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zeolitic imidazolate framework
Enzyme and Microbial Technology 129 (2019) 109347
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Immobilization of high temperature-resistant GH3 β-glucosidase on a magnetic particle Fe3O4-SiO2-NH2-Cellu-ZIF8/zeolitic imidazolate framework ⁎⁎
Xuejia Shia,1, Jin Xua,1, Changning Lua,b, Zhenzhong Wangc, Wei Xiaoc, , Linguo Zhaoa,b,
T
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a
Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, 159 Long Pan Road, Nanjing 210037, China College of Chemical Engineering, Nanjing Forestry University, 159 Long Pan Road, Nanjing 210037, China c Jiangsu Kanion Pharmaceutical Co., Ltd., 58 Haichang South Road, Lianyungang 222001, Jiangsu Province, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Magnetic particle β-Glucosidase High-temperature stability
The magnetic particle Fe3O4-SiO2-NH2-Cellu-ZIF8 (FSNCZ8) was used to immobilize the high-temperature resistant GH3 β-glucosidase (Tpebgl3) from the Thermomotoga petrophila DSM 13995. The Tpebgl3 has great potential in the catalytic conversion of pharmaceutically active components. The magnetic carrier (FSNCZ8) which provided stronger adsorption capacity, magnetic and high-temperature stability than the previously discussed Fe3O4-NH2-Cellu-ZIF8 without tetraethyl silicate coated. The properties of FSNCZ8-Tpebgl3 (FSNCZ8-T) were as follows: The optimal temperature and pH were 90 °C and 5.5, respectively; the highest activity of FSNCZ8-T approached 2672 U/g; Fe2+ enabled immobilized enzyme to increase its relative activity to 194%. The main characteristics of FSNCZ8-T, including thermal stability, pH stability, and glucose tolerance, were greatly enhanced by adding Fe2+, which was also superior to free enzymes; moreover, the residual activity of FSNCZ8-T was 74% of the initial activity at the end of 10 repeated cycles.
1. Introduction High temperature-resistant recombinant GH3 β-glucosidase (Tpebgl3, EC 3.2.1.21) from Thermomotoga petrophila DSM 13995 [1] could be widely used in the enzymatic catalysis of medicinally-active components of herbs that contain flavonoids and saponins [2–5]. For example, Tpebgl3 provides high catalytic efficiency and high specificity and can transform the major ginsenosides Rb1 and Rd into the minor ginsenoside 20(S)-Rg3, which has better pharmacological value than Rb1 or Rd [6–9]. Due to the characteristics of the enzyme itself, the bioactive enzymes require the ability to withstand extreme conditions and variable environments, such as extreme pH, high temperatures and high sugar concentrations, to meet industrial demands [10,11]. In addition, the cost of preparation and lack of reusability of the free enzyme are major barriers to industrial applications. Many methods are available for using enzymes in industrial applications, and the immobilization of enzymes on a solid support is a general method that is often used to maintain high activity and specificity and to enhance the properties of the enzyme in extreme environments [12]. Magnetic microspheres
that are composed of Fe3O4 nanomaterials have excellent magnetic adsorption capacity and a large specific surface area that can support and fix most common biological macromolecules to provide higher enzyme activity and are environmentally acceptable [13]. In addition, studies have shown that solid phase carriers allow for repeated and continuous operations and can improve the thermal and pH stabilities of the enzymes [14,15]. Fe3O4 nanoparticles, because of their magnetic and metal–organic framework properties, can promote the effective absorption of enzymes and their separation from reaction mixtures, and enhance the reusability of the biocatalyst [16]. Previous work has indicated that the magnetic force of Fe3O4 nanoparticles can be strengthened after being modified with functional groups. For example, when graphene oxide was integrated into Fe3O4 nanoparticles, the surface area of modified Fe3O4 nanoparticles could be increased with retention of the functional groups, and the magnetic force of the particles was maintained. However, the barriers regarding the acidification and poor high temperature stability of Fe3O4 must be overcome. Studies have shown that cellulose combined with a zeolitic imidazolate framework (ZIF-8) may be a
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Corresponding author at: College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China. Corresponding author. E-mail addresses: [email protected] (W. Xiao), [email protected] (L. Zhao). 1 These authors equally contributed to this work. ⁎⁎
https://doi.org/10.1016/j.enzmictec.2019.05.004 Received 20 February 2019; Received in revised form 8 May 2019; Accepted 13 May 2019 Available online 14 May 2019 0141-0229/ © 2019 Published by Elsevier Inc.
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2.2.3. Preparation of Fe3O4-NH2-Cellu-ZIF8 and Fe3O4-SiO2-NH2-CelluZIF8 Fe3O4-NH2-Cellu-ZIF8 (FNCZ8) was prepared as follows: 1 g of FN was added to 200 mL of an aqueous solution containing 12% urea and 7% sodium hydroxide, the solution was precooled at −20 °C for more than 1 h and then 0.67 g of microcrystalline cellulose was added to the mixture which was frozen for an hour until the cellulose was dissolved. Cellulose-coated Fe3O4-NH2-Cellu was obtained by washing the reaction mixture repeatedly with ethanol and deionized water [26]. Then, 100 mg of the above product was dissolved in 20 mL of a zinc nitrate hexahydrate solution (40 mM), stirred for 20 min, then treated with 20 mL of 2-methylimidazole (160 mM) for 3 h. The desired product, FNCZ8, was washed and dried. This procedure was also conducted using Fe3O4-SiO2-NH2-Cellu-ZIF8 (FSNCZ8) and FSN as staring materials.
promising candidate for use as a surface material [17]. The hydroxyl groups of cellulose that are coated onto metal–organic frameworks can improve the acidification [18], poor high temperature stability and organic solvent tolerance [19–21]. Moreover, microparticles obtained by functionalizing Fe3O4 nanomaterials with silica also exhibit significantly enhanced enzymatic properties [4,22–24]. In addition, the effects of metal cations on the properties of free and immobilized enzymes have been evaluated, including improved catalytic efficiency and reusability, thermal stability and substrate tolerance. Based on the Lewis acid-base theory, when accepting an electron pair, the metal ion is normally a Lewis acid that can generate a force that can change the active site of the enzyme. In this study, our goal was to enhance the loading of recombinant GH3 β-glucosidase (Tpebgl3) onto Fe3O4-SiO2-NH2-Cellu-ZIF8 to improve upon the low activity that is obtained with Tpebgl3 immobilized on the carrier resin NKA-9 [25]. This metal–organic framework, compared with Fe3O4-NH2-Cellu-ZIF8 magnetic particles, has excellent high temperature stability and acid resistance. Large-scale preparation of Tpebgl3 would be required for its use in commercial applications.
2.2.4. Determination of enzyme activity and protein concentration The activities of the free and immobilized forms of the Tpebgl3 enzyme were measured. The assay mixture (200 μL) contained 1 mM pnitrophenyl-β-D-glucopyranoside (pNPG), citric acid/Na2HPO4 buffer (100 mM, pH 5.0), and an appropriate amount of free or immobilized Tpebgl3. After incubating at 90 °C in a shaking water bath for 5 min, the reaction was terminated by the addition of 300 μL of 1 M Na2CO3, and the absorbance at 405 nm was measured using a microplate reader (SpectraMax190). The activities of the free and immobilized enzymes, defined as a unit of enzyme activity [U], involved the release of 1 μmol pNP from 1 mM pNG. The protein concentration was determined using a Bradford Protein Assay Kit (Sangon Biotech, Shanghai, China) [25].
2. Materials and methods 2.1. Materials Tpebgl3 is from Thermomotoga petrophila DSM 13995 and is expressed in E.coli BL21 (DE3) [1]. The nano-Fe3O4 was prepared using FeCl3,·6H2O, FeCl2·4H2O, ethanol and 25% ammonia water as starting materials, which were purchased from Sinopharm Group Chemicals Co., Ltd. (Shanghai, China). The materials that were used for the surface modification of Fe3O4 include 98% 3-aminopropyltriethoxysilane (APTES) from Sinopharm Group Chemicals Co., Ltd. (Shanghai, China), tetraethyl silicate (TEOS) from Macleans (Shanghai, China), 2-methylimidazole from In Aladdin (Shanghai, China), and zinc nitrate hexahydrate from Nanjing Chemical Reagent Co., Ltd. (Naning, China). Glutaraldehyde was purchased from Sinopharm Chemical RCo., Ltd. (Shanghai, China).
2.2.5. Immobilization of Tpebgl3 and determination of its surface characteristics The support carriers (0.1 g) were incubated in a solution (10 mL) of 10 mM citric acid/Na2HPO4 buffer, pH 4.5, with 35 U/mL of Tpebgl3 for 24 h. The total protein mass in the reaction solution was approximately 1.2 mg. Glutaraldehyde was added and allowed to react for 2 h. After each step, the reaction mixture was washed with deionized water to remove unbound enzymes and other excess components, including glutaraldehyde, using vacuum filtration. The properties of the modified solid supports were investigated using Fourier transform infrared spectroscopy (FTIR, Bruker, Germany) and X-ray diffraction (XRD, Bruker, Germany)
2.2. Methods 2.2.1. Preparation of nano-Fe3O4 A co-precipitation method was used to prepare nano-Fe3O4. FeCl3·6H2O (8.1 g) was dissolved in deionized water in a 250 mL conical flask using a magnetic stirrer. The mixture was slowly heated to 70 °C, then 4.4 g of FeCl2·4H2O dissolved in deionized water was added to the flask. The reaction solution was stirred and its volume was maintained at 150 mL during the process. The solution was filtered rapidly through a 0.45 μm membrane and 5% aqueous ammonia (NH3·H2O) was then added dropwise to the acquired filtrate with stirring at pH 10. The temperature of the solution was gradually increased to and maintained at 85 °C for 1 h. The desired product was obtained by washing the precipitate that was adsorbed to a magnet four times with absolute ethanol and deionized water.
