| HS Code | 851850 |
| Productname | Keratinase |
| Type | Proteolytic enzyme |
| Source | Microorganisms (e.g., Bacillus spp.) |
| Molecularweight | 30-50 kDa |
| Optimalph | 7.0-9.0 |
| Optimaltemperature | 40-60°C |
| Enzymeactivity | ≥5000 U/g |
| Substrate | Keratin proteins |
| Modeofaction | Hydrolysis of disulfide and peptide bonds in keratin |
| Stability | Stable at 4°C for 6 months |
| Inhibitors | Metal ions (e.g., Hg2+, Cu2+) |
| Applications | Feed, leather, cosmetics, and waste management |
As an accredited Keratinase factory, we enforce strict quality protocols—every batch undergoes rigorous testing to ensure consistent efficacy and safety standards.
| Packing | Keratinase powder, 25 kg net weight, packed in sealed polyethylene-lined fiber drums with tamper-evident closures. |
| Container Loading (20′ FCL) | Keratinase shipped as 20′ FCL in sealed containers, with moisture-proof packaging and proper ventilation to ensure stability and safe transport. |
| Shipping | Keratinase is shipped as a lyophilized powder or sterile-filtered solution, typically in sealed vials. Transport requires cold-chain conditions: dry ice for frozen aliquots or gel packs for refrigerated enzyme solutions. Proper labeling, moisture-resistant packaging, and immediate storage at -20°C or 4°C upon receipt are essential to preserve enzymatic activity. |
| Storage | Store Keratinase at –20°C, preferably lyophilized, in a tightly sealed, desiccated container protected from light and moisture. Avoid repeated freeze-thaw cycles; aliquot into single-use portions if in solution. For working solutions, keep at 2–8°C for short periods. Always handle with proper PPE and follow the safety data sheet. |
| Shelf Life | Keratinase should be stored lyophilized at -20°C, retaining activity for at least six months under recommended conditions. |
Where sodium sulfide is removed from beamhouse operations, keratinase is introduced as part of a soak-and-paint or pit-unhairing sequence. The enzyme preparation is dosed at 0.5–1.5% w/w on green hide mass, equivalent to 80–250 U keratinase activity per kilogram of salted hide, in a recirculating drum with a charge factor of 2.8–3.2 and rotational speed not exceeding 4–6 rpm. Process water is pre-adjusted to pH 9.0–10.0 with sodium carbonate/bicarbonate buffer, and temperature is held at 30–34°C for 6–10 h. Hair looseness is checked by pull-test at 30-min intervals after hour 4; the endpoint is reached when wool release occurs under finger pressure without grain slip. The dehairing liquor after enzyme-only processing is analyzed for residual dissolved sulfide according to ISO 10530:1992, with a target below 20 mg/L; conventional paint-dehairing liquors frequently exceed 2,000 mg/L, though published data for hide-specific sulfide baselines is limited. Finished leather after deliming and bating is tested for pH and difference number according to ISO 4045:2018 and for hide substance retention according to IULTCS/IUC 10. The main operational boundary is that keratinase does not fully replace lime as a swelling agent. Salted hides stored longer than 6 months or those with heavy subcutaneous fat may require 0.2–0.5% sodium sulfide as a sensitizer; otherwise dehairing time extends beyond 16 h and bacterial growth risk increases. Terminal leather retains intact grain and is suited to subsequent deliming, bating, and wet-end processing without the effluent load associated with full-sulfide unhairing.
In liquid heavy-duty detergent systems, keratinase is co-formulated with subtilisin only after the neutralisation loop has stabilized the base at pH 7.8–8.3. The keratinase dose ranges from 0.15–0.40 wt% of a 50,000 U/g liquid enzyme product, giving 75–200 U/g in the finished detergent. Post-dosing temperature is maintained at 35–38°C; addition to a surfactant slurry above 45°C causes rapid activity loss. Stabilizers include 5–8 wt% propylene glycol, 0.05–0.2 mM calcium chloride, and 0.1–0.3 wt% sodium formate. The enzyme is added after the main high-shear mixing step; subsequent agitation is limited to 50–70 rpm side-scrape blending for 10–15 min. Wash performance is evaluated under ASTM D4265-14 using cotton swatches soiled with keratinaceous material; typical main-wash conditions are 2.5–4.0 g/L detergent at 30°C for 20 min. Retained activity after 12 weeks at 37°C must exceed 80% for batch release. Liquid detergent pH above 9.5 at storage temperature leads to activity retention below 55% and is avoided in production. The terminal product is a liquid heavy-duty laundry detergent or unit-dose gel with keratin-soil removal functionality. Compliance for enzyme labelling and safety data follows EC 648/2004, Annex VII. The main incompatibility is direct premixing with linear alkylbenzene sulfonate above 8% active, which denatures the enzyme before the stabilizer matrix is fully distributed.
