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. 2020 Aug 6;5(32):20062–20069. doi: 10.1021/acsomega.0c01625

Immobilization of Pectinase onto Porous Hydroxyapatite/Calcium Alginate Composite Beads for Improved Performance of Recycle

Danping Qi 1, Min Gao 1, Xiaoyuan Li 1, Jiangli Lin 1,*
PMCID: PMC7439264  PMID: 32832760

Abstract

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Pectinase is an industrially important enzyme widely used in juice production, food processing, and other fields. The use of immobilized enzyme systems that allow several reuses of pectinase is beneficial to these fields. Herein, we developed mechanically strong and recyclable porous hydroxyapatite/calcium alginate composite beads for pectinase immobilization. Under the optimal immobilization parameters of 40 °C, pH 4.0, 5.2 U/L pectinase concentration and 4 h reaction time, pectinase showed the highest enzymatic activity (8995 U/mg) and immobilization yield (91%). The thermal stability and pH tolerance of the immobilized pectinase were superior to those of free pectinase. The storage stability of the free and immobilized pectinase for 30 days retained 20 and 50% of their initial activity, respectively. Therefore, these composite beads might be promising support for the efficient immobilization of industrially important enzymes.

1. Introduction

Pectinases are a group of enzymes that include polygalacturonase, endo-polygalacturonase, pectin esterase, pectin lyase, and others.1,2 These are widely used for fruit juice extraction and clarification, plant fiber processing, coffee and oil extraction, pectin-containing wastewater treatment, and tea leaf maceration.3,4 However, soluble pectinases present some drawbacks, such as poor stability, the impossibility of reuses, and difficult product recovery.5 To overcome these problems, researchers proposed enzyme immobilization to increase the number of reuses, save production cost, and improve the catalytic features of the enzyme.6 Enzyme immobilization is a kind of technology that uses solid materials to bind or confine an enzyme in a certain area for a specific catalytic reaction, improve operational stability and easy separation from reaction systems, and reduce the price of using enzyme in large-scale applications.7 The nature of the carrier for enzyme immobilization is a key factor for the preparation of enzyme biocatalysts with high mechanical properties and operational stability. Typically, a carrier should have a high surface area to allow the immobilization of significant amounts of enzyme and a low solubility to avoid product contamination.2

Porous hydroxyapatite (HAp), with a chemical formula of Ca10(PO4)6(OH)2, has a high specific surface area and superior mechanical properties. It has an excellent biocompatibility and bioactivity and is widely applied in the delivery of drugs, the separation of proteins, and the removal of heavy metal pollutants.8,9 In this perspective, it is an ideal carrier for the immobilized enzyme.10−13 In these studies, enzyme immobilized onto the porous HAp supports are highly stable.14 Although immobilized enzymes have many advantages, they still have disadvantages. Because HAp immobilized enzyme is a powder, the process of filtering them from a liquid phase was tedious, and the loss of the enzyme in the separation process is inevitable, especially in industrial production. As such, porous HAp should be covered with suitable biocompatible compounds to facilitate the recovery of the immobilized enzyme.15

Sodium alginate (SA) is a polyelectrolyte polysaccharide that possesses a high negative charge density. It is also water-soluble, nontoxic, biodegradable, and biocompatible16,17 and is often used as a carrier for immobilized enzymes. However, its application in this field has been limited because the mechanical property of this material is not good in the process of repeated use, and the carrier will collapse, leading to the leakage of the enzyme, which cannot play the role of an immobilized enzyme carrier. However, SA has unique colloidal properties, including thickening and film forming.18−22 A good strategy is the preparation of immobilized enzyme composite carrier by coating SA to HAp because of the film forming and thickening properties of SA. This strategy not only solves the problem of recovering powdered HAp but also solves the poor mechanical properties of SA.

In this study, porous HAp with a high surface area and superior mechanical properties was synthesized and covered with SA to form porous HAp/SA composite beads. In the presence of calcium ions, it can form porous HAp/calcium alginate (CA) composite beads with them.18,19 The composite beads with a hard surface can be used as the carrier to immobilize pectinase. In pectinase immobilization, electrostatic adsorption and covalent bonding are involved, resulting in highly stable interactions for immobilization. This work mainly aimed to evaluate the effect of immobilization variables, namely, immobilization time, enzyme concentration, pH, and reaction temperature of immobilization. The immobilization parameters were also optimized via an orthogonal test. The kinetic behavior, pH and temperature profile, storage stability, and reusability of the immobilized pectinase were investigated.

