New 
Proteases hydrolyze proteins via peptide bonds and are classified into serine, cysteine, aspartate, and metalloprotease families . They are found in all living organisms, including microbes, animals, and plants. Bacillus has a high capacity for extracellular protease secretion that is active at neutral and alkaline pH; therefore, it has considerable potential as a microbial protease source , . Bacillus strains capable of producing proteases can grow on inexpensive nutrient media, which is important because media components cost up to 40% of the cost of the enzyme itself . Bacillary proteases are tolerant to high temperatures, organic solvents, and detergents , which is one reason for their use in detergents. Furthermore, these proteases are used in the food industry , cheese making , baking , pharmaceutical industry, stereoisomeric compound synthesis , cosmetics, leather industry , wastewater treatment, and feed industry . Most proteolytic enzymes secreted by Bacillus have broad substrate specificity and can hydrolyze phosphoproteins (such as caseins) and albumins (including bovine serum albumin (BSA) and ovalbumin). Of particular interest are proteases capable of hydrolyzing fibrillar proteins (such as collagen, myosin, and keratin), which are present in large quantities in animal husbandry and meat processing industries. Feathers are composed of more than 90% proteins, including keratin, azelon, and other insoluble proteins . Keratin is a structural protein densely packed into a β-sheet polypeptide chain intensely cross-linked by disulfide bonds and stabilized by non-covalent interactions . According to their secondary structure, proteins of the keratin family are divided into two groups: α-keratins, which have a conformation in the form of tight coils around the long axis of the molecule, known as α-helix (the basis of mammalian hair, wool, horns, claws, and hooves), and β-zigzag polypeptide chains known as β-sheets (claw bases, lizard scales, turtle shells, feathers, beaks, and claws of birds) . The abundance of disulfide and hydrogen bonds, as well as hydrophobic interactions, provides a keratin structure that is resistant to protease action . Keratin is difficult to cleave using known proteases such as pepsin, trypsin, and papain , and specific proteases with keratinolytic activity are required for its hydrolysis. Keratinases are proteases that can cleave keratin and belong to a family of serine proteases or metalloproteases . In practice, keratinases have an advantage over proteases because they can render feathers an inexpensive feed, amino acid, and peptide source . Enzymatic hydrolysis optimally retains the nutritional content of the products and greatly enhances their solubility and digestion. Recently, many studies have focused on keratinases isolated from Bacillus – . Nondirected mutagenesis is used to create mutant strains with enhanced keratinase activity . The high biotechnological potential of bacterial keratinases has attracted considerable attention owing to their numerous industrial applications in the development of environmentally friendly processes . Here, we describe the isolation and identification of Bacillus paralicheniformis T7, a strain with high proteolytic and keratinolytic activities isolated from soil. When cultured on feather medium, the proteolytic and keratinolytic features of B. paralicheniformis T7 were investigated qualitatively and quantitatively using biochemistry, zymography, mass spectrometry, proteomics, and spectrophotometry. Chicken feather, horn, hoof, and cattle wool hydrolysis was conducted using this strain, and the hydrolysates were studied using scanning electron microscopy. The correlation between the genome, proteome, and biochemical activities of the strain was investigated. Moreover, the possibility of obtaining an enzyme preparation through submerged fermentation with B. paralicheniformis strain T7 in a bioreactor was shown. 