| Liquid detergent pH | Retained keratinase activity after 28 days at 37°C | Phase separation observed |
|---|---|---|
| 7.5 | 82% | None |
| 8.5 | 76% | None |
| 9.5 | 54% | Trace |
| 10.5 | 18% | Visible |
Feather meal digestibility improvements are contingent on autoclave condensate management and post-hydrolysis drying temperature. Poultry feathers are washed, autoclaved at 121°C for 20 min, and flash-cooled to 55°C before entering a jacketed ribbon blender. The hydrolysis vessel operates at low-shear axial mixing of 10–20 rpm; pH is adjusted with 3 M sodium hydroxide to 8.0–8.2, and moisture is held at 60–65%. Keratinase is dosed at 800–1,800 U/kg dry feather, frequently with a food-grade protease co-additive at 0.05–0.10% w/w to increase free amino nitrogen. The reaction proceeds for 14–18 h at 50°C; endpoint pH falls to 7.5–7.8. The resulting slurry is dried in a ring dryer with inlet temperature 180°C and product temperature not exceeding 75°C, because lysine availability declines rapidly above 80°C. Pepsin digestibility is determined according to AOAC 971.09; enzyme-hydrolyzed feather meal typically reaches 72–85%, compared with 50–60% for untreated feather meal. Feed safety is verified by absence of Salmonella in 25 g according to ISO 6579-1, and the material is classified as Category 3 under EC 1069/2009. The process output falls under the zootechnical additive framework of EU 1831/2003 when used as a feed material or additive; registration requirements differ by member state. The terminal product is enzyme-hydrolyzed feather meal with reduced particle size, higher pepsin digestibility, and improved free amino nitrogen content for monogastric feed blending.
For leave-on emulsions, enzyme stability data are constrained by two competing variables: water activity above 0.90 required for enzyme hydration and preservative load below 0.8 wt% needed to avoid inhibition. The enzyme is added at 0.05–0.15% w/w of a 10,000 U/g liquid preparation, yielding 5–15 U/g in the final O/W cream. The formulation base is cooled to 32°C after high-shear rotor-stator emulsification; agitation is then switched to side-scraped anchor mixing at 25–35 rpm. The enzyme is injected below the liquid surface through a 0.2 μm inline filter to remove aggregated protein. Formula pH is maintained at 6.8–7.2 with citrate buffer; activity retention falls below 30% of nominal after 7 days at 25°C when pH is below 5.5. Preservative efficacy is validated by ISO 11930:2019 acceptance criterion A; residual enzyme activity after challenge must remain above 70%. Microbial limits for cosmetic products are checked by ISO 17516:2014. Cosmetic claims are governed by EC 1223/2009; any claim indicating hair growth or removal shifts the product toward a medical-device or drug classification. The terminal product is a keratolytic foot mask, rough-skin exfoliant, or callus-softening cream. Formulation incompatibility includes direct combination with alpha-hydroxy acids at pH below 4.5, which destabilizes the enzyme and promotes phase separation in O/W systems.
Composting and rendering side streams receive keratinase only after thermal prehydrolysis, because native feather keratin remains water-recalcitrant at atmospheric pressure. Whole feathers are charged into a closed reactor at a 1:2 feather-to-water ratio and held at 90°C for 60 min; the resulting slurry is cooled to 50°C and adjusted to pH 8.5–9.0. Keratinase is dosed at 0.8–1.2% w/w dry feather, with intermittent low-shear recirculation for 24–36 h. The endpoint is measured by formol titration; free amino nitrogen typically reaches 1,500–2,500 mg N/L, corresponding to a degree of hydrolysis of 35–50%. Total nitrogen is determined by ISO 16634-1:2008; the liquid digestate commonly contains 8–10% total N on a dry matter basis, but published data for farm-specific feedstock blends is limited. The resulting product is classified under the animal by-product rules of EC 1069/2009 and can be placed on the EU market as a component material under EU 2019/1009, provided the ABP end-point conditions are met. The critical processing boundary is ammonia volatilization: at pH above 9.5 and temperature above 50°C, ammonia stripping can remove more than 5% of total nitrogen within 12 h. Off-gas scrubbing with sulfuric acid or a closed reactor is required. The terminal output is a liquid amino acid fertilizer or fertigation concentrate suitable for drip irrigation after filtration through 200 μm mesh.
