*Review* **Recent Progress on Sulfated Nanozirconia as a Solid Acid Catalyst in the Hydrocracking Reaction**

**Serly Jolanda Sekewael 1, Remi Ayu Pratika <sup>2</sup> , Latifah Hauli <sup>2</sup> , Amalia Kurnia Amin <sup>2</sup> , Maisari Utami <sup>3</sup> and Karna Wijaya 2,\***


**Abstract:** Zirconia has advantageous thermal stability and acid–base properties. The acidity character of ZrO2 can be enhanced through the sulfation process forming sulfated zirconia (ZrO2-SO4). An acidity test of the catalyst produced proved that the sulfate loading succeeded in increasing the acidity of ZrO2 as confirmed by the presence of characteristic absorptions of the sulfate group from the FTIR spectra of the catalyst. The ZrO2-SO4 catalyst can be further modified with transition metals, such as Platinum (Pt), Chromium (Cr), and Nickel (Ni) to increase catalytic activity and catalyst stability. It was observed that variations in the concentrations of Pt, Cr, and Ni produced a strong influence on the catalytic activity as the acidity and porosity of the catalyst increased with their addition. The activity, selectivity, and catalytic stability tests of Pt/ZrO2-SO4, Cr/ZrO2-SO4 and Ni/ZrO2-SO4 were carried out with their application in the hydrocracking reaction to produce liquid fuel. The percentage of liquid fractions produced using these catalysts were higher than the fraction produced using pure ZrO2 and ZrO2-SO4 catalyst.

**Keywords:** catalyst; zirconia; sulfated; acidity

#### **1. Introduction**

Zirconium dioxide, known as zirconia, is a crystalline oxide of zirconium that found form in the mineral baddeleyite. Zirconia is a white material that does not react with water or another solvent, and that has acid–base properties and excellent thermal dan chemical stabilization. ZrO2 materials are of wide interest and development in their application in various fields, such as heterogeneous catalyst, optics, electronics, magnetics, and ceramics owing to the high melting point (≥2700 ◦C), low thermal conductivity, corrosion resistance, and good thermal and mechanical strength [1–3].

Modification of zirconia to increase its catalytic activity has been developed. Many studies have showed that modification zirconia such as sulfated process or metal supported was effective during the chemical process [3–5]. Sulfated zirconia catalyst via hydrothermal treatment for hydrocracking of LDPE plastic waste into liquid fuels was examined by Utami et al. [6]. The total acidity of zirconia increased after the sulfation process, thus increasing the amount of liquid yield. However, the catalytic activity of sulfated zirconia catalyst during the hydrocracking reaction at high temperature decreases due to the deactivation catalyst. This process therefore requires an appropriate catalyst to increase the catalytic activity of sulfated zirconia, as well as the acidity and liquid yield [7,8].

Supported noble metals such as Platinum (Pt), Chromium (Cr), and Nickel (Ni) as a promoter have shown good catalytic activity in the hydrocracking reaction. The synthesis

**Citation:** Sekewael, S.J.; Pratika, R.A.; Hauli, L.; Amin, A.K.; Utami, M.; Wijaya, K. Recent Progress on Sulfated Nanozirconia as a Solid Acid Catalyst in the Hydrocracking Reaction. *Catalysts* **2022**, *12*, 191. https://doi.org/10.3390/ catal12020191

Academic Editors: Elisabeth Egholm Jacobsen, Simona M. Coman and Madalina Tudorache

Received: 17 December 2021 Accepted: 27 January 2022 Published: 3 February 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

of Pt/sulfated zirconia [9], Cr/sulfated zirconia [10], and Ni/sulfated zirconia [11] catalyst in the hydrocracking reaction reported the enhance of acidity of sulfated zirconia and the liquid product of hydrocracking after the addition of promoter Pt, Cr, and Ni, respectively.

#### **2. Zirconium Dioxide (ZrO2)**

Zirconium dioxide (ZrO2) is a polymorphic material with three crystalline phases, namely the monoclinic, tetragonal, and cubic phases, as shown in Figure 1. The monoclinic and tetragonal phases are stable up to temperatures of 1170 and 2360 ◦C, respectively, while the cubic phase is stable at temperatures above 2680 ◦C. ZrO2 as a catalyst is normally used in its monoclinic and metastable tetragonal crystalline phases [12–14]. Transformation of the crystalline phases of ZrO2 is driven by changes in temperature. ZrO2 calcined at <800 ◦C forms monoclinic and metastable tetragonal phases [15,16].

The transformations of one ZrO2 phase to another are accompanied by changes in lattice parameters. The rate of phase transformation of ZrO2 is also influenced by the particle size of the ZrO2 precursor used. The larger the ZrO2 particle size, the faster the phase transformation occurs. Nano-sized particles have a high surface area, allowing for more of the atoms of the particles to interact and form bonds [15,17,18]. Based on surface properties and polymorphic form, ZrO2 is also often used as a catalyst or carrier material because it has acidic and basic properties [19,20]. In addition, ZrO2 has a structure with vacant sites on its surface that can allow cations to easily enter [21,22].

**Figure 1.** Crystal structures of (**a**) monoclinic, (**b**) tetragonal, and (**c**) cubic ZrO2. Copyright Elsevier, reprinted from Ref. [23].

#### **3. ZrO2-SO4 Catalyst**

Zirconia is a catalyst containing high Lewis acid sites. Ore [15] mentioned that ZrO2 can be modified with acid or base to achieve, based on the intended application, the appropriate and desired strong acid or base characteristics. Sulfation is a method that can increase the strength of ZrO2 acid through the formation of Brønsted acids on the zirconia substrate that consequently increases its catalytic activity. ZrO2-SO4 can be prepared from sulfate precursors such as H2SO4, (NH4)2SO4, (NH4)2S2O3, and (NH4)2S) [24–26].

The formation of ZrO2-SO4 occurs through the chelation of the zirconium cations (Zr4+) with sulfate ions. The SO4 <sup>2</sup><sup>−</sup> ions acts as ligands that donate their lone pair of electrons from the O atom, thus forming a coordination bond with two Zr4+ as the central atoms and causing the acid molecule to release two protons simultaneously. After relaxation, adsorptive complex molecules are produced from the coordination of SO4 <sup>2</sup><sup>−</sup> ions onto the ZrO2 surface through the sharing of two O atoms [27–29]. Saravan et al. [30] illustrate the surface model of ZrO2-SO4 with the Lewis and Brønsted acid sites shown as shown in Figure 2.

**Figure 2.** Brønsted and Lewis acid sites on ZrO2-SO4. Copyright MDPI, reprinted from Ref. [17].

Modification of zirconia to ZrO2-SO4 produces materials with high Lewis and Brønsted acid strengths. Wang et al. [29] presented an illustration of the ZrO2-SO4 surface as presented in Figure 3. The high acid strength of the Brønsted acid site is associated with the location of Zr4+ ions that are adjacent to the S=O bond which attracts electrons from the bisulfate to form the Brønsted acid site.

**Figure 3.** ZrO2-SO4 surface model of bidentate chelate type. Reprinted from ref. [29].

#### *3.1. Functional Group Characterization for ZrO2-SO4*

Research conducted by Utami et al. [31] reported the preparation of sulfated zirconia catalysts with various H2SO4 concentrations and calcination temperatures. Figure 4 shows the same absorption peak at wave number 424–741 cm<sup>−</sup>1, indicating the Zr–O–Zr bond [32]. Absorptions at 3426–3449 and 1636 cm−<sup>1</sup> refer to the O-H stretching and bending vibrations of the water molecules adsorbed on the material [33]. In addition, according to Ore et al. [15], the broadband in the absorption region of 3400 cm−<sup>1</sup> signifies the bridge between the hydroxyl group with two or three Zr atoms.

**Figure 4.** FTIR spectra of SZ catalyst at various sulfate concentrations and calcination temperatures. Reprinted with permission from Dr. Utami, Ref. [31]. Copyright 2019 Trans Tech Publication.

The presence of SO4 <sup>2</sup><sup>−</sup> ions on the surface of ZrO2 can be confirmed by the formation of a new peak at 995-1404 cm−<sup>1</sup> which is typical of the bidentate SO4 <sup>2</sup><sup>−</sup> chelate ion covalently bonded to the Zr4+ cation [7,27]. The absorption peaks at 995–1003, 1049–1096, 1134–1157, and 1227 cm−<sup>1</sup> are S–O symmetric vibrations, S–O asymmetric vibrations, S=O symmetry vibrations, and S=O asymmetric vibrations [34–36]. The peak with low intensity in the area of 1404 cm−<sup>1</sup> is the stretching vibration of S=O, indicating the formation of SO3 species on the surface of ZrO2 [37]. The presence of characteristic bands of ZrO2-SO4 proves that the impregnation of H2SO4 on ZrO2 has been successfully carried out.

The use of too high a concentration of H2SO4 can lead to loss of several absorption bands of the SO4 <sup>2</sup><sup>−</sup> ion which is covalently bonded to Zr4+ cations as the ZrO2 structure is degraded [9,37]. Such a case implies that an appropriate or optimum concentration of H2SO4 is needed for the ZrO2 activation process. The absorption intensity of ZrO2-SO4 increased at a temperature range of 500–600 ◦C but decreased at temperatures of 700–800 ◦C. Calcination treatment at 600 ◦C (SZ-0.8-600) was found to be optimum resulting in the highest SO4 <sup>2</sup><sup>−</sup> dispersion. According to Ore [15], the maximum calcination temperature in the sulfation process is 650 ◦C. Temperatures above 650 ◦C cause the decomposition of SO4 <sup>2</sup><sup>−</sup> ions, thereby reducing the acidity and reactivity of the catalyst.

The acidity test of the catalyst was carried out by the gravimetric method based on the amount of NH3 vapor absorbed by the catalyst. Table 1 shows the results of the catalyst acidity test. ZrO2 has a total acidity of 0.18 mmol/g. The acidity of ZrO2 comes from the Zr4+ cations that act as Lewis acid sites [7,8]. After modification with H2SO4, the acidity of ZrO2 increased. SZ-0.8-600 catalyst showed the highest acidity value of 1.06 mmol/g. Ore et al. [15] reported that the number of Brønsted and Lewis acid sites depends on the SO4 2− concentration present on the catalyst surface. Sulfation with optimum sulfate concentration can increase the catalytic activity of the catalyst, while the decrease in catalyst acidity with increasing temperature occurs due to dehydration of protonic sites and loss of SO4 2− groups on the surface of the catalyst [38–40].