2.2.6. Temperature and pH optima for immobilized Tpebgl3 The optimum temperature for immobilized and free Tpebgl3 activities were determined at the following temperatures: 65, 70, 75, 80, 85, 90 and 95 °C. The optimum pH for the immobilized enzyme was determined using a pH gradient method (pH 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 and 8.0) at 90 °C. 2.2.7. Effect of metal cations on the catalytic activity of free and immobilized Tpebgl3 The effect of metal cations on the catalytic activity of the immobilized enzyme were determined. The metal cations included Fe2+, Li2+, Al3+, K+, Ni2+, Mn2+, Ca2+, Zn2+ and Mg2+. The kinetic constants of the purified Tpebgl3 (0.6 μg) and immobilized enzyme (0.6 μg) were measured by determining the initial rates at different pNPG ending concentrations (0, 0.1, 0.2, 0.3, 0.6, 1, 1.5 and 2 mM pNPG) at the optimal conditions.
2.2.2. Preparation of Fe3O4-SiO2-NH2 and Fe3O4 -NH2 Fe3O4-SiO2 was prepared as follows: 1 g of Fe3O4 was dispersed in 80% ethanol-water (100 mL) and mixed by ultrasonication, 1.6 mL of 25% NH3·H2O was then added followed by the addition of 2 mL of TEOS. The mixture was stirred and maintained at 40 °C for 4 h to produce the desired products. The Fe3O4-SiO2 and nano-Fe3O4 were added to 50% ethanol-water solutions (80 mL) and stirred rapidly. APTES (4 mL) was then added and the mixture was rotated at 200 rpm at 40 °C for 24 h. The reaction products, Fe3O4-SiO2-NH2 (FSN) and Fe3O4 -NH2 (FN), were washed 3 times with ethanol and then dried.
2.2.8. Determination of the thermal and pH stabilities of immobilized Tpebgl3 The reaction solution (1 mL) contained 0.01 g of FSNCZ8-T (approximately 0.1133 mg of protein) and 35 U of Tpebgl3 (approximately 0.12 mg of protein) in 100 mM citric acid/Na2HPO4 buffer, pH 5.5. FSNCZ8-T and free enzyme were incubated at 65, 70, 75, 80, 85 and 90 °C in a water bath for 3 h and the thermal stability of the 2
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immobilized and free enzymes were determined. The pH stability of the immobilized or free enzymes was investigated after the immobilized and free enzymes were maintained in 100 mM citric acid/Na2HPO4 buffer (pH 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 and 8.0) for 3 h at room temperature; the protein concentrations were similar to the solutions described above. The effects of metal ions on the thermal and pH stabilities at high temperature of the free and immobilized enzymes were determined using the reaction systems described above as the control.
90 °C; however, the residual activity of the microspheres FSNCZ8-T remained at 45% and the structure was relatively intact. This result visually demonstrates that the magnetic microspheres FSNCZ8 encapsulated with TEOS is structurally stable. Moreover, it can be maintained at a higher temperature and as a fixed support carrier for Tpebgl3.
2.2.9. Effect of glucose concentrations on the catalytic activity of the free and immobilized enzymes The effects of glucose concentration on the activities of free and immobilized enzymes were determined with and without Fe2+. The reactions were conducted with initial concentrations of monosaccharide ranging from 10 to 400 mM at 90 °C and at pH 5.0.
The immobilized enzyme FSNCZ8T displayed a stable structure and provided a fixed support carrier for Tpebgl3. The optimal reaction temperature and pH of FSNCZ8-T were determined. Samples were exposed to temperatures ranging from 65 to 95 °C at pH 5.0 to determine the effect of reaction temperatures on the immobilized enzyme FSNCZ8-T and on free enzyme and to determine the optimal reaction temperature conditions. The results are shown in Fig. 2A. The optimum temperature for FSNCZ8-T was 90 °C, which was the same as that for the free enzyme. Furthermore, there was no difference in relative activities between the immobilized enzyme and free enzyme at different temperatures. The optimum pH and the effect of different reaction temperatures on the activity of FSNCZ8-T and free enzyme were determined at 90 °C, and the results are shown in Fig. 2B. The optimum pH of FSNCZ8-T increased by 0.5 pH units, as the FSNCZ8T exhibited maximum activity at pH 5.5. This result also demonstrated that the relative activity of the immobilized enzyme FSNCZ8-T, compared to free Tpebgl3, was reduced significantly under acidic conditions, which may be due to acidolysis of the magnetic material itself. The activity of the magnetic microspheres was significantly higher than that of the free enzyme under alkaline conditions. The selection of an alkaline buffer system may better reflect the value and significance of immobilizing the Tpebgl3 with magnetic microspheres.
3.2. Optimal conditions of FSNCZ8-T
2.2.10. Reusability of FSNCZ8-T The activity of FSNCZ8-T was measured at 90 °C for 5 min. When the reaction ended, the solid supports were washed with 100 mM citric acid/Na2HPO4 buffer, pH 5.5. This process was repeated 10 times to measure the reusability of the immobilized enzymes. 3. Results and discussion 3.1. Effect of chemical modification on FSNCZ8-immobilized Tpebgl3 Amino groups were activated to provide functional groups on the magnetic Fe3O4 supports. In addition, the magnetic properties of the particles were enhanced after they were encapsulated with microcrystalline cellulose and tetraethyl silicate (TEOS). Because the metal organic framework zeolitic imidazolate frameworks-8 (ZIF8) exhibited high stability in protein denaturing solvents and at high temperatures, FNC and FSNC were coated with zinc nitrate hexahydrate and a ZIF-8 framework grew around the solid. The magnetic microspheres were named FNCZ8 and FSNCZ8, both of which have protective shells that provide for high adsorption and activity of the enzyme and that also improve its stability. The FTIR in Fig. 1A shows an intense peak at 1066 cm-1 that is attributed to the SiO2 network. The XRD diffraction peak at 2θ = 23.8° (Fig. 1B) indicates the formation of amorphous silica. Both methods demonstrate the effectiveness of the synthesis of FSNCZ8. Both FNCZ8 and FSNCZ8 were used to adsorb Tpebgl3. The results are shown in Fig. 1C, D and F. As shown in Fig. 1C, the highest adsorption rate and enzyme activity obtained with FSNCZ8 as support were 94.44% (11.33 mg protein/g FSNCZ8) and 3173 U/g after 24 h of reaction, respectively, which were significantly higher than that obtained with FNCZ8 (52.87%, 1355.2 U/g). Furthermore, to improve the repeatability of the immobilized materials, the experiments were conducted using glutaraldehyde to enable covalent coupling between free aldehyde groups and free amino groups on the protein [27]. The results are presented in Fig. 1D and show the effect of different concentrations of glutaraldehyde on the immobilized enzyme. The results indicate that as the concentration of glutaraldehyde increases, the activity of the prepared magnetic particle immobilized enzyme is reduced. This reduction in activity may be due to a weakening of the adsorption and immobilization of the magnetic microsphere or to an inhibition of the activity of the enzyme by the high concentration of glutaraldehyde. The selected glutaraldehyde concentration was 0.5%, and the activity of the magnetic particle Fe3O4-SiO2-NH2-Cellu-ZI8-immobilized Tpebgl3 (FSNCZ8-T) was 2672 U/g. The activity of magnetic particle Fe3O4NH2-Cellu-ZI8-immobilized TpeRha (FNCZ8-T) was 1612 U/g. The thermal stability of the magnetic microspheres prepared with immobilized enzyme is shown in Fig. 1D. The residual activities of the two immobilized enzymes were compared after three hours of incubation at high temperature, and the results indicated that the immobilized enzyme FNCZ8-T was completely inactivated. In addition, the appearance of the microspheres changed and the magnetic force was lost at
3.3. Effect of metal ions on the activity of FSNCZ8-T The effect of various metal cations on the catalytic efficiency of immobilized enzymes has not been studied thoroughly [28–30]. Based on known interactions between free enzymes and metal ions, the effect of metal cations on the activities of FSNCZ8-T and free Tpebgl3 were determined at pH 5.5 and at 90 °C. The final concentration of metal cations in the reaction solutions was 2 mM. The results presented in Table 1 show that the activity of free Tpebgl3 is inhibited by Fe2+, Li+ and Al3+. Three metal cations, K+, Mn2+ and Zn2+, have a weak positive effect on the activity of free Tpebgl3. However, except for Fe2+, it was found that several common metal cations had no inhibitory effect on the activity of FSNCZ8T. Although Zn2+, Mn2+ and K+ can effectively improve the enzyme activity of FSNCZ8-T, the combination of FSNCZ8-T and Fe2+ (2 mM) increases the catalytic activity of FSNCZ8T 1.94-fold, which is the most significant effect of all metal cations tested on FSNCZ8-T. However, increased Fe2+ concentrations do not enhance further the catalytic activity of FSNCZ8-T (Fig. 3A). Moreover, the catalytic activity of free enzyme was inhibited by high Fe2+ concentrations. The kinetic equation was obtained by adding different substrate concentrations to the solution at the optimal conditions and a reaction time of 5 min. Km and Kcat values of 0.559 mM and 11840 s−1, respectively, were obtained for free Tpebgl3 with no added Fe2+; and 0.384 mM and 9786 s−1, respectively, for free Tpebgl3 with 2 mM Fe2+ under optimal conditions. Thus, the catalytic efficiency of free Tpebgl3 with Fe2+ was considerably lower than that without Fe2+. In addition, the dependence of the rate of the enzymatic reaction on the substrate concentration followed Michaelis-Menten kinetics, the Km of FSNCZ8-T was 0.303 mM without Fe2+, and 0.403 mM in the presence of metal ion. Thus, the addition of 2 mM Fe2+ significantly increases the binding between the enzyme active site and the substrate, which may be due to an alteration in the affinity of the active site toward substrate binding due to limited diffusion [31]. The Kcat in the presence of 2 mM Fe2+ 3
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Fig. 1. Determination of the characteristics of magnetic materials FNCZ8 and FSNCZ8, and immobilization of Tpebgl3. A. Structural analysis of FNCZ8 and FSNCZ8 by FITR (IR KBr (cm−1): 3420 (amino, N-H), 2928 (aromatic, CeH), 1634 (aromatic frame), 578 (Fe3O4) and 1066 (SiO2). B. XRD results for FNCZ8 and FSNCZ8. FNCZ8 (Fig. 1b–B) produced five distinct peaks at 2θ = 30.3°, 35.3°, 43.5°, 57.7° and 63° that correspond to the (220), (311), (400), (500) and (440) crystal planes of Fe3O4. It was shown that amino group modification had no effect on the Fe3O4 diffraction peak. The XRD pattern for FSNCZ8 is also shown in Fig. 1b–A. Compared to FNCZ8, FSNCZ8 produced a diffraction peak at 2θ = 23.8°, which indicated the formation of amorphous silica. Moreover, SiO2 and Fe3O4 were shown to be separated, and Fe3O4 did not show any change in its crystal structure after coating with SiO2. C. Ability of two magnetic microspheres to adsorb Tpebgl3. D. Effect of glutaraldehyde concentration on the two magnetic microspheres. F. Thermostability of FNCZ8-T and FSNCZ8-T.
