Enhancing Catalytic Activity and Stability of Yeast Alcohol

Article pubs.acs.org/IECR

Enhancing Catalytic Activity and Stability of Yeast Alcohol Dehydrogenase by Encapsulation in Chitosan-Calcium Phosphate Hybrid Beads Pingping Han,†,‡ Xiaokai Song,†,‡ Hong Wu,*,†,‡ Zhongyi Jiang,†,‡ Jiafu Shi,‡,§ Xiaoli Wang,†,‡ Wenyan Zhang,†,‡ and Qinghong Ai†,‡ †

Key Laboratory for Green Chemical Technology of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China ‡ Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072, China § School of Environment Science and Engineering, Tianjin University, Tianjin 300072, China S Supporting Information *

ABSTRACT: A kind of calcium phosphate-mineralized chitosan beads (chitosan−CaP) was prepared via a one-pot method by adding droplets of Ca2+-containing chitosan aqueous solution into phosphate-containing sodium tripolyphosphate aqueous solution. The chitosan beads formed immediately coupled with in situ precipitation of calcium phosphate on the surface. The antiswelling properties of hybrid beads were greatly improved with the swelling degree as low as 5%. The morphology of the resultant chitosan−CaP hybrid beads was observed by scanning electron microscopy (SEM). Yeast alcohol dehydrogenase (YADH) was encapsulated in the hybrid beads with an about 40% lower enzyme leakage compared with that in the pure chitosan beads. The optimal temperature and pH value for enzymatic conversion catalyzed by YADH immobilized in the chitosan−CaP beads were 30 °C and 7.0, respectively, which were identical to those for free YADH. The immobilized YADH displayed obviously higher thermal stability, pH stability, recycling stability, and storage stability than the free YADH counterpart.

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INTRODUCTION Enzymes are versatile catalysts due to their superior chemo-, regio-, and stereospecificity.1−3 However, the large-scale application of free enzymes is economically limited since it is difficult to separate and recover the free enzymes from the mixture of substrates, products, and possible byproducts, and the enzymes are liable to denature.4 Enzyme immobilization offers an effective solution to overcome these drawbacks by rendering the immobilized enzymes with enhanced stability, repeated usability, and facile separation from reaction mixtures to prevent enzyme contamination in products.5,6 Reuse of immobilized enzymes provides a cost advantage for industrial application of enzyme-catalyzed processes. Various carriers have been used for enzyme immobilization such as polyamide,7,8 gelatin,9,10 alginate,11,12 and chitosan.13−16 Natural polymers, especially chitosan-based materials, have been extensively explored for enzyme immobilization because of their low toxicity, high hydrophilicity, good biocompatibility, and easy availability. However, the mechanical strength of pure chitosan hydrogels is often very low, which leads to the leakage and instability of immobilized enzyme. The polymer−inorganic hybrid carriers are a new generation of materials for enzyme immobilization, owing to their unique, tunable, and superior physicochemical and mechanical properties.17−19 So far, two main kinds of configurations of polymer−inorganic hybrid carriers for enzyme immobilization have been developed: mixed−matrix configuration20 and core−shell configuration.21 The mixed-matrix configuration refers to the homodistribution of polymer and inorganic materials, while the core−shell configuration refers to the heterodistribution of polymer and © 2014 American Chemical Society

inorganic materials. Compared to the mixed-matrix configuration, the core−shell configuration can provide the immobilized enzyme with more favorable microenvironment22 by using hydrophilic, biocompatible polymers as core materials, and hydrophilic, robust inorganic particles as shell materials. Recently, Chu et al.23 have fabricated a type of core−shell capsules through a coextrusion minifluidic approach and a biosilicification method for enzyme immobilization. In our previous work, organic−inorganic hybrid capsules have been fabricated through biomimetic silicification.24 The formation of the silica shell is induced by protamine which is preabsorbed on the Ca-alginate core. Chitosan is a natural cationic polysaccharide which has been proved to be an effective structuredirecting agent for inorganic minerals. Calcium phosphate, a principal component of hard tissues such as tooth enamel and bone,25,26 is of superior mechanical stability, insolubility, and biocompatibility. Calcium phosphate can be prepared under mild conditions, which is suitable for enzyme immobilization.27 Zeng et al.28 have immobilized enzymes on the CaHPO4/αamylase hybrid nanoflowers with enhanced enzymatic activity. We have immobilized catalase on the chitosan/calcium pyrophosphate hybrid microflowers and obtained high catalytic activity.29 In this study, calcium phosphate-mineralized chitosan beads with a core−shell configuration were fabricated and employed Received: Revised: Accepted: Published: 597