2. Results and Discussion

2.1. Preparation of Porous HAp/CA Composite Beads and Immobilization of Pectinase

HAp was synthesized through a wet chemical precipitation method (Figure S1), and porous HAp with a pore size of 7.2 nm (Figure S2) was synthesized with polystyrene (PS) as a pore-forming agent and polyvinyl alcohol (PVA) as a binder via high-temperature calcining. Finally, PS and PVA filler were converted into carbon dioxide and water under aerobic conditions. The formation of a uniform porous HAp material was promoted by the disappearance of the filler and the release or liberation of gas.23

Pectinase was loaded on composite beads through physical adsorption and covalent binding. Pectinase was bound to SA in composite beads through electrostatic adsorption. Because HAp mainly contains Ca2+ and PO43–, these ions can be used to chelate with groups in the side chain of the enzyme.2,13 A covalent bond was generated because of the chelation between the carboxylic acid group present in pectinase and the calcium ion present in HAp.

2.2. Characterization of Porous HAp and Porous HAp/CA Beads

The formation of porous HAp/CA beads was confirmed by comparison of their Fourier transform infrared (FT-IR) spectra with those of HAp, CA beads, and porous HAp/CA beads from 400 to 4000 cm–1. In the FT-IR spectra of CA beads (Figure 1c), the peaks at 3422.4, 2919.7, 1620.0, 1401.0, and 1028.7 cm–1 were attributed to the presence of OH– (stretching), C–H (stretching), C=O (asymmetric stretching), C=O (symmetric stretching), and C–O–C (stretching) vibrations, respectively.8 For the porous HAp (Figure 1a), the characteristic peaks at 3570.0 and 632.0 cm–1 were assigned to the stretching and bending vibration of OH–, respectively. The characteristic peaks at 1091.0, 1033.2, 961.0, 602.1, 569.3, and 473.0 cm–1 were assigned to the stretching and bending vibration of PO43–.23 As shown in Figure 1b, the incorporation of SA into HAp resulted in the appearance of the peaks at 602.1 and 569.3 cm–1, which could be attributed to the addition of HAp. The results showed that HAp was combined with SA.

Figure 1.

Figure 1

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FT-IR spectra of (a) HAp, (b) HAp/CA composite beads, and (c) CA beads.

The HAp and composite beads were analyzed though scanning electron microscopy (SEM). Figure 2b shows that HAp has a porous structure. In Figure 2c, the surface of CA beads was almost entirely surrounded by distinct folds. For the HAp/CA composite beads (Figure 2d), many pores formed on the surface of the beads. By contrast, after pectinase was loaded, the surface of the HAp/CA composite beads lacked pores, suggesting that pores were filled with pectinase (Figure 2e).

Figure 2.

Figure 2

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SEM analysis of (a) HAp, (b) porous HAp, (c) CA beads, (d) porous HAp/CA composite beads, and (e) porous HAp/CA composite bead- immobilized pectinase.

The mechanical property and morphology of the composite beads were analyzed. As shown in the stress–strain curve (Figure 3), as the content of HAp increased, the compressive strength of the composite beads also increased. When the HAp-to-SA ratio reached 0.4:0.4, the compressive strength of the composite beads could reach 30 kPa (Figure 3). Moreover, when an external force was released, the porous HAp/CA composite beads could quickly recover their original shape. At the same time, the porous HAp/CA beads were characterized by SEM. It can be seen from Figure 4a that the porous HAp/CA composite beads retain their porous structure, and the morphology was intact. Moreover, the composite beads with a HAp-to-SA ratio of 0.6:0.4 were also characterized by SEM. As shown in the Figure 4b, the pore distribution on the surface of the porous HAp/CA composite beads changes, and the morphology becomes incomplete. It showed that with the increase of hydroxyapatite content, the compressive strength and mechanical properties of the porous HAp/CA composite beads decreased (Figure 3). This result demonstrated that the composite beads 0.4:0.4 had superior mechanical properties. Therefore, the composite beads with the HAp-to-SA ratio of 0.4:0.4 were selected to study the immobilized pectinase.

