Showing 25 results for Protease
Volume 3, Issue 2 (11-2012)
Abstract
Papain (EC2.22.4.3) is a thiol protease with high level of activity that has widespread industrial applications. The use of immobilized papain provides many advantages over its free form. In many applications, cysteine must be added as an activator. On the other hand, certain bivalent metal ions including Ca2+ behave as the inhibitors of papaein. In the present study, after preparation of Sepharose 6B with CNBr, a 5 mg/ml-protein solution was added to activate the gel for covalent attachment of enzyme and, subsequently, 2M glycine solution was added to block the remaining active groups on the gel. The immobilization process brought about significant enhancement of storage, thermal stability, stability at extreme pHs, and resistance against the inhibitory effect of bivalent metal ions with respect to papain. The optimum temperature of papain was increased by 20 °C (from 60 to 80 °C) and its optimum pH was shifted from 7 to 8.0 upon immobilization. Also km and kcat of the enzyme altered due to the immobilization process.These results are important in particular if one considers that the major problem in enzyme immobilization is the loss of enzyme activity and catalytic efficiency.
Volume 3, Issue 4 (12-2014)
Abstract
Entomopathogenic fungi produce a variety of degrading enzymes, including proteases, chitinases and lipases, to facilitate their entry through the massive barriers of insect cuticle. Isolates of the entomopathogenic fungi vary considerably in their proteolytic activity and virulence. The proteolytic activity of different isolates has been hypothesized to reflect their virulence toward the host. In this study, we evaluated the virulence and proteolytic activity of 17 Beauveria bassiana sensu lato isolates collected from different geographical regions in Iran. The selective medium D0C2 was used for isolating B. bassiana from soil samples. Casein substrate was used for protease assay. Total mortalities caused by different B. bassiana isolates through the dipping method, ranged from 25 to 60% with the highest and lowest rates for isolates BA and MITE, respectively. Our results revealed a wide variation in both proteolytic activity and virulence among the studied isolates. Additionally, we found a strong positive correlation between the proteolytic activity on Casein substrate and virulence of the isolates against the Khapra beetle, Trogoderma granarium. This finding will facilitate the screening and selection process of virulent fungal isolates as efficient agents for use in biological control programs of insect pests.
Mohammad Hosseinpour, Valiollah Jafari, Abdolmajid Abdolmajid, Abbasali Zendebodi,
Volume 4, Issue 1 (6-2015)
Abstract
The effect of simultaneous application of probiotic and digestive enzyme on the intestinal digestive activity of the whiteleg shrimp, Litopenaeus vannamei, was evaluated. For this purpose, the juvenile shrimps were fed for 30 days with a dietary probio-enzyme (containing a combination of six exogenous enzymes and four probiotic bacterial strains) at four concentrations of 0, 0.25, 0.5, and 1 g kg-1 feed. Shrimps (5.04±0.39 g ind-1) were randomly distributed in 12 aquaria (4 treatments × 3 replications); each aquarium contained 15 individual shrimps. Results indicated significantly (p≤0.05) higher growth performance, amylase and lipase activity at 0.5 g kg-1 treatment as compared to other treatments. Protease activity was, however, significantly (p≤ 0.05) higher at 1.0 g kg-1treatment as compared to other groups. Results also indicated that increase in the concentration of probiotic and enzymes supplementation was not associated with increase in all the digestive enzyme activity. In other words, probiotics and enzymes onlywithin specific range can have positive effect on growth performance and digestive enzyme activity of L. vannamei, above or below.