Fig. 2. Optimal reaction conditions for free Tpebgl3 and FSNCZ8-T. a, optimum temperature; b, optimum pH.
was 24454 s-1, which was 2.17 times higher than that without Fe2+. The catalytic efficiency (Kcat/Km) in the presence of 2 mM Fe2+ was 60680 mM-1 s−1 (Table 2), which was increased by 1.63 times compared to that in the absence of metal ions. However, Fe2+ had little effect on the activity of free Tpebgl3, possibly because the metal cation altered the structure of the magnetic microspheres or the interaction between the carrier and the bound enzyme [32,33].
3.4. Effect of Fe2+ on thermal stability and pH stability of FSNCZ8-T The catalytic activity of free Tpebgl3 at high temperatures requires the immobilized enzyme to possess multiple features: high temperature structural stability, pH stability, avoiding the enzyme configuration, preventing rapid deactivation of the enzyme, and prolonging the reaction in a continuous high temperature reaction. The effect of Fe2+ on the thermal stability of FSNCZ8-T was determined. The free enzyme and FSNCZ8-T were incubated separately at 65, 70, 75, 80, 85 and 90 °C for 3 h. The results in Fig. 3B show that the immobilized enzyme in the 4
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Table 1 The effect of different metal ions on the catalytic activity of immobilized enzyme FSNCZ8-T.
Table 2 Kinetic equation parameters for free Tpebgl3 and FSNCZ8-T with and without Fe2+.
Metal cation
Free enzyme
FSNCZ8- Tpebgl3
Materials
free Tpebgl3
FSNCZ8-T
Fe2+ Li+ Al3+ K+ Ni2+ Mn2+ Ca2+ Zn2+ Mg2+
88.4 ± 1.0 88.1 ± 1.0 90.6 ± 1.0 110.2 ± 1.2 97.1 ± 1.1 113.5 ± 1.2 100.4 ± 1.1 108.4 ± 1.2 98.6 ± 1.1
194.92 ± 11.2 97.47 ± 5.82 95.20 ± 3.12 126.89 ± 0.75 109.46 ± 4.50 128.07 ± 2.48 104.73 ± 6.86 124.74 ± 6.22 98.55 ± 0.38
Fe2+ concentration (mM)
0 mM
2 mM
0 mM
2 mM
Km (mM) Kcat (s−1) Kcat/Km (mM1 -1 s )
0.559 ± 0.07 11840 ± 65 21181
0.384 ± 0.06 9786 ± 42 25484
0.303 ± 0.03 11267 ± 37 37184
0.403 ± 0.03 24454 ± 42 60680
compared to that in the presence of Fe2+. The pH stability of the immobilized enzyme was relatively greater, and the relative activity remained at 90% or greater under alkaline conditions.
presence of Fe2+ has significantly increased thermal stability compared to the free enzyme. However, the stability of FSNCZ8-T at 80 °C or higher is reduced in the presence or absence of metal ions, possibly due to the leaching of enzyme from the magnetic microspheres at high reaction temperatures. The adsorption of magnetic microspheres is weakened at high temperatures, resulting in loss of enzyme. The pH stability of free enzyme and FSNCZ8-T was determined. The enzymes were incubated at room temperature in different pH value buffer systems for 3 h and the activity of the enzymes was then measured. The results are shown in Fig. 3C. The residual activity of FSNCZ8-T was basically unchanged under alkaline conditions. The residual activity of FSNCZ8-T in the absence of Fe2+ was increased by more than 50% compared to the free enzyme at pH 8.0. The activity of FSNCZ8-T was slightly decreased under acidic conditions (pH 3.5), due to the instability of the carrier itself under acidic conditions. Furthermore, the relative activity of the free enzyme decreased significantly. This may be because immobilized Tpebgl3 can maintain the spatial structure and active site of the enzyme more effectively and resist the destruction of the enzyme protein by extremes of pH. In addition, there was no significant change in the pH stability of the free enzyme
3.5. Effect of Fe2+ on the glucose tolerance of immobilized enzyme β-glucosidase is subject to feedback inhibition by glucose during the hydrolysis of the substrate, leading to a decrease of the conversion rate and the formation of product. Immobilization is expected to improve the tolerance of the enzyme to glucose [34,35]. The tolerance of free enzyme and FSNCZ8-T to glucose was determined between 0 and 400 mM glucose. The results shown in Fig. 3D indicate that as the glucose concentration is gradually increased, the relative activity of free Tpebgl3 decreases rapidly. When the glucose concentration is greater than 100 mM, the relative enzyme activity is less than 15%. At a glucose concentration of 400 mM, the free enzyme is completely inhibited, but the residual activity of immobilized enzyme remains at 25% of the initial activity, or greater. A reasonable explanation is that the spatial configuration of the magnetic microspheres provides a wall that resists inhibition by high concentrations of glucose. The results indicate that the use of the magnetic microsphere FSNCZ8 to immobilize Tpebgl3 can
Fig. 3. Characteristics of FSNCZ8-T. a, effect of Fe2+ concentration on the activity of free Tpebgl3 and FSNCZ8-T; b, high temperature stability of free Tpebgl3 and FSNCZ8-T; c, pH stability of free Tpebgl3 and FSNCZ8-T; d, glucose tolerance of free Tpebgl3 and FSNCZ-T. 5
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Fig. 4. Effect of Fe2+ on the reusability of FSNCZ8-T.
increase the glucose tolerance of the GH3 β-glucosidases (Tpebgl3). Furthermore, after the addition of 2 mM Fe2+, the glucose tolerance of the immobilized enzyme was greatly improved. At a glucose concentration of 400 mM, a residual activity of approximately 40% was retained by immobilized enzyme, and the relative activity in the presence of Fe2+ was increased by approximately 15% compared to that in the absence of Fe2+. This may be because Fe2+ acts as an enzyme modifier in the reaction system [36]. This could increase the reaction velocity by enzyme activators by favoring a productive interaction between enzyme and substrate to form an enzyme-substrate complex by creating an enzyme-substrate-activator complex.
In addition, The Fe2+ enabled the immobilized enzyme to exhibit a higher catalytic activity than that of metal ion-free enzyme. The properties of FSNCZ8-TpeRha, including thermal stability, pH stability, and glucose tolerance, were greatly enhanced by metal cations, which can be of great significance in industrial applications of Tpebgl3. Acknowledgements This work was supported by the National Key R&D Program of China (2016YFD0600805), the Forestry Achievements of Science and Technology to Promote Projects ([2017] 10) and the Science and Technology project of Jangsu Province (SZ-XZ2017023)
3.6. Effect of Fe2+ on the reusability of FSNCZ8-T References The reusability of the immobilized enzyme is essential for the repeated use of the enzyme, for reducing the amount of enzyme used, and for reducing enzyme production and consumption. Due to the high specificity and inseparability of free Tpebgl3, the reusability of FSNCZ8-T was determined and the results are shown in Fig. 4. First, 2 mM Fe2+ was added to the mixture containing immobilized enzyme to verify the effect of the presence or absence of metal ions on the reusability of FSNCZ8-T. The results shown in Fig. 4A demonstrate that metal ions cannot form stable bonds or interact with immobilized enzymes. The activity of the immobilized enzyme in the presence of metal ions was recorded as 100% for the first reaction cycle, and then FSNCZ8-T was washed with deionized water, and the activity was then determined without metal ions. Repeating the reaction, the relative activity of the first cycle was found to be 1.5 times the relative activity of the two cycles. In addition, the relative activity of the third repeat cycle with added Fe2+ was also higher than that of the metal ion-free enzyme (2 and 4 times). This may be because Fe2+ activates the catalytic activity of FSNCZ8-T. Therefore, to increase the activity of the FSNCZ8-T, it is necessary to add Fe2+. As shown in Fig. 4B, FSNCZ8-T was reacted 10 times in the presence or absence of Fe2+, and the relative activity of the immobilized enzyme in the presence of Fe2+ was maintained above 74%. This is due to leakage and denaturation of the free enzyme and does not constitute a major obstacle. This represents a significant improvement for industrial applications of thermostable βglucosidase TpebgL3.