Figure 3.

Figure 3

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Stress–strain curve.

Figure 4.

Figure 4

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SEM analysis of (a) HAp/CA beads (HAp 0.4/SA 0.4) and (b) HAp/CA beads (HAp 0.6/SA 0.4).

2.3. Kinetic Parameters

Km indicates the affinity of a substrate to an enzyme. A high Km implies that the affinity of a given enzyme to its substrate is apparently low. In this study, Km and Vmax of the free pectinase were 11.35 mg/mL and 0.43 mg/(mL·min), respectively, whereas Km and Vmax of the immobilized pectinase were 14.74 mg/mL and 0.27 mg/(mL·min), respectively. The turnover number (kcat) is the rate constant for conversion of the enzyme–substrate complex to the product. When substrate concentration is excessive, kcat tends to Vmax.26 Therefore, the kcat of free and immobilized enzymes was 0.060 and 0.040 s–1, respectively, the kcat/Km of the free and immobilized pectinase was 0.005 and 0.003 mg–1·s–1·mL, respectively. Vmax, kcat, and kcat/Km decreased possibly because the substrate could not bind to the active sites of the immobilized pectinase because of the increased diffusion limitation.24−26 An increase in Km after immobilization indicated a reduction of the affinity of pectinase to its substrate, possibly because of the negative effect of immobilization in terms of the increased steric hindrance of the active site and the loss of enzyme flexibility necessary for complete substrate binding.

Activation energy (Ea) of the free and immobilized pectinase was calculated from the Arrhenius formula. Using 1000/T (K) as the x-coordinate and ln k (logarithm of % residual activity) as the y-coordinate, the regression equations of the free and immobilized pectinase were y = 53.491x + 1.8198 and y = 44.635x + 1.8531, respectively (Figure 5). The slope of the equation is Ea/R, so we can use this formula to figure out Ea. The result showed that the Ea of the free and immobilized pectinase shifted from 1024.19 to 854.62 kJ·mol–1, which led to a higher catalytic efficiency of the immobilized pectinase.27,28

Figure 5.

Figure 5

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Arrhenius plots for the free and immobilized pectinase.

2.4. Activity of the Immobilized Pectinase in Porous HAp/CA Composite Beads

The activity of the immobilized pectinase in composite beads was investigated at a pH 4.0, 40 °C, 4 h reaction time, and enzyme concentration of 5.2 U/L. In Figure 6, the enzyme activity increased markedly as the HAp-to-SA mass ratio in the composite carrier increased. The maximum enzyme activity was reached at 0.4:0.4. At this ratio, the immobilization yield was 91%. However, this yield declined subsequently because the polymeric matrix, which is composed of beads, has pores where the enzyme can be adsorbed. These pores can also allow the entry and exit of a substrate solution. Hence, the total activity is the summation of both internal and external activities. Thus, the combination of 0.4:0.4 HAp and SA might be the ratio at which such an activity is potentialized because of the kind of pores that are formed. Another situation is correlated with the loading capacity of the beads toward the enzyme. However, amounts above the loading capacity might decrease the enzymatic activity because of steric impediment between them.29

Figure 6.

Figure 6

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Effect of the HAp to SA mass ratio in the composite on the immobilization efficiency.

2.5. Analysis of Immobilization Parameters

The immobilized time, pH, reaction temperature, and concentration of pectinase were included in the analysis of the immobilization parameters.

The effect of pH on enzyme immobilization is shown in Figure 7a. The optimum pH of pectinase immobilization was obtained at pH 4.0. The activity of pectinase was different under various acidic conditions.6 When pH was too high or too low, protein molecules might denature, so enzyme activity was lost or reduced.

Figure 7.

Figure 7

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Effect of immobilization conditions on enzyme activity (a) pH (b) time (c) enzyme concentration (d) temperature.

