Effective Enzyme Immobilization onto a Magnetic Chitin Nanofiber

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Effective Enzyme Immobilization onto a Magnetic Chitin Nanofiber Composite Wen-Can Huang, Wei Wang, Changhu Xue, and Xiangzhao Mao ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b01150 • Publication Date (Web): 29 May 2018 Downloaded from http://pubs.acs.org on May 29, 2018

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ACS Sustainable Chemistry & Engineering

Effective Enzyme Immobilization onto a Magnetic Chitin Nanofiber Composite

Wen-Can Huanga§, Wei Wanga§, Changhu Xuea,b, Xiangzhao Maoa,b* a

College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China

b

Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, China

* Corresponding author: Professor Xiangzhao Mao Address: Yushan Road 5, College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China Tel.: +86-532-82032660 Fax: +86-532-82032272 E-mail: [email protected]

§ These authors contributed equally.

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ABSTRACT In this study, a novel biocompatible magnetic chitin nanofiber composite (MCNC) was developed as a support for enzyme immobilization, and the enzyme-immobilizing ability was elucidated using chymotrypsin (CT) as a model enzyme. Chitin nanofibers (CNFs) were prepared via 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-mediated oxidation of chitin and then further modified with magnetic nanoparticles. Glutaraldehyde was used to cross-link the additional CT molecules and aggregate them onto the MCNCs. The CNFs were characterized by transmission electron microscopy, and Fourier transform infrared spectroscopy. The results showed that the CNFs were properly formed and that the CT molecules immobilized on the MCNCs presented excellent properties. After heating the composites at 60 °C for 3 h, the non-cross-linked and cross-linked immobilized CTs retained 51.6% and 70.7% of the initial activity, respectively, while the free CTs retained only 29.6% of the initial activity. In addition, non-cross-linked and cross-linked immobilized CTs retained 85.7% and 84.9% of the initial activity, respectively, after 20 days, whereas the free CTs retained only 18.8% of the initial activity. When the MCNCs were used to immobilize the CT molecules, the enzyme loading capacity was enhanced up to 6.3-fold upon cross-linking. Moreover, the immobilized CTs could be easily separated and recycled from the reaction system by a magnetic force.

KEYWORDS: chitin, nanofiber, enzyme immobilization, chymotrypsin, cross-linking, magnetic nanoparticle

INTRODUCTION Enzymes are excellent biocatalysts that are applied in various areas, including pharmaceutical chemistry, food modification, and energy production.1-4 However, the application of enzymes in industry is seriously hampered due to their low operational stability


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and difficulties in recovery and reuse.5-7 Thus, enzyme immobilization, the restriction of enzyme mobility on solid materials,7 is a requisite for the application of enzymes in industries as biocatalysts since immobilization enhances the enzyme stability and enables their separation from the reaction mixture for reutilization.5, 8-11 Basically, there are three methods for enzyme immobilization including support binding, entrapment, and cross-linking.8 To date, different nanostructured supports have been developed for enzyme immobilization, such as nanoparticles, nanotubes and nanofibers.12 Among the various kinds of nanostructured supports, nanofibers are considered to be promising supports for enzyme immobilization due to the advantages of reduced mass transfer limitation, high dispersibility in reaction solutions, easy recovery after reaction, high surface to volume ratio, highly porous mesh characteristics, as well as advanced dimensional and mechanical characteristics.13-16 Thus, development of nanofiber-based supports for enzyme immobilization is an important subject due to their unique dimensional and mechanical characteristics, which offer many promising properties for advanced supports for enzyme immobilization. Chitin, a natural polysaccharide,17 is one of the most plentiful organic resources.18-20 It is a main component of the exoskeleton of crustaceans, such as crabs, shrimp, and insects.18-19, 2122