[1] J. Xie, D. Zhao, L. Zhao, J. Pei, W. Xiao, G. Ding, Z. Wang, Overexpression and characterization of a Ca2+ activated thermostable β-glucosidase with high ginsenoside Rb1 to ginsenoside 20(S)-Rg3 bioconversion productivity, J. Ind. Microbiol. Biotechnol. 422 (2015) 839–850. [2] Q. Yan, W. Zhou, X. Shi, P. Zhou, J.U. Dianwen, M. Feng, Biotransformation pathways of ginsenoside Rb1 to compound K by β-glucosidases in fungus Paecilomyces Bainier sp. 229, Process Biochem. 45 (2010) 1550–1556. [3] K.H. Chang, N.J. Mi, K.T. Kim, H.D. Paik, Evaluation of glucosidases of Aspergillus niger strain comparing with other glucosidases in transformation of ginsenoside Rb1 to ginsenosides Rg3, J. GINS Res. 38 (2014) 47–51. [4] R. Riedel, Nanoscaled inorganic materials by molecular design, Chem. Soc. Rev. 41 (2012) 5029–5031. [5] L. Li, S.J. Lee, Q.P. Yuan, T.I. Wan, C.K. Sun, N.S. Han, Production of bioactive ginsenoside Rg3(S) and compound K using recombinant Lactococcus lactis, J. GINS Res. 42 (2018) 412–418. [6] J.T. Hwang, M.S. Lee, H.J. Kim, M.J. Sung, H.Y. Kim, M.S. Kim, D.Y. Kwon, Antiobesity effect of ginsenoside Rg3 involves the AMPK and PPAR-gamma signal pathways, Phytother. Res. 23 (2010) 262–266. [7] H.S. Kim, E.H. Lee, S.R. Ko, K.J. Choi, J.H. Park, D.S. Im, Effects of ginsenosides Rg3 and Rh2 on the proliferation of prostate cancer cells, Arch. Pharm. Res. 27 (2004) 429–435. [8] R. Ye, G. Zhao, X. Liu, Ginsenoside Rd for acute ischemic stroke: translating from bench to bedside, Expert Rev. Neurother. 13 (2013) 603–613. [9] L. Hae-Ung, B. Eun-Ah, H. Myung Joo, K. Dong-Hyun, Hepatoprotective effect of 20(S)-ginsenosides Rg3 and its metabolite 20(S)-ginsenoside Rh2 on tert-butyl hydroperoxide-induced liver injury, Biol. Pharm. Bull. 28 (2005) 1992–1994. [10] S. Hu, D. Wang, J. Hong, A Simple method for β-glucosidase immobilization and its application in soybean isoflavone glycosides hydrolysis, Biotechnol. Bioprocess Eng. 23 (2018) 39–48. [11] P.G. Vazquez-Ortega, M.T. Alcaraz-Fructuoso, J.A. Rojas-Contreras, J. LópezMiranda, R. Fernandez-Lafuente, Stabilization of dimeric β-glucosidase from Aspergillu s niger via glutaraldehyde immobilization under different conditions, Enzyme Microb. Technol. 110 (2018) 38–45. [12] Y. Carvalho, Almeida JMAR, P.N. Romano, K. Farrance, P.D. Carà, N. Pereira, J.A. Lopez-Sanchez, E.F. Sousa-Aguiar, Nanosilicalites as support for β-glucosidases covalent immobilization, Appl. Biochem. Biotechnol. 182 (2017) 1619–1629. [13] S. Peirce, M.E. Russo, R. Perfetto, C. Capasso, M. Rossi, R. Fernandez-Lafuente, P. Salatino, A. Marzocchella, Kinetic characterization of carbonic anhydrase immobilized on magnetic nanoparticles as biocatalyst for CO2 capture, Biochem. Eng. J. 138 (2018) 1–11. [14] M. Defaei, A. Taheri-Kafrani, M. Miroliaei, P. Yaghmaei, Improvement of stability and reusability of α-amylase immobilized on naringin functionalized magnetic nanoparticles: a robust nanobiocatalyst, Int. J. Biol. Macromol. 113 (2018) 354–360.
4. Conclusion High temperature-resistant recombinant GH3 β-glucosidase (Tpebgl3, EC 3.2.1.21) from Thermomotoga petrophila DSM 13995 has great potential for the catalytic conversion of pharmaceutically active components. The Tpebgl3 was immobilized on a synthetic high temperature magnetic carrier (FSNCZ8) that provided stronger adsorption capacity and magnetic and high temperature stabilities than the previously described FNCZ8, which was not coated with tetraethyl silicate. 6
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[26] Yen-Ning Kuo, J. Hong, Investigation of solubility of microcrystalline cellulose in aqueous NaOH, Polym. Adv. Technol. 16 (2005) 425–428. [27] F.F. Freitas, L.D.S. Marquez, G.P. Ribeiro, G.C. Brandão, V.L. Cardoso, E.J. Ribeiro, Optimization of the immobilization process of β-galatosidade by combined entrapment cross-linking and the kinetics of lactose hydrolysis, Braz. J. Chem. Eng. 29 (2012) 15–24. [28] Z. Zhai, T. Yang, B. Zhang, J. Zhang, Effects of metal ions on the catalytic degradation of dicofol by cellulase, J Environ. Sci. 33 (2015) 163–168. [29] Ruinan Zhao, Mancheng Hu, Shuni Li, Quanguo Zhai, Yucheng Jiang, Immobilization of chloroperoxidase in metal organic framework and its catalytic performance, Acta Chim. Sin. 75 (2017) 293–299. [30] S. Fennouh, V. Casimiri, A. Geloso-Meyer, C. Burstein, Kinetic study of heavy metal salt effects on the activity of L-lactate dehydrogenase in solution or immobilized on an oxygen electrode, Biosens. Biolectron. 13 (1998) 903–909. [31] J. Grewal, R. Ahmad, S.K. Khare, Development of cellulase-nanoconjugates with enhanced ionic liquid and thermal stability for in situ lignocellulose saccharification, Bioresour. Technol. Rep. 242 (2017) 236–243. [32] L. Fernandez-Lopez, R. Bartolome-Cabrero, M. Daniela Rodriguez, Stabilizing effects of cations on lipases depend on the immobilization protocol, RSC Adv. 5 (2015) 83868–83875. [33] N.T. Wright, K.M. Varney, K.C. Ellis, J. Markowitz, R.K. Gitti, D.B. Zimmer, D.J. Weber, The three-dimensional solution structure of Ca2+ -bound S100A1 as determined by NMR spectroscopy, J. Mol. Biol. 353 (2005) 410–426. [34] H. Cao, Y. Zhang, P. Shi, R. Ma, H. Yang, W. Xia, Y. Cui, H. Luo, Y. Bai, B. Yao, A highly glucose-tolerant GH1 β-glucosidase with greater conversion rate of soybean isoflavones in monogastric animals, J. Ind. Microbiol. Biotechnol. 45 (2018) 369–378. [35] R.J. You, H.Y. Shin, Y.S. Song, S.B. Kim, S.W. Kim, Enhancement of immobilized enzyme activity by pretreatment of β-glucosidase with cellobiose and glucose, J. Ind. Eng. Chem. 18 (2012) 702–706. [36] José Carlos Santos Salgado, Luana Parras Meleiro, Sibeli Carli, Richard John Ward, Glucose tolerant and glucose stimulated β-glucosidases – a review, Bioresour. Technol. Rep. 267 (2018) 704–713.
[15] G. Josep, Z. Yue, T. Hannah, C. Oscar, M.E. Webb, Z. Dejian, Multilayer enzymecoupled magnetic nanoparticles as efficient, reusable biocatalysts and biosensors, Nanoscale 3 (2011) 3721–3730. [16] H.J. Park, A.J. Driscoll, P.A. Johnson, The development and evaluation of β-glucosidase immobilized magnetic nanoparticles as recoverable biocatalysts, Biochem. Eng. J. 133 (2018) 66–73. [17] A. Schejn, T. Mazet, V. Falk, L. Balan, L. Aranda, G. Medjahdi, R. Schneider, Fe3O4@ ZIF-8: magnetically recoverable catalysts by loading Fe3O4 nanoparticles inside a zinc imidazolate framework, Dalton Trans. 44 (2015) 10136–10140. [18] S.L. Cao, H. Xu, L.H. Lai, W.M. Gu, P. Xu, J. Xiong, H. Yin, X.H. Li, Y.Z. Ma, J. Zhou, Magnetic ZIF-8/cellulose/Fe3O4 nanocomposite: preparation, characterization, and enzyme immobilization, Bioresour. Bioprocess. 4 (2017) 56. [19] M. Jian, L. Bao, G. Zhang, R. Liu, X. Zhang, Adsorptive removal of arsenic from aqueous solution by zeolitic imidazolate framework-8 (ZIF-8) nanoparticles, Colloid Surf. A 465 (2015) 67–76. [20] V. Chiaradia, N.S. Soares, A. Valério, D.D. Oliveira, P.H.H. Araújo, C. Sayer, Immobilization of Candida antarctica lipase B on magnetic poly(Urea-Urethane) nanoparticles, Appl. Biochem. Biotechnol. 180 (2016) 1–18. [21] S.A. Mohamed, M.H. Al-Harbi, Y.Q. Almulaiky, I.H. Ibrahim, H.A. Salah, M.O. ElBadry, A.M. Abdel-Aty, A.S. Fahmy, R.M. El-Shishtawy, Immobilization of trichoderma harzianum α-amylase on PPyAgNp/Fe3O4 -nanocomposite: chemical and physical properties, Artif. Cells Nanomed. Biotechnol. 26 (2018) 1–6. [22] S. Shi, J. Yang, S. Liang, M. Li, Q. Gan, K. Xiao, J. Hu, Enhanced Cr(VI) removal from acidic solutions using biochar modified by Fe3O4@SiO2-NH2 particles, Sci. Total Environ. 628–629 (2018) 499–508. [23] V.G. Pérez, C. Márquez-Álvarez, R.M. Blanco, Successful encapsulation of β-glucosidase during the synthesis of siliceous mesostructured materials, J. Chem. Technol. Biotechnol. 93 (2018) 2625–2634. [24] V. Califano, F. Sannino, A. Costantini, et al., Wrinkled silica nanoparticles: efficient matrix for β-glucosidase immobilization, J. Phys. Chem. C 122 (2018) 8373–8379. [25] X. Shi, L. Zhao, J. Pei, G. Lin, P. Wan, Z. Wang, X. Wei, Highly enhancing the characteristics of immobilized thermostable β-glucosidase by Zn2+, Process Biochem. 66 (2018) 89–96.
