New Method for the Immobilization of Pullulanase onto Hybrid

(24) At pullulanase/chitosan ratio = 2:1, the formation of soluble complex was .... In run 2, the immobilized enzyme was prepared by simply mixing mag...
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New Method for the Immobilization of Pullulanase onto Hybrid Magnetic (Fe3O4−κ-Carrageenan) Nanoparticles by Electrostatic Coupling with Pullulanase/Chitosan Complex Jie Long,†,‡ Zhengzong Wu,†,‡ Xingfei Li,† Enbo Xu,†,‡ Xueming Xu,†,‡ Zhengyu Jin,*,†,‡ and Aiquan Jiao*,†,‡ †

The State Key Laboratory of Food Science and Technology, School of Food Science and Technology and ‡Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China ABSTRACT: We present a simple method to immobilize pullulanase onto hybrid magnetic (Fe3O4−κ-carrageenan) nanoparticles, involving the in situ synthesis of magnetic carrageenan nanoparticles and the formation of pullulanase/chitosan complex. The complex behavior of pullulanase with chitosan as a function of pH and protein−polysaccharide ratio was studied by turbidimetric titration. Then, the as-prepared immobilized enzymes were characterized by vibrating-sample magnetometer, transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray diffractometer, and thermogravimetric analysis. It was found that the activity retention of immobilized pullulanase and amount of enzyme loaded reached 95.5% and 96.3 mg/g, respectively, under optimal conditions. The immobilized enzyme exhibited great operational stability (retaining approximately 61% residual activity after ten consecutive reuses), demonstrating that enzyme leakage during the catalysis reaction was efficiently reduced. Furthermore, the activity of immobilized pullulanase was significantly (p < 0.01) higher than that of free pullulanase in a low pH range (pH < 3.0) and temperature over 60 °C, and the immobilized enzymes retained 45% of their initial activity after 5 h at 60 °C, compared to 21% for the free enzyme. These results indicated that immobilized pullulanase was efficient in terms of catalytic activity and can be applied to continuous starch processing applications in the food industry. KEYWORDS: magnetic Fe3O4−κ-carrageenan nanoparticle, pullulanase, immobilization, stability, pullulanase/chitosan complex

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INTRODUCTION Pullulanase, an important biocatalyst that cleaves the α-1,6glucosidic linkages of pullulan, amylopectin, and other amylaceous polysaccharides, has drawn much attention in the starch processing industry because of its applications in the preparation of glucose, maltose, maltotriose syrup, and amylose.1 However, commercial pullulanase as a biocatalyst always presents some drawbacks that limit its popularization in food industry, such as easy deactivation at higher temperature, inability of reutilization, and product contamination.2 Enzyme immobilization is a common approach to addressing these problems because it enables the continuous use of enzymes, preserves their catalytic features against several forms of denaturation, and facilitates substrate and product recovery.3 Much effort has been made to produce, stabilize, and reuse pullulanase.4 To date, pullulanase has been immobilized onto different polymeric materials, such as agarose, casein, etc.4,5 Nanoparticles are usually considered to be ideal enzyme carriers. They offer a high specific surface area for enzyme attachment, which results in a higher concentration of the biocatalyst.6 They have a large surface-area−volume ratio, allowing enzyme immobilization to be carried out under moderate conditions, thus retaining the catalytic activity of the enzyme.7 Furthermore, diffusion hindrance can be reduced because of the small size of the support.8 However, the major drawback of nanoparticles as carriers is the low efficiency of nanoparticle separation or recovery from a mixed system. The supports are difficult to separate after catalytic reaction without ultracentrifugation. By the introduction of magnetism, this © 2015 American Chemical Society

problem can be overcome, and the process can be carried out continuously.9 Therefore, magnetic nanoparticles are promising materials for enzyme immobilization.10 In situ synthesis of magnetic nanoparticles is carried out in aqueous systems under ambient conditions without surfactants and is therefore more environmentally friendly and efficient than traditional synthesis methods.11 To control particle size, polymers are extensively used in the synthesis process because the polymeric functional groups can chelate iron ions and thus act as stabilizers to prevent the spontaneous agglomeration of nanoparticles.12 The resultant functional nanomaterials are good candidates for use in the pharmaceutical and biomedical fields because of their nontoxicity and biocompatibility.13 Carrageenans (CRG), a group of linear, sulfated natural polysaccharides, have been widely used as gelling agents in the food and pharmaceutical industries.13,14 At present, there have been no reports of enzyme immobilization using Fe3O4/ carrageenan nanoparticles prepared by in situ method. Chitosan (CS) is a widely distributed biopolymer with high molecular weight and is composed of glucosamine and N-acetylglucosamine.15 It is often used as an adsorbent for protein, dye, metal, and lipid because of its functional groups, which include amino, hydroxyl, and N-acetyl-reactive groups.16 CRG and CS are polyelectrolytes of opposite charge that when Received: Revised: Accepted: Published: 3534

