Thermolysin

Thermolysin ⁽¹⁾, a proteinase (EC 3. 4. 24. 4) produced by Bacillus thermoproteolyticus Rokko ⁽²⁾ is characterized by its excellent heat stability and substrate specificity.

Optimum activity pH: 7.0-9.0 (casein-Folin method)
Optimum stability pH: 5.5-7.5
Remaining activity after 1 hr at 80 degrees C: 50% (in water containing calcium ion)
Substrate activity: Amino sites of Leu, Ileu, Val and Phe were hydrolyzed using cytochrome C ⁽¹⁾, insulin ^(3,4), and tabacco mosaic virus protein ⁽³⁾ as substrates.
Elastolytic property: Substrate specificity of Thermolysin was exactly the same as that of the Pseudomonas aeruginosa elastase, but the elastolytic activity of Thermolysin was about ten times greater than that of the P. aeruginosa elastase and about four times that of pancreatic elastase ⁽⁴⁾.
Activators: No activator is required. However, calcium ion enhances the thermostability and usually M/500 is sufficient. Sodium ion has no effect. In order to expect the full activity of Thermolysin, the enzyme solution should be adjusted to pH 8.0 before it is brought in contact with substrate, but no duration is required.
Inactivators: Calcium blocking agents such as EDTA, polyphosphate, etc., and non-specific protein denaturants. Calcium precipitating salts such as phosphate also reduces the stability. SH reagents, soybean trypsin inhibitor and potato inhibitor do not inactivate Thermolysin. 0.1M sodium chloride selectively inhibits the elastolytic activity without diminishing the proteolytic activity ⁽⁴⁾.
Molecular weight: 37,500 ⁽⁹⁾
Extinction coefficient: E (280nm, 1%) = 17.65 ⁽⁹⁾ at pH 7.0
Activity: 7,000 PU/mg of enzyme protein
Amino acid composition ⁽⁹⁾: Asp43 Glu20 Thr23 Ser23 Pro8 Gly36 Ala28 Val24 Leu17 Ileu18 Cys-Met2 Tyr 29 Phe10 Try5 Lys12 His9 Arg10 (NH3) 38
Solubility: About 0.02% in dilute buffer solution at the neutral pH, about 4 % in 40-50 % glycerin. Freeze dried preparation is freely soluble in water, but crystallization is shortly to occur in a thick solution.
Uses
(1) Studies of the primary structure of proteins
(2) Studies of the structure of Thermolysin in relation to enzymatic activity and thermostability
(3) Digestion of proteinous substances
References:
1. Matsubara, H., Singer, A., Sasaki, R., and Jukes, T.H., Biochem. Biophys. Res. Commun., 21, 242 (1965)
2. Endo, S., J. Fermentation Tech., 40, 346 (1962)
3. Matsubara, H., Sasaki, R., Singer, A., and Jukes, T.H., Arch. Biochem. Biophys., 115, 324 (1966)
4. Morihara, K., and Tsuzuki, H., Biochem. Biophys. Acta, 118, 215 (1966)
5. Ohta, Y., and Ogura, Y., J. Biochem., 58, 607 (1965)
6. Nagai, Y., Sakaibara, S., Noda, H., and Akabori, S., Biochem. Biophys. Acta, 37, 566 (1960)
7. Matsubara, H., Biochem. Biophys. Res. Commun., 24, 427 (1966)
8. Morihara, K., and Ebata, M., J. Biochem., 61, 149 (1967)
9. Ohta, Y., Ogura, Y., and Wada, A., J. Biol. Chem., 241, 5919 (1966)
10. Daiwa Kasei Tech. Bull, No. 13
11. Hagihara, B., Ann. Rep. Sci. Osaka Univ., 2, 35 (1954)
12. Anson, M.L., J. Gen. Physiol., 22, 79 (1938)