**Table 1.** Acidity test for the SZ catalyst at various sulfate concentrations and calcination temperatures [31].

Qualitatively, the number of Brønsted and Lewis acid sites can be observed absorption spectra intensity that denotes the interaction between the catalyst acid sites and NH3 [41]. Figure 5 presents the FTIR spectra of the catalyst after the acidity test. The absorption peak at 1119-1126 cm−<sup>1</sup> indicates the presence of NH3 coordinated to the Lewis acid site. The peak at 1404 cm−<sup>1</sup> confirmed the presence of NH4+ ions formed by proton transfer from the Brønsted acid site to NH3 [42,43]. The higher the intensity of the absorption band, the higher the number of Brønsted and Lewis acid sites. It was found that the SZ-0.8-600 catalyst had the highest acid site intensities. The absorption intensities of the acid sites decreased with the increase in calcination temperature. Calcination treatment at high temperature decreases the acidity of the catalyst due to the decrease in the number of acid sites on the surface of the catalyst [44,45].

**Figure 5.** FTIR spectra of SZ catalyst at various sulfate concentrations and calcination temperatures after acidity test. Reprinted with permission from Dr. Utami, Ref. [31]. Copyright 2019 Trans Tech Publication.

#### *3.2. ZrO2-SO4 Crystal Structure Characterization*

Figure 6 presents the diffraction pattern of the SZ catalyst at various sulfate concentrations and calcination temperatures. The main diffraction peaks appear at 2θ = 28.34◦ (d-111) and 31.64◦ (d111), referring to the ZrO2 monoclinic crystalline phase [6,15]. In general, the diffraction pattern showed stable crystallinity even after the addition of acid and calcination treatment. However, the intensity of the ZrO2 monoclinic diffraction peak decreased after acid treatment. SZ-0.8-600 catalyst with the highest total acidity showed the lowest monoclinic peak intensity. The addition of a high concentration of H2SO4 causes a large number of SO4 <sup>2</sup><sup>−</sup> ions to cover the surface of ZrO2, decreasing crystallinity [37,38]. The intensities of the monoclinic peaks at temperatures of 800 and 900 ◦C were higher than those at 600 and 700 ◦C. This occurred because the high calcination temperature caused SO4 <sup>2</sup><sup>−</sup> ions to decompose from the catalyst surface, increasing the crystallinity of the catalyst.

**Figure 6.** Diffraction patterns of SZ catalyst at various sulfate concentrations and calcination temperatures. Reprinted with permission from Dr. Utami, Ref. [31]. Copyright 2019 Trans Tech Publication.

ZrO2-SO4 calcined at a temperature of <800 ◦C can exist in a metastable tetragonal phase and a monoclinic phase. Similar results were reported by Ore et al. [15] which stated that ZrO2-SO4 calcined at a temperature of 600 ◦C consists of a mixture of metastable tetragonal and monoclinic phases. The ZrOCl2·8H2O amorphous precursor used made it possible for transformation to a metastable tetragonal structure to occur. However, the ZrO2 used in this study is commercial ZrO2, which contains the monoclinic structure of high stability and crystallinity, hindering it from undergoing phase transformation [46]. The catalyst diffraction pattern in the research by Utami et al. [6] demonstrated the ZrO2 catalyst and its modifications consisting only of monoclinic structure.

#### **4. Platinum/Sulfated Zirconia (Pt/ZrO2-SO4) Catalyst**

A heterogeneous catalyst is a catalyst material composed of two components, namely the doping and carrier components. The metal catalyst, when used in its pure form, has low thermal stability and tends to sinter that can leading to a decrease in surface area and deactivation [47–49]. Appropriate distribution of metal catalysts on the carrier material having acid–base sites and a large surface area is necessary to avoid sintering [50,51]. The ZrO2-SO4 material has many Brønsted and Lewis acid sites in which, despite its high acidity, this catalyst can be rapidly deactivated. The addition of Pt metal can increase the stability of the catalyst with the simultaneous presence of hydrogen gas (H2) [52–54].

The distribution mechanism of H2 through the Pt surface on the ZrO2-SO4 carrier is illustrated in Figure 7. The H2 molecule dissociates on the surface of the Pt particle homolytically to form two H radicals which then bind to the unpaired electrons in the 5d orbitals. The H+ ions released from Pt are distributed on the ZrO2-SO4 carrier and migrate to the electron-rich O atomic sites, forming Brønsted acid sites [55,56]. Figure 8 shows the physical appearance of ZrO2-SO4 catalyst which a white powder and after the impregnation with Pt metal as a Pt/ZrO2-SO4 catalyst, solid powder which is darker in color is formed due to the presence of the Pt metal that impregnated to the ZrO2-SO4 catalyst [6,9].

**Figure 7.** Mechanism of H2 distribution through the Pt surface on ZrO2-SO4. Reprinted and modified with permission from Dr. Utami, Ref. [9]. Copyright 2019 The Royal Society of Chemistry.

**Figure 8.** ZrO2-SO4 (**right**) and Pt/ZrO2-SO4 (**left**) catalyst. Reprinted with permission from Dr. Utami, Ref. [42].

#### *4.1. FTIR and Acidity Characterization of Pt/ZrO2-SO4 Catalyst*

Utami et al. [6] researched the synthesis of Pt-promoted zirconia (Pt/SZ) catalyst and its application in hydrocracking LDPE plastic into liquid fuel. The FTIR spectra of the sulfated zirconia impregnated with Pt metal can be seen in Figure 9. Overall, the Pt1/SZ, Pt2/SZ, and Pt3/SZ spectra showed the same absorption peaks as ZrO2 and SZ. Pt metal impregnated catalysts had characteristic peaks of ZrO2-SO4 at wave number 1065–1126 cm<sup>−</sup>1. The addition of Pt metal caused some of the absorption peaks of ZrO2-SO4 to disappear. This is an early indication that Pt metal had been impregnated on nanoSZ [9].

**Figure 9.** FTIR spectra of ZrO2, SZ, Pt1/SZ, Pt2/SZ, and Pt3/SZ. Reprinted with permission from Dr. Utami, Ref. [6]. Copyright 2019 Elsevier.

Table 2 shows the total acidity of the catalysts of the present study. Pt metal impregnation on sulfated zirconia was proven to increase the total acidity of the catalyst significantly (from 1.06 to 10.75 mmol/g). Pt1/SZ, Pt2/SZ, and Pt3/SZ catalysts showed increasing acidity values with increasing concentrations of Pt metal. The increase in the acidity of the catalyst occurs because the Pt metal provides vacant orbitals that can act as electron-pair acceptors (Lewis acid sites) and the presence of unpaired electrons in the d orbitals that form Brønsted acid sites [57,58].


**Table 2.** Acidity test results of ZrO2, SZ, Pt1/SZ, Pt2/SZ, and Pt3/SZ [6].

FTIR spectra interpretations of ZrO2, SZ, Pt1/SZ, Pt2/SZ, and Pt3/SZ are shown in Figure 10. Increasing concentration of Pt metal produced increased adsorption of NH3 bound to the Brønsted and Lewis acid sites as shown at wavenumbers of 1396–1404 and 1119 cm<sup>−</sup>1, indicating that the higher the concentration of Pt metal, the higher the number of acid sites contained in the catalyst. The Pt3/SZ catalyst showed the highest intensity of Brønsted and Lewis acid absorptions. Based on the results of the acidity test of the catalyst, the Pt3/SZ catalyst was confirmed to have the highest acidity value.

**Figure 10.** FTIR spectra of ZrO2, SZ, Pt1/SZ, Pt2/SZ, and Pt3/SZ after acidity test. Reprinted with permission from Dr. Utami, Ref. [6]. Copyright 2019 Elsevier.

#### *4.2. XRD and GSA Characterizations of Pt/ZrO2-SO4 Catalyst*

SZ catalysts are shown in Figure 11. Based on crystal identification, all samples showed the presence of the monoclinic phase. According to Ore [15], SO4 <sup>2</sup><sup>−</sup> species can be thermally crystallized through the calcination process and undergo a crystalline phase transformation, further stabilizing the ZrO2 crystalline phase. The addition of Pt metal to SZ would not have caused changes in the crystal structure of the material. A decrease in the intensity of the diffraction peak after the addition of Pt metal was observed. This phenomenon indicated that Pt metal had been successfully impregnated on the SZ surface, where a higher concentration of the impregnated Pt metal would cause the intensity of the monoclinic peak to decrease [6,9].

**Figure 11.** Diffraction patterns of ZrO2, SZ, Pt1/SZ, Pt2/SZ, and Pt3/SZ. Reprinted with permission from Dr. Utami, Ref. [6]. Copyright 2019 Elsevier.

Based on the catalyst diffraction pattern, Pt metal characteristic peaks were not detected. This is because the concentration of impregnated Pt metal was relatively low. This is by research conducted by Aboul-Gheit et al. [59] that showed similar results whereby the diffraction peak of Pt metal was not identified after the addition of 0.6% Pt metal to ZrO2-SO4. A relatively low concentration of Pt metal was used in the present study to prevent agglomeration of Pt particles on the ZrO2-SO4 surface, which could cause a decrease in catalytic activity.

Characterization carried out by GSA (Table 3) showed an increase in surface area and pore volume along with the increasing concentration of Pt metal impregnated onto SZ. The Pt1/SZ catalyst had a surface area and pore volume of 13.49 m2/g and 0.05 cm3/g, respectively, which saw an increase to 20.23 m2/g and 0.06 cm3/g for Pt2/SZ and 29.48 m2/g and 0.08 cm3/g for Pt3/SZ. Based on the data reported in the study by Utami et al. [9], the increase in the surface area and pore volume of the SZ catalyst can be attributed to the inhibition of the agglomeration process related to the high presence of Pt metal.


