Microorganisms
The strains Bacillus cytotoxicus LT-1 and B. cytotoxicus Oll-15, previously isolated from samples collected from the hot springs of Los Tachos and Las Olletas in the Domuyo geothermal area, were used in this study. They are deposited on the Microbiological Culture Collection of CINDEFI-CONICET Institute, La Plata, Buenos Aires, Argentina, and maintained on nutrient agar slants or glycerol stock (20% w/v) at 4 and – 80 °C.
The strains were previously identified as B. cytotoxicus not only by their 16S rRNA gene sequences but also by the amplification of cytK-1 gene [10, 11].
Growth conditions and protease production
Submerged fermentations were performed using 250-ml Erlenmeyer flasks with 50 ml of minimal mineral media (MMM) supplemented with 10 g·l-1 of whole chicken feather. MMM was composed of 2.486 g·l-1 K2HPO4, 0.496 g·l-1 NaH2PO4, 0.01 g·l-1 MgCl2, 0.016 g·l-1 FeCl3, 0.1 mg·l-1 CaCl2, and 0.013 g·l-1 ZnCl2, with pH 6.0 [12]. Chicken feathers were obtained from a local slaughterhouse and were washed with 0.1% (w/v) sodium dodecyl sulfate (SDS) and with 1:1 (v/v) methanol and water with shaking for 18 h before use.
MMM supplemented with whole chicken feathers were inoculated with 1 ml of a 24-h nutrient broth culture (ODi 600 0.5) of each bacterial strain and incubated at 50 °C, 150 rpm for 2 days. Samples were withdrawn in sterile conditions periodically. Biomass and other residues were separated by centrifugation at 10,000 × g for 10 min at 4 °C, and supernatants were used as crude extracts for the subsequent analysis.
Production of feather protein hydrolysates (FPHs)
FPHs were obtained inoculating the bacterial strains on MMM supplemented with 10 g·l-1 of whole chicken feathers and incubating them at 50 °C, 150 rpm until feather degradation was completed (2 days). Then, cultures were centrifuged at 6000 × g for 15 min, filtered through a 0.54-μm cellulose acetate filter, and the supernatants were preserved at – 20 °C until the performance of antioxidant assays.
Enzyme activity
Proteolytic activity was determined using azocasein (Sigma Co., USA) as substrate following the protocol described by Cavello et al. [12]. Briefly, 100 μl of suitable dilution of each enzyme preparation were added to 250 μl of the substrate (10 g·l-1, Tris-HCl buffer 20 mM; pH 7.0). The admixture was incubated for 1 h at 50 °C, and after that, the reaction was stopped by the addition of 1 ml of trichloroacetic acid (TCA; 10%, w/v). After centrifugation (10,000 × g for 15 min, 4 °C), equal volumes of the supernatant and NaOH (1.0 M) were mixed. The absorbance at 440 nm was measured. Blanks were performed for each reaction. One unit (U) of protease activity was defined as the amount of enzyme that produced an increase in 0.01 absorbance units under the experimental assay conditions described [12].
Protein determination
The protein concentration of the samples were performed by the Bradford method [13], using bovine serum albumin (SIGMA) as standard.
Effect of pH on the enzyme activity
Optimum pH was determined by incubating each enzymatic extract with azocasein as substrate at different pH levels (6.0–12.0, MES-Tris-Glycine buffers 20 mM each). After 1 h of incubation at 50 °C, residual activity was performed, and percentage relative activity was calculated considering 100% of the activity was displayed with the optimum pH.
Effect of temperature on the activity and stability
Effect of temperature on enzyme activity was studied by incubating the reaction admixture (enzyme + substrate) at different temperatures (20–80 °C). Enzyme activity has been expressed as percentage relative activity.
Activation energies (Ea) of the enzymes were calculated by plotting the residual activity after the incubation at different temperatures according to the linearized Arrhenius equation (1):
$$ \ln K=\frac{\mathrm{Ea}}{R}\ \frac{1}{T}+\ln A, $$
(1)
where K is the enzymatic activity rate at the correspondent temperature (T, in Kelvins), Ea is the activation energy, R is the gas constant (8.314 J·K-1·mol-1), and A is a constant.
Thermal stability was determined by incubating the extracts at different temperatures between 30 and 50 °C. Aliquots were withdrawn at different times, and residual activities were measured considering 100% of activity was displayed by the non-treated enzyme.
Effect of protease inhibitors, metal ions, and organic solvents on protease activity
The nature of the proteases present on the enzymatic extracts was studied by evaluating the effect of different protease inhibitors: phenylmethylsulfonyl fluoride (PMSF, 1 mM), ethylenediaminetetraacetic acid (EDTA, 1 mM), 1,10-phenanthroline (1 mM), iodoacetamide (1 mM), and pepstatin A (5 μM). Extracts were incubated with each inhibitor separately for 1 h at 20 °C, and after that, standard assay of proteolytic activity was performed. The activity obtained by the enzyme without any inhibitor-incubation was taken as 100% of activity.
