Antibacterial peptide production: study on the effect of bioprocess variables by ‘one-factor at-a-time method’
Various bioprocess parameters affecting maximum peptide production under submerged fermentation were studied by one-factor at-a-time method using CD medium. The parameters included temperature, inorganic nitrogen source, organic nitrogen sources, carbon source, initial pH, sodium chloride, incubation period, inoculum concentration, agitation, metal ions and additional inorganic nitrogen source. The effect of each parameter for maximal peptide production was evaluated and incorporated the same variable at its optimized level in the subsequent experiment to optimize next parameter. In each case, samples were analyzed for antibacterial activity and protein concentration using standard protocol unless otherwise mentioned (Enan et al. 1996; Bradford 1976). All the experiments were carried out in triplicates.
Time course experiment was conducted with the Czapek- Dox medium and optimized medium after one- factor- at - a - time method to ascertain the rate of peptide production. The optimized medium with selected conditions included 30oC, sodium nitrate (0.3%) as inorganic nitrogen source, sucrose (3%) as carbon source, initial pH 7, sodium chloride (0.5%), inoculum concentration (0.4%), Ca2+ as metal ion (2mM), ammonium phosphate (1%) as additional inorganic nitrogen source incubated at 150 rpm for 168 h. Samples were taken at a regular interval of 24 h for antibacterial activity analysis.
Physico-chemical parameters on the stability and activity of peptide
The stability of the MAFP9 has been studied using various physico-chemical parameters. Effect of various non-ionic and ionic detergents such as triton X-100, tween 80, tween 20, SDS and CTAB (0.1% each w/v) on peptide activity was determined by incubating the purified peptide in each detergent for 1 h. Effect of various metal ions was evaluated by incubation along with 1 mM concentrations of various metals ions such as Na+, Ca2+, Mg2+, Fe3+, Mn2+, Ni2+, , Ba2+, Cd2+, Zn2+, Cu2+, Co2+, Cr2+ and Al3+ respectively for 1 h. The effect of reducing agents on the activity was studied by incubating the MAFP9 for 1 h with β-mercaptoethanol at a concentration of 20, 40, 60, 80 & 100 mM and dithiothreitol at a concentration of 2, 4, 6, 8 & 10 mM. Impact of oxidizing agents on the activity of peptide was studied by incubating the purified peptide with dimethyl sulfoxide (1-5%, v/v) and sodium hypochlorite at a concentration of 20, 40, 60, 80 and 100 mM for 1 h. Effect of amino acid modifiers on the activity of peptide was studied by incubating the purified peptide with diethylpyrocarbonate (0.1 M Tris HCl, pH- 7) at 30°C for 30 min, PMSF (0.05 M Tris HCl, pH- 7.8 ) at 25°C for 120 min, iodoacetamide (0.1 M Tris HCl, pH- 8) at 35 °C for 60 min at a concentration of 5, 10, 15, 20 and 25 mM and N- Bromosuccinamide (0.1 M Tris HCl, pH- 7) at concentrations of 1, 2, 3, 4 and 5 mM at 30 °C for 30 min followed by testing the antibacterial activity. Each parameter studied followed a dialysis against 0.01 M phosphate buffer (pH 7.5). The samples were then lyophilized and residual activity was estimated.
In vitro antiproliferative studies of peptide on cultured cell lines
Cell culture maintenance
L929 fibroblast (Normal cell line) and A549 (lung carcinoma cell line) were the cell lines used for this study and were purchased from National Centre for Cell Sciences (NCCS), Pune, India. Dulbecco’s modified Eagles media (HiMedia) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, USA) was used for cell line maintenance and grown to confluency at 37°C in 5 % CO2 (NBS, Eppendorf, Germany) in a humidified atmosphere in a CO2 incubator.
Procedure
The cells were trypsinized (500 µL of 0.025% Trypsin in PBS/ 0.5 mM EDTA solution (HiMedia)) for 2 min and passaged to T flasks in complete aseptic conditions. Peptide sample was added to grown cells at a final concentration of 6.25 µg/ml, 12.5 µg/mL, 25 µg/mL, 50 µg/mL and 100 µg/mL from a stock of 0.1 mg/mL and incubated for 24 h. The percentage difference in cell viability was determined by standard MTT assay after 24 h of incubation (Arung et al. 2006). The morphological features of cells were imaged using inverted phase contrast microscope (Olympus CKX41, Japan) with Optika Pro5 CCD camera.