Chitin has recently received increased attention and has been considered a promising

material for enzyme immobilization because of the characteristics of biodegradability, biocompatibility, renewability, sustainability, high affinity to proteins, availability of reactive functional groups for chemical modification, and mechanical stability.23-27 Due to these advantages, chitin and chitin-based materials are used in a wide range of applications, such as water filtration, medicine, catalysis, and biosensing.28-30 However, chitin is not dispersible in water and most organic solvents due to its highly hierarchical crystalline structure with strong intramolecular hydrogen bonding between chitin molecules;17,


31-33

thus, the diffusional


limitation seriously limits the application of chitin as a support for enzyme immobilization. Many approaches have been developed to disrupt the intramolecular hydrogen bonding of chitin,34 and there have been many reports using chitin-based materials for enzyme immobilization.35-41 For instance, Xu et al.40 prepared chitosan/poly(vinyl alcohol) composite nanofibrous membranes for laccase immobilization, and the results showed that immobilized enzymes exhibited higher stability and reusability than free enzymes. Pharm et al.41 reported the immobilization of β-galactosidases onto chitin via a chitin-binding domain, and the results showed that the enzymes immobilized on chitin were more stable than the corresponding native enzymes. Despite these efforts, chitin-based supports for enzyme immobilization with a high loading capacity, activity, biocompatibility, reusability, and recyclability are still required.34 In this study, a novel biocompatible magnetic chitin nanofiber composite (MCNC) was developed as a support for enzyme immobilization and its efficiency in enzyme immobilization was investigated. Chymotrypsin (CT) was selected as a model enzyme to test the efficiency of chitin nanofiber-based supports. CTs immobilized on MCNCs were further cross-linked with additional CTs via glutaraldehyde. The activity, loading, stability, and reusability of the immobilized enzymes were evaluated.

EXPERIMENTAL SECTION Preparation of chitin nanofibers (CNFs). To prepare CNFs, 2,2,6,6-tetramethylpiperidine1-oxyl (TEMPO)-mediated oxidation of chitin was performed.42 Chitin (20 g) was added and suspended in deionized water containing 1 mM TEMPO and 10 mM sodium bromide. A NaClO solution (4.5%, 330 mL) was added into the slurry to initiate the TEMPO-mediated oxidation of the chitin followed by gentle agitation at room temperature, and the pH of the chitin slurry was maintained at 10. After the reaction, the pH was adjusted to 7 by adding a ACS Paragon Plus Environment

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0.5 M HCl solution, and the mixture was centrifuged at 10000 rpm for 10 min to remove the supernatant. Next, the oxidized chitin was thoroughly washed with deionized water via centrifugation, and then, the chitin nanofibers were freeze-dried and stored at 4 °C for further use.

Characterization. TEM images were recorded using a transmission electron microscope (TEM; FEI Tecnai G2 F30) at an accelerating voltage of 120 kV. The samples (0.02% (w/v)) were mounted dropwise onto a carbon-coated TEM grid. A drop of 2% phosphotungstic acid stain was added to each sample, and then, the samples were dried naturally at room temperature. Sodium carboxylate groups in the CNFs were converted to free carboxyl groups via immersion of the CNFs into a 0.01 M HCl solution, followed by rigorous washing with deionized water. Then, all the samples were freeze-dried, and Fourier transform infrared (FTIR) spectra of the samples were measured in the range of 4000 to 400 cm-1 by an FT-IR spectrometer (Thermo Scientific Nicolet iS10).

Enzyme immobilization. To covalently immobilize the enzymes onto the surface of the supports, CNFs were modified via EDC/NHS activation. The prepared CNFs were suspended in a MES (100mM pH 6) buffer containing EDC (434 mM) and NHS (53.2 mM) and incubated under shaking for 3 h at room temperature. Then, the excess EDC and NHS were removed by washing thoroughly with deionized water three times. CT was selected as the model enzyme because it can be inactivated via autolysis.43 To immobilize the CTs onto the CNFs, EDC/NHS-activated CNFs were added into an aqueous solution of CT. The resulting mixture was incubated for 2 h at 20 °C under shaking



