High uptake carboxyl-functionalized porous β-cyclodextrin polymer for selective extraction of lysozyme from egg white

Accepted Manuscript Title: High Uptake Carboxyl-Functionalized Porous ␤-cyclodextrin Polymer for Selective Extraction of Lysozyme from Egg White Authors: Hui-Ling Duan, Qi-Le Niu, Jun Wang, Shi-Yao Ma, Jing Zhang, Zhi-Qi Zhang PII: DOI: Reference:

S0021-9673(19)30441-8 https://doi.org/10.1016/j.chroma.2019.04.056 CHROMA 360202

To appear in:

Journal of Chromatography A

Received date: Revised date: Accepted date:

26 February 2019 19 April 2019 21 April 2019

Please cite this article as: Duan H-Ling, Niu Q-Le, Wang J, Ma S-Yao, Zhang J, Zhang Z-Qi, High Uptake Carboxyl-Functionalized Porous ␤-cyclodextrin Polymer for Selective Extraction of Lysozyme from Egg White, Journal of Chromatography A (2019), https://doi.org/10.1016/j.chroma.2019.04.056 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

High Uptake Carboxyl-Functionalized Porous β-cyclodextrin Polymer for Selective Extraction of Lysozyme from Egg White Hui-Ling Duan, Qi-Le Niu, Jun Wang, Shi-Yao Ma, Jing Zhang, Zhi-Qi Zhang

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Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, and Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry of Ministry of Education, School of Chemistry and Chemical

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Engineering, Shaanxi Normal University, Xi’an 710062, China

Corresponding

author. Fax: +86 29 81530792. E-mail address: [email protected]. 1

Highlights This paper develops a specific adsorbent for lysozyme with high uptake and selectivity

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Carboxyl-functionalized porous β-cyclodextrin polymer (P-CDP-COO-) was first prepared

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The maximum adsorption capacity of 1520 mg g-1 is far higher than other adsorbents P-CDPCOO- could effectively and selectively extract lysozyme from egg white

The extraction involves nonspecific adsorption and electrostatic attractions

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ABSTRACT

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Lysozyme is widely used in medical, food and industrial fields due to its bacteriolytic effect and thus

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it is significant to design and develop a specific adsorbent with high adsorption capacity and selectivity

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for lysozyme. Inspired by the high uptake capacity of tetrafluoroterephthalonitrile-crosslinked porous

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β-cyclodextrin polymers (P-CDPs) and the noncovalent interaction between lysozyme and carboxyl groups, the carboxyl-functionalized P-CDPs (P-CDP-COO-) were synthesized by base-catalysed

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hydrolysis of nitrile group in P-CDPs to carboxyl. Porous structure, large extent of carboxylic

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functional groups, cyclodextrin's preventing aggregation, and negatively charged make P-CDP-COOpossess an outstanding adsorption capability for lysozyme. The maximum saturated adsorption

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capacity reaches 1520 mg g-1, which is much better than the parent polymer and other reported materials. The as-prepared material was successfully utilized for the selective extraction of lysozyme from egg white. Key words: Carboxyl-functional modification, porous β-cyclodextrin polymer, dispersive solid-phase extraction, lysozyme. 2

1. Introduction Lysozyme, a kind of alkaline globulin, is widely existed in various tissues of human body, secretions of mammals, and egg white of poultry. Egg white lysozyme is a monomeric protein of 129

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amino acids with the molecular size of 3.0×3.0×4.5 nm [1, 2]. Lysozyme is of great application value in the industry and medical treatment field. It can be used as a natural food preservative with the anti-

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bacterial, anti-viral and anti-tumor effects. Meanwhile, it is an essential tool enzyme for cell fusion in gene and cell engineering [3]. Therefore, the lysozyme separation and preparation have received

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considerable attention recently. Some adsorbents have been applied for separation of lysozyme, such

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as lysozyme-imprinted poly-dopamine layer on a fibrous SiO2 micro-sphere [4], poly(sodium 4-

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styrenesulfonate) modified magnetic nanoparticles (PSS–MNPs) [5], sulphonyl and carboxyl

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functionalized stearyl alcohol grafted epichlorohydrin [6], magnetic ionic liquid-molecularly

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imprinted polymers [3], carbonylated cellulose nanofibrous membranes [7], multi-walled carbon nanotubes [8] and so on. However, its special cyclic structure and a large molecular size lead to a lower

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adsorption capacity.

