Subtilisin, subtilisin/ryanase, is a bacterial serine protease. Subtilisin could be utilized as a detergent additive with thermal stability, pH tolerance, and calcium-dependent stability.

Physicochemical Properties

Molecular Weight	0
CAS #	9014-01-1
PubChem CID	3086051
Appearance	Light-colored, free-flowing powders Light-colored prills
Density	1.3
Hydrogen Bond Donor Count	13
Hydrogen Bond Acceptor Count	15
Rotatable Bond Count	18
Heavy Atom Count	70
Complexity	1860
Defined Atom Stereocenter Count	8
SMILES	N#CC1=CC=C(CCl)C=C1
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Toxicity/Toxicokinetics	Interactions Binary or tertiary mixtures of Alcalase (9014011), Savinase (9014011), Termamyl (9000902), or Lipolase (9001621) were administered at concentrations of 0 to 0.3 ug once a wk for 10 wk by intratracheal instillation. Alcalase and Savinase were proteases, Termamyl an alpha-amylase, and Lipolase a lipase. The mixtures always contained either Alcalase or Savinase along with Termamyl, Lipolase or both. Some mixtures contained Alcalase that had been inactivated by pretreatment with hexamethylene-isocyanate or diisopropylfluorophosphate. Control animals were administered single enzymes. The animals were observed for signs of respiratory distress such as periodic diaphragmatic spasms or retractions. Retroorbital blood samples were collected before and 4, 5, 6, 8, or 10 wk after the start of exposure. The sera were separated and analyzed for circulating immunoglobulin(Ig)-G1 homocytotropic antibodies (HARs) by the passive cutaneous anaphylaxis assay. The number of guinea-pigs with respiratory symptoms and the severity of the symptoms of animals exposed to the Alcalase or Savinase containing mixtures were not different from the symptoms of animals exposed to either Alcalase or Savinase alone. HAR titers to Lipolase and Termamyl were significantly increased when they were administered in mixtures with Alcalase or Savinase. HAR titers to Alcalase or Savinase were not significantly increased by the presence of other enzymes in the mixtures. The enhanced responses produced in mixtures containing Alcalase or Savinase were abolished when the inactivated forms of the enzymes were used in the mixtures. Additional guinea-pigs inhaled aerosols containing 500 ug/cu m Lipolase alone or with 2,000 ug/cu m Alcalase 20 minutes daily for 5 days. They were challenged with the same aerosols 14 or 21 days after the last exposure. Blood samples were collected and analyzed for Lipolase specific IgG antibodies by an enzyme linked immunosorbent assay. Coexposure to Alcalase caused a significant increase in Lipolase IgG antibodies compared to Lipolase alone. The authors conclude that the presence of proteolytic enzymes such as Alcalase and Savinase in a mixture enhances antibody responses to other enzymes in the mixture. This should be taken into account when establishing exposure guidelines for protease containing enzyme mixtures. Non-Human Toxicity Values LD50 Rat oral 3700 mg/kg
References	[1]. Philipp M, et al. Kinetics of subtilisin and thiolsubtilisin. Mol Cell Biochem. 1983;51(1):5-32. [2]. Azrin NAM, et al. Versatility of subtilisin: A review on structure, characteristics, and applications. Biotechnol Appl Biochem. 2022 Dec;69(6):2599-2616.
Additional Infomation	Bacillomycin has been reported in Bacillus subtilis and Saccharomyces cerevisiae with data available.

Solubility Data

Solubility (In Vitro)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Solubility (In Vivo)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.  (Please use freshly prepared in vivo formulations for optimal results.)