Wrinkled Silica Nanoparticles: Efficient Matrix for Î²-Glucosidase

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Wrinkled Silica Nanoparticles: Efficient Matrix for #-Glucosidase Immobilization Valeria Califano, Filomena Sannino, Aniello Costantini, Joshua Avossa, Stefano Cimino, and Antonio Aronne J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.8b00652 • Publication Date (Web): 23 Mar 2018 Downloaded from http://pubs.acs.org on March 25, 2018

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The Journal of Physical Chemistry C is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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The Journal of Physical Chemistry

Wrinkled Silica Nanoparticles: Efficient Matrix for βglucosidase Immobilization Valeria Califano,a Filomena Sannino,b Aniello Costantini,c* Joshua Avossa,c Stefano Cimino,d Antonio Aronnec a

Istituto Motori-CNR, via G. Marconi 4, 80125 Napoli, Italy

c

Department of Agricultural Sciences Università degli Studi di Napoli Federico II, Via

Università 100, 80055 Portici (Na), Italy b

Department of Chemical Engineering, Materials and Industrial Production, Università

degli Studi di Napoli Federico II, P.le Tecchio 80, 80125 Napoli, Italy d

Istituto Ricerche Combustione CNR, P.le Tecchio 80, 80125, Naples Italy

*Corresponding authors email: [email protected] , tel: +390817682596

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ABSTRACT: β-Glucosidase (BG) was immobilized by adsorption on wrinkled silica nanoparticles (WSNs) giving an active and stable biocatalyst for the hydrolysis of cellobiose. WSNs exhibiting both a central-radial pore structure and a hierarchical trimodal micro/mesoporous pore size distribution were synthesized. They were used as matrix to immobilize BG, obtaining a biocatalyst (BG/WSNs) containing 150 mg of enzyme for gram of matrix. A complete textural and morphological characterization of BG/WSNs performed by Brunauer–Emmett–Teller (BET) method, Thermogravimetric (TG), Fourier Transform Infrared (FT-IR) and Transmission Electron Microscopy (TEM) analyses showed that this matrix can generate a microenvironment particularly suitable for this enzyme. The immobilization procedure used allowed preserving most of the secondary structure of the enzyme and, consequently, its catalytic activity. The kinetic parameters of the cellobiose hydrolysis performed with the biocatalyst were determined and compared with those of the free enzyme. It was found that the apparent KM value of the biocatalyst was slightly lower than that of the free enzyme, indicating that the enzymesubstrate affinity was increased. A complete hydrolysis of cellobiose was observed for four consecutive runs, showing a high operational stability of the biocatalyst.

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1.

INTRODUCTION

In the past few years, the exploitation of lignocellulosic biomass has attracted great attention for its role in the production of biofuels and chemicals and its potential as a promising alternative, at low environmental impact, to reduce dependence on fossil fuel reserves.1 The enzymatic hydrolysis of cellulosic polymers and the consequent glucose fermentation to generate bioethanol is an ecologically sustainable and cost-effective process. In particular, the bioconversion of cellulose involves a set of enzymes, collectively referred to as cellulase, which act sequentially and synergistically:2 endo1,4- β glucanases EC 3.2.1.4; exo-1,4-β-glucanase (EC 3.2.1.91) and β-glucosidase (EC3.2.1.21). β-Glucosidase (BG) plays a key role in the enzymatic degradation of cellulose, hydrolyzing cellobiose to two glucose molecules.3 Since cellobiose acts as inhibitor for both endo and exo-glucanase activities, its hydrolysis is the rate-limiting factor for the whole process of the enzymatic degradation of cellulose.4 Even if the enzymatic hydrolysis takes place under mild conditions, allowing a lower energy expense with respect to the chemical processes, the enzyme preparation cost is an issue, since recycling of free enzymes is not industrially possible.5 For this reason, enzyme immobilization has emerged as an alternative for improving enzyme stability.6,7 Protein immobilization methods can be classified into two main approaches: physical adsorption and covalent binding. Both approaches have their advantages and shortcomings.6,7 The advantage of adsorption is that cross-linking reagents or activation steps are not usually required. As a result, adsorption is cheap, easily carried out, and tends to be less destructive towards the enzyme than covalent binding.8 To facilitate the adsorption of the enzyme on the matrix, the matrix surface should have high affinity for the enzymes, leading to a high enzymatic activity. For this reason, the immobilization of enzymes on mesoporous silica offers many advantages.7,

