Influence of Lipase Immobilization Mode on Ethyl Acetate Hydrolysis

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Influence of lipase immobilization mode on ethyl acetate hydrolysis in a continuous solid-gas biocatalytic membrane reactor Giuseppe Vitola, Rosalinda Mazzei, Teresa Poerio, Giuseppe Barbieri, Enrica Fontananova, Dominic Büning, Mathias Ulbricht, and Lidietta Giorno Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.9b00463 • Publication Date (Web): 16 Jul 2019 Downloaded from pubs.acs.org on July 20, 2019

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Bioconjugate Chemistry

Influence of lipase immobilization mode on ethyl acetate hydrolysis in a continuous solid-gas biocatalytic membrane reactor

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Giuseppe Vitola1, Rosalinda Mazzei1,*, Teresa Poerio1,*, Giuseppe Barbieri1, Enrica

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Fontananova1, Dominic Büning2, Mathias Ulbricht2, Lidietta Giorno1

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1 Institute

on Membrane Technology, National Research Council, ITM-CNR, via P. Bucci, 17/C, I87030 Rende (Cosenza), Italy

2 Lehrstuhl

für Technische Chemie II, Universität Duisburg-Essen, 45117 Essen, Germany

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*:

corresponding author; Tel.: +39 0984 492076; fax: +39 0984 402103; e-mail address: [email protected]; [email protected]

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ACS Paragon Plus Environment

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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

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Abstract

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Solid-gas biocatalysis was performed in a specially designed continuous biocatalytic membrane

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reactor (BMR). In this work, lipase from Candida rugosa (LCR) and ethyl acetate in vapour phase

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were selected as model enzyme and substrate, respectively, to produce acetic acid and ethanol. LCR

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was immobilized on functionalized PVDF membranes by using two different kinds of chemical

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bond: electrostatic and covalent. Electrostatic immobilization of LCR was carried out using a

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membrane functionalized with amino groups, while covalent immobilization was carried out using

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membrane, with or without surface-immobilized polyacrylamide (PAAm) microgels, functionalized

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with aldehyde groups. These biocatalytic membranes were tested in a solid-gas BMR and compared

10

in terms of enzyme specific activity, catalytic activity and volumetric reaction rate. Results

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indicated that lipase covalently immobilized is more performant only when the immobilization is

12

mediated by microgels, showing a catalytic activity doubled with respect to the other system with

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covalently bound enzyme (4.4 v.s. 2.2 µmol h-1). Enzyme immobilized by ionic bond, despite a

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lower catalytic activity (3.5 vs 4.4 µmol h-1), showed the same specific activity (1.5 mmol·h-1·g-

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1 ENZ)

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analogously improved enzyme hydration. Using the optimized operating conditions regarding

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immobilized enzyme amount, ethyl acetate and molar water flow rate, all the three BMRs showed a

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continuous catalytic activity for about 5 months. On the contrary, the free enzyme (in water/ethyl

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acetate emulsion), at 50 °C resulted completely inactive and at 30°C (temperature optimum) it has a

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specific activity of two orders of magnitude lower (8.4·10-2 mmol h-1g-1) than the solid-gas

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biocatalytic membrane reactor. To the best of our knowledge, this is the first example of solid-gas

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biocatalysis, working in gaseous phase in which a biocatalytic membrane reactor, with the

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enzyme/substrate system lipase/ethyl acetate, was used.

of the system using microgels, due to a higher enzyme degree of freedom coupled with an

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Keywords: solid-gas biocatalysis, biocatalytic membrane reactor, continuous membrane process, lipase from Candida rugosa.


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INTRODUCTION

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Immobilized enzymes are used in industry for synthesizing fine chemicals, pharmaceuticals and

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other valuable products 1. Most of times enzymatic reactions were carried out in aqueous solution,

4

organic solvent, two phase water/organic mixtures or microemulsions either for homogeneous or

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heterogeneous catalysis2, 3. Interestingly, biocatalysis can also be applied to gas phase systems, in

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which substrates and products are in gaseous/vapour phase and enzymes are in solid phase. This

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approach, named solid-gas biocatalysis4, was proposed with the aim to develop new technologies

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for applications in chemical compounds production5,6, waste treatment7 and biosensing8,9. By this

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technology, the ability of cells, lyophilized enzymes or enzymes immobilized on solid support to