Reagents and media Ovalbumin was purchased from MP Biomedicals (Santa Ana, CA, USA). BSA, hemoglobin, and casein sodium salt were obtained from Sigma-Aldrich (St. Louis, MO, USA). Phenylmethylsulfonyl fluoride (PMSF), pepstatin A, ethylenediaminetetraacetic acid (EDTA), E64, and other chemical reagents were purchased from Sigma-Aldrich and AppliChem (Darmstadt, Germany). Gelatin and its constituents were purchased from Titan Biotech Ltd. (Delhi, India). Feathers were collected at the poultry farm of “CAPITAL PROJECTS LTD” LLP, located in the village of Akmol, Tselinograd district, Akmola region, Republic of Kazakhstan. The following liquid nutrient media were used for cultivation: nutrient broth [0.5% (w/v) peptone, 0.5% (w/v) NaCl, 0.15% (w/v) yeast extract, and 0.15% (w/v) beef extract, pH 7.4] and feather broth [(g/L) NaH 2 PO 4 (0.3), Na 2 HPO 4 (0.35), and feather powder (7.5), pH 7.0]. The agar media used for isolating and screening the strain included nutrient agar [nutrient broth with 1.5% (w/v) agar], skim milk agar [2% (w/v) skim milk, 0. 1% (w/v) NaCl, 1% (w/v) tryptone, 1% (w/v) agar], gelatin agar [0.4% (w/v) peptone, 0.1% (w/v) yeast extract, 1.5% (w/v) gelatin, 1.5% (w/v) agar], and feather agar [(g/L) NaH 2 PO 4 (0.3), Na 2 HPO 4 (0.35), feather powder (7.5), and agar (15), pH 7.0]. Isolation and identification of the microorganism For isolation, 1 g of soil was suspended in 9 mL of 0.9% (m/vol.) NaCl and the suspension was diluted 10-fold. The third 10-fold dilution was seeded onto the agarized medium. Next, 100 µL was seeded on a nutrient agar dish and incubated at 37 °C for 48 h. The proteolytic and keratinolytic activities of the strains were tested by culturing on dishes containing milk, gelatin, and feather agar. Transparent zones around the colonies confirmed the enzymatic activity of the strain. Colony purity was checked using gram staining and light microscopy (Primo star, Carl Zeiss, Goettingen, Germany). Strains were identified by proteomic profiling of ribosomal proteins and sequencing of a conserved DNA locus, as previously described , . Proteomic analysis of the strain was performed by time-of-flight mass spectrometry with matrix-assisted laser desorption and ionization (MALDI-TOF) on a Biotyper Microflex LT instrument (Bruker Daltonics, Bremen, Germany). For sequencing, the genomic DNA of the strain was isolated using a Genomic Wizard Purification Kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol. The 16 S rRNA gene fragment was amplified by PCR using universal primers 27 F and 1492R. The amplified DNA fragment was sequenced by the Sanger method using BigDye™ Terminator v3.1 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. DNA fragments were separated using an ABI 3730xl automated sequencer (Applied Biosystems, Foster City, CA, USA). Chromatograms were analyzed and compared with the reference sequence using Vector NTI software version 11 (Thermo Fisher Scientific) and the NCBI database ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ). Preparation of extracellular enzymatic extract The enzymatic extract used to evaluate activity was obtained by first culturing the cells of the strain in 5 mL of Luria broth for 16 h at 37 °C and shaking at 170 rpm in a thermostated shaker. Next, the culture was used to inoculate 150 mL of feather broth, and the cells were cultured for 48 h under the same conditions. Subsequently, the supernatant was clarified by centrifugation at 10,000 × g for 10 min at 4 °C and filtered through a 0.22-µm pore-sized membrane to remove microparticles and bacterial cells. The enzymatic extract was stored on ice under aseptic conditions without freezing. Preparation of azo-labeled substrates Casein was prepared from skimmed cow milk by acid precipitation, followed by drying and grinding into a fine powder. Keratin was prepared from ground chicken feathers according to a previously described protocol . Azocasein and azokeratin were prepared as previously described . Determination of the enzymatic activity of the extract Proteolytic activity was determined according to Kembhavi et al. using azocasein as a substrate. Keratinase activity was determined as previously described , , using azokeratin as the substrate. Effect of temperature and pH on enzymatic activity The effect of temperature on enzymatic activity was determined by conducting the reaction in a temperature range of 30–80 °C (with an interval of 10 °C). Maximum enzymatic activity was defined as 100%. To determine thermostability, the enzymatic extract was pre-incubated for 2 h at 50, 60, and 70 °C in 0.1 M Tris buffer (pH 8.5), and then the residual activity was measured at 60 °C in 0.1 M Tris-HCl buffer (pH 8.5). The results are expressed as a percentage relative to the activity of the extract without temperature incubation, which was taken as 100%. The enzymatic activity was measured over a pH range of 3.0 to 11.0. The following buffer systems were used: citrate buffer (pH 3.0–6.0), sodium phosphate buffer (pH 6.0–7.5), Tris-HCl (pH 7.5–9.0), and glycine-NaOH (pH 9.0–11.0). Values were converted to relative units with a maximum value of 100%. Effect of metal ions and protease-inhibiting chemicals on enzymatic activity The effects of metal ions (Na + , K + , Ca 2+ , Ni 2+ , Mg 2+ , Mn 2+ , Cu 2+ , Co 2+ , Cd 2+ , Ba 2+ , and Fe 3+ ) were determined by using salts: NaCl, KCl, CaCl 2 , NiCl 2 , MgCl 2 , MnCl 2 , CuCl 2 , CoCl 2 , CdCl 2 , BaCl 2 , FeCl 3 . Reducing agents [β-mercaptoethanol and dithiothreitol (DTT)], protease inhibitors [phenylmethylsulfonyl fluoride (PMSF), ethylenediaminetetraacetic acid (EDTA), pepstatin A, E64], and detergents [1% Tween 20, Triton X-100, sodium dodecyl sulfate (SDS)] on enzymatic activity were determined by adding the reagents to the reaction mixture. Thereafter, enzymatic activity was evaluated at 60 °С in 0.1 M Tris buffer (pH 8.5). Activity values were considered 100% in the absence of metal ions or chemicals. Zymography The ability of the enzymatic extract to degrade various protein substrates was determined using SDS-polyacrylamide gel electrophoresis (PAGE) in a 4–10% gel copolymerized with (w/v) sodium caseinate (0.1%), BSA (0.1%), gelatin (1%), and keratin (0.7%). The enzymatic extract was added to SDS-PAGE sample buffer (125 mM Tris-HCl pH 6.8, 0.002% bromophenol blue, 4% SDS, and 20% glycerol) at a ratio (sample: buffer) of 4:6. PMSF (5 mM), EDTA (5 mM), pepstatin A (0.035 mM), and E64 (0.01 mM) were added as serine, metalloprotease, asparagine, and cysteine protease inhibitors, respectively. The samples were not boiled, and no β-mercaptoethanol or DTT was added before electrophoresis. Thereafter, the gels were washed twice for 10 min each in 2.5% Triton X-100 and incubated in 500 mM Tris-HCl (pH 8.5) at 50 °С for 20 h. Next, the gels were stained with 0.08% Coomassie Brilliant Blue G-250 (AppliChem, Darmstadt, Germany) in 20% ethanol with 1.6% orthophosphoric acid and 8% ammonium sulfate for 4 h and then clarified in MilliQ water for 3–4 h until the hydrolysis zones were completely clarified. A protein marker (Color Prestained Protein Standard, cat#P7719S; New England Biolabs, Ipswich, MA, USA) was used to determine the molecular weight. Hydrolysis of protein substrates Hemoglobin, gelatin, keratin, casein, ovalbumin, and BSA were tested as substrates for hydrolysis using the enzymatic extract. Each substrate was dissolved in 1 mL 50 mM Tris-HCl (pH 8.5) to a concentration of 1 mg/mL and treated with enzymatic extract at 60 °C for 30 min with periodic sampling for analysis. A substrate without enzymatic extract treatment was used as a control. The hydrolysis products were analyzed using 12% SDS-PAGE. The gels were stained with 0.08% Coomassie Brilliant Blue R-250 (AppliChem) in 50% ethanol and 10% acetic acid. A protein marker (Color Prestained Protein Standard, cat#P7719S; New England Biolabs) was used for molecular weight determination. Hydrolysis of keratinous raw materials The ability of the B. paralicheniformis strain T7 to degrade keratinous animal waste (chicken feathers, horns, hooves, hides, and wool) was investigated. B. paralicheniformis strain T7 was cultured in 15 mL of nutrient broth in a shaker incubator at 37 °C and 170 rpm for 18 h. Thereafter, 1 mL inoculum was added to glass tubes containing horn (0.777 g), hoof (1.405 g), hide (1.055 g), wool (0.254 g), and feather (0.25 g) fragments in 10 mL of sodium phosphate buffer (pH 7.0). Next, the samples were degraded in a shaker incubator at 37 °C and 250 rpm for 7 days. The degree of degradation of the samples was determined by measuring the dry weights of unhydrolyzed samples. For this purpose, the culture was passed through a pre-weighed filter paper, and the remaining residue was washed twice with distilled water and dried at 60 °C to a constant weight. The results are expressed as a percentage of the initial weight of the samples, which was taken as 100%. Scanning electron microscopy of feather hydrolysis products Feather samples were inoculated with 1 mL of sterile enzymatic extract in 10 mL of 0.1 M Tris-HCl buffer (pH 8.5) supplemented with 0.02% sodium azide. Incubation was conducted for 2–7 d at 37 °C. To evaluate the effect of B. paralicheniformis T7 on the degradation process, hydrolysis was performed using an unfiltered enzymatic extract without sodium azide under the same conditions. Samples prepared under the same conditions but without enzymatic extracts were used as controls. After incubation, the samples were analyzed using scanning electron microscopy (SEM) to evaluate the degree of degradation. The samples were placed on stubs and sputtered with gold (10 nm). Images were captured using an Auriga CrossBeam 540 Scanning Electron Microscope (Carl Zeiss, Jena, Germany) at 3 kV. Whole-genome sequencing and mass spectrometric identification of extracellular proteases The genome of B. paralicheniformis T7 was sequenced using the Oxford Nanopore method on a MinION sequencer (Oxford Nanopore Technologies Ltd., Oxford, UK). Genomic DNA libraries were constructed using an Oxford Nanopore Technologies Sequencing Kit (SQK-LSK109). This involved DNA fragmentation, followed by adapter ligation (Ligation Sequencing Kit, SQK-LSK109; Oxford Nanopore Technologies Ltd). The resulting libraries were quantified using Qubit 2.0 (Invitrogen, Carlsbad, CA, USA) and subjected to sequencing on a MinION platform with an FLO-MIN106D flow cell (R9; Oxford Nanopore Technologies Ltd.). The annotated genome was deposited in the NCBI GenBank. A library of amino acid sequences for all strains was generated based on the annotated genome of B. paralicheniformis T7. The enzymatic extract was concentrated 150-fold on a Pierce™ Protein Concentrator (10 K MWCO: 10 kDa molecular weight cutoff; Thermo Fisher Scientific) for mass spectrometric analysis of the secretory proteome. Protein separation was performed using 12% SDS-PAGE. Proteins were extracted from the gel and treated with trypsin (Promega) to form peptides. Subsequently, the peptides were separated on an Acclaim Pep-Map RSLC column (Thermo Fisher Scientific) using an acetonitrile gradient. An unmodified CaptiveSpray ion source was used to interface the HPLC system with a Maxis Impact II Instrument (Bruker Daltonics). The mass range of the MS scan was set to m/z 150–2,200 in positive ion polarity mode. B. paralicheniformis T7 extracellular proteases were identified on the Mascot platform using an amino acid sequence library of the strain. Fermentation of B. Paralicheniformis T7 in a bioreactor and enzyme preparation A 10-L Biostat Bioreactor (Sartorius, Göttingen, Germany) was used to test the ability of B. paralicheniformis T7 to produce proteolytic enzymes during submerged fermentation. Submerged fermentation was performed according to the . A single colony was inoculated into 5 mL of nutrient broth and cultured at 37 °C in a shaker incubator at 180 rpm for 18 h. Afterward, the culture was transferred to 200 mL of feather medium supplemented with 0.2% yeast extract and grown for 24 h under the same conditions. In a 10-L fermenter, the culture was inoculated into 6 L of sterile feather medium supplemented with 0.2% yeast extract. The conditions for submerged fermentation were as follows: temperature, 37 °C; agitation, 450 rpm; aeration, 6–10 L/min; and culturing time, 48 h. Samples were taken periodically to determine the number of colony-forming units and to measure protease activity. The culture was cleared of cells and substrate residues by centrifugation at 11,000 × g and sterilized by microfiltration on a UPIRO-018 bench filtration apparatus (Vladisart, Vladimir, Russia) using an MKM46020 membrane polyethersulfone module (Vladisart) with a cutoff threshold of 0.22 μm. Next, 150 mL of sterile culture was concentrated by evaporation for 1 h on an RV05 basic rotary evaporator (IKA-Werke, Staufen, Germany) at 55 °C and 50 rpm under vacuum (4.9 kPa) until a volume of 3 mL was reached. Subsequently, 450 mL of sterile culture was frozen at -80 °С in a U570 Ultra-low freezer (New Brunswick Scientific, Enfield, CT, USA) and dried in a BETA 2–8 LDplus (Christ, Osterode, Germany) at -90 °С under vacuum (0.028 mBar) for 48 h in accordance with . The lyophilized enzyme was ground into a powdered state, and the remaining sterile culture was spray-dried on an OM-1500 A spray dryer (Shanghai Pilotech Instrument & Equipment Co., Ltd., Shanghai, China) under the following conditions: chamber inlet temperature, 55 °C; chamber outlet temperature, 40 °C; spray pulse duration, 1 s; extract delivery rate, 4.5 mL/min. Software tools, bioinformatics, and statistical analysis The chromatograms were analyzed after capillary sequencing using the Vector NTI Advance 11 program (Thermo Fisher Scientific). After nanopore sequencing, the sequences were annotated using Prokka Rapid Prokaryotic Genome Annotation Software (Prokka) version 1.13.14 ( https://github.com/tseemann/prokka ) and DNA Features Viewer software version 3.1.3 ( https://github.com/Edinburgh-Genome-Foundry/DnaFeaturesViewer ) to rapidly annotate the prokaryote genome. The Mascot platform was used for mass spectrometric proteins. The online resource Peptide Signal IP 5.0 ( http://www.cbs.dtu.dk/services/SignalP/ ) was used for signal peptide prediction. All activity experiments were performed in triplicate. Enzymatic activity measurement data were obtained through independent activity assays, with mean values, standard deviations (SD), and p-values calculated using GraphPad Prism version 8.0.1 (GraphPad Software, La Jolla, CA, USA, www.graphpad.com ). All data are presented as the mean ± SD ( n = 3). Statistical significance between two groups was determined using the Student’s t-test, with a significance threshold of P < 0.05. 