Volume 6, Issue 1 (10-2015)
Abstract
In this study, a Bacillus species was identified from the Dosarri mineral spring in Jiroft microflora. This strain produce clear halo in casein agar media. It has been identified as Bacillus Pumilus (KHB3) based on biochemical tests and 16S rRNA gene sequencing. To enzyme production, this strain was cultured in specific medium for 48 h. Supernatant was partially purified after precipitation with ammonium sulphate (85 %), dialysis and ion exchange chromatography (Q-Sepharose). KHB3 protease was characterized in the presence of different pHs, ions and detergents. Results indicated that the enzyme showed maximum activity and stability in pH 8.0. This enzyme retained about 100 % of its activity in the presence of 1.0 and 1.5 M NaCl. KHB3 protease showed 33 and 10 % increase in protease activity in the presence of MnSO4 and FeSO4. KHB3 protease retained at least 45 % of its activity and stability in the presence of commercial detergents. In addition, it show 12 % increase in enzyme activity in the presence of Banoo detergent. Activity and stability in alkaline pH, organic solvents and detergent compounds show that this protease has high value capacity in detergent industry.
A. Zamani, A. Moafi ,
Volume 7, Issue 1 (3-2018)
Abstract
Aims: Increasing the aquatic consumption, developing aquaculture, and the need for aquatic food production will make unclear the availability to fish oil in the future. The aim of this study was to investigate the effect of replacement of fish oil by grape seed oil on growth indices and protease enzymes activity in Rainbow Trout.
Materials & Methods: This study was conducted on 450 Rainbow Trout during a 60-day period. In this study, control diet (A) containing 100% fish oil and 25% (B), 50% (C), 75% (D), and 100% (E) grape seed oil were used instead of fish oil. The data were analyzed by Graph pad prism and SPSS 20 software, using one-way ANOVA test.
Findings: The highest final weight and weight gain was in diet C and the lowest was in D, having a significant difference. Specific Growth Rate (SGR) and Protein Efficiency Ratio (PER) had no significant difference. The highest and lowest feed conversion ratio (FCR) was observed in diets E and C, respectively, with a significant difference. The highest fat efficiency was in diet C. The highest feed efficiency was in diets C and D and the lowest was in E, and the diets were not significantly different. The most activity of pepsin and trypsin was observed in pyloric additions in diet C and in intestine in C and D. The optimal amount of fish oil replacement was satisfied by grape seed oil 50% (diet C).
Conclusion: The diet containing 50% fish oil and 50% grape seed oil is effective in improving the growth indices and activity of pepsin and trypsin enzymes in Rainbow Trout.
F. Movahedrad , A. Hajimoradloo , A. Zamani, H. Kolangi ,
Volume 7, Issue 3 (9-2018)
Abstract
Aims: In this study, the replacement effect of dietary fish meal with AquPro (Processed soybean meal) was assessed on growth performance, body composition and total protease activity of rainbow trout (Oncorhynchus mykiss) over a period of 6 weeks.
Materials and Methods: In this experimental study, at the beginning of the trial, 5 experimental diets were formulated with fish meal as control (A) and diets with 25% (B), 50% (C), 75% (D) and 100% (E) AquPro, respectively. At the end of the feeding trial, growth performance, body composition and total protease activity from pyloric caeca and intestine were measured.
Findings: The results indicated that the WG, SGR and ADG in experimental diet C were the highest and showed a significant difference with other treatments (p<0.05). However, the status index in experimental diet C was lower than other treatments, but did not show significant differences (p>0.05). The moisture content, fat, and body ash were respectively, significantly higher in experimental diets A, B and E than those of other treatments (p<0.05), But the highest amount of protein was observed in the experimental diet of C, which did not show any significant difference with other treatments (p>0.05). The highest total protease activity from pyloric caeca and the intestine was observed in the fish fed with diet C which showed a significant difference with other diets (p<0.05).
Conclusion: Based on the results of growth performance, body composition, and total protease activity was revealed that the diet containing 50% AquPro could be appropriate for rainbow trout growth.