9-12

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Mesoporous silicates offer high porosity, large surface area, easy functionalization and thermal, chemical and biological stability. Besides, they can promote enhanced enzyme stability, due to a limited exposure to environmental factors and to the constrains of polypeptide conformational freedom as a consequence of the interactions with the pore walls.7 β-glucosidase from almonds was physically immobilized on monomodal Santa Barbara Amorphous 15 (SBA-15) obtaining a catalytic activity similar to that of the free enzyme.13 Recently, spherical silica nanoparticles with central-radial pore structures have been synthesized (wrinkled silica nanoparticles WSNs).14, 15 These nanoparticles exhibit several ameliorated characteristics with respect to SBA-15: i) smaller particle size allowing easy dispersion and reducing diffusion limitation; ii) an open pore structure, in which the radial pore channel size increases going from the interior to the surface, enhancing the accessibility of such a big enzyme (67.5 KDa) inside the pores; iii) the large pore entrance reduces the pore block observed for SBA-15.16 WSNs were used as efficient matrix for the immobilization of lipase obtaining an oleic acid conversion rate higher respect to free enzyme.17 The better performance of the immobilize enzyme was supposedly due to the radially aligned mesopores of WSNs, allowing dispersing active catalytic sites on large internal surface and pores. In this work, β-glucosidase was immobilized by physical adsorption onto WSNs and its catalytic performance in the cellobiose hydrolysis was evaluated by a kinetic study. The results indicate that the immobilized enzyme activity is similar to that of the free enzyme, and the immobilization enhances the affinity between the enzyme and its substrate.

2.

MATERIAL AND METHODS

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2.1 Materials. Tetraethylorthosilicate (TEOS), cetylpyridinium bromide (CPB), urea, cyclohexane, iso-propanol, acetone, hydrochloric acid solution 37%, βglucosidase from almond (molecular weight 135000 Da for the dimer), citric acid, sodium hydroxide and glucose oxidase-peroxidase (GOD–POD) assay kit were purchased from Sigma-Aldrich (Milan, Italy). The activity of BG was ≥ 6 U/mg, where 1 U corresponds to the amount of enzyme that liberates 1 µmol glucose per minute at pH 5.0 and 37 °C (salicin as substrate). 2.2. WSNs Synthesis. Wrinkled silica nanoparticles were synthesized following the procedure of Moon and Lee,15 with a more accurate surfactant extraction procedure. First, 8.96 g of cetylpyridinium bromide and 5.38 g of urea were dissolved in 268 mL of water. Subsequently, 268 mL of cyclohexane and 8.24 mL of iso-propanol were added to the solution. TEOS was added dropwise to the stirred solution for a final volume of 24.0 mL. After continuous stirring for 30 min at room temperature, the reaction mixture was heated up to 70 °C and kept for 16 h at this temperature in a close system to avoid the evaporation of the solvent. Afterwards, the reaction mixture was centrifuged at 12000 rpm for 6 min, dispersed in acetone by sonication, then centrifuged again and dispersed twice in acetone : water = 2 : 1 volume ratio. The surfactant removal was obtained by rinsing the particles in 500 mL of ethanol and 40 mL of acid chloride solution for 24 hours. Then, the particles were centrifuged at 12000 rpm for 3 min and sonicated in ethanol for 8 times to remove small nanoparticles. Finally, the complete removal of the surfactant from silica-gel nanoparticles was obtained by rinsing in 500 mL of ethanol and 40 mL of hydrochloric acid solution for 24 hours and subsequent thrice washing in ethanol. 2.3. BG Immobilization Procedure. Adsorption was carried out by adding a colloidal solution of WSNs of 2 mg/mL (solution A) to a β-glucosidase 0.03 mM buffer solution at pH= 5 of (solution B). The volume ratio B: A was 0.25. The resulting 5



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mixture was kept under stirring overnight at room temperature. The nanoparticles containing BG (BG/WSNs) were removed by centrifugation at 11000 rpm for 10 minutes, washed twice with the buffer solution to remove from the solid support the enzyme that had not been adsorbed, and immediately used in the catalytic assays. 2.4. Structural Characterization. Thermogravimetric analysis (TGA) was performed in a thermogravimetric apparatus, TA Instrument Q600SDT, with a heating rate of 10 C/min under N2 atmosphere, between 25 and 900 °C. Samples of initial weight of about 10 mg were set in platinum pans. The morphology of WSNs and BG/WSNs was observed by transmission electron microscopy (TEM) (PHILIPS EM208S microscope equipped with a Mega View camera for digital acquisition of images). The textural properties of WSNs were determined by N2 adsorption at -196 °C with a Quantachrome Autosorb 1-C, after degassing for 4 h at 150 °C. The Brunauer– Emmett–Teller (BET) method was adopted for the calculation of the specific surface area, while pore size distribution was evaluated by means of Barrett-Joyner-Halenda (BJH) adsorption method for mesopores and Dubinin-Astakov (DA) method for micropores. Fourier transform infrared (FT-IR) transmittance spectra were recorded in the 4000-400 cm-1 range, using a Nexus FT-IR spectrometer equipped with a DTGS KBr (deuterated triglycine sulphate with potassium bromide windows) detector. A spectral resolution of 2 cm-1 was chosen and each spectrum represents an average of 32 scans, corrected for the spectrum of the blank KBr pellet. Samples for FT-IR analysis were prepared by mixing KBr and dried WSNs and BG/WSNs powders (0.5% wt) and pressing into pellets of 13 mm diameter. To minimize the water contribution at 1640 cm-1, the BG/WSNs in KBr pellet were kept in a dryer under slight vacuum overnight.

