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Vol. 8. Issue 4.
Pages 3644-3652 (July - August 2019)
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Vol. 8. Issue 4.
Pages 3644-3652 (July - August 2019)
Original Article
DOI: 10.1016/j.jmrt.2019.06.002
Open Access
Clay/chitosan biocomposite systems as novel green carriers for covalent immobilization of food enzymes
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Ilaria Cacciottia, Claudio Lombardellib, Ilaria Benuccib,
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ilaria.be@unitus.it

Corresponding author.
, Marco Estib
a Department of Engineering, University of Rome “Niccolo Cusano”, Via Don Carlo Gnocchi 3, 00166 Rome, Italy
b Department of Agriculture and Forestry Science (DAFNE), Tuscia University, Via S. Camillo de Lellis snc, 01100 Viterbo, Italy
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Tables (2)
Table 1. Thermal properties of clay-free, OPT-70, OPT-80, SMP-70 and SMP-80 samples, related to the DSC first heating scan.
Table 2. Immobilization yield (IY), maximum velocity (Vmax), Michaelis–Menten constant (KM), turnover number (kcat) and affinity constant (Ka) of bromelain, immobilized on clay/CS composite systems loaded with different nanoclays (SMP or OPT) at various concentrations: 70, 75 and 80% w/w. Bromelain covalently linked on the clay-free chitosan carrier is reported as reference. Kinetic parameters were determined toward Bz-Phe-Val-Arg-pNA substrate (0–750μM), in model wine (tartaric acid/sodium tartrate solution 0.03M, pH 3.2, containing 12% v/v of ethanol at 20°C). Immobilized enzyme parameters are mean of triplicate measurements.
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Abstract

In this work, innovative green composite systems, based on high amounts of montmorrilonite (MMT) and low amounts of chitosan in different relative ratios (70:30, 75:25, 80:20, % w/w), were proposed as potential alternatives to the free nanoclay particles, commonly used as carriers for enzymes covalent immobilization. In details, two different MMT were selected, i.e. SMP (a high purity unmodified MMT) and Optigel (OPT, an activated food-grade MMT). The chitosan was used both to make the clays appropriate supports for the enzymes covalent binding, and to maintain the clay particles together in a unique structure, acting as a binder. The mechanical, thermal, and chemical properties of the obtained composite systems were studied. Moreover, the catalytic properties of a protease covalently immobilized on these carriers were investigated in a model wine-like medium toward a synthetic substrate. OPT based composite systems presented higher σmax and Young modulus values with respect to the SMP based ones, due to OPT better distribution and interaction with the chitosan. Irrespective of nanoclays amount, SMP and OPT composite systems appeared suitable carriers for the covalent immobilization of protease, with an immobilization yield of 18% and 14–17%, respectively. The highest product release velocity was detected when the protease was immobilized on OPT carriers (Vmax 10.49-10.74 mIUmg−1IP), and the greatest apparent affinity when it was linked to SMP composite systems (Ka 2.19–3.10min−1μM).

Keywords:
Clay
Chitosan
Food enzymes
Microstructure
Kinetic parameters.
Full Text
1Introduction

In recent years, clay minerals have been applied as attractive inorganic supports for enzyme immobilization [1], due to their low cost, chemical inertia, thermal stability, well-defined layered structure and ion-exchange ability [2]. Clay minerals, and smectites in particular, are characterized by high protein adsorption affinity due to the presence of exchangeable cations (i.e. Na+ or Ca2+) in the interlayer space. These cations can be replaced by positively-charged enzyme molecules through a cation exchange reaction [1].

For this reason, various authors have used Na-montmorillonite (Na-MMT) and Ca-montmorillonite (Ca-MMT) for the covalent and physical immobilization of enzyme molecules, such as catalase [3], glucoamylase [4], α-amylase [5], lipase [6], laccase [7] and invertase [8]. Despite non-covalent adsorption onto clay minerals be simple and cost effective immobilization technique, its main criticism is the limited stability of adsorbed enzymes, due to the weak interactions between enzyme and support, as well as to the environmental conditions, i.e. pH, temperature, ionic strength and biomolecule concentration [9].