at 200 rpm. Amine-functionalized magnetic nanoparticles were prepared according to a previous report with a slight modification.44 Then, 1 mg of amine-functionalized magnetic nanoparticles was added into the mixture and shaken at room temperature for 1 h. The crosslinked immobilization of CT onto the MCNCs was carried out as follows. EDC/NHSactivated CNFs were added into 10 mL of a CT solution, and the mixture was incubated for 2 h at 20 °C under shaking at 200 rpm. Next, 1 mg of amine-functionalized magnetic nanoparticles and glutaraldehyde (0.4%w/v) were added to the solution, followed by incubation with shaking at room temperature for 1 h. Then, the samples were collected using a magnet, washed with a citric acid–sodium citrate buffer solution, and stored at 4 °C for further use.

Evaluation of the catalytic performance of free and immobilized CTs. The catalytic performance of free and immobilized CTs was evaluated via the hydrolysis of casein. Briefly, free and immobilized CTs (the amount of CTs immobilized on the MCNCs was the same as that of free CTs) were incubated in casein in a 50 mM Tris-HCL (pH 8.0) solution at 37 °C.45

Stability and reusability of immobilized CTs. To evaluate the thermal stability, the free and immobilized CTs were incubated in a Tris-HCl (50 mM, pH 8.0) solution at 60 °C, and the residual activity of free and immobilized CTs was measured at each time point. Storage stability was investigated by incubating the free and immobilized CTs in a 50 mM Tris-HCl (pH 8.0) solution at 4 °C for 20 days, and residual activity was measured at each time point. The pH stability was examined by measuring the residual activity of free and immobilized CTs after the free and immobilized CTs were incubated in a phosphate buffer solution with the pH range of 3 to 10 at 37 °C for 60 min. The reusability of the immobilized CTs was assessed by investigation of the relative activity changes after repeated usage for hydrolysis ACS Paragon Plus Environment

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of casein. After each hydrolysis cycle, the immobilized CTs were separated by a magnetic force and washed with deionized water to remove residual substrates and product for reuse in the next cycle.

Determination of enzyme loading. The amount of CTs immobilized onto the MCNCs was determined by measuring the initial and final concentrations of CT in the immobilization medium. The enzyme loading was calculated as follows: Enzyme loading (mg/g) = [(C - C0)V / W] × 100%

(1)

where C0 and C are the initial and final concentrations (mg/mL) of enzyme within the immobilization medium, respectively, V is the volume (mL) of the immobilization medium, and W is the weight (g) of the support.

RESULTS AND DISCUSSION Characterization of the CNFs. Figure 1 shows the TEM images of TEMPO-oxidized chitin. As shown in the figure, chitin was converted into individual fibrils with a nanoscale length. Needle-shaped CNFs with a nearly uniform size are clearly formed and are well individualized. When sufficient amounts of NaClO, which is used as the primary oxidant, is applied to chitin, the TEMPO-mediated oxidation selectively oxidizes the C6 primary hydroxyl groups of chitin to carboxylate groups. As a result, the hydrogen bonds between the chitin fibers are broken, and crystalline chitin nanocrystals are converted to the corresponding polyuronic acid, which is dispersible in water, as supported by the FT-IR spectra. The FT-IR spectra of TEMPO-oxidized chitin show an absorption peak at 1738 cm-1, which corresponds to the free carboxyl groups (Figure 2). These results indicated that CNFs with free carboxyl groups were successfully prepared by TEMPO-mediated oxidation.