Ovalbumin was purchased from MP Biomedicals (Santa Ana, CA, USA). BSA, hemoglobin, and casein sodium salt were obtained from Sigma-Aldrich (St. Louis, MO, USA). Phenylmethylsulfonyl fluoride (PMSF), pepstatin A, ethylenediaminetetraacetic acid (EDTA), E64, and other chemical reagents were purchased from Sigma-Aldrich and AppliChem (Darmstadt, Germany). Gelatin and its constituents were purchased from Titan Biotech Ltd. (Delhi, India). Feathers were collected at the poultry farm of “CAPITAL PROJECTS LTD” LLP, located in the village of Akmol, Tselinograd district, Akmola region, Republic of Kazakhstan. The following liquid nutrient media were used for cultivation: nutrient broth [0.5% (w/v) peptone, 0.5% (w/v) NaCl, 0.15% (w/v) yeast extract, and 0.15% (w/v) beef extract, pH 7.4] and feather broth [(g/L) NaH 2 PO 4 (0.3), Na 2 HPO 4 (0.35), and feather powder (7.5), pH 7.0]. The agar media used for isolating and screening the strain included nutrient agar [nutrient broth with 1.5% (w/v) agar], skim milk agar [2% (w/v) skim milk, 0. 1% (w/v) NaCl, 1% (w/v) tryptone, 1% (w/v) agar], gelatin agar [0.4% (w/v) peptone, 0.1% (w/v) yeast extract, 1.5% (w/v) gelatin, 1.5% (w/v) agar], and feather agar [(g/L) NaH 2 PO 4 (0.3), Na 2 HPO 4 (0.35), feather powder (7.5), and agar (15), pH 7.0]. 

For isolation, 1 g of soil was suspended in 9 mL of 0.9% (m/vol.) NaCl and the suspension was diluted 10-fold. The third 10-fold dilution was seeded onto the agarized medium. Next, 100 µL was seeded on a nutrient agar dish and incubated at 37 °C for 48 h. The proteolytic and keratinolytic activities of the strains were tested by culturing on dishes containing milk, gelatin, and feather agar. Transparent zones around the colonies confirmed the enzymatic activity of the strain. Colony purity was checked using gram staining and light microscopy (Primo star, Carl Zeiss, Goettingen, Germany). Strains were identified by proteomic profiling of ribosomal proteins and sequencing of a conserved DNA locus, as previously described , . Proteomic analysis of the strain was performed by time-of-flight mass spectrometry with matrix-assisted laser desorption and ionization (MALDI-TOF) on a Biotyper Microflex LT instrument (Bruker Daltonics, Bremen, Germany). For sequencing, the genomic DNA of the strain was isolated using a Genomic Wizard Purification Kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol. The 16 S rRNA gene fragment was amplified by PCR using universal primers 27 F and 1492R. The amplified DNA fragment was sequenced by the Sanger method using BigDye™ Terminator v3.1 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. DNA fragments were separated using an ABI 3730xl automated sequencer (Applied Biosystems, Foster City, CA, USA). Chromatograms were analyzed and compared with the reference sequence using Vector NTI software version 11 (Thermo Fisher Scientific) and the NCBI database ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ). 

The enzymatic extract used to evaluate activity was obtained by first culturing the cells of the strain in 5 mL of Luria broth for 16 h at 37 °C and shaking at 170 rpm in a thermostated shaker. Next, the culture was used to inoculate 150 mL of feather broth, and the cells were cultured for 48 h under the same conditions. Subsequently, the supernatant was clarified by centrifugation at 10,000 × g for 10 min at 4 °C and filtered through a 0.22-µm pore-sized membrane to remove microparticles and bacterial cells. The enzymatic extract was stored on ice under aseptic conditions without freezing. 

Casein was prepared from skimmed cow milk by acid precipitation, followed by drying and grinding into a fine powder. Keratin was prepared from ground chicken feathers according to a previously described protocol . Azocasein and azokeratin were prepared as previously described . 

Proteolytic activity was determined according to Kembhavi et al. using azocasein as a substrate. Keratinase activity was determined as previously described , , using azokeratin as the substrate. 

The effect of temperature on enzymatic activity was determined by conducting the reaction in a temperature range of 30–80 °C (with an interval of 10 °C). Maximum enzymatic activity was defined as 100%. To determine thermostability, the enzymatic extract was pre-incubated for 2 h at 50, 60, and 70 °C in 0.1 M Tris buffer (pH 8.5), and then the residual activity was measured at 60 °C in 0.1 M Tris-HCl buffer (pH 8.5). The results are expressed as a percentage relative to the activity of the extract without temperature incubation, which was taken as 100%. The enzymatic activity was measured over a pH range of 3.0 to 11.0. The following buffer systems were used: citrate buffer (pH 3.0–6.0), sodium phosphate buffer (pH 6.0–7.5), Tris-HCl (pH 7.5–9.0), and glycine-NaOH (pH 9.0–11.0). Values were converted to relative units with a maximum value of 100%. 

The effects of metal ions (Na + , K + , Ca 2+ , Ni 2+ , Mg 2+ , Mn 2+ , Cu 2+ , Co 2+ , Cd 2+ , Ba 2+ , and Fe 3+ ) were determined by using salts: NaCl, KCl, CaCl 2 , NiCl 2 , MgCl 2 , MnCl 2 , CuCl 2 , CoCl 2 , CdCl 2 , BaCl 2 , FeCl 3 . Reducing agents [β-mercaptoethanol and dithiothreitol (DTT)], protease inhibitors [phenylmethylsulfonyl fluoride (PMSF), ethylenediaminetetraacetic acid (EDTA), pepstatin A, E64], and detergents [1% Tween 20, Triton X-100, sodium dodecyl sulfate (SDS)] on enzymatic activity were determined by adding the reagents to the reaction mixture. Thereafter, enzymatic activity was evaluated at 60 °С in 0.1 M Tris buffer (pH 8.5). Activity values were considered 100% in the absence of metal ions or chemicals. 

The ability of the enzymatic extract to degrade various protein substrates was determined using SDS-polyacrylamide gel electrophoresis (PAGE) in a 4–10% gel copolymerized with (w/v) sodium caseinate (0.1%), BSA (0.1%), gelatin (1%), and keratin (0.7%). The enzymatic extract was added to SDS-PAGE sample buffer (125 mM Tris-HCl pH 6.8, 0.002% bromophenol blue, 4% SDS, and 20% glycerol) at a ratio (sample: buffer) of 4:6. PMSF (5 mM), EDTA (5 mM), pepstatin A (0.035 mM), and E64 (0.01 mM) were added as serine, metalloprotease, asparagine, and cysteine protease inhibitors, respectively. The samples were not boiled, and no β-mercaptoethanol or DTT was added before electrophoresis. Thereafter, the gels were washed twice for 10 min each in 2.5% Triton X-100 and incubated in 500 mM Tris-HCl (pH 8.5) at 50 °С for 20 h. Next, the gels were stained with 0.08% Coomassie Brilliant Blue G-250 (AppliChem, Darmstadt, Germany) in 20% ethanol with 1.6% orthophosphoric acid and 8% ammonium sulfate for 4 h and then clarified in MilliQ water for 3–4 h until the hydrolysis zones were completely clarified. A protein marker (Color Prestained Protein Standard, cat#P7719S; New England Biolabs, Ipswich, MA, USA) was used to determine the molecular weight. 