December March 21, March 22, March 23,

10, 2014 2015 2015 2015 DOI: 10.1021/jf505981t J. Agric. Food Chem. 2015, 63, 3534−3542

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Journal of Agricultural and Food Chemistry

chitosan solution (w/v). Pullulanase was diluted with sodium acetate buffer (10 mM) to a final concentration of 1 mg/mL. Each solution was adjusted to a pH of 2.8. Turbidity titration of pullulanase/chitosan mixture was conducted by first mixing the two solutions at different ratios, ranging from 10:1 to 1:1, then slowly adjusting the pH to 8.0 with 0.1 M NaOH. Homogenous pullulanase (1 mg/mL) and chitosan solutions (0.1%, w/v) were used as blanks.18 Measurement of Turbidity. A UV−vis spectrophotometer (Shimadzu, Japan) with a 0.5 cm cuvette was used to measure turbidity at 600 nm. By the addition of gradated NaOH solutions (1, 0.5, 0.1 M), turbidimetric titration was carried out in increments of ∼0.1 pH unit up to a final pH of 8.0 under gentle stirring. Titrations were carried out at 25.0 ± 1.0 °C, and the pH (±0.01 pH unit) was monitored with a Mettler Toledo Delta 320 pH meter.19 Enzyme Immobilization. Fe3O4−κ-carrageenan nanoparticles were incubated with the pullulanase/chitosan complex solutions (pH 4.8) at 40 °C for 4 h under gentle stirring. After immobilization, successive washings with the buffer (pH 4.8) were conducted to remove the excess enzyme until no more activity was detected in the washing fractions. Then, the immobilized enzyme was further incubated with 0.1% (w/v) carrageenan solution (pH 4.8) at 40 °C for 1 h under gentle stirring and washed with the buffer several times. The immobilized activity was calculated by taking the difference between the applied and the recovered activities. The amount of the immobilized protein was determined by subtracting the amount of protein remaining in the supernatant and washing solutions after 4 h of stirring from the amount of protein added to immobilization, according to the Bradford protein assay method reported previously.20 The activity retention (AR) and enzyme loading efficiency were calculated according to the following equations:

combined form polyelectrolyte complexes by molecular entanglements, ionic forces, hydrogen bonding, or hydrophobic forces.17 The assembly of the protein-loaded CS/CRG magnetic nanoparticles was mediated by polyelectrolyte complexation under mild conditions, thus avoiding harmful organic solvents or high shear forces and retaining protein activity during immobilization. Currently, because of high costs and complex methodology, enzyme immobilization has not yet been widely employed in industrial applications and is instead conducted either for the purpose of basic research or for use in technical processes.9 Therefore, there is a need for the development of simple and green methods to facilitate the use of enzyme immobilization in industry. In the present work, we describe a simple, green, and novel method for the immobilization of pullulanase by adsorption. Chitosan was utilized as a flocculating agent to prepare pullulanase/chitosan complexes, which were then electrostatically coupled with Fe3O4/carrageenan nanoparticles. To prevent the adsorbed enzymes from leaking during repeated uses, which would restrict their application in enzyme immobilization, we further coated the immobilized enzymes with carrageenan by electrostatic interaction, expecting the interaction of chitosan, enzymes, and carrageenan to enhance the stability of the biocatalysts. Such structure was supposed to provide the advantage of high residual activity, besides the merit of enzyme recovery. To achieve these aims, the Fe3O4−κcarrageenan nanoparticles were first prepared in situ under mild conditions without any surfactants. Pullulanase/chitosan mixtures were brought to complexing conditions by the slow addition of NaOH, and the effects of pH and protein/chitosan ratio on turbidity were investigated. The hybrid magnetic nanoparticles were then mixed with the pullulanase/chitosan complexes to obtain the immobilized enzymes and further coated with carrageenan. The effects of support/enzyme ratio on enzyme activity and protein loading were studied. Finally, the properties of the immobilized enzyme, such as kinetic behavior, pH and temperature profiles, thermal and operational stability, were also investigated.