**Table 3.** Textural characteristics of ZrO2, SZ, Pt1/SZ, Pt2/SZ, and Pt3/SZ [6].

The increase in the surface area of the catalyst can also be attributed to the smaller crystal size dimensions. Crystal size results of ZrO2, SZ, Pt1/SZ, Pt2/SZ, and Pt3/SZ (Table 4) presented a decrease in size after metal impregnation of Pt. This indicated that the Pt metal impregnated by the reflux technique was evenly dispersed on the surface and pores of SZ [9]. In contrast, Aboul-Gheit et al. [59] impregnated Pt metal on ZrO2-SO4 through the wet impregnation method and reported an increase in the concentration of Pt metal causing the surface area and pore volume to increase due to the Pt metal being not evenly distributed and covering most of the ZrO2-SO4 pores.


**Table 4.** Crystal sizes of ZrO2, SZ, Pt1/SZ, Pt3/SZ Pt2/SZ and Pt3/SZ [6].

#### *4.3. Elemental Composition Characterization Using EDXRF*

Table 5 shows the concentrations of elements contained in the catalysts. The concentrations of Pt metals identified in the Pt1/SZ, Pt2/SZ, and Pt3/SZ samples were 0.35, 0.90, and 1.19%. The metal content of Pt in SZ was strongly influenced by the dispersion ability on the surface and pores of the carrier material. The reduction treatment with H2 gas flow was carried out after the calcination step which aimed to obtain Pt0 particles that would result in dispersion [14].


**Table 5.** Elemental compositions of ZrO2, SZ, Pt1/SZ, Pt2/SZ dan Pt3/SZ [6].

Based on the elemental compositions above, the concentrations of Pt that were observed were lower than the theoretical concentration of Pt metal by the addition of PtCl4 solution used in the impregnation process. This discrepancy occurred due to competition between impregnated Pt metals, causing the formation of multilayer stacking of the active metal in the pore mouth area of the carrier material [60,61]. The active Pt metal that sat at the top position would have weak interaction and would experience easier desorption [62]. Because of this, the amount of active metal in the pore area of the carrier material was observed to be less.

#### *4.4. Pt metal Composition Identification Using XPS*

Subsequent research related to the synthesis of platinum-loaded sulfated zirconia catalysts using the hydrothermal method was reported by Utami et al. [9]. XPS spectra were used to determine the composition of Pt in the samples based on a comparison of binding energy values. The peaks generated from XPS are not single peaks, so that deconvolution of the peaks was needed to identify the multiple peaks that made up each peak. Figure 12 presents the XPS spectra along with the deconvolution of Pt 4f peaks from Pt/nano ZrO2-SO4 consisting of Pt<sup>0</sup> 4*f* 7/2, Pt0 4*f* 5/2, and Pt2+ 4*f* 7/2. The spectra indicated the interaction of electrons between the Pt particles and the nanoZS surface. Table 6 shows the relative area of the deconvoluted peaks of Pt<sup>0</sup> 4*f* 7/2, Pt0 4*f* 5/2, and Pt2+ 4*f* 7/2 in the Pt/nano ZrO2-SO4 sample. The data obtained showed that the detected Pt<sup>0</sup> composition was 81.82%. The results indicated that the reduction treatment with H2 gas flow at the metal impregnation stage succeeded in forming Pt<sup>0</sup> particles.

**Figure 12.** XPS spectra of Pt/nano ZrO2-SO4 in the 4*f* region and the catalyst model. Reprinted and modified with permission from Dr. Utami, Ref. [9]. Copyright 2019 The Royal Society of Chemistry.

**Table 6.** Percentage of relative areas of Pt 4*f* deconvoluted peaks [4].


#### *4.5. Thermal Stability Characterization with TG/DTA*

The TG/DTA curve provides information about changes in thermal conditions with mass changes in the sample. Figure 13 shows the TGA curves for nano ZrO2, nano ZrO2- SO4, and Pt/nano ZrO2-SO4 samples analyzed at temperatures of 30–900 ◦C. The TGA curve of nano ZrO2 did not indicate mass decrease indicating that nano Z had good thermal stability [63]. Mass decrease in nano ZrO2-SO4 and Pt/nano ZrO2-SO4 in the range of 50–200 ◦C by 2.41 and 1.24% were associated with the elimination of water molecules physically adsorbed on the material. At 500–700 ◦C, decreases in a mass of 5.69 and 2.48%, indicating the decomposition of SO4 <sup>2</sup><sup>−</sup> ions bound to the ZrO2 surface. The decomposition of SO4 <sup>2</sup><sup>−</sup> ions at 600–1000 ◦C affected the structural changes of ZrO2-SO4, causing a decrease in catalytic activity [64]. The decomposition of H2SO4 occurs through a two-stage endothermic process at high temperatures according to Equations (1) and (2).

$$\text{H}\_2\text{SO}\_4\text{ (aq)} \rightarrow \text{H}\_2\text{O}\_{\text{(l)}} + \text{SO}\_3\text{(g)}\tag{1}$$

$$\text{SO}\_{3\text{ (g)}} \rightarrow \text{SO}\_{2\text{ (g)}} + \frac{1}{2}\text{ O}\_{2\text{ (g)}}\tag{2}$$

**Figure 13.** TGA curves of (a) nano ZrO2, (b) nano ZrO2-SO4, (c) Pt/nano ZrO2-SO4. Reprinted and modified with permission from Dr. Utami, Ref. [9]. Copyright 2019 The Royal Society of Chemistry.

#### *4.6. Activity and Selectivity of Pt/ZrO2-SO4 Catalyst in LPDE Hydrocracking Application*

Utami et al. [6] reported the activity and selectivity of the Pt/ZrO2-SO4 catalyst applied in LPDE hydrocracking. The hydrocracking liquid fraction of the LDPE plastic waste thermal cracking is shown in Figure 14. Physically, the hydrocracking liquid fraction has a yellow color and pungent odor, which indicate the success of the hydrocracking process. The percentages of liquid fractions produced using the Pt1/SZ, Pt2/SZ, and Pt3/SZ catalysts were higher than that of the SZ catalyst. The percentage of liquid fraction obtained with the SZ catalyst was 57.92%, while with Pt1/SZ, Pt2/SZ, and Pt3/SZ the liquid fractions were 70.58, 71.15, and 74.60%, respectively. Through MS data, it was found that the hydrocarbon compounds in the gasoline range (C5–C12) were more commonly found in the liquid fraction samples that had used Pt3/SZ (catalyst with the highest acidity). Table 7 shows the gasoline fraction percentages from the hydrocracking reaction using Pt1/SZ, Pt2/SZ, and Pt3/SZ catalysts, which were 48.76, 64.22, and 67.51 *w*/*w*%, respectively. This cased that besides affecting the amount of hydrocracking liquid fraction produced, the addition of Pt metal can also increase selectivity towards the gasoline fraction [65].

**Figure 14.** The physical appearance of the hydrocracking liquid fraction (**a**) without catalyst, and with catalyst (**b**) ZrO2, (**c**) SZ, (**d**) Pt1/SZ, (**e**) Pt2/SZ3, (**f**) Pt3/SZ. Reprinted with permission from Dr. Utami, Ref. [14].

**Table 7.** Distribution of hydrocracking products using different catalysts (T = 250 ◦C, t = 60 m, catalyst to feed ratio = 1% *w*/*w*) [6].


The percentage of hydrocarbon compounds in the C5–C12 range can be seen to be greater than that of C13–C20 hydrocarbons. This shows that the hydrocracking liquid fraction of LDPE plastic waste had a higher gasoline fraction than the diesel fraction and that the four types of catalysts used had good selectivity towards the hydrocracking reaction that produces liquid fuel fraction (gasoline fraction).

Figure 15 shows the proportion of hydrocarbon compounds contained in the hydrocracked liquid fraction in the gasoline range, namely olefins, paraffin, isoparaffins, and naphthenes, with a total composition of 56.36 and respective amounts of 20.07, 14.60, and 6.81% *w*/*w* with use of the Pt3/SZ catalyst. Aromatic compounds in small amounts were also produced with a composition of 0.70% *w*/*w*, and only 1.46% *w*/*w* was indicated to be non-hydrocarbon compounds. Overall, olefin (unsaturated/double-bonded compound) was dominantly produced from the hydrocracking. This is because LDPE, as the plastic feed used, consists of olefin in which, during the hydrocracking reaction, not all olefins react with the hydride to become paraffin (saturated/single bond compound) [6,9].

#### *4.7. Stability Test of Pt/ZrO2-SO4*

A stability test of the Pt/nano ZrO2-SO4 catalyst, along with nano ZrO2 and nano ZrO2-SO4 for comparison, was carried out by Utami et al. [9] through a hydrocracking reaction of LDPE plastic waste with a catalyst/feed ratio 1% *w*/*w* and a temperature of 250 ◦C for 60 min. The catalyst stability test was carried out for six cycles with the same reaction conditions. Figure 16 shows that ZrO2, SZ, and Pt3/SZ had good catalytic performances when first used. The hydrocracking reaction with the ZrO2 catalyst showed a significant decrease in the percentage of liquid fraction produced in the fourth cycle, while in SZ this significant reduction occurred in the second cycle.

**Figure 15.** The composition of liquid yield in the gasoline fraction from the hydrocracking reaction of LDPE plastic waste using Pt3/SZ at 250 ◦C. Reprinted with permission from Dr. Utami, Ref. [6]. Copyright 2019 Elsevier.

**Figure 16.** Hydrocracking liquid fraction graphs of (a) nano ZrO2, (b) nano ZrO2-SO4, and (c) Pt/nano ZS3 Pt/nanoZS3-600. Reprinted and modified with permission from Dr. Utami, Ref. [9]. Copyright 2019 The Royal Society of Chemistry.

The catalytic activity of Pt3/SZ showed very good stability up to the sixth cycle. Aboul-Gheit et al. [59] reported that the catalytic activity of ZrO2-SO4 in the n-pentane isomerization reaction decreased in the fourth cycle and became inactive in the eighth cycle. In contrast, the Pt/ZrO2-SO4 catalyst showed stable activity until the tenth cycle. The Pt/ZrO2-SO4 catalyst showed high resistance to the deactivation related to the removal of coke from the catalyst surface, thus increasing the stability of the catalyst [36,37].