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FT-IR data analysis was performed by means of Grams32 software. The amide I band was analyzed in terms of a linear combination of Gaussian components. In the fitting, the number of components and the initial values of their peak positions were taken from the second derivative spectrum. The initial half-bandwidth was automatically generated according to the number of peaks. 2.5. Catalytic Assay. β-Glucosidase activity was determined in 80 mM citric acid/sodium hydroxide buffer (pH 5.0) at 50 °C using 8 mM cellobiose as substrate under gentle stirring. The amount of BG was 0.15 mg/mL for both free and immobilized BG. At fixed time (10, 30, 60, 90, 120 and 240 min) aliquots of the solution were withdrawn. The reactions were stopped by thermal inactivation of the enzyme, by heating the mixture at 100 °C for 10 min. In the presence of immobilized β-glucosidase (BG/WSNs), the samples were also centrifuged for 7 min at 11000 rpm. Each aliquot of the solution was frozen before glucose concentration analysis. Glucose (GO) assay kit was used for measuring glucose concentration. The amount of glucose was determined by incubating an appropriate amount of quenched reaction mixture (1mL), previously diluted, with 2 ml of glucose-measuring reagent at 37 °C for 30 min, based on the D glucose oxidase–peroxidase method.18 Absorbance (OD) was measured at 540 nm using a spectrometer (Perkin Elmer Instruments, Lambda 25 UV/Vis). Each experiment was performed in triplicate. The kinetic parameters were evaluated from the time course of the reaction using the integrated rate equation:19

1  Si  Vmax Si − S ln  = − t  S  KM t ⋅ Km

where Si and S are the initial concentration and the concentration at time t of cellobiose.

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By plotting the first term as a function of (Si-S)/t a straight line was obtained with slope -1/KM and intercept Vmax/KM. In literature19, this procedure is reported as an alternative to the one based on the initial velocity at several substrate concentrations. This methodology requires only one experiment. However, the experimental conditions have to be carefully controlled to avoid enzyme inactivation and inhibition by reactants and products. For these reasons, we chose cellobiose concentration 8 mM (cellobiose concentration higher than 10 mM is known to inhibit BG 20). Finally, immobilized BG was evaluated at 50 °C by carrying out the hydrolysis of cellobiose under standard assay conditions to explore the reusability. After each 24 h cycle, the mixture reaction was subjected to centrifugation at 11000 rpm for 7 min. The supernatant was analyzed for glucose concentration. The solid was washed with the buffer solution and reused, by dispersing it in a fresh cellobiose solution. The yield (mole of glucose / 2

initial mole of cellobiose) of the reaction promoted by the

immobilized enzyme after the first cycle was defined as the control and attributed a value of 1.

3.

RESULTS AND DISCUSSION 3.1.

Textural and Morphological Characterization. The BET surface area

and the pore size distribution of the synthesized WSNs were evaluated from the nitrogen adsorption–desorption isotherm (Figure not shown). The BET surface area is 580 m2/g, while the total pore volume is 1.72 cm3/g. The pore size distribution indicates the presence of mesopores in the range 5-50 nm, which corresponds to inter-wrinkled distances having a mean value of 12.2 nm. Furthermore, a second distribution is in the range of 2-4 nm, suggesting that these particles have a 8


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mesoporous structure in addition to wrinkles. Moreover, adsorption points acquired at low p/p0 (down to 10-5) indicate the occurrence of additional microporosity in the range of 1-2 nm. This indicates a hierarchical porous structure. This trimodal pore distribution was unexpected, as it was never observed adopting this synthesis procedure. This result could be due to a more accurate surfactant removal. Hierarchically porous systems are supposed to possess more advantages than monomodal porous systems.21 In particular; hierarchical micro/mesoporous structure can enhance diffusion of small molecules (i.e. glucose and cellobiose) because hierarchical combinations of multiple-scale pores would allow for accessible mass transport paths within inorganic networks. TGA analysis was carried out on dried samples WSNs, BG/WSNs, a mix of BG and WSNs (blended in the solid state) containing 15 wt% of BG, and lyophilized BG. Before the analysis, the first two samples (WSNs, BG/WSNs) were dried in air at room temperature for 48 h. The TGA curves and derivative spectra (DTG) are displayed in Figure 1. For sake of clarity only the TGA curves of WSNs and BG/WSNs are displayed in Figure 1a.