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activity retention =

A im × 100 A0

(1)

where A0 is the specific activity of free pullulanase and Aim is the specific activity of the immobilized enzyme.

enzyme loading efficiency =

C0V0 − C iVi × 100% C0V0

(2)

where C0 and V0 are the protein concentration and the volume of the free pullulanase solution added to immobilization, respectively, and Ci and Vi are the protein concentration and the volume of the supernatant solution after immobilization, respectively. Characterization. A Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) with a He/Ne laser of 633 nm wavelength was used to determine size distribution of freshly prepared nanoparticles; each sample was diluted to the appropriate concentration with ultrapure water, then placed in the electrophoretic cell at 20 °C. A highperformance digital imaging transmission electron microscopy (TEM) machine (JEM-2100 (HR), JEOL Ltd., Tokyo, Japan) was used to determine the particle size and morphology of the magnetic nanoparticles. Image-Pro Plus 6.0 (Media Cybernetics, Inc., Bethesda, Maryland, USA) was used to analyze the TEM micrographs to obtain the size distribution of nanoparticles. An average result of nanoparticle size was obtained by analyzing TEM images of 20 (total counts of particles: 200). Fourier transform-infrared (FT-IR) spectra were recorded using a Thermo Nicolet iS10 FT-IR Spectrometer (Thermo Electron Corp., Madison, WI, USA). X-ray diffraction (XRD) measurements were carried out with a BRUKER AXS GMBH D8 (Karlsruhe, Baden-Wuerttemberg, Germany) instrument; a continuous scan mode was used to collect 2θ data from 10 to 80°, at a constant rate of 4°/min. A vibrating-sample magnetometer (VSM, Lakeshore7304, Westerville, Ohio, USA) was used to obtain magnetization curves at room temperature. Thermogravimetric analysis (TGA) was carried out with a Mettler Toledo SDTA 851e (Zürich, Canton of Zürich, Switzerland) instrument at a heating rate of 10 °C/min from 30 to 700 °C. Activity Assay of Pullulanase. The activities of free and immobilized pullulanase were determined by the 3,5-dinitrosalicylic acid (DNS) method using pullulan as substrate.2 In brief, 2 g of

MATERIALS AND METHODS

Materials. Pullulanase from Klebsiella pneumoniae and pullulan were purchased from Sigma-Aldrich. κ-Carrageenan (CRG, commercial grade) and chitosan (degree of deactylation = 90%) were obtained from Aladdin Industrial Co., Ltd. Ferric chloride hexahydrate (FeCl3· 6H2O), ferrous sulfate heptahydrate (FeSO4·7H2O), and sodium hydroxide (NaOH) were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All other chemicals were analytic-grade and available from commercial sources. Preparation of Hybrid Magnetic (Fe3O4−κ-Carrageenan) Nanoparticles. In situ synthesis of magnetic Fe3O4−κ-carrageenan nanoparticles was carried out as reported previously with minor modification.12 In brief, 0.5 g of κ-carrageenan was dissolved in 100 mL of deoxygenated water, stirred until dissolved completely at 60 °C, and cooled to 40 °C. After cooling, 2 mL of FeCl3 solution (0.2 M), freshly prepared in deoxygenated water, and 65 mg of FeSO4 were immediately added to 20 mL of κ-carrageenan solution. To account for the possible oxidation of Fe2+, ferrous ion was added in slight excess of the magnetite stoicheometric molar ratio (Fe3+/Fe2+ = 2:1). After stirring the mixture for 15 min, 2.2 mL of 1 M sodium hydroxide was added, giving a final pH of over 10. The mixture immediately turned black, indicating that the Fe3O4−κ-carrageenan composite was successfully prepared. Preparation of Pullulanase/Chitosan Complex Solutions. Chitosan powder was dispersed in 0.1% acetic acid solution (v/v) under magnetic stirring for 4 h at room temperature to prepare a 0.1% 3535