August 19, 2014 December 18, 2014 December 26, 2014 December 26, 2014 DOI: 10.1021/ie503294a Ind. Eng. Chem. Res. 2015, 54, 597−604

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Industrial & Engineering Chemistry Research

of Polymer-Inorganic Hybrid Beads. Finally, the concentration of residual YADH in TPP solution ([YADH]s) was determined also by the micro-Bradford method. The encapsulation efficiency of beads was calculated as follows (eq 2).

to encapsulate YADH for effective conversion of formaldehyde to methanol. The hybrid chitosan beads were fabricated by a one-pot method. The formation of the calcium phosphatemineralized chitosan beads via electrostatic interactions was coupled with the controlled precipitation of calcium phosphate arisen from the counter-diffusion of ions across the polysaccharide interface. In order to enhance mechanical strength and reduce the leakage of enzyme, the enzymecontaining chitosan beads were cross-linked by sodium tripolyphosphate and coated with calcium phosphate to form chitosan−CaP core−shell hybrid beads. The enzyme leakage, activity retention as well as temperature, pH, storage, and recycling stability of the encapsulated YADH were studied extensively.

Encapsulation efficiency (%) [YADH]s × Vs = 100 − × 100 [YADH]i × Vi

where Vs and Vi represented the volume of TPP and chitosan solution, respectively. The enzyme leakage behavior was investigated by immersing the YADH-containing pure chitosan beads and calcium phosphate-mineralized hybrid beads into phosphate buffer (50 mM, pH 7.0) at room temperature, respectively. The changes of YADH concentration released from the beads into solution were detected by a UV−vis spectrophotometer at 280 nm. The leakage ratio was calculated by following equation (eq 3):

2. MATERIALS AND METHODS 2.1. Materials. Yeast alcohol dehydrogenase (YADH, EC1.1.1.1, from Saccharomyces cerevisiae, Mw 14−15 kDa, pI 5.2−5.6), nicotinamide adenine dinucleotide (NADH, grade I, 98%) and chitosan (viscosity 20−200 cps, deacetylation degree 75−85%) were purchased from Sigma-Aldrich. Sodium phosphate dibasic dodecahydrate (Na2HPO4·12H2O), calcium chloride dehydrate (CaCl2·2H2O), and sodium tripolyphosphate (Na5P3O10) were purchased from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China). All the other chemicals were of analytical reagent grade. 2.2. Preparation of Polymer-Inorganic Hybrid Beads. Chitosan was dissolved in deionized water to get a concentration of 2% (w/v) unless otherwise noted, and then CaCl2 was dissolved into the above chitosan solution to get a specific concentration in the range of 0.05−0.1 M. Then YADH was dissolved into the CaCl2−chitosan solution. Calcium phosphate-mineralized hybrid beads with encapsulated enzyme were fabricated by adding the above mixture into sodium tripolyphosphate (50 mM) and Na2HPO4 (60 mM) solution dropwise using a 0.9 mm diameter needle attached to a 10 mL syringe. The droplets were incubated for 30 min in the sodium tripolyphosphate solution, and then the droplets were taken out and washed by deionized water. Pure chitosan beads were prepared according to the same procedure except that CaCl2 was not added to the chitosan solution. All processes were conducted at pH 7.0. 2.3. Characterization. The beads were lyophilized and gold coated, and then the morphology of the beads was observed by SEM (Nanosem 430). The surface elemental composition of the beads was analyzed by energy-dispersive spectroscopy (EDX) attached to the SEM. Circular Dichroism (CD) spectra of enzymes were obtained by Jasco J810 spectropolarimeter (Toyko, Japan). 2.4. Swelling Property. The fresh pure chitosan beads and calcium phosphate-mineralized hybrid beads were weighed (Wi) and then immersed into PBS solution (50 mM, pH 7.0) at room temperature. At specified time intervals, the weights of beads reached a constant value (Ws). The swelling degree (Sw) was calculated using the following equation (eq 1). Sw = (Ws − Wi )/Wi × 100%

