ABSTRACT

A novel adsorbent based on peanut shells modified with glutaraldehyde and succinic anhydride was prepared. Factors affecting the adsorption capacity, such as the pH, temperature, adsorption time, initial cytochrome c (cyt c) concentration and NaCl ionic strength, were extensively investigated. The results showed that the maximum adsorption capacity of the modified peanut shells (MPSs) was 432.6 mg/g when 10 mL of cyt c solution was adsorbed by 20 mg of MPSs at pH 5.0 for 3 h. In contrast, the adsorption capacities of the unmodified peanut shells (PSs), alkaline peanut shells (APSs) and crosslinked peanut shells (CPSs) were only 100.6, 180.3, and 173.0 mg/g, respectively, 4.3-, 2.4-, and 2.5-fold lower, respectively, than that of the modified shells. The desorption rate reached 89.9% with 1.5 mol/L NaCNS as an eluent, because the electrostatic attraction between the positive charges of the protein and the negative charges of the MPSs was reduced when the ionic strength was increasing. The MPSs were used to separate and purify cytochrome c from pig myocardium. A purification of 13.5-fold in a single step with a total enzyme activity recovery of 74.0% was achieved.

1. INTRODUCTION

Cyt c, a type of spherical electronic-transfer protein in the biological respiratory chain that has high biological stability, is not only an alkaline respiratory enzyme with an iron porphyrin group but is also an important cell respiration activator. It is readily soluble in water and acidic solutions and is abundant in yeast and animal myocardium. Typically, crude cyt c is obtained from animal myocardium by salting-out and then further purified by column chromatography. However, this process is time consuming and has a low throughput. In contrast, adsorption techniques using adsorbents with affinity ligands allow a high throughput and a short purification circle. Recently, experiments on the purification cyt c with different adsorbents have been reported. These adsorbents include polyhydroxylethyl methylacrylic acid bonded with dimethyleneimine [1], Fe₃O₄ containing nanomaterials modified with Cl-NH₂ [2] and magnetic materials bonded with Cu²⁺ [3] None of these adsorbents is a biomaterial, and they all have a low adsorption capacity for cyt c. In contrast, we demonstrated that affinity ligandmodified peanut shells have much better adsorption properties for cyt c. PSs are used to treat wastewater in development research pertaining to agricultural waste [4]. The use of similar agricultural wastes in deodorisation [5] and in the adsorption of oil [6] and hyperoxide [7] have also been reported. However, the use of chemically modified peanut shells to adsorb bioactive substances has not been studied previously. The primary components of peanut shells are cellulose and hemicellulose. Therefore, there are a large number of hydroxyls located on the surface of peanut shells that can be effectively activated and easily modified with carboxyl, amidogen and affinity dye ligands. The affinities of peanut shells for cyt c differ based on the modified group. Based on this, we can achieve the selective adsorption of cyt c and lysozyme over haemocyanin and ovalbumin. In this study, we prepared a novel adsorbent from peanut shells modified with succinic anhydride and then compared its adsorption of cyt c with that of unmodified shells. In addition, using optimised parameters, cyt c was purified from a solution crude extract of pig myocardium using this novel adsorbent.

2. MATERIALS AND METHODS

2.1. Materials

Glutaraldehyde was purchased from Jingchun Chemical Co., and cytochrome c was obtained from Sigma (USA). All other chemicals were of analytical grade. Pig myocardium and peanut shells were purchased from a farmer’s market in Wuhan, China. The collected biomaterial was extensively washed with tap water to remove soil and dust, sprayed with distilled water and then dried in an oven at 50˚C to a constant weight. The dry biomass was crushed to form a powder and sieved with 100 - 140 mesh.

2.2. Preparation of the Adsorbent

PSs (7.5 g) were mixed with 200 mL of NaOH solution (20 wt%) with stirring at 25˚C for 16 h. The mixture was then filtered, rinsed with water until the pH was 7, ovendried and stored in a desiccator. This protocol yielded the APSs.

To obtain CPSs, 1.5 g APSs, 2 mL 50% glutaraldehyde and 48 mL distilled water were added to a flask and shaken for 12 h at 30˚C. Then, the mixture was rinsed with distilled water. Afterwards, the filter residue was dried under vacuum at 60˚C to a constant weight.

Then, 1.0 g CPSs was added to 50 mL pyridine in which 3.0 g succinic anhydride had been dissolved. The mixture was reacted with agitation at 75˚C for 24 h. Then, the products were rinsed, leached and oven-dried to obtain MPSs. The MPSs were treated with saturated NaHCO₃ for one hour and then oven-dried, weighed (1.45 g) and stored in a desiccator. The ligand-binding rate (45%) was calculated by weighing the peanut shells before and after modification.