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catalyse transformations of gaseous substrates is exploited [10]. Many advantages are presented by

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solid-gas biocatalysis over traditional biocatalysis in aqueous systems4,10. They include a higher

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thermostability of the dehydrated enzyme with respect to the hydrated form, reduction of microbial

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contamination, reduction of by-products formation, as well as improvements in mass transfer and

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product recovery10,11. In literature, several bioreactor configuration (fluidized bed, membrane

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bioreactor, air lift etc10,11), for solid-gas biocatalysis are reported, in which different biocatalysts

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(hydrogenase12, alcol oxidase13, carbonic anhydrase14, lipases from different sources and cells)

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immobilized on different supports10 (alumina silicate, molecular sieves, glass beads resins and

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polymeric supports) or free in solution were used.. The design of a solid-gas bioreactor is quite

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simple and an important parameter in these systems is the water-enzyme interaction15. Early

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attempts to operate solid-gas biocatalysis employed batch bioreactors where enzyme powder was

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well mixed with the gaseous substrate5, 6. Thereafter, tubular reactors containing packed enzymatic

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sample or cells, which were percolated by gaseous carrier, water vapour and substrates, were

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adopted16,17. However, optimal supports for solid-gas biocatalysis are porous materials that allow

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high flow rate and low pressure drop4. In particular, porous membranes could be good supports, due

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to their properties of high specific surface area and the possibility to combine separation with

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biochemical reaction by biocatalytic membrane reactor technology18. Different biocatalytic ACS Paragon Plus Environment

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membrane reactors are used to carry out CO2 hydration thanks to immobilized carbonic anhydrase

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19, 20, 21, 22, 23,

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configuration used, so the transport through the membrane mainly occur by diffusion. To the best of

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our knowledge, no studies on solid-gas biocatalysis in which immobilized lipase on membrane and

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ethyl acetate as substrate are present. Therefore, we investigated the possibility to use

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biofunctionalized polymeric membranes for the hydrolysis of gaseous ethyl acetate. A crucial role

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in solid-gas bioreactor is played by the hydrophobic-hydrophilic character of the supporting

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material. The materials hydrophobicity is a valuable property for the membranes employed in solid-

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gas bioreactor, where pores wetting by water shall be prevented to avoid pore blocking by

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condensed water and thus to ensure high flow rates. With this aim, a strongly hydrophobic

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membrane made of polyvinylidene fluoride (PVDF) was used. The PVDF was functionalized in two

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different ways to allow electrostatic or covalent binding of enzyme within the membrane. Covalent

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bond was carried out directly on the membrane activated membrane surface or mediated by surface-

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immobilized hydrophilic microgel particles. Then three different BMRs were developed in which

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the enzyme was immobilized on the membrane by the different mentioned immobilization

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procedures. The performance of the heterogenized biocatalysts was studied with the model system

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enzyme/gaseous substrate lipase from Candida rugosa (LCR)/ethyl acetate. Parameters such as

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effect of LCR loading, ethyl acetate and water vapours molar flow rates on the catalytic

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performance were studied. The optimized system showed a continuous performance as a function of

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time and long term stability, demonstrating the potentiality of solid-gas BMR.

but in these systems a biocatalytic membrane gas-liquid contactor is the main

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RESULTS AND DISCUSSION

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LCR immobilization on pre-functionalized porous PVDF membranes

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LCR was immobilized on three PVDF membranes, functionalized in different ways and named in

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following as PVDF-DAMP, PVDF-DAMP-GA and PVDF-DAMP-GA-MG-GLY-GA, according to

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the different functionalization procedure reported in Materials and Methods section. ACS Paragon Plus Environment

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LCR immobilization on PVDF-DAMP membranes was based on electrostatic interactions between

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protonated amino groups and net negatively charged protein (see also Results and Discussion for

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zeta potential below). PVDF-DAMP-GA membrane was obtained by means of covalent bonds

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between the aldehyde groups introduced on the membrane and the amino groups of the enzyme.

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LCR immobilization on PVDF-DAMP-GA-MG-GLY-GA membrane was mediated by microgels.

