Understanding the silica-based sol-gel encapsulation mechanism of Thermomyces lanuginosus lipase: The role of polyethylenimine

Molecular Catalysis 449 (2018) 106–113

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Understanding the silica-based sol-gel encapsulation mechanism of Thermomyces lanuginosus lipase: The role of polyethylenimine

T

Sindy Escobara, Claudia Bernalb,c, Juan M. Bolivard, Bernd Nidetzkyd, Fernando López-Gallegoe,f, ⁎ Monica Mesaa, a Grupo Ciencia de los Materiales, Instituto de Química, Facultad de Ciencias Exactas y Naturales, Universidad de An-tioquia – UdeA, Calle 70 No. 52–21, Medellín, Colombia b Grupo Tecnología Enzimática para Bioprocesos, Departamento de Ingeniería de Alimentos, Universidad de La Serena, Raul Bitran 1305, La Serena, Chile c Instituto Multidisciplinario de Ciencia y Tecnología, Universidad de La Serena, Raul Bitran 1305, La Serena, Chile d Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, Petersgasse 12/l, A-8010 Graz, Austria e Heterogeneus Biocatalysis Group, CIC BiomaGUNE, Paseo Miramon 182, San Sebasitan-Donostia, 20014, Spain f IKERBASQUE, Basque Foundation for Science, Bilbao, Spain

A R T I C L E I N F O

A B S T R A C T

Keywords: Thermomyces lanuginosus lipase Polyethylenimine Sol-gel process Encapsulation

Silica-based sol-gel enzyme immobilization aided by polyamines has been used for generating active and stable heterogeneous biocatalysts. However, the encapsulation mechanism and its influence on the characteristics of the resulting biocatalysts have not been fully understood yet. This work aimed to shed light on how Thermomyces lanuginosus lipase, siliceous species and the additive polyethylenimine (PEI) interact during enzyme encapsulation, by establishing a correlation between addition of PEI, material formation and enzyme encapsulation. The addition of PEI appears to increase enzyme incorporation producing a biocatalyst with high encapsulation efficiency in terms of percentage of encapsulated protein (90%) and activity (47%). Furthermore, we observed that the addition of PEI resulted in a biocatalyst with high catalytic activity (600 U/g) and thermal stability (247-fold more stable than the soluble enzyme at 65 °C). The formation of silica particles was followed over time, showing a homogeneous incorporation of the enzyme in its active form. Intrinsic fluorescence spectroscopy suggested that the addition of PEI during the encapsulation process resulted in a conservation of the structural and functional features of the enzyme, suggesting a stabilizing role of the additive. The integration of the results of this work allowed proposing a polycondensation/enzyme encapsulation mechanism for silicabased sol-gel encapsulation mediated by PEI. These new evidences will contribute to the design of novel and more efficient silica-based biocatalysts achieved by sol gel encapsulation methodology.

1. Introduction The enzymes immobilization allows their thermal stabilization, resistance against chemicals or inhibitors, and the re-use in different process, as it has been shown in different reviews [1,2,3–5]. Spite the immobilization processes can change the selectivity and specificity of the enzyme [6], the positive effects for the enzyme activation [7,8] and purification [9] increase the interest in these processes. Silica sol-gel encapsulation has been widely used to immobilize a great variety of industrially relevant enzymes [10–12]. Enzyme encapsulation in silica by sol-gel is an one-step process, based on the condensation of the silica precursors around the enzyme molecules, at low temperatures [11,13], furthermore this process offers a immobilization strategy with high yield, low enzyme lixiviation, high mechanical stability and suitable

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catalytic properties (expressed activity, thermal stability). In this process, enzyme molecules are entrapped during silica sol (colloidal particles dispersed in a liquid medium) and gel formation (polycondensation). This polycondensation is modulated by temperature, pH, silica source, reaction medium, and the presence of additives, affecting the molecular structure of the enzymes entrapped into the silica and consequently, the enzyme activity of the generated biocatalyst [14–16]. In order to conserve the enzyme activity from the sol-gel conditions, the enzyme preinmobilization in an organic support was carried out [17]. However, the encapsulation in silica in one-step using additives to protect the enzyme is simpler. Additives such as sugars, surfactants, and polyamines are described to modulate enzyme encapsulation, enabling high immobilization yield, expressed activity, mechanical and molecular stability [18–22], taking

Corresponding author. E-mail addresses: fl[email protected] (F. López-Gallego), [email protected] (M. Mesa).

https://doi.org/10.1016/j.mcat.2018.02.024 Received 27 September 2017; Received in revised form 20 February 2018; Accepted 21 February 2018 2468-8231/ © 2018 Elsevier B.V. All rights reserved.