Volume 8, Issue 1 (1-2019)
Abstract
Efficacy of quercetin on α-amylase, lipase and protease activities via crustacean cardioactive peptide (CCAP) content of the midgut of the diamondback moth, Plutella xylostella (L.) was investigated. Fresh cabbage leaf discs were dipped in quercetin solution at different concentrations (100, 500 and 1000ppm) for 10 seconds. Third instar larvae of P. xylostella were fed on leaf dipped in quercetin solutions for 5 days. α-Amylase, lipase and protease activities were evaluated for 5 days. Quercetin significantly decreased lipase, protease and α-amylase activities in the midgut. The results of competitive ELISA showed that different concentration of quercetin had no effect on short neuropeptide F, tachykinin-4 and allatostatin content of the midgut, but it was shown that quercetin (500 and 1000ppm) decreased CCAP content of the midgut. Moreover, incubation of dissected midgut with CCAP increased α-amylase, lipase and protease activities. The injection of CCAP into the hemocoel clearly increased α-amylase, lipase and protease activities. Here, for the first time, it was confirmed that feeding on leaf dipped in quercetin, decreases CCAP content in the midgut of P. xylostella, that itself led to decrease of α-amylase, protease and lipase activities.
Volume 8, Issue 29 (5-2011)
Abstract
In comparison with traditional extraction methods, aqueous enzymatic extraction of oil from oilseeds is a recent clean technology. This paper reports work performed at laboratory scale to extract soybean oil by aqueous enzymatic extraction method.
In the present work the influence of enzymes concentration, extraction time, dilution ratio, particle size and 3 interactions in the final yield are evaluated and process parameters have been optimized by Taguchi method.16 extraction experiments carried out, statistical analysis showed that particle size was the most significant variable in oil extraction. Themaximal oil extraction yield was 61.42%.
Volume 9, Issue 4 (12-2018)
Abstract
Aims: Alkaline protease is one of the most important groups of industrial enzymes with many applications. The aim of this study was to determine the physicochemical parameters affecting the production of alkaline protease enzyme produced by Bacillus pseudofirmus MSB22 by one-factor-at-a-time (OFAT) method and optimize the production of this enzyme by the response surface methodology (RSM) in the form of a rotatable central composite design.
Materials and Methods: In the present experimental study, the isolation of microorganism producing alkaline protease from wastewater from sausage and lunch meat factories in Isfahan was carried out. The morphological and biochemical characteristics of the strain were performed according to the Bergey's book and amplification of 16S rRNA gene sequences. Detection of metalloproteinase gene and alkaline serine protease was done by polymerase chain reaction (PCR) reaction and enzyme activity measurement was performed by Folin reagent. Screening of variables effective in enzyme production was done, using one-factor-at-a-time method and optimization was performed by response surface methodology. MEGA 6 software was used for phylogenetic analyses. To analyze the data, the Design Expert 7 software and the one-way analysis of variance were used.
Findings: The maximum protease production, which was 1.85 times higher than that of OFAT method and 3.45 times higher than unoptimized conditions was obtained, using 1% w/v xylose, 3% w/v beef extract, 4% v/v inoculation size, pH 10, and 30°C. The established quadratic model had a great ability to predict responses to new observations due to a high value of the predicted determination coefficient.
Conclusion: OFAT and RSM strategies are useful screening and optimization methods, respectively and sub I and sub II genes (alkaline serine protease genes) are detected in Bacillus pseudofirmus MSB22.
Volume 10, Issue 1 (3-2019)
Abstract
The fruit of has a lot of acidic proteases and its extract has been used in cheese manufacturing. However, there are few studies about purification and characterization of this enzyme. must be satisfied for the enzyme to be used in industry: 1- stability of enzymes against metal ions and 2- Ability to sustain proper function and stability in the absence of metal ion. Accordingly, in this investigation, the effect of various ions different concentrations activity, stability and somewhat on structural properties of the purified protease were studied. Based on the results, it was shown that the enzyme was relatively stable against NaCl and CaCl2, but by increment of these salts, stability and activity of enzyme . Also, the enzyme was stable against low concentration of various metal ions and only Hg2+ reduces enzyme stability and activity. By studying the role of 2+ of , it was found that 2+ have any role in thermal stability of enzyme at 67˚C. Likewise, by observing the effect of metal ions on of it was that all tested ions increased intensity of emission and caused to shift toward lower wave length. In all, of these showed that the purified enzyme from bad is very stable against various metal heavy metals and it is favorable for industrial application.