The effect of different metal ions (MgCl2, CoCl2, CaCl2, MnCl2, ZnCl2, NaCl, KCl, and HgCl2, 1 mM) on the enzymatic activity was evaluated incubating each extract for 60 min at 20 °C. After that, the residual activity was determined, and the activity of the enzyme without metal ion was considered as 100%.
Concerning to the effect of some organic solvents on enzyme activity, methanol, ethanol, and isopropanol (1% v/v) were incubated with extracts for an hour at 20 °C. After that, the residual enzymatic activity was determined and expressed as a percentage of the non-incubated enzyme.
Stability and compatibility with laundry detergents
Stability and compatibility of B. cytotoxicus extracts with solid (Drive, Ariel, Ace or Skip, 7 mg·l-1) and liquid (Ace, Ala, Ariel, or Skip, 1% v/v) laundry detergents were studied, preincubating each laundry detergent with each enzymatic extract for 1 h at room temperature. After incubation, the remaining activity was determined under standard assay conditions, and residual activity was calculated considering 100% of the activity was displayed by the enzyme incubated without the addition of any detergent. Before the assay, endogenous enzymes present on the commercial detergents were inactivated by a heat treatment of 65 °C for 1 h.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymogram
SDS-PAGE coupled to a zymogram was performed as described before by García-Carreño et al. [14], with some modifications. A 5% w/v stacking gel and 12% w/v separating gel were prepared as described by Laemmli [15]. To carry out the zymogram, after electrophoresis, SDS was removed from the gel flooding it with a solution of Triton X-100 (2.5% w/v Tris-HCl 20 mM pH 7.0) under agitation for 1 h. Then, the gel was washed three times with Tris-HCl 20 mM and incubated with casein 10.00 g·l-1 for 1 h at 50 °C. After incubation, the gel was stained with Coomassie Brilliant Blue G-250 following the Coomassie Colloidal method [16]. The presence of protease activity was evidenced by a clear zone in a blue background. For the SDS-PAGE, a broad range molecular weight marker was used (Thermo Fisher Scientific, #26612).
Antioxidant activity
Reducing power assay
To determine the ability of the feather protein hydrolysates to reduce Fe (III), 100 μl of sample were incubated with 250 μl of phosphate buffer (0.2 M, pH 6.6) and 250 μl of K3Fe(CN)63H2O 10.00 g·l-1 at 50 °C for 30 min. After that, 250 μl of 10% w/v TCA were added. The admixture was centrifuged at 16,000 × g for 10 min, and 250 μl of the supernatant was mixed with 250 μl of water and 50 μl of FeCl3 (0.1% w/v). After 10 min, absorbance at 700 nm was measured, and reducing power was identified in samples that produced an increase of the absorbance. Results were expressed as absorbance at 700 nm. As positive control, the reducing power of ascorbic acid was also determined (0–3.0 g·l-1). Blanks were performed using not inoculated media instead of FPH [17].
ABTS radical scavenging activity
ABTS (2,20-azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid) radical cation (ABTS*+) was produced by reacting ABTS stock solution (3.60 g·l-1) with potassium persulfate (0.66 g·l-1) allowing the mixture to stand in the dark for at least 12 h at 20 °C before use [18]. For the assay, a fresh solution of ABTS*+ was prepared by dilution with 5 mM phosphate-buffered saline (PBS, pH 7.4) to reach an absorbance of 0.7 ± 0.05 at 734 nm. Sample (10 μl) was mixed with ABTS*+ solution (1 ml), and the absorbance (734 nm) was taken after 4 min of reaction. A calibration curve with Trolox (6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) was performed (0–5.0 mM). Results were expressed as the milliequivalents (mEq) of Trolox displayed per gram of feather protein hydrolysate.
The radical scavenging activity of the sample was calculated as shown in equation 2:
$$ \mathrm{ABTS}\ \mathrm{radical}\ \mathrm{scavenging}\ \mathrm{activity}\ \left(\%\right)=\frac{A_B-{A}_S}{A_B}\times 100 $$
(2)
where AB is the absorbance at 734 nm of the blank, and AS is the absorbance at 734 nm of the sample reaction. Blank reactions were performed using non-inoculated media instead of FPH.
Half maximal effective concentration (EC50) values were calculated from the percentage radical scavenging against FPH concentration plots and represent the concentration of FPH resulting in 50% radical scavenging.
Nucleotide sequence accession number
Sequences of 16S rRNA gene of strains LT-1 and Oll-15 are deposited in the GenBank database under accession numbers KR559937 and KR559942, respectively [11].
Statistical analysis
Results were expressed as an average of three independent experiments with the correspondent standard deviation. P values less or equal to 0.05 were considered as statistically significant (LSD test of ANOVA).