Figure 16 illustrates the proposed origin of the catalytic stability of ZrO2-SO4 and Pt/nanoZS3-600 nanoscales. Based on the activity and catalytic selectivity data, the conversion of LDPE using nano ZrO2-SO4 produced the highest amount of coke. In addition, this material had a high initial activity but low resistance to the deactivation process as its catalytic properties decreased rapidly in its consequent cycle of use. The formation of a coke can causes the pores and the active sites of the catalyst to be closed and thus reduce activity [44], i.e., the deactivation process that occurred in the ZrO2-SO4 nanocatalyst would have been difficult to control. To restore the activity of nanoZS3-600, catalyst regeneration, i.e., coke removal, is indispensable, especially in large-scale industrial applications. Promisingly, the Pt/nanoZS3-600 catalyst showed good activity, selectivity, and stability even after repeated use.

#### **5. Cr/ZrO2-SO4 Catalyst**

The metals that are widely used for bifunctional catalysts are usually transition metals with have incomplete orbitals that function as Lewis acid sites. One of the said transition metals is chromium (Cr) [66]. Cr can be doped on a carrier to enhance the catalytic activity of the host [67,68]. The addition of Cr metal to sulfated zirconia can also increase the acidity of the catalyst as the metal would contribute to the presence of Lewis acid sites [69]. The presence of Cr metal on sulfated zirconia also affects increasing the surface area of the catalyst [70,71]. Figure 17 illustrates the reaction mechanism of sulfated zirconia impregnated with Cr metal when interacting with ammonia during an acidity test. Hauli et al. [71] stated that the addition of chromium metal to sulfated zirconia not only increases the surface area of the catalyst but also stabilizes pure zirconia at high calcination temperatures. Increased temperature can thus remove sulfate groups and damage the porosity of the structure, causing the catalyst to deactivate. The physical appearance of the ZrO2-SO4 and Cr/ZrO2-SO4 catalyst (Figure 18) shows solid particle color change after the impregnation of Cr metal to the ZrO2-SO4 catalyst. The gray solid particle is formed due to the presence of Cr metal.

**Figure 17.** Proposed model of ammonia interaction on metal-embedded sulfated zirconia. Reprinted from Ref. [68]. Copyright Elsevier.

**Figure 18.** ZrO2-SO4 (**left**) and Cr/ZrO2-SO4 (**right**) catalyst. Reprinted with permisission from Dr. Hauli, Ref. [71].

#### *5.1. FITR and Acidity Characterizations of Cr/ZrO2-SO4 Catalyst*

Research on Cr/ZrO2-SO4 catalyst has been reported by Hauli et al. [72]. FTIR results of ZrO2, SZ 0.8-600 (SZ), 0.5% Cr/SZ (Cr1/SZ), 1.0% Cr/SZ (Cr2/SZ), and 1.5% Cr/SZ (Cr3/SZ) are shown in Figure 19. The spectra showed no specific differences between the spectra of Cr-embedded zirconia catalyst and sulfated zirconia. However, the specific absorption peak of the sulfate group at 1053–1224 cm−1, decreased with the presence of Cr. This is because the heating process involved in the loading of the metal had allowed sulfates to be released from the ZrO2 surface. The presence of metal produced not sulfate decomposition but rather the release of the sulfate groups from the surface of ZrO2 during the heating process [72]. Hanifah et al. [38] reported similar phenomena, namely that the presence of monometals and bimetals on ZrO2-SO4 release of sulfate group materialized whilst its decomposition did not.

**Figure 19.** FTIR spectra of ZrO2, SZ, Cr1/SZ, Cr2/SZ, Cr3/SZ. Reprinted with permission from Dr. Hauli, Ref. [72]. Copyright 2019 Trans Tech Publication.

The presence of empty orbitals in Cr metal allows electrons from other atoms to take their place in the orbital in what would then contribute to higher acidity of the Crcontaining material. Acidity test results of Cr-containing catalysts are presented in Table 8 confirming this. The acidity of the catalyst increased after the addition of Cr metal. The Cr1/SZ catalyst was the catalyst with the highest acidity value with 8.22 mmol/g. The low acidity value at high Cr-metal concentration was caused by the presence of metal particle aggregates that had covered the active metal sites on the carrier material. The more Cr metal that was embedded, the greater the amount of metal not accommodated in the pores of the carrier material, leading to the formation of aggregates.


**Table 8.** Acidity values of the catalysts [72].

The acidity test of the catalyst was carried out to determine the total acidity value of each catalyst. The FTIR spectra after the acidity test results for the ZrO2, SZ, Cr1/SZ, Cr2/SZ, and Cr3/SZ catalysts are shown in Figure 20. The results show the same absorption peaks at wave numbers 1119 and 1404 cm−1, confirming the presence of the Lewis and Brønsted acid sites on each catalyst. Cr-embedded sulfated zirconia catalyst showed a higher intensity of Lewis and Brønsted acid sites than before Cr metal was added. The spectra of the Cr/SZ catalyst had the highest acid site intensity, indicating that Cr2/SZ had a high acidity value. The results of the acidity test of the catalysts confirmed that Cr2/SZ had the highest acidity value of 8.22 mmol/g.

**Figure 20.** FTIR spectra of SZ, Cr1/SZ, Cr2/SZ, and Cr3/SZ after acidity test. Reprinted with permission from Dr. Hauli, Ref. [72]. Copyright 2019 Trans Tech Publication.

#### *5.2. XRD Characterization of Cr/ZrO2-SO4 Catalyst*

The diffraction patterns of ZrO2, SZ, Cr1/SZ, Cr2/SZ, and Cr3/SZ catalysts are presented in Figure 21. The bearing of Cr metal on sulfated zirconia provided no change in the crystal phase structure of ZrO2. The main peaks were found in the 2θ region around 28◦, 31◦, and 50◦ denoting plane distances of d-111 (3,2 Å), d111 (2,8 Å), d220 (1,8 Å) with the highest intensity in the 28◦ region. Sulfate and Cr impregnation on ZrO2 have a lower intensity peak than ZrO2. This is due to the presence of sulfate on ZrO2 and to Cr metal covering the surface of SZ, inhibiting the growth of ZrO2 crystals and thereby decreasing the crystallinity of the materials [73,74].

**Figure 21.** Diffraction patterns of ZrO2, SZ, Cr1/SZ, Cr2/SZ, Cr3/SZ. Reprinted with permission from Dr. Hauli, Ref. [72]. Copyright 2019 Trans Tech Publication.

#### *5.3. SAA Characterization of Cr/ZrO2-SO4 Catalyst*

Characterization of pore characteristics including surface area, pore diameter, and pore volume was carried out by SAA analysis. The results of the SAA measurements of the catalysts are shown in Table 9. As can be seen, the addition of Cr metal to sulfated zirconia increased the surface area, pore diameter, and pore volume of the catalyst. This can be attributed to the uniform distribution of Cr metal on the catalyst surface [69,70]. The surface area of the catalyst, however, decreased upon the addition of higher concentrations of Cr metal, namely at Cr3/ZS, due to the entry of Cr metal into the catalyst pores causing agglomeration of metal atoms that covered the catalyst pores [74].


**Table 9.** Pore characteristics of the catalysts [72].

The adsorption and desorption isotherm patterns for ZrO2, SZ, Cr1/SZ, Cr2/SZ, and Cr3/SZ catalysts are shown in Figure 22. Based on the IUPAC classification, all catalysts showed a type IV isotherm pattern, which is characteristic of the isotherm pattern for mesoporous materials with pore diameter sizes of 2–50 nm. The adsorption–desorption isotherm patterns of the Cr1/SZ and Cr2/SZ catalysts demonstrated monolayer absorption of nitrogen gas on the surface when *P*/*P*<sup>0</sup> < 0.2. At a relative pressure of 0.2 < *P*/*P*<sup>0</sup> < 1, the isotherm curve experienced a sharp increase in volume representing a multilayer arrangement. In the Cr3/SZ catalyst, the absorption of a monolayer of nitrogen gas on the surface occurred when *P*/*P*<sup>0</sup> < 0.4 and experienced a sharp increase in volume at 0.4 < *P*/*P*<sup>0</sup> < 1. The ZrO2, Cr2/SZ, and Cr3/SZ catalyst had a type H4 hysteresis, while SZ and Cr1/SZ had type H3. Type H3 hysteresis showed no absorption limit at high *P*/*P*0. Type H4 hysteresis is associated with narrow slit pores [4,72]. These three types of catalysts exhibit the characteristics of porous materials. This form of porous material is composed of aggregates of particles such as plates that form pore gaps [10].

**Figure 22.** Adsorption desorption isotherm patterns of ZrO2, SZ, Cr1/SZ, Cr2/SZ, and Cr3/SZ. Reprinted with permission from Dr. Hauli, Ref. [72]. Copyright 2019 Trans Tech Publication.

#### *5.4. Activity and Selectivity Tests of Cr/ZrO2-SO4 Catalyst*

A catalytic activity test of the Cr/ZrO2-SO4 catalyst in the hydrocracking reaction of LDPE into liquid fuel has been carried out by Hauli et al. [72]. The hydrocracking reaction was carried out at a temperature of 250 ◦C using ZrO2, SZ, Cr1/SZ, Cr2/SZ, and Cr3/SZ catalysts. The percentages of conversion yields of the hydrocracking of each catalyst are presented in Table 10. The presence of sulfate and Cr metal in ZrO2 can increase the yield of liquid products from the hydrocracking reaction. The liquid product increased along with the increase in the amount of Cr metal deposited on ZrO2. This can be attributed to the role of acid sites in the catalyst (from sulfate and metal Cr) which contribute to the active sites of the catalyst, thereby increasing its catalytic activity [50].