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In particular, the enzyme was covalently immobilized on the membrane by Schiff base formation

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with the aldehyde groups introduced on the microgels. According to literature data24, 25the lipase

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immobilization on the hydrophobic functionalized surface occurs in two steps. In the first step the

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enzyme adsorption on the hydrophobic support promotes the interfacial activation, followed in the

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second step, by the formation of electrostatic (on PVDF-DAMP) -or covalent bond (PVDF-DAMP-

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GA and PVDF-DAMP-GA-MG-GLY-GA). However, it is noteworthy to underline that in the case

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of enzyme immobilization “glutaraldehyde mediated” (PVDF-DAMP-GA and PVDF-DAMP-GA-

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MG-GLY-GA ) the contribution of ionic groups due to the protonated amino-groups of DAMP has

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also to be considered in the bonds formation. Literature data confirm our hypothesis in similar

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systems, in fact at low ionic strength; a rapid ionic interaction, due to ionic groups, is followed by

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the covalent bond formation26.

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Various amounts of LCR were immobilized on the three functionalized PVDF membranes by

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changing the contact time between the enzyme solution and the functionalized membranes (Fig. 1).

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Results evidenced that the amount of immobilized LCR increased with the increase of contact time

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until 14 hours and after this time the saturation amount for all the membranes was reached.

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Moreover, from the Fig. 1 is possible to observe that the maximum amount of LCR immobilized on

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the three membranes was quite similar, with a slightly higher enzyme concentration (14.56 mg/cm3)

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when the PVDF-DAMP-GA-MG-GLY-GA membrane was used. This outcome can be explained

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taking into account that the microgels loaded on the membrane provide a larger reactive surface for

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enzyme binding compared to direct immobilization onto functionalized PVDF.


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Fig. 1. Immobilized enzyme amount on functionalized membranes obtained by immersing the

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different membranes in the biocatalyst solution for different time (contact time). Membrane reactor

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volume= 0.2 cm3.

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Surface characterization of biofunctionalized membranes

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The three biocatalytic membranes, containing the saturation amounts of LCR, were characterized in

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terms of surface properties by means of static water contact angle, surface zeta potential and FT-IR

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measurements.

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The data of the water contact angle measurement before and after enzyme immobilization are

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reported in Table 1. The results indicate that native PVDF is a hydrophobic polymer and the

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treatment with DAMP causes a slight decrease of the contact angle due to the grafting of polar

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amino groups. The subsequent derivatization of amino groups with glutaraldehyde does not change

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substantially the wettability of the membrane. A slight decrease of the water contact angle is also

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observed after the immobilization of the hydrophilic microgel; but also in this case the membrane

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largely remains in the hydrophobic range. This was due to the fact that the microgels are randomly


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distributed on the surface (Fig. 2) and inside the membrane pores as clusters (Fig 2B and 2C);

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therefore, their influence on the membrane surface wettability is mitigated as intended. Considering

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the static water contact angles values, it can be stated that the three functionalization protocols as

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well as the subsequent biofunctionalization are able to preserve the membrane hydrophobicity. This

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is an important goal for biocatalytic membranes that have to be used in solid-gas bioreactor because

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the poor wettability permits to prevent water absorption in the membrane.

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Table 1. Static water contact angle of modified PVDF membranes before and after LCR

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immobilization (measurements referred to membranes loaded with saturation amounts of LCR; cf.

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Fig. 1).

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Membrane

Contact angle (°) Before LCR immobilization

After LCR immobilization

PVDF

134 ± 4

-

PVDF-DAMP

125 ± 2

129 ± 2

PVDF-DAMP-GA

127 ± 3

132 ± 3

PVDF-DAMP-GA-MG-GLY-GA

115 ± 4

124 ± 2

12 13 14 15


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investigated. These parameters were studied keeping constant temperature (50°C) and carrier gas

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(nitrogen) molar flow rate (0.29 mol h-1).