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concentration of 0.2–4.7%v/v. The obtained silica sol was kept under orbital agitation (250 rpm) at 25 °C during 30 min. The gelation was carried out in sealed vessels, during 24 h at 50 °C. This temperature was settled at 50 °C based on preliminary assays of surface response analysis, as described in Supporting information. Finally, the obtained heterogeneous biocatalysts were thoroughly rinsed (three times) with 25 mM phosphate buffer solution (pH 7) in order to eliminated physisorbed molecules of enzymes and PEI. The samples were stored at 4 °C.

specially relevance the polyamines [22–24], since the primary amine groups from of this kind of molecules regulate the silicification process by catalyzing the hydrolysis and the polycondensation of silica species that triggers the particle aggregation [25]. One of the interesting polyamines is the polyethylenimine (PEI), which could improve the efficiency on enzymes encapsulation and the final expressed activity [26,27], especially for those enzymes with low content of e-amines (lysines), facilitating the incorporation of proteins during the encapsulation [25]. In addition, the use of PEI enables the immobilization of proteins via anion exchange adsorption [26] and the stabilization of enzymes through PEI coating [28]. PEI also affects the microenvironment of immobilized enzymes [25]. On the other hand, the interaction between enzyme, siliceous species and additives influence the enzymatic behavior, since the entrapped protein by sol-gel process can exhibit a different performance from those on the native environment [15,29]. Hence, the development of robust biocatalyst depends on the understanding of conformational dynamics of silica-additives-enzyme and the accessibility of the entrapped proteins on siliceous matrix, whereby these should be closely monitored. Previous studies have mainly focused either on studying the effect of PEI on the morphological and textural properties of the siliceous matrix or their biocatalytic properties [30,31], whereas the relationship of both aspects has yet to be properly studied. In this work, the effect of PEI addition during sol-gel enzyme encapsulation was characterized using Lipase from Thermomyces lanuginosus (TLL) as a case study. TLL is an enzyme derived from a thermophilic microorganism that has shown optimal activity towards hydrolysis and esterification at temperatures between 40−60° C and a wide pH range in the basic region [32]. Key studied parameters were structure, material formation yield, protein yield and quality of the resulting biocatalysts. Enzyme incorporation and silica formation kinetics were followed in situ by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM), whereas enzyme structural changes were studied by UV and intrinsic fluorescence spectroscopy. The thermal behavior of the resulting biocatalysts was also assessed as a quality parameter of the process. The integration of the results of this work allowed to propose a polycondensation/enzyme encapsulation mechanism for silica-based sol-gel encapsulation mediated by PEI. This work contribute to the development of an one-pot encapsulation process in silica in the presence of PEI, avoiding the multistep pathway involved during postsynthesis immobilization pathways.

2.2.1.1. Activity assay. Enzymatic activity was measured by monitoring the release rate of p-nitrophenol using UV–vis spectroscopy at 348 nm after adding 50 μl of TLL suspension to 2.5 ml of 25 mM phosphate buffer with 20 μl of 50 mM p-nitrophenil butyrate (pNPB) substrate. The measurement was made at pH 7.0 and 25 °C under agitation, in a Perkin Elmer Lambda35 UV–vis Spectrophotometer. The expressed activity and recovered activity were the International Units per ml (U/ml) for the soluble enzyme (Ac) or per gram of support (U/g) for the encapsulated form (Ai) respectively. Here, 1 Unit (U) is defined as the amount of enzyme required to hydrolyze one micromole of pNPB per minute. 2.2.1.2. Encapsulation efficiency. The encapsulation yield in terms of activity (YA) was calculated by Eq. (1)

YA =

AI *100 Ac

(1)