(2)

leakage (%) =

[YADH]solution × Vsolution × 100 total encapsulation in beads

(3)

where [YADH]solution was the concentration of YADH in the phosphate buffer solution, Vsolution represented the volume of the phosphate buffer solution. 2.6. Enzyme Activity Assay. The catalytic activity of YADH was evaluated by the hydrogenation reaction of formaldehyde into methanol, which is coupled with the oxidation reaction of NADH into NAD+ YADH

HCHO + NADH + H+ ←⎯⎯⎯→ CH3OH + NAD+

The standard assay was conducted in the Tris-HCl buffer solution (pH 7.0) containing HCHO (10 mM) and NADH (133 μM) under the conditions of temperatures (30−60 °C) and pH values (4.0−10.0). The enzyme activity was measured by the decrease of NADH absorbance at 340 nm using UV−vis spectrophotometry, and each measurement was repeated three times. The definition of one unit was the quantity of YADH required to convert 1.0 μmol of NADH to NAD+ per min at pH 7.0 and 25 °C. The kinetic parameter, Michaells constant (Km), for the enzymatic conversion of HCHO into CH3OH by either free enzyme or encapsulated enzyme was measured and determined using the Dalziel equation. The activity assay was applied to different CHOH concentrations (from 5 to 250 mM) and NADH concentrations (88 to 260 μM). Enzyme activity was determined at 30 °C in Tris-HCl (50 mM, pH 7.0). 2.7. Thermal and pH Stability. The thermal stability was evaluated by the residual activity, which was measured at optimum conditions, after heating the Tris-HCl buffer solution containing free and encapsulated YADH for 2 h at different temperatures (30−60 °C). The thermal denaturation kinetics of YADH was expressed by the first order exponential equation,31,32 and the thermal denaturation constants (kd) were calculated according to eq 4.

(1)

A = A 0 exp( −kdt )

2.5. Encapsulation Efficiency and Leakage Ratio. The encapsulation efficiency was measured as follows. First, the concentration of YADH in chitosan solution ([YADH]i) was measured by the micro-Bradford method30 using a UV spectrophotometer and then YADH was encapsulated into the polymer-inorganic hybrid beads as described in Preparation

(4)

where A0 was the activity of YADH before incubation, A was the activity of YADH after incubation for a certain time at a certain temperature, and t was the incubation time. The half-life (t1/2) value for YADH thermal denaturation was calculated by eq 5. 598

DOI: 10.1021/ie503294a Ind. Eng. Chem. Res. 2015, 54, 597−604

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Industrial & Engineering Chemistry Research

t1/2 = ln 2/kd

Scheme 1. Preparation Procedure of Mineralized ChitosanCaP Hybrid Beads (Chitosan−CaP)a

(5)

The activation energy (Ed) for YADH thermal denaturation was calculated based on the Arrhenius equation (eq 6). ln kd = Ed /RT + ln C

(6)

The Ed was calculated by the plot of log denaturation rate constants (ln kd) versus reciprocal of the absolute temperature (T) from (eq 7). slope = −Ed /R

(7)

The enthalpy (ΔH°, kJ mol −1) for YADH thermal denaturation was calculated by eq 8, ΔH ° = Ed − RT

(8)

where R was the gas constant (8.3145 J mol−1 K−1) and T was the corresponding absolute temperature.33 Similarly, the pH stability was evaluated by preincubating YADH in buffer solution with a pH ranging from 4.0 to 10.0 for 2 h and then the residual activity was determined at optimum conditions. The activity of YADH without incubation was taken as 100%. 2.8. Recycling Stability and Storage Stability. The recycling stability of YADH, which was expressed in terms of recycling efficiency, was evaluated by the activity of YADH in each successive reaction cycle after YADH was recovered and rinsed from each batch. The initial activity of YADH was assumed to be 100% (eq 9).

a

The chitosan beads formed immediately through cross-linking between chitosan and sodium tripolyphsphate due to the interfacial complexation of the positively charged chitosan and negatively charged sodium tripolyphosphate. Meanwhile, counter-diffusion of Ca2+ and phosphate anions across the polysaccharide interface led to the in situ precipitation of calcium phosphate.