Immobilization of enzymes. A schematic diagram of the synthesis and immobilization is illustrated in Figure 3. The effect of the CT concentration on the relative activity and enzyme loading was investigated. (Figure 4). As shown in Figure 4a, when the CT concentration was less than 0.5 mg/mL, the relative activity of non-cross-linked immobilized CTs increased as the CT concentration increased up to 0.5 mg/mL. The relative activity reached a maximum at the concentration of 0.5 mg/mL, and when the concentration exceeds 0.5 mg/mL, a decrease in the relative activity was observed. A similar phenomenon was observed on cross-linked immobilized CTs (Figure 4b). When the CT concentration was less than 1 mg/mL, the relative activity of cross-linked immobilized CTs continued to increase as the CT concentration increased. The maximum relative activity was obtained at 1 mg/mL of CTs, and beyond this value, the relative activity decreased because the excess enzyme loading could result in crowding or agglomeration of the enzyme molecules onto the support surface. As a result, dispersion and transmission of the immobilized enzymes were restrained, and the relative activity decreased. The immobilization capacity of the support was assessed by measuring the CT loading. The enzyme loading of non-cross-linked immobilized CTs was 92.4 mg/g support at the relative activity of 100% (Figure 4a). In comparison, the enzyme loading of cross-linked immobilized CTs reached 581.84 mg/g support at the relative activity of 100% (Figure 4b), corresponding to 6.3 times higher than that of the non-cross-linked immobilized CTs. This high enzyme loading capacity of MCNCs can contribute to the large surface area for the immobilization of enzymes provided by CNFs.13 Moreover, glutaraldehyde cross-linking significantly increased the enzyme loading capacity since glutaraldehyde treatment could cross-link additional enzyme molecules and aggregate them onto the covalently attached enzymes that are immobilized on the CNFs.13 Consequently, more enzymes were


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immobilized on the CNFs upon cross-linking, revealing an improvement in the enzyme loading capacity.

Catalytic efficiency of immobilized CTs. The catalytic performance of the immobilized CTs was investigated by measuring the amount of hydrolyzed casein. For comparison, free CTs were also used to catalyze hydrolysis of casein, and default reaction conditions were the same as those given for the immobilized CTs. Figure 5 shows the time courses of the hydrolysis of casein by free and immobilized CTs. After 8 h of reaction, the conversion of casein catalyzed by non-cross-linked and cross-linked immobilized CTs was 1.28 and 1.31 times higher than that of free CTs, respectively. Enzyme immobilization via covalent attachment onto the MCNCs can effectively protect the enzyme molecules from being denatured or inactivated during the overall reaction. Moreover, casein hydrolysis was saturated after 120 min of treatment with the free and non-cross-linked CTs, while casein hydrolysis continued to increase up to 240 min when catalyzed by cross-linked CTs, suggesting that the cross-linking by glutaraldehyde effectively protected the enzyme molecules from denaturation, inactivation or leaching during the reaction.13, 46

Stability and reusability. Thermal, storage, and pH stability is important for enzyme immobilization. The thermal stability of free and immobilized CTs is shown in Figure 6a. The MCNC immobilizing system presents a significant resistance to thermal inactivation compared with the free enzyme. At 60 °C, the non-cross-linked and cross-linked immobilized CTs retained 51.6% and 70.7% of the initial activity after heating for 3 h, respectively, while the free CTs retained only 29.6% of the initial activity. Figure 6b presents the storage stability of free and immobilized CTs, revealing that this parameter was greatly improved by immobilization onto MCNCs. In comparison with free CTs, the immobilized CTs presented a