Where AI is the expressed activity by the encapsulated biocatalyst per gram of support, and AC is the offered enzyme activity per gram of support. The immobilization yield in terms of percentage of encapsulated protein (YP) and mg of protein per g of support (mg/g) were calculated by Eqs. (2) and (3), respectively

YP =

QPT − QPs *100 QPT

mgprotein gsupport

=

QPT − QPs g

(2)

(3)

Where QPT is the total quantity total of offered protein, QPs is the quantity of protein in the supernatant and g support is the gram of support. The protein was measured by the Bradford method [33]. 2.3. Characterization of the biocatalysts

2. Materials and methods

2.3.1. Thermal stability Inactivation profiles were obtained by incubation of biocatalysts under non-reactive conditions in 25 mM potassium phosphate buffer at pH 7.0 and 65 °C. Samples were withdrawn at different times and residual enzyme activity was measured. The inactivation curves were fitted to first-order deactivation models [34,35] by iterative non-linear regression method, using the Solver tool of Microsoft Office Excel. The stabilization factor (SF) was calculated by Eq. (4).

2.1. Materials Thermomyces lanuginosus Lipase (TLL, Sigma), Triton X-100 surfactant (Sigma), Potassium phosphate (Merck), Tetraethyl orthosilicate (TEOS, Sigma), p-Nitro phenyl butyrate (pNPB, Sigma), Acetonitrile (Merck), and Hydrochloric acid (Merck), Polyethylenimine (PEI, Mw:25000, Sigma), Dimethyl sulfoxide (DMSO, Sigma), Rhodamine B (Merck), 1-ethyl-3-(3-dimetilaminopropyl) carbodiimide (EDC, sigma), N-Hydroxysuccinimide (NHS, Sigma), and Fluorescein Isothiocyanate (FITC, Sigma) were acquired commercially. All reactants were used without previous purification.

SF =

t 1 encapsulated enzyme 2

t 1 soluble enzyme 2

(4)

Where the half-time (t1/2) is the time required for losing 50% of the initial activity of the encapsulated and soluble enzymes. To evaluate the structural changes of the TLL due to the thermal unfolding, the soluble and encapsulated enzymes were characterized by intrinsic tryptophan fluorescence measurements. This fluorescence was measured before and after the incubation of the biocatalysts (4 mg of protein per measurement) at 65 °C for 2 h under non-reactive conditions. The excitation was at 280 nm and emission was recorded from 300 to 400 nm using Varioskan TM Flash Multimode Reader (ThermoScientific).

2.2. Methods 2.2.1. TLL encapsulation In a typical procedure, 1 g of Tetraethyl orthosilicate (TEOS) was dissolved in 2 ml of HCl (approximately pH 3.5). This solution was mixed in a 1:1 vol ratio with a buffered enzymatic solution (phosphate buffer pH 7 25 mM until final pH 5.5), containing different concentrations of Thermomyces lanuginosus Lipase (TLL) enzyme (0.2–0.6 mg/ml) and 1 × 10−3 M Triton X-100. After that, PEI was added to a final 107

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2.3.2. Biocatalyst morphology The morphology of biocatalysts was determined by Scanning Electron Microscopy (SEM) with a JEOL JSM-6490LV SEM equipment. 2.4. Monitoring TLL encapsulation by confocal laser scanning microscopy (CLMS) 2.4.1. Encapsulation kinetic of the TLL on the silica matrix TLL labelled with FITC and further encapsulated in silica/PEI particles were analyzed by CLMS. FITC labelling was carried out by dissolving the protein in 0.1 M sodium bicarbonate solution at pH 9.5 and mixing with a solution of FITC in a 1:1 molar ratio (protein: FITC). The mixed solution was incubated for 1 h, in the dark, at 22 °C and under stirring. The excess of FITC was eliminated by ultrafiltration with Amicon® ultra centrifugal filters. The labelled protein was encapsulated following the procedure described above. Encapsulation kinetic was followed by measuring the fluorescence intensity of encapsulated enzymes at different times (0.5 h, 1 h, 6 h, and 24 h). Each sample was washed and resuspended in 25 mM buffer phosphate pH 7.0 in a 1:40 w: v ratio, and one aliquot of 200 μl was analyzed by CLMS at 480 nm (excitation) and 525 nm (emission). 2.4.2. Distribution of the TLL and PEI on the silica matrix For these experiments, encapsulation was performed using FITClabelled TLL and rhodamine-labelled PEI. PEI was labelled in a similar way described for the enzyme, using 0.2 g of PEI was dissolved in sodium carbonate buffer (20 ml, pH 9.5), and activating its carbonyl groups with 0.5 g 1-ethyl-3-(3-dimetilaminopropyl) carbodiimide (EDC) and N-Hydroxysuccinimide NHS (0.15 g), to attach the rhodamine B (0.15 g). Conditions were controlled and the reaction monitored with UV–vis spectroscopy to reach approximately 10% labelling efficiency, to avoid effects on the PEI interactions with the silica species during the encapsulation process.

