Purification Technology and Antimicrobial Activity Analysis of Antimicrobial Peptide from Ovotransferrin

Transcription

1 CHEM. RES. CHINESE UNIVERSITIES 2011, 27(3), Purification Technology and Antimicrobial Activity Analysis of Antimicrobial Peptide from Ovotransferrin ZHANG Tie-hua, ZHENG Jian, YE Hai-qing, YU Ya-li, ZHAO Ping and LIU Jing-bo * College of Light Industry and Economics & Management,Jilin University, Changchun , P. R. China Abstract Antibacterial peptides mixture purified from Ovotransferrin by pepsin digest was used as the raw material. Peptide sections with good antibacterial activity were determined after bacteriostasis experiments, its molecular weight and amino acid composition were analyzed. The results of experiments indicate that with Sephadex G-50 and distilled water as mobile phase, detection wavelength 220 nm, flow rate 1.5 ml/min, sample density 0.2 g/ml, and volume 0.2 ml are the optimal conditions. Bacteriostasis experiments of the fraction of purified peaks were carried out and the result was: peak 1>peak 3>peak 2; the molecular weight of peak 1 was about 3015 by high performance liquid chromatography; active peptide possessed positive charges by amino acid analysis, its cationic characteristics are in accordance with the nature of antimicrobial peptides. Keywords Ovotransferrin; Purification; Molecular weight; Amino acid Article ID (2011) Introduction In recent years, antibiotics have played a significant role in controlling and treating diseases, and resisting pathogenic microorganisms [1,2]. As more and more use of antibiotics, the antibiotic resistance rises and the effect of them weakens. To solve this problem, alternative antimicrobials are being sought. The antimicrobial peptides are the new potential source of antibiotics [3,4]. In contrast to the immune system of animals, which produces antimicrobial polypeptides if need be, the avian egg albumin could efficiently resist the invasion of microorganisms. Avian egg albumin constitutes the second defense line against invading bacteria. Various studies have indicated that antimicrobial peptide from Ovotransferrin, is the key factor in the chemical defense line of the egg albumin [5,6]. Column chromatography technology on peptide separation and purification is a common method in industry. Hermine et al. [7] purified antimicrobial peptides derived from Lactobacillus acidophilus nv Er 317/402 strain Narine with Sephadex G-15; Mário et al. [8] purified antimicrobial peptides from Bacillus licheniformis wiht Sephadex G-100; Liu et al. [9] separated and purified antimicrobial peptides derived from the Hardware salivary glands of ticks with Sephadex G-50; Wang et al. [10] purified antimicrobial peptides with Sephadex G-25; Song et al. [11] purified antimicrobial peptides from the skin secretion of Fejervarya cancrivora with Sephadex G-50; Thomas et al. [12] purified the skin antimicrobial peptides of rana from northern United States with Sephadex G-25. Antimicrobial peptides as the main immune and antibacterial substances have become a research hotspot in recent years, and there have been few reports about the application of enzymolysis technology to separate and produce antimicrobial peptides from egg protein. Therefore, according to the above scholars who studied the separation and purification of peptides, we also used the Sephadex G-50 as the separation medium of peptides, optimized better purification parameters, collected purified products, studied the antibacterial activity of the active peptide which have been purified, and identified the molecular weight of the peptide, according to the amino acid composition analysis, determined its feature in agreement with cationic nature. 2 Materials and Methods 2.1 Materials Ovotransferrin was prepared from ammonium sulfate by ion exchange chromatography method [13]. Trifluoroacetic acid (chromatography pure), acetonitrile(chromatography pure), methanol(chromatography pure), cytochrome C, insulin and bacitracine were purchased from Sigma Chemicals Co.; Sephadex G-50 was from Pharmacia Co.; peptone, LP002Yeast extract, agar powder and beef extract were purchased from Beijing Aobo Star Biotechnology Co., Ltd.(China); E. coli *Corresponding author. ljb168@sohu.com Received December 1, 2010; accepted February 10, Supported by the Applied Basic Fund of Jilin Provincial Science and Technology Department(No ) and the Frontiers of Science of Jilin University and Innovation Fund of Interdisciplinary, China(No ).