The prepared particle was spherical with a diameter of ∼3 mm. The surface morphologies of the pure chitosan beads and the chitosan−CaP hybrid beads were shown in Figure 1. It could be clearly found that the chitosan−CaP beads had an intact and smooth surface structure, and in comparison, the pure chitosan beads had a wrinkled surface structure which was ascribed to the shrink of the polymer during the lyophilization process. Moreover, as shown in Figure 1 (panels f−h), the core−shell structure could be observed by the SEM crosssection images as specifically indicated by the red line in Figure 1g. The shell layer was relatively dense with a thickness of about 45−55 μm. Energy dispersive X-ray (EDX) analysis of the surface of the chitosan and chitosan−CaP beads was performed and compared in Figure 2. The C, O, N, P, and Na elements were clearly detected by EDX for the chitosan beads. Compared with the nonmineralized chitosan beads, the elemental peaks of Ca appeared after mineralization, confirming the successful deposition of CaP on the surface of the chitosan beads via a biomimetic mineralization process. Moreover, the elemental peaks of C, O, N, P, and Na still existed after CaP deposition, indicating that the surface of the chitosan−CaP beads was actually a hybrid coating consisting of both chitosan and CaP instead of a complete pure CaP layer. It could be found that the surface morphologies of the chitosan−CaP hybrid beads became smoother and smoother with an increased CaCl2 concentration to 50 mM, and then with the further increase of CaCl2 concentration, the surface morphology of the chitosan−CaP hybrid beads became more wrinkled. 3. 2. Swelling Property. Swelling behavior is a characteristic property for evaluating the stability and lifetime of hydrogels. Since swelling may reduce mechanical strength, loading efficiency, and recycling stability, the swelling behavior was a serious challenge for hydrogels used as carriers for enzyme immobilization. In this study, no breakage was found for all of the beads during immersion in phosphate buffer at room temperature, even when a swelling equilibrium was achieved. As shown in Figure 3, the swelling degree of the

recycling efficiency (%) enzyme activity in the n th reaction cycle = × 100 initial enzyme activity in the 1st reaction cycle (9)

Free and encapsulated YADH were stored at 4 °C. The residual activity was analyzed to investigate the storage stability, which was compared by storage efficiency, the ratio of the residual activity to the initial activity (eq 10). storage efficiency (%) enzyme activity after storage for n days = × 100 initial enzyme activity

(10)

3. RESULTS AND DISCUSSION 3.1. Fabrication and Characterization of Hybrid Beads. Mineralized chitosan beads (chitosan−CaP) were prepared via a one-pot method, as illustrated in Scheme 1. The calcium phosphate-mineralized hybrid beads were prepared by adding droplets of Ca2+-containing chitosan solution [2% (w/v)] into phosphate-containing sodium tripolyphosphate solution. Due to the interfacial complexation of the positively charged chitosan and negatively charged sodium tripolyphosphate, the chitosan beads formed immediately through cross-linking between chitosan and sodium tripolyphsphate. At the same time, counter-diffusion of Ca2+ and phosphate anions across the polysaccharide interface led to the in situ precipitation of calcium phosphate (see Figure S1 of the Supporting Information). The beads became stiffer and stiffer owing to further cross-linking between chitosan and sodium tripolyphosphate as well as the deposition of calcium phosphate on the bead surface. 599

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Figure 2. EDX analysis of the chitosan and chitosan−CaP beads’ surface: (a) chitosan beads’ surface and (b) chitosan−CaP beads’ surface.

Figure 1. SEM images of surface morphology of (a) pure chitosan beads. The inset optical image was the pure chitosan beads with diameter of ∼3 mm. SEM images of the surface morphology of the chitosan−CaP bead prepared in the presence of (b) 25 (inset: optical image was chitosan−CaP beads with diameter of ∼3 mm), (c) 50, (d) 75, and (e) 100 mM CaCl2. SEM images of (f) the cross section of pure chitosan beads, and (g) the cross section of chitosan−CaP beads. (h) Partial magnified SEM images of the cross section of chitosan− CaP beads. The small particles in the red circle were calcium phosphate. Figure 3. Swelling ratio of beads in phosphate buffer (50 mM, pH 7.0). Each data was repeated three times.

