before and after enzyme immobilization, and of the EnzSP magnetically dispersed on the membrane was obtained by means of scanning electron microscopy (SEM) with a high resolution (SEM FEI QUANTA 200 SEM). Samples were air dried prior to the SEM analysis for 24 h.

Enzyme activity measurements in batch

The activity of enzyme immobilized on PVDF membranes, and on the surface of MNP (EnzSP) was measured in batch (25°C) under static condition by following the p-nitrophenol (the reaction product) formation as a function of time using UVVisible spectrophotometric analysis. Blanc experiments carried out using EnzSP suspended in Tris/HCl buffer, confirmed that any color development was observed (Fig. S3). Hence, the developed color was solely due to the desired biocatalysis. The concentration of the p-nitrophenol product was calculated from the absorbance value at 405 nm (ε= 19920 M-1cm-1). The reaction mixture is composed of 850 µL H2O, 50 µL Tris/HCl buffer pH 8.5 (0.4 M) and 100 µL paraoxon-ethyl (10 mM) to keep the initial concentration of paraoxon-ethyl at 1 mM. When enzyme was grafted on the membrane, a thin strip of PVDF-DAMPGA-Enz was placed along the cuvette wall in such a way that it did not interfere with the UV-Vis path; when the enzyme was attached to the MNP, EnzSP were placed at the bottom of the cuvette. The reaction rate was obtained from the slope of p-nitrophenol concentration versus time plot while the enzyme activity was obtained from the slope of mass of p-nitrophenol versus time plot. The specific activity was calculated by normalizing the enzyme activity by the mass of enzyme.


Page 22 of 26

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Biocatalytic membrane reactor experimental set-up

The efficiency of the system with both methods of enzyme immobilization was measured in a biocatalytic membrane reactor (BMR). In the first case (Fig.1a), the chemically grafted (PVDF-DAMP-GA-Enz) membrane (4.5 cm diameter) was mounted in a dead-end filtration module. The system was then filled with 30 mL of the reaction mixture using a paraoxon-ethyl concentration ranging from 0.0625 mM to 1 mM. A N2 source was used to apply constant pressure, to force the reaction mixture to pass through the membrane. While

for

the

magnetic

responsive

system,

since

the

EnzSP

exhibit

superparamagnetism, it allows for the EnzSP to be initially homogeneously dispersed in the bulk reaction mixture and then deposited onto the membrane surface by applying an external magnetic field (PVDF-EnzSP). The EnzSP layer on the membrane surface permitted to reversibly immobilize the enzyme over the membrane surface to build the BMR shown in Fig.1b. In both configurations, the system performance was evaluated by measuring flux and amount of p-nitrophenol product collected in the permeate. The reaction rate in both cases was calculated considering mass balance according to (20):

!∙#$

=

2

where vr is the volumetric reaction rate (g/L h), c is p-nitrophenol concentration (g/L), V is the reaction volume (L) and t is the time (h). Hence, V was derived from the linear section of C vs t plot.



a

b

Fig. 1: Schemes of the biocatalytic membrane reactors containing: (a) a chemically grafted membrane; PVDF-DAMP-GA-Enz and (b) a magnetic nanoparticle EnzSP layers on the membrane surface; PVDF-EnzSP.

Acknowledgments

The authors acknowledge the European Union, FESR, MIUR, MSE for the financial support to the project Biodefensor - PON01_01585, within the framework PON Ricerca e Competitività 2007−2013. We also would like to acknowledge the Education, Audiovisual and Culture Executive Agency (EACEA) - European Commission within the Programme “Erasmus Mundus Doctorate in Membrane Engineering” (EUDIME, FPA 2011-0014).


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References

(1)

(2)

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10)

(11)

(12)

(13)