much smaller rate of inactivation at 4 °C. The free CTs retained only 18.8% of the initial activity after 20 days, while the non-cross-linked and cross-liked immobilized CTs retained 85.7% and 84.9% of the initial activity, respectively. Figure 6c shows the pH stability of the free and immobilized CTs. The maximum activity of the free and immobilized CTs was observed at pH 7. The immobilized CTs sustained higher relative activity over a pH range of 3 to 10 in comparison with the free CTs. The relative activity values of cross-linked CTs and non-cross-linked CTs were 28.1% and 34.5% at pH 3, 96.9% and 94.1% at pH 7, and 78.1% and 79.2% at pH 10, respectively, in comparison with the values of 8%, 90% and 73% for the free CTs at the respective pH levels. In addition to the thermal, storage, and pH stability, the reusability of the immobilized enzyme is another important aspect for practical applications. Figure 7 presents the reusability of free and immobilized CTs. The cross-linked immobilized CTs showed a significantly higher reusability than that of the non-cross-linked immobilized CTs. After 5 and 10 cycles, the cross-linked CTs immobilized on MCNCs retained 78.6% and 54.7% of the initial activity, while the non-cross-linked immobilized CTs retained 63.5% and 46.3% of the initial activity, respectively. The high stability and reusability of immobilized CTs could be attributed to the preservation of the structure and active sites of the enzyme by immobilization onto the CNFs.47 Meanwhile, compared with non-cross-linked immobilized CTs, cross-linked immobilized CTs presented a higher thermal stability and reusability, revealing that the noncross-linked CTs immobilized onto the MCNCs may also be attached via adsorption in addition to covalent interactions. Moreover, cross-linkages produce a very strong multipoint covalent attachment.48 In this case, multipoint cross-linkages could effectively remain relative positions of enzyme groups that are involved in cross-linking because one group cannot move without moving the others.48 As a result, compared with free and non-cross-linked enzymes,


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cross-linked immobilized enzymes can retain their structure under much harsher conditions. These results indicated that cross-linkage by glutaraldehyde can significantly increase the stability and prevent leaching of the immobilized CTs.

CONCLUSIONS A novel biocompatible MCNC was successfully synthesized and was shown to be a potential support for enzyme immobilization. CT was used as a model enzyme, and it was successfully immobilized onto the MCNCs. The immobilized CTs exhibited a significantly enhanced stability compared with the free CTs. The loading capacity of the MCNCs was greatly improved upon cross-linking. Moreover, the artificial biocatalytic system exhibited excellent recyclability. These results suggested that the MCNC has great potential for use as a support for the immobilization of various enzymes in biotechnological applications.

ASSOCIATED CONTENT Supporting Information Materials, preparation of magnetic nanoparticles, experimental details for additional characterizations.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (31471607), China Agriculture Research System (CARS-48), Major Special Science and Technology Projects in Shandong Province (2016YYSP016) and Applied Basic Research Program of Qingdao (16-5-1-18-jch).



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Figure 1. TEM images of the CNFs. 353x173mm (72 x 72 DPI)


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Figure 2. FT-IR spectra of (a) chitin and (b) the CNFs. 272x208mm (300 x 300 DPI)



Figure 3. Schematic diagram of the immobilization of CTs onto the MCNCs. 555x478mm (72 x 72 DPI)


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Figure 4. Effect of the CT concentration on the relative activity and enzyme loading of (a) non-cross-linked CTs immobilized onto the MCNCs and (b) cross-linked CTs immobilized onto the MCNCs. 363x543mm (72 x 72 DPI)



Figure 5. Time courses of the hydrolysis of casein using the free CTs, non-cross-linked CTs immobilized onto the MCNCs (CTs-MCNCs), and cross-linked CTs immobilized onto the MCNCs (cross-linked CTs-MCNCs). 272x208mm (300 x 300 DPI)


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Figure 6. (a) Thermal stability, (b) storage thermal stability and (c) pH stability of the free CTs, non-crosslinked CTs immobilized onto the MCNCs (CTs-MCNCs), and cross-linked CTs immobilized onto the MCNCs (cross-linked CTs-MCNCs). 363x807mm (72 x 72 DPI)



Figure 7. Reusability of non-cross-linked CTs immobilized onto the MCNCs (CTs-MCNCs), and cross-linked CTs immobilized onto the MCNCs (cross-linked CTs-MCNCs). 272x208mm (300 x 300 DPI)


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A novel biocompatible magnetic chitin nanofiber composite (MCNC) was developed as a support for enzyme immobilization and that the enzyme molecules immobilized on the MCNCs presented excellent properties. 475x322mm (72 x 72 DPI)


Effective Enzyme Immobilization onto a Magnetic Chitin Nanofiber

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