mineralized hybrid chitosan−CaP beads was less than 5%, whereas that of the nonmineralized chitosan beads was almost 25%, 5-fold higher. The result inferred that the rigid inorganic layer could effectively prevent the mineralized beads from severe swelling. Moreover, the swelling degree of the beads was directly associated with the concentration of CaCl2. The swelling degree decreased at first and then increased with the increase of CaCl2 concentration and the lowest value was obtained at the CaCl2 concentration of 50 mM. At higher CaCl2 concentrations, a large part of the free amine groups on the chitosan molecules were consumed in the mineralization process, thus the quantity of amino groups for cross-linking with sodium tripolyphosphate considerably decreased, resulting in a loose network structure. At lower CaCl2 concentrations,

most of the free amine groups were used to cross-link with the sodium tripolyphosphate, resulting in a weakened ability to induce the mineralization, thereby, the antiswelling property was reduced. The optimal combined effects of cross-linking and mineralization on the antiswelling property was achieved at the CaCl2 concentration of 50 mM. 3.3. Encapsulation Efficiency and Enzyme Leaching Behavior. To evaluate the encapsulation capacity of the beads, the encapsulation efficiency was investigated (Figure 4a). It was found that the encapsulation efficiency remained 100% (i.e., all the enzyme molecules were immobilized when the YADH 600

DOI: 10.1021/ie503294a Ind. Eng. Chem. Res. 2015, 54, 597−604

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Industrial & Engineering Chemistry Research

Figure 4. (a) Encapsulation efficiency of the chitosan−CaP hybrid beads. (b) The leakage of YADH from chitosan beads and chitosan− CaP beads. Each data was repeated three times.

concentration was below 12 mg/mL). When the YADH concentration further increased, the encapsulation efficiency decreased but only slightly, for example, at the YADH concentration of 24 mg/mL, the encapsulation efficiency was still as high as 96%. Although the encapsulation efficiency showed a downward trend with the increasing enzyme concentration, the loading efficiency kept a proportional increasing trend. At the YADH concentration of 24 mg/mL, the loading capacity of the hybrid beads was about 1150 mg YADH/g chitosan beads, which was comparable to other types of immobilization carriers.34,35 To assess the leakage behavior of YADH from the chitosan and chitosan−CaP beads, both the pure chitosan beads and the chitosan−CaP hybrid beads were immersed in deionized water, and the amount of enzyme leached from the beads into the water was monitored. As shown in Figure 4b, over 70% of the immobilized YADH leaked out from the pure chitosan beads into the soaking solution within a period of 6 h. In contrast, the YADH immobilized in the chitosan−CaP hybrid beads exhibited a notably lowered leakage ratio by about 2 folds during the same period of immersion time of 6 h. The in situ precipitation of calcium phosphate on the chitosan bead surface significantly inhibited the enzyme leakage. 3.4. Thermal and pH Stability of YADH. Activity assays were conducted to investigate the effect of temperature and pH on the activity and stability of YADH. Thermal stability of free and encapsulated YADH incubated at different temperatures were shown in Figure 5a. The relative activity was defined as the ratio of the activity of YADH after being incubated to the highest activity of YADH under its optimal condition, which was taken as 100%. The results showed that the optimal

Figure 5. (a) The relative activity of free and immobilized YADH at different temperatures. (b) The relative activity of free and immobilized YADH at different pH values. Each data was repeated three times.

temperature for encapsulated YADH was 30 °C, which was consistent with that of the free YADH. The YADH immobilized in chitosan−CaP beads expressed higher relative activity than free YADH and YADH immobilized in the pure chitosan beads at the same reaction temperature, indicating that hybrid beads provided the immobilized enzyme with a more benign environment, which prevented enzyme from heatinduced denaturation. At the temperature as high as 60 °C, the free enzyme and YADH immobilized in the pure chitosan beads lost all of its activity, while YADH immobilized in the chitosan−CaP beads still remained approximately 25% of the initial activity. Compared with the enzyme in immobilized form, the enzyme in soluble form is more likely to suffer thermal inactivation. Due to the aggregation and formation of scrambled structures, the thermal inactivation of the enzyme started with the unfolding of the protein molecule followed by irreversible changes.36,37 The half-lives (t1/2) and thermal denaturation constants (kd) for YADH were calculated and listed in Table 1. The kd values for both free and immobilized enzyme increased with the increasing incubation temperature, and the t1/2 decreased with the increasing incubation temperature. The result indicated that the denaturation became quicker with the increase of incubation temperature for both free and immobilized 601

DOI: 10.1021/ie503294a Ind. Eng. Chem. Res. 2015, 54, 597−604

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Industrial & Engineering Chemistry Research Table 1. Thermal Denaturation Kinetic Parameters of Free and Immobilized YADH kd*(h−1)

t1/2* (h)

temperature (°C)

free YADH

chitosan beads

mineralized beads

free YADH

chitosan beads

mineralized beads

30 35 40 50 60

0.0068 0.0116 0.0749 0.5684 1.2664

0.0032 0.0106 0.0510 0.4904 1.1984

0.0015 0.0031 0.0263 0.2542 0.6952

102 60.0 9.25 1.22 0.547

217 65.0 13.6 1.41 0.578

450 225 26.4 2.73 0.997

*

The error is ±5%. Each data was repeated three times.

