Thermolysin is a heat-stable metalloprotease produced by Geobacillus stearothermophilus (Former: Bacillus stearothermophilus). Thermolysin specifically hydrolyzes peptide bonds on the amino side of bulky hydrophobic residues such as Leu, Ile, Val, and Phe. Thermolysin has one zinc ion necessary for activity and four calcium ions for stability. The amino acid sequence and three-dimensional structure of this enzyme have been determined.
Features
- Amorphous powder, freeze-dried (Purity: Two times crystallized)
- Optimum temperature: 65 - 70°C
- Optimum pH: 8.0
- Stable pH range: 5.0 - 8.5
- Stable temperature: Considerably stable at below 70°C
- Inhibitors: EDTA, SMPI (Streptomyces metalloprotease inhibitor)
- Solubility: approx. 0.02% in dilute buffer solution; approx. 4% in 40-50% glycerin
- Molecular weight: 34,600
- E1% 1cm (280nm): 17.65 at pH 7.0
The data for genera enzyme features and enzyme activity are based only on our laboratory experiment results. Therefore, different experiment conditions may yield different results.
Specification
Protein digestive activity (pH 7.2): Not less than 7,000 PU/mg.
Assay method
This method is based on the Hagihara modification of the Anson method.
Unit definition
One protease unit (PU) is defined as the amount of the enzyme that will liberate non-proteinous digestion products from milk casein (final concentration 0.5%) to give a Folin's color equivalent to 1μg of tyrosine per minute at the early stage of the reaction at 35°C, pH
7.2.The assay method is available on request.
Reagents
- Substrate: 1% milk casein (according to Hammarsten) containing 1/15M Na₂HPO₄-KH₂PO₄ buffer (pH 7.55). The pH value of this solution should be 7.2.
- TCA mixture: 0.1M trichloroacetic acid (TCA) containing 0.2M sodium acetate and 0.3M acetic acid.
- 0.4M Na₂CO₃
- Diluted Folin’s reagent: 5-fold
- Enzyme diluents: 0.01M borate buffer, pH 8.0, containing 0.002M CaSO₄ and 0.005% Triton X-100.
- 42% Glycerin (pH 8.0): 42% glycerin adjusted to pH 8.0 with 0.01N NaOH, containing 0.005% Triton X-100.
Test preparation
Weigh accurately about 100mg of Thermolysin, dissolve it in 42% glycerin (pH 8.0), and bring to 100ml with 42% glycerin (pH 8.0). Dilute it with enzyme diluents so that 1ml of the final dilution will contain between 5 and 10 PU.
Procedure
- Pipette 1.0ml of the test preparation into a test tube, and equilibrate it to 35°C.
- Add 1.0ml of substrate pre-warmed to 35°C, and mix.
- After exactly 10 minutes, add 2.0ml of TCA mixture to stop the reaction and incubate the mixture for a further 30 minutes at 35°C.
- Filter the mixture with filter paper.
- Pipette 1.0ml of the filtrate into a test tube, add 5.0ml of 0.4M Na₂CO₃ and then 1.0ml of diluted Folin’s reagent, mix, and incubate the mixture for 20 minutes at 35°C.
- Read the optical density at 660nm (ODₜ)
- Run a blank in which the order of adding substrate and TCA mixture is reversed (OD<sub>b</sub>)
Calculation
Activity(PU/ml)=(ODₜ−OD<sub>b</sub>)×4/A×10
4: Total volume (ml)
10: Reaction time (min)
A: Optical density of 1μg/ml tyrosine solution (dissolved in 0.2N HCl) according to procedures 5 and 6
Handling caution
- Do not inhale.
- In case of direct contact with skin or eyes, immediately wash or rinse with plenty of water.
- Please refer to MSDS for details.
General properties
The following results were obtained for the enzyme solution in some buffers. Please check appropriate conditions for your applications.
ZJ26F0333342