Hemoglobin, gelatin, keratin, casein, ovalbumin, and BSA were tested as substrates for hydrolysis using the enzymatic extract. Each substrate was dissolved in 1 mL 50 mM Tris-HCl (pH 8.5) to a concentration of 1 mg/mL and treated with enzymatic extract at 60 °C for 30 min with periodic sampling for analysis. A substrate without enzymatic extract treatment was used as a control. The hydrolysis products were analyzed using 12% SDS-PAGE. The gels were stained with 0.08% Coomassie Brilliant Blue R-250 (AppliChem) in 50% ethanol and 10% acetic acid. A protein marker (Color Prestained Protein Standard, cat#P7719S; New England Biolabs) was used for molecular weight determination. 

The ability of the B. paralicheniformis strain T7 to degrade keratinous animal waste (chicken feathers, horns, hooves, hides, and wool) was investigated. B. paralicheniformis strain T7 was cultured in 15 mL of nutrient broth in a shaker incubator at 37 °C and 170 rpm for 18 h. Thereafter, 1 mL inoculum was added to glass tubes containing horn (0.777 g), hoof (1.405 g), hide (1.055 g), wool (0.254 g), and feather (0.25 g) fragments in 10 mL of sodium phosphate buffer (pH 7.0). Next, the samples were degraded in a shaker incubator at 37 °C and 250 rpm for 7 days. The degree of degradation of the samples was determined by measuring the dry weights of unhydrolyzed samples. For this purpose, the culture was passed through a pre-weighed filter paper, and the remaining residue was washed twice with distilled water and dried at 60 °C to a constant weight. The results are expressed as a percentage of the initial weight of the samples, which was taken as 100%. 

Feather samples were inoculated with 1 mL of sterile enzymatic extract in 10 mL of 0.1 M Tris-HCl buffer (pH 8.5) supplemented with 0.02% sodium azide. Incubation was conducted for 2–7 d at 37 °C. To evaluate the effect of B. paralicheniformis T7 on the degradation process, hydrolysis was performed using an unfiltered enzymatic extract without sodium azide under the same conditions. Samples prepared under the same conditions but without enzymatic extracts were used as controls. After incubation, the samples were analyzed using scanning electron microscopy (SEM) to evaluate the degree of degradation. The samples were placed on stubs and sputtered with gold (10 nm). Images were captured using an Auriga CrossBeam 540 Scanning Electron Microscope (Carl Zeiss, Jena, Germany) at 3 kV. 

The genome of B. paralicheniformis T7 was sequenced using the Oxford Nanopore method on a MinION sequencer (Oxford Nanopore Technologies Ltd., Oxford, UK). Genomic DNA libraries were constructed using an Oxford Nanopore Technologies Sequencing Kit (SQK-LSK109). This involved DNA fragmentation, followed by adapter ligation (Ligation Sequencing Kit, SQK-LSK109; Oxford Nanopore Technologies Ltd). The resulting libraries were quantified using Qubit 2.0 (Invitrogen, Carlsbad, CA, USA) and subjected to sequencing on a MinION platform with an FLO-MIN106D flow cell (R9; Oxford Nanopore Technologies Ltd.). The annotated genome was deposited in the NCBI GenBank. A library of amino acid sequences for all strains was generated based on the annotated genome of B. paralicheniformis T7. The enzymatic extract was concentrated 150-fold on a Pierce™ Protein Concentrator (10 K MWCO: 10 kDa molecular weight cutoff; Thermo Fisher Scientific) for mass spectrometric analysis of the secretory proteome. Protein separation was performed using 12% SDS-PAGE. Proteins were extracted from the gel and treated with trypsin (Promega) to form peptides. Subsequently, the peptides were separated on an Acclaim Pep-Map RSLC column (Thermo Fisher Scientific) using an acetonitrile gradient. An unmodified CaptiveSpray ion source was used to interface the HPLC system with a Maxis Impact II Instrument (Bruker Daltonics). The mass range of the MS scan was set to m/z 150–2,200 in positive ion polarity mode. B. paralicheniformis T7 extracellular proteases were identified on the Mascot platform using an amino acid sequence library of the strain. 

B. Paralicheniformis T7 in a bioreactor and enzyme preparation A 10-L Biostat Bioreactor (Sartorius, Göttingen, Germany) was used to test the ability of B. paralicheniformis T7 to produce proteolytic enzymes during submerged fermentation. Submerged fermentation was performed according to the . A single colony was inoculated into 5 mL of nutrient broth and cultured at 37 °C in a shaker incubator at 180 rpm for 18 h. Afterward, the culture was transferred to 200 mL of feather medium supplemented with 0.2% yeast extract and grown for 24 h under the same conditions. In a 10-L fermenter, the culture was inoculated into 6 L of sterile feather medium supplemented with 0.2% yeast extract. The conditions for submerged fermentation were as follows: temperature, 37 °C; agitation, 450 rpm; aeration, 6–10 L/min; and culturing time, 48 h. Samples were taken periodically to determine the number of colony-forming units and to measure protease activity. The culture was cleared of cells and substrate residues by centrifugation at 11,000 × g and sterilized by microfiltration on a UPIRO-018 bench filtration apparatus (Vladisart, Vladimir, Russia) using an MKM46020 membrane polyethersulfone module (Vladisart) with a cutoff threshold of 0.22 μm. Next, 150 mL of sterile culture was concentrated by evaporation for 1 h on an RV05 basic rotary evaporator (IKA-Werke, Staufen, Germany) at 55 °C and 50 rpm under vacuum (4.9 kPa) until a volume of 3 mL was reached. Subsequently, 450 mL of sterile culture was frozen at -80 °С in a U570 Ultra-low freezer (New Brunswick Scientific, Enfield, CT, USA) and dried in a BETA 2–8 LDplus (Christ, Osterode, Germany) at -90 °С under vacuum (0.028 mBar) for 48 h in accordance with . The lyophilized enzyme was ground into a powdered state, and the remaining sterile culture was spray-dried on an OM-1500 A spray dryer (Shanghai Pilotech Instrument & Equipment Co., Ltd., Shanghai, China) under the following conditions: chamber inlet temperature, 55 °C; chamber outlet temperature, 40 °C; spray pulse duration, 1 s; extract delivery rate, 4.5 mL/min. 

The chromatograms were analyzed after capillary sequencing using the Vector NTI Advance 11 program (Thermo Fisher Scientific). After nanopore sequencing, the sequences were annotated using Prokka Rapid Prokaryotic Genome Annotation Software (Prokka) version 1.13.14 ( https://github.com/tseemann/prokka ) and DNA Features Viewer software version 3.1.3 ( https://github.com/Edinburgh-Genome-Foundry/DnaFeaturesViewer ) to rapidly annotate the prokaryote genome. The Mascot platform was used for mass spectrometric proteins. The online resource Peptide Signal IP 5.0 ( http://www.cbs.dtu.dk/services/SignalP/ ) was used for signal peptide prediction. All activity experiments were performed in triplicate. Enzymatic activity measurement data were obtained through independent activity assays, with mean values, standard deviations (SD), and p-values calculated using GraphPad Prism version 8.0.1 (GraphPad Software, La Jolla, CA, USA, www.graphpad.com ). All data are presented as the mean ± SD ( n = 3). Statistical significance between two groups was determined using the Student’s t-test, with a significance threshold of P < 0.05. 