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In recent years, advance in materials science has led to the rapid emergence of a large number of functional porous adsorbent materials, from traditional activated carbon and zeolites to modern metal

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organic frameworks (MOFs) [9], covalent organic frameworks (COFs) [10], polymers of intrinsic microporosity (PIMs) [11] and so on. Nevertheless, low adsorption capacity and slow adsorption rate hinder the rapid development of these materials to adsorb and separate organics [12]. Hearteningly, tetrafluoroterephthalonitrile-crosslinked porous β-cyclodextrin polymers (P-CDPs) have attracted much public attention owing to the rapid extraction rate and high uptake capacity for organic 3

micropollutants [13-15]. P-CDPs functionalized with cotton fabric for capturing organic pollutants [16] and magnetic microspheres for extraction of microcystins [17] have also been reported. However, the potential for lysozyme separation is unknown and little has been published on the preparation of carboxyl-functionalized P-CDPs via chemical modification.

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The purpose of this paper is to design and develop a specific adsorbent for lysozyme with high uptake ability, and to apply this material to selective extraction of lysozyme from egg white (Figure

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1). Since P-CDPs is of the high uptake capacity and β-CD could suppress aggregation of lysozyme [18, 19], P-CDPs was chosen as the adsorbent parent. Moreover, the introduction of carboxyl groups can

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enhance the nonspecific adsorption of lysozyme [20, 21] and deprotonation of carboxyl can enhance

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surface negative charge of material. It is expected that carboxyl-functionalization of P-CDPs

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converting nitrile group to carboxyl by base-catalyzed hydrolysis can further improve the adsorption

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capacity for lysozyme via nonspecific adsorption and electrostatic attractions.

2. Material and methods

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2.1 Chemicals and materials

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Lysozyme and ovalbumin were obtained from GBCBIO Technologies (Guangzhou, China). HPLC-grade acetonitrile was purchased from Fisher (New York, USA). HPLC-grade trifluoroacetic

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acid was purchased from Aladdin Chemical Reagent Co. (Shanghai, China). All of other reagents were of analytical grade. Deionized water was prepared with a Milli-Q water purification system (Millipore, USA). Conalbumin was obtained from Sigma-Aldrich and used as received. Chicken eggs were purchased from a local market.

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2.2 Synthesis of Carboxyl-functionalized P-CDPs(P-CDP-COO-） The P-CDPs were synthesised using a modification for the method of Alsbaiee et al [13] (Supporting Information). Then, P-CDP-COO- was prepared by base-catalysed hydrolysis of P-CDPs. In detail, P-CDPs powder (0.2 g) and 30% NaOH solution (H2O/Ethanol = 1/1, 20 mL) were joined to

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a round bottom flask equipped. The mixture was refluxed at the oil bath with continuous stirring in

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different reaction conditions. The polymer solids were collected through centrifugal filtration, then dispersed in water, adjusted pH to 4-5 by dropwise adding HCl solution and sonicated for 10 min.

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Finally, the products were washed with water until neutral and dried by freeze-drying for overnight.

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2.3 Characterization

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Fourier transform infrared spectra (FT-IR) were recorded in the 400-4000 cm-1 region with an

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EQUINX 55 FTIR spectrometer (Bruker Inc., Germany). Elemental analysis was carried out on an

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Elementarvario EL III Analyzer. The zeta potentials were measured with a Delsa Nano C (Beckman Coulter). Thermogravimetric Analysis (TGA) was performed under a nitrogen atmosphere with a

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Q600SDT System (TA Inc., America) at 10 oC min-1. N2 adsorption isotherms were measured at 77 K

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using an ASAP 2020M micropore physisorption analyser (Micromeritics, Norcross, GA, USA). SEM images were observed on a Hitachi TM3030 scanning electron microscope. Field transmission electron

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microscope (TEM) images were recorded with FEI Tecnai G2 F20 microscope instrument (FEI, USA).