Fig. 1. Immobilization yield (a), recovered activity and specific activity (b) for TLL/silicaPEI biocatalysts prepared in presence of PEI (0–4.7%v/v) at pH 5.5, 50 °C.

The recovered activity stopped to increase after 1% PEI meanwhile the specific activity was constant only at PEI concentrations higher than 3%. At the same time, a direct relationship between PEI concentration and quantity of silica (Table 1S) is clear, because PEI acts as catalyst for silica polycondensation [36], improving the quantity of the obtained solid biocatalyst. In the range of 1–3%PEI, both specific activity (Fig. 1b) and silica quantity (Table 1S) increase, therefore the recovered activity will be constant as it was calculated as the International Units per gram of support (U/g). Moreover, a full coating of TLL surface with PEI ≥ 3% could also explain the non-dependence between catalytic activity and PEI concentration. As summary, we observed an improvement on silica yield, enzyme load and recovered activity when the PEI is added in suitable proportion; however, the optimal PEI concentrations could be different for different enzymes especially the specific activity parameter [25]. The obtained values of recovered activity and immobilization yield are at this point competitive with values to those obtained with other immobilization methodologies of TLL [37,38], but with the advantages of the silica sol-gel process. By increasing the enzyme concentration, in presence of PEI (3%), both the recovered activity (U/gsolid) and the enzyme load (mgTLL/ mgsolid) is maximized when 0.4 mg/ml of TLL are in the sol-gel reaction mixture. It leads to a biocatalyst exhibiting 600 U/gsolid and 150 mgTLL/ gsolid (Fig. 2a). Regardless of enzyme concentration, immobilization yields in terms of activity (YA) and protein (YP) remain constant (47% and 90%, respectively, Fig. 2b). According to our results, the biocatalyst with the best balance between immobilization yield, recovered activity and specific activity was obtained using 0.4 mg/ml TLL, in the presence of 3% PEI at pH 5.5 and 50 °C during 24 h.

3. Results and discussion 3.1. PEI-mediated sol-gel TLL encapsulation in silica The encapsulation of TLL in presence of PEI was optimized, evaluating the effect of PEI and enzyme concentration on recovered activity and immobilization yield. The gelation for enzyme immobilization was carried out at 50 °C based on preliminary assays in the absence of PEI that shows that this temperature did not affect the catalytic behavior of the TLL (Fig. S1). These preliminary assays were analyzed by One Factor at a Time (OFAT) and Response Surfaces methods. The optimization of pH, temperature and offered enzyme during immobilization process permitted to establish that at 50 °C, 24 h of gelation, pH 5.5, and offered enzyme of 100 U, the TLL was effectively immobilized, with high expressed activity (125 U/g) (Fig. S1, a,b and c), but low immobilization yield (19%, Fig. S1, d,e and f). Taking into account that this temperature did not affect the catalytic behavior of the enzyme and, in order to improve the immobilization yield, we proposed to make the immobilization at this temperature and evaluating the effect of PEI and enzyme concentration on recovered activity and immobilization yield. Recovered activity was defined as enzyme activity of biocatalysts per gram of support, and immobilization yield in terms of activity (Eqs. (1)–(3). These parameters show how active is the biocatalyst and how efficient is the encapsulation process. When the encapsulation of TLL by sol-gel process was carried out in the absence of PEI, low recovered activity and immobilization yield are obtained (125 U/g and 19% respectively, Fig. 1). The addition of PEI increases immobilization yield, reaching maximum yield at 3% PEI by using 0.2 mg/ml TLL at 50 °C and pH 5.5 (YA, Fig. 1a). Further addition of PEI does not result in an increased immobilization yield. On the other hand, Fig. 1b shows the PEI has a positive effect on the recovered and specific activities in a narrow range of concentrations.