2.3. Equipment and Characterisation Methods

XPS (VGMultilab 2000 X-ray photoelectron spectrometer) was used to analyse the surfaces of the different types of biomass: PSs, APSs, CPSs and MPSs. The analysis was performed with an Mg X-ray source to determine the percentages of C, O and N atoms on the surface of the samples. During each measurement, the pressure in the analysis chamber was maintained at less than 10⁻⁸ Torr. All binding energies were referenced to the neutral C (1 s) peak at 284.6 eV to compensate for the surface charge effects.

Infrared spectra of PSs, CPSs and MPSs were obtained using an FT-IR spectrophotometer (Nicolet NEXUS 470, Nicolet Co., Ltd., USA) with KBr disks. A Systronics microprocessor pH meter (pHS-3C, Shanghai Leizi Instrument Factory, China) was used to take the pH measurements. A temperature-controlled water bath flask shaker (SHZ-03, Shanghai Kanxin Instrument Factory, China) was used to mix all solutions. The concentration of cyt c in each solution was determined using a UV-vis spectrophotometer (LAMBDA B10 35, PerkinElmer, USA).

2.4. Adsorption and Desorption Performance

The adsorption of cyt c in aqueous solution was performed by adding 20 mg PSs, APSs, CPSs or MPSs to each of a series of 50 mL conical flasks containing 10 mL of single cyt c solution (0.5 mg/mL). Then, the flasks were placed in a flask shaker at 120 rpm for 3 h before centrifugation. The cyt c concentration was determined with a UV-vis spectrophotometer at the wavelength of 408 nm. The amount of adsorbed cyt c was determined by calculating the concentration difference between the initial and residual solutions. The cyt c adsorption rate or adsorption capacity of the adsorbent was calculated as follows.

(1)

(2)

where E is the adsorption rate; Q is the adsorption capacity; C₀ and C_e are the cyt c concentrations before and after adsorption, respectively; m is the mass of the biomass; and V is the volume of the cyt c solution.

After adsorption, the solution was centrifuged, and the supernatant was discarded. The desorption of cyt c was performed by putting 10 mL eluent into each flask, and the flasks were then shaken at 120 rpm for 3 h. The concentration and the desorption rate of cyt c were determined.

2.5. Cytochrome c Purification from Pig Myocardium

The method for the preparation of the crude pig myocardium extract for the purification of cyt c can be found in Zhang [8].

2.6. Cytochrome c Activity Measurement

The cyt c activity was determined based on its protoheme activity toward hydrogen peroxide according to the method of Li [9]. One cyt c unit was defined as the amount of enzyme that caused an increase or decrease of 0.001 per minute in the absorbance at 426 nm using 20 mmol/L o-diaminobenzene and 20% H₂O₂ as the substrate.

3. RESULTS AND DISCUSSION

3.1. Adsorbent Characterisation

3.1.1. FT-IR Spectra

Infrared spectroscopy provided information on the chemical structure of the adsorbent materials. As shown in Figure 1, the 3360/cm band was attributed to the stretching vibrations of the hydroxyl group (-OH); the 2930/cm

band was attributed to the stretching vibrations of -CH; the 1380/cm band was attributed to the deformation vibration of -CH in cellulose and hemicellulose; the 1260/cm band was attributed to the vibration of C-O in lignose; and the 1060/cm band was attributed to the stretching vibrations of C-O in cellulose and hemicellulose [10-12]. Compared with that of CPSs, the FT-IR spectrum of MPSs contained a new peak at 1740/cm that was due to the stretching vibrations of carboxyl groups (C=O) in carboxylates [13]. The results indicate that succinic anhydride was grafted on to the shells, leading to an increase in the weight of the biomass.

3.1.2. X-Ray Photoelectron (XP) Spectra

The C:O:N ratios on the surfaces of the PSs, APSs, CPSs and MPSs were 72.31:24.94:2.75, 73.07:24.32:2.62, 70.91: 27.09:2.0 and 65.42:33.33:1.26, respectively. There was a significant 6.24% increase in the percentage of oxygen atoms and a 5.49% decrease in the percentage of carbon atoms in the MPSs compared with the CPSs. Moreover, we found that the area ratio of the O(1 s) spectra for the MPSs was higher than that for the unmodified shells. These results confirmed that the biomass was indeed modified by succinic anhydride through the modification reaction.

3.2. Adsorption Experiments

3.2.1. Effect of pH on Adsorption

Figure 2 illustrates the effect of pH on cyt c adsorption. This adsorption primarily depends on the physicochemical properties of cyt c and its degree of ionisation under specific pH conditions. When the pH was between 4.0 and the pI of cyt c (10.7), cyt c was positively charged, whereas the carboxyl groups on the surface of the adsorbent dissociated to form -COO^–, allowing cyt c to be