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Journal of Agricultural and Food Chemistry pullulan was added into 100 mL of sodium acetate buffer (0.04 M, pH 4.4) to obtain a homogeneous pullulan solution (2%, w/v). Then 0.1 mL of free (50 μg/mL) or immobilized pullulanase (0.5 mg.d.s.mL−1) solution was incubated with 1 mL of pullulan solution at a water bath of 50 °C for 30 min. The reaction was stopped by adding 1.5 mL of 3, 5-dinitrosalicylic acid (DNS). After incubation, the reaction mixtures were kept in boiling water for 10 min and then immediately cooled to room temperature. The reducing sugars were determined at an absorbance of 540 nm by the ultraviolet−visible (UV−vis) spectrophotometer. One unit of pullulanase activity was defined as the amount of enzyme that produced 1 μmol of reducing sugars (as maltotriose equivalents) in 1 min at 50 °C. Kinetic Parameters of Free and Immobilized Enzyme. The kinetic properties of free and immobilized pullulanase were determined by using different substrate concentrations of 1−10 mg/ mL. The kinetic constants Km and Vm were calculated according to the Lineweaver−Burk plots.21 pH Stability. Stabilities of free and immobilized enzyme against pH were evaluated in light of the catalytic activity of enzyme by incubating enzyme at a pH range of 2.5−6.4 for 30 min at 50 °C, and their residual activities were determined. Reusable Stability. The operational stability was assessed by incubating immobilized enzyme with pullulan (2%, w/v) in a water bath of 50 °C for 30 min, after which the activity was assayed immediately. After each reaction period, the immobilized pullulanase were removed from the reaction medium and washed with sodium acetate buffer (40 mM, pH 4.4). Then, the fresh pullulan solution was added again to start the reaction. The activity of the first run was defined as 100%. Thermal Stability. The thermostabilities of free and immobilized pullulanases were tested in light of the optimum temperature and thermal inactivation courses. For the optimum temperature, enzymes were incubated in sodium acetate buffer (pH 4.4, 10 mM) in a water bath (20−65 °C) for 30 min, and their residually activities were determined. For the thermal inactivation courses, enzymes were incubated at 60 °C in 40 mM sodium acetate buffer (pH 4.4) for 6 h, and samples were withdrawn after incubation for 10, 20, 30, 60, 180, and 300 min, then immediately assayed for residual activity. Statistical Analysis. The data expressed in various studies were plotted using Origin-8.5. Experiments were triplicated. Data represent the mean of three independent assays carried out in duplicate, and the results are presented as mean ± SD. Significant differences were determined statistically using one-way analysis of variance (ANOVA) posthoc Tukey test. Statistical significance was established at p < 0.05.

pullulanase and chitosan. Figure 1 shows the turbidity curves for the pullulanase/chitosan system. Below pHc (the pH at

Figure 1. Turbidity curves for the pullulanase/chitosan system (Cp = 1 mg/mL) as a function of pullulanase/chitosan mixing ratio. Enzyme and chitosan (CS) solution at their corresponding concentrations were used as controls. The values of pHc and pHφ1 were determined graphically as the intersection point of two tangents to the curve: pHc corresponds to the pH value at which soluble complexes form, and pHφ1 corresponds to the pH value at which insoluble complexes form. Results are the mean of triplicates.

which soluble complexes form because of the local negative charges carried by proteins), the turbidity remained at the baseline because the electrostatic repulsive forces prohibited the formation of complexes. An abrupt increase in turbidity was observed with further addition of NaOH (at pHφ1), indicating the formation of insoluble complex particles. Before complexation with proteins, the chitosan solution was transparent until the pH increased to the pKa of its amine (pH 6.3), where a protonated effect was observed. Therefore, the turbidity curve (Figure 1) for chitosan showed an abrupt increase at pKa, followed by a maximum at around pH 7.0, and then reached the plateaus of turbidity, corresponding to the insolubility of chitosan. Meanwhile, the pullulanase/chitosan interaction also can be affected by the mixing ratio. The decrease in pullulanase/ chitosan ratio resulted in less negative charges being carried by proteins and more positive charges being carried by chitosan; as the consequence, pHφ1 shifted to higher pH values to achieve the charge neutralization.24 At pullulanase/chitosan ratio = 2:1, the formation of soluble complex was distributed in a wide pH range from 4.0 (pHc) to 5.27 (pHφ1). Ratios such as 5:1 and 10:1 had narrower pH ranges for soluble complex formation, whereas the 1:1 ratio yielded weak pullulanase−chitosan interactions that occurred near the deprotonation point of chitosan (pH 6.3). Therefore, the most flexible pH range for pullulanase−chitosan complex formation is seen using a mixing ratio of 2:1. From the view of charge neutralization (obtained at pHφ1), the negative charge of pullulanase (−12.5 mV, pH 4.8) was insufficient to neutralize the positive charge of chitosan solution (45.2 mV, pH 4.8) at the concentration of 0.1% (w/v), and the zeta potential of the complex was about 15.6 mV (pH 4.8), indicating the possibility that all the proteins were adsorbed in the chain of chitosan. Moreover, increased pH values led to the formation of insoluble complexes (pH 5.27), and there was almost no activity in the supernatant after centrifugation. These observations mean that the pullulanase was completely absorbed by chitosan at pH 5.27, indicating the