2.4 Adsorption and desorption experiments Aqueous stock solutions of 8 mg mL-1 lysozyme were prepared in deionized water. Dilute solutions at the specified pH were prepared using 20 mM phosphate buffer. For adsorption experiments, about 4 mg sorbent and the dilute solution (1.5 mg mL-1, pH 9.0) were mixed in a 10 mL plastic 5

centrifuge tube, and shaken at 200 rpm for 45 min using a thermostatted rotary shaker. Then the mixture was centrifuged at 10,000 rpm for 3 min, and the absorbance of supernatant was determined at 280 nm (Figure S1) on an Evolution 220 spectrometer (Thermo Fisher Scientific Inc., America). Each experiment was carried out in triplicate and the average value was calculated. The adsorbed

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amount of lysozyme per unit mass of adsorbent at equilibrium (qe, mg g-1) was calculated with the following equation: (C o  C e )  V

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qe 

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where C0 and Ce (mg L-1) are the concentrations of lysozyme at initial and equilibrium, respectively.

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V (L) is the solution volume, and m (g) is the adsorbent mass used.

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For desorption experiments, the lysozyme-adsorbed particles were added into 5.0 mL of NaCl

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solution (1.0 M), and oscillated at room temperature for 10 min to elute lysozyme. The eluted lysozyme solution was taken for UV determination. The adsorbent was regenerated by soaking and rinsing in

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order with NaH2PO4 and deionized water.

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2.5 Extraction of lysozyme from egg white

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The chicken egg whites were treated by the method of Chen et al [5] (Supporting Information). Then 5 mL egg whites solution and 30 mg P-CDP-COO- were mixed and stirred at 200 rpm for 2 h at

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room temperature. The extracted lysozyme on adsorbent was eluted with phosphate buffer (20 mM, pH 7.0) containing 1.0 M NaCl. The initial egg white solution, the supernatant after extraction, and the eluate were analyzed using HPLC. HPLC analysis was performed by using a Shimadzu LC-20A HPLC system with SPD-20A detection (Shimadzu Co., Kyoto, Japan) and an analytical reversed-phase C18 column (5 µm, 4.6 mm 6

× 250 mm). The sample injection volume was 20 µL. The mobile phases were water (A) and acetonitrile (B) solutions of trifluoroacetic acid (0.1%, v/v), respectively. The gradient procedure was performed as 0-20 min from 25% to 60% B, 21-25 min from 60% to 25% B, and 25-30 min holding

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25% B at flow-rate of 1.0 ml min-1. The UV-vis detector was set at 280 nm.

3. Results and discussion

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3.1 Synthesis of P-CDP-COO-

The nitrile group in organic compounds could be transformed into other groups including

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carboxylic acid, amine and ethanolamine [22-25]. In order to obtain the carboxyl-functionalized

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absorbent, P-CDPs were hydrolyzed at base condition to transform nitrile group into carboxylic acid

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and the adsorption capacity for lysozyme was evaluated. As can be seen in Figure 2, the yield of as-

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made material and adsorption amount of lysozyme increase as the raise of reaction temperature and

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the extension of reaction time within 48 h. This can be considered that the hydrolysis is more complete and more carboxyl functional groups are generated with longer reaction time and higher temperature.

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In view of the solvent nature of ethanol and water, the reaction temperature was set at 120 °C. Beyond

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48 h of reaction time, the yield and adsorption are not obviously changed, suggesting that the hydrolysis is basically completed. Therefore, the later experiments were carried out at 120 °C

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hydrolysis for 48 h.

3.2 Characterization The characterization results of FT-IR spectroscopy are shown in Figure 3. In the spectrum of parent P-CDPs, there is an absorption peak at 2243 cm-1, corresponding to the nitrile stretch. In the 7

spectrum of carboxyl-functionalized product, the absorption of nitrile disappears, and carbonyl bands at 1604 and 1676 cm-1 appear. These results indicate nitrile group being converted to carboxyl group [22]. Elemental analysis indicates that the contents of C (43.13) and N (2.27) in the P-CDP-COO- are

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lower than those of C (46.67) and N (6.28) in the P-CDPs. Oppositely, the content of H (5.03) in P-

confirme nitrile groups being transformed into carboxylic groups.