100

(a) Weigth loss (%)

95 (2) 90

85 (1) 80 0

200

400

600

800

Temperature (°C) 0,18 Derivative wt/T (%/°C)

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


T max=435

Tmax=235

0,12

(b)

Tmax=330 T max=290

0,06

T max=560

(1) (2) (3) (4)

0,00 200

400

600

800

Temperature (°C)

9



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Figure 1. (a) TGA curves of BG/WSNs (1) and WSNs (2) and (b) DTG of BG/WSNs (1), WSNs (2) lyophilized BG (3) and BG-WSNs solid mix (4). In the 25-180 °C range, a weight loss occurs for both WSNs and BG/WSNs biocatalyst related to water desorption. This effect is enhanced in the TG curve of WSNs (Tmax =135 °C) indicating its higher hydrophilicity with respect to the BG/WSNs biocatalyst. The second weight loss is more pronounced for the BG/WSNs (Tmax = 435 °C) than WSNs (Tmax = 560 °C) and it was related to different phenomena: the condensation of surface Si-OH to form Si-O-Si bonds for WSNs;22, 23 the degradation of the sample, i.e. the progressive deamination, decarboxylation and depolymerization arising from the breaking of polypeptide bonds for BG/WSNs biocatalyst.24 After the TGA runs, both samples appeared white and were made of pure silica, as ascertained by FT-IR (see later). Therefore, we have estimated the amount of BG adsorbed in the BG/WSNs sample as the difference of the total weight loss between the TGA curves displayed in Figure 1 (a) without considering the loss of water. It is equal to 150 mg/g of matrix. This value is the same of that obtained for a mix of BG and WSNs (blended in the solid state) containing 15 wt% of BG. Moreover, this result is congruent with the one reported in literature13 (140 mg/g) for the immobilization of β-glucosidase from almonds on mesoporous silica SBA-15 with a comparable surface area, carried out at a pH 5.5. The DTG of lyophilized BG shows two maxima at Tmax=235 °C and Tmax=290 °C, indicative of a distribution of conformations.24 The displacement to higher Tmax in the DTG of BG/WSNs (Tmax=330 °C and Tmax=435 °C) suggests that the thermal stability of the immobilized enzyme is increased with respect to the free one. This result indicates an interaction between the enzyme and the matrix through a densification of

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hydrogen bonds.24 The peak at Tmax=560 °C is related to transitions of the support (i.e. Si-OH condensation). TEM images of WSNs and BG/WSNs are displayed in Figures 2a and 2b, respectively.

Figure 2. TEM images of WSNs before (a) and after BG adsorption (b) at different magnifications. Scale bars = 200 nm (a, top), 100 nm (b, top) and 50 nm (a and b bottom. TEM images of WSNs disclose colloidal particles with diameters of 200-250 nm, whose internal morphology is composed by silica fibers or wrinkles coming out from the centre of the particles. The wrinkles spread uniformly in all directions, forming central-radial pores that widen radially outward. TEM images of BG/WSNs show that the diameters of the nanoparticles are similar, whereas their morphology is markedly different. The WSN appears composed by a dense dark silica core and a transparent, less dense wrinkled region. The images of the BG/WSN show that the dark region is much more extended, indicating a higher density in the wrinkle region, due to the presence of the protein. Although the wrinkled structure is no longer visible, a more 11



transparent outer crown can be seen, in which the protein can be present at lower concentration. Therefore, it can be inferred that the BG is in the interior of the nanoparticle and there is no evidence of its presence on the outer surface. In Figure 3a the FT-IR spectra of lyophilized BG (curve 1) BG/WSNs before (curve 2) and after (curve 3) a TG run are reported.

(1) BG (2) BG/WSNs (3) BG/WSNs 900°C in air

(a) Absorbance

1,0

(3)

0,5

(2) (1) 0,0 500

1000

1500

20 00

2500

3000

3500

4 000

-1

Wavenumber (cm )

0,15

(1) BG/WSNs (2) BG liophylized

(b)

0,20

(1) BG/WSNs (2) BG liophylized

(c)

0,15

0,10

Absorbance

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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(1)

0,10

0,05

(1) 0,05 (2)

0,00 1500

1600

1700

1800

(2) 0,00 2 500

-1

Wavenumber (cm )

3000

3500

40 00

-1

Wavenumber (cm )

Figure 3. FT-IR spectra of lyophilized BG, BG/WSNs and BG/WSNs after a TGA run (a), lyophilized BG and BG/WSNs in the 1480-1850 cm-1 range (b) and 2500-4000 cm-1 range (c).