2 362 CHEM. RES. CHINESE UNIVERSITIES Vol.27 ATCC and staphylococcus aureus ATCC were provided by College of Animal Science and Veterinary Medicine, Jilin University(China); 0.5 and 3 mol/l HCl standard solution, sodium chloride and other reagents were prepared by our lab. Membrane: Millipore company; Freeze dryer FD-1: Beijing Medical Health Technology Expo(China); SPX-250B-D oscillation incubator: Shanghai Motion Limited Medical Equipment Factory(China); TH2-92B desktop temperature shock box: Shanghai Yuejin Medical Instrument; 8037-SVP intelligent pressure steam sterilizer: Changchun(China), 100 Ocean Biological Instrument Co., Ltd.; LC-2010 liquid analyzer: Shimadzu(Japan); TSK-G2000-SWXL column: Japan TSK Company; CXG-1 constant temperature cabinet computer tomography: Shanghai Qingpu Huxi Instrument(China); Hitachi automatic amino acid L-8800 analyzer: Changchun Dacheng Industrial Group Co., Ltd. Ovotransferrin was digested with pepsin, then the hydrolyzate went through 5000(molecular weight) of cellulose acetate membrane, and filtered liquid separated and purified by Sephadex G-50. The specifications of column chromatography were 16 mm(i.d.) 80 cm. With sample concentration and sample volume as the study factors of the experiment, five levels were set each factor. 2.2 Sample Concentration The samples were prepared into 0.05, 0.10, 0.20, 0.25 and 0.30 g/ml(table 1), the sample volume was 0.2 ml, the detective wavelength was 220 nm, speed was 1.5 ml/min. 2.3 Sample Volume According to the experimental studies of sample concentration, the determined optimum concentration of the sample was 0.2 g/ml, and then the sample volumes 0.1, 0.2, 0.4, 0.5 and 1 ml were investigated(table 1). Table 1 Levels of factors * Sample concentration/(g ml 1 ) Sample volume/ml * The detective wavelength was 220 nm, speed was 1.5 ml/min. When studying sample concentration, the sample volume is 0.2 ml; after determining the sample concentration, the sample volume was examined. 2.4 Antibacterial Activity Tests Disk diffusion assay used 0.1 ml suspension of any tested bacterium containing about 10 7 cfu/ml spotted onto agar plates with medium using sterile swab. The 6 mm filter paper which contained the sample was placed on the surface of agar plate, the tests were performed in triplicate. Plates were incubated at 37 C for 24 h and then the inhibition zones were measured in diameter [14,15]. liquid sample was hydrolyzed with 6 mol/l hydrochloric acid for 22 h, then 3 ml of the sample was taken and followed by adding the machine buffer, which was determined on the machine [16]. The amino acid content of sample was calculated. 3 Results and Discussion 3.1 Search on Experimental Conditions with Water as Mobile Phase 2.5 Standard Curve, Sample Purity and Molecular Weight Assay According to the Light Industry Standard QB/T of People s Republic of China, the molecular weight range of peptides was determined by high performance gel filtration chromatography. We used 1421(molecular weight) of bacitracine, 5733(molecular weight) of bovine Insulin and (molecular weight) of cytochrome C to determine the molecular weight of standard preparations and antimicrobial peptide on a Shimadzu LC-2010 high performance liquid chromatography gel instrument. According to the relationship between peak time and molecular weight, we drew out the standard curve of standard molecular weight vs. peak time with the help of mathematical processing software. Liquid condition: Sample concentration 0.2 mg/ml; Mobile phase: acetonitrile-0.1% trifluoroacetic acid(volume ratio 10:90); temperature 25 C; the fluid rate 0.5 ml/min; the injected volume 10 μl; wavelength 220 nm. 2.6 Amino Acid Analysis of Active Peptide A Hitachi L-8800 automatic amino acid analyzer was used. Typically 1 ml(or weigh a certain amount of solid sample) of According to Section 2.3, the experimental results are shown in Fig.1. As can be seen in Fig.1, the degree of the sample separation gradually enlarges with the increasing of sample concentration. As the concentration was up to 0.1 g/ml, two peaks appeared. As the sample concentration was up to 0.2 g/ml, three peaks of sample appeared by Sephadex G-50; but as the concentration further increased, the separation weakened, and only two peaks were isolated due to the sample itself had a certain viscosity. When the concentration increased, the viscosity of the sample increased, resulting in Sephadex G-50 could not play well, so 0.2 g/ml was the best concentration of the sample. According to Section 2.4, the experimental results are shown in Fig.2. As can be seen in Fig.2, the function of Sephadex G-50 on the sample is seriously affected as the amount of sample volume increased. When the sample volume was up to 0.2 ml, chromatographic peaks were obvious; therefore, 0.2 ml was the optimal volume. From the above experimental results, we know that water as the mobile phase was the best choice for the separation and purification of samples. Other impurities could not be introduced, and the follow-up processing and application of samples would not bring up adverse effects, so water was chosen to be the mobile phase of sample separation.

