Production of Copper and Cobalt Aluminate Spinels and Their Application As Supports for Inulinase Immobilization

Spinel structure oxides constitute one of the most interesting classes of advanced ceramic materials due their intrinsic physical and chemical properties. Among the spinel oxides, copper (CuAl2O4) and cobalt (CoAl2O4) aluminates possess interesting properties for technological application. Specifically, copper aluminate (CuAl2O4) possesses important applications in various fields such as gases sensor1, catalyst in the oxidation reaction of benzyl alcohol2,3, methanol steam reforming4 and hydrogenolysis of glycerol5, thermoelectric properties6, photocatalyst for degradating the pollutants in aqueous solution7-10, solar absorber11, oxygen carrier in chemical-looping combustion12 and optical and dielectric properties2,13. Cobalt aluminate oxide (CoAl2O4) has been employed in various fields such as ceramic pigment14,15, optical and magnetic properties16,17, catalysts in reactions such as soot combustion with NOx/O2 [18], oxidation of CO[19], carbon monoxide hydrogenation20, and reforming of methane21, and sensors for gas22, humidity23 and volatile organic compounds1,24. The use of immobilized enzymes has been increased in the last years due the many fields application. In the immobilization process enzymes are trapped in a solid matrix, therefore, is possible the reuse and it will not be subjected to denaturation25. Several materials with different characteristics such as natural clay26, grafted alginate beads27, aminated non-porous silica nanoparticles28, chitin29, alginate–chitosan beads30, poly(EGDMA) cryogels31 and carbon nanotubes32 have been used as supports for inulinase immobilization by adsorption process. The immobilization process presents some advantages compared with free enzyme, which are the easily removed of the products from immobilized enzymes and possibility the continuous mode of operation33. Immobilized inulinases have been used for many processes, especially for the sugar production through inulin hydrolysis, yielding fructose and fructooligosaccharides, which could be used as functional ingredient26. In this work a new application for copper and cobalt aluminate oxides was proposed, i.e., as support for inulinase immobilization. Copper aluminate spinel (CuAl2O4) has been prepared by several techniques such as microwave combustion method3, co-precipitation10, electrochemical process11, solvothermal20, microemulsion method34, sonochemical9,35, solid state reaction4,36, sol-gel7,37, modified sol–gel2,38, sputtering13,39, Pechini method40 and metallic alkoxide41. Cobalt aluminate spinel (CoAl2O4) has been prepared by reverse microemulsion process16, sol–gel17, hydrothermal42,43, microwave combustion method44, sonochemical45, via glycine chelated precursors46, via polyacrylamide gel method47, double decomposition reaction between solid LiAlO2 and molten KCoCl3 [48], low temperature combustion49, complexation method50, reaction-sintering process51, polymeric precursors method52, polymeric-aerosol pyrolysis53, freeze–drying54, sol–gelhydrothermal method55, ultrasonic-assisted-hydrothermal method56, polymerized complex technique57, chemical vapor deposition58, and via supercritical water conditions59. In this work, both copper and cobalt aluminates were prepared by route using chitosan as template. This technique has as advantages obtain a material with high porosity and surface area60. These characteristics are important for enzymes Production of Copper and Cobalt Aluminate Spinels and Their Application As Supports for Inulinase Immobilization


Introduction
Spinel structure oxides constitute one of the most interesting classes of advanced ceramic materials due their intrinsic physical and chemical properties.Among the spinel oxides, copper (CuAl 2 O 4 ) and cobalt (CoAl 2 O 4 ) aluminates possess interesting properties for technological application.Specifically, copper aluminate (CuAl 2 O 4 ) possesses important applications in various fields such as gases sensor 1 , catalyst in the oxidation reaction of benzyl alcohol 2,3 , methanol steam reforming 4 and hydrogenolysis of glycerol 5 , thermoelectric properties 6 , photocatalyst for degradating the pollutants in aqueous solution [7][8][9][10] , solar absorber 11 , oxygen carrier in chemical-looping combustion 12 and optical and dielectric properties 2,13 .Cobalt aluminate oxide (CoAl 2 O 4 ) has been employed in various fields such as ceramic pigment 14,15 , optical and magnetic properties 16,17 , catalysts in reactions such as soot combustion with NO x /O 2 [18] , oxidation of CO [19] , carbon monoxide hydrogenation 20 , and reforming of methane 21 , and sensors for gas 22 , humidity 23 and volatile organic compounds 1,24 .
The use of immobilized enzymes has been increased in the last years due the many fields application.In the immobilization process enzymes are trapped in a solid matrix, therefore, is possible the reuse and it will not be subjected to denaturation 25 .Several materials with different characteristics such as natural clay 26 , grafted alginate beads 27 , aminated non-porous silica nanoparticles 28 , chitin 29 , alginate-chitosan beads 30 , poly(EGDMA) cryogels 31 and carbon nanotubes 32 have been used as supports for inulinase immobilization by adsorption process.The immobilization process presents some advantages compared with free enzyme, which are the easily removed of the products from immobilized enzymes and possibility the continuous mode of operation 33 .Immobilized inulinases have been used for many processes, especially for the sugar production through inulin hydrolysis, yielding fructose and fructooligosaccharides, which could be used as functional ingredient 26 .In this work a new application for copper and cobalt aluminate oxides was proposed, i.e., as support for inulinase immobilization.
Hence, in the present study, copper and cobalt aluminates were prepared by the route using chitosan as template.The spinel oxides were prepared at two different temperatures, and their physical properties were determined.Both oxides were evaluated as supports for inulinase immobilization.