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The spectra of BG/WSNs and BG/WSNs after the TGA run show bands related to Si-O-Si vibrational modes at 500 (δSi―O―Si), 800 (νsym Si―O―Si)

, with a shoulder at 1200 cm-1 (ν LO asym

Si―O―Si)

Si―O―Si)

and 1090 cm-1 (ν ΤΟ

asym

. The positions of these bands are

indicative of a dense silica network.25 The bands at 560 (δSi―OH) and 960 cm-1 (νSi―OH) indicate the presence of surface Si-OH groups.26,27 As expected, the absorption bands related to BG are seen only in the curve 2; in particular, the band at 1540 and 1650 cm-1 are related to the amide I and amide II bands, respectively.28,

29

Furthermore, in the

2750-3700 cm-1 range a broad band appears with features at 2850, 2920 and 2960 cm-1. These features are due to asymmetric and symmetric C-H stretching of methylene and methyl groups30 found in aliphatic side chains of the polypeptide, whereas the broad band is due to the overlapping of the protein backbone N-H vibration (amide A) and to the stretching modes of Si-OH and H-OH. The spectra of BG/WSNs and lyophilized BG are displayed in two ranges: the 1480-1850 cm-1 range, where amide I and amide II bands occur (Figure 3b) and in the 2500-4000 cm-1 range, where the amide A takes place (Figure 3c). The absorption bands related to the immobilized BG occur at about the same position of that of lyophilized enzyme, except for a slight broadening of amide I toward lower wavenumbers. The secondary structure of the enzyme was studied by the fitting of the amide I band

into

Gaussian

components.

During adsorption,

proteins

can

undergo

conformational changes due to either interaction with the matrix surface or proteinprotein interaction. This can cause denaturation and/or aggregation, which implies the formation of intermolecular hydrogen bonding (intermolecular β-sheets). FT-IR technique is very useful to establish the secondary structure of proteins, which is composed by different structural elements, such as α-helices, β-sheets, β-turns and disordered structures, kept together by hydrogen bonds. The polypeptide backbone gives rise to a series of vibrational bands, some of which are very sensitive to the unique 13



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molecular geometry and hydrogen bonding pattern of the various secondary structure elements.28 Amide I, due mainly to the C=O stretching vibration mode, is the most useful in determining the secondary structure of a protein, since the C=O stretching vibration frequency is very sensitive to the secondary structure where the carbonyl is located. The fitting procedure described in the experimental section was applied to both lyophilized and immobilized BG. In Figure 4 the experimental and calculated curves with best-fit Gaussian components of amide I band of BG/WSNs are displayed.

0.7

0

Figure 4. Experimental and calculated curves with best-fit Gaussian components of amide I band from BG/WSNs FT-IR spectrum. The experimental and calculated curves are fully superimposed as a consequence of the fit accuracy. The position and the assignment of the Gaussian components of the amide I band of the BG/WSN and of the lyophilized BG are compared with the literature data in table 1.31-33 The amount of the different secondary structure elements was estimated by the area underneath each Gaussian band/the total area underneath amide I band ratio (table 2).

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Except for a small amount of aggregation and the corresponding decrease of α-helices and increase of β-sheets, the conformational structure of BG is fundamentally preserved upon adsorption. However, since the biocatalyst was dried prior to FT-IR spectrum acquisition, this slight conformational rearrangement could be due to drying rather than to adsorption. Actually, this behavior is typical of proteins that have undergone a freezedrying process, where the removal of water molecules is believed to be at least partially responsible for protein aggregation.34 Table 1. Band Position and Assignment of the best-fit Gaussian Components of the Amide I band Literature31 BG/WSNs BG lyophilized Attribution 31, 32 1612 1615 Aggregates 1624 1626 β-sheets β-sheets 1632 1628 1629 1642 1637 1638 β-sheets 1651 1646 1647 α-helices 1657 1656 1653 α-helices/unordered 33 1667 1670 1667 β-turns β-turns 1673 1676 1680 1683 Aggregates 1690 1688 1688 β-sheets/turns

Table 2. The Amount (%) of the Different Secondary Structure Elements Structure Literature31 BG/WSNs BG lyophilized α-helices 34 20.4 28.9 β-sheets 30 34.2 13.9 β-turns 25 25.2 35.4 Unordered 11 10.8 16.4 Aggregates 9.4 5.4 Overall, the secondary structure of the adsorbed protein is better preserved than in its lyophilized form, where the conformational changes are nevertheless reversible. In addition, the lyophilized BG shows a certain degree of aggregation. This can be the origin of the higher temperature peak in the DTG curve of the free enzyme.