The structural and chemical characteristics of layered clay minerals could be easily improved by the addition of organic and polymeric biomolecules. Among them, chitosan (CS) can be used to supply additional functional groups (i.e. hydroxyl and amino groups), in order to render the clays suitable supports for the covalent binding of enzymes. In this regard, α-amylase, β-amylase, glucoamylase and β-glucosidase have been covalently immobilized on clay/CS composites, showing an improved stability when applied at acidic pH [10,11].

Clay/CS composite membranes have been also investigated for application in pharmaceutical industry as controlled drug-delivery vehicles [12,13], and in food industry as packaging materials [14–16]. One of the first attempts to form an organo-mineral complex involving CS was carried out by Clapp and Emerson [17]. For this purpose, they used homoionic smectites (Ca-MMT) and found that the positively charged CS was adsorbed at the same extent as the most strongly adsorbed neutral species. The mechanisms that control smectite–biopolymer interactions depend on the nature of both the involved biopolymers and the clay mineral hosts. Given that diluted acid solutions are required to dissolve CS, this biopolymer behaves in solution as a positively charged polyelectrolyte, being able to intercalate MMT and other smectites by ion-exchange reactions [18]. The intercalation mechanism is mainly driven by electrostatic interaction of the positively charged amino groups of CS chains with the negatively charged sites in the clay mineral layers. Besides, hydrogen bonding and other types of interaction may contribute to the adsorption mechanism. As proved by Darder et al. [19] when the starting amount of CS is lower than the cationic exchange capacity of the clay, the intercalation of CS in MMT takes place in monolayer configuration.

In the present work SMP (a high purity unmodified MMT) and Optigel (OPT, an activated MMT) were selected, on the basis of previous results related to the production of CS nano-composite films loaded with different kinds of nanoclays (organomodified MMT, bentonite and sepiolite) in low concentration (1–5% wt) [20]. Indeed, among the tested nanoclays, SMP allowed to improve the mechanical properties of CS based films, whereas OPT appeared interesting as food-grade material, since it is the only nanoclay authorized to be used in contact with food. Thus, the novelty and significance of this paper consist in combining the selected MMT powders with low amounts of chitosan, in different relative ratios (MMT:CS 70:30, 75:25 and 80:20, % w/w), providing novel green carriers for the immobilization of food enzymes. Indeed, the chitosan addition allowed to make the clays appropriate supports for the covalent binding of enzymes, providing them with additional functional groups (i.e. hydroxyl and amino groups), and to maintain the clay particles together in a unique structure, acting as a binder. The obtained compact novel clay/CS composite systems were able to provide a favorable microenvironment for the enzyme molecules and allowing them the retention of catalytic activity [21,22].

The aim of the present study was to evaluate the potential application of the obtained clay/CS systems as novel carriers for enzyme covalent immobilization. The mechanical, thermal and chemical properties of the produced composite systems, as well as the catalytic properties of a proteolytic enzyme (bromelain from pineapple stem) covalently immobilized on these carriers, were investigated. Finally, biocatalysts were tested in model wine like medium toward a synthetic substrate.

2Materials and methods2.1Materials

Shellfish derived chitosan (CS) powder (low molecular weight 50–190kDa; percentage of deacetylation 75%), pineapple stem bromelain (BR, EC 3.4.22.32), glycerol (≥99.5%), glutaraldehyde (GDH, 25% v/v) were purchased from Sigma-Aldrich (Milan, Italy). Two different nanoclays, i.e. high purity unmodified montmorillonite (SMP) and activated food-grade montmorillonite (Optigel, OPT), were kindly provided by Zhejiang Fenghong New Material Co., Ltd (China) and BYK Additives GmbH (Germany), respectively. The selected tripeptide chromogenic substrate, i.e. Bz-Phe-Val-Arg-p-nitroaniline (pNA), was purchased from Bachem (Germany).

