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Official Full Name
Thermolysin is a thermostable neutral metalloproteinase enzyme produced by the Gram-positive bacteria Bacillus thermoproteolyticus. It requires one zinc ion for enzyme activity and four calcium ions for structural stability. Thermolysin specifically catalyzes the hydrolysis of peptide bonds containing hydrophobic amino acids. However thermolysin is also widely used for peptide bond formation through the reverse reaction of hydrolysis. Thermolysin is the most stable member of a family of metalloproteinases produced by various Bacillus species. These enzymes are also termed 'neutral' proteinases or thermolysin-like proteinases (TLPs).
thermolysin; Bacillus thermoproteolyticus neutral proteinase; thermoase; thermoase Y10; TLN; EC

Product Name
EC No.
CAS No.9073-78-3
CAS No.9073-78-3
SourceGeobacillus ste...
CAS No.9073-78-3
SourceBacillus thermo...
Related Reading


Thermolysin is a thermostable neutral metalloprotease produced by the Gram-positive bacterium Proteus thermophilus. One zinc ion is required for enzyme activity, and four calcium ions are required for structural stability. Thermolysin can specifically catalyze the hydrolysis of peptide bonds containing hydrophobic amino acids. However, Thermolysins are also widely used to form peptide bonds through the reverse reaction of hydrolysis. Thermolysins are the most stable member of the metalloprotease family produced by various Bacillus species. These enzymes are also known as "neutral" proteases or thermolysin-like proteases (TLN).


Thermophilic proteases are firstly pro-enzymes synthesized by bacteria. Thermolysin is synthesized as a proenzyme consisting of a 28 amino acid long signal peptide, a 204 amino acid long propeptide and a 316 amino acid long mature enzyme. The signal peptide signals the translocation of protohemolysin to the plasma membrane of bacterial cells. Then in the periplasm, prolysin is processed into prolysin by signal peptidase. This presequence then acts as a molecular chaperone and results in the automatic cleavage of the peptide bond connecting the presequence and the mature sequence. Then secrete the mature protein into the extracellular medium.


The molecular weight of thermolysin is 34,600 Da. Its overall structure consists of two roughly spherical domains, with deep fissures traversing the middle of the molecule to separate the two domains. The secondary structure of each domain is quite different. The N-terminal domain is mainly composed of β-sheets, while the structure of the C-terminal domain is mostly α-helix. The two domains are connected by a central alpha helix, spanning amino acids 137-151. In contrast to many proteins that undergo conformational changes during heating and denaturation, thermolysin does not undergo any major conformational changes until at least 70°C.

Properties of TLN

The thermal stability of TLN series members is measured by the T50 temperature. Incubation at this temperature for 30 minutes will reduce the enzyme activity by half. The T50 value of thermolysin is 86.9°C, making it the most thermally stable member of the TLN series. Research on the contribution of calcium to the stability of Plasmin shows that after heat inactivation, a single calcium ion is released in the molecule. By changing the binding site to prevent the calcium from initially binding to the molecule, the stability of thermolysin will be reduced by 7°C. However, although calcium binding plays an important role in stabilizing thermophilins, what is more critical for stability is a small cluster of N-terminal domain amino acids located on the surface of the protein. In particular, phenylalanine (F) at amino acid position 63 and proline (P) at amino acid position 69 have a significant contribution to the stability of the heat-soluble protein. In unstable thermophilin-like proteases produced by Bacillus stearothermophilus (TLN-ste), these amino acids were changed to threonine (T) and alanine (A), respectively, resulting in decreased stability 7 °C (F63→T) and 6.3 °C (P69→A), the stability decreases by 12.3°C.


  1. Endo, S. Studies on protease produced by thermophilic bacteria. J. Ferment. Technol. 1962, 40: 346–353.

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