As shown in Figure 7b, the immobilization time to achieve the maximum activity was 4 h. After 4 h, the enzyme activity gradually decreased possibly because the pectinase molecules at the bottom of the carrier could not migrate to the surface and bind to the substrate with the extension of immobilization time.30

As shown in Figure 7c, the optimal enzyme concentration was 5.2 U/L. However, when the enzyme concentration exceeded 5.2 U/L, the enzyme activity was almost unchanged likely because of the oversaturation of the pore space of the support with the enzyme. As a result, the diffusion of enzyme molecules was relatively limited.29

The optimal temperature of pectinase immobilization onto HAp/CA beads was 40 °C (Figure 7d). As the temperature further increased, the activity of the immobilized pectinase decreased. If the temperature was too high, the enzyme structure changed, and enzyme activity was gradually lost.

Four influencing factors, namely, pH, reaction temperature, enzyme concentration, and reaction time were optimized via an orthogonal experiment to load pectinase onto porous HAp/CA composite beads (Table 1).

Table 1. Result of the Orthogonal Experiment for Immobilized Pectinase.

test temperature (°C) pH time (h) enzyme concentration (U/L) vacant column enzyme activity/U·g–1
1 1(35) 1(4.5) 1(5) 1(5.2) 1 8.391
2 1(35) 2(4.0) 2(4) 2(5.0) 2 7.881
3 1(35) 3(3.5) 3(3) 3(5.4) 2 8.346
4 2(40) 1(4.5) 3(3) 1(5.2) 3 7.762
5 2(40) 2(4.0) 2(4) 1(5.2) 1 8.995
6 2(40) 3(3.5) 1(5) 2(5.0) 2 7.671
7 3(45) 1(4.5) 3(3) 2(5.0) 3 8.307
8 3(45) 2(4.0) 1(5) 3(5.4) 3 7.598
9 3(45) 3(3.5) 2(4) 3(5.4) 1 7.337
K1 24.618 25.693 23.660 23.490 24.723  
K2 24.428 23.241 22.980 23.859 23.898  
K3 23.242 23.354 25.648 24.939 23.667  
k1 8.206 8.564 7.887 7.830 8.241  
k2 8.143 7.747 7.660 7.953 7.966  
k3 7.747 7.785 8.549 8.313 7.889  
R 0.459 0.817 0.889 0.483 0.275  
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Variance analysis confirmed the following optimal combination of reaction time, pH, enzyme concentration, and reaction temperature obtained via the orthogonal experiment: 4 h, pH 4.0, 5.2 U/L, and 40 °C, respectively. These results were consistent with those of single-factor test optimization (Table 2).31

Table 2. Analysis of Variance.

source of variance sum of squares of deviation df mean square F F0.05
reaction temperature (°C) 0.370 2 0.1853 5.4154 5.14
pH 1.277 2 0.6387 18.6509 5.14
reaction time (h) 1.281 2 0.6407 18.7106 5.14
enzyme concentration (U/L) 0.378 2 0.1890 5.5196 5.14
vacant column (error) 0.2054 2 0.03424    
total 3.299 10      
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2.6. Effect of pH and Temperature on the Immobilized and Free Pectinase

In Figure 8a, the free and immobilized pectinase had an optimal pH of 4.5 and 4.0, respectively. The shift in the optimal pH was most probably attributed to the favorable adaptability of the immobilized pectinase against environmental acidity. In the pH range of 3.0–6.0, the activity of the immobilized pectinase was greater than that of the free pectinase. This finding indicated that the immobilized pectinase was more resistant to pH changes than the free pectinase.32

Figure 8.

Figure 8

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Stability of free and immobilized pectinase at different (a) pH, (b) temperature, (c) cycle time, and (d) storage time.

The optimum temperature of the free and immobilized pectinase was 45 °C (Figure 8b). The activity of the immobilized pectinase was higher than that of free pectinase at 40–60 °C. In particular, the immobilized enzyme retained about 95% of its activity at 60 °C, whereas the activity of the free enzyme was less than 90%. These results indicated that the thermal stability of the immobilized pectinase improved. Furthermore, as summarized in Table 3, the HAp/CA composite beads presented a preferable activity than that described in other reports on immobilized enzymes in a wider range of pH and temperature.7

Table 3. Stability of Immobilized Pectinase in the Current Work and Other Reportsa.

  pH stability
 thermal stability
 storage stability
 recycling stability
  
support tested range of pH (RA > 80%) pH range tested range of temperature (°C) temperature range (RA > 80%) days residual activity (%) recycle time residual activity (%) refs
HAp     35–50   30   4 43 (13)
HAp/CA composite beads 3.0–6.0 3.0–6.0 30–60 30–60 30 50 10 40 current work
CA composite beads 3.0–8.0 4.0–5.0 20–60 40–50 30 70 6 37 (33)
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a

RA: relative activity.