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Immobilization of high temperature-resistant GH3 β-glucosidase on a magnetic particle Fe3O4-SiO2-NH2-Cellu-ZIF8/zeolitic imidazolate framework ⁎⁎
Xuejia Shia,1, Jin Xua,1, Changning Lua,b, Zhenzhong Wangc, Wei Xiaoc, , Linguo Zhaoa,b,
T
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a
Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, 159 Long Pan Road, Nanjing 210037, China College of Chemical Engineering, Nanjing Forestry University, 159 Long Pan Road, Nanjing 210037, China c Jiangsu Kanion Pharmaceutical Co., Ltd., 58 Haichang South Road, Lianyungang 222001, Jiangsu Province, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Magnetic particle β-Glucosidase High-temperature stability
The magnetic particle Fe3O4-SiO2-NH2-Cellu-ZIF8 (FSNCZ8) was used to immobilize the high-temperature resistant GH3 β-glucosidase (Tpebgl3) from the Thermomotoga petrophila DSM 13995. The Tpebgl3 has great potential in the catalytic conversion of pharmaceutically active components. The magnetic carrier (FSNCZ8) which provided stronger adsorption capacity, magnetic and high-temperature stability than the previously discussed Fe3O4-NH2-Cellu-ZIF8 without tetraethyl silicate coated. The properties of FSNCZ8-Tpebgl3 (FSNCZ8-T) were as follows: The optimal temperature and pH were 90 °C and 5.5, respectively; the highest activity of FSNCZ8-T approached 2672 U/g; Fe2+ enabled immobilized enzyme to increase its relative activity to 194%. The main characteristics of FSNCZ8-T, including thermal stability, pH stability, and glucose tolerance, were greatly enhanced by adding Fe2+, which was also superior to free enzymes; moreover, the residual activity of FSNCZ8-T was 74% of the initial activity at the end of 10 repeated cycles.
1. Introduction High temperature-resistant recombinant GH3 β-glucosidase (Tpebgl3, EC 3.2.1.21) from Thermomotoga petrophila DSM 13995 [1] could be widely used in the enzymatic catalysis of medicinally-active components of herbs that contain flavonoids and saponins [2–5]. For example, Tpebgl3 provides high catalytic efficiency and high specificity and can transform the major ginsenosides Rb1 and Rd into the minor ginsenoside 20(S)-Rg3, which has better pharmacological value than Rb1 or Rd [6–9]. Due to the characteristics of the enzyme itself, the bioactive enzymes require the ability to withstand extreme conditions and variable environments, such as extreme pH, high temperatures and high sugar concentrations, to meet industrial demands [10,11]. In addition, the cost of preparation and lack of reusability of the free enzyme are major barriers to industrial applications. Many methods are available for using enzymes in industrial applications, and the immobilization of enzymes on a solid support is a general method that is often used to maintain high activity and specificity and to enhance the properties of the enzyme in extreme environments [12]. Magnetic microspheres
that are composed of Fe3O4 nanomaterials have excellent magnetic adsorption capacity and a large specific surface area that can support and fix most common biological macromolecules to provide higher enzyme activity and are environmentally acceptable [13]. In addition, studies have shown that solid phase carriers allow for repeated and continuous operations and can improve the thermal and pH stabilities of the enzymes [14,15]. Fe3O4 nanoparticles, because of their magnetic and metal–organic framework properties, can promote the effective absorption of enzymes and their separation from reaction mixtures, and enhance the reusability of the biocatalyst [16]. Previous work has indicated that the magnetic force of Fe3O4 nanoparticles can be strengthened after being modified with functional groups. For example, when graphene oxide was integrated into Fe3O4 nanoparticles, the surface area of modified Fe3O4 nanoparticles could be increased with retention of the functional groups, and the magnetic force of the particles was maintained. However, the barriers regarding the acidification and poor high temperature stability of Fe3O4 must be overcome. Studies have shown that cellulose combined with a zeolitic imidazolate framework (ZIF-8) may be a
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Corresponding author at: College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China. Corresponding author. E-mail addresses: [email protected] (W. Xiao), [email protected] (L. Zhao). 1 These authors equally contributed to this work. ⁎⁎
https://doi.org/10.1016/j.enzmictec.2019.05.004 Received 20 February 2019; Received in revised form 8 May 2019; Accepted 13 May 2019 Available online 14 May 2019 0141-0229/ © 2019 Published by Elsevier Inc.
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2.2.3. Preparation of Fe3O4-NH2-Cellu-ZIF8 and Fe3O4-SiO2-NH2-CelluZIF8 Fe3O4-NH2-Cellu-ZIF8 (FNCZ8) was prepared as follows: 1 g of FN was added to 200 mL of an aqueous solution containing 12% urea and 7% sodium hydroxide, the solution was precooled at −20 °C for more than 1 h and then 0.67 g of microcrystalline cellulose was added to the mixture which was frozen for an hour until the cellulose was dissolved. Cellulose-coated Fe3O4-NH2-Cellu was obtained by washing the reaction mixture repeatedly with ethanol and deionized water [26]. Then, 100 mg of the above product was dissolved in 20 mL of a zinc nitrate hexahydrate solution (40 mM), stirred for 20 min, then treated with 20 mL of 2-methylimidazole (160 mM) for 3 h. The desired product, FNCZ8, was washed and dried. This procedure was also conducted using Fe3O4-SiO2-NH2-Cellu-ZIF8 (FSNCZ8) and FSN as staring materials.
promising candidate for use as a surface material [17]. The hydroxyl groups of cellulose that are coated onto metal–organic frameworks can improve the acidification [18], poor high temperature stability and organic solvent tolerance [19–21]. Moreover, microparticles obtained by functionalizing Fe3O4 nanomaterials with silica also exhibit significantly enhanced enzymatic properties [4,22–24]. In addition, the effects of metal cations on the properties of free and immobilized enzymes have been evaluated, including improved catalytic efficiency and reusability, thermal stability and substrate tolerance. Based on the Lewis acid-base theory, when accepting an electron pair, the metal ion is normally a Lewis acid that can generate a force that can change the active site of the enzyme. In this study, our goal was to enhance the loading of recombinant GH3 β-glucosidase (Tpebgl3) onto Fe3O4-SiO2-NH2-Cellu-ZIF8 to improve upon the low activity that is obtained with Tpebgl3 immobilized on the carrier resin NKA-9 [25]. This metal–organic framework, compared with Fe3O4-NH2-Cellu-ZIF8 magnetic particles, has excellent high temperature stability and acid resistance. Large-scale preparation of Tpebgl3 would be required for its use in commercial applications.
2.2.4. Determination of enzyme activity and protein concentration The activities of the free and immobilized forms of the Tpebgl3 enzyme were measured. The assay mixture (200 μL) contained 1 mM pnitrophenyl-β-D-glucopyranoside (pNPG), citric acid/Na2HPO4 buffer (100 mM, pH 5.0), and an appropriate amount of free or immobilized Tpebgl3. After incubating at 90 °C in a shaking water bath for 5 min, the reaction was terminated by the addition of 300 μL of 1 M Na2CO3, and the absorbance at 405 nm was measured using a microplate reader (SpectraMax190). The activities of the free and immobilized enzymes, defined as a unit of enzyme activity [U], involved the release of 1 μmol pNP from 1 mM pNG. The protein concentration was determined using a Bradford Protein Assay Kit (Sangon Biotech, Shanghai, China) [25].
2. Materials and methods 2.1. Materials Tpebgl3 is from Thermomotoga petrophila DSM 13995 and is expressed in E.coli BL21 (DE3) [1]. The nano-Fe3O4 was prepared using FeCl3,·6H2O, FeCl2·4H2O, ethanol and 25% ammonia water as starting materials, which were purchased from Sinopharm Group Chemicals Co., Ltd. (Shanghai, China). The materials that were used for the surface modification of Fe3O4 include 98% 3-aminopropyltriethoxysilane (APTES) from Sinopharm Group Chemicals Co., Ltd. (Shanghai, China), tetraethyl silicate (TEOS) from Macleans (Shanghai, China), 2-methylimidazole from In Aladdin (Shanghai, China), and zinc nitrate hexahydrate from Nanjing Chemical Reagent Co., Ltd. (Naning, China). Glutaraldehyde was purchased from Sinopharm Chemical RCo., Ltd. (Shanghai, China).
2.2.5. Immobilization of Tpebgl3 and determination of its surface characteristics The support carriers (0.1 g) were incubated in a solution (10 mL) of 10 mM citric acid/Na2HPO4 buffer, pH 4.5, with 35 U/mL of Tpebgl3 for 24 h. The total protein mass in the reaction solution was approximately 1.2 mg. Glutaraldehyde was added and allowed to react for 2 h. After each step, the reaction mixture was washed with deionized water to remove unbound enzymes and other excess components, including glutaraldehyde, using vacuum filtration. The properties of the modified solid supports were investigated using Fourier transform infrared spectroscopy (FTIR, Bruker, Germany) and X-ray diffraction (XRD, Bruker, Germany)
2.2. Methods 2.2.1. Preparation of nano-Fe3O4 A co-precipitation method was used to prepare nano-Fe3O4. FeCl3·6H2O (8.1 g) was dissolved in deionized water in a 250 mL conical flask using a magnetic stirrer. The mixture was slowly heated to 70 °C, then 4.4 g of FeCl2·4H2O dissolved in deionized water was added to the flask. The reaction solution was stirred and its volume was maintained at 150 mL during the process. The solution was filtered rapidly through a 0.45 μm membrane and 5% aqueous ammonia (NH3·H2O) was then added dropwise to the acquired filtrate with stirring at pH 10. The temperature of the solution was gradually increased to and maintained at 85 °C for 1 h. The desired product was obtained by washing the precipitate that was adsorbed to a magnet four times with absolute ethanol and deionized water.