3 No.3 ZHANG Tie-hua et al. 363 Fig.1 Chromatograms of samples with different concentrations Concentration of sample/(g ml 1 ): (A) 0.05; (B) 0.10; (C) 0.20; (D) 0.25; (E) Fig.2 Chromatograms of samples with different volumes Sample volume/ml: (A) 0.1; (B) 0.2; (C) 0.4; (D) 0.5; (E) Bacteriostasis Capability Analysis of Antimicrobial Peptide According to Section 2.5, the fractions of three peaks were purified by Sephadex G-50 on a freeze-drying machine; then the concentrations of the samples of the three peaks were 1 mg/ml separately. From Table 2 it can be seen that after the antibacterial activity tests(e. coli and S. aureus), the three peaks, which were purified by Sephadex G-50, shows the antibacterial activity in the order: peak 1>peak 3>peak 2. The inhibition zone diameter of peak 1 resisting E. coli is (21.62± 0.43) mm; the inhibition zone diameter of peak 1 resisting Staphylococcus aureus is (24.26±0.41) mm, then the antibacterial activity of S. aureus is higher than that of E. coli. We know that peak 1 had the antibacterial activity against S. aureus and E. coli, so we prepared different sample concentrations to further study the antibacterial activity of peak 1 against S. aureus. Sample concentrations: 0.5, 0.3, 0.2, 0.1, 0.08, 0.06 and Table 2 Bacteriostatic activity of purified samples * Diameter of inhibition zone/mm Test strain Peak 1 Peak 2 Peak 3 S. aureus ATCC ± ± ±0.52 E. coli ATCC ± ± ±0.51 * Each value is expressed as mean±sd of triplicate measurements.

4 364 CHEM. RES. CHINESE UNIVERSITIES Vol mg/ml. The results are shown in Table 3. So we know that the minimum inhibitory concentration is 0.06 mg/ml against S. aureus, and 0.1 mg/ml against E. coli. Table 3 Antibacterial activity of sample of peak 1 after purified * Sample concentration/ (mg ml 1 ) Diameter of inhibition zone/mm Against S. aureus Against E. coli ± ± ± ± ± ± ± ± ± ± * Each value is expressed as mean±sd of triplicate measurements. 3.3 Molecular Weight Analysis of Active Peptide From the gel chromatogram of HPLC analysis of bacitracin standard of 1421(molecular weight), bovine insulin standard of 5733(molecular weight), cytochrome C standard of 12384(molecular weight), the peak time was , and min, respectively, and they were washed off in turn according to different molecular weight. Based on the relationship between peak time and molecular weight of the three standards, the regression curve was obtained by mathematical analysis software. The standard curve regression equation: Y = X , the relative coefficient: R 2 =0.9989(Fig.3). retention time, the molecular weight was about 3015 and its purity was 99.2%. 3.4 Analysis of Amino Acids Composition of Active Peptide The amino acid contents of samples were obtained on a Hitachi L-8800 automatic amino acid analyzer with the results shown in Table 4 and Fig.5. Table 4 Amino acid content Amino acid Content/(mg ml 1 ) Amino acid Content/(mg ml 1 ) Asp 0.63 Met 0.59 Thr 0.56 Ile 0.67 Ser 0.61 Leu 0.79 Glu 0.30 Tyr 0.60 Pro Phe 0.23 Gly 0.66 Lys 1.00 Ala 0.78 His 0.28 Cys 1.20 Arg 0.60 Val 0.27 Fig.3 Standard curve of molecular weight vs. appearance time of standard preparation As can be seen in Fig.4, peak 1 with better antibacterial activity is purified by Sephadex G-50. Its molecular weight was determined by gel chromatography, the composition of the peptide was single; the peak time was min. According to the regression equation determined by molecular weight and Fig.4 HPLC chromatogram of sample after purification Fig.5 Chromatogram of amino acid Based on Table 4, the basic amino acids were lysine(lys), arginine(arg) and histidine(his), which accounts for 18.8%, according to Schibli [17] report, and the basic amino acid content of classical defensin HBD-1 is about 20%, which is higher than that of Ovotransferrin antimicrobial peptide. Yu et al. [18] investigated that peptide chain, finding that basic amino acid content of the peptide chain in frog skin antimicrobial peptide a was 14.7%, which is lower than that of Ovotransferrin antimicrobial peptide. Positively charged amino acid of Ovotransferrin antimicrobial peptide included Lys(10%), His(2.8%) and Arg(6.0%), while negatively charged amino acid included Asp(6.3%) and Glu(3.0%), so Ovotransferrin antimicrobial peptide possessed excessive positive charges, which is fit to cationic nature. This feature is consistent with antimicrobial peptide, and peptide chain of frog skin antimicrobial peptide a, and antimicrobial peptides purified by Hisham et al. [6]. In conclusion, the results obtained in the present study have shown that the Ovotransferrin antimicrobial peptide can effectively resist the bacteria including gram-positive and gram-negative bacteria. The mechanism of antimicrobial peptide is generally believed that the cationic characteristics of antimicrobial peptide and bacterial cell membrane cause the membrane perforation by electrostatic force when they combine with each other,