**Table 10.** Distribution of LDPE plastic hydrocrack products on various catalysts [72].

The highest conversion of liquid product was produced using the Cr2/SZ catalyst, which was 40.99%, with a lesser amount of 37.51% with the use of the Cr3/SZ catalyst. Moreover, the Cr2/SZ catalyst had a higher acidity value than Cr3/SZ. In addition, the surface area of the catalyst also affects the catalytic activity of the catalyst in the hydrocracking of LDPE plastics [75,76]. Accordingly, the Cr2/SZ had a larger surface area, resulting in greater conversion. The modification of Cr on sulfated zirconia demonstrated a reduction in coke formation, thereby increasing the feed interaction on the active sites of the catalysts.

The selectivity of the catalysts towards liquid products in the hydrocracking reaction are shown in Figure 23. All the catalysts showed higher selectivity in the gasoline fraction than the diesel fraction. The presence of sulfate and Cr in sulfated zirconia increased the selectivity towards the gasoline fraction (C5–C12) and decreased the selectivity towards the diesel fraction (C13–C20), as expected. The highest selectivity of the gasoline fraction was obtained from the use of the Cr2/SZ catalyst, which was at 93.42%.

**Figure 23.** Liquid product selectivity of ZrO2, SZ, Cr1/SZ, Cr2/SZ, and Cr3/SZ catalysts. Reprinted with permission from Dr. Hauli, Ref. [72]. Copyright 2019 Trans Tech Publication.

#### **6. Ni/ZrO2-SO4 Catalyst**

#### *6.1. FITR Characterization of Ni/ZrO2-SO4 Catalyst*

Research on the ZrO2-SO4 catalyst was also carried out about its application in the hydrocracking of used cooking oil into liquid fuel. Modification of ZrO2-SO4 with Ni metal has been reported by Aziz et al. [73]. Figure 24 show the FTIR spectra of Ni-SZ 1, Ni-SZ 2, and Ni-SZ 3. Impregnated Cr metal to SZ caused the presence of a new peak in the area of 1103 and 1141 cm<sup>−</sup>1, confirming the S-O-S stretching of from SO4 ion from SZ coordinated with Ni metal [76,77]. However, the sulfate spectra at 1002–1219 cm−<sup>1</sup> disappear due to the Ni metal coverage on the SZ surface and the higher calcination temperature.

**Figure 24.** FTIR spectra of SZ (a), Ni-SZ 1 (b), Ni-SZ 2 (c), and Ni-SZ 3 (d) catalysts. Reprinted with permission from Aziz, Ref. [73]. Copyright 2020 Budapest University.

#### *6.2. Acidity and SAA Analysis of Ni/ZrO2-SO4 Catalyst*

The total acidity and pore characteristic of Ni-SZ catalysts is shown in Table 11. It can be seen that the sulfation of ZrO2 (SZ catalyst) and Ni metal impregnated to SZ catalysts successfully increase the total acidity of the catalyst. Ni metal has a vacant p orbital that will accepting electron pair and acts as a Lewis acid site [78]. The Ni-SZ 3 catalyst was the catalyst with the highest total acidity of 4.235 mmol/g.


**Table 11.** The acidity and pore characteristics of the Ni-SZ catalysts [73].

The pore characteristic of the catalyst shows the increase in surface area after the impregnation of Cr metal onto the SZ catalyst due to the highly dispersion of Ni metal on the pore and surface of the SZ catalyst [79]. However, the presence of Ni causing the form of pore-blocking that significantly decreases the pore diameter of Ni-SZ catalysts [72].

The selectivity of the Ni-ZrO2-SO4 catalyst is shown in Table 12. ZrO2 and SZ catalysts were observed to have lower activity and selectivity than Ni-SZ due to the lowest acidity and surface area that will produce considerable amounts of coke (block the active site) and decreases the amount of liquid product [80,81]. The addition of Ni metal (Ni-SZ 3 catalyst) increased the acidity (4.23 mmol/g) and surface area (11.68 m2/g) of ZrO2, thereby increasing its activity and selectivity in the hydrocracking process [82,83]. The largest amount of liquid product produced was gasoline with the highest selectivity was produced by the Ni-SZ 3 catalyst with the diesel fraction (C5–C12) reached 100%.

**Table 12.** Selectivity of ZrO2, SZ, Ni-SZ 1, Ni-SZ 2, and Ni-SZ 3 catalysts in the hydrocracking reaction of used cooking oil into liquid fuel [73].


#### **7. Conclusions**

Zirconia and its modified heterogeneous catalyst forms hold great potential in hydrocracking reaction applications to convert LDPE waste into liquid fuels with their excellent activity, selectivity, and stability. The sulfation process on ZrO2 with various concentrations of sulfuric acid and calcination temperatures succeeded in increasing the acidity of ZrO2. The ZrO2-SO4 catalyst treated with Platinum (Pt) and Chrome (Cr) transition metals had significantly increased acidity. Characterization analyses confirmed that Pt and Cr metals had been successfully impregnated on the SZ surface. The percentage of liquid fraction obtained with the use of the Pt/ZrO2-SO4 catalyst proofed better activity, selectivity, and stability than Cr/ZrO2-SO4 and ZrO2-SO4. The optimum amount of liquid fraction produced from the hydrocracking reaction LDPE plastic waste with the Pt/ZrO2-SO4 catalyst was 67.515%, while that from the Cr/ZrO2-SO4 catalyst was 40.99%. The development of Ni on the ZrO2-SO4 catalyst also demonstrated an increase in selectivity in the hydrocracking reaction of used cooking oil into liquid fuel. The selectivity of the Ni-SZ catalyst was found to be better than that of SZ. The percentages of gasoline fractions produced were 100%.

#### **8. Future Suggestion**

Zirconia-based nanocatalysts have bright prospects for application in various industrial areas such as petroleum cracking, biofuel synthesis, pharmaceutical, and the synthesis of various organic materials. The potential application of sulfated nanozirconia catalysts is shown in the Figure 25. Due to characteristics such as being non-toxic and easy to regenerate, as well as having a large surface area and high thermal and structural resistance, nanozirconia catalysts also have the potential to be used in the pharmaceutical industry. Preliminary studies in our laboratory have shown that this catalyst has the potential to be used as a solid acid catalyst in the synthesis of nitrobenzene from benzene.

**Figure 25.** Potential application of sulfated nanozirconia catalysts.

**Author Contributions:** Conceptualization, R.A.P., S.J.S., M.U., L.H. and A.K.A.; validation, R.A.P., M.U., L.H. and A.K.A.; investigation, R.A.P.; resources, R.A.P.; data curation, R.A.P., M.U., L.H. and A.K.A.; writing—original draft preparation, R.A.P. and S.J.S.; writing—review and editing, R.A.P.; visualization, K.W.; supervision, R.A.P. and K.W.; project administration, K.W.; funding acquisition, K.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Postdoctoral Research Grant Universitas Gadjah Mada (Contract Number: 6144/UN1/DITLIT/DIT-LIT/PT/2021).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**Honghai Wang 1,2, Wenda Yue 1,2, Shuling Zhang 1,2, Yu Zhang 1,2, Chunli Li 1,2 and Weiyi Su 1,2,\***


**Abstract:** Silica xerogels have been proposed as a potential support to immobilize enzymes. Improving xerogels' interactions with such enzymes and their mechanical strengths is critical to their practical applications. Herein, based on the mussel-inspired chemistry, we demonstrated a simple and highly effective strategy for stabilizing enzymes embedded inside silica xerogels by a polydopamine (PDA) coating through in-situ polymerization. The modified silica xerogels were characterized by scanning and transmission electron microscopy, Fourier tranform infrared spectroscopy, X-ray diffraction, X-ray photoelectron spectroscopy and pore structure analyses. When the PDA-modified silica xerogels were used to immobilize enzymes of *Candida antarctica* lipase B (CALB), they exhibited a high loading ability of 45.6 mg/gsupport, which was higher than that of immobilized CALB in silica xerogels (28.5 mg/gsupport). The immobilized CALB of the PDA-modified silica xerogels retained 71.4% of their initial activities after 90 days of storage, whereas the free CALB retained only 30.2%. Moreover, compared with the immobilization of enzymes in silica xerogels, the mechanical properties, thermal stability and reusability of enzymes immobilized in PDA-modified silica xerogels were also improved significantly. These advantages indicate that the new hybrid material can be used as a low-cost and effective immobilized-enzyme support.

**Keywords:** *Candida antarctica* lipase B; silica xerogel; enzyme immobilization; polydopamine; modification

#### **1. Introduction**

Biocatalysts play a vital role in various scientific fields due to their unique advantages, such as high substrate specificity, outstanding catalytic ability and mild reaction conditions. Biocatalysis, applied in ester synthesis, is useful and its synthetic products can be identical to natural products. Recently, a transesterification reaction catalyzed by lipase (triacylglycerol ester hydrolase, EC 3.1.1.3) has been performed to produce esters [1]. Lipase-catalyzed reactions have been applied to the synthesis of chiral drugs [2], wax esters [3], structural lipids [4] and biodiesel [5]. However, the main bottlenecks of enzyme application are its low thermal stability, poor operational stability and the difficulty of reusing enzymes. Therefore, significant effort has been devoted to exploiting immobilization strategies to stabilize enzymes and endow them with greater stability and reusability [6,7].

In immobilizing enzymes, it is necessary to select an appropriate support material, which can improve the properties of enzymes. Lipases are widely recognized to have a hydrophobic domain [8]. The hydrophobic immobilization of a lipase can act upon its domains, to increase its activity and stability, by interfacial activation [9,10]. Thus, materials comprised with ordered mesoporous organosilica, in which organic hydrophobic groups are homogeneously distributed within their frameworks, may be ideal supports for lipase immobilization. Silica xerogel, thanks to a high specific surface area, good mechanical strength, inertness and stability at high temperature, has attracted much attention in

**Citation:** Wang, H.; Yue, W.; Zhang, S.; Zhang, Y.; Li, C.; Su, W. Modification of Silica Xerogels with Polydopamine for Lipase B from *Candida antarctica* Immobilization. *Catalysts* **2021**, *11*, 1463. https:// doi.org/10.3390/catal11121463

Academic Editors: Elisabeth Egholm Jacobsen, Simona M. Coman and Madalina Tudorache

Received: 5 November 2021 Accepted: 27 November 2021 Published: 30 November 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

enzyme immobilization [11,12]. The xerogel synthesis of entrapped enzymes based on silicon-containing compounds has been widely used [13]. The formation principle of a silicon sol−gel matrix for enzyme immobilization consists in the transition of a silicon alkoxide sol into a gel as a consequence of hydrolysis and polycondensation reactions, with the subsequent transformation into a monolithic xerogel, powder or film coating [14]. This method retains the inherent structures biocomposites, showing enzymatic activity and an expanded range of conditions for catalysis [15]. Enzymes' inclusion in xerogel structures allows increasing the resistances thereof to different physical and chemical factors, such as temperature, pH, radiation and aggressive compounds. In general, xerogel-encapsulated enzyme technology is a method for preparing bioactive nanocomposites [16].