2.2Preparation of clay/CS composite systems by solvent casting

Composite systems were obtained by solvent casting method, using low molecular weight CS powder blended with glycerol in the weight ratio CS:glycerol 75:25, as described in literature [20,23]. Two different nanoclay types, SMP and OPT, were tested, in various w/w percentages with respect to the chitosan (i.e. 70, 75 and 80% w/w), obtaining three different compositions for each clay. Firstly, various amounts of nanoclays were ultrasonicated in aqueous solution (Ultrasonicator Sonics Vibracell CV33, 750W, 20kHz, amplitude 30%, time 30min) and then the proper contents of acetic acid (2% v/v), low molecular weight CS and glycerol were added; the obtained suspensions were magnetically stirred overnight. The CS solutions were laid down onto plastic Petri dishes, placed in a fume hood at room temperature for 48h to complete the solvent evaporation and the composite system formation. Furthermore, as a reference, a system composed of only CS and glycerol was produced following the same procedure. The obtained samples were designed as Clay-free for the CS/glycerol based film and SMP-x and OPT-x in the case of clay-loaded composites (where x is the percentage w/w of the used nanoclay).

2.3Characterization of clay/CS composite systems

To identify the functional groups of the produced samples, Fourier Transform Infrared (FT-IR) spectra were acquired by means of a FT-IR spectrometer (Jasco, FT/-6600) equipped with an attenuated total reflectance (ATR), in the following conditions: wavenumber range 600–4000cm−1, spectral resolution 4cm−1 and scans number 32.

The morphology of the obtained supports was investigated by Field-Emission Gun Scanning Electron Microscope (FEG-SEM, Cambridge Leo Supra 35, Carl Zeiss). The thermal properties of the prepared systems were studied by differential scanning calorimetry (DSC, TAInstruments Q2000) measurements that were performed under the following conditions: sample weight ∼5mg, nitrogen atmosphere (N2 flow rate 50cm3min−1), range temperature 25–400°C, heating rate 10°Cmin−1. Uniaxial tensile tests were performed on dog-bone specimens (width 4.8mm, length 22.25mm), following the ASTM D1708 and ASTM D882 standards, at 1.2mmmin−1, by an electromechanical apparatus equipped with a 50N load cell (Lloyd LRX Lloyd Instruments). Eight specimens were tested for each sample. All mechanical properties were calculated considering the nominal specimen cross-section.

2.4Enzyme covalent immobilization on clay/CS composite systems

Before the immobilization, clay/CS composite systems were neutralized by shaking overnight in 26% (v/v) ethanol–NaOH 2M solution, in order to render them water insoluble [24]. Membranes were then cut into squares (10mm×10mm) with a razor blade. The immobilization procedure was performed activating the surface of each clay/CS composite system square with 1mL of GDH (3% v/v), used as cross-linker. The suspension was kept under constant agitation for 2h at room temperature, and then profusely washed with distilled water. Thereafter, 1mL of stem BR preparation (2.2mg proteinmL−1), previously solubilized in the immobilization buffer (tartaric acid/sodium tartrate 0.03M, pH 3.2), was added to the activated clay/CS composite system square, shaking it (150rpm, at 20°C) overnight.

The applied immobilization procedure is based on the use of chitosan (as carrier) containing amino groups in its structure, and GDH (as cross-linker), which reacts with the amino group of chitosan to form a Schiff base. Thereafter, the covalent linkage occurs between the activated carrier and the amino groups of Lys residues in the enzyme structure [25].

At the end of the incubation time, samples were washed three times with immobilization buffer and then left to stand for 20min in a 0.1M glycine solution. At the end of the immobilization procedure, biocatalysts were washed three times with 2M NaCl solution to remove all non-covalently bound proteins and the composite systems were resuspended in the immobilization buffer. All supernatants were collected and diluted with buffer solution to a constant final volume to determine the bound protein. The immobilization yield (IY, %) was determined by Bradford's method [26], using Coomassie brilliant blue reagent and measuring absorbance at 595nm. Bovine serum albumin (BSA) was used as standard. The percentage of bound protein was indirectly determined as the difference between the amount of protein in solution before and after immobilization.