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RESULTS AND DISCUSSION Pullulanase/Chitosan Complexing Behavior as a Function of pH and Mixing Ratio. Chitosan is a wellknown biopolymer commonly used in the formation of protein−polysaccharide complexes. Complex coacervates have many applications in the biotechnology, pharmaceutical, and food industries.22 Amino groups in chitosan chains intensively interact with the carboxyl groups of enzymes largely by electrostatically driven forces, which in turn leads to the formation of a complex.23 By controlling the conditions, this protein−polysaccharide complex could form nanoscale particles in soluble form in aqueous solution. For this reason, we first prepared pullulanase/chitosan complexes, then incubated them with magnetic nano−κ-carrageenan particles to prepare the nanoscale immobilized enzyme, and further coated them with carrageenan, expecting the interaction of biopolymers and enzymes to further prevent the adsorbed enzymes from leaking. The complexing behavior of pullulanase/chitosan was affected by pH changes. The increase in absorbance at 600 nm upon addition of NaOH to the pullulanase/chitosan mixtures was indicative of the formation of colloidal dispersions via the self-assembly of the oppositely charged polyelectrolytes 3536

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Figure 2. (a) Particle size distribution of the pullulanase/chitosan complex and (b) immobilized pullulanase.

Characterization of Immobilized Enzymes. Magnetic Properties. At the first stage, the in situ synthesis of magnetic carrageenan nanoparticles involves the chelation of ferric (and ferrous) ions with the sulfate and hydroxyl groups of carrageenan at low pH, promoting the physical cross-linking of the carrageenan chains and formation of microgel; upon increasing pH, self-capped iron oxide nanoparticles are formed involving biopolymer molecules, with a number of the sulfate groups released. However, some sulfate groups still remain chelated to the nanoparticle surfaces, resulting in the high colloidal stability of this system.12 This high colloidal stability results in a weak magnetic response of the nanoparticles to an external magnetic field. After the addition of the pullulanase/ chitosan complex, the charge of the sulfate groups was neutralized, and the problem of weak magnetic response was solved. As shown in Figure 4, within the external magnetic field, immobilized enzymes aggregated completely in 15 s, whereas Fe3O4−κ-carrageenan nanoparticles moved slowly and finally aggregated completely in 8 min. The hysteresis loops of Fe3O4−κ-carrageenan nanoparticles and immobilized enzymes were also calculated by a vibratingsample magnetometer. As can be seen from Figure 4, both magnetic nanoparticles and immobilized enzymes exhibited superparamagnetic behavior. The saturation magnetization (Ms) of the immobilized enzymes was higher (42.6 emu/g) than that of the Fe3O4−κ-carrageenan nanoparticles (37.8 emu/g), whereas pure Fe3O4 was 98.1 emu/g. The excellent response to an applied magnetic field suggests that the immobilized enzymes meet the requirements for reuse in the food industries. Morphology and Particle Size Distribution. Figure 5 shows a typical TEM image of immobilized enzymes. As can be seen from the picture, the average diameter of the immobilized enzymes was 156 nm, which is consistent with the result obtained on the zetasizer (Figure 2b). These results indicate that mixing of pullulanase/chitosan complex and magnetic carrageenan nanoparticles showed feasibility in the preparation

high content of protein adsorbed by chitosan at pH 4.8 before phase separation. In our research, the pullulanase/chitosan complexes formed at the ratio of 2:1 (pH 4.9) possessed an average diameter of 3.5 nm (Figure 2a), and after incubation of the complex with Fe3O4−κ-carrageenan nanoparticles, the immobilized enzyme showed an average diameter of 152 nm (Figure 2b). This indicated that the pullulanase/chitosan complexes take advantage of forming immobilized enzyme on the nanoscale because the diameter of immobilized enzyme was not in nanoscale when chitosan and pullulanase were added separately. Schematics for the fabrication process and the structure of the hybrid magnetic nanoparticles with immobilized pullulanase are shown in Figure 3.

Figure 3. Schematic diagram of experimental design. 3537

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Figure 4. Magnetization curves for pure Fe3O4 (★), Fe3O4− carrageenan nanoparticles (○), and immobilized pulluanase (●). The inset shows the photograph of Fe3O4/carrageenan (A) and immobilized pullulanase (B) dispersion in water (left) and their magnetic response when placed in external magnetic field (right).