In biomedical scaffold hydrolysis, the control variables shift from cost per ton to endotoxin burden, molecular weight distribution, and residual enzyme removal. Soluble wool keratin is dissolved in 0.5 M urea or a reduced cysteine buffer at pH 7.4; keratinase is added at 10–50 U/g keratin, and incubation proceeds at 37°C for 2–6 h. The hydrolysate is clarified by depth filtration and passed through a 10 kDa MWCO polyethersulfone tangential flow filter to separate the enzyme and reduce the peptide fraction to a defined molecular weight window. Retentate flux is maintained at 20–40 L/m²/h; transmembrane pressure is held below 1.0 bar to limit gel-layer formation. The permeate is lyophilized at a shelf temperature of −40°C and chamber pressure of 0.1 mbar, producing a white soluble peptide powder. Cytotoxicity is evaluated according to ISO 10993-5:2009; bacterial endotoxin is determined by USP <85> with a limit appropriate for the intended scaffold material. Residual enzyme activity in the peptide fraction is monitored by casein zymography; published data for specific scaffold configurations is limited, and each cell-contact claim must be supported by ISO 10993-12 extraction testing. The terminal product is a soluble keratin peptide fraction below 10 kDa, used in hydrogel precursors and surface-modification solutions. The operational boundary is that the process does not provide a sterile product without terminal filtration or aseptic lyophilization.
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Keratinase KER-101 is a thermostable serine endopeptidase preparation standardised for hydrolysis of β-keratin-rich industrial by-products. The product is supplied as a spray-dried, dust-suppressed powder with a declared activity of 50,000 U/g ± 5,000 U/g by the azokeratin method at pH 8.5 and 40°C; the method is maintained under ISO 17025:2017 clause 7.2 for personnel competence and method validation. Moisture is controlled to ≤ 8.0% when tested by ISO 6496:1999, and total viable count is ≤ 10,000 CFU/g by ISO 4833-1:2013. The preparation uses a nontoxigenic Bacillus licheniformis production strain; the active enzyme is a subtilisin-class serine protease with a molecular mass of 28 kDa and an isoelectric point of pH 8.2. This differentiates it from trypsin-class enzymes and from cysteine proteases such as papain, which require reducing equivalents for full activity. The product’s keratin-binding module imparts measurable adsorption to feather β-sheet surfaces, whereas conventional subtilisin A preparations show limited adsorption to intact feather keratin.
| Parameter | Method | Specification |
|---|---|---|
| Declared activity | Azokeratin, pH 8.5, 40°C, ISO 17025:2017 | 50,000 U/g ± 5,000 U/g |
| Moisture | ISO 6496:1999 | ≤ 8.0% |
| Bulk density | USP <616> Method I | 0.55–0.65 g/cm³ |
| Particle size | ISO 2591-1:2008 | ≥ 90% through 150 µm |
| Total viable count | ISO 4833-1:2013 | ≤ 10,000 CFU/g |
| pH of 1% solution | Electrometric method, 25°C | 7.8–9.0 |
Intact feather keratin contains approximately 7%–10% of cysteine residues engaged in interchain disulphide bonds, which restrict access of the catalytic serine to the polypeptide backbone. In a 5,000 L jacketed stirred reactor with dual Rushton turbines operating at 0.35 kW/m³, a 10 wt% feather slurry treated with 200 U/g dry feather at 60°C and pH 9.0 reaches 38%–42% soluble protein in 4 h when no reducing agent is present. Addition of 0.3 wt% sodium sulphite before enzyme dosing increases soluble protein to 78%–84% under identical mixing and temperature, because sulphite cleaves cystine disulphide bonds and exposes β-sheet surfaces. This is a critical process conflict: the enzyme’s substrate specificity does not eliminate the need for reductive pre-treatment. The sulphite should be dosed 30 min before enzyme addition; without this sequence, batch-to-batch variation in feather sources can produce differences of up to 12 percentage points in soluble protein content.
The hydrolysis kinetics follow a two-phase pattern. An initial rapid phase from 0–90 min releases 55%–60% of final soluble protein, while a slower phase from 90–240 min is controlled by particle surface renewal. Turbine tip speeds below 1.8 m/s permit a hydrated feather mass to segregate at the liquid surface; tip speeds above 2.5 m/s reduce enzyme affinity through air entrainment and oxidative deactivation. Operators should maintain dissolved oxygen below 2 mg/L during the first 60 min because the catalytic serine is subject to air-liquid interface denaturation in low-solids hydrolysates.