2.5Enzymatic activity determination

Proteolytic activity toward the selected substrate (Bz-Phe-Val-Arg-pNA) was tested in wine-like acidic medium, designated as model wine, which was a 0.03M tartaric acid/sodium tartrate solution, pH 3.2, containing ethanol (12% v/v). Protease activity was determined measuring the cleavage of the substrate, resulting in free pNA release, which was spectrophotometrically (UV-visible spectrophotometer, Shimadzu UV, Milan, Italy) detected evaluating the change in absorbance (at 410nm) vs time for 10min. Specific activity, calculated in mIU of pNA produced ɛ mM8.480mM−1cm−1 for pNA [27], was expressed as mIUmg−1 of immobilized protein (mIUmg−1IP). A blank correction was made using a sample that did not contain enzyme. All measurements were performed in triplicate.

2.6Kinetic study of immobilized protease

A kinetic study of pineapple stem bromelain immobilized on clay/CS composite systems, by means of GDH cross-linking, was carried out in model wine, fortified with Bz-Phe-Val-Arg-pNA substrate (0–750μM).

Kinetic parameters (KM, kcat and Ka=kcat/KM) were determined according to Michaelis–Menten equation, fitting experimental data by a non-linear regression procedure (GraphPad Prism 5.01, GraphPad software, Inc.). The goodness of-fit of each data set to its best-fit theoretical kinetic curve was assessed as the square of the correlation coefficient (R2).

Vmax represents the maximum velocity at which enzyme catalyzes the reaction, KM (Michaelis–Menten constant) is equal to the substrate concentration when the initial velocity is one-half of the Vmax, indicating the catalysis efficiency. kcat (turnover number) relates to the number of substrate molecules converted into product, in a unit of time, by an enzyme molecule. Moreover, affinity constant (Ka=kcat/KM) reflects the affinity of the enzyme toward substrate, and it refers on both reaction steps and expresses the overall catalytic efficiency.

2.7Statistical analysis

Data were analyzed by one-way completely randomized Analysis of Variance (ANOVA) with the EXCEL® Add-in macro DSAASTAT, followed by Tukey Honestly Significant Difference (Tukey HSD) post hoc test (P=0.05) for multiple comparisons of samples.

3Results and discussion3.1Microstructural, thermal and mechanical properties of chitosan/clay systems

The morphology of the produced systems was investigated by means of FESEM analysis. It is interesting to note that all systems based on OPT were characterized by a very uniform and homogeneous surface, whereas the SMP addition led to the presence of several agglomerates and defects (Fig. 1). The occurred immobilization of bromelain on the surface of the produced films was confirmed by observation at SEM. As an example, in Fig. 2, the SEM micrographs of SMP-70 and OPT-70, before and after the enzyme immobilization, are compared, evidencing the presence of a continuous and uniform organic layer on the film surface, ascribable to the enzyme immobilization.

Fig. 1.

FE-SEM micrographs of the plane view surfaces of OPT-70, OPT-80, SMP-70 and SMP-80 systems.

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Fig. 2.

FE-SEM micrographs of the plane view surfaces of OPT-70 and SMP-70 systems before and after bromelain immobilization.

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In order to investigate the possible CS/clays interaction, FTIR/ATR spectra were acquired on all produced systems. In Fig. 3 the ATR spectra of the clay-free film and of films loaded with 80% clays are compared, revealing the typical chitosan vibration modes [28] in all cases. In addition, the clay loaded films spectra reveal the typical vibrational modes of clays, including the characteristic absorption bands at ∼3622cm−1 (νOH,e stretching vibration of AlOH and SiOH), ∼3416cm−1 (νOH, stretching vibration of H2O), ∼1628cm−1 (δHOH, bending vibration of H2O), ∼1118cm−1 and at ∼980cm−1 (νSiO, stretching vibration of SiO), ∼912cm−1 (δAlAlOH, bending vibration of AlAlOH), ∼843cm−1 (δAlMgOH, bending vibration of AlMgOH) [29,30].

