جستجوی مقالات مرتبط با کلیدواژه "thermolysin" در نشریات گروه "زیست شناسی"

Thermolysin- like proteases (TLPs) belong to subset of Zn-metalloproteases superfamily. The family includes a zinc ion which plays a critival role in the function of enzyme and some different calcium ions to improve stability of the structure. The enzyme function of this family depends on specific conformational motions of active site. The active site in this family is located between N-terminal and C-terminal domain and it has been proposed that TLPs endure a hinge-bending motion during catalysis resulting in “closure” and “opening” of the active-site cleft and raise the probability of structural changes during catalytic process. In this study, the activity of thermolysin compared with elastase achieved from Pseudomonas aeruginosa was investigated and structural changes of the enzymes were investigated by molecular dynamics simulation. The results disclosed that, on one hand- due to reduced hinge-angle- thermolysin tends to greater affinity to the substrate, however, the open mouth of active site represents more freedom of movements of catalytic residues in elastase and therefore catalytic rate constant (kcat) is higher. In general, catalytic efficiency of two enzymes does not reveal much difference at 60 °C.

Thermolysin, obtained from bacillus thermoproteolyticus, is a thermophilic protease having application in peptide synthesis. Previous studies have revealed that salt activates the enzyme. Thermal stability of TLN is critically dependent on four calcium ions bound in N- and C-terminal domains. The aim of present study is unraveling the role of calcium ions in the salt activation of enzyme. On the presence of NaCl 2 M, measurement of the activity of enzyme in the absence and presence of CaCl2 10 mM using FAGLA as substrate showed activation of enzyme using FAGLA and in the presence of CaCl2 10 mM increased 2.5 times. Circular dichroism and fluorescence spectroscopies were used to understand the calcium dependent changes in the enzyme structure. Circular dichroism spectra suggest that the overall structure of thermolysin has not changed during salt and calcium activation. However, fluorescence studies revealed that the active site has changed under calcium-dependent salt activation. According to the results of the present study, the binding of calcium ions might influence the activation of thermolysin by salt.

Extensive research has been conducted on the stability of proteases in organic solvents, because they have numerous applications in organic media. Thermolysin from Bacillus thermoproteolyticus is a highly thermostable Zn-metalloprotease and is applied in industry for peptide synthesis, a reaction essentially performs in organic media. However, thermolysin has low stability at organic solvents.In contrast, its homologous enzyme, Pseudomonas aeruginosa elastase (PAE) is an organic solvent stable protease. In the present study, recombinant PAE was purified and compared with thermolysin in different organic solvents. For this purpose, activity of enzymes was measured in ethanol, methanol, isopropanol, dimethylformamide, glycerol and ethylenglycol (0-50 % (V/V)). Except isopropanol and ethanol, not only the elastase activity was not decreased, but also strongly increased in organic solvents. However, activity of thermolysin was abolished in all organic solvents. In the presence of DTT 1mM, the PAE activity was notably decreased in organic solvents. Considering that PAE contains two disulfide bonds at N- and C-terminal domains, it may suggest that disulfide bonds play a role in organic solvent stability of PAE.

Thermolysin is a thermostable protease produced by Bacillus thermoproteolyticus. This enzyme is industrially applicable especially for peptide synthesis. Due to industrial applications، numerous investigations have been performed on thermolysin. In the present study، the role of calcium on thermal، acidic pH، denaturant (urea and SDS) and salt stabilities was studied. In the absence of calcium، t1/2 at 80، 85 and 90 ÛC was 7، 3 and 1 min، respectively. However، in the presence of 10 mM calcium، t1/2 at the same temperatures was >95، 45 and 16 min، respectively. On the other hand، calcium had marginal effect on the pH stability of enzyme. The analyses revealed that SDS and urea contrarily affect on the enzyme stability. The concentration of SDS by which the enzyme activity diminished by half was 0. 1 and 0. 9 (%W/V) in the presence and absence of calcium، respectively. Oppositely، urea did not reduced and even promoted the enzyme activity in both conditions. Stability of thermolysin in NaCl up to 3M was slightly increased in the presence of calcium while slightly decreased in the absence of calcium. At higher concentrations of NaCl، however، the stability was decreased in both conditions. These results reveal that thermal and SDS stability of thermolysin are strongly Ca-dependent، stability against NaCl and urea are moderately Ca-dependent، and its acidic pH stability is Ca-independent.