Preparation of Zinc Tungstate ( ZnWO 4 ) Particles by Solvo-hydrothermal Technique and their Application as Support for Inulinase Immobilization

ZnWO4 particles were synthesized as a single-phase by a simple and easy solvo-hydrothermal technique using water-ethylene glycol mixture as solvent, without using surfactant. Physical properties of produced particles were analyzed by X-ray diffraction (XRD), infrared spectroscopy (FTIR), surface area (BET), particles size distribution and atomic force microscopy (AFM). This material was used as support for inulinase immobilization by physical adsorption and the influence of temperature (30 and 50 oC) was evaluated. Material with mesoporous characteristic and with a surface area of 35.5 m2.g-1 was obtained. According to the findings, ZnWO4 present a satisfactory inulinase adsorption, and the better result was 605 U.g-1 support at 30 oC. Therefore, ZnWO4 particles prepared by one-step solvo/ hydrothermal route provide a new potential support for inulinase immobilization.


Introduction
Metal tungstates with formula MWO 4 (where M is a divalent metal ion) are important ceramic materials that have high application potential in various technological fields 1,2 .Specifically zinc tungstate (ZnWO 4 ) have attracted attention due to its unique physical and chemical properties, possessing a high application potential in various fields, such as scintillator material 3 , photoluminescence 4 , electronic and optical properties 5 , photovoltaic property 6 , humidity sensor 7 , hydrogen sensor 8 , ether sensor 9 , photocatalyst 10 and high-power lithium-ion batteries 11 .In this work, a new application is proposed for the ZnWO 4 oxide, as support for enzymes immobilization.
The use of enzymes has been increased in the last years due the variety application such as food production, medicine, textile and pharmaceutical 12 .Immobilization presents some advantages such as lowering downstream purification requirements because the products are easily removed from the immobilized enzymes 13 and lowering the costs because enzyme can be reuse.Enzymes are supported in solid matrix for the immobilization by a variety of methods such as physical and chemical mechanisms 12 .The physical adsorption of enzyme 12,14 is entrapment on porous matrix, and in the chemical immobilization enzyme is attachment by covalent bonds 15 and cross-linking between enzyme and matrix 16 .The immobilization by adsorption usually preserves the catalytic activity of the enzyme 17 , therefore, sometimes during its use the immobilized enzyme can be lost when the interactions between adsorbent and enzyme are relatively weak 17,18 , and in this case, the support can be reused.Inulinases are enzymes useful on industrial processes, which can be applied for the production of sugars.It may produce high fructose syrups by enzymatic hydrolysis, and are used for the production of fructooligosaccharides, which are functional food ingredients.Inulinase has been immobilized by adsorption on different supports such as grafted alginate beads 19 , aminated non-porous silica 20 and chitin 21 .However, the inulinase immobilization using the ZnWO 4 oxide as support has not been explored yet.
In this context, we aimed prepare ZnWO 4 particles by one-step solvo-hydrothermal route and investigate their ability as support for inulinase immobilization.