Figure 6. FT-IR spectra of chitosan (a, black trace), carrageenan (b, blue trace), immobilized pullulanase (c, red trace), and Fe3O4 (d, pink trace).

both chitosan and carrageenan. The spectra confirmed the successful formation of an immobilized enzyme. XRD Patterns. XRD analysis of Fe3O4 and immobilized enzymes is shown in Figure 7. According to Joint Committee

Figure 5. TEM micrographs of the immobilized pullulanase.

of nanoscale immobilized enzymes. The uniform and flexible environment of the pullulanase/chitosan complexes and magnetic carrageenan nanoparticles facilitated the homogeneous distribution of the enzymes. FT-IR Analysis. FT-IR spectra of chitosan, carrageenan, immobilized enzymes, and Fe3O4 are shown in Figure 6. As can be seen from the figure, chitosan showed the typical peaks of amide I at 1651 cm−1, amide II at 1597 cm−1, and glycosidic bonds at 1080 cm−1.25 As for κ-carrageenan, the characteristic peaks at 1262, 847, 1068, and 928 cm−1 corresponded to the sulfate groups, galactose-4-sulfate, glycosidic linkage, and 3,6anhydrogalactose, respectively.26 The FT-IR spectrum of the immobilized enzymes exhibited the characteristic peaks of carrageenan at 1224, 1065, 929, and 845 cm−1 and the characteristic amide peak of chitosan, which converted into a single band at 1636 cm−1. This verified the presence of carrageenan and chitosan in the biocatalyst. In the spectrum of the immobilized enzyme, the characteristic peak around 580 cm−1 was attributed to vibrations of the Fe−O bond of Fe3O4. Moreover, upon complexation of chitosan, carrageenan, and pullulanase, a new absorption band at 1530 cm−1, assigned to NH3+ groups, was observed, which was absent in the spectra of

Figure 7. Wide-angle XRD patterns of immobilized pullulanase.

on Powder Diffraction Standards, JCPDS, entry 19-629, the powder XRD of immobilized enzymes matches the typical diffraction patterns observed for magnetite, which can be assigned to the six characteristic peaks at 2θ = 30.1, 35.4, 43.1, 53.2, 56.9, and 62.5°, corresponding to (220), (311), (400), (422), (511), and (440), respectively. Especially, the presence of the characteristic peak at 35.4° represents the formation of the spinel structure of Fe3O4, indicating that the immobilized enzyme still maintains its natural crystalline structure of Fe3O4 with no phase change. Thermal Degradation. The thermal properties of pure Fe3O4, Fe3O4−κ-carrageenan nanoparticles, and immobilized enzymes studied by TGA are shown in Figure 8. Fe3O4 was thermally stable, and there was no weight loss in the interval of 200−800 °C (Figure 8a). For the Fe3O4−κ-carrageenan nanoparticles and immobilized enzymes, the derivative of 3538

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pullulanase was assayed in 10 batches, and the same assay conditions were used for all batches. Activity of the first run was defined as 100%. In run 1, the immobilized enzyme was prepared as discussed in this paper. In run 2, the immobilized enzyme was prepared by simply mixing magnetic carrageenan nanoparticles with pullulanase. In run 3, the latter immobilized enzyme was further coated with carrageenan. As illustrated in Figure 9, the immobilized enzyme prepared in this paper (run

Figure 8. Thermogravimetric analysis of Fe3O4 (a, blue trace), Fe3O4− carrageenan nanoparticles (b, black trace), and immobilized pullulanase (c, red trace). The inset shows the derivative weight loss curves for Fe3O4−carrageenan nanoparticles and immobilized pullulanase.