Isolation and identification of proteolytic strain B. paralicheniformis T7 A strain with proteolytic activity was isolated from soil samples collected near Taraz City, Kazakhstan (42.9 N 71.36667 E). After testing the seven isolates for proteolytic activity, T7 was found to have the highest proteolytic and keratinolytic activity (Fig. ). Taxonomic and morphological analyses of the colonies revealed that isolate T7 formed irregularly shaped colonies with turbid and opaque areas. These colonies were milky in color, irregularly shaped, 2–4 mm in diameter, and had glossy surfaces. When the T7 isolate was cultured in nutrient broth for 24 h, the strain exhibited average growth. During the growth process, the medium became turbid, and bacterial flakes formed in the culture medium. A hard-to-break film was formed at the liquid–air interface. Gram staining of the strain revealed the presence of gram-positive straight rod-shaped bacteria, characteristic of bacteria belonging to the genus Bacillus . Bacterial cells were found alone or in chains. Schaeffer–Fulton staining demonstrated the ability to produce subterminal spores. Sanger sequencing of 16 S rRNA and complete genome sequencing using the Oxford Nanopore method revealed that strain T7 belonged to the species B. paralicheniformis . The effect of pH and temperature on the enzymatic activity of the extract The dependence of the activity of the enzymatic extract of B. paralicheniformis T7 on temperature at 30–80 °C was studied using azocasein and azokeratin. The extract showed maximum activity at 60 °C on both substrates (Fig. ). The proteolytic enzymes in the extract retained 60% activity at 40–70 °C. Figure shows the residual proteolytic activity of the enzymatic extract after incubation for 2 h at 50, 60, and 70 °C. The extract retained 100% activity throughout incubation at 50 °C; after incubation at 60 °C, the residual activity was 83%. When incubated at 70 °C, the residual activity after 15 min was 16.5%; after 30 min, it was less than 0.5%. The pH dependence of the proteolytic activity of the enzymatic extract of B. paralicheniformis T7 against azocasein and azokeratin was studied in the pH ranges of 6–11 and 4–10, respectively. The results showed that the proteolytic enzymes were active over a wide pH range and retained more than 60% of their activity at pH 6.0–10.0. The optimum conditions for all activities were achieved in Tris-HCl buffer with a pH of 9.0 (Fig. ). Effect of metal ions, detergents, reducing agents, and inhibitors on enzymatic stability A study on the effect of metal ions on the activity of the proteolytic enzymes of B. paralicheniformis T7 showed that Na⁺ and K⁺ did not affect the proteolytic and keratinolytic activities of the enzymatic extract (Table ). Ca 2 ⁺ at a concentration of 10 mM reduced proteolytic and keratinolytic activities by 25 and 12%, respectively. At concentrations of 5 and 10 mM, Ni 2+ reduced proteolytic activity by 19 and 57%, respectively, but did not inhibit caseinolytic activity. Mg 2+ did not affect either proteolytic or keratinolytic activity. At a concentration of 10 mM, Mn 2+ reduced proteolytic activity by 69%. Cu 2+ at concentrations of 5 and 10 mM reduced proteolytic activity by 24% and increased keratinolytic activity by 21 and 55%, respectively. The addition of Co 2+ reduced the proteolytic activity of the extract by 60–30% and increased keratinolytic activity 2.7–3.7-fold. Cd 2+ inhibited both proteolytic (50–78%) and keratinolytic activity (up to 40%). No pronounced effect was observed for Ba 2+ ions, whereas Fe 3+ ions were strong inhibitors of proteolytic activity. Protease inhibitors, detergents, and reducing agents were tested for their effects on the keratinolytic and proteolytic activities of the enzymatic extract. The results showed that β-mercaptoethanol, at concentrations of 5 and 10 mM, increased the yield of azokeratin hydrolysis products but did not affect the enzyme’s ability to break down azocasein (Table ). In addition to increasing the production of azokeratin hydrolysis products, 5 and 10 mM DTT decreased the proteolytic activity of the enzymatic extract by 19 and 28%, respectively. Tween 20 reduced the proteolytic and keratinolytic activities of the extract by 13–30%. Triton X-100 reduced proteolytic activity by 20%. The addition of 1% Triton X-100 increased keratinolytic activity by 16%, and 5% Triton X-100 reduced keratinolytic activity by 20%. SDS is a strong inhibitor of keratinolytic activity; after adding 1% SDS, the residual activity of the enzymatic extract was 13%. The addition of protease inhibitors to the enzymatic extract revealed that 5 mM PMSF reduced both the proteolytic and keratinolytic activities by 90%, whereas increasing the PMSF concentration to 10 mM completely inhibited both extract activities. EDTA also had an inhibitory effect, reducing proteolytic and keratinolytic activity by 60 and 22%, respectively. Pepstatin A slightly reduced proteolytic activity but did not affect the keratinolytic activity of the extract. E64 also showed a minor inhibitory activity on the proteolytic activity of the extract. Substrate specificity Zymographic analysis revealed that the enzymatic extract of B. paralicheniformis T7 contained proteases that hydrolyzed casein, keratin, gelatin, and BSA (Fig. ). PMSF inhibited the activity of the enzymatic extract of B. paralicheniformis T7 in reactions with all substrates, indicating that most proteases in the enzymatic extract belonged to the serine protease class. In gelatin zymography, enzymes with masses of 32, 40, and 45 kDa were inhibited by EDTA, indicating that they belong to the class of metalloproteases. Hydrolysis of hemoglobin, gelatin, keratin, casein, BSA, and ovalbumin The hydrolysis rate, which depends on the nature of the substrate, is critical for protein cleavage. To study the hydrolysis rate, the following proteins were selected: hemoglobin, gelatin, keratin, casein, BSA, and ovalbumin. All substrates were hydrolyzed at 60 °C in 0.1 M Tris-HCl buffer at pH 8.5. At the end of the experiment, electrophoretic separation of the hydrolysis products was performed by SDS-PAGE (Fig. ). The rate of hydrolysis of different substrates by the proteolytic enzymes in the enzymatic extract of B. paralicheniformis T7 differed remarkably. Casein was hydrolyzed faster than the other substrates (Fig. ), being hydrolyzed after 15 s of treatment. One minute was sufficient to hydrolyze ovalbumin, while BSA, hemoglobin, and gelatin were hydrolyzed within 5 min. Feather keratin was most resistant to the hydrolytic action of B. paralicheniformis T7 enzymes; 1 mg of keratin was hydrolyzed in 30 min. Degradation of horn, hoof, hide, wool, and feather samples Treatment of horn, hoof, hide, wool, and chicken feather samples with B. paralicheniformis T7 culture showed the degradative properties of the strain in all samples. Figure shows the dynamics of horn, hoof, hide, wool, and feather sample degradation (Table ). The enzymes of the strain hydrolyzed the studied substrates differently, with the type of protein substrate used also affecting enzyme activity. Scanning electron microscopy Changes in the microstructure of chicken feathers during hydrolysis by B. paralicheniformis T7 enzymes were observed using scanning electron microscopy. Figure shows images of feathers subjected to hydrolysis. Feather stems with beards and beards with hooks were clearly observed in the images of the untreated feathers (Fig. A). Damaged nicks were visible after 48 h of culture (Fig. B). The hooks and beards were almost completely decomposed after 72 h (Fig. C). B. paralicheniformis T7 cells clearly adhered to feather surfaces. Besides enzyme action, the adhesion of live B. paralicheniformis T7 bacteria to feather substrates is important in feather destruction. Scanning electron microscopy enabled the visualization of bacterial cells and confirmed the direct involvement of B. paralicheniformis T7 bacteria in feather destruction (Fig. C). The importance of live bacteria in feather hydrolysis was confirmed in feather destruction experiments with live bacteria, an enzymatic extract obtained by culture filtration through a 0.22-µm filter, and sodium azide addition. Feather destruction in samples containing live bacteria was faster than that in samples without microorganisms. Mass spectrometric and genomic analyses of the secretory proteome The sequenced B. paralicheniformis T7 genome comprised 4,359,207 bp and 4,388 protein-coding sequences. In the closely related strain B. paralicheniformis MKU3, the genome has 4,370,039 bp encoding 4872 protein-coding genes . The genome of B. licheniformis strain T5 also isolated from this soil sample has 4,247,430 bp encoding 5391 protein-coding genes . The complete B. paralicheniformis T7 genome has been deposited in NCBI GenBank under accession number CP124861. Seven peptidases with molecular masses of 154.8, 86.2, 41.2, 53.3, 60.6, 30.5, and 26.8 kDa were identified in the enzymatic extract using HPLC-Q/TOF mass spectrometry and Mascot analysis against all B. paralicheniformis T7 proteins (Table ). Fermentation of B. paralicheniformis T7 in a bioreactor and production of proteolytic preparation To obtain proteolytic enzymes by the biotechnological method, B. paralicheniformis T7 was cultivated by submerged fermentation in a 10-L bioreactor. The density of the culture during fermentation was 0.3 × 10 9 , 1 × 10 9 , and 5.3 × 10 9 CFU/mL after 18, 24, and 48 h of cultivation, respectively. Figure shows the protease activity per unit volume as a function of fermentation time in the bioreactor. The results indicate that the maximum protease and keratinase activities were 249.20 ± 7.88 and 116.17 ± 8.10 U/mL, respectively. The following methods were used to obtain enzyme preparations from the culture liquid after B. paralicheniformis T7 fermentation: concentration by vacuum evaporation at 55 °C for 1 h; lyophilic drying at -52 °C under vacuum for 63 h; spraying drying in an air stream at 55 °C. The enzymatic extract was concentrated by volume 50-fold by evaporation under negative pressure. The protease and keratinase activities of the obtained preparations were measured and presented in Table . 