15



3.2. Kinetic Parameters and Reusability Study. Given the importance of BG’s conversion of cellobiose to the potential production of bioethanol, several authors have investigated the kinetics of cellobiose hydrolysis promoted by BG immobilized with several methods on non-porous35,

36

and porous matrices with different pore size and

surface areas.37-39 In the present work, the kinetics of the reaction promoted by the BG/WSNs system was studied in the cellobiose hydrolysis to glucose. The immobilized enzyme, like the free enzyme, followed a Michaelis-Menten behavior, as illustrated in Figure 5.

BG

10

Glucose Concentration (mM)

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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BG/WSNs

8

6

4

2

0

0

50

100

150

200

250

Time (min)

Figure 5. Time course of the enzyme catalyzed reaction: product concentration versus time of reaction catalyzed by free BG and immobilized BG. The experimental points were linearized following the integrated rate equation19 as specified in the experimental section. The linearized experimental data were nicely fitted with a straight line (correlation coefficient R= 0.99982 and 0.99963 for the immobilized and free enzyme respectively), from which Vmax and KM were obtained. 16


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The kinetic parameters are KM = 5.4 ± 0.2 mM and 4.3 ± 0.2 mM and Vmax= 43 µmol/min·mg and 41 µmol/min·mg for free and immobilized BG, respectively. The standard deviation calculated for the KM values, obtained by a statistical analysis of the data, confirmed that the KM values of the free and immobilized enzyme were statistically different. The Vmax did not vary after immobilization while the apparent KM slightly decreased, contrary to what reported in literature, where an increase in the KM value was often observed.35,

37-40

However, in some instances, a decrease of KM has been

reported.41, 42 A decrease of the apparent KM, indicates that the immobilized enzyme has either an apparently higher affinity for its substrate or that there is an interaction between the substrate and the matrix enhancing the substrate concentration near the active sites. Furthermore, the decrease of KM indicates that diffusion problems of the substrate are avoided, possibly due to the peculiar morphology of the matrix, composed by silica fibers spreading outward with an open porosity and a hierarchical porous structure. The time course of reaction promoted by immobilized BG (BG/WSNs) shows a slight decrease in the rate of the reaction after 60 min with respect to the free enzyme (BG) (Figure 5). It can be inferred that the reaction catalyzed by BG/WSNs is a little more inhibited, probably due to the accumulation of glucose inside the matrix. This indicates that diffusion limitations exist for the reaction product, possibly due to its interaction with the matrix. Operational stability of the immobilized biocatalyst was assessed by repeated use in several consecutive 24 h batch hydrolysis runs at 50 °C (Figure not shown). There was no loss of activity after three batch runs. In the fourth, the yield reduced to 0.8 and to 0.4 with the fifth reuse. A contribution to the decrease of the reaction yield could be due to the physical loss of small amounts of biocatalyst during the transfer procedures required for its reuse, as detected by visual inspection. 17



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4. CONCLUSIONS β-Glucosidase was successfully immobilized on wrinkled silica nanoparticles by adsorption. The conformational analysis performed pointed out a preserved enzyme native conformation. The kinetic parameters of BG/WSNs were determined in the hydrolysis of cellobiose to glucose and demonstrated that the immobilized BG had an apparent KM value slightly lower than the free enzyme, indicating an increased enzymesubstrate affinity. In conclusion, the current study resulted in the efficient entrapment of BG within nanoparticles with improved reusability, allowing the effective hydrolysis of cellobiose for the biofuel production.

Acknowledgments This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. The authors would like to sincerely express their appreciation to Prof. Dianna Pickens Centro Linguistico di Ateneo (CLA), Università degli Studi di Napoli Federico II, for the careful editing of the manuscript.

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REFERENCES 1. Cherubini, F. The biorefinery concept: Using biomass instead of oil for producing energy and chemicals. Energy Convers. Manage. 2010, 51, 1412–1421. 2. Taherzadeh, M. J.; Karimi, K. Enzyme-based hydrolysis process for ethanol from lignocellulosic materials: a review. BioResources 2007, 2 (4), 707-738. 3. Sørensen, A.; Lübeck, M.; Lübeck, P. S.; Ahring, B. K. Fungal beta-glucosidases: a bottleneck in industrial use of lignocellulosic materials. Biomolecules 2013, 3, 612-631. 4. Alftrén, J.; Hobley, T. J. Covalent immobilization of β-glucosidase on magnetic particles for lignocellulose hydrolysis. Appl. Biochem. Biotechnol. 2013, 169 (7), 20762087. 5. Addorisio, V.; Sannino, F.; Mateo, C.; Guisan, J. M. Oxidation of phenyl compounds using strongly stable immobilized-stabilized laccase from Trametes versicolor. Process Biochem. 2013, 48, 1174-1180. 6. Sheldon, R. A. Enzyme immobilization: the quest for optimum performance. Adv. Synth. Catal. 2007, 349, 1289-1307. 7. Tran, N. D.; Balkus, K. J. Perspective of recent progress in immobilization of enzymes. Catalysis 2011, 1, 956-968. 8. Jesionowski, T.; Zdarta, J.; Krajewska. B. Enzyme immobilization by adsorption: A review. Adsorption 2014, 20, 801-821. 9. Mateo, C.; Palomo, J. M.; Fernandez-Lorente, G.; Guisan, J. M.; Fernandez-Lafuente, R. Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microb. Technol. 2007, 40, 1451–1463.

