Subtilisin targets peptide bonds in proteins and peptide amides, catalyzing their hydrolysis. As a serine protease, it contains a catalytic triad (Ser, His, Asp) that is essential for its proteolytic activity. The enzyme has broad substrate specificity towards native and denatured proteins. Its primary targets are protein substrates, which it cleaves to produce smaller peptides and amino acids. No specific pharmacological receptor targets have been identified for this enzyme.

In vitro, subtilisin exhibits proteolytic activity by hydrolyzing proteins and peptide amides. The enzyme has a bioactivity of ≥200 U/mg powder and demonstrates robust activity, ease of production via fermentation, and compatibility with various chemical environments. It is known for its thermal stability and catalytic efficiency. The enzyme is used in peptide synthesis and protein modification applications. In research settings, subtilisin is used as a model enzyme for studying protease mechanisms.

In vivo, subtilisin has been studied for its immunogenic properties. Subtilisin (1 μg; s.c. or i.n.; twice for sensitization and twice for challenge; whole experimental cycle of 22 days) induces prototypic immune responses in animal models. Exposure to subtilisin can cause "washing powder lung," a form of hypersensitivity pneumonitis. The enzyme is used in industrial applications rather than as a therapeutic agent.

Non-cellular assays for subtilisin would involve measuring its proteolytic activity using synthetic substrates such as N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (SAAPF-pNA) or casein. Enzyme activity is measured spectrophotometrically by detecting the release of p-nitroaniline or other chromogenic products. Kinetic parameters such as Km, Vmax, and kcat can be determined using varying substrate concentrations. Inhibition studies can be performed using serine protease inhibitors such as PMSF or aprotinin.

Cell-based assays for subtilisin are not typically performed, as the enzyme is primarily used in industrial and biochemical applications. However, its effects on cells can be studied in the context of protease activity and protein degradation. The enzyme's immunogenic properties can be evaluated in immune cell activation assays. Cytotoxicity assays may be performed to assess its effects on cell viability.

In vivo animal studies for subtilisin typically involve sensitization and challenge models to study its immunogenic properties. Subtilisin (1 μg; s.c. or i.n.; twice for sensitization and twice for challenge; whole experimental cycle of 22 days) induces prototypic immune responses. These models are used to study hypersensitivity pneumonitis and other immune responses to protease exposure. The enzyme is also studied in the context of occupational exposure and respiratory health.

Pharmacokinetic data for subtilisin are not applicable, as it is an enzyme used in industrial applications rather than a therapeutic agent. When inhaled, subtilisin can cause respiratory sensitization and immune responses. The enzyme is not administered systemically for therapeutic purposes. Its biological activity is primarily localized to the site of application or exposure.

Interactions
Alcalase (9014011), Savinase (9014011), Termoyl (9000902), or Lipolase (9001621) in binary or ternary mixtures at concentrations of 0 to 0.3 μg were administered weekly via intratracheal infusion for 10 weeks. Alcalase and Savinase are proteases, Termoyl is an α-amylase, and Lipolase is a lipase. The mixture always contains either Alcalase or Savinase, or Termoyl, Lipolase, or both. Some mixtures contain Alcalase inactivated by pretreatment with hexamethylene isocyanate or diisopropyl fluorophosphate. Control animals were given only the single enzyme. Animals were observed for signs of respiratory distress, such as periodic diaphragmatic spasms or retractions. Blood samples were collected from the retro-orbital venous plexus before exposure and at 4, 5, 6, 8, or 10 weeks after exposure. Serum was isolated and circulating immunoglobulin (Ig)-G1 isotropic antibodies (HARs) were analyzed by passive cutaneous anaphylaxis. The number and severity of respiratory symptoms in guinea pigs exposed to mixtures containing Alcalase or Savinase were not significantly different from those exposed to Alcalase or Savinase alone. HAR titers against Lipolase and Termonyl were significantly increased when mixed with Alcalase or Savinase. The presence of other enzymes in the mixture did not significantly increase HAR titers against Alcalase or Savinase. The enhanced response produced in mixtures containing Alcalase or Savinase disappeared when inactivated forms of the enzymes were used in the mixture. Other guinea pigs were inhaled daily for 20 minutes for 5 days with a mixture of 500 μg/m³ lipase or lipase and 2000 μg/m³ alkaline protease. Challenge tests were performed again with the same aerosol 14 or 21 days after the last exposure. Blood samples were collected, and lipase-specific IgG antibodies were analyzed by enzyme-linked immunosorbent assay (ELISA). Co-exposure to alkaline protease significantly increased the level of lipase IgG antibodies compared to exposure to lipase alone. The authors concluded that the presence of proteolytic enzymes such as alkaline protease and saverase in the mixture enhances antibody responses to other enzymes in the mixture. This should be considered when developing exposure guidelines for mixtures containing proteases.

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Non-human toxicity values
Oral LD50 in rats: 3700 mg/kg

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Subtilisin can cause respiratory sensitization and hypersensitivity pneumonitis, known as "washing powder lung," upon inhalation exposure. It meets the specifications laid down by the Joint FAO/WHO Expert Committee on Food Additives and the Food Chemicals Codex. Lead content is less than 5 mg/kg, and total viable count is less than 10,000 CFU/g. Appropriate safety precautions should be taken when handling this enzyme to avoid inhalation exposure.

[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.

Reports indicate that Bacillus subtilis and Saccharomyces cerevisiae contain bacillus toxins, and relevant data is available for reference.

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Subtilisin is an industrial enzyme, not an approved pharmaceutical drug. It is an extracellular alkaline serine protease that catalyzes the hydrolysis of proteins and peptide amides. The enzyme is produced by Bacillus species and is commonly applied in laundry detergents, leather processing, peptide synthesis, and food protein modification. Subtilisin is known for its robust activity, ease of production via fermentation, and compatibility with various chemical environments. It is a valuable tool in biochemistry for studying protease mechanisms and enzyme structure-function relationships.

0

988.487

9014-01-1

3086051

Light-colored, free-flowing powders
Light-colored prills

1.3

13

15

18

70

1860

8

N#CC1=CC=C(CCl)C=C1

VLKSXJAPRDAENT-OWGHDAAGSA-N

InChI=1S/C45H68N10O15/c1-3-4-5-6-7-8-10-26-20-36(61)49-30(21-34(46)59)41(66)51-29(19-25-12-14-27(58)15-13-25)40(65)52-31(22-35(47)60)45(70)55-18-9-11-33(55)43(68)50-28(16-17-37(62)63)39(64)53-32(23-56)42(67)54-38(24(2)57)44(69)48-26/h12-15,24,26,28-33,38,56-58H,3-11,16-23H2,1-2H3,(H2,46,59)(H2,47,60)(H,48,69)(H,49,61)(H,50,68)(H,51,66)(H,52,65)(H,53,64)(H,54,67)(H,62,63)/t24-,26?,28+,29-,30+,31-,32-,33+,38+/m1/s1

3-[(3R,6R,9S,16S,19R,22S,25S)-3,9-bis(2-amino-2-oxoethyl)-16-[(1R)-1-hydroxyethyl]-19-(hydroxymethyl)-6-[(4-hydroxyphenyl)methyl]-13-octyl-2,5,8,11,15,18,21,24-octaoxo-1,4,7,10,14,17,20,23-octazabicyclo[23.3.0]octacosan-22-yl]propanoic acid

2934.99.9001

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.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

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

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.

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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).

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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)

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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).

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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

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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.

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 (Please use freshly prepared in vivo formulations for optimal results.)