3.2. Morphological characterization of materials containing encapsulated TLL The effect of PEI during enzyme silica-based sol gel encapsulation was studied at the structural level. The morphology of biocatalysts 108

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3.3. Enzyme and PEI distribution within silica matrix The distribution of TLL and PEI on the silica matrix and the colocalization of both protein and polymer within the silica particles was studied by CLMS, using both FITC labelled TLL and rhodamine B- labelled PEI. Results show that in most cases, both TLL and PEI are coentrapped into the silica particles (Fig. 4a–b). It can be observed that both the cationic polymer and the enzyme interact inside the silica matrix and during the encapsulation process. This interaction leads to the formation of conjugates that are co-entrapped, but such polymerenzyme association is not observed in 100% of the sample (Fig. 4c). This experimental evidence offers interesting insights on encapsulation dynamics of TLL in presence of PEI, highlighting how the PEI-mediated biosilification occurs. 3.4. In situ monitoring of TLL silica sol-gel encapsulation by CLMS Enzyme distribution and material formation were monitored by CLSM over time during the encapsulation process. Silica formation is promoted by hydrogen bonds between silanol groups and deprotonated amines, favoring the precipitation of silica nanoparticles and entrapment of other species in solution [39]. Particle morphology and the distribution of the FITC-labelled TLL at different times across the formed silica particles are shown in Fig. 5. The formation of small particles (< 1 μm) is observed during the first 30 min. After 24 h, particles aggregate to form larger and denser amorphous particles (50–100 μm). The protein is homogeneously distributed across the particles at the different growth stages (Fig. 5). These results show that silica formation and enzyme incorporation occur simultaneously, indicating that TLL-PEI conjugates interact with silica species during the polycondensation process. It is possible that TLL-PEI conjugates, TLL, and PEI species interact with silica monomers leading to the nucleation process, which was also observed by SEM (Fig. 3) and CLMS images (Fig. 4).

Fig. 2. Effect of concentration of offered enzyme on the encapsulation of TLL in presence of PEI. (a) Recovered activity and Enzyme loading (b) Immobilization yields in terms of activity (YA) and protein (YP) for TLL/silica-PEI biocatalysts.

prepared using TLL-PEI/silica conjugates (with 3% PEI) differ from those prepared using only TLL/silica (Fig. 3). The biocatalyst obtained through PEI addition exhibits small aggregates of spherical and porous particles formed through anisotropic growth (Fig. 3a), while the material prepared in the absence of PEI is more compact and its morphology is less homogenous (Fig. 3b). These results can be explained because the PEI catalyzes the polycondensation of silica species, [39] as it is corroborated by the amount of formed silica (70 mg in presence and 40 mg in absence of polyethylenimine, supporting information Table S1).

3.5. Structure-function relationship study of biocatalysts The encapsulation in the absence of PEI leads to biocatalyst with low recovered activity and immobilization yield (Fig. 1a–b). Nevertheless, the addition of 3% of PEI results in a biocatalyst with high recovered activity and yield immobilization (600 U/g and 47% respectively). It is possible that these differences are associated to structural features of the enzyme, due the presence of PEI during the encapsulation process. Changes in intrinsic fluorescence of soluble and encapsulated TLL,

Fig. 3. SEM images for (a) TLL/silica-PEI in presence of 3% PEI and (b) TLL/silica biocatalysts prepared at pH 5.5, 50 °C and 0.4 mg/ml enzyme.

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Fig. 4. CLMS images of TLL/silica-PEI showing the distribution of the (a) Bright field (b) FITC-labelled enzyme, (c) rhodamine B-labelled PEI and (d) superposition of A and B.