19



Page 20 of 24

10. Magner, E. Immobilization of enzymes on mesoporous silicate materials. Chem. Soc. Rev. 2013, 42, 6213-6222. 11. Zhao, X. S.; Bao, X. Y.; Guo, W.; Lee, F. Y. Immobilizing catalysts on porous materials. Materials today 2006, 9 (3), 32-39. 12. Hartmann, M.; Jung, D. Biocatalysis with enzymes immobilized on mesoporous hosts: the status quo and future trends. J. Mater. Chem. 2010, 20, 844-857. 13. Gómez, J. M.; Romero, M. D.; Fernández, T. M.; García, S. Immobilization and enzymatic activity of β-glucosidase on mesoporous SBA-15 silica. J. Porous Mater. 2010, 17, 657–662. 14. Zhang, H.; Li, Z.; Xu, P.; Wu, R.; Jiao, Z. A facile two step synthesis of novel chrysanthemum-like mesoporous silica nanoparticles for controlled pyrene release. Chem. Commun. 2010, 46, 6783–6785. 15. Moon, D.; Lee, J. Tunable synthesis of hierarchical mesoporous silica nanoparticles with radial wrinkle structure. Langmuir 2012, 28, 12341-12347. 16. Gómez, J. M.; Romero, M. D.; Fernández, T.M.; García, S.; Díez, E. Immobilization of β-glucosidase in fixed bed reactor and evaluation of the enzymatic activity. Bioprocess Biosyst. Eng. 2012, 35, 1399-1405. 17. Pang, J.; Zhou, G.; Liu, R.; Li, T. Esterification of oleic acid with methanol by immobilized lipase on wrinkled silica nanoparticles with highly ordered, radially oriented mesochannels. Mater. Sci. Eng. C 2016, 59, 35-42. 18. Bergmeyer, H. U.; Bernt, E. Methods of Enzymatic Analysis. H.U. Bergmeyer, Ed., New York, Academic Press, 2nd Edition, 1974. 19. Illanes, A. Enzyme biocatalysis: principles and applications; Springer Science & Business Media, Amsterdam, 2008. 20. Woodward, J. Immobilized cellulases for cellulose utilization. J. Biotechnol. 1989, 11, 299-312. 20


Page 21 of 24 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


21. Du, X.; He, J. Hierarchically mesoporous silica nanoparticles: extraction, aminofunctionalization, and their multipurpose potentials. Langmuir 2011, 27, 2972–2979. 22. Pan, D.; Yuan, P.; Zhao, L.; Liu, N.; Zhou, L.; Wei, G.; Zhang, J., Ling, Y.; Fan, Y.; Wei, B.; et al. New understanding and simple approach to synthesize highly hydrothermally stable and ordered mesoporous materials. Chem. Mater. 2009, 21 (22), 5413–5425. 23. Mukherjee, I.; Mylonakis, A.; Guo, Y.; Samuel, S. P.; Li, S.; Wei, R. Y.; Kojtari, A.; Wei, Y. Effect of nonsurfactant template content on the particle size and surface area of monodisperse mesoporous silica nanospheres. Microporous Mesoporous Mater. 2009, 122, 168–174. 24. Dandurand, J.; Samouillan, V.; Lacoste-Ferre, M. H.; Lacabanne, C.; Bochicchio, B.; Pepe, A. Conformational and thermal characterization of a synthetic peptidic fragment inspired from human tropoelastin: signature of the amyloid fibers. Pathologie Biologie 2014, 62, 100-107. 25. Bloisi, F.; Califano, V.; Perretta, G.; Nasti, L.; Aronne, A.; Di Girolamo, R.; Auriemma, F.; De Rosa, C.; Vicari, L. R. M. Lipase immobilization for catalytic applications obtained using fumed silica deposited with MAPLE technique. Appl. Surf. Sci. 2016, 374, 346-352. 26. Aronne, A.; Marenna, E.; Califano, V.; Fanelli, E.; Pernice, P.; Trifuoggi, M., Vergara, A. Sol–gel synthesis and structural characterization of niobium-silicon mixedoxide nanocomposites. J. Sol-Gel Sci. Technol. 2007, 43 (2), 193–204. 27. Silvestri, B.; Guarnieri, D.; Luciani, G.; Costantini, A.; Netti, P. A.; Branda, F. Fluorescent (rhodamine), folate decorated and doxorubicin charged, PEGylated nanoparticles synthesis. J. Mater. Sci.: Mater. Med. 2012, 23, 1697-1704. 28. Barth, A. Infrared spectroscopy of proteins. Biochim. Biophys. Acta 2007, 1767, 1073-1101. 21