weight-loss curves clearly indicated the inflection point of thermal decomposition, which was observed around 100, 240, 330, 620, and 750 °C (inset Figure 8). The weight loss below 200 °C was attributed to the loss of residual water.27 The polysaccharide units of chitosan and carrageenan began to decompose at 200 °C.28 In the interval of 200−800 °C, the degradation of carrageenan caused a weight loss of 18.41% estimated from the TGA curve of Fe3O4−κ-carrageenan nanoparticles (Figure 8b). At least three thermal events were observed: (i) From 200 to 300 °C, the degradation of carrageenan is associated with sulfur dioxide leaving and carbohydrate backbone fragmentation with weight loss of 5.80%. (ii) When the temperature increased to 450 °C, the degradation of carrageenan is related to polysaccharide decomposition with weight loss of 3.65%. (iii) The residual carrageenan or the intermediate compounds degradation caused a 8.96% weight loss that is continued until approximately 800 °C. Although in the interval of 200−800 °C, the degradation of chitosan, pullulanase and carrageenan (further coated) caused a 43.86% weight loss estimated from the TGA curves of Fe3O4−κ-carrageenan nanoparticles (Figure 8b) and immobilized enzymes (Figure 8c). At least three thermal degradations were also observed: an 8.28% weight loss from 200 to 300 °C, an 11.10% weight loss from 300 to 450 °C, and a 24.48% weight loss from 450 to 800 °C. Moreover, a new inflection point in the derivative weight-loss curve of immobilized enzymes was observed (inset Figure 8), which may be due to the complexation of chitosan, carrageenan, and pullulanase. Reusability of Immobilized Pullulanase. Reusability of the immobilized enzyme is very important from the viewpoint of practical applications, and an improved stability makes the immobilized enzyme more advantageous than its free counterpart. However, as for absorption method, the leakage of enzymes from support in practice should be considered. To compare the stability of the immobilized pullulanase prepared as discussed in this paper with that of immobilized enzyme prepared by simply mixing magnetic carrageenan nanoparticles with pullulanase, we determined the reusability of the different immobilized enzymes. The activity of the immobilized

Figure 9. Reusability of the immobilized pullulanase. Experiments were triplicated, and the results are presented as mean ± SD.

1) showed the best reusability, retaining more than 76 and 61% of its original activity after 5 and 10 consecutive reuses, respectively. Pullulanase immobilized by simply mixing with magnetic carrageenan nanoparticles (run 2) exhibited a loss in activity of approximately 53 and 75% after 5 and 10 batch reactions, respectively. The immobilized enzyme prepared in run 3 also experienced a large loss in activity after 5 and 10 batch reactions (residual activity ≈ 52 and 34% of the initial value, respectively). Moreover, these enzymes undergo a fast decline in catalytic activity (residual activity ≈ 54 and 69% of the initial value) after the first use. In contrast, the immobilized enzyme prepared in this paper maintained its activity well (92% of the initial activity) after the first use. Obviously, the leakage of enzymes was remarkably abated. Enzyme Immobilization and Loading Capacity. Activity retention, amount of protein immobilized, and loading efficiency after immobilization for different amounts of enzyme were summarized in Table 1. The amount of protein Table 1. Activity Parameters of the Free and Immobilized Pullulanasea applied activity (U.g.d.s.−1)b 37910 63183 126367 189550 379100 505467

amount of protein immobilized (mg/g) 40.3 51.2 70.0 76.4 96.4 102.6

± ± ± ± ± ±

2.4 1.2 1.5 2.0 2.1 2.3

protein loading efficiency (%) 70.7 54.9 40.8 29.3 23.9 20.4

± ± ± ± ± ±

4.4 3.8 2.1 2.3 2.8 2.6

specific activity (u/ mg) 182.5 222.1 260.0 290.2 340.8 320.6

± ± ± ± ± ±

3.9 2.6 6.0 7.6 6.4 5.6

activity retention (%) 51.2 62.3 72.9 81.4 95.6 89.9

± ± ± ± ± ±

1.5 2.5 0.7 1.5 0.8 0.9

Experiments were triplicated, and the results are presented as mean ± SD. bd.s. corresponds to dry support. a

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Journal of Agricultural and Food Chemistry immobilized increased when the loading efficiency decreased because saturation of the support was being achieved. The maximum quantity of pullulanase immobilized on 1 g of magnetic nanoparticles was 102 mg. Similarly, the activity retention increased from 51.19 to 95.58%, which implies that the activity of the enzyme was preserved well by this mild method; however, activity retention decreased with further increase of the bound protein. These results suggest that when a small amount of protein is immobilized on the support, its activity is totally expressed; however, when more proteins are attached, the enzyme molecules either become aggregated, forming a multilayer of enzyme molecules at the particle surface and blocking the active site, or the presence of neighboring enzyme molecules reduces the accessibility of substrate to the active sites,29 resulting in decreased activity retention. Effect of Immobilization on the pH Profile of Pullulanase. The effect of pH on the activity of free and immobilized pullulanase was estimated in the range of pH 2.5− 6.4 (Figure 10a). The maximum pullulanase activity was

Figure 11. Lineweaver−Burk plots for the pullulan reduction with free and immobilized pullulanase (pH 4.4 at 50 °C). Results are the mean of triplicates.