2.7. Reusability Analysis

The reusability of the immobilized pectinase is an important characteristic for its potential industrial application. In Figure 8c, the immobilized pectinase maintained about 40% of its original activity after 10 consecutive operations. The activity loss of the immobilized pectinase could be due to the conformational changes in the enzyme and the falling off of pectinase from the surface of the carrier.24,30

2.8. Storage Stability Analysis

The free and immobilized pectinase were stored at 4 °C for 30 days. The activity was measured every 5 days. The immobilized pectinase retained about 50% of its initial activity after 30 days, whereas the free pectinase lost more than 80% of its initial activity.

The results indicated that the storage stability of the immobilized pectinase with the composite bead as a carrier greatly improved (Figure 8d).

3. Conclusions

Porous HAp/CA composite beads with good mechanical properties were prepared to immobilize pectinase. The immobilized pectinase prepared with these composite beads as a carrier could be used to save time and effort and was convenient for recycling. After 10 cycles of recycling, the enzyme activity remained 40%. Furthermore, the thermal stability and pH tolerance of the immobilized pectinase were superior to those of the free pectinase. The immobilized pectinase activity remained about 50% after 30 days of storage at 4 °C. Conversely, only about 20% of the free pectinase remained, indicating that the residual enzyme activity of the immobilized pectinase was higher than that of the free pectinase. With these good properties, the immobilized pectinase might show potential for application in the food industry and enzymatic catalysis in the juice industry.

4. Materials and Methods

4.1. Chemicals

Commercially available pectinase, SA, and pectin were purchased from Yuhua Biotechnology Co. Ltd. (Shanghai, China). The chemicals used were Ca(OH)2, H3PO4, and CaCl2. PVA (Mw = 1750), styrene, azobisisobutyronitrile, Tween-20 polysorbate, and NaHCO3 were obtained from Tianjin Beilian Technology Co., Ltd. Ultrapure water was used throughout the experiments.

4.2. Synthesis of Porous HAp and HAp/CA Composite Beads

HAp was synthesized in accordance with previously described methods.23,34−38 Porous HAp was synthesized with HAp as a precursor, and PS microspheres as pore-forming agents were synthesized in accordance with previously reported methods.8 A solution containing 0.5 g of HAp and 6.0 mL of ultrapure water was added to a solution composed of 4.0 mL of PS and 8.0 mL of PVA, stirred for 3 h, dried, and calcined in a muffle furnace at 900 °C for 10 h to remove residual organic matter.

Porous HAp/CA composite beads with different mass ratios of HAp to SA (0:0.4, 0.2:0.4, 0.4:0.4, 0.6:0.4, and 0.8:0.4) were prepared by using a CaCl2-hardening method.39 Porous HAp was added to the SA solution under stirring. The resulting mixture was added dropwise to the CaCl2 solution (0.01%) by using a syringe, cured for 2 h; HAp/CA composite beads with a hard surface were formed, which were then removed, rinsed with deionized water several times, and stored at 4 °C.

4.3. Characterization of Porous HAp and Porous HAp/CA Beads

The synthesized porous HAp and porous HAp/CA beads were then analyzed through field emission SEM (SU8010, Hitachi Ltd., Japan) and FT-IR spectroscopy (VERTEX 70).

4.4. Enzyme Immobilization

In this procedure, 1.0 g of porous HAp/CA composite beads was added to 2.0 mL of citrate buffer solution with pH 4.0 and enzyme concentration of 5.2 U/L and then reacted at 40 °C for 4 h. Subsequently, the beads were separated and washed thrice with the buffer solution to remove unabsorbed pectinase. The immobilized pectinase was stored at 4 °C before use. The immobilization yield was calculated using eq 1.28

4.4. 1

4.5. Pectinase Activity Assay

The activity of the immobilized pectinase was determined using the 3,5-dinitrosalicylic acid (DNS) method.7,40 A total of 1 unit of pectinase activity was defined as the amount of enzyme required to catalyze the formation of 1 μmol of a reducing sugar per minute under the described conditions. The immobilized pectinase (1.0 g) was added to 1.0 mL of 0.01 g/mL pectin solution (citrate buffer, pH 4.0) and incubated at 50 °C for 0.5 h. After 3.0 mL of DNS was added by heating in a boiling water bath for 5 min, the reaction was stopped, immediately cooled to room temperature, and diluted with 15.0 mL of water. The reducing sugar content in the resulting reactant was determined by measuring the absorbance at 540 nm using a UV-1780 spectrophotometer (Shimadzu, Japan).