Application to hide unhairing requires different mixing and pH boundaries. For bovine hides in a mix drum with float ratio 1:2 at 35°C–38°C, Keratinase KER-101 is dosed at 0.1–0.3 wt% of green hide weight. The unhairing effect is achieved through degradation of the basement membrane and follicular keratins, not through collagenolysis. Hydroxyproline release under these conditions remains below 0.5 mg/g hide, indicating minimal damage to the collagen triple helix. Conventional subtilisin-type alkaline protease powders remove hair in 18–24 h under the same conditions and can show hydroxyproline release of 1.2–2.0 mg/g; KER-101 reduces processing time to 14–16 h while maintaining hydroxyproline below 0.5 mg/g because its keratin-binding module restricts adsorption to collagen fibrils. Sodium sulphide load in the liming step can be reduced from 2.5 wt% to 0.8 wt%, but complete replacement of sulphide is not recommended because residual disulphide-stabilised keratin in the root sheath may remain intact.
| Property | Keratinase KER-101 | Subtilisin A alkaline protease | Papain |
|---|---|---|---|
| Azokeratin activity | 50,000 U/g | 1,800–2,500 U/g | 1,200–1,600 U/g |
| Effective pH range | 8.0–10.5 | 8.0–10.5 | 6.0–8.0 |
| Feather solubilisation, 4 h, 60°C, 0.3% Na₂SO₃ | 78%–84% | 42%–55% | 30%–38% |
| Bovine hide unhairing time | 14–16 h | 18–24 h | 20–26 h |
| Hydroxyproline release | ≤ 0.5 mg/g | 1.2–2.0 mg/g | 1.0–1.8 mg/g |
The enzyme retains 85% residual azokeratin activity after 60 min at 65°C in 50 mM Tris-HCl buffer at pH 9.0. At 70°C, residual activity falls to 52% under the same conditions, and at 75°C the half-life is 11 min. This defines a processing window that must be controlled within ±3°C when feather hydrolysis is operated at 65°C. Spray-dried powder stored in sealed foil-laminated bags at 25°C and 50% RH retains 92% activity after 12 months. Storage at 35°C and 70% RH reduces activity to 68% after 6 months, primarily through moisture-induced aggregation. Facilities without climate-controlled warehousing should pre-dry storage rooms to RH < 60%. The product should not be combined with cationic surfactants above 0.1 wt% or with available chlorine above 0.05% unless the enzyme is encapsulated; these agents strip the water shell from the catalytic serine and accelerate deactivation. The powder disperses in water at 25°C in less than 120 s under stirring at 200 rpm, but direct addition into a high-shear mixer without prior 1:10 water pre-dispersion can form enzyme-rich agglomerates that reduce effective activity by up to 18%.
For soluble keratin peptide production, the substrate is first reduced with 0.5 wt% sodium sulphite and heated to 60°C for 2 h, then KER-101 is dosed at 500 U/g dry substrate. The resultant hydrolysate reaches an average molecular weight of 1.2–1.5 kDa after 4 h as measured by gel permeation chromatography; residual intact keratin above 10 kDa is below 6% of total peptide area. This differentiates the product from papain, which under the same reducing conditions produces a broader distribution of 2.5–4.5 kDa and leaves 18%–24% of material above 10 kDa. The narrower cleavage pattern reduces the need for membrane diafiltration when low molecular weight improves solution clarity and casting behaviour. Downstream filtration uses a 0.45 µm polyethersulfone membrane; without reductive pre-treatment, membrane blinding occurs because intact feather barbs compress into a mat with specific resistance of 3.2 × 10¹² m/kg.
When Keratinase KER-101 is substituted for subtilisin-type alkaline protease in feather meal coating for aquafeed extrusion, the enzyme must not be injected into the extruder barrel. A twin-screw extruder with L/D 32:1 and barrel zones above 90°C produces localised deactivation of unencapsulated enzyme; post-extrusion coating at ≤ 70°C is required. Aquafeed pellets coated with hydrolysate produced by KER-101 at 3 wt% inclusion show crude protein digestibility of 88.7% in rainbow trout when measured by inert-marker trial protocols; published data for this specific configuration is limited, but production-scale observations indicate reduced nitrogen excretion compared with untreated feather meal. The product is intended for industrial use in leather processing, feed hydrolysate production, and detergent compounding; it is not intended for direct human ingestion or as a pharmaceutical API.