5 No.3 ZHANG Tie-hua et al. 365 which is involved in the formation of ion channels with consequent the leakage of cell contents, and the osmotic pressure inside and outside cell would change, leading to eventual cell death [19,20]. The inhibition mechanism study of lactoferrin peptide has proved this point. Lactoferrin peptide with a positive charge has a strong affinity for negatively charged phospholipids and lipopolysaccharide, hence it has an activity of affinity membrane, thus increasing the permeability of cell membranes [21]. This function is similar to the affinity membrane activity of polymyxin B with antibacterial activity produced by bacillus more sticky [22,23]. Therefore, the lethal effect of iron-containing peptides to microorganisms including Ovotransferrin antimicrobial peptide, mainly is the complete destruction of the physiological function of cell membrane because of the release of lipopolysaccharide. As the theory of antimicrobial peptide and the study of application deepen, broad-spectrum bactericidal of antimicrobial peptide without damage to the normal cells of body, as a new drug instead of antibiotic which has antibacterial and anti-infective activity, has potential development prospects. 4 Conclusions The best purified condition of Ovotransferrin antimicrobial peptide was mobile phase water, detective wavelength 220 nm, fluid rate 1.5 ml/min, sample concentration 0.2 g/ml, sample volume 0.2 ml, three types of compositions were obtained, and peak 1 had strong antibacterial activity, the minimum inhibitory concentration is 0.06 mg/ml against S. aureus, 0.1 mg/ml against E. coli. The molecular weight of antimicrobial peptide was about 3015, which was determined by high performance liquid chromatography, and the purity was 99.2%; amino acid composition analysis indicated that active peptide after purification possessed positive charges, and cationic nature, consistent with the nature of antimicrobial peptides. References [1] Alanis A. J., Arch. Med. Res, 2005, (36), 697 [2] Marr A. K., Gooderham W. J., Hancock R. E., Curr. Opin. Pharmacol., 2006, 6, 468 [3] Yeaman M. R., Yount N. Y., Nat. Rev. Microbial., 2007, (5), 727 [4] Fu F., Wu Y. M., Liu J. X., Protein Expression and Purification, 2010, 5(12), 1 [5] Hisham R. I., Md. Imranul H., Takayoshi A., International Journal of Biological Macromolecules, 2007, (41), 631 [6] Hisham R. I., Yasushi S., Takayoshi A., Biochemical et Biophysical Acta, 2000, (1523), 196 [7] Hermine M., Simon G., Sibylle H., Mire Z., Hamidreza K. L., International Journal of Antimicrobial Agents, 2010, 35(3), 255 [8] Mário L. T., Florencia C. O., Juliana S., Adriano B., Food Research International, 2009, (42), 63 [9] Liu Z. G., Liu H., Liu X. Y., Wu X. L., Comparative Biochemistry and Physiology, 2008, (149), 557 [10] Wang L., Lai C. E., Wu Q. F., Process Biochemistry, 2008, (43), 1124 [11] Song Y. Z., Lu Y., Wang L. J., Peptides, 2009, (30), 1228 [12] Thomas H., Yousef J. B., Floyd C. K., Conlon J. M., Peptides, 2000, 21, 469 [13] Zhang T. H., the 9th China Egg Science and Technology Congress, Changchun, 2010, 353 [14] Zhang D. S., Pang G. C., Journal of Food Science and Biotechnology, 2005, 24(2), 1 [15] Hu Z. H., Dong Y., Pang G. C., Food Science, 2005, 26(8), 99 [16] Zhao F. L., Research on the Controllable Enzyme Disassemble Conditions of High F Value Oligopeptides from Egg White, Jilin University, Changchun, 2007 [17] Schibli D. J., Hunter H. N., Aseyev V., Biol. Chem., 2002, 277(10), 8279 [18] Yu P. C., Li Y. Y., Tu Q., Jian S. Q., Journal of Nanchang University(Natural Science), 2008, 32(1), 75 [19] Park Y., Lee D. G.Janq S. H., Woo E. R., Jeonq H. G., Choi C. H., Hahm K. S., Biochimica et Biophysica Acta, 2003, 1645, 172 [20] Huey W. H., Biochimica et Biophysica Acta, 2006, 1758, 1292 [21] Shimazaki K. I., Isuda H., Tomita M., Experta Medica International Congress Series, Amsterdam, Elsevier., 1998, 1195 [22] Richard M. E., Hans J. V., Biochimica et Biophysica Acta, 1999, 1462, 11 [23] Kang P., Yin Y. L., Huang R. L., China Feed., 2006, 15, 29