Fig. 3.

FTIR/ATR spectra of clay-free, OPT-80 and SMP-80 systems.

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The occurred electrostatic interaction between the chitosan NH3+ groups and the negatively charged sites of the clay [19] was corroborated by the identification of a slight shift of the peaks at 1574cm−1 toward a lower frequency (around 1554cm−1), in agreement with Silva et al. [31]. Indeed, this peak is ascribed to the vibrational modes of chitosan protonated amine group (δNH3)[19] and its shift toward lower wavenumbers is an indication of the ionic exchange between chitosan and montmorillonite and, thus, of the intercalation of chitosan within the montmorillonite structure. The secondary amide band (ν1) at 1645cm−1 of chitosan is overlapped with the δHOH bending vibration band at 1628cm−1 of the water molecules associated with the chitosan/clay films, which are present as in the starting clay, as expected for a biopolymer with high water retention capability [32,33]. Comparing the spectra of SMP and OPT based samples, it is possible to conclude that the interaction of the chitosan with both clays (SMP and OPT) is similar, as expected, due to the comparable chemical composition.

Furthermore, the influence of the nanoclay fillers on the chitosan thermal properties was investigated by means of DSC measurements. In Fig. 4 the DSC first heating run curves of all the chitosan based systems were compared. In most samples a broad endothermic peak was observed at approximately 105–120°C (Table 1), caused by the dissociation process of interchain hydrogen bonding of CS (TIHB) [34], as well as an exothermic band at approximately 285–315°C ascribed to CS decomposition [35–37]. It is interesting to note that the nanoclays based systems were characterized by a decreased hydrogen bonds dissociation temperature value with respect to the clay-free sample (121°C), as well as a decrement of the related enthalpies, particularly for SMP based samples. As regards to chitosan degradation temperature (Td), remarkably enhanced temperature values were detected for the composite systems compared to the clay-free sample (287°C), demonstrating that the nanoclays are able to act as thermal barrier, as expected.

Fig. 4.

DSC first heating curves of clay-free, OPT-70, OPT-80, SMP-70 and SMP-80 systems.

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Table 1.

Thermal properties of clay-free, OPT-70, OPT-80, SMP-70 and SMP-80 samples, related to the DSC first heating scan.

Sample  TIHB (°C)  ΔHmI (J/g)  Td 
Clay-free  121  259  287 
OPT-70  116  331  311 
OPT-80  118  442  306 
SMP-70  107  259  316 
SMP-80  106  293  313 

To analyze the mechanical properties of the obtained systems, uniaxial tensile tests were performed (Figs. 5–7). Higher σmax values were detected in the case of OPT based composite systems with respect to the SMP based ones (Fig. 5), due to its better distribution and interaction with the chitosan chains, as evident from the reported SEM micrographs (Fig. 1). Furthermore, in the case of OPT samples the σmax values were comparable independently of the OPT amounts, whereas in the case of SMP composites a decrement was observed with the increasing SMP percentage, due to the increment of the agglomerates. Similarly, Young modulus (E) appeared to be remarkably improved in the case of OPT based systems particularly for OPT-80 (Fig. 6). Consequently, a significant decrease in elongation at break was observed (Fig. 7), due to the presence of an inorganic component in very high concentration.

Fig. 5.

Ultimate tensile stress (σmax) of clay/CS composite systems obtained from chitosan films added with ultrasonicated clays at high contents (70, 75 and 80% (w/w) with respect to the chitosan). The clay-free chitosan carrier is reported as reference. Reported values are mean±95% confidence interval of triplicate measurements. The used roman letters (i.e. a, b, c, ab, bc and abc) indicate significant differences (Tukey's test, P<0.05) in the mechanical results among the investigated samples.

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Fig. 6.

