Biochemical and molecular identification
The FUM125 strain was isolated from a poultry field around Mashhad, and identified according to morphological, biochemical and molecular methods. The DNA extraction was conducted using CinnaGen kit (Cinnagen, Iran) according to the supplier's instructions. PCR was conducted using Amplicon kit (Amplicon Co, Denmark) and 27F and 1492R universal primers. The PCR products were electrophoresed on 1% agarose gel.
The PCR products were sequenced and data were analyzed using NCBI and Ez Taxon databases. Next, MEGA software (version 7), MUSCLE software and Neighbor-joining model were used for phylogenetic analysis and to generate phylogenetic tree.
The feather meal broth (FMB) medium was prepared as follows: NaCl (0.5 g/l), MgCl2.6H2O (0.1 g/l), K2HPO4 (0.4 g/l), KH2PO4 (0.3 g/l), powder feather (1 g/l), distilled water (1l) and adjusted to pH 7.5. The feather used in these media were washed and prepared according to the Mazotto method .
Assay of keratinolytic activity
The overnight culture of the FUM125 isolate (2% v/v), adjusted to 0.5 McFarland turbidity standard, was inoculated in the FMB medium and incubated at 37°C for 5 days with shaking (150 rpm). At 24 h intervals, 1 ml of medium was centrifuged at 10,000 rpm for 5 minutes, and the supernatant was used as the crude enzyme. Then, 100 µl of the crude enzyme was added to 900 µl of Tris-HCl buffer (50 mM, pH 8) containing 10 mg of Azokeratin substrate, incubated at 37°C for 1 h with shaking and finally, to stop the keratinolytic activity, the solution was placed in ice water for half an hour. Then, the sample was filtered to remove the feather residues, and its optical density (OD) versus the control sample was measured at a wavelength of 450 nm . The Azokeratin was synthesized according to Herzog protocol . The medium without bacterial inoculation was used as the control sample. One unit (U) of keratinolytic activity is defined as the amount of enzyme that causes 0.01 absorbance increase at 450 nm within 1 h under standard assay conditions . The experiments were conducted in two replicates and the average was reported as the results.
Optimization of keratinase production
Optimization of the culture conditions was conducted using one factor at a time method. The factors including temperature (20, 30, 37, 40, 45 and 50°C), pH (3 to 12.5 with a difference of 0.5 units), feather substrate concentration (0.5, 1, 2, 3, 4, 5, 6 and 7), shaking speed (125, 150, 175, 200 and 225 rpm), additional carbon source (glucose, fructose, starch, dextrose and sucrose 1%), additional nitrogen source (organic sources of peptone, tryptone, yeast extract and mineral sources of ammonium nitrate, ammonium chloride, ammonium sulfate and potassium nitrate), aeration (25, 50, 75 and 85%) and bacterial inoculation (1, 2, 4, 8 and 16%) were studied step by step. Data were statistically analyzed by SPSS software version 16 at 95% confidence level.
The feather and the bacterial residues were removed by filtration, and the supernatant was separated by centrifugation at 10,000 rpm for 10 min. The protein was precipitated by addition of 85% saturated ammonium sulfate and the precipitated protein was kept at 4°C for 24 h and collected by centrifugation at 10,000 rpm at 4ºC for 30 min. Next, the precipitate was dissolved in sodium phosphate buffer (25 mM, pH 6.5) and dialyzed at 4°C for 24 h changing buffer twice during the procedure. The dialyzed sample was loaded onto an equilibrated SP-Sepharose column using 25 mM sodium phosphate buffer (pH 6.5), and the column was eluted with NaCl at the concentrations of 0.1-1.0 M using 20 mM sodium phosphate buffer (pH 6.5). The fractions were collected and their OD values were measured at 280 nm for the presence of proteins. All fractions were assayed for the maximum keratinolytic activity and the molecular weight. Protein concentrations were calculated using the Bradford method .
SDS-PAGE polyacrylamide gel electrophoresis
The molecular weight of the pure enzyme was determined by SDS-PAGE using 15% gel according to the Laemmli method . The gel was stained by Coomassie Brilliant Blue G250 and silver nitrate dyes, and the molecular weight of the sample was estimated by a protein marker (Vivantis protein ladder; California USA).