Page 22 of 24

29. Kong, J.; Yu, S. Fourier transform infrared spectroscopic analysis of protein secondary structures. Acta Biochim. Biophys. Sin. 2007, 39 (8), 549–559. 30. Long, G.; Ji, Y.; Pan, H.; Sun, Z.; Li, Y.; Qin, G. Characterization of thermal denaturation structure and morphology of soy glycinin by FTIR and SEM. Int. J. Food Prop. 2015, 18, 763–774. 31. Perez-Pons, J. A.; Pardos, E.; Querol, E. Prediction and Fourier-transform infraredspectroscopy estimation of the secondary structure of a recombinant β-glucosidase from Streptomyces sp. (ATCC 11238). Biochem. J. 1995, 308, 791-794. 32. Bruins, M. E.; Meersman, F.; Janssen, A. E. M.; Heremans, K.; Boom, R. M. Increased susceptibility of β-glucosidase from the hyperthermophile Pyrococcus furiosus to thermal inactivation at higher pressures. FEBS Journal 2009, 276, 109–117. 33. van Stokkum, I. H. M.; Linsdell, H.; Hadden, J. M., Hais, P. I.; Chapman, D.; Bloemendal, M. Temperature-induced changes in protein structures studied by Fourier transform infrared spectroscopy and global analysis. Biochemistry 1995, 34, 1050810518. 34.

Souillac,

P.

O.;

Middaugh,

C.

R.;

Rytting,

J.

H.

Investigation

of

protein/carbohydrate interactions in the dried state. 2. Diffuse reflectance FTIR studies. Int. J. Pharm. 2002, 235, 207–218. 35. Dekker, R. F. H. Application of a magnetic immobilized β-glucosidase in the enzymatic saccharification of steam-exploded lignocellulosic residues. Appl. Biochem. Biotechnol. 1990, 23, 25-39. 36. Romero, M. D.; Aguado, J.; Rodriguez, L.; Calles, J. A. Hydrolysis of cellobiose using β-glucosidase from Penicilliurn funiculosum kinetic analysis. Acta Biotechnol. 1999, 19 (1), 3-16.

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Page 23 of 24 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


37. Figueira, J. A.; Sato, H. H.; Fernandes, P. Establishing the feasibility of using β-glucosidase entrapped in lentikats and in sol−gel supports for cellobiose hydrolysis. J. Agric. Food Chem. 2013, 61, 626-634. 38. Verma, M. L.; Rajkhowa, R.; Wang, X.; Barrow, C. J.; Puri, M. Exploring novel ultrafine Eri silk bioscaffold for enzyme stabilization in cellobiose hydrolysis. Bioresour. Technol. 2013, 145, 302-306. 39. Ahmed, S. A.; El-Shayeb, N. M. A.; Hashem, A. M.; Saleh, S. A.; Abdel-Fattah, A. F. Biochemical studies on immobilized fungal β-glucosidase. Braz. J. Chem. Eng. 2013, 30 (4), 747-758. 40. Keerti, Gupta, A.; Kumar, V.; Dubey, A.; Verma, A. K. Kinetic characterization and effect of immobilized thermostable ߚ-glucosidase in alginate gel beads on sugarcane juice. ISRN Biochemistry 2014. 41. Iborra, J. L.; Castellar, M. R.; Cáinovas M.; Manjón, A. Picrocrocin hydrolysis by immobilized β-glucosidase. Biotech. Letters 1992, 14 (6), 475-480. 42. Fan, G.; Xu, Y.; Zhang, X.; Lei, S.; Yang, S., Pan. S. Characteristics of immobilised β-glucosidase and its effect on bound volatile compounds in orange juice. Int. J. Food Sci. Technol. 2011, 46, 2312–2320.

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TOC GRAPHIC β-glucosidase pH=5, 24h

Immobilized β-glucosidase

Wrinkled silica nanoparticles Cellulase

Cellobiose

Glucose

Cellulose

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Wrinkled Silica Nanoparticles: Efficient Matrix for Î²-Glucosidase

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