Tensile modulus (E) of clay/CS composite systems obtained from chitosan films added with ultrasonicated clays at high contents (70, 75 and 80% (w/w) with respect to the chitosan). The clay-free chitosan carrier is reported as reference. Reported values are mean±95% confidence interval of triplicate measurements. The used roman letters (i.e. a, b, d, bc, cd and bcd) indicate significant differences (Tukey's test, P<0.05) in the mechanical results among the investigated samples.

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Fig. 7.

Elongation at break (ɛmax) of clay/CS composite systems obtained from chitosan films added with ultrasonicated clays at high contents (70, 75 and 80% (w/w) with respect to the chitosan). The clay-free chitosan carrier is reported as reference. Reported values are mean±95% confidence interval of triplicate measurements. The used roman letters (i.e. a, b, c, d, and bc) indicate significant differences (Tukey's test, P<0.05) in the mechanical results among the investigated samples.

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Based on the collected mechanical data (Figs. 5–7) and SEM micrographs of the sample surfaces (Fig. 1), it is possible to conclude that the used chitosan is able to act as binder and to entrap the dispersed inorganic nanoparticles, obtaining compact films. Moreover, the better mechanical behavior of OPT based composites could be ascribed to the higher compatibility between the used nanoclays and the chitosan, as corroborated by DSC data (Table 1), and by the different morphology and agglomeration tendency of the considered nanoclays. Indeed, both SMP and OPT are platelet-like particles, but OPT ones resulted homogeneously and better disagglomerated within the polymeric matrix (Fig. 1), suggesting a good wettability and compatibility, with consequent enhanced mechanical behavior.

3.2Kinetic properties of protease immobilized on chitosan/clay nanocomposite films

The addition of both nanoclays always affected the IY, resulting the amount of covalently bonded protein lower for all clay/CS membranes with respect to the clay-free carrier. Similar results have been reported by other authors [6], who proved that the addition of Na-bentonite into CS beads resulted in a lower immobilization yield for lipase. For both SMP and OPT composite systems, the percentage of immobilized proteins did not significantly change using different nanoclay concentrations, as testified by the comparable IY values (18% for SMP-composite system and 14–17% for OPT-composite system).

The kinetic study, carried out on bromelain covalently immobilized on the novel produced clay/CS composite systems, allowed us to investigate the effect of carrier modification (due to the addition of high nano-clay concentrations) on the catalytic properties of the reference enzyme. All the kinetics followed the hyperbolic behavior described by the Michaelis–Menten equation (Fig. 8) and the catalytic properties of biocatalysts are reported in Table 2.

Fig. 8.

Kinetic curves of bromelain immobilized on clay/CS composite systems loaded with different nanoclays (SMP or OPT) at various concentrations: 70 (●), 75 (□) and 80 (▴) % w/w. Bromelain covalently linked on the clay-free chitosan carrier is reported as reference. Proteolytic activity was determined toward Bz-Phe-Val-Arg-pNA substrate (0–750μM), in model wine (tartaric acid/sodium tartrate solution 0.03M, pH 3.2, containing 12% v/v of ethanol at 20°C).

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Table 2.

Immobilization yield (IY), maximum velocity (Vmax), Michaelis–Menten constant (KM), turnover number (kcat) and affinity constant (Ka) of bromelain, immobilized on clay/CS composite systems loaded with different nanoclays (SMP or OPT) at various concentrations: 70, 75 and 80% w/w. Bromelain covalently linked on the clay-free chitosan carrier is reported as reference. Kinetic parameters were determined toward Bz-Phe-Val-Arg-pNA substrate (0–750μM), in model wine (tartaric acid/sodium tartrate solution 0.03M, pH 3.2, containing 12% v/v of ethanol at 20°C). Immobilized enzyme parameters are mean of triplicate measurements.