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Effect of LCR amount

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Generally, the amount of biocatalyst immobilized on membrane is a critical parameter to be

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optimized during the development of a BMR, since it influences the enzyme activity as well as the

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transport of substrate and product through the membrane. Indeed, the membrane saturation

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coverage or the highest amount of immobilized enzyme, not always guarantees the highest catalytic

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performance, since other phenomena such as protein aggregation or crowding can occur, that may

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drastically reduce the enzyme activity32.For this reason the catalytic tests were carried out also

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using the membranes prepared with a lower immobilized enzyme amount respect to the saturation

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point. Fig. 4 shows the volumetric reaction rate as a function of the amount of immobilized LCR for

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the three different BMRs. Results show that considering the reaction rate trend of all the three

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biocatalytic membranes, the higher the amount of immobilized enzyme, the higher is the reaction

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rate. Besides, this trend indicates that the reaction, for all the three membranes, is not mass transfer

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limited in the investigated LCR loading range, permitting all enzyme molecules to participate in the

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hydrolysis reaction. This behaviour is in good agreement with literature data, which report for solid-

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gas systems negligible or completely absent diffusion and mass transfer limitations, thanks to the

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low viscosity and high diffusion coefficients of gases compared to liquids10. On the basis of these

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results, the immobilized enzyme amount used in the following experiments was the one in which

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the maximum reaction rate is obtained (11 ± 0.6 mg cm-3, 12 ± 3 mg·cm-3 and 14 ± 4 mg cm-3 for

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PVDF-DAMP-LCR,

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respectively), which also corresponds to the maximum amount that is possible to immobilize on the

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functionalized membranes (see also Fig. 1).

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The comparison of immobilized lipase on PVDF-DAMP-GA and on PVDF-DAMP evidenced that

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although the amount of immobilized enzyme is about the same (Fig. 1) a higher catalytic (3.49

PVDF-DAMP-GA-LCR,

and

PVDF-DAMP-GA-MG-GLY-GA-LCR,


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±0.13 vs 2.21 ±0.10 µmol h-1) and specific activity (1.50 ±0.1 vs 0.90 ± 0.03 mmol h-1gENZ-1) are

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obtained when the immobilization was carried out by the electrostatic interaction. The lower

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performance in case of the covalent immobilization is due to the presence of covalent bonding sites

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in the active center. The chemical reaction between membrane ligands and enzyme causes a

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decreased availability of the catalytic site altering enzyme structure and activity. This was recently

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demonstrated by Zhang H et al.33, which compared the covalent and electrostatic immobilization of

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lipase from Aspergillus Oryzae, using a liquid substrate.

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Fig. 4. Volumetric reaction rate as a function of immobilized LCR on functionalized membrane

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(membrane reactor volume: 0.2 cm3). Operating conditions: 50 °C, nitrogen molar flow rate: 0.29

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mol h-1, ethyl acetate molar flow rate 12.30 mmol h-1, water molar flow rate 15.33 mmol h-1.

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Effect of ethyl acetate molar flow rate

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The influence of ethyl acetate molar flow rate on the volumetric reaction rate was investigated for

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all the three BMRs, keeping constant the water molar flow rate (15.33 mmol h-1). As can be noted

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from Fig. 5a the reaction rate increases until the ethyl acetate molar flow rate reached the value of ACS Paragon Plus Environment

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12.30 mmol h-1. Further increases do not generate increases in reaction rate, suggesting a complete

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biocatalyst saturation at the ethyl acetate molar flow rate of 12.30 mmol h-1. Therefore, to study the

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influence of the others parameters on the BMRs productivity, ethyl acetate molar flow rate was

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fixed to 12.30 mmol h-1.

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A

6


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B

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Fig. 5. Volumetric reaction rate as a function of: A) ethyl acetate (nitrogen molar flow rate: 0.29

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mol h-1, water flow rate 15.33 mmol h-1) and B) water molar flow rate (nitrogen molar flow rate:

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0.29 mol h-1, ethyl acetate molar flow rate: 12.30 mmol h-1).

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Effect of water molar flow rate

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In solid-gas biocatalysis, the degree of biocatalyst hydration is a crucial parameter because it deeply

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affects activity and stability of the biocatalyst within the water-restricted microenvironment

34.

In

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literature, the effect of water on biocatalyst performance has been thoroughly investigated and

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represented the main aspect in most of the published works concerning solid–gas biocatalysis [12].

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In this work, the effect of water molar flow rate on the reaction rate of the studied systems was

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investigated by varying it from 5.11 to 30.67 mmol h-1. Fig. 5b shows that the reaction rate, in a

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similar way for the three different BMRs, slightly increases when the water molar flow rate is

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increased up to 15.33 mmol h-1; then it becomes constant because the complete hydration of the

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immobilized enzyme on the hydrophobic membrane was reached. This finding is in good agreement


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with literature data 10, 11, 15. On the other hand, water could have a negative effect on the biocatalyst

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because, at a high temperature, it can denaturate it9. However no enzyme denaturation can be

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supposed in the studied systems, since for the investigated molar flow rate range, no decreasing of

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reaction rate was observed. Based on these results, water molar flow rate of 15.33 mmol h-1 was

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selected for subsequent experiments to test enzyme stability.