Remarkably, TLL encapsulated in presence of PEI shows a half-life 247 times higher than that of the soluble enzyme at 65 °C and, also higher than the TLL/silica biocatalyst prepared in the absence of PEI, corroborating the positive effect of PEI (Table 1). The high thermal stabilization of TLL entrapped into the silica matrix may relate to the structure changes and chemical environment of the enzyme trapped in the silica matrix. These results are comparable with TLL biocatalysts prepared by adsorption (SF: 125 at 60 °C) [40], or covalent immobilization methodologies (SF=21 at 70 °C) [41]. However, the biocatalysts achieved in this work exhibit the advantages of encapsulation by sol-gel process (fast and simple immobilization method). The analysis of the intrinsic fluorescence, performed after 2 h of incubation at 65 °C, shows that the signal intensity for the soluble enzyme decreases 7.0% after incubation at same temperature (Fig. 6a), which suggests that structural changes may occur during the thermal incubation. A similar behavior is observed for the TLL/silica biocatalyst (Fig. 6b). These results are in accordance with the short t1/2 values measured for these biocatalysts (Table 1). Conversely, the fluorescence intensity for the TLL/silica-PEI biocatalyst remains constant (Fig. 6c) but the λmax blue-shifts 5 nm. These data indicate that encapsulation in presence of PEI could prevent protein unfolding, although some structural rearrangements still occur. Therefore, the encapsulation of TLL with cationic polymers possibly preserves the native conformation of this enzyme, although the conformational changes triggered by the thermal incubation at short times could generate a more active TLL

by excitation of the tryptophan (Trp) residues in the TLL structure (8 residues) allows to identify effects of encapsulation on the structure of the enzyme (Fig. 6). These analyses were made before and after heating at 65 °C for the soluble and encapsulated TLL. The emission spectrum (taken before thermal treatment) for the encapsulated TLL in the absence of PEI shows that the process generates conformational changes on the enzyme structure, as evidenced by the dramatic decrease on emission intensity, the blue-shift of the maximum and the widening of fluorescence signal, compared to the spectrum for the soluble enzyme before heating (Fig. 6b). On the other hand, the dramatic changes observed in the fluorescence spectrum after the encapsulation without PEI can explain the low recovered activity of such encapsulated enzymes. These changes suggests that the enzyme could be partially unfolded during the sol-gel process, which deteriorates the activity of the encapsulated enzymes (Fig. 1). On the contrary, when PEI is incorporated, the fluorescence spectrum presents similar width, the same maximum emission wavelength (λmax = 330 nm) but with a maximum intensity 1.2 times lower than for the soluble enzyme (Fig. 6C). These results indicate that the presence of PEI helps to preserve the TLL structure during encapsulation process and allow obtaining a biocatalyst with high recovered activity (Fig. 1). The obtained results are also related with the effect of PEI on the thermal stability of the encapsulated TLL which was studied by comparing residual activity of TLL/silica and TLL/silica-PEI biocatalysts incubated at 65 °C and pH 7.0, under non-reactive conditions.

Fig. 5. CLMS images of the TLL/silica-PEI during different stages of encapsulation, in 25 mM buffer phosphate pH 7.0 in a 1:40 w: v ratio at 480 nm (excitation) and 525 nm (emission).

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Fig. 6. Intrinsic Tryptophan fluorescence spectra for the (a) soluble (b) TLL/silica and (c) TLL/silica-PEI biocatalysts before (solid) and after (dashed) heating at 65 °C for 2 h.

the enzyme during encapsulation both functionally and structurally, evidenced by an increased recovered activity in the resulting heterogeneous biocatalyst. PEI coating prevent the enzyme surface to directly interact with the sol-gel, protecting the native structure of TLL and consequently stabilizing it against increased temperature. Other authors have been probed that enzyme-PEI electrostatic interactions are enough strong, that can modify and improve the biocatalytic behavior of enzyme immobilized by post-synthesis processes after the PEI excess was watched with water [43–45]. On the other hand, the incorporation of the PEI in siliceous matrix have been probed in silicas synthesized by biomimetic synthesis, where the PEI induces the silicification process. These authors had experimental evidences, from different techniques, showing the hybrid nature of the PEI/silica materials [36,46], which inspire us to use this PEI, under similar conditions, where the enzyme and PEI could be co-encaspulated during the silica matrix formation. Considering the findings of this work, a TLL/silica-PEI encapsulation mechanism through a sol-gel process is proposed (Fig. 7): during the encapsulation process the enzyme is surrounded by PEI, establishing electrostatic interactions between the carboxyl residues of the enzyme (Isoelectric point 4.5) and the protonated primary amine groups of the PEI (pKa 7.6) [47]. These TLL-PEI conjugates interact with the silica species, forming nucleation points. Subsequently, the PEI triggers and catalyzes the silica growth by forming hydrogen bonds between the amine groups of the polymers and silanol groups of the silica species [48]. Finally, the resulting hybrid nanoparticles start growing over time through a polycondensation reaction that forms larger particles [49] which encapsulate active enzymes resulting in a stable heterogeneous biocatalyst. The suggested catalytic role of PEI in the sol-gel process is supported by the higher mass yield of the