some H+ ions to some extent, regulating the OH− ions content inside the bead. By means of buffering function, the change of pH in the microenvironment where the encapsulated YADH actually located was imagined to be much smaller than that taken place in the bulk solution. 3.5. Free and Immobilized Enzyme Activity under Optimal Conditions. The activity of free and encapsulated YADH in chitosan−CaP beads was studied under optimal conditions (30 °C and pH 7.0). As shown in Figure 6a, the

YADH. Compared with free YADH and YADH encapsulated in the pure chitosan beads, YADH encapsulated in the chitosan− CaP beads showed a lower kd value and a higher t1/2 value under the same incubating temperature, indicating a better stability against heat treatment. The ΔH° values for both free and immobilized YADH are listed in Table 2. As the incubating Table 2. Change in Enthalpy (ΔH°) for the Thermal Denaturation of Free and Immobilized YADH ΔH°*(kJ mol−1)

temperature (°C) 30 35 40 50 60

free YADH

chitosan beads

mineralized beads

155.059 155.017 154.976 154.893 154.810

168.891 168.850 168.808 168.725 168.642

180.035 179.993 179.952 179.868 179.785

*

The error is ±5%. Each data was repeated three times.

temperature increased, the ΔH° values for free and immobilized YADH decreased, indicating that the inactivation of YADH required less energy at higher incubating temperatures. Therefore, it was easy for both free and immobilized YADH to undergo denaturation at higher incubating temperatures. However, at the same temperature, the ΔH° values for YADH immobilized in the chitosan−CaP beads were higher than that for free YADH and YADH encapsulated in the pure chitosan beads, indicating that the immobilized YADH required more energy to denature compared with free YADH. In other words, the encapsulated YADH was in fact more thermally stable than free YADH at the same incubating temperature. As shown in Figure 5b, the influence of pH on activity was investigated at different pH values (4.0 to 10.0). Both of the encapsulated enzymes displayed a much higher relative activity than its free counterpart under identical pH conditions. The highest activity for immobilized enzyme was achieved at pH 7.0, identical with that of free enzyme, which further confirmed the well-preserved conformation of the enzyme after immobilization. Furthermore, the immobilized enzyme showed a considerably broadened pH stability profile. For example, the immobilized YADH in the chitosan−CaP beads retained 57% and 16% of its maximum activity at pH 4.0 and at pH 10.0 conditions respectively, whereas the free YADH retained only 33% and 5%, correspondingly. This enhanced tolerance to acidic and alkaline changes of encapsulated YADH was tentatively accounted for the buffering function of the chitosan−CaP beads. In acidic medium, the NH/NH2 buffer pairs on the chitosan could attract and consume H+ ions to a certain degree, resulting in a less acidic microenvironment for the enzymes inside. On the contrary, in alkaline medium, the −NH2/−NH3+ buffer pairs on the chitosan would release

Figure 6. (a) NADH conversion with reaction time (30 °C, pH 7.0). Each data was repeated three times. (b) CD spectra of YADH.

reaction was monitored by determining the conversion of NADH into NAD+. The reaction rate and the final conversion rate for free and immobilized YADH were almost the same. The equilibrium conversion ratio obtained by free YADH was 94% in 3 min, whereas that for encapsulated YADH was 97% in 10 min. The enzyme activity unit was defined as the quantity of YADH required to convert 1.0 μmol of NADH to NAD+ per min at pH 7.0 and 25 °C. The specific activity of free YADH was 416 U/mg and that for encapsulated YADH was 129 U/ mg. The secondary structures of YADH before and after encapsulation were analyzed by CD spectra. As shown in Figure 6b, compared with free YADH in deionized water, the negative 602