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Comparison and stability of BMRs using selected operating conditions

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The BMRs performance was evaluated in term of specific and catalytic activity, during continuous

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long run experiments (Fig. 6). In order to investigate the BMRs stability, the experiments were

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carried out continuously for 40 hours at fixed ethyl acetate (12.30 mmol h-1) and water (15.33

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mmol h-1) molar flow rate and then once a month (each reaction cycle lasted 8 hours) over a period

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of 5 months. . The lowest enzyme specific activity and catalytic activity were obtained when LCR

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was immobilized by covalent bond on PVDF-DAMP-GA. These results can be attributed both to

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the type of interaction between enzyme and support and to the nature of the support itself. In fact,

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either by PVDF-DAMP-GA or PVDF-DAMP-GA-MG-GLY-GA a covalent immobilization of

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LCR, via reaction with a glutaraldehyde-functionalized support, is established, but the system

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containing the microgels showed a doubled catalytic activity, despite the immobilized enzyme

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amount was just 13 % more (Table 2). The hydrophilic microgels improve the interaction between

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the LCR and the water required for the hydrolysis reaction, creating a hydrated microenvironment

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which enhances the LCR activity.

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Same specific activity for PVDF-DAMP-GA-MG-GLY-GA-LCR and PVDF-DAMP-LCR

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biocatalytic membranes was obtained (Fig.6) and the difference in catalytic activity (20 % more for

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PVDF-DAMP-GA-MG-GLY-GA-LCR) was only due to the higher amount of immobilized enzyme

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(20% more) in the PVDF-DAMP-GA-MG-GLY-GA-LCR system. Indeed the increasing in

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catalytic activity was directly proportional to the LCR amount. This is probably due to the fact that

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electrostatic bond is a type of interaction which permitted an increased range of motion35. ACS Paragon Plus Environment

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Fig. 6. Specific and catalytic activity of the three BMRs as a function of time. Operating conditions:

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50 °C, Nitrogen molar flow rate 0.29 mol h-1, ethyl acetate molar flow rate 12 mmol h-1, water

3

molar flow rate 15.33 mmol h-1.

4 5

On the other hand, although the PVDF-DAMP-GA-MG-GLY-GA membrane is produced with a

6

longer procedure, it can permit the increase of the biocatalyst loading by increasing the immobilized

7

microgels content and consequently the active surface area able to bind the LCR.

8 9

Table 2. Immobilized enzyme amount and catalytic activity of biocatalytic membranes Immobilized enzyme

Catalytic activity

(mg cm-3)

(µmol h-1)

PVDF-DAMP-LCR

11.6 ± 0.6

3.5 ± 0.3

PVDF-DAMP-GA-LCR

12.7 ± 0.6

2.2 ± 0.3

PVDF-DAMP-GA-MG-GLY-GA-LCR

14.6 ± 0.7

4.4 ± 0.4

Biocatalytic membrane

10 11

The stability of the three BMRs was also evaluated, as previously mentioned, for five reaction

12

cycles and for a total observation period of 5 months. The enzyme specific and catalytic activity

13

remained constant in all the three different systems, indicating a continuous performance and a high

14

stability as a function of time, independently from the biocatalytic membrane used (Fig. 7). On the

15

contrary the free enzyme has no activity at 50 °C, and at 30°C it shows a specific activity (8.4-2

16

mmol·h-1g-1ENZ) which is two orders of magnitude lower than the ones obtained in the solid-gas

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BMRs (Fig. 6).


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Fig. 7. Enzyme specific and catalytic activity as a function of time for the different BMRs (ethyl

3

acetate molar flow rate 12 mmol h-1, water molar flow rate 15.33 mmol h-1).