However, the limitation of xerogels' entrapping of enzymes is the shrinkage of their structures, which is not conducive to entrapping enzyme due to the large capillary force caused by continuous internal shrinkage [17]. In our previous work, we proposed a strategy of producing an immobilized enzyme-containing xerogel coating on metal packings for reactive distillation, but there was still a relatively weak interaction between the enzyme and the surface of the support, which often lead to the leakage of the enzyme from the support [18]. In addition, mesoporous silica shows a certain brittleness [19]. When a xerogel is subjected to an external force, the partial or complete rupture of its skeletal structure will also lead to the loss and leakage of an entrapped enzyme. Many studies have been conducted to improve the interactions between enzymes and supports, or the mechanical properties of the support. To date, polyacrylamide [20] and glutaraldehyde [21] have been used as crosslinking agents to increase enzyme loading, while glycerol has been used to prevent xerogel cracking [22]. In fact, a simple and effective method of the two problems latter is to coat an active protective layer on the surfaces of pre-xerogel polymers. Inspired by mussel adhesion proteins, polydopamine (PDA) technology has attracted extensive research [23,24]. Dopamine molecules have been shown to self-polymerize under alkaline conditions, leading to a facile deposition of PDA coating on a material's surface [25]. More importantly, the residual quinone on the surface of polydopamine or an intermediate displays a nucleophilic amino reaction that can be covalently connected with nucleophilic biological molecules, producing a polydopamine coating that is robust and durable [26]. This provides a new way of improving the interactions between enzymes and supports, and of enhancing the mechanical properties of xerogels. However, to the best of our knowledge, there are few reports on dopamine self-polymerization deposition on the surfaces of xerogel supports aimed at improving the mechanical properties of and interactions between enzymes and supports.

Surface modification with polydopamine has already become an efficient and feasible method of endowing inorganic materials with biological functionality since Messersmith et al. pioneered the single-step formation of polymer film-based dopamine on various substrates [27]. Meanwhile, this method is not involved in complex linkers and is free of organic solvents, making it suitable for biomaterial applications. Furthermore, the abundant functional groups (i.e., catechol and amine) existing on such modified surfaces could enhance enzymes' binding abilities [28]. Therefore, in the present work and based on this idea, we design a new hybrid support by modifying, with polydopamine, the surfaces of silica xerogels. Specifically, CALB was chosen as a model enzyme. Firstly, CALB was encapsulated in silica xerogels by the sol–gel method, denoted as SiO2–CH3–CALB. Second, in order to prevent enzyme leakage and improve enzyme stability, the polymer networks in xerogels were coated with polydopamine (denoted as SiO2–CH3–CALB@PDA). Finally, the xerogels' mechanical properties and the enzyme-immobilizing ability of SiO2- CH3–CALB and SiO2–CH3–CALB@PDA were investigated in detail. The results show that SiO2–CH3–CALB@PDA had a significant CALB-embedding ability. Compared with the SiO2–CH3–CALB, the results showed that PDA-modified SiO2–CH3–CALB had better mechanical properties, thermal stability, storage stability and reusability. This indicated that the new hybrid silica xerogel could be used as a low-cost and relatively effective immobilized-enzyme support.

#### **2. Results and Discussion**

#### *2.1. Characterization*

Figure 1a shows a FTIR spectral comparison of SiO2–CH3–CALB and SiO2–CH3– CALB@PDA. The adsorption peaks at 777 cm−<sup>1</sup> and 445 cm−<sup>1</sup> corresponded to the Si−O−Si group [29]. The band at 1277 cm−<sup>1</sup> was assigned to the characteristic peak of Si–CH3, which proved that MTMS successfully deposited a methyl polymer layer on the silica surface. Other bands, at 3388 cm−<sup>1</sup> and 1643 cm−1, belonged to the stretching and bending vibrations of −OH [30]. After modification by polydopamine, the vibration absorption peak at 3388 cm−<sup>1</sup> was significantly enhanced and widened, which was related to the catechol composition of polydopamine [31]. In addition, SiO2–CH3–CALB@PDA showed a peak at 1508 cm−1, which was ascribed to the bending vibrations of indolequinone groups [32]. The XRD patterns of SiO2–CH3–CALB and SiO2–CH3–CALB@PDA are illustrated in Figure 1b. There was a relatively wide peak at 2*θ* = 22◦, which is characteristic of amorphous silica [33]. The peak at 2*θ* < 10◦ was due to the siloxane network and the xerogel's structure, composed of ordered organic layers [34]. After modification by polydopamine, the intensity of the characteristic peak at 10◦ became weak, implying the microstructure of SiO2–CH3–CALB had changed due to the uniformly distributed deposition of polydopamine within the structure of the xerogel [35]. These results indicate that a polydopamine coating formed on the silica xerogel through self-polymerization.

**Figure 1.** Physical properties of SiO2–CH3–CALB and SiO2–CH3–CALB@PDA. (**a**) FTIR spectra and (**b**) XRD 2*θ* scans.

Microstructural images of SiO2–CH3–CALB@PDA are shown in Figure 2. Figure 2a,b shows the SEM images of the SiO2–CH3–CALB and SiO2–CH3–CALB@PDA prepared in this work, respectively by panel. They were constituted by the agglomeration of many silica clusters in uniform shape. Compared with SiO2–CH3–CALB, the surface of SiO2–CH3– CALB@PDA was rougher and looser between clusters, indicating that a polydopamine layer had formed on the Si−O−Si surface. In this structure, the formation of a protective enzyme barrier can absorb and disperse most of the energy from external forces, preventing the xerogel from breaking [36]. TEM images of monodispersed SiO2–CH3–CALB@PDA showed that its particles have a relatively uniform, nano-scale size (Figure 2c); a more intuitive expression is shown in Figure 2d. The shape of the SiO2–CH3–CALB@PDA particles irregularly spherical. Additionally, the SiO2–CH3–CALB@PDA surface had openframework channels (Figure 2e) that facilitated the diffusion of the substrate and product molecules [37]. Elemental mapping analysis (Figure 2g−j) demonstrated that PDA was uniformly distributed within the xerogel (as these contained nitrogen), and oxygen, carbon

and silicon were also found in the SiO2–CH3–CALB@PDA surface. Notably, the oxygen content was high.

**Figure 2.** SEM images of (**a**) SiO2–CH3–CALB, (**b**) SiO2–CH3–CALB@PDA, (**c**–**e**) TEM images of SiO2–CH3–CALB@PDA and (**f**–**j**) elemental mapping analysis of SiO2–CH3–CALB@PDA (TEM).

XPS measurements in Figure 3 confirmed the existence of polydopamine coating on SiO2–CH3–CALB@PDA. XPS spectra of SiO2–CH3–CALB@PDA show the presence of C, N, O and Si (Figure 3a). The different chemical states of C, O and N in the regional spectra reveal the complex properties of polydopamine on SiO2–CH3–CALB@PDA (Figure 3b−d). The main peaks of C 1s spectra (Figure 3b) at 283.9, 284.5, 285.7, 286.4 and 287.2 eV, respectively, corresponding to C−C, C−N, C−O, C=O bands, O−C=O and feature for aromatic carbon species in the polydopamine. In the O1s peak (Figure 3c), two peaks were observed at 531.9 eV and 532.7 eV, respectively, which belonged to O atoms of polydopamine in the form of quinone and catechol [38]. The high-resolution spectra of N 1's peak are shown in Figure 3d. The main peak, at 399.4 eV, indicated the existence of R2NH and RNH2, while the peak at 401.2 eV was attributed to R3N [39]. This result indicates that an adhesive polydopamine coating formed on the surface of the Si−O−Si network structure of the xerogel by self-polymerization.

Figure 4a−c and Figure 4d−f show the sol–gel process and finished products, SiO2– CH3–CALB and SiO2–CH3–CALB@PDA, respectively. They were no significant differences in the solution phase (sol), showing a slightly yellow liquid (Figure 4a,d). Then, they entered the gel phase, the SiO2–CH3–CALB showed a milky white gel block, while the SiO2–CH3–CALB@PDA showed a black transparent gel block (Figure 4b,e). This may be explained by dopamine's having begun to self-polymerize into polydopamine on the

gel network. After the final drying stage, The final samples of SiO2–CH3–CALB and SiO2–CH3–CALB@PDA were obtained by grinding (Figure 4c,f).

**Figure 3.** XPS analyses of SiO2–CH3–CALB@PDA. (**a**) Survey scan, (**b**) C 1s, (**c**) O 1s and (**d**) N 1s.

With the aim of further prove the PDA can delay shrinkage of xerogel, analysis of BET of SiO2–CH3–CALB and SiO2–CH3–CALB@PDA were taken into account. It can be seen from their adsorption–desorption curves, in Figure 4g, that they had strong interaction with N2 at low pressure and presented typical Langmuir type IV curves. The H2 hysteresis loops were also observed, indicating the mesoporous structure and the characteristics of 'ink bottle' pores [40]. From the pore size distribution curve in Figure 4h, it can be seen that the pore size (15.02 nm) and BET surface area (165.84 m2·g<sup>−</sup>1) of SiO2–CH3–CALB@PDA were larger than those of SiO2–CH3–CALB (13.52 nm and 121.67 m<sup>2</sup>·g−1), which we believe to be due to the PDA coatings and deposits on the surface of the Si−O−Si network structure during the sol–gel process, delaying the gel shrinkage [41].