DOI: 10.1021/ie503294a Ind. Eng. Chem. Res. 2015, 54, 597−604

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Industrial & Engineering Chemistry Research

hybrid beads, the reduced enzyme leakage led to the substantially improved recycling stability. This result also proved the importance of inhibiting leaching for reuse. The storage stability of the free and immobilized enzymes was also tested over a period of 50 days. Taking the initial activity to be 100%, the relative activity of the YADH encapsulated in mineralized beads retained a relative activity as high as 80% after the encapsulated YADH in mineralized beads was stored for 50 days (Figure 7b). On the contrary, the free YADH lost its activity completely within 50 days. Due to the suitable microenvironment offered by chitosan and the unique encapsulation process, the encapsulated YADH displayed an obvious merit over free YADH during long-time storage and the chitosan beads could closely imitate the effects of crowding and confinement in a living cell. The conformational transition of YADH from folded state to unfolded state, which may lead to the denaturalization of YADH, was efficiently suppressed by the electrostatic interaction between the positively charged chitosan and the negatively charged YADH under a neutral pH value. Moreover, chitosan, which was hydrophilic and biocompatible, provided YADH with a favorable environment to confront the unfavorable effect caused by the storage environment.

peak of free YADH in the chitosan solution was red-shifted by 2.5 nm (from 220 to 222.5 nm). After encapsulation in pure chitosan beads and chitosan−CaP beads, the negative peak of YADH was red-shifted by 2 nm (from 220 to 222 nm). These slight red-shifts indicated changes of conformation, which might exert an effect on enzyme activity. Besides the conformational change of enzyme, diffusion resistance was another key factor influencing enzyme activity after encapsulation in beads. The Michaelis constants (KmB) for free enzyme and encapsulated enzyme were calculated by the Dalziel equation. The KmB was 10.06 mmol/L for the free enzyme and 81.11 mmol/L for the encapsulated enzyme. The KmB value suggested the affinity of the enzyme to the substrate. The higher KmB of the encapsulated enzyme indicated the lower affinity between YADH and HCHO, which was attributed to the diffusion resistance created by the beads. In this study, the reduced apparent enzyme activity was due to both the conformational change and the additional diffusion resistance. 3.6. Recycling Stability and Storage Stability. In the current work, the recycling stability of the encapsulated YADH was investigated by a repetitive batch procedure. The relative activity was defined as the ratio of the activity of YADH after circulation to the initial activity of encapsulated YADH in beads (100%). As shown in Figure 7a, after the fifth reaction cycles,

4. CONCLUSIONS A facile method for fabricating chitosan−calcium phosphate hybrid beads as carriers for effective enzyme immobilization was presented. Compared with pure chitosan beads, the swelling degree of the hybrid beads was dramatically decreased. The biocompatible chitosan liquid core offered the YADH enzyme a mild and suitable microenvironment, and the outer calcium phosphate shell effectively avoided enzyme leakage. The incorporation of inorganic calcium phosphate particles into hydrophilic and biocompatible chitosan polymer led to higher thermal and pH stability of the encapsulated enzyme than its free counterpart. Moreover, both the recycling stability and the storage stability of the encapsulated YADH were also remarkably enhanced. It is expected that the present approach can be extended to preparing a variety of organic−inorganic hybrid materials for diverse applications in pharmacy, biomedical, biotechnology, and biosensor fields.

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ASSOCIATED CONTENT

S Supporting Information *

XRD profiles of beads (Figure S1) and absolute activity of free and YADH in chitosan−CaP beads at different pH (Table S1). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Address: School of Chemical Engineering & Technology, Tianjin University, 92 Weijin Road, Nankai District, Tianjin 300072, China. Tel.: +86-22-23500086. Fax: +86-22-23500086. E-mail: [email protected].

Figure 7. (a) Recycling stability of encapsulated YADH in chitosan− CaP and chitosan beads. (b) Storage stability of free and immobilized YADH. Each data was repeated three times.

Notes

the YADH encapsulated in pure chitosan beads only retained 30% of its initial activity, whereas in the mineralized beads it still retained 81% of its initial activity. The remarkably lowered activity of YADH in the pure chitosan beads was attributed to the severe leakage of YADH from the beads during the repeated separation, rinsing, and soaking procedures after each reaction cycle. For the YADH encapsulated in the mineralized

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the financial support from the National Science Foundation of China (Grant 21076145), the Program for New Century Excellent Talents in University (Grant 603

DOI: 10.1021/ie503294a Ind. Eng. Chem. Res. 2015, 54, 597−604

Article

Industrial & Engineering Chemistry Research

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NCET-10-0623), Specialized Research Fund for the Doctoral Program of Higher Education (Grant 20130032110023), Program of Introducing Talents of Discipline to Universities (Grant B06006).

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DOI: 10.1021/ie503294a Ind. Eng. Chem. Res. 2015, 54, 597−604