Experimental 2.1. Synthesis of oxides
Synthesis procedure of copper and cobalt aluminates was based in a previous work 62  ), aqueous solutions of aluminum nitrate (15 g in 30 mL distilled water) and copper nitrate (4.82 g in 8 mL distilled water) were added into an acetic acid aqueous solution (5% v/v) (80 mL) containing 2.5 g of chitosan previously dissolved, under magnetic stirring.The mixture was ultrasonicated at 30 °C for 30 min in an ultrasonic bath (Unique Inc., model USC 1800A, 35 kHz Brazil).After that, the resulting solution was slowly added to an ammonia aqueous solution (50% v/v, 200 mL) under magnetic stirring.The formed precipitates were separated from the solution and further dried at ambient temperature for 48 h.Then, the solids were calcined in air at 650 °C and 900 °C for 4 h, using a conventional muffle-furnace.Cobalt aluminate powders were prepared by same procedure previously described, but the amounts used for the synthesis were 15 g of aluminum nitrate and 4.76 g of cobalt chloride.

Characterization techniques of oxides
The phase identification of the samples was carried out by XRD (Rigaku Miniflex 300 diffractometer) using Cu Kα radiation at 30 kV and 10 mA, with a step size (2θ) of 0.03° and a count time of 0.9 s per step.The average crystallite size of each sample was determined through the Scherrer equation 63 (silicon powder was used as a standard reference material): D = K.λ /( h 1/2 .cosθ), where D is the average crystallite size, K the Scherrer constant (0.9), λ the wavelength of incident X-rays (0.1541 nm), h 1/2 the peak width at half height and θ corresponds to the peak position (2θ = 36.88°,lattice plane of {311} for CuAl 2 O 4 and 36.74°,lattice plane of {311} for CoAl 2 O 4 ).N 2 adsorption-desorption isotherms measurements were carried out at 77 K using an ASAP 2020 apparatus.Before analysis the samples were degassed at 200 °C under vacuum.Silica-alumina pellets (Part No. 004-16821-00) were used as a reference material to test instrument performance.

Enzyme immobilization and activity essays
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)and it was purchased from Sigma-Aldrich (São Paulo, Brazil).The temperature was fixed at 30 °C and the inulinase adsorption was performed using a batch technique.Typically, 0.025 g of oxide was placed in glass flasks containing different inulinase concentrations in a sodium acetate buffer (pH 4.8) solution.The effects of enzyme concentration (0.5; 1.0 and 1.5% v/v) and adsorbent:adsorbate ratio (1:300-1:500) on the adsorption were evaluated.The solution was maintained under agitation at 150 rpm, and then an aliquot of the aqueous solution was collected after reaching the adsorption equilibrium time (90 min), and then filtered through the polyvinylidene difluoride (PVDF) membrane (0.22 μm) before analysis.For the enzyme activity essays, an aliquot of 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 64 .A separate blank was set up for each sample to correct 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 immobilization efficiency was expressed in terms of inulinase loading capacity (Q T : U g -1 ) through the Equation 1.
where A O and A T (U L -1 ) are the inulinase activities at t = 0 and time t, respectively; V (L) is the volume of solution, and m (g) is the mass of support.
All the immobilization experiments were carried out in duplicate and only the mean values were reported.The maximum deviation observed was about ± 6.0%.