Table 1 Half-live (t1/2) and stabilization factors (SF) for soluble and encapsulated biocatalysts at 65 °C and pH 7.0. The t1/2 were calculated from the deactivation curves reported in Fig. S2 (Supplementary information). Biocatalysts

t1/2 (h)

SF

Soluble TLL/silica TLL/silica-PEI

0.3 26.7 74.2

– 92 247

conformation that may explain the hyperactivation observed after short incubation times. These results evidence a correlation between functional and structural observations for TLL sol gel encapsulation.

3.6. Role of polyethylenimine Considering the role of PEI in the biosilification process proposed by Siddharth V. Patwardhan et al. and Betancourt L. et al. (polyamines facilitate the condensation of silica species), the reports in literature proposing amine polymer additives as promising candidates for enzyme encapsulation [42], and the results obtained in this work, it is possible to suggest that the PEI has an important role during silica-based sol gel TLL encapsulation: (1) The morphology and compactness of silica particles grown with or without PEI is remarkably different (Figs. 3 and 5), and the mass yield of the resulting sol-gel silica (Table S1, supporting information); (2) The incorporation of enzyme into the silica particles correlates with the amount of PEI used, affecting protein load, immobilization yield (YP, YA Fig. 2b) and; (3) PEI appears to stabilize

Fig. 7. Scheme of the proposed mechanism for PEI-mediated silica-based sol gel encapsulation of TLL.

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polycondensation reaction at higher PEI concentrations under otherwise identical reaction conditions.

[13]

4. Conclusions

[14] [15]

In this work, the optimization of the silica-based sol-gel encapsulation of Thermomyces lanuginosus lipase mediated by polyethylenimine was achieved while studying the role of the additive polyethylenimine (PEI) in the polycondensation process. An optimal encapsulation occurs in the presence of 3% PEI, at pH 5.5 and 50 °C, achieving high encapsulation efficiency, high recovered activity and high thermal stability at 65 °C. The morphology of the biocatalysts (Thermomyces lanuginosus lipase – polyethylenimine – silica particles) was characterized by SEM and the spatial localization of both enzyme and polyethylenimine was determined by CLSM. The formation of silica particles was followed over time, showing a homogeneous incorporation of the enzyme in its active form. Intrinsic fluorescence spectroscopy suggested that the addition of PEI during the encapsulation process resulted in a conservation of the structural and functional features of the enzyme, suggesting a stabilizing role of the additive. The integration of the results of this work allowed to propose a polycondensation/enzyme encapsulation mechanism for silica-based sol-gel encapsulation mediated by PEI. We hope that these new evidences help to elucidate sol gel encapsulation mechanisms and contribute to the design of novel and more efficient silica-based biocatalysts.

[16] [17]

[18]

[19]

[20]

[21]

[22] [23]

Acknowledgments

[24]

We thank Universidad de Antioquia for the financial support, through the 2014-622 CODI Project: “Estudio de los factores involucrados en la preparación de biocatalizadores heterogéneos de lipasa de Thermomyces lanuginosus mediante inmovilización por adsorción y encapsulación en soportes silíceos porosos” and the scholarship program “Becas Doctorado Universidad de Antioquia 2013”. We also thank IKERBASQUE foundation for funding Dr. F. López-Gallego and the support of COST Action CM1303 Systems Biocatalysis.

[25] [26] [27]

[28]

Appendix A. Supplementary data

[29]

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.mcat.2018.02.024.

[30]

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