Table 2. Kinetic Parameters of Free and Immobilized Pullulanase enzyme type free enzyme immobilized enzyme

Km (mg/mL)

Vm (mg/(mL min))

enzymatic catalytic efficiency (Vm/ Km)

6.073 8.867

1.942 1.594

0.319 0.179

both partitioning and diffusional effects; Vm reflects the scale of mass transportation.30 The Km value of the immobilized enzyme (8.867 mg/mL) was higher than that of the free enzyme (6.073 mg/mL), and the Vm value of the immobilized enzyme (1.594 mg/(mL min)) was lower than that of the free enzyme (1.942 mg/(mL min)). The above results imply that the immobilized enzyme has an apparent lower affinity between enzyme and substrate, suggesting that it was more difficult for the substrate to diffuse and access the enzyme and that a higher concentration of substrate is needed when utilizing the immobilized enzyme. The lower affinity may be due to the negative effect of immobilization in terms of increased steric hindrance of the active site or substrate diffusion resistance.31 Moreover, the lower affinity of the immobilized enzyme may be caused by the loss of enzyme flexibility necessary for substrate binding.32 Therefore, a decrease in Vm of immobilized pullulanase was observed. Thermal Stability of the Immobilized Pullulanase. The activity of free and immobilized pullulanase was assayed at temperatures in the range of 20−65 °C (pH 4.4). The maximum pullulanase activity was defined as 100%. As shown in Figure 12A, immobilization had no effect on the optimum temperature (50 °C) of pullulanase. A dramatic decrease in enzyme activity was observed in both systems when temperature went above 60 °C; however, the immobilized pullulanase showed higher activity than the free enzyme (p < 0.01). For example, at 65 °C, the relative activity of immobilized pullulanase was 66.7%, whereas that of free enzyme was only 25.5%. This result indicated that the immobilized enzyme possesses a better tolerance to heat. The effect of temperature on the activity of free and immobilized enzyme at 60 °C versus the reaction time was also investigated. The initial activity was defined as 100%. As shown

Figure 10. Effect of pH on pullulanase activity. Experiments were triplicated, and the results are presented as mean ± SD.

defined as 100%. It was observed that the activity of both free and immobilized pullulanase was greatly affected by pH, and the maximum activity was observed at pH 4.4 for both free and immobilized pullulanase. Compared with free pullulanase, the immobilized enzyme exhibited a better pH resistance at pH ≤ 3.0 and pH > 6. For example, at pH ≤ 3.0, there was a significant difference (p < 0.01) in enzyme activity: for the free enzyme, there almost no activity was detected, whereas for the immobilized enzyme, the relative activity was 21.05 and 30.14% at pH 2.5 and 3.0, respectively. Mechanisms of the decreased pullulanase activity might be attributed to the changed protein folding and binding modes (electrostatic or via hydrogen bonds).7 Catalytic Property (Activity and Kinetic Parameters) of Immobilized Pullulanase. Catalytic kinetics of the free and immobilized enzymes were further investigated. The Lineweaver−Burk plot for pullulan reduction (1−10 mg/mL) with free and immobilized pullulanase is shown in Figure 11. The apparent Michaelis constant, Km, and the maximum reaction rates, Vm, of pullulanase calculated from the equations of these plots were summarized in Table 2. Km relates to the affinity of substrate molecule and enzyme that depends upon 3540

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ACKNOWLEDGMENTS We are grateful to Dr. Qingqing Liu for her technical assistance. This study was supported by the National Twelfth Five-Year Plan for Science & Technology Support of China (nos. 2012BAD37B02 and 2012BAD37B06).

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Figure 12. (A) Effect of temperature on activity of free and immobilized enzyme. (B) Thermal inactivation courses of free and immobilized enzyme (60 °C, pH 4.4). Experiments were triplicated, and the results are presented as mean ± SD.

in Figure 12B, the immobilized enzyme presented a higher thermal stability compared to the free enzyme. The rate of inactivation of the free enzyme was markedly higher than that of the immobilized enzyme. After 5 h, the immobilized enzyme retained more than 45% of its initial activity, whereas the free enzyme retained only 21% of its initial activity. This implies that immobilization had a positive effect on the thermal stability of pullulanase and was able to protect the active conformation of the enzyme from damage.33 With this stabilizing effect, immobilized pullulanase exhibits an advantage in continuous applications in food industry.

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REFERENCES

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Corresponding Authors

*Phone:+86-510-85913299. Fax: +86-510-85913299. E-mail: [email protected]. *Phone:+86-510-85320225. Fax: +86-510-85320225. E-mail: [email protected]. Notes

The authors declare no competing financial interest. 3541

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