B. paralicheniformis T7 A strain with proteolytic activity was isolated from soil samples collected near Taraz City, Kazakhstan (42.9 N 71.36667 E). After testing the seven isolates for proteolytic activity, T7 was found to have the highest proteolytic and keratinolytic activity (Fig. ). Taxonomic and morphological analyses of the colonies revealed that isolate T7 formed irregularly shaped colonies with turbid and opaque areas. These colonies were milky in color, irregularly shaped, 2–4 mm in diameter, and had glossy surfaces. When the T7 isolate was cultured in nutrient broth for 24 h, the strain exhibited average growth. During the growth process, the medium became turbid, and bacterial flakes formed in the culture medium. A hard-to-break film was formed at the liquid–air interface. Gram staining of the strain revealed the presence of gram-positive straight rod-shaped bacteria, characteristic of bacteria belonging to the genus Bacillus . Bacterial cells were found alone or in chains. Schaeffer–Fulton staining demonstrated the ability to produce subterminal spores. Sanger sequencing of 16 S rRNA and complete genome sequencing using the Oxford Nanopore method revealed that strain T7 belonged to the species B. paralicheniformis . 

The dependence of the activity of the enzymatic extract of B. paralicheniformis T7 on temperature at 30–80 °C was studied using azocasein and azokeratin. The extract showed maximum activity at 60 °C on both substrates (Fig. ). The proteolytic enzymes in the extract retained 60% activity at 40–70 °C. Figure shows the residual proteolytic activity of the enzymatic extract after incubation for 2 h at 50, 60, and 70 °C. The extract retained 100% activity throughout incubation at 50 °C; after incubation at 60 °C, the residual activity was 83%. When incubated at 70 °C, the residual activity after 15 min was 16.5%; after 30 min, it was less than 0.5%. The pH dependence of the proteolytic activity of the enzymatic extract of B. paralicheniformis T7 against azocasein and azokeratin was studied in the pH ranges of 6–11 and 4–10, respectively. The results showed that the proteolytic enzymes were active over a wide pH range and retained more than 60% of their activity at pH 6.0–10.0. The optimum conditions for all activities were achieved in Tris-HCl buffer with a pH of 9.0 (Fig. ). 

A study on the effect of metal ions on the activity of the proteolytic enzymes of B. paralicheniformis T7 showed that Na⁺ and K⁺ did not affect the proteolytic and keratinolytic activities of the enzymatic extract (Table ). Ca 2 ⁺ at a concentration of 10 mM reduced proteolytic and keratinolytic activities by 25 and 12%, respectively. At concentrations of 5 and 10 mM, Ni 2+ reduced proteolytic activity by 19 and 57%, respectively, but did not inhibit caseinolytic activity. Mg 2+ did not affect either proteolytic or keratinolytic activity. At a concentration of 10 mM, Mn 2+ reduced proteolytic activity by 69%. Cu 2+ at concentrations of 5 and 10 mM reduced proteolytic activity by 24% and increased keratinolytic activity by 21 and 55%, respectively. The addition of Co 2+ reduced the proteolytic activity of the extract by 60–30% and increased keratinolytic activity 2.7–3.7-fold. Cd 2+ inhibited both proteolytic (50–78%) and keratinolytic activity (up to 40%). No pronounced effect was observed for Ba 2+ ions, whereas Fe 3+ ions were strong inhibitors of proteolytic activity. Protease inhibitors, detergents, and reducing agents were tested for their effects on the keratinolytic and proteolytic activities of the enzymatic extract. The results showed that β-mercaptoethanol, at concentrations of 5 and 10 mM, increased the yield of azokeratin hydrolysis products but did not affect the enzyme’s ability to break down azocasein (Table ). In addition to increasing the production of azokeratin hydrolysis products, 5 and 10 mM DTT decreased the proteolytic activity of the enzymatic extract by 19 and 28%, respectively. Tween 20 reduced the proteolytic and keratinolytic activities of the extract by 13–30%. Triton X-100 reduced proteolytic activity by 20%. The addition of 1% Triton X-100 increased keratinolytic activity by 16%, and 5% Triton X-100 reduced keratinolytic activity by 20%. SDS is a strong inhibitor of keratinolytic activity; after adding 1% SDS, the residual activity of the enzymatic extract was 13%. The addition of protease inhibitors to the enzymatic extract revealed that 5 mM PMSF reduced both the proteolytic and keratinolytic activities by 90%, whereas increasing the PMSF concentration to 10 mM completely inhibited both extract activities. EDTA also had an inhibitory effect, reducing proteolytic and keratinolytic activity by 60 and 22%, respectively. Pepstatin A slightly reduced proteolytic activity but did not affect the keratinolytic activity of the extract. E64 also showed a minor inhibitory activity on the proteolytic activity of the extract. 

Zymographic analysis revealed that the enzymatic extract of B. paralicheniformis T7 contained proteases that hydrolyzed casein, keratin, gelatin, and BSA (Fig. ). PMSF inhibited the activity of the enzymatic extract of B. paralicheniformis T7 in reactions with all substrates, indicating that most proteases in the enzymatic extract belonged to the serine protease class. In gelatin zymography, enzymes with masses of 32, 40, and 45 kDa were inhibited by EDTA, indicating that they belong to the class of metalloproteases. 

The hydrolysis rate, which depends on the nature of the substrate, is critical for protein cleavage. To study the hydrolysis rate, the following proteins were selected: hemoglobin, gelatin, keratin, casein, BSA, and ovalbumin. All substrates were hydrolyzed at 60 °C in 0.1 M Tris-HCl buffer at pH 8.5. At the end of the experiment, electrophoretic separation of the hydrolysis products was performed by SDS-PAGE (Fig. ). The rate of hydrolysis of different substrates by the proteolytic enzymes in the enzymatic extract of B. paralicheniformis T7 differed remarkably. Casein was hydrolyzed faster than the other substrates (Fig. ), being hydrolyzed after 15 s of treatment. One minute was sufficient to hydrolyze ovalbumin, while BSA, hemoglobin, and gelatin were hydrolyzed within 5 min. Feather keratin was most resistant to the hydrolytic action of B. paralicheniformis T7 enzymes; 1 mg of keratin was hydrolyzed in 30 min. 

Treatment of horn, hoof, hide, wool, and chicken feather samples with B. paralicheniformis T7 culture showed the degradative properties of the strain in all samples. Figure shows the dynamics of horn, hoof, hide, wool, and feather sample degradation (Table ). The enzymes of the strain hydrolyzed the studied substrates differently, with the type of protein substrate used also affecting enzyme activity. 

Changes in the microstructure of chicken feathers during hydrolysis by B. paralicheniformis T7 enzymes were observed using scanning electron microscopy. Figure shows images of feathers subjected to hydrolysis. Feather stems with beards and beards with hooks were clearly observed in the images of the untreated feathers (Fig. A). Damaged nicks were visible after 48 h of culture (Fig. B). The hooks and beards were almost completely decomposed after 72 h (Fig. C). B. paralicheniformis T7 cells clearly adhered to feather surfaces. Besides enzyme action, the adhesion of live B. paralicheniformis T7 bacteria to feather substrates is important in feather destruction. Scanning electron microscopy enabled the visualization of bacterial cells and confirmed the direct involvement of B. paralicheniformis T7 bacteria in feather destruction (Fig. C). The importance of live bacteria in feather hydrolysis was confirmed in feather destruction experiments with live bacteria, an enzymatic extract obtained by culture filtration through a 0.22-µm filter, and sodium azide addition. Feather destruction in samples containing live bacteria was faster than that in samples without microorganisms. 

