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 enzyme is a heat-resistant metalloproteinase enzyme that binds (EC 3.4.24.4) a peptide containing hydrophobic amino acid leucine and phenylalanine. This enzyme`s immobilization is very important. Enzyme immobilization enhances the stability and reuse of the enzyme and improves the properties of the enzyme and facilitates the enzyme readiness of the product. In order to study the adsorption in the presence of ethanol, different concentrations of ethanol were investigated with enzyme. To investigate the presence of CMC, the enzyme was mixed with different percentages of CMC solution and the casein (substrate) was mixed with different amounts. The effect of CMC and alcohol on the enzymatic solution of CMC was obtained. The effect of CMC and alcohol was investigated. The effect of CMC 2%, 4%, 6%, 8% and 10% with 1% casein on the enzyme was investigated. In this study, it was shown that thermolysin increases activity and stability against temperature rise and then decreases at very high temperatures, and alcohol causes enzyme instability. CMC increases the activity and stability of the thermolysin enzyme, but the presence of CMC can increase the stability and resistance of the thermolysin enzyme to alcohol. CMC has been shown to stabilize thermolysin.

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.

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 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.

Enzyme-assisted hydrolysis of the proteins from foodsources is an effective way to generate peptides with various bioactive properties. Furthermore, enzyme immobilization is a way to recycle enzymes for thefuture uses.The objective of this study was to investigate the effects of neutrase and thermolysin immobilization on the enzyme properties and in vitroanti-diabetic properties of intestinal digests achieved by the simulated digestion of egg white protein hydrolysates.

Neutrase and thermolysin were immobilized on cellulose-coated magnetite nanoparticles. Then, enzyme activity, thermal resistance, reusability and optimum conditions of the egg white protein hydrolysis were assessed. Egg white protein hydrolysates were then digested in vitroand inhibitory activities of the intestinal digests against dipeptidyl peptidase IV (DPP-IV) and α-glucosidase were investigated.

Enzymes immobilizationresulted in increasesin the thermal stabilities of them. Optimum temperatures for the egg white protein hydrolysis increased by 4.0 and 3.2 °Cfor neutrase and thermolysin, respectively.Digests from the hydrolysates of free neutrase effectively inhibited DPP-IV and α-glucosidase by 17.9 and 29.7%, respectively. These values for the hydrolysates released by the free thermolysin were higher (37.2 and 35.1%, respectively). The enzyme immobilization resulted in a 4.4% decrease in DPP-IV inhibitory activities of the digests for the hydrolysates from neutrase and a 28.6% decrease for those from thermolysin. Decreases in α-glucosidase inhibition due to the immobilization included 9.8% for neutrase and 12.2% for thermolysin for the digests from the hydrolysates. Based on the results from the current study, hydrolysates from the egg white proteins achieved by the free and immobilized neutrase and thermolysin can be used in formulations of the functional foods and nutraceuticals with multifunctional properties.

Pyruvate kinase is a key enzyme in glycolytic pathway that catalyzes the transphosphorylation between phosphoenolpyruvate and ADP to yield ATP and Pyruvate. Geobacillus stearothermophillus has a stable pyruvate kinase with determined crystal structure that composed of four separate domains. Given that limited proteolysis experiments can be successfully used to probe conformational features of proteins, in this study we obtained useful information on Geobacillus pyruvate kinase using limited proteolysis with two proteases that have different substrate specificity and optimum temperature of activity, trypsin and thermolysin. Proteolytic patterns at different temperatures indicate that resulting fragments were the same but the rate of digestion increased with temperature. In the next step, Sucrose and Glycine were used to examine the effects of additives on stability and activity of pyruvate kinase. Limited proteolysis was carried out at 37 °C by trypsin and at 30, 55 and 60 °C in presence of thermolysin, in the absence and presence of different concentrations of sucrose (0– 1.5 M) and glycine (0–1.5 M). We observed that stabilization of pyruvate kinase by this osmolytes is concentration dependent and the rate of limited proteolysis in presence of additives, at temperatures above 60 °C decrease; however, there was no any effect on proteolytic patterns. In all experiments the activity of pyruvate kinase was determined with a couple assay methods by luciferase. A clear correlation was observed between proteolytic digestion and enzyme activity. This study reveals a number of flexible and protease-prone regions of pyruvate kinase that exist regardless of the environmental conditions.