Characterization of oxides
The XRD patterns of the copper and cobalt aluminates prepared at different temperatures are shown in Figure 1 (profiles a and b, respectively  44-0160, respectively.On the basis of the Scherrer equation, the crystallite size was calculated to be ca.7.5 for both CuAl 2 O 4 samples, and ca.6.0 and 8.0 nm for the CoAl 2 O 4 samples prepared at 650 and 900 °C, respectively.
Figure 2 shows the adsorption-desorption isotherms of N 2 of cobalt and copper oxides prepared at 650 and 900 °C.
All samples present type-IV isotherms (Figure 2a), in accordance with the International Union of Pure and Applied Chemistry (IUPAC) classification 65 .This adsorption behavior is characteristics of mesoporous structure.The desorption branches present H1-like hysteresis loops at high relative pressures, indicative of the presence of mesoporosity.The corresponding pore size distribution curves (Figure 2b) were measured from the adsorption branches according to the Barrett-Joyner-Halenda (BJH) method.All samples display a similar unimodal distribution, with peaks centered in the mesoporous region (between 20 and 500 Å).These features are of great importance for immobilization purposes because it allows for a greater accessibility of enzymes molecules to the support.Surface physical parameters of all samples are shown in Table 1.It can be observed that specific surface area decreases with increasing calcination temperature.Thus samples prepared at 650 °C present higher surface area.This can occur due to sintering of material when it is treated at higher temperatures, resulting in reducing the void spaces and consequently, its surface area 66 .
For comparison purposes, surface area values of CuAl 2 O 4 and CoAl 2 O 4 powders found in literature are shown in Table 2. From Table 2, it can be seen that each method results in materials with different surface areas.Therefore, the preparation route strongly affects their physical properties.The results revealed that the route presented in this work generates materials with a high surface area.So preparation of single phase spinel with porous structure and high surface area makes this method technically easier, simpler and low cost because it does not need sophisticated procedures and requires inexpensive precursors.As cobalt-and copper-based oxides are colored materials, so this detail was evidenced from visual examination (Figure 3), whose images were obtained by a commercial digital camera (Nikon Coolpix).The powders were compacted on a glass sample holder.These results aimed provide additional information regarding the physical characteristics of produced materials, i.e., their intrinsic color.The samples containing cobalt exhibit a bright blue color whereas samples containing copper are greenish/ yellowish.Cobalt aluminate has been used as blue ceramic pigment 14,15 , whereas copper-based oxides as green/yellow/turquoise pigments [79][80][81] .

Enzyme immobilization
The enzyme immobilization assays were performed using only the copper aluminate and cobalt aluminate samples at 650 o C, because in this temperature was obtained the highest surface area (see Table 1).Table 3 shows the results for inulinase loading capacity using copper and cobalt aluminates.Copper aluminate sample presented a loading capacity of 8,510 U g -1 using an inulinase concentration of 0.5% (v/v) and 1:500 of adsorbent:adsorbate ratio, whereas cobalt aluminate sample presented similar loading capacity (8,343 U g -1 ) under the same adsorbent:adsorbate ratio (1:500), but using an inulinase concentration of 1.5% (v/v).The results showed that the cobalt aluminate presents more capacity for inulinase adsorption, which could be explained by the surface area value, being higher when compared to the cupper aluminate.Table 4 shows the inulinase loading capacity of different supports found in literature.According to the results founded it is possible to observe that both aluminates presented a satisfactory inulinase loading capacity.This result could be explained by the high surface area values of both samples prepared in this work.Microwave assisted glycothermal 18 181 Sol-gel 1 10 Hydrothermal 42 29 Sol-gel 2 10 Sonochemical 45 75 Modified sol-gel 2 41 Low temperature combustion 49 77 Solid-state reaction 4 13 Autoignition 67 16 Sonochemical 9 110 Colloidal sol-gel 68 156 Dry soaking 74 30 Combustion 69 188 Microwave combustion 75 87 Microwave 70 94 Conventional combustion 75 44 Coprecipitation 71 169 Mixed chelates thermolysis 76 86 Solution-based combustion 72 53 Soft chemical approach 77 5 Malonate 73 23 Copper-laden sludge thermally treated 78 2

Conclusions
Copper and cobalt aluminates were successfully synthesized using chitosan as template.The powders produced presented high surface area and mesoporous structure.These features are of great importance for immobilization purposes because it allows a greater accessibility of enzyme molecules to the support.Both aluminates presented a satisfactory loading capacity for inulinase immobilization.Therefore, these results revealed that copper and cobalt aluminates can be used as alternative supports for enzymes immobilization.

Figure 3 .
Figure 3. Images of (a) CoAl 2 O 4 and (b) CuAl 2 O 4 samples obtained at different temperatures.

Table 1 .
Surface physical characteristics of oxide samples prepared at different conditions.

Table 2 .
Comparing the surface area values of different synthesis methods found in the literature.