The sequenced B. paralicheniformis T7 genome comprised 4,359,207 bp and 4,388 protein-coding sequences. In the closely related strain B. paralicheniformis MKU3, the genome has 4,370,039 bp encoding 4872 protein-coding genes . The genome of B. licheniformis strain T5 also isolated from this soil sample has 4,247,430 bp encoding 5391 protein-coding genes . The complete B. paralicheniformis T7 genome has been deposited in NCBI GenBank under accession number CP124861. Seven peptidases with molecular masses of 154.8, 86.2, 41.2, 53.3, 60.6, 30.5, and 26.8 kDa were identified in the enzymatic extract using HPLC-Q/TOF mass spectrometry and Mascot analysis against all B. paralicheniformis T7 proteins (Table ). 

B. paralicheniformis T7 in a bioreactor and production of proteolytic preparation To obtain proteolytic enzymes by the biotechnological method, B. paralicheniformis T7 was cultivated by submerged fermentation in a 10-L bioreactor. The density of the culture during fermentation was 0.3 × 10 9 , 1 × 10 9 , and 5.3 × 10 9 CFU/mL after 18, 24, and 48 h of cultivation, respectively. Figure shows the protease activity per unit volume as a function of fermentation time in the bioreactor. The results indicate that the maximum protease and keratinase activities were 249.20 ± 7.88 and 116.17 ± 8.10 U/mL, respectively. The following methods were used to obtain enzyme preparations from the culture liquid after B. paralicheniformis T7 fermentation: concentration by vacuum evaporation at 55 °C for 1 h; lyophilic drying at -52 °C under vacuum for 63 h; spraying drying in an air stream at 55 °C. The enzymatic extract was concentrated by volume 50-fold by evaporation under negative pressure. The protease and keratinase activities of the obtained preparations were measured and presented in Table . 