Trantidou, T., Friddin, M., Elani, Y., Brooks, N. J., Law, R. V., Seddon, J. M., and Ces, O. (2017) Engineering Compartmentalized Biomimetic Microand Nanocontainers. ACS nano 11, 6549-6565. Mazzei, R., Drioli, E., and Giorno, L. (2012) Enzyme membrane reactor with heterogenized β-glucosidase to obtain phytotherapic compound: Optimization study. Journal of Membrane Science 390–391, 121-129. Gebreyohannes, A. Y., Mazzei, R., Curcio, E., Poerio, T., Drioli, E., and Giorno, L. (2013) Study on the in Situ Enzymatic Self-Cleansing of Microfiltration Membrane for Valorization of Olive Mill Wastewater. Industrial & Engineering Chemistry Research 52, 10396-10405. Ji, C., Hou, J., Wang, K., Zhang, Y., and Chen, V. (2016) Biocatalytic degradation of carbamazepine with immobilized laccase-mediator membrane hybrid reactor. Journal of Membrane Science 502, 11-20. Mazzei, R., Piacentini, E., Gebreyohannes, A. Y., and Giorno, L. (2017) Membrane bioreactors in food, pharmaceutical and biofuel applications: state of the art, progresses and perspectives. Current Organic Chemistry 21, 1-1. Barbosa, O., Torres, R., Ortiz, C., Berenguer-Murcia, Á., Rodrigues, R. C., and Fernandez-Lafuente, R. (2013) Heterofunctional Supports in Enzyme Immobilization: From Traditional Immobilization Protocols to Opportunities in Tuning Enzyme Properties. Biomacromolecules 14, 2433-2462. Koo, H., Huh, M. S., Ryu, J. H., Lee, D.-E., Sun, I.-C., Choi, K., Kim, K., and Kwon, I. C. (2011) Nanoprobes for biomedical imaging in living systems. Nano Today 6, 204-220. Crespilho, F. N., Emilia Ghica, M., Florescu, M., Nart, F. C., Oliveira, O. N., and Brett, C. M. A. (2006) A strategy for enzyme immobilization on layer-bylayer dendrimer–gold nanoparticle electrocatalytic membrane incorporating redox mediator. Electrochemistry Communications 8, 1665-1670. Shekh, A. Y., Krishnamurthi, K., Mudliar, S. N., Yadav, R. R., Fulke, A. B., Devi, S. S., and Chakrabarti, T. (2012) Recent Advancements in Carbonic Anhydrase–Driven Processes for CO2 Sequestration: Minireview. Critical Reviews in Environmental Science and Technology 42, 1419-1440. Gebreyohannes, A. Y., Giorno, L., Vankelecom, I. F. J., Verbiest, T., and Aimar, P. (2017) Effect of operational parameters on the performance of a magnetic responsive biocatalytic membrane reactor. Chemical Engineering Journal 308, 853-862. Gebreyohannes, A. Y., Mazzei, R., Poerio, T., Aimar, P., Vankelecom, I. F. J., and Giorno, L. (2016) Pectinases immobilization on magnetic nanoparticles and their anti-fouling performance in a biocatalytic membrane reactor. RSC Advances 6, 98737-98747. Vitola, G., Büning, D., Schumacher, J., Mazzei, R., Giorno, L., and Ulbricht, M. (2016) Development of a Novel Immobilization Method by Using Microgels to Keep Enzyme in Hydrated Microenvironment in Porous Hydrophobic Membranes. Macromolecular Bioscience. Gebreyohannes, A. Y., Bilad, M. R., Verbiest, T., Courtin, C. M., Dornez, E., Giorno, L., Curcio, E., and Vankelecom, I. F. J. (2015) Nanoscale tuning of enzyme localization for enhanced reactor performance in a novel magnetic25 ACS Paragon Plus Environment


(14)

(15)

(16)

(17)

(18) (19)

(20)

responsive biocatalytic membrane reactor. Journal of Membrane Science 487, 209-220. Vitola, G., Mazzei, R., Fontananova, E., and Giorno, L. (2015) PVDF membrane biofunctionalization by chemical grafting. Journal of Membrane Science 476, 483-489. Militano, F., Poerio, T., Mazzei, R., Piacentini, E., Gugliuzza, A., and Giorno, L. (2016) Influence of protein bulk properties on membrane surface coverage during immobilization. Colloids and surfaces. B, Biointerfaces 143, 309-17. Del Giudice, I., Coppolecchia, R., Merone, L., Porzio, E., Carusone, T. M., Mandrich, L., Worek, F., and Manco, G. (2016) An efficient thermostable organophosphate hydrolase and its application in pesticide decontamination. Biotechnol Bioeng 113, 724-34. Vitola, G., Mazzei, R., Fontananova, E., Porzio, E., Manco, G., Gaeta, S. N., and Giorno, L. (2016) Polymeric biocatalytic membranes with immobilized thermostable phosphotriesterase. Journal of Membrane Science 516, 144-151. Eisenthal, R., Danson, M. J., and Hough, D. W. (2007) Catalytic efficiency and kcat/KM: a useful comparator? Trends in Biotechnology 25, 247-249. Ross, G., Watts, J., Hill, M., and Morrissey, P. (2000) Surface modification of poly (vinylidene fluoride) by alkaline treatment1. The degradation mechanism. Polymer 41, 1685-1696. Bailey, J. E., and Ollis, D. F. (1986) Biochemical Engineering Fundamentals, McGraw-Hill, New York.


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Phosphotriesterase-Magnetic Nanoparticle Bioconjugates with

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