2.2.6. Temperature and pH optima for immobilized Tpebgl3 The optimum temperature for immobilized and free Tpebgl3 activities were determined at the following temperatures: 65, 70, 75, 80, 85, 90 and 95 °C. The optimum pH for the immobilized enzyme was determined using a pH gradient method (pH 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 and 8.0) at 90 °C. 2.2.7. Effect of metal cations on the catalytic activity of free and immobilized Tpebgl3 The effect of metal cations on the catalytic activity of the immobilized enzyme were determined. The metal cations included Fe2+, Li2+, Al3+, K+, Ni2+, Mn2+, Ca2+, Zn2+ and Mg2+. The kinetic constants of the purified Tpebgl3 (0.6 μg) and immobilized enzyme (0.6 μg) were measured by determining the initial rates at different pNPG ending concentrations (0, 0.1, 0.2, 0.3, 0.6, 1, 1.5 and 2 mM pNPG) at the optimal conditions.
2.2.2. Preparation of Fe3O4-SiO2-NH2 and Fe3O4 -NH2 Fe3O4-SiO2 was prepared as follows: 1 g of Fe3O4 was dispersed in 80% ethanol-water (100 mL) and mixed by ultrasonication, 1.6 mL of 25% NH3·H2O was then added followed by the addition of 2 mL of TEOS. The mixture was stirred and maintained at 40 °C for 4 h to produce the desired products. The Fe3O4-SiO2 and nano-Fe3O4 were added to 50% ethanol-water solutions (80 mL) and stirred rapidly. APTES (4 mL) was then added and the mixture was rotated at 200 rpm at 40 °C for 24 h. The reaction products, Fe3O4-SiO2-NH2 (FSN) and Fe3O4 -NH2 (FN), were washed 3 times with ethanol and then dried.
2.2.8. Determination of the thermal and pH stabilities of immobilized Tpebgl3 The reaction solution (1 mL) contained 0.01 g of FSNCZ8-T (approximately 0.1133 mg of protein) and 35 U of Tpebgl3 (approximately 0.12 mg of protein) in 100 mM citric acid/Na2HPO4 buffer, pH 5.5. FSNCZ8-T and free enzyme were incubated at 65, 70, 75, 80, 85 and 90 °C in a water bath for 3 h and the thermal stability of the 2
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immobilized and free enzymes were determined. The pH stability of the immobilized or free enzymes was investigated after the immobilized and free enzymes were maintained in 100 mM citric acid/Na2HPO4 buffer (pH 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 and 8.0) for 3 h at room temperature; the protein concentrations were similar to the solutions described above. The effects of metal ions on the thermal and pH stabilities at high temperature of the free and immobilized enzymes were determined using the reaction systems described above as the control.
90 °C; however, the residual activity of the microspheres FSNCZ8-T remained at 45% and the structure was relatively intact. This result visually demonstrates that the magnetic microspheres FSNCZ8 encapsulated with TEOS is structurally stable. Moreover, it can be maintained at a higher temperature and as a fixed support carrier for Tpebgl3.
2.2.9. Effect of glucose concentrations on the catalytic activity of the free and immobilized enzymes The effects of glucose concentration on the activities of free and immobilized enzymes were determined with and without Fe2+. The reactions were conducted with initial concentrations of monosaccharide ranging from 10 to 400 mM at 90 °C and at pH 5.0.
The immobilized enzyme FSNCZ8T displayed a stable structure and provided a fixed support carrier for Tpebgl3. The optimal reaction temperature and pH of FSNCZ8-T were determined. Samples were exposed to temperatures ranging from 65 to 95 °C at pH 5.0 to determine the effect of reaction temperatures on the immobilized enzyme FSNCZ8-T and on free enzyme and to determine the optimal reaction temperature conditions. The results are shown in Fig. 2A. The optimum temperature for FSNCZ8-T was 90 °C, which was the same as that for the free enzyme. Furthermore, there was no difference in relative activities between the immobilized enzyme and free enzyme at different temperatures. The optimum pH and the effect of different reaction temperatures on the activity of FSNCZ8-T and free enzyme were determined at 90 °C, and the results are shown in Fig. 2B. The optimum pH of FSNCZ8-T increased by 0.5 pH units, as the FSNCZ8T exhibited maximum activity at pH 5.5. This result also demonstrated that the relative activity of the immobilized enzyme FSNCZ8-T, compared to free Tpebgl3, was reduced significantly under acidic conditions, which may be due to acidolysis of the magnetic material itself. The activity of the magnetic microspheres was significantly higher than that of the free enzyme under alkaline conditions. The selection of an alkaline buffer system may better reflect the value and significance of immobilizing the Tpebgl3 with magnetic microspheres.
3.2. Optimal conditions of FSNCZ8-T
2.2.10. Reusability of FSNCZ8-T The activity of FSNCZ8-T was measured at 90 °C for 5 min. When the reaction ended, the solid supports were washed with 100 mM citric acid/Na2HPO4 buffer, pH 5.5. This process was repeated 10 times to measure the reusability of the immobilized enzymes. 3. Results and discussion 3.1. Effect of chemical modification on FSNCZ8-immobilized Tpebgl3 Amino groups were activated to provide functional groups on the magnetic Fe3O4 supports. In addition, the magnetic properties of the particles were enhanced after they were encapsulated with microcrystalline cellulose and tetraethyl silicate (TEOS). Because the metal organic framework zeolitic imidazolate frameworks-8 (ZIF8) exhibited high stability in protein denaturing solvents and at high temperatures, FNC and FSNC were coated with zinc nitrate hexahydrate and a ZIF-8 framework grew around the solid. The magnetic microspheres were named FNCZ8 and FSNCZ8, both of which have protective shells that provide for high adsorption and activity of the enzyme and that also improve its stability. The FTIR in Fig. 1A shows an intense peak at 1066 cm-1 that is attributed to the SiO2 network. The XRD diffraction peak at 2θ = 23.8° (Fig. 1B) indicates the formation of amorphous silica. Both methods demonstrate the effectiveness of the synthesis of FSNCZ8. Both FNCZ8 and FSNCZ8 were used to adsorb Tpebgl3. The results are shown in Fig. 1C, D and F. As shown in Fig. 1C, the highest adsorption rate and enzyme activity obtained with FSNCZ8 as support were 94.44% (11.33 mg protein/g FSNCZ8) and 3173 U/g after 24 h of reaction, respectively, which were significantly higher than that obtained with FNCZ8 (52.87%, 1355.2 U/g). Furthermore, to improve the repeatability of the immobilized materials, the experiments were conducted using glutaraldehyde to enable covalent coupling between free aldehyde groups and free amino groups on the protein [27]. The results are presented in Fig. 1D and show the effect of different concentrations of glutaraldehyde on the immobilized enzyme. The results indicate that as the concentration of glutaraldehyde increases, the activity of the prepared magnetic particle immobilized enzyme is reduced. This reduction in activity may be due to a weakening of the adsorption and immobilization of the magnetic microsphere or to an inhibition of the activity of the enzyme by the high concentration of glutaraldehyde. The selected glutaraldehyde concentration was 0.5%, and the activity of the magnetic particle Fe3O4-SiO2-NH2-Cellu-ZI8-immobilized Tpebgl3 (FSNCZ8-T) was 2672 U/g. The activity of magnetic particle Fe3O4NH2-Cellu-ZI8-immobilized TpeRha (FNCZ8-T) was 1612 U/g. The thermal stability of the magnetic microspheres prepared with immobilized enzyme is shown in Fig. 1D. The residual activities of the two immobilized enzymes were compared after three hours of incubation at high temperature, and the results indicated that the immobilized enzyme FNCZ8-T was completely inactivated. In addition, the appearance of the microspheres changed and the magnetic force was lost at
3.3. Effect of metal ions on the activity of FSNCZ8-T The effect of various metal cations on the catalytic efficiency of immobilized enzymes has not been studied thoroughly [28–30]. Based on known interactions between free enzymes and metal ions, the effect of metal cations on the activities of FSNCZ8-T and free Tpebgl3 were determined at pH 5.5 and at 90 °C. The final concentration of metal cations in the reaction solutions was 2 mM. The results presented in Table 1 show that the activity of free Tpebgl3 is inhibited by Fe2+, Li+ and Al3+. Three metal cations, K+, Mn2+ and Zn2+, have a weak positive effect on the activity of free Tpebgl3. However, except for Fe2+, it was found that several common metal cations had no inhibitory effect on the activity of FSNCZ8T. Although Zn2+, Mn2+ and K+ can effectively improve the enzyme activity of FSNCZ8-T, the combination of FSNCZ8-T and Fe2+ (2 mM) increases the catalytic activity of FSNCZ8T 1.94-fold, which is the most significant effect of all metal cations tested on FSNCZ8-T. However, increased Fe2+ concentrations do not enhance further the catalytic activity of FSNCZ8-T (Fig. 3A). Moreover, the catalytic activity of free enzyme was inhibited by high Fe2+ concentrations. The kinetic equation was obtained by adding different substrate concentrations to the solution at the optimal conditions and a reaction time of 5 min. Km and Kcat values of 0.559 mM and 11840 s−1, respectively, were obtained for free Tpebgl3 with no added Fe2+; and 0.384 mM and 9786 s−1, respectively, for free Tpebgl3 with 2 mM Fe2+ under optimal conditions. Thus, the catalytic efficiency of free Tpebgl3 with Fe2+ was considerably lower than that without Fe2+. In addition, the dependence of the rate of the enzymatic reaction on the substrate concentration followed Michaelis-Menten kinetics, the Km of FSNCZ8-T was 0.303 mM without Fe2+, and 0.403 mM in the presence of metal ion. Thus, the addition of 2 mM Fe2+ significantly increases the binding between the enzyme active site and the substrate, which may be due to an alteration in the affinity of the active site toward substrate binding due to limited diffusion [31]. The Kcat in the presence of 2 mM Fe2+ 3
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Fig. 1. Determination of the characteristics of magnetic materials FNCZ8 and FSNCZ8, and immobilization of Tpebgl3. A. Structural analysis of FNCZ8 and FSNCZ8 by FITR (IR KBr (cm−1): 3420 (amino, N-H), 2928 (aromatic, CeH), 1634 (aromatic frame), 578 (Fe3O4) and 1066 (SiO2). B. XRD results for FNCZ8 and FSNCZ8. FNCZ8 (Fig. 1b–B) produced five distinct peaks at 2θ = 30.3°, 35.3°, 43.5°, 57.7° and 63° that correspond to the (220), (311), (400), (500) and (440) crystal planes of Fe3O4. It was shown that amino group modification had no effect on the Fe3O4 diffraction peak. The XRD pattern for FSNCZ8 is also shown in Fig. 1b–A. Compared to FNCZ8, FSNCZ8 produced a diffraction peak at 2θ = 23.8°, which indicated the formation of amorphous silica. Moreover, SiO2 and Fe3O4 were shown to be separated, and Fe3O4 did not show any change in its crystal structure after coating with SiO2. C. Ability of two magnetic microspheres to adsorb Tpebgl3. D. Effect of glutaraldehyde concentration on the two magnetic microspheres. F. Thermostability of FNCZ8-T and FSNCZ8-T.