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CDP-COO- is higher than that of H (3.20) in P-CDPs. The decreased N content and increased H content

The zeta potentials of P-CDPs and P-CDP-COO- are negative in the pH range at 2.0 to 12.0, and

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increase with the solution pH up (Figure 4A). More negative zeta potential of hydrolysis products than

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groups of the parent polymer have changed.

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the parent polymer should be attributed to the production of carbonyl. This result also declares that the

Thermogravimetric analysis (TGA) curves for P-CDP-COO- and parent polymer P-CDPs are

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shown in Figure 4B. The weight losses below 100°C may be attributed to the adsorbed moisture both

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for P-CDPs and P-CDP-COO-. Compared to the parent polymer, the degradation temperature for P-

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CDP-COO- is reduced from 260 to 200 °C, indicating the change of polymer groups and the presence of carboxylate salt (ammonium and sodium) in the polymer [22]. To evaluate the porous structure of P-CDP-COO-, nitrogen sorption isotherms were investigated.

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As shown in Figure 4C, reversible sorption profile is observed with typical type II sorption characteristics. The pore size distribution is hierarchical with an average pore size of 12.7 nm. The BET micropore area is 4.3 and external surface area is 18.7 m2 g-1. The surface morphology characterization (Figure 4D) showed that P-CDP-COO- is irregular in the micron-level size, which is 8

similar to P-CDPs (Figure S2). It is can be seen from TEM characterization (Figure 4E) that the polymer crosslinks to form mesopores with different pore sizes, while the enlarged TEM image shows the micro-porous structure of the polymer matrix (Figure 4F).

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3.3 Effect of pH and NaCl on adsorption The influences of pH and ionic strength on adsorption capacity of as-prepared absorbent for

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lysozyme were investigated.

The sample solution pH from 2 to 12 was adjusted by using 0.1 M HCl or NaOH solution. As

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shown in Figure 5A, the adsorption capacity of adsorbent increases gradually with the solution pH

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raising from 2.0 to 9.0, maintains constant from pH 9.0 to 11.0, and then decreases rapidly beyond pH

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11.0. Notably, this is a classic pH dependent adsorption.

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The lysozyme is positively charged below pH 11.0. The deprotonation of carboxyl in P-CDP-

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COO- and the stability of polymer are enhanced with the solution pH raising and more negative charges are generated on the surface. The maximum adsorption capacity of adsorbent is obtained at the pH of

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9.0-11.0 for lysozyme, suggesting that electrostatic interactions dominated affinity between the

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adsorbent and lysozyme. Beyond pH 11.0, lysozyme is negatively charged and the electrostatic repulsion would prevent lysozyme from adsorbing on the negatively charged nanoparticles [5], but it

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is still maintained high adsorption capacity, indicating other interactions also play roles on attraction. The adsorption amount of lysozyme is affected by ionic strength (Figure 5B). Sodium ions can

compete with the lysozyme and interact with the P-CDP-COO- preferentially, thus leading to the decrease of the adsorption amount of lysozyme with the increase of ionic strength. It further confirms that the main driving force for the adsorption of lysozyme on P-CDP-COO- is electrostatic interaction, 9

which also means that NaCl in high concentration is conducive to elution of the loaded lysozyme from P-CDP-COO-.

3.4 Adsorption Kinetics

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The adsorption of lysozyme on P-CDP-COO- with time variation is shown in Figure 5C. With the extending of contact time, the adsorption amount of lysozyme rapidly increases in 10 min, slowly

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increases from 10 to 45 min, and then remains basically constant. Significantly, 85.0% of lysozyme is extracted from the aqueous solution within 10 minutes, and extraction efficiency reaches up to 97.1%

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at 45 minutes.