Microbial proteases are in demand in various fields, including food and feed processing and detergent and pharmaceutical production. The cost of microbial enzymes is significantly lower than that of eukaryotic analogs because microorganisms produce enzymes faster than mammalian and plant cells. Furthermore, enzyme production does not depend on climatic conditions or seasonal changes and does not violate ethical or environmental standards . Among proteases, keratinases (EC 3.4.21/24/99) can hydrolyze strong fibrillar proteins such as collagen and keratin, depending on their substrate specificity . Keratinases efficiently cleave keratinous materials, such as feathers, nails, and wool, as well as soluble protein substrates, such as casein, whey proteins, and BSA . A promising source of cheap proteinaceous raw materials is keratinous waste from animal husbandry, including feathers, horns, hooves, wool, and hides. For example, chicken feathers account for approximately 5–7% of the total weight of a chicken , and hides, horns, and hooves account for 11% of cattle weight. The high proteolytic activity of keratinases enables them to hydrolyze highly resistant keratins, which is why they are considered the most promising proteolytic enzymes in the industry , . Bacillus has an efficient enzymatic system, including various proteolytic enzymes that enable it to occupy different ecological niches . Bacillus spp. are ubiquitous in various soil types, compost, and landfills. They are predominantly mesophilic microorganisms; however, several of their enzymes show increased thermostability , . The productive properties of Bacillus have allowed this taxon to occupy a dominant position in the microbial synthesis industry . Among the keratin-degrading microorganisms, bacilli are known keratinase producers – , some of which have been isolated from soil and poultry waste , . Strain T7 was isolated from Kazakhstani soil and identified as B. paralicheniformis based on culture, morphological, proteomic, molecular genetic, and genomic characteristics. Studies have shown that when cultured on milk, keratin, and gelatin agar, this strain produces proteolytic enzymes that hydrolyze the corresponding substrates, forming characteristic clear zones. B. paralicheniformis T7 can grow on feather medium with added salts, in which feather keratin is the only source of organic matter. Studies on the biochemical characteristics of the enzymatic extract of B. paralicheniformis T7 indicated that the proteolytic enzymes it produces have an optimum pH of 9.0. Compared with the described proteases, most bacillary proteases and keratinases are alkaline (Table ), except for proteases from B. velezensis Y1 , Bacillus sp. ZG20 , and keratinases from B. licheniformis dcs1 . B. paralicheniformis T7 proteases and keratinases display maximum activity at 60 °C. Table shows that, among bacillary proteases and keratinases, many enzymes have similar temperature characteristics , – . The thermostability study revealed that the proteolytic enzymes in the enzymatic extract exhibited enhanced thermostability. The proteases retained activity after a 2-h preincubation at 50 °C, and residual activity was lowered by 17% after preincubation at 60 °C. Following a preincubation period of 70 °C, the proteases exhibited reduced stability. In this instance, 83.5% of extract protease activity was lost after 15 min, and less than 0.5% remained after 30 min. Nevertheless, the thermostability of B. paralicheniformis T7 enzymes was greater than those of Bacillus sp. A.5.3 . Additionally, B. paralicheniformis T7 proteases are more thermostable than those of B. velezensis Y1, which lose over 80% of their activity following preincubation for 2 h at 55 °C and 10 min at 60 °C . Single-charged ions were shown not to affect the proteolytic and keratinolytic activities of the enzymatic extract of B. paralicheniformis T7. Among doubly charged ions, Ca 2 ⁺, Mg 2 ⁺, and Ba 2 ⁺ have relatively neutral effects. Ni 2 ⁺, Mn 2 ⁺, and Cd 2 ⁺ at a concentration of 10 mM strongly inhibited proteolytic activity. At a concentration of 10 mM, Cu 2 ⁺ and Cd 2 ⁺ negatively affected keratinolytic activity. Co 2⁺ at a concentration of 5 mM did not reduce proteolytic activity, only at a concentration of 10 mM did it slightly reduce proteolytic activity by 13%. At the same time, a decrease in keratinolytic activity by 59 − 48% was observed at both 5 mM and 10 mM Co 2⁺ . Fe 3⁺ had the greatest inhibitory effect; the addition of 5 mM residual protease and keratinase activity was 23% and 25%, respectively. And 10 mM FeCl 3 completely inactivated proteolytic activity and reduced keratinolytic activity by 95%. This sensitivity to metal ions is typical for metalloproteases. Therefore, keratinases in the extract had a higher tolerance to metal ions than that of proteases. The increase in the yield of keratin hydrolysis products following the addition of reducing agents can be explained by their reducing effect on the disulfide bonds between cysteine residues within keratin . β-mercaptoethanol at 5 and 10 mM concentrations has no significant effect on proteolytic activity ( P > 0.05), but causes a slight increase in keratinolytic activity, with significant P values (0.0158 and 0.038). In contrast, dithiothreitol (DTT) at 5 and 10 mM significantly decreases proteolytic activity ( P < 0.001) but increases keratinolytic activity ( P < 0.01). Ionic detergents such as Tween 20 and Triton X-100 lead to a decrease in proteolytic activity, as evidenced by significant P values (0.0416 for Tween 20 and 0.0004 for Triton X-100). However, their effect on keratinolytic activity varies: Twin 20 reduces activity with a high level of significance ( P < 0.05), while Triton X-100 at a concentration of 1% causes no significant change ( P = 0.0917). Among the protease inhibitors, the strongest inhibition was observed in the presence of PMSF and EDTA, where enzyme activity was significantly inhibited at both concentrations ( P < 0.001). These data confirm that the enzyme systems are sensitive to these inhibitors. Qualitative and quantitative experiments on the effects of the protease inhibitors PMSF, EDTA, pepstatin A, and E64 showed that the proteolytic and keratinolytic activities of the enzymatic extract were primarily attributed to serine proteases. Metalloproteases also participate in protein hydrolysis but contribute less to the total activity, which is probably due to their low level of expression and possibly low specific activity. The action of asparagine and cysteine proteases is negligible. The inhibitors Pepstatin A and E64 have less pronounced effects; the P value indicates insignificant changes in activity. The proteolytic activity of the enzymatic extracts of Bacillus sp. A5.3 and other Bacillus representatives has been shown to be strongly inhibited by PMSF , – , and the negative effects of EDTA on proteolytic enzyme activity have also been reported for proteases from B. halodurans PPKS-2 , Bacillus sp. MKR5 , and Chryseobacterium sp. kr6 . Zymographic analysis of casein, keratin, gelatin, and BSA belonging to different protein structural classes revealed their hydrolysis by proteases of different molecular masses. Similar zymographic data were obtained for the extracts of B. subtilis SCL, B. subtilis 1271, and Bacillus sp. A5.3 , , . The zymograms show that BSA is hydrolyzed by one serine protease with an estimated mass of 26 kDa, which is also visible in the zymogram with casein, along with two other proteases. Five proteases were involved in gelatin hydrolysis. Protein hydrolysis showed that milk casein was the most susceptible to hydrolysis; 1 mg was completely hydrolyzed in 15 s. Ovalbumin was the second most susceptible to hydrolase action, with 1 mg being hydrolyzed in 1 min. BSA, hemoglobin, and gelatin were more resistant to the action of B. paralicheniformis T7 proteases, requiring 5 min for hydrolysis. B. paralicheniformis T7 keratinases hydrolyzed 1 mg of keratin in 30 min. Compared with that of Bacillus sp. A5.3, B. paralicheniformis strain T7 extract is comparable in terms of keratinolytic activity but superior in proteolytic activity. B. paralicheniformis T7 enzymes required much less time to hydrolyze casein, ovalbumin, and BSA than proteases from Bacillus sp. A5.3 . The presence of several clear zones in all the zymograms indicated that the enzymatic extract was a cocktail of proteases of different molecular masses in range 20–200 kDa. A similar zymogram with many bands has been reported for B. subtilis SCL, B. subtilis 1271, B. paralicheniformis MKU3 , , . B. paralicheniformis T7 enzymes are characterized by high proteolytic activity against keratin- and collagen-containing substrates. The proteases of this strain can hydrolyze substrates such as wool, hide, feathers, horns, and hooves, which are resistant to the action of most proteases. β-keratin, a component of feathers, and α-keratin (horns, hooves, and wool) have high mechanical strength and resistance to the action of most proteases , which requires the synergistic action of enzymes . Comparative analysis revealed that the horn was the most resistant to protease action; more than 96% of horns remained unhydrolyzed after 7 days of co-culture with B. paralicheniformis T7 cells. Chicken feathers and cattle hides were the least resistant to hydrolysis. Hooklet and barbule degradation was observed on the feathers after 2 days. Barb degradation began on the third day, and feathers were completely hydrolyzed by the end of the fourth day. The dermis of the hide was also highly sensitive to B. paralicheniformis T7 proteases, and at the end of incubation, only wool remnants were observed in the sample, with the culture losing transparency and acquiring a milky color. The P value for proteolytic activity indicated statistically significant differences for hoof, hide, and horn, while the values for wool and feathers were less significant. For most substrates except wool, the P value for keratinolytic activity was significant. The resistance of the keratinous substrates to the proteolytic action of B. paralicheniformis T7 was in the order of horn > hoof > wool > hide > feather. B. paralicheniformis T7 degrades feathers 120 h faster than does Bacillus sp. A5.3 . The keratinous substrate hydrolysis results indicated that feathers and hides are promising inexpensive substrates for producing protein hydrolysates. A comparative analysis of the protease and keratinase activities of B. paralicheniformis T7 cultured on different substrates revealed that wool and feathers effectively induced protease activity, whereas hooves and wool stimulated keratinase activity. The lowest values for both activities were observed when hides were used. These findings indicate that wool and feathers are excellent stimulators of proteolytic and keratinolytic enzyme production, respectively. Previously, the determining effect of chicken feathers as a substrate on the production of keratinases of B. paralicheniformis strain MKU3 was also noted . In contrast to B. paralicheniformis MKU3, which showed no collagenase activity, this activity was observed for B. paralicheniformis strain T7, as confirmed by a zymographic test on gelatin and more than 97% skin hydrolysis. The SEM visual analysis of hydrolyzed chicken feathers revealed that their hydrolysis was much more efficient using bacterial culture than using enzymatic extract alone. The SEM results showed that B. paralicheniformis T7 cells tightly adhered to the feather surface, intensifying feather degradation at the attachment sites, consistent with previous results , , . Without live bacterial cells, the extracellular keratinolytic activity of Bacillus sp. A5.3 and B. licheniformis RG1 strains did not result in complete feather degradation . Taken together, this evidence suggests that the adhesion of bacterial cells to the substrate surface plays an important role in the efficient and rapid hydrolysis of keratinous raw materials. The mass spectrometric analysis of the secretory proteome of B. paralicheniformis T7 identified seven proteases, four of which belong to serine peptidase families S8 and S41, and three belong to metallopeptidase families M14, M42, and M55. Previously, 11 serine proteases of the S8 clan were established in the genome of B. paralicheniformis MKU3 . By the LC-MS/MS method, 2 extracellular serine proteases and 2 metalloproteases were established in B. paralicheniformis MKU3 , while 4 serine proteases and 3 metalloproteases were established in the secretory proteome of B. paralicheniformis T7. Notably, 2 proteases from B. paralicheniformis MKU3 with molecular masses of 87 kDa and 60 kDa correspond to the proteases S8 family serine peptidase and M14 family metallocarboxypeptidase detected in the secretory proteome of B. paralicheniformis T7. Further analysis showed that all identified serine peptidases and M14 carboxy metallopeptidase had signal peptides 28–36 amino acids in length. A specific contribution to the keratinolytic activity of this strain is likely attributable to an enzyme identified as an S8 serine peptidase 378 amino acids long with a molecular mass of 41.2 kDa. Sequence alignment revealed that this enzyme has high homology with the KerA keratinase from B. licheniformis . A high score indicated that this peptidase was highly expressed in the secretory proteome of B. paralicheniformis T7. Submerged B. paralicheniformis T7 fermentation on feather medium in a bioreactor revealed that under the specified conditions, the bacteria efficiently produced proteolytic enzymes after 7 h of fermentation. After 24 h of fermentation, protease and keratinase activities reached a maximum. Comparison with other bacillary strains (Table ) showed that B. paralicheniformis T7 cultured on feather medium had production properties similar to those of Bacillus sp. CL33A but requires less time for fermentation. A short fermentation cycle was observed for B. paralicheniformis strain MKU3 , which also showed a maximum keratinase production at 24 h of fermentation. Feather medium is inexpensive and effective for protease production, and supplementation of the basic substrate with yeast extract or peptone positively affects the production properties of bacillary strains – . Freeze-drying is a common but time-consuming method for obtaining concentrated enzyme preparations. However, the ability of B. paralicheniformis T7 proteases to withstand heating at 50–60 °C for up to an hour or more enables the preparation of concentrated proteolytic preparations by vacuum evaporation and spray-drying in heated air. Protease and keratinase activities in the concentrate, lyophilizate, and spray-dried powders were 4,940 U/mL, 148,670 U/g, and 193,670 U/g and 2,493 U/mL, 83,670 U/g, and 83,170 U/g, respectively. Overall, B. paralicheniformis T7 fermentation and the concentration and drying of proteolytic enzymes indicate the potential of B. paralicheniformis T7 as a producer of thermostable proteases and keratinases. Further studies on cloning the corresponding genes and producing recombinant proteases and keratinases for characterization and determining their individual and synergistic involvement in protein substrate hydrolysis would be of interest. 

This study describes a soil strain, B. paralicheniformis T7, with high proteolytic and keratinolytic activities. The enzymatic extract of this strain contains thermostable alkaline proteolytic enzymes from the serine peptidase and metallopeptidase families with distinct keratinase activity. Culturing B. paralicheniformis T7 on keratinous raw materials indicated that the strain completely degraded chicken feathers in 4 days and cattle hide in 7 days. The degradation rates for horns, hooves, and wool for this period were 3.6, 29.6, and 34.5%, respectively. SEM data showed that B. paralicheniformis T7 cells were highly and tightly adhered to the feather surface, accelerating the degradation process. Proteomic and genomic studies identified seven peptidases in the bacterial extract. Submerged fermentation of B. paralicheniformis T7 on feather raw material for 24 h yielded a protease activity of 249.1 U/mL. The milk-clotting activity of the strain was determined, in addition to its protease and keratinase activities. These results demonstrate that B. paralicheniformis T7 has the potential to produce thermostable proteolytic and keratinolytic enzymes. Future studies will aim at focusing on each identified protease and keratinase enzyme extract of the strain. 

Supplementary Material 1 

