Subtilisin

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Products
Physical properties and Specificity
Activity Determination and Optimal Conditions
Storage and Stability
Solubility and Solution Stability
References

Synonyms: Subtilisin Carlsberg, Subtilopeptidase A, Bacterial Alkaline Protease
CAS #: 9014-01-1
EC #: 3.4.21.62

Sigma's Subtilisins are produced from fermentation of Bacillus licheniformis. We offer several grades and formulations including high purity lyophilized powders as well as economical industrial grade solutions.

Physical properties and Specificity
MW: Subtilisin is a non-glycosylated single polypeptide chain without disulfide bonds and a of MW 27 KDa.²
Specificity: Subtilisin A is a member of the Serine S8 Endoproteinase family. It has broad specificity with a preference for a large uncharged residue in the P1 position. It hydrolyzes native and denatured proteins, and is active under alkaline conditions.
Isoelectric point: pI = 9.4
E^1%(280nm) = 8.6
Sedimentation coefficient: S_20,w = 2.77³
Alcalase® (Prod Code P4860) Liquid Density: 1.25 g/ml

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Activity Determination and Optimal Conditions
Unit definition: One Sigma Unit will hydrolyze casein to produce color equivalent to 1.0 mmole (181 µg) of tyrosine per minute at pH 7.5 at 37 °C (color by Folin-Ciocalteu reagent).

The activity Alcalase is reported in Anson units. (One Anson unit is defined as the amount of enzyme which, under specified conditions, digests urea-denatured hemoglobin at an initial rate such that there is liberated an amount of TCA-soluble product per minute which gives the same color with Folin-Ciocalteu Phenol reagent as one milliequivalent of tyrosine at 25 °C at pH 7.50.)² Based on average values, one Anson unit = 370 Sigma units.¹

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Storage and Stability
Subtilisin A (Prod Code P5380) is supplied as a powder, essentially free of buffer salts. Supplier studies indicate that the solid will lose less than 10% of its activity per year if stored at 4 °C. Long-term storage in excess of 20 °C is to be avoided.

Alcalase 2.4L (Prod Code P4860) is supplied as an aqueous solution. It is stable for at least one year when stored at 2-8 °C and 3 months when stored at 25 °C.

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Solubility and Solution Stability
Sublilisins are typically soluble in water at all concentrations which occur in normal usage. Sigma tests P5380 in 10 mM sodium acetate buffer, containing 5 mM calcium acetate, pH 7.5, for assay purposes, and obtains a clear solution.¹
This enzyme is stable 1-2 days at 4 °C as a 100-200 mg/mL solution in 0.1 M borate, pH 8.0, containing 0.1 M calcium chloride.³
Subtilisin A is reportedly active in some organic solvents such as dry octane.⁴

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Products

References

1. Sigma quality control.
2. Supplier information.
3. P. Boyer, The Enzymes, ed., vol. III, 3rd ed., p. 564.
4. Zaks, A. and Klibanov, A.M., J. Biol. Chem., 263, 3194?3201 (1988).

Additional References
Neidhart, D.J. and Petsko, G.A., Protein Engineering, 2, 271-276 (1988). Crystal structure of subtilisin Carlsberg.
Jacobs, Myra, et al., Nucleic Acids Research, 13, 8913-8926 (1985). Cloning, sequencing and expression of subtilisin Carlsberg.
Ottesen, M. and Svendsen, I., Methods in Enzymology, XIX, 199. Subtilisins.
Hannappel, E., et al., Archives of Biochem. Biophysics, 214, 293-298 (1982). Limited proteolysis of aldolases.

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