Practical Protein Chemistry - A. Darbre 1989

Fragmentation of Polypeptide Chains by Enzymatic Methods
Proteases with Low Specificity
Thermolysin

Thermolysin is isolated from Bacillus thermoproteolyticus. The enzyme is widely used in protein sequencing studies. Thermolysin is commercially available from Calbiochem and several other suppliers. Detailed information regarding the Applications of this enzyme can be found in [41, 65].

3.6.2.1. Specificity and Hydrolysis conditions.

Specificity. The enzyme hydrolyzes N-terminal peptide bonds involving hydrophobic amino acid residues of the -X-H- type, where H is most commonly Val, Leu, Ile, Phe, Tyr, or Trp. However, the Specificity of the enzyme is considerably broader, as it also cleaves Alanine peptide bonds at a fairly high rate. Hydrolysis at Asn, Thr, His, and Gly residues is observed quite rarely. Among numerous studies on thermolysin specificity, works [1, 41] deserve special attention. The Pro-Ile- segment is susceptible to the enzyme's action, whereas the -X-Phe- bond in sequences of the -X-Phe-Pro- type remains resistant. Thermolysin hydrolysis is inhibited by adjacent α-amino or carboxyl groups; therefore, the enzyme lacks exopeptidase activity. Amino and carboxyl groups in the side chains of amino acid residues do not exert a significant effect on Enzyme Specificity.

Hydrolysis conditions. Thermolysin exhibits activity within the pH range of 7.0–8.0 and requires the presence of Ca²⁺ ions. Hydrolysis is typically performed in 100 mM NH₄HCO₃ containing 5 mM CaCl₂. Most commercial enzyme preparations contain a substantial amount of Ca²⁺, which is generally sufficient for enzymatic activity. The enzyme is inhibited in the presence of EDTA. Treating Proteins at 37 °C for 4 h at an Enzyme-to-substrate ratio of 1 : 50 yields satisfactory results, although additional optimization studies may sometimes be required. Thermolysin is thermostable, retaining 100% of its activity upon incubation at 60 °C for 1 h and a significant fraction at 80 °C [65]. The enzyme is stable in 8 M urea, as well as in 20% ethanol or methanol; however, under these conditions, the likelihood of thermal Denaturation increases, and consequently, hydrolysis should be carried out at 20 °C.