4.6. Optimization of the Immobilization Parameters

The immobilization parameters were studied in detail by using the method of control variables. Immobilization parameters included immobilization time, enzyme concentration, pH of the enzyme solution, and reaction temperature. The immobilization times (1–6 h) were studied under the following conditions: pH 4.0, 50 °C, and enzyme concentration of 5.0 U/L. The immobilization time was 4 h, and other conditions were unchanged; different pH between 3.0 and 6.0 were investigated. Different enzyme concentrations between 4.2 and 5.6 U/L at an immobilization time of 4 h were used for immobilization to determine the proper enzyme concentration of the immobilization solution, and other conditions were kept constant. The reaction temperatures (30–60 °C) were studied under the conditions of pH 4.0, immobilization time 4 h, and enzyme concentration of 5.0 U/L. An orthogonal experiment was performed to optimize the immobilization parameters.

4.7. Optimum pH and Temperature of Free and Immobilized Pectinase

The pH stability of the free and immobilized pectinase was studied at 45 °C with a variation in the pH of the reaction mixture in the range of 3.0–6.0 by using the citrate buffer. Other conditions remained unchanged. The thermal stability of the free and immobilized pectinase was determined at a reaction temperature range of 30–60 °C by using citrate buffer at pH 4.0. Other conditions were kept constant. For better comparison, the enzyme activity at the optimal pH and temperature was 100%, and relative activity was compared.7 The relative activity was calculated by using eq 2

4.7. 2

4.8. Storage Stability of Free Pectinase and Immobilized Pectinase

The storage stability of the free and immobilized pectinase was evaluated by calculating the residual percentage of the enzymatic activity of each specimen stored at 4 °C for 5, 10, 15, 20, 25, and 30 days. For better comparison, the initial enzyme activity was 100%, and relative activity was compared. The relative activity was calculated by using eq 2.

4.9. Reusability of the Immobilized Pectinase

The reusability of the immobilized pectinase was evaluated by measuring its activity for 10 recycles. In detail, 1.0 g of the immobilized pectinase was added to 2 mL of the pectin solution (0.01 g/mL) with pH 4.0 and incubated at 45 °C for 30 min. After each cycle, the supernatant was taken, and enzyme activity was measured. Immobilized pectinase was rinsed with the sodium citrate buffer solution thrice and then repositioned into the new pectin solution. For better comparison, the initial enzyme activity was 100%, and relative activity was compared. The relative activity was calculated by using eq 2.

4.10. Kinetic Parameters and Activation Energy

Michaelis–Menten kinetics (Km) and maximum reaction velocity (Vmax) were used to calculate the enzymatic activities of the free and immobilized pectinase at different concentrations of the pectin solution (0.2–2.5 g/mL). These activities were evaluated through the classical Michaelis–Menten kinetics.

4.10. 3

where V mg/(mL·min) is the initial reaction rate, [s] (mg/mL) is the pectin concentration, Vmax mg/(mL·min) is the maximum reaction velocity obtained at an infinite substrate concentration, and Km (mg/mL) is the Michaelis–Menten constant.7Vmax and Km were calculated by the line graphic method, respectively.

Ea of the free and immobilized pectinase was calculated from the Arrhenius formula.27,28 It can be seen that the drawing of ln k–1/T is a straight line with slope Ea/R, as given in the following equation:

4.10. 4

R is the gas constant (8.314 J·mol–1·K–1).

Acknowledgments

This work was financially supported by the Xinjiang Uygur Autonomous Region Natural Science Joint Fund Project (grant no. 2019D01C085).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c01625.

  • XRD results and BET data of hydroxyapatite (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao0c01625_si_001.pdf (90.3KB, pdf)

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