Preparation and characterization of ZnWO 4
ZnWO 4 support was prepared by solvo/hydrothermal method using sodium tungstate (Na 2 WO 4 .2H 2 O) and zinc chloride (ZnCl 2 ) as starting materials.For the material synthesis, 0.14 g of sodium tungstate was dissolved in 20 mL of solution containing deionized water and ethylene glycol (1:1, v/v), under magnetic stirring by 30 min.The same procedure was taken to ZnCl 2 , however using 0.13 g.The sodium tungstate solution was added into the zinc chloride solution under magnetic stirring.Then the resulting homogeneous solution was transferred into Teflon-lined stainless-steel autoclave.This autoclave was sealed and maintained at 180 ºC for 24 h and then cooled to room temperature.The obtained white powers were collected and washed with deionized water and ethanol for several times to remove impurities, and then the product was dried at 110 ºC for 4 h.
ZnWO 4 particles were characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), BET surface area measurement, particles size distribution and atomic force microscopy (AFM).X-ray diffraction patterns were obtained using a Rigaku Miniflex 300 diffractometer.The X-ray source was Cu-Kα radiation, powered at 30 kV and 10 mA.Data were collected over the 2θ range 10-70º with a step size of 0.03º and a count time of 0.9 s per step.By means of infrared spectroscopy, Fourier transform infrared spectra (FTIR) for all samples pressed into KBr pellets (10 mg zinc tungstate/300 mg KBr) were recorded by a Shimadzu IR-Prestige-21 spectrometer.IR spectra were measured in the range 3700-475 cm -1 .Nitrogen adsorption-desorption isotherms were obtained from nitrogen adsorption isotherms at 77 K, carried out on an ASAP 2020 apparatus at relative pressure (P/P 0 ) ranging from 0 to 0.99.The particle size distribution of sample was measured using a laser particle size analyzer (Mastersizer 2000).The morphology of particles was examined by atomic force microscopy (AFM) (Agilent Technologies 5500 equipment).Before analysis, the sample was sonicated in acetone for 15 min to break up the possible agglomerates, and then dropped onto a freshly cleaved mica substrate.AFM image was acquired at room temperature, in non-contact mode using high resolution probes SSS-NCL (Nanosensors, force constant = 48 N.m -1 , resonance frequency = 154 kHz).Image was captured and analyzed using PicoView 1.14.4 software (Molecular Imaging Corporation, USA).

Enzyme immobilization assays
Adsorption experiments were carried out to investigate the inulinase immobilization from aqueous solution.Commercial inulinase was obtained from Aspergillus niger (fructozyme, exo-inulinase EC 3.2.1.80and endo-inulinase EC 3.2.1.7)was purchased from Sigma-Aldrich.The influence of temperature (30 and 50 o C) on the immobilization process was investigated.The adsorption of inulinase was performed using a batch technique.Typically, ZnWO 4 (0.025 g) were placed in Erlenmeyers flasks containing of inulinase solution (1.3 % v/v) in sodium acetate buffer (pH 4.8) and 1:400 of adsorbent:adsorbate ratio.The resulting solution was maintained under agitation (150 rpm), and then an aliquot of the aqueous solution was taken at various time intervals and filtered through a polyvinylidene difluoride (PVDF) membrane (0.22 μm) before analysis.The inulinase activity in the aqueous solution was determined according to section 2.3.

Inulinase Activity Assay
An aliquot of the enzyme (0.5 mL) was incubated with sucrose solution (4.5 mL, 2% w/v) in sodium acetate buffer (0.1 M, pH 4.8) at 50 °C.Released reducing sugars were measured by the 3.5-dinitrosalicylic acid method 36 .A separate blank was set up for each sample to correct for the non-enzymatic release of sugars.One unit of inulinase activity was defined as the amount of enzyme necessary to hydrolyze 1 µmol of sucrose per minute under the mentioned conditions (sucrose as a substrate).The inulinase immobilization capacity (Q t ) was determined using the Equation (1).

] g
Where: A o and A t (U.mL -1 ) are the inulinase activities at t = 0 and time t, respectively; V (mL) is the volume of solution, and m (g) is the mass of support.