Fig. 2. Optimal reaction conditions for free Tpebgl3 and FSNCZ8-T. a, optimum temperature; b, optimum pH.
was 24454 s-1, which was 2.17 times higher than that without Fe2+. The catalytic efficiency (Kcat/Km) in the presence of 2 mM Fe2+ was 60680 mM-1 s−1 (Table 2), which was increased by 1.63 times compared to that in the absence of metal ions. However, Fe2+ had little effect on the activity of free Tpebgl3, possibly because the metal cation altered the structure of the magnetic microspheres or the interaction between the carrier and the bound enzyme [32,33].
3.4. Effect of Fe2+ on thermal stability and pH stability of FSNCZ8-T The catalytic activity of free Tpebgl3 at high temperatures requires the immobilized enzyme to possess multiple features: high temperature structural stability, pH stability, avoiding the enzyme configuration, preventing rapid deactivation of the enzyme, and prolonging the reaction in a continuous high temperature reaction. The effect of Fe2+ on the thermal stability of FSNCZ8-T was determined. The free enzyme and FSNCZ8-T were incubated separately at 65, 70, 75, 80, 85 and 90 °C for 3 h. The results in Fig. 3B show that the immobilized enzyme in the 4
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Table 1 The effect of different metal ions on the catalytic activity of immobilized enzyme FSNCZ8-T.
Table 2 Kinetic equation parameters for free Tpebgl3 and FSNCZ8-T with and without Fe2+.
Metal cation
Free enzyme
FSNCZ8- Tpebgl3
Materials
free Tpebgl3
FSNCZ8-T
Fe2+ Li+ Al3+ K+ Ni2+ Mn2+ Ca2+ Zn2+ Mg2+
88.4 ± 1.0 88.1 ± 1.0 90.6 ± 1.0 110.2 ± 1.2 97.1 ± 1.1 113.5 ± 1.2 100.4 ± 1.1 108.4 ± 1.2 98.6 ± 1.1
194.92 ± 11.2 97.47 ± 5.82 95.20 ± 3.12 126.89 ± 0.75 109.46 ± 4.50 128.07 ± 2.48 104.73 ± 6.86 124.74 ± 6.22 98.55 ± 0.38
Fe2+ concentration (mM)
0 mM
2 mM
0 mM
2 mM
Km (mM) Kcat (s−1) Kcat/Km (mM1 -1 s )
0.559 ± 0.07 11840 ± 65 21181
0.384 ± 0.06 9786 ± 42 25484
0.303 ± 0.03 11267 ± 37 37184
0.403 ± 0.03 24454 ± 42 60680
compared to that in the presence of Fe2+. The pH stability of the immobilized enzyme was relatively greater, and the relative activity remained at 90% or greater under alkaline conditions.
presence of Fe2+ has significantly increased thermal stability compared to the free enzyme. However, the stability of FSNCZ8-T at 80 °C or higher is reduced in the presence or absence of metal ions, possibly due to the leaching of enzyme from the magnetic microspheres at high reaction temperatures. The adsorption of magnetic microspheres is weakened at high temperatures, resulting in loss of enzyme. The pH stability of free enzyme and FSNCZ8-T was determined. The enzymes were incubated at room temperature in different pH value buffer systems for 3 h and the activity of the enzymes was then measured. The results are shown in Fig. 3C. The residual activity of FSNCZ8-T was basically unchanged under alkaline conditions. The residual activity of FSNCZ8-T in the absence of Fe2+ was increased by more than 50% compared to the free enzyme at pH 8.0. The activity of FSNCZ8-T was slightly decreased under acidic conditions (pH 3.5), due to the instability of the carrier itself under acidic conditions. Furthermore, the relative activity of the free enzyme decreased significantly. This may be because immobilized Tpebgl3 can maintain the spatial structure and active site of the enzyme more effectively and resist the destruction of the enzyme protein by extremes of pH. In addition, there was no significant change in the pH stability of the free enzyme
3.5. Effect of Fe2+ on the glucose tolerance of immobilized enzyme β-glucosidase is subject to feedback inhibition by glucose during the hydrolysis of the substrate, leading to a decrease of the conversion rate and the formation of product. Immobilization is expected to improve the tolerance of the enzyme to glucose [34,35]. The tolerance of free enzyme and FSNCZ8-T to glucose was determined between 0 and 400 mM glucose. The results shown in Fig. 3D indicate that as the glucose concentration is gradually increased, the relative activity of free Tpebgl3 decreases rapidly. When the glucose concentration is greater than 100 mM, the relative enzyme activity is less than 15%. At a glucose concentration of 400 mM, the free enzyme is completely inhibited, but the residual activity of immobilized enzyme remains at 25% of the initial activity, or greater. A reasonable explanation is that the spatial configuration of the magnetic microspheres provides a wall that resists inhibition by high concentrations of glucose. The results indicate that the use of the magnetic microsphere FSNCZ8 to immobilize Tpebgl3 can
Fig. 3. Characteristics of FSNCZ8-T. a, effect of Fe2+ concentration on the activity of free Tpebgl3 and FSNCZ8-T; b, high temperature stability of free Tpebgl3 and FSNCZ8-T; c, pH stability of free Tpebgl3 and FSNCZ8-T; d, glucose tolerance of free Tpebgl3 and FSNCZ-T. 5
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Fig. 4. Effect of Fe2+ on the reusability of FSNCZ8-T.
increase the glucose tolerance of the GH3 β-glucosidases (Tpebgl3). Furthermore, after the addition of 2 mM Fe2+, the glucose tolerance of the immobilized enzyme was greatly improved. At a glucose concentration of 400 mM, a residual activity of approximately 40% was retained by immobilized enzyme, and the relative activity in the presence of Fe2+ was increased by approximately 15% compared to that in the absence of Fe2+. This may be because Fe2+ acts as an enzyme modifier in the reaction system [36]. This could increase the reaction velocity by enzyme activators by favoring a productive interaction between enzyme and substrate to form an enzyme-substrate complex by creating an enzyme-substrate-activator complex.
In addition, The Fe2+ enabled the immobilized enzyme to exhibit a higher catalytic activity than that of metal ion-free enzyme. The properties of FSNCZ8-TpeRha, including thermal stability, pH stability, and glucose tolerance, were greatly enhanced by metal cations, which can be of great significance in industrial applications of Tpebgl3. Acknowledgements This work was supported by the National Key R&D Program of China (2016YFD0600805), the Forestry Achievements of Science and Technology to Promote Projects ([2017] 10) and the Science and Technology project of Jangsu Province (SZ-XZ2017023)
3.6. Effect of Fe2+ on the reusability of FSNCZ8-T References The reusability of the immobilized enzyme is essential for the repeated use of the enzyme, for reducing the amount of enzyme used, and for reducing enzyme production and consumption. Due to the high specificity and inseparability of free Tpebgl3, the reusability of FSNCZ8-T was determined and the results are shown in Fig. 4. First, 2 mM Fe2+ was added to the mixture containing immobilized enzyme to verify the effect of the presence or absence of metal ions on the reusability of FSNCZ8-T. The results shown in Fig. 4A demonstrate that metal ions cannot form stable bonds or interact with immobilized enzymes. The activity of the immobilized enzyme in the presence of metal ions was recorded as 100% for the first reaction cycle, and then FSNCZ8-T was washed with deionized water, and the activity was then determined without metal ions. Repeating the reaction, the relative activity of the first cycle was found to be 1.5 times the relative activity of the two cycles. In addition, the relative activity of the third repeat cycle with added Fe2+ was also higher than that of the metal ion-free enzyme (2 and 4 times). This may be because Fe2+ activates the catalytic activity of FSNCZ8-T. Therefore, to increase the activity of the FSNCZ8-T, it is necessary to add Fe2+. As shown in Fig. 4B, FSNCZ8-T was reacted 10 times in the presence or absence of Fe2+, and the relative activity of the immobilized enzyme in the presence of Fe2+ was maintained above 74%. This is due to leakage and denaturation of the free enzyme and does not constitute a major obstacle. This represents a significant improvement for industrial applications of thermostable βglucosidase TpebgL3.
