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.
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 [
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.
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.
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).
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.
where E is the adsorption rate; Q is the adsorption capacity; C0 and Ce 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.
The method for the preparation of the crude pig myocardium extract for the purification of cyt c can be found in Zhang [
The cyt c activity was determined based on its protoheme activity toward hydrogen peroxide according to the method of Li [
Infrared spectroscopy provided information on the chemical structure of the adsorbent materials. As shown in
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 [
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.