Results and discussion
The XRD pattern of the ZnWO 4 prepared through solvo/hydrothermal process is shown in Figure 1.The XRD peaks of ZnWO 4 sample can be assigned to monoclinic where D is the average crystallite size, K is the Scherrer constant (0.90), λ is the wavelength of the X-ray radiation (0.1541 nm for Cu-Kα ), h 1/2 is the peak width at half height and θ corresponds to the peak position (in the current study, 2θ = 30.44o ).The average crystallite size of ZnWO 4 sample was 11 nm.
The nitrogen adsorption-desorption isotherms and pore size distribution corresponding to ZnWO 4 support are depicted in Figure 2. The nitrogen adsorption-desorption isotherms (Fig. 2a) showed weak adsorption at low relative pressure and a H1-type hysteresis loop at higher relative pressure (P/P 0 = 0.70-0.90).This suggests that the material presents mesoporosity, which can be attributed to the interparticle pores due to the crystallites agglomeration.According to the IUPAC classification, the isotherms are type IV and typical of mesoporous solids.Pore size distribution (Figure 2b) consisted of one wide peak centered at around 15 nm.The Brunauer Emmett-Teller (BET) surface area, average pore size and total pore volume of the ZnWO 4 sample were 35.5 m 2 .g - , 12.4 nm and 0.112 cm 3 .g - , respectively.These  physical characteristics regarding the pores structure are essential for immobilization purposes by adsorption process.
The particle size distribution pattern for the zinc tungstate obtained by the solvo-hydrothermal technique was expressed on a logarithmic scale, as shown in Figure 3.The particles size for the oxide sample range between 0.30 µm and 240 µm, resulting in an average size of 26 µm.These particle sizes in micrometric scale can explain the mesoporosity of material due to a variety of accumulated pore voids among the particles formed by the agglomeration of crystallites.Thus this mesoporous structure can be interesting for enzyme immobilization purposes.
Figure 4 shows the morphology of some isolated particles of ZnWO 4 oxide measured by atomic force microscopy (AFM).The average size of particles was around 0.30 µm.Also, it is possible to observe that the particles present an irregular shape.In order investigate the immobilization of enzyme on the ZnWO 4 support, FTIR spectra of ZnWO 4 support, immobilized enzyme on the support and free enzyme were recorded (Figure 5).The ZnWO 4 support (Figure 5a) shows main absorption bands between 475 and 1000 cm −1 38 .The bands to 820 and 880 cm −1 are due to the stretching modes of W-O bonds.The bands at 600 and 700 cm −1 are assigned to Zn-O-W bonds.Bands at 1600 and 3400 cm -1 are associated to presence of water absorbed on the ZnWO 4 sample.These results indicate the formation of ZnWO 4 single phase, corroborating to the results from XRD analysis.Bands around 2300 cm -1 are assigned to the adsorbed atmospheric CO 2 .Inulinase free (Figure 5c) shows bands associated with amino groups (CONH) at 1400-1600 cm -1 39,40 .Bands around 1000 cm -1 correspond to -C = O binding of enzyme on the support.These bands are also displayed in the spectrum of the immobilized enzyme on the support (Figure 5b), confirming the immobilization of inulinase on the ZnWO 4 support.
According to the results of inulinase immobilization shown in Figure 6, it is possible to observe that the adsorption equilibrium was obtained in 120 min for both temperatures (30 and 50 o C). Figure 6 also demonstrates that the increase of the temperature had a negative effect in the improvement of enzyme adsorption, with loading capacity of 605 U.g -1 support and 264 U.g -1 support at 30 and 50 o C, respectively.For comparison purposes, Missau et al. 16 found similar results regarding the inulinase immobilization on alginate-chitosan beads, achieving 668 U.g -1 gel beads at 50 o C. Grafted alginate beads showed an inulinase loading capacity of 530 U.g -1 gel beads 19 .Chitin 21 and silica 41 were used as inulinase supports, reaching 291 U.g -1 chitin and 43 U.g -1 silica, respectively.Therefore, these findings indicate that the ZnWO 4 particles present satisfactory inulinase immobilization, which can be attributed to their porous structure.

Conclusions
ZnWO 4 powders were successfully synthesized through one-step solvo-hydrothermal crystallization at a mild temperature, without using additives.ZnWO 4 particles presented porous structure with surface area of 35.5 m 2 .g - .
Inulinase could be successfully immobilized using ZnWO 4 particles.Temperature had a significant effect on enzyme immobilization process.In the best condition, the enzyme loading capacity was 605 U.g -1 at 30 o C using 1.3% (v/v) enzyme concentration and a 1:400 adsorbent:adsorbate ratio.Therefore, the ZnWO 4 support prepared in this work shows attractive physical characteristics for the potential application on inulinase immobilization.

Figure 3 :
Figure 3: Particle size distribution plot for zinc tungstate prepared by the solvo-hydrothermal route.

Figure 4 :
Figure 4: AFM image of some isolated particles of ZnWO 4 sample after sonication.