Zymography was employed to confirm the keratinolytic activity and the purity of the enzyme using gelatin as a substrate with 0.2% concentration in 15% resolving gel, and then other steps such as SDS gel were performed . The clear zone was considered as protease activity.
Biochemical properties of the purified enzyme
Effect of temperature and pH on enzyme activity and stability
Four buffering systems used in this study included sodium acetate buffer (pH=4.5-5), sodium phosphate buffer (pH=6-8), Tris-HCl buffer (pH=8.5-9) and NaOH-glycine buffer (pH=9.5-11) at a concentration of 50 mM. Using Azokeratin as a substrate, the enzyme activity was measured in the pH range of 4.5-11 with 0.5-unit intervals at 37°C for 60 min. The effect of pH on enzyme stability was investigated by exposing the enzyme to the appropriate buffer at 4°C for 60 min and then, the residual activity for each sample relative to the initial enzyme activity was measured. The maximum activity of the enzyme in a buffer solution was considered as 100%, and other samples were assayed accordingly.
keratinolytic activity at the temperature range of 10-90°C was measured using optimal buffer. And the maximum activity of enzyme at a defined temperature was considered as 100% activity, and the enzyme activity at different temperatures was measured accordingly. The temperature effect on the enzyme stability was investigated by storing the enzyme in the mentioned temperature range for 60 min and then, the enzyme activity at the optimal temperature was measured using Azokeratin as a substrate. Finally, the residual activity of the sample was calculated relative to the initial enzyme activity at the optimal temperature.
Effect of metal ions and organic solvent on the enzyme activity
The effect of various metal ions including Ca2+, Fe2+, Ba2+, Co2+, Zn2+, Mg2+, Cu2+, Hg+, Na+, and K+ at the concentrations of 2, 5, and 10 mM, and the effect of organic solvents including methanol, ethanol, acetone, isopropanol, chloroform, hexane, and toluene using optimal buffer at the concentrations of 10, 20, and 40% were investigated on the enzyme activity incubating the pure enzyme in the presence of mentioned materials for 60 min at the optimal temperature and pH. The enzyme activity in the absence of metal ions or organic solvents was considered as 100%, and other results were compared accordingly.
Effect of detergents, inhibitors, and other chemical agents on the enzyme activity
The effects of inhibitors including phenylmethylsulfonyl fluoride (PMSF) as a serine protease inhibitor, beta-mercaptoethanol as a cysteine protease inhibitor, and 1,10-phenanthroline and Ethylenediaminetetraacetic acid (EDTA) as metalloprotease inhibitors at the concentrations of 2 and 5 mM using optimal buffer were investigated on the enzyme activity. The effect of chemical surfactants including Tween-80 and Triton X-100 (both neutral) at the concentrations of 1 and 5% (v/v), cationic and anionic detergents including cetyltrimethylammonium bromide (CTAB) and SDS at the concentrations of 0.5, 1 and 1.5% (w/v), and reducing and oxidizing agents including dithiothreitol (DTT) and hydrogen peroxide (H2O2) at the concentrations of 1 and 5% (v/v) using optimal buffer were tested on the enzyme activity. The enzyme activity in the absence of any chemical agents was considered as 100%.
The appropriate substrates for the keratinolytic activity were determined incubating pure enzyme in the presence of keratin, feather meal, gelatin, casein, bovine serum albumin, and Azokeratin at 1% (w/v) for one hour at the optimal temperature. The enzyme activity was measured in the presence of each substrate versus the control sample (having substrate but lacking enzyme). Maximum enzyme activity in the present of a defined substrate was considered as 100%, and the rest of the results were compared accordingly.
The half-life and kinetic parameters determination
Three different temperatures at which the enzyme showed the maximum activity were selected to determine the half-life of the enzyme. The purified enzyme using optimal buffer was incubated at the temperatures 50, 60 and 70 °C for 3 h and subsequently, 100 µl of the samples were taken every 30 min to measure the enzyme activity according to the standard method. Then, the residual activity of the enzyme was measured relative to the baseline (time zero), and a logarithmic diagram of the residual activity was drawn versus the time. In order to compute the values of Km and Vmax, the enzyme was exposed to different substrate concentrations (2, 4, 6, 8, 10, 12 and 14 mg/ml) at the optimal temperature and pH. The data were analyzed using GraphPad prism7 software, and the Km and Vmax values were calculated.