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To analyze the kinetics of lysozyme sorption on the P-CDP-COO-, the kinetic sorption process

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are simulated by pseudo-first-order rate equation and pseudo-second-order rate equation (Supporting

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Information), and the results are showed in Figure 5D. The correlation coefficient of pseudo-second-

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order equation is higher than that of pseudo-first order equation (Table S1), indicating that the adsorption process is well fitted the pseudo-second-order rate model. The pseudo-second-order rate

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model is based on the fact that the rate-limiting step is very close to chemisorption. It can be concluded

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that the main adsorption form of lysozyme on P-CDP-COO- is chemical adsorption or surface complexation.

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3.5 Adsorption Isotherms The adsorption isotherm of lysozyme on P-CDP-COO- is performed to investigate the saturated

adsorption behavior and capacity. Langmuir, Freundlich and Temkin isotherm models are used to analyze the equilibrium data (Supporting Information). The results shows that the data is better fitted the Langmuir (Figure 5E) than Freundlich (Figure S3) and Temkin (Figure S4) models, implying that 10

the adsorption of lysozyme may take place on homogeneous adsorption sites and incline to monolayer adsorption. The maximum saturated adsorption capacity of P-CDP-COO- is as high as 1520 mg g-1 for lysozyme calculated from Langmuir isotherm (Table S2), which is about 2.5 times more than that of

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P-CDPs parent polymer (615 mg g-1).

3.6 Desorption and regeneration studies

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In practical application, desorption of lysozyme and regeneration of adsorbent are important. Considering the activity of lysozyme, different concentration of NaCl in phosphate buffer (20 mM, pH

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7.0) was chose as the eluting solvent to investigate the influence of ionic strength (Figure S5). The

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results indicate that the desorption efficiency of lysozyme increases with the addition of NaCl below

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1.0 M, but desorption efficiency is modestly improved beyond 1.0 M. Therefore, the eluting solvent

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of phosphate buffer containing 1.0 M NaCl is optimal and 96.5% of lysozyme can be desorbed and

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removed from adsorbent.

From the view of the electrostatic interaction between lysozyme and adsorbent, acidic elution

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solvent is benefit to the protonation of carboxyl, decrease of negative charges on adsorbent surface,

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and complete elution of lysozyme. Thus, NaH2PO4 and deionized water are selected as eluents to regenerate adsorbent. The adsorption capacity of P-CDP-COO- slightly reduces with the increase of

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reuse times, but 78.8% of adsorption capacity is remained after five regenerations (Figure 5F). 3.7 Adsorption selectivity of P-CDP-COO- for lysozyme To evaluate the selectivity of as-prepared polymer, the adsorption capacity of P-CDP-COO- for ovalbumin and conalbumin, two abundant proteins in egg white, was investigated at different pH. As shown in Figure 5A, the adsorption capacity for lysozyme is significantly higher than that for 11

ovalbumin or conalbumin at any pH, suggesting that as-made polymer can selectively extract lysozyme from egg white. The selectivity may contribute to multiple interactions including electrostatic interaction and size effect.

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3.8 Extraction of lysozyme from egg white There are various proteins in egg white, mainly including ovalbumin (54%), conalbumin (12%)

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and lysozyme (3.4%). Because of high lysozyme content, the as-prepared P-CDP-COO- was applied to extraction of lysozyme from chicken egg white. The chromatograms of diluted egg white solutions

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before and after treatment with P-CDP-COO- are shown in Figure 6. It can be seen that there are three

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main proteins in the diluted egg white solution, two proteins in the supernatant after extraction, and

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only lysozyme in the eluted solution. Results shows that P-CDP-COO- can selectively adsorb the

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lysozyme from egg white, and the extracted lysozyme is of high purity. The recovery rate for once

of literature [26, 27].