4 5

CONCLUSIONS

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In this study the use of immobilized LCR within PVDF membranes for the bioconversion of ethyl

7

acetate in gaseous phase was explored. Three continuous solid-gas BMRs, differing each other for

8

the enzyme immobilization strategy used (ionic, covalent and covalent mediated by microgels),

9

have been developed. The BMR in which LCR immobilization was carried out by hydrophilic

10

microgel mediation exhibited the highest catalytic activity, but comparable specific activity of the

11

system in which ionic bond for the enzyme immobilization was used (1.5 mmol·h-1g-1ENZ). Hence,

12

the ionic immobilization of lipase for this application seems the most appropriate, since it permits

13

the production of biocatalytic membranes with the same specific activity of the system compared

14

with those mediated by microgels, but by an easier production process. However a higher catalytic

15

activity can be achieved in the BMR with microgels, since the immobilized enzyme amount could


17


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be increased with the increase of microgels loading. A high stability and a continuous catalytic

2

activity were observed for all the three biocatalytic membranes reactors developed (at 50 °C),

3

confirming the improvement of enzyme performance in controlled/restricted water environment. On

4

the contrary, the free enzyme is inactive at 50°C and it showed two orders of magnitude lower

5

specific activity at 30°C, compared to the newly developed BMR.

6 7

MATERIALS AND METHODS

8

Chemicals

9

Lipase from Candida rugosa (LCR, 65 kDa), 1,5-diamino-2-methylpentane (DAMP),

10

glutaraldehyde (GA), acrylamide (AAm), = =R

%$11

monooleate (Span80),

12

potassium chloride, sodium hypochlorite solution (NaOCl, available chlorine 10%–15%), isopropyl

13

alcohol, glycine as well as the salts to prepare phosphate buffer and carbonate buffer were

14

purchased from Sigma-Aldrich. Ethyl acetate and ethanol were purchased from VWR international.

15

The bicinchoninic acid assay (BCA) kit (Thermo Scientific) was used to evaluate the concentration

16

of protein in solutions. Flat sheet PVDF membranes were used as support for LCR immobilization.

17

The membrane was supplied by GVS S.p.A (code M09G0020). It is characterized by a nominal

18

pore size of 0.2 S% a water breakthrough pressure higher than 1.8 bar, and a thickness ranging

19

from 150 to 200 S%

R$ 4 ' *

%$(

(

bisacrylamide (MBAAm), sorbitane

. (AIBN), cyclohexane, sodium chloride,

20 21

Microgels preparation

22

The PAAm microgels were synthesized by the inverse miniemulsion polymerization technique and

23

then they were amino-functionalized by Hoffman reaction (mean particle size 242 ±8 nm,

24

polydispersity index, PDI 0.55 ±0.3) as in detail described in our previous work

25

disperse phase was prepared by dissolving 0.094 g of NaCl, 0.8 g of AAm and 0.04 g of MBAAm

26

in 3.2 g of water. The disperse phase was mixed for 90 min with the continuous phase prepared by ACS Paragon Plus Environment

36.

Briefly, a

18

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1

dissolving 1.2 g of Span 80 in 50 g of cyclohexane. Afterward, the thus obtained pre-emulsion was

2

ultrasonicated for 2 min to obtain the inverse miniemulsion which was then heated at 65 °C in an oil

3

bath. Finally, polymerization was started by adding the initiator AIBN, and it was carried out for 2

4

h. Afterwards amino groups were created in the polymeric network by means of Hofmann reaction.

5

With this aim a dispersion of PAAm microgels in water (50 mL, 1 g LT ) was mixed with 30 mL of

6

a solution consisting of 23·10T M NaOCl and 10.7 M NaOH kept at -10 °C. After 90 min, 70 mL of

7

8.29 M NaOH solution was added to the mixture, the temperature was maintained at -10 °C for

8

further 30 min, then it was increased to 0 °C and the reaction was carried out for 17 h.

9 10

Membranes biofunctionalization

11

Membrane functionalization with amino groups, used to promote Coulombic interaction between

12

membrane and LCR, was carried out by using the method reported in Vitola et al.37. Briefly, the

13

PVDF membrane was cut into disk (4.7 cm in diameter) and soaked into 20 mL of 1,5-diamino-2-

14

methylpentane solution (DAMP, 2M) in carbonate buffer pH 11, for 6 h at 50 °C to introduce amino

15

groups onto the PVDF surface (PVDF-DAMP).