The immobilization capacity of SiO2–CH3–CALB@PDA was evaluated by investigating the CALB loading. As shown in Figure 4i, the amount of CALB immobilized on SiO2–CH3–CALB@PDA increased with increasing CALB concentration. When the CALB concentration was 14.5 mg/mL, the CALB loading increased to 45.6 mg/g. However, when the CALB concentration was more than 14 mg/mL, a decline in the activity recovery of the immobilized CALB was observed. The loading reached a maximum at a high enzyme concentration (~16 mg/mL), and there is a slightly continuous decrease in the enzyme activity when the enzyme concentration exceeds 14.5 mg/mL. This can be explained by the fact that excess CALB loading will easily lead to the congestion of the enzyme molecules. Therefore, the resulting spatial constraint can increase the mass transfer resistance of the

substrate and product, which is expressed as reducing activity [42]. Therefore, the optimum CALB concentration was chosen as 14.5 mg/mL. In this case, the CALB loading is efficient (activity recovery higher than 93%) without sacrificing excess enzyme to unnecessary use. Meanwhile, compared with the enzyme loading of 28.5 mg/g on pristine SiO2–CH3–CALB at an initial CALB concentration of 14.5 mg/mL, the enzyme loading on SiO2–CH3–CALB@PDA reached as high as 45.6 mg/g, nearly twice as high as that on SiO2–CH3–CALB. As mentioned above, the modification of PDA provided a barrier for the enzyme, and covalent linking enhanced the interaction between the enzyme and the support, effectively preventing enzyme leakage.

**Figure 4.** Sol−gel process of (**a**–**c**) SiO2–CH3–CALB, (**d**–**f**) SiO2–CH3–CALB@PDA, (**g**,**h**) pore structure and (**i**) enzyme loading.

#### *2.2. Strategy for Immobilizing CALB and Possible Mechanism*

Figure 5 shows the internal microstructure of the SiO2–CH3–CALB- and SiO2–CH3– CALB@PDA-immobilized enzyme and the mechanism of the polydopamine-modified immobilized enzyme. The Si−O−Si polymer network skeleton was obtained by hydrolysis and a condensation reaction with TMOS and MTMS as co-precursors, and the enzyme molecules were embedded in the Si−O−Si network. In hydrolysis reaction, the methyl group of MTMS was not involved in hydrolysis, replacing and cross-linking with hydroxyl groups on Si−O−Si network, which provided a necessary condition for the development of hydrophobic properties [43]. The polydopamine-modified immobilized enzyme was based on the synergistic sol–gel mechanism [44]. In short, dopamine nanoparticles were uniformly mixed into the sol. In this system, dopamine hydrochloride was self-polymerized into PDA under alkaline conditions and deposited on the surface of Si−O−Si network. Moreover, the residual quinone functional groups presented in the polydopamine coat-

ing were reactive toward nucleophilic groups, and CALB could couple covalently with polydopamine through Michael-type addition or Shiff-based formation [45,46]. We expected that the resulting SiO2–CH3–CALB@PDA xerogels would have excellent mechanical strengths and enzyme activity stabilities.

**Figure 5.** The internal microstructures of SiO2–CH3–CALB and SiO2–CH3–CALB@PDA and the possible mechanism of the polydopamine-modified immobilized enzyme.

#### *2.3. Mechanical Properties*

In practical applications, xerogel is prone to deformation under external force, resulting in enzyme leakage or inactivation. Therefore, strength is crucial for the application of xerogel in organic catalysis. We experimentally compared the strengths of SiO2–CH3–CALB and SiO2–CH3–CALB@PDA. The compressive stress–strain curves for SiO2–CH3–CALB and SiO2–CH3–CALB@PDA are presented in Figure 6. A macroscopic compression experiment showed that the SiO2–CH3–CALB@PDA xerogel model could withstand higher pressures (12.55 Mpa) than that of SiO2–CH3–CALB (9.00 Mpa), and the strain of SiO2– CH3–CALB@PDA (9.64%) was greater than that of SiO2–CH3–CALB (9.07%). In addition, the fracture modes of the two materials were also significantly different. The fracture mode of SiO2–CH3–CALB was similar to that of brittle materials, while the fracture mode of SiO2–CH3–CALB@PDA was similar to that of viscoelastic materials [47,48]. This can be ascribed to two factors. On the one hand, PDA was deposited on the surface of the Si−O−Si network, which reduced the capillary force generated by the shrinkage of the xerogel during drying [29]. On the other hand, the surface of the PDA contained a large number of functional groups that could interact with Si−O−Si chains, serving as crosslinking sites to increase the mechanical strength of the SiO2–CH3–CALB@PDA xerogel, preventing it from breaking under pressure [32]. Overall, the internal network structure of the xerogel and polydopamine coatings played a key role in the whole compression process, confirming the formation of a stable xerogel. After modification by PDA nanoparticles, the mechanical properties of the xerogels were improved. This occurred because polydopamine can interact with the xerogel matrix, increasing xerogel hardness and improving brittleness.

**Figure 6.** Stress–strain curves of SiO2–CH3–CALB and SiO2–CH3–CALB@PDA.

#### *2.4. Stability of Immobilized CALB*

The free and immobilized CALB was incubated at 60 ◦C for a certain time to investigate their thermal stability. The influence of temperature towards the stability of CALB is illustrated in Figure 7a. With the increasing of incubation time, the hydrolytic activity of free CALB and SiO2–CH3–CALB decreased, and free CALB was entirely deactivation after 3 h treatment. However, the SiO2–CH3–CALB@PDA exhibited better stability, which maintained 36.5% of its activity after 6 h of incubation. These results revealed that the better thermal stability of SiO2–CH3–CALB@PDA among free CALB and SiO2–CH3–CALB was attributed to the strong covalent bonds that formed through the reaction between the amine in the enzyme and the electrophilic groups in the PDA [49]. In addition, the PDA layer provides a stiffer external backbone to protect the CALB molecule from high temperatures [50]. Improvements in thermal stability will expand the range of applications for immobilized enzymes.

In order to investigate the storage stability of SiO2–CH3–CALB and SiO2–CH3–CALB@PDA, the examination was carried out at room temperature for 90 days. As shown in Figure 7b, SiO2–CH3–CALB exhibited 66.3% of its initial activity after 90 days, while SiO2–CH3– CALB@PDA exhibited approximately 71.4% under the same conditions. The high storage stability exhibited by CALB can be explained by the protective effect of the Si−O−Si network in the silica structure, which protects the enzyme activity inside, and further enhances its structural stability. The interactions of different geometries of the enzyme and support may have a significant influence on the enzyme activity. Generally, it is accepted that the highly curved surface reduces the possibility of enzyme denaturation and inhibits lateral interactions between adjacent enzymes, further leading to the structural stability and persistent activity of the adsorbed enzyme [51,52]. Additionally, multiple points of binding were observed between the PDA support and CALB in SiO2–CH3–CALB@PDA, which formed the PDA coating on the surface of the polymer network inside the xerogels, acting in a protective role [53,54]; this could explain their greater activity in external environments.

**Figure 7.** Thermal stability at 60 ◦C (**a**) and storage stability at 25 ◦C (**b**) of free CALB, SiO2–CH3–CALB and SiO2– CH3–CALB@PDA.

#### *2.5. Transesterification and Reusability*

Some enzymes have been used as biocatalysts to synthetize ester compounds, among which CALB can form high value-added ester products by transesterification reactions. As one of the major biocatalysts for ester synthesis, CALB can catalyze the transesterification of *n*-butanol with ethyl acetate to produce butyl acetate, which is an excellent organic solvent. Figure 8 shows the CALB-catalyzed synthesis of butyl acetate by transesterification of *n*-butanol with ethyl acetate. The reaction is a solvent-free system, and was carried out in a batch reactor at 70 ◦C. In a solvent-free system, the enzyme directly acts on the substrate, increases the substrate concentration, improving the reaction rate and selectivity and reducing the damage of organic solvents to the enzyme [55]. Therefore, we chose ethyl acetate as a reactant, as it also acts as a solvent in the CALB-catalyzed synthesis of butyl acetate.

The transesterification of *n*-butanol with ethyl acetate was selected as a target reaction to evaluate the conversion efficiency and reusability of immobilized CALB in the present work. The conversion of *n*-butanol and the reusability of SiO2–CH3–CALB and SiO2–CH3– CALB@PDA were compared under optimal active conditions. As shown in Figure 9, in the first cycle, the conversion of *n*-butanol of SiO2–CH3–CALB and SiO2–CH3–CALB@PDA retained 52.42% and 57.67%, respectively. For SiO2–CH3–CALB, CALB molecules were encapsulated in the xerogel polymer network by physical adsorption, Virgen-Ortíz et al. have reported some substrates/product may produce the enzyme's release from physically absorbed enzymes, so the leakage of CALB was prone to denaturation or inactivation during the reaction [56]. The decrease in conversion was observed in the first five cycles. After the fifth cycle, the activity began a slow deceleration state, lasting for the next three cycles. After eight cycles, SiO2–CH3–CALB@PDA retained more than a 30.84% conversion of *n*-butanol. SiO2–CH3–CALB retained a 25.04% conversion of *n*-butanol. The conversion

of *n*-butanol loss could be due to enzyme leakage during washing and enzyme deactivation during repeated uses [57]. As the reaction produces a by-product of ethanol in the batch reactor system, resulting in enzyme inhibition, inhibition will reduce lipase activity. High concentrations of *n*-butanol inhibit the synthesis of butyl acetate catalyzed by immobilized CALB. This inhibitory effect has been found in the reaction among butyric acid and lauric acid with ethanol [58,59]. Therefore, operational stability of the enzyme is not too high. Considering SiO2–CH3–CALB@PDA had better reusability, storage stability and mechanical strength, SiO2–CH3–CALB@PDA is more applicable for practical applications.