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extraction reaches to 93.6% calculated from the chromatogram peak areas, which is higher than that

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3.9 P-CDP-COO- as unique material for lysozyme extraction

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Compared to reported sorbents, P-CDP-COO- possesses an outstanding adsorption performance

for lysozyme (Table 1). Firstly, cyclodextrin-lysozyme interaction is relying on non-bonding

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electrostatic, van der Waals and hydrophobic interactions. Meanwhile, β-CD could not form complex with lysozyme and could prevent aggregation occurring upon refolding, which causes the loss of biological activity of lysozyme [19, 28, 29]. Secondly, the surface charge has significant effect on the adsorption capacity. Lysozyme has an electropositive character below isoelectric point, and carboxylfunctionalized material has more negative surface charge than the parent polymer. The electrostatic 12

attraction drives the adsorbate close to the sorbent. Thirdly, carboxyl groups enhance the nonspecific adsorption between lysozyme and sorbents. Finally, the structural and textural properties of P-CDPCOO- are superiority. The hierarchical structure with an average pore size of 12.7 nm is available for lysozyme entering. All these factors make P-CDP-COO- an efficient adsorbent for lysozyme isolation

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and extraction.

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4. Conclusion

The carboxyl-functionalized P-CDPs, a novel adsorbent, was synthesized by base-catalyzed

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hydrolysis nitrile group into carboxyl. The introduction of carboxyl dramatically enhances the

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adsorption capacity for lysozyme. The maximum lysozyme adsorption amount from 615 mg g-1 of P-

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CDPs raises to 1520 mg g-1 of P-CDP-COO-. The adsorption process is mainly limited by

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chemisorption and the adsorption equilibrium is fitted to the Langmuir model. The as-prepared

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material was successfully applied to selective extraction of lysozyme from egg white. In brief, the P-

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CDP-COO- material is of outstanding adsorption capacity and high selectivity for lysozyme, and could be a potential adsorbent for the extraction and preparation of lysozyme.

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Acknowledgements

This work was financially supported by National Natural Science Foundation of China (No.

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21775100).

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Figure Captions Figure 1 Schematic illustration of the process for synthesis of P-CDP-COO- and selective extraction of lysozyme from egg white

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Figure 2 Influence of hydrolysis condition on the yield of P-CDP-COO- and the

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adsorption amount of lysozyme

Figure 3 FTIR spectra of P-CDPs (a) and P-CDP-COO- (b)

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Figure 4 (A) Zeta potentials and (B) TGA curves of P-CDPs and P-CDP-COO-, (C)

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Nitrogen adsorption-desorption isotherms curve and (D) SEM image of P-CDP-COO-,

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(E) TEM image of P-CDP-COO-, and (F) HR-TEM image of P-CDP-COO-

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Figure 5 Effect of (A) pH value, (B) ionic strength, and (C) extraction time, (D) Plots

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of pseudo first-order (a) and pseudo second-order (b) equations for the adsorption of lysozyme on P-CDP-COO-, (E) Langmuir adsorption isotherm of lysozyme on P-CDP-

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COO-, and (F) Influence of reuse on adsorption mount Figure 6 Chromatograms of diluted egg white solutions before and after treatment with

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P-CDP-COO, and eluted solution

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Adsorption Capacity (mg g-1)

References

Magnetic carboxymethyl chitosan nanoparticles

256.4

[26]

Nitrogen-doped carbon materials

447.0

[30]

Fe3O4/polymethyl methacrylate/SiO2 nanorattles

400.0

[31]

Poly(hydroxyethyl methacrylate) based magnetic nanoparticle

376.1

[32]

Magnetic poly(lactic-co-glycolic composite microspheres

/Fe3O4

497.0

Dye attached poly(2-hydroxyethyl methacrylate) nanoparticle

630.7

Oligo(ethylene glycol)-based molecularly imprinted magnetic nanoparticles

204.1

Lysozyme-imprinted polydopamine layer on a Fe3O4@ fibrous-SiO2 microsphere

700.0

Carbonylated cellulose nanofibrous membranes

160

[7]

Poly(sodium 4-styrenesulfonate) modified magnetic nanoparticles

476.2

[5]

Sulphonyl and carboxyl functionalized stearyl alcohol grafted epichlorohydrin

394.7 and 379.7

[6]

Magnetic ionic polymers

liquid-molecularly

213.7

[3]

P-CDP-COO-

1520

This work

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imprinted

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[27]

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acid)

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Materials

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Table 1 Comparison of various adsorbents for lysozyme adsorption.

[33] [34] [4]

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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