[1] J. Xie, D. Zhao, L. Zhao, J. Pei, W. Xiao, G. Ding, Z. Wang, Overexpression and characterization of a Ca2+ activated thermostable β-glucosidase with high ginsenoside Rb1 to ginsenoside 20(S)-Rg3 bioconversion productivity, J. Ind. Microbiol. Biotechnol. 422 (2015) 839–850. [2] Q. Yan, W. Zhou, X. Shi, P. Zhou, J.U. Dianwen, M. Feng, Biotransformation pathways of ginsenoside Rb1 to compound K by β-glucosidases in fungus Paecilomyces Bainier sp. 229, Process Biochem. 45 (2010) 1550–1556. [3] K.H. Chang, N.J. Mi, K.T. Kim, H.D. Paik, Evaluation of glucosidases of Aspergillus niger strain comparing with other glucosidases in transformation of ginsenoside Rb1 to ginsenosides Rg3, J. GINS Res. 38 (2014) 47–51. [4] R. Riedel, Nanoscaled inorganic materials by molecular design, Chem. Soc. Rev. 41 (2012) 5029–5031. [5] L. Li, S.J. Lee, Q.P. Yuan, T.I. Wan, C.K. Sun, N.S. Han, Production of bioactive ginsenoside Rg3(S) and compound K using recombinant Lactococcus lactis, J. GINS Res. 42 (2018) 412–418. [6] J.T. Hwang, M.S. Lee, H.J. Kim, M.J. Sung, H.Y. Kim, M.S. Kim, D.Y. Kwon, Antiobesity effect of ginsenoside Rg3 involves the AMPK and PPAR-gamma signal pathways, Phytother. Res. 23 (2010) 262–266. [7] H.S. Kim, E.H. Lee, S.R. Ko, K.J. Choi, J.H. Park, D.S. Im, Effects of ginsenosides Rg3 and Rh2 on the proliferation of prostate cancer cells, Arch. Pharm. Res. 27 (2004) 429–435. [8] R. Ye, G. Zhao, X. Liu, Ginsenoside Rd for acute ischemic stroke: translating from bench to bedside, Expert Rev. Neurother. 13 (2013) 603–613. [9] L. Hae-Ung, B. Eun-Ah, H. Myung Joo, K. Dong-Hyun, Hepatoprotective effect of 20(S)-ginsenosides Rg3 and its metabolite 20(S)-ginsenoside Rh2 on tert-butyl hydroperoxide-induced liver injury, Biol. Pharm. Bull. 28 (2005) 1992–1994. [10] S. Hu, D. Wang, J. Hong, A Simple method for β-glucosidase immobilization and its application in soybean isoflavone glycosides hydrolysis, Biotechnol. Bioprocess Eng. 23 (2018) 39–48. [11] P.G. Vazquez-Ortega, M.T. Alcaraz-Fructuoso, J.A. Rojas-Contreras, J. LópezMiranda, R. Fernandez-Lafuente, Stabilization of dimeric β-glucosidase from Aspergillu s niger via glutaraldehyde immobilization under different conditions, Enzyme Microb. Technol. 110 (2018) 38–45. [12] Y. Carvalho, Almeida JMAR, P.N. Romano, K. Farrance, P.D. Carà, N. Pereira, J.A. Lopez-Sanchez, E.F. Sousa-Aguiar, Nanosilicalites as support for β-glucosidases covalent immobilization, Appl. Biochem. Biotechnol. 182 (2017) 1619–1629. [13] S. Peirce, M.E. Russo, R. Perfetto, C. Capasso, M. Rossi, R. Fernandez-Lafuente, P. Salatino, A. Marzocchella, Kinetic characterization of carbonic anhydrase immobilized on magnetic nanoparticles as biocatalyst for CO2 capture, Biochem. Eng. J. 138 (2018) 1–11. [14] M. Defaei, A. Taheri-Kafrani, M. Miroliaei, P. Yaghmaei, Improvement of stability and reusability of α-amylase immobilized on naringin functionalized magnetic nanoparticles: a robust nanobiocatalyst, Int. J. Biol. Macromol. 113 (2018) 354–360.
4. Conclusion High temperature-resistant recombinant GH3 β-glucosidase (Tpebgl3, EC 3.2.1.21) from Thermomotoga petrophila DSM 13995 has great potential for the catalytic conversion of pharmaceutically active components. The Tpebgl3 was immobilized on a synthetic high temperature magnetic carrier (FSNCZ8) that provided stronger adsorption capacity and magnetic and high temperature stabilities than the previously described FNCZ8, which was not coated with tetraethyl silicate. 6
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[26] Yen-Ning Kuo, J. Hong, Investigation of solubility of microcrystalline cellulose in aqueous NaOH, Polym. Adv. Technol. 16 (2005) 425–428. [27] F.F. Freitas, L.D.S. Marquez, G.P. Ribeiro, G.C. Brandão, V.L. Cardoso, E.J. Ribeiro, Optimization of the immobilization process of β-galatosidade by combined entrapment cross-linking and the kinetics of lactose hydrolysis, Braz. J. Chem. Eng. 29 (2012) 15–24. [28] Z. Zhai, T. Yang, B. Zhang, J. Zhang, Effects of metal ions on the catalytic degradation of dicofol by cellulase, J Environ. Sci. 33 (2015) 163–168. [29] Ruinan Zhao, Mancheng Hu, Shuni Li, Quanguo Zhai, Yucheng Jiang, Immobilization of chloroperoxidase in metal organic framework and its catalytic performance, Acta Chim. Sin. 75 (2017) 293–299. [30] S. Fennouh, V. Casimiri, A. Geloso-Meyer, C. Burstein, Kinetic study of heavy metal salt effects on the activity of L-lactate dehydrogenase in solution or immobilized on an oxygen electrode, Biosens. Biolectron. 13 (1998) 903–909. [31] J. Grewal, R. Ahmad, S.K. Khare, Development of cellulase-nanoconjugates with enhanced ionic liquid and thermal stability for in situ lignocellulose saccharification, Bioresour. Technol. Rep. 242 (2017) 236–243. [32] L. Fernandez-Lopez, R. Bartolome-Cabrero, M. Daniela Rodriguez, Stabilizing effects of cations on lipases depend on the immobilization protocol, RSC Adv. 5 (2015) 83868–83875. [33] N.T. Wright, K.M. Varney, K.C. Ellis, J. Markowitz, R.K. Gitti, D.B. Zimmer, D.J. Weber, The three-dimensional solution structure of Ca2+ -bound S100A1 as determined by NMR spectroscopy, J. Mol. Biol. 353 (2005) 410–426. [34] H. Cao, Y. Zhang, P. Shi, R. Ma, H. Yang, W. Xia, Y. Cui, H. Luo, Y. Bai, B. Yao, A highly glucose-tolerant GH1 β-glucosidase with greater conversion rate of soybean isoflavones in monogastric animals, J. Ind. Microbiol. Biotechnol. 45 (2018) 369–378. [35] R.J. You, H.Y. Shin, Y.S. Song, S.B. Kim, S.W. Kim, Enhancement of immobilized enzyme activity by pretreatment of β-glucosidase with cellobiose and glucose, J. Ind. Eng. Chem. 18 (2012) 702–706. [36] José Carlos Santos Salgado, Luana Parras Meleiro, Sibeli Carli, Richard John Ward, Glucose tolerant and glucose stimulated β-glucosidases – a review, Bioresour. Technol. Rep. 267 (2018) 704–713.
[15] G. Josep, Z. Yue, T. Hannah, C. Oscar, M.E. Webb, Z. Dejian, Multilayer enzymecoupled magnetic nanoparticles as efficient, reusable biocatalysts and biosensors, Nanoscale 3 (2011) 3721–3730. [16] H.J. Park, A.J. Driscoll, P.A. Johnson, The development and evaluation of β-glucosidase immobilized magnetic nanoparticles as recoverable biocatalysts, Biochem. Eng. J. 133 (2018) 66–73. [17] A. Schejn, T. Mazet, V. Falk, L. Balan, L. Aranda, G. Medjahdi, R. Schneider, Fe3O4@ ZIF-8: magnetically recoverable catalysts by loading Fe3O4 nanoparticles inside a zinc imidazolate framework, Dalton Trans. 44 (2015) 10136–10140. [18] S.L. Cao, H. Xu, L.H. Lai, W.M. Gu, P. Xu, J. Xiong, H. Yin, X.H. Li, Y.Z. Ma, J. Zhou, Magnetic ZIF-8/cellulose/Fe3O4 nanocomposite: preparation, characterization, and enzyme immobilization, Bioresour. Bioprocess. 4 (2017) 56. [19] M. Jian, L. Bao, G. Zhang, R. Liu, X. Zhang, Adsorptive removal of arsenic from aqueous solution by zeolitic imidazolate framework-8 (ZIF-8) nanoparticles, Colloid Surf. A 465 (2015) 67–76. [20] V. Chiaradia, N.S. Soares, A. Valério, D.D. Oliveira, P.H.H. Araújo, C. Sayer, Immobilization of Candida antarctica lipase B on magnetic poly(Urea-Urethane) nanoparticles, Appl. Biochem. Biotechnol. 180 (2016) 1–18. [21] S.A. Mohamed, M.H. Al-Harbi, Y.Q. Almulaiky, I.H. Ibrahim, H.A. Salah, M.O. ElBadry, A.M. Abdel-Aty, A.S. Fahmy, R.M. El-Shishtawy, Immobilization of trichoderma harzianum α-amylase on PPyAgNp/Fe3O4 -nanocomposite: chemical and physical properties, Artif. Cells Nanomed. Biotechnol. 26 (2018) 1–6. [22] S. Shi, J. Yang, S. Liang, M. Li, Q. Gan, K. Xiao, J. Hu, Enhanced Cr(VI) removal from acidic solutions using biochar modified by Fe3O4@SiO2-NH2 particles, Sci. Total Environ. 628–629 (2018) 499–508. [23] V.G. Pérez, C. Márquez-Álvarez, R.M. Blanco, Successful encapsulation of β-glucosidase during the synthesis of siliceous mesostructured materials, J. Chem. Technol. Biotechnol. 93 (2018) 2625–2634. [24] V. Califano, F. Sannino, A. Costantini, et al., Wrinkled silica nanoparticles: efficient matrix for β-glucosidase immobilization, J. Phys. Chem. C 122 (2018) 8373–8379. [25] X. Shi, L. Zhao, J. Pei, G. Lin, P. Wan, Z. Wang, X. Wei, Highly enhancing the characteristics of immobilized thermostable β-glucosidase by Zn2+, Process Biochem. 66 (2018) 89–96.
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