16

The PVDF membrane having aldehyde groups (PVDF-DAMP-GA), which is able to covalently

17

bind LCR, was produced reacting the amino groups introduced on the PVDF-DAMP membrane

18

with 20 mL of 10% (v/v) glutaraldehyde solution at 25 °C for 2 h (Schiff base formation

19

mechanism). The use of glutaraldehyde as crosslinker is widespread and it is generally known that

20

biomolecules bind on glutaraldehyde activated supports mainly by means of the free amino groups

21

of lysine residues38, 39.

22

The PVDF membrane containing hydrophilic PAAm microgels (MG) was prepared by soaking the

23

PVDF-DAMP-GA membrane in aqueous microgels dispersion (50 mL, 0.2 mg mLT ) for 16 h,

24

according to the scheme in Fig. 8. In this way, the aldehyde groups grafted on the PVDF membrane

25

were reacted with amino groups of microgels by Schiff base formation (PVDF-DAMP-GA-MG).

26

The membrane was then soaked in a glycine solution (25 mL, 1 M, in 50 mM phosphate buffer pH ACS Paragon Plus Environment

19


Page 20 of 29

1

7) at 25 °C for 2 h to quench unreacted aldehyde groups (PVDF-DAMP-GA-MG-GLY). Afterward,

2

the amino groups of the microgels were activated by glutaraldehyde treatment by soaking the

3

PVDF-DAMP-GA-MG-GLY membrane into 20 mL of 10% (v/v) glutaraldehyde solution at 25 °C

4

for 2 h (PVDF-DAMP-GA-MG-GLY-GA membrane).

5

LCR immobilization on PVDF-DAMP, PVDF-DAMP-GA and PVDF-DAMP-GA-MG-GLY-GA

6

membranes was performed by soaking the membranes in a LCR solution (0.76 ± 0.06 mg mL-1, 25

7

mL phosphate buffer pH 7.5) at 25 °C. Different contact times between the membranes and the

8

LCR solution (0.5, 1 and 14 h) were used in order to increase the immobilized LCR amount.

9

In order to verify that the amount of immobilized enzyme is chemically bonded to the membrane,

10

the biocatalytic membranes, after the LCR immobilization on PVDF-DAMP-GA and on PVDF

11

PVDF-DAMP-GA-MG-GLY-GA were washed with buffers at different pH (5.5-7-8.5) and ionic

12

strength, showing no release of protein in the collected fraction. This step was necessary to check

13

the removal of eventual traces of enzyme reversibly linked on the membrane (e.g. adsorbed by

14

electrostatic interaction). In the case of lipase immobilized on PVDF-DAMP, unbonded enzyme

15

was removed by washings with the buffer previously used for enzyme assay. Afterward, the

16

membrane was dried over-night at room temperature and stored in a desiccator over silica gel until

17

its use.

18

The protein concentration in the initial, final and washing solutions were determined by BCA

19

The amount of immobilized LCR was determined by mass balance according equation (1).

20 21

CiVi = CfVf + CwsVws + m

(1)

22

Here, m indicates the immobilized protein mass in the membrane, C and V represent the

23

concentration (mg cm-3) and volume (cm3), respectively; the subscripts i, f, and ws indicate the

24

initial, final, and washing solutions, respectively. The immobilized enzyme amount was then

25

normalized by the membrane reactor volume, which is 0.2 cm3.


20

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Page 22 of 29

1

Apparatus, method and operating conditions of biocatalytic tests

2

A reactor at a laboratory scale, for the gas-phase hydrolysis of ethyl acetate was designed and built

3

(Fig. 9). The reactor consisted of a cell made of stainless steel with an arrangement to hold the

4

biocatalytic membrane (active surface area 12 cm2). The carrier gas, nitrogen, was supplied by a

5

mass

6

polytetrafluoroethylene (PTFE) tube to the biocatalytic membrane. Ethyl acetate and water were

7

supplied by means of a syringe pump (Kent Scientific Genie Touch Syringe Pump). The hydrolysis

8

products (ethanol and acetic acid) and the unconverted reagents (ethyl acetate and water) were

9

recovered in a trap immersed in an ice bath. Valves, flow meters (Brooks instrument), and pressure

10

gauges (Wika, DG-10) were used to control and monitor the operating conditions. The membrane

11

reactor cell was placed in a thermostated chamber to control reaction temperature. Ethyl acetate,

12

water and nitrogen were heated up inside the same chamber and then mixed before they were fed to

13

the biocatalytic membrane. The principle for water and ethyl acetate evaporation was based on the

14

“liquid injection method”, which is an effective method used in fuel cell system for the gas

15

humidification40. It consists in the injection of liquid water into a gas stream, at fixed temperature

16

and optimized axial flow rate, which permits the evaporation of the water droplets.