*2.6. Comparison of Butyl Acetate Production Using Previous Lipase Biocatalysts*

The prepared catalyst of SiO2–CH3–CALB@PDA possessed the advantages of biocompatibility, environmental friendliness, operating convenience and safety. Compared with previous lipase catalysts, such as SiO2–CH3–CALB and Novozyme 435, the catalytic efficiency of SiO2–CH3–CALB@PDA (57.67%) in the transesterification reaction system was slightly higher than those of SiO2–CH3–CALB (52.42%) and Novozyme 435 (55.30%) [60]. Although the improvement in operational stability and catalytic performance is not obvious, the polydopamine modification strategy is worth adopting; it can improve the immobilized enzyme loading and balance the mechanical properties of the supports, which expands the application range of immobilized enzymes in some special cases.

#### **3. Experimental Section**

#### *3.1. Materials*

Tetramethoxysilane (TMOS), methyltrimethoxysilane (MTMS), methanol (MeOH), sodium fluoride (NaF), polyethylene glycol (PEG, MW 400), dopamine hydrochloride, 4-nitrophenyl palmitate (*P*-NPP), Coomassie Brilliant Blue G250, bovine serum albumin (≥96%), *n*-butanol, ethyl acetate, *n*-propanol and sodium-phosphate buffer (PBS, 0.1 M, pH 7.5) were purchased from Aladdin (Shanghai, China). *Candida antarctica* lipase B (CALB) was provided from Novozymes (Copenhagen, Denmark) with a free enzyme activity of 510 U·g−1. All reagents except bovine serum albumin were analytically pure without further purification.

#### *3.2. Preparation of SiO2–CH3–CALB*

SiO2–CH3–CALB was prepared by sol–gel method. Firstly, TOMS (0.54 g), MTMS (1.934 g), methanol (3.39 g), PEG (0.14 g), NaF solution (0.49 g, 1 M), water (1.26 g) and CALB enzyme solution (3.39 g) were mixed and stirred at 0 ◦C, and the mixture was transferred to a clean petri dish. Then, the petri dish was sealed and placed at room temperature for 2 h to form a gel network. Finally, the petri dish was opened to evaporate the water and solvent in the gel completely, and then dried at room temperature for 48 h.

#### *3.3. Preparation of SiO2–CH3–CALB@PDA*

Dopamine hydrochloride (0.02 g) was dispersed in methanol (3.39 g), then NaF solution (0.98 g, 1 M) was added and mixed for 10 min. The use of NaF solution rather than Tris buffer was due to the fact that primary amine group in Tris can covalently interact with PDA, which could affect the deposition of PDA and the continuous coupling of CALB with PDA.

To the obtained mixture we added TMOS (0.54 g) and 1.934 g of MTMS (1.934 g), PEG (0.14 g), water (1.26 g) and CALB enzyme solution (3.39 g), which was then mixed and stirred at 0 ◦C, and the mixture was transferred to a clean petri dish. Then, the petri dish was sealed and placed at room temperature for 4 h to form a gel network. Finally, the petri dish was opened to evaporate the water and solvent in the gel completely and then dried at room temperature for 48 h.

#### *3.4. Characterization*

The microstructures of the samples were observed using a transmission electron microscope (TEM, Talos F200S, Hillsboro, FL, USA) and scanning electron microscopy (SEM, Nova Nano SEM 450, Hillsboro, FL, USA). Fourier transform infrared (FT–IR) spectra of the samples were collected from 4000 to 400 cm−<sup>1</sup> on a Bruker Tensor 27 analyzer (Bremen, Germany) using KBr pellets method. X-ray diffraction (XRD) patterns were measured by a Bruker D8 Discover (Bremen, Germany) with scanning rate of 6◦ min−<sup>1</sup> under Cu Kα radiation (λ = 0.154056 nm). Samples were mounted on a low background silicon substrate and diffraction scans covered a 2*θ* range of 5◦ to 80◦. X-ray photoelectron spectra (XPS, Al-Kα) were recorded on an X-ray photoelectron spectrometer (ESCALAB 250Xi, Hillsboro, FL, USA), and the C 1 s of 284.8 eV was referred to for calibrating the binding energy. The N2 adsorption–desorption isotherms were measured by a pore sizespecific surface area analyzer (SSA–6000, Beijing, China) at 77 K. The pore size distribution and surface area were determined through calculating N2 adsorption–desorption according to the Brunauer–Emmett–Teller (BET) method. A spectrophotometer (UV-2600, Shimadzu, Kyoto, Japan) was used to analyze the concentration and activity of the enzyme.

#### *3.5. Determination of Enzyme Loading*

The Bradford method was used to determine enzyme embedding in the silica xerogels by measuring of the protein concentrations in the initial enzyme solutions and immobilized enzyme phosphate detergents. A calibration curve was plotted, using Coomassie Brilliant Blue G-250 solutions as standards. The enzyme concentration in the solution was able to be determined with UV-vis spectrophotometry, by measuring the absorbance at 595 nm. The amount of enzyme embedded in silica xerogels was calculated by the following equation:

$$\text{enzymeLoading} = \frac{\mathcal{C}\_0 - \mathcal{C}\_1}{\mathcal{C}\_0} \times 100\% \tag{1}$$

where *C*<sup>0</sup> is the initial enzyme concentration (mg/g), *C*<sup>1</sup> is the enzyme concentration in phosphate detergent (mg/g).

#### *3.6. Properties of Free CALB and the Immobilized CALB*

#### 3.6.1. Assay of the CALB Activity

The free CALB and samples of immobilized CALB activities were determined by using *p*-NPP (5 mg/mL in ethanol) as the substrate. Typically, 200 μL of *p*-NPP solution was added to the solution consisting of the samples (2 mg) and PBS (0.1 M, pH 7.5, 3 mL). After reaction for 3 min, the filtrate of the reaction that contained 4-nitrophenol (*p*-NP), and

the concentration of *p*-NP was quantified via absorbance at 410 nm on a spectrophotometer. One unit (U) of lipase hydrolytic activity was regard as the lipase mass that liberates 1 nmol of *p*-NP under these test conditions per minute. The relative enzymatic activity was related to a percentage of this highest activity (100% means the highest enzymatic activity). The activity recovery was calculated from the value of the activity of the initial CALB solution divided by the activity value of immobilized CALB obtained immediately after the immobilization procedure.

#### 3.6.2. Thermal and Storage Stability of the Free CALB and Immobilized CALB

Free CALB, SiO2–CH3–CALB and SiO2–CH3–CALB@PDA were incubated in PBS (50 mM, pH 7.5) at 70 ◦C for 6 h to examine their thermal stabilities. The *p*-NPP assay was employed to measure residual activity as described in Section 3.6.1. To evaluate the storage stability, the residual activity of SiO2–CH3–CALB and SiO2–CH3–CALB@PDA was tested after a given treatment duration at 25 ◦C, respectively. The residual activity of each sample under treatment was measured at given time intervals and used for comparison with the original activity.

#### *3.7. Mechanical Performance Tests*

The mechanical performances of SiO2–CH3–CALB and SiO2–CH3–CALB@PDA were tested using a microcomputer control electron universal testing machines (CMT6104, Shenzhen, China) with a 5000-N load cell. To facilitate testing, samples were made into rectangular specimens. Compression strain tests of the samples (lengths, 23 mm; widths, 13.28 mm; thicknesses, 6 mm) were performed at a compression rate of 2 mm/min.

#### *3.8. Transesterification and Reusability*

The reaction for the transesterification of *n*-butanol with ethyl acetate was performed in a glass three-necked reactor with a volume of 250 mL at 343 K and 101.3 kPa. The electric stirring was controlled up to 3000 rpm to achieve uniform mixing of the reactive mixture. In the experiment, the mixture of reactants ethyl acetate and *n*-butanol (molar ratio of ethyl acetate to *n*-butanol was 1:1) were heated to 343 K in a water bath, then the catalysts (the catalyst dosage was 10% of the mass of *n*-butanol, and the catalysts were SiO2–CH3–CALB and SiO2–CH3–CALB@PDA) were set in the reactor to start the reaction. Samples were withdrawn from the reactor every 30 min with a syringe during the reaction for composition analysis until the 5 h. Finally, the catalysts were washed with PBS (0.1 M, pH 7.5) buffer and dried for 12 h before next cycle.

The composition of the product was analyzed by gas chromatography (GC-2010 Pro, Shimadzu, Kyoto, Japan) equipped with a flame ionization detector (FID) and an InertCap FFAP capillary column (30 m × 0.25 mm × 0.25 mm). Typically, *n*-propanol was used as the internal standard substance. N2 with purity of 99.99 wt% was used as carrier gas at 1 mL/min. The temperature of the injection port and the detector were controlled at 473 K and 493 K, respectively. 0.4 μL sample was injected each time.

#### **4. Conclusions**

In this work, the immobilization of CALB in PDA-modified silica xerogels was successfully prepared by the self-polymerization of dopamine on the Si−O−Si network surfaces of silica xerogels. The modified silica xerogels showed an excellent embedding ability for CALB compared with conventional silica xerogels. They exhibited a high capacity of 45.6 mg/gsupport for CALB encapsulation. The mechanical strength and thermal and storage stability of the immobilized CALB were greatly elevated. Moreover, the immobilization of an enzyme in PDA-modified silica xerogels was utilized in the transesterification between *n*-butanol with ethyl acetate, which retained 30.84% conversion of *n*-butanol after eight cycles. In short, the SiO2–CH3–CALB@PDA catalyst was prepared by a simple and practical method, which is expected to overcome the related problems of shrinkage and

weak binding force in conventional silica xerogels, and it has great application potential in the field of industrial catalysis.

**Author Contributions:** Conceptualization, H.W. and W.S.; methodology, H.W. and W.S.; validation, H.W., W.Y. and S.Z.; formal analysis, H.W.; investigation, H.W., W.Y.; resources, H.W.; data curation, Y.Z.; writing—original draft preparation, W.Y.; writing—review and editing, H.W., W.Y., Y.Z., C.L. and W.S.; visualization, H.W. and W.Y.; supervision, W.S.; project administration, W.S.; funding acquisition, H.W., W.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by financial support of National Natural Science Foundation of China (No. 21878066 and No. 21878068), National Natural Science Foundation of Hebei Province (No. B2020202015) and Special Correspondent Project of Tianjin (No. 18JCTPJC56500).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