Determination of apoptosis and necrosis by acridine orange (AO) and ethidium bromide (EB) double staining method
DNA-binding dyes AO and EB (Sigma, USA) were used for the morphological detection of apoptotic and necrotic cells (Attari et al. 2009). AO is taken up by both viable and non-viable cells and emits green fluorescence if intercalated into double stranded nucleic acid (DNA). Ethidium bromide is taken up only by non-viable cells and discharges red fluorescence by intercalation into DNA. A549 cells were washed by cold PBS and then stained with a mixture of AO (100 μg/mL) and EB (100 μg/mL) at room temperature for 10 min. The stained cells were washed twice with 1X PBS and analyzed using a fluorescence microscope in blue filter (Olympus CKX41 with Optika Pro5 camera). The cells were segregated into living cells (normal green nucleus), late apoptotic (orange-stained nuclei with chromatin condensation or fragmentation), early apoptotic (bright green nucleus with condensed or fragmented chromatin), and necrotic cells (uniformly orange-stained cell nuclei).
Antioxidant activity
DPPH radical scavenging by purified MAFP9 was estimated using the method of Liyana-Pathirana and Shahidi, 2005). A solution of 0.135 mM DPPH in methanol was prepared and 1.0 mL of this solution was mixed with 100 μL of peptide at different concentrations (0.8, 1.6, 2.4, 3.2 and 4 μg). The standard antioxidant ascorbic acid in the same concentrations as that of peptide was used as positive controls. The reaction mixture was vortexed and kept in the dark at room temperature for 30 min. The optical density of each sample was measured at 517 nm. The ability to scavenge DPPH radical was calculated by the following equation:
Free radical scavenging activity (%) = A Control – A Sample x 100
AControl
where Acontrol is the absorbance of the free radical solution (DPPH) + methanol, and Asample is the absorbance of the free radical solution with peptide/standard antioxidant.
Determination of in vitro anti-inflammatory effect of MAFP9 on cultured THP1 cell lines
The anti-inflammatory effect of MAFP9 was determined by assessing the inhibition of cycloxygenase enzyme (COX-2) when THP1 (Human monocytic cell lines) THP1 treated with LPS (Raheema et al. 2014). Cell line was cultured in RPMI 1640 [HiMedia] media, supplemented with 10% heat inactivated FBS, antibiotics (Penicillin and Streptomycin) and 1.5% sodium bicarbonate. The media was filtered by 0.2 µm pore sized cellulose acetate filter (Sartorius) in totally aseptic conditions. The cells were then grown till 60% confluency followed by activation with 1 µl LPS (1 µg/mL). LPS stimulated THP 1 cells were exposed with different concentrations of peptide samples (6.25 µg/ml, 12.5 µg/mL 25 µg/mL, 50 µg/mL and 100 µg/mL) from a stock of 1mg/ml dissolved in 1% DMSO and incubated for 24 hours.
The crude enzyme isolation was done by spinning at 6000 rpm for 10 minutes. Supernatant was discarded and 200 µl of cell lysis buffer (1MTris HCl, 0.25M EDTA, 2M NaCl, 0.5% Triton) was added .The incubation was done for 30 minutes at 4°C and enzyme assay was done in pellet suspended in a small amount of supernatant.
Assay of cycloxygenase (COX-2) activity
The assay mixture contained 100 mM Tris- HCl buffer (pH. 8), 5 mM glutathione, 5 μM hemoglobin and crude enzyme supernatant. The reaction was started by the addition of 200 μM arachidonic acid and incubated at 37ºc for 20 min. Reaction was terminated by adding 0.2 mL of 10% TCA in 1N HCl, mixed and 0.2 mL of 1 % thiobarbituric acid was added. Reaction mixture kept in a boiling water bath for 20 min, cooled and centrifuged at 1000 rpm for 3 min. The absorbance was measured at 632 nm for COX activity.