Sample  IY (%)  Vmax (mIUmg−1IPKM (μM)  kcat (min−1Ka (min−1μM)  R2 
Clay-free  23a  3.91b  28.70b  139.00f  4.90a  0.99 
SMP-70  18bc  3.05b  45.01b  139.71f  3.10b  0.97 
SMP-75  18b  3.15b  65.75b  144.15e  2.19cd  0.96 
SMP-80  18bc  3.21b  59.95b  147.04d  2.45bc  0.98 
OPT-70  15c  10.74a  312.70a  555.84b  1.78cde  0.98 
OPT-75  14c  10.50a  351.60a  572.27a  1.63de  0.98 
OPT-80  17bc  10.49a  378.00a  495.15c  1.31e  0.98 

For each parameter, values with different roman letters are significantly different (Tukey's test, P<0.05).

Despite clay/CS composite systems approximately linked the same amount of protein, significant differences were revealed in the catalytic performance of biocatalysts. Protease on SMP membranes showed Vmax and KM values similar to those observed for the clay-free biocatalyst (Table 2). Contrariwise, bromelain on OPT carriers showed the highest Vmax and kcat. These values were about 3-fold and 3.5-fold higher (respectively) in comparison to the clay-free enzyme as well as the SMP composite systems, thus suggesting a greater product release velocity. An increased Vmax can be induced by conformational changes of biocatalysts, which can occur after the linkage to the support, as observed for other enzymes immobilized on clay/CS carriers, such as α-amylase and β-amylase [10]. However, bromelain on OPT composite systems showed a greater KM (about 10-fold higher than the clay-free biocatalyst and about 6-fold higher with respect to the SMP biocatalysts), thus indicating a lower enzyme–substrate complex formation. An increase of KM values can be caused by diffusion limitations or steric hindrance due to the clay mineral particles, as evidenced by other authors who immobilized enzymes on MMT or modified CS–clay supports [10,38,39]. Furthermore, for both SMP and OPT, the addition of various nanoclay amounts did not significantly affect either Vmax or KM.

Overall, the decrease of Ka revealed for all the biocatalysts on clay/CS membranes in comparison to the clay-free sample, indicated a lower apparent affinity of the protease for the synthetic substrate. These results could be ascribed to the increased rigidity of the carrier, which could limit the mobility of immobilized enzyme, as already proved [20]. Among the clay/CS biocatalysts, bromelain on SMP showed higher Ka values with respect to OPT composite systems. Comparing the two different nanoclay types, the greater apparent affinity of the protease for the synthetic substrate was revealed immobilizing bromelain on SMP-70 (Ka value 3.10min−1μM) or on OPT-70 (Ka value 1.78min−1μM).

4Conclusions

Clay/CS composite systems, based on low amounts of chitosan, used as binder, and high amounts of nanoclays, were produced using two different kinds of montmorillonite, i.e. SMP (a high purity unmodified MMT) and Optigel (an activated MMT), in order to propose them as novel carriers for the covalent enzyme immobilization. Compact systems were obtained in all cases, characterized by a very uniform and homogeneous surface for OPT based composites and by several agglomerates and defects for SMP ones. The occurred electrostatic interaction between the chitosan NH3+ groups and the negatively charged sites of the clay was demonstrated by means of FTIR/ATR investigation. Decreased hydrogen bonds dissociation temperature and remarkably enhanced chitosan degradation temperature values were detected for the composite systems compared to the clay-free sample, demonstrating that the nanoclays are able to act as thermal barrier. Higher σmax and Young modulus values were revealed in the case of OPT based composite systems with respect to the SMP based ones, due to OPT better distribution and interaction with the chitosan chains, as evident from the reported SEM micrographs.

Both SMP and OPT composite systems appeared to be suitable carriers for the covalent immobilization of bromelain and the addition of various nanoclay amounts did not affect neither the protein binding capacity nor the catalytic properties of the enzyme. Protease immobilized on OPT carriers showed the highest product release velocity, despite the fact that the apparent affinity was greater using SMP composite systems.

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgement

This work was supported by BioEnBi project “Biotecnologie enzimatiche innovative per processi di chiarifica sostenibili nel settore birrario” (Grant 85-2017-15362), funded by Lazio Innova Spa, Lazio Region (Italy) in the context of Progetti Gruppi di Ricerca, Lazio Innova 2018–2020.

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