17

The axial flow rate of the carrier gas nitrogen was 2.0·10-2 m/s, while the ones of the water and the

18

ethyl acetate were 1.7·10-6 and 7.0 10-6 m/s, respectively.

19

The temperature of the thermostated chamber was set at 50 °C and the samples of recovered

20

substrate (not converted) and products were collected every two hours. Prior to start measurements

21

the reactor was stabilized for one hour under the reaction conditions. In order to investigate the

22

BMRs stability, the experiments were carried out continuously for 40 hours at fixed ethyl acetate

23

(12.30 mmol h-1) and water (15.33 mmol h-1) molar flow rate and then once a month over a period

24

of 5 months. Between each reaction cycle the biocatalytic membranes were stored in a desiccator

25

over silica gel. Each reaction cycle lasted 8 hours.

flow

controller

(Brooks

instrument,

model

0254)

and

flowed

through

a

26 ACS Paragon Plus Environment

22

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Page 24 of 29

1

The catalytic activity (µmol h-1) was calculated according to the concentration of ethanol in the

2

aqueous phase as a function of time:

3

The reaction rate of the BMR was calculated taking into account that transport mainly occurs by

4

convective flow, so the product was continuously removed from the reaction site. Then the system

5

worked as a plug flow reactor at the steady-state. Since the product was continuously removed from

6

the reaction site, the accumulation term in mass balance equation is zero42, and the reaction rate was

7

calculated as follows:

vr

8

F (Cf Cp ) V

(2)

9

Here, vr is the volumetric reaction rate (mmol cm-3h-1), F is the permeate flow rate (cm3 h-1), C is

10

the substrate concentration (mmol cm-3), V is the reactor volume (cm3 ); the indices f and p

11

indicate feed and permeate, respectively.

12

The reactor volume, in this system, is represented by the membrane void volume, which is the

13

fraction of the membrane volume (where the reaction occurred) not occupied by the polymer.

14 15 16 17 18

Acknowledgements

19

The authors acknowledge the project PON01_01585 “Innovative products for monitoring and

20

detoxification/decontamination of nerve agents and explosives in the environment and/or for

21

handling of emergency” (BIODEFENSOR, PON Ricerca e Competitività 2007-2013) for the

22

financial support.

23 24 25 26 ACS Paragon Plus Environment

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1

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Croissant J. G., Cecchi J.L., Rempe S. B., Brinker C.J., (2018), Ultra-thin enzymatic liquid

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J.M., (1993), Preparation of activated supports containing low pK amino groups. A new tool for

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protein immobilization via the carboxyl coupling method, Enzyme Microb. Technol., 15, 546–550. ACS Paragon Plus Environment

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Barbosa O., Ortiz C., Fernandez-Lafuente R., (2019) Immobilization of lipases on hydrophobic

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supports: immobilization mechanism, advantages, problems, and solutions, Biotechnol. Adv.

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C., Fernandez-Lafuente R., Guisan J.M., (2006), Different mechanisms of protein immobilization

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on glutaraldehyde activated supports: Effect of support activation and immobilization conditions,

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Enzyme Microb. Technol., 39, 877–882.

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measurement as a diagnostic tool in enzyme immobilisation. Colloids Surf B Biointerfaces. 66, 39-

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from Candida rugosa on cellulose and its influence on lipolytic activity, Enz. Microb. Technol., 14,

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ionic liquids for lipase immobilization: Enzyme activity in catalyzing esterification. J. Mol. Catal.,

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graphene oxide support for high-efficiency biocatalysis. Appl. Mat. Today, 5, 200–208.

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protein bulk properties on membrane surface coverage during immobilization. Colloids Surf B

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Microenvironment in Porous Hydrophobic Membranes. Macromol. Biosci. 17, 1600381.

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Continuous solid-gas biocatalytic membrane reactor +

-+ --

+

+ + + + -- +

Electrostatic immobilization

Covalent immobilization

Covalent immobilization microgels-mediated


Influence of Lipase Immobilization Mode on Ethyl Acetate Hydrolysis

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