Volume 10, Issue 1 (3-2019)
Abstract
Enzymes of marine organisms are ideal candidates for biomonitoring of pollution in marine environments. For the widespread use of enzymes in industrial processes, carried out under certain physico-chemical conditions, their stability must be improved. In this study, for the first time, chitosan nanoparticles were used as matrices for augmenting the stability of Penaeus vannamei (Whiteleg shrimp)-derived purified proteases against metallic ions. For the electrostatic binding of the enzyme to the chitosan nanoparticles, the protein solution at a concentration of 7mg/ml was added to the nanoparticles, and incubated for 4 hours at 10°C. After 3 times rinsing with phosphate buffer of pH=7.5, the nano-enzyme was dissolved in 1ml phosphate buffer, and used for further studies. The results of this study showed that Fe2+ and Mn2+ significantly increased the enzyme activity, whereas a strong inhibitory effect was observed in the presence of Cd2+, Hg2+, Co2+, Ni2+, Cu2+ and Zn2+, and a weak inhibitory effect in the presence of Na+ and K+. The immobilized enzyme exhibited greater resistance to metal ions than its free counterpart. The free enzyme was susceptible to the presence of metal ions, and with the increment of their concentrations, enzyme activity declines. From this nexus, it could be inferred that the high stability of immobilized enzyme is due to the presence of chitosan nanoparticles. Stability retention of the immobilized enzyme at high concentrations of metal ions indicates the efficacy and utility of the immobilization method in industrial enzyme technology.
Volume 10, Issue 1 (3-2019)
Abstract
The Stability of protease in organic solvent media has been widely discussed for more than two decades. Proteases can catalyze synthetic reactions in organic media, by this way solvent stabilities of proteases are very important. In this study, we reported a bacterium isolated from hot spring of Geinarje, Iran producing an organic solvent stable protease. Protease producing bacteria were screened on skim milk agar and the formation of a clear zone around the bacterial colony was investigated. Proteolytic activity was assayed by a modified caseinolytic method using casein as a substrate. The best alkaline protease producing bacterium was selected and identified on the basis of 16S rDNA gene sequencing and morphological and biochemical characteristics. The effect of organic solvents, temperature, pH, and NaCl on proteolytic activity were examined. According to phylogenetic analysis, morphological and physiological tests, isolated, the bacterium was identified as a new strain of Brevibacillus borstelensis. This strain was able to produce an extracellular organic solvent-stable protease with 0.53U/ml enzyme activity. After 2 hour incubation at 30°C the protease of Brevibacillus borstelensis AMN was active in wide ranges of organic solvents, and its activity was enhanced in the presence of 25% (V/V) isopropanol. The biochemical properties of the enzyme revealed that the optimal pH and temperature for protease activity were 9.0 and 60°C, respectively. Our finding indicated that these robust properties of protease, like outstanding activity and stability in organic solvents and alkaline medium, might be applicable for various industrial biotechnologies.
Volume 10, Issue 4 (12-2019)
Abstract
The use of enzymes in organic solvents represents an important area of industrial and biotechnological development. However, organic solvents often cause protein denaturation, thereby reducing the activity and stability of enzymes. Use of stabilizing additives, protein engineering and chemical modification of enzymes are common strategies to overcome this problem. In this study, a cysteine protease from the latex of Ficus johannis was purified and the activity and stability of the protease were investigated in the presence of different organic solvents. The effect of trehalose, sorbitol, and sucrose on the enzyme activity was also studied in the presence of organic solvents. The results showed that the enzyme activity was elevated in the presence of low concentrations of organic solvents increased, while it was decreased with increasing concentration of organic solvents. However, the enzyme still retained 60% of its activity at 30% organic solvent concentration. The enzyme was considerably stable in the presence of organic solvents, maintaining almost 90% of its stability in the presence of 50% of all solvents. As stabilizing additives, sugars enhanced the catalytic activity and stability of the enzyme, and trehalose was the most effective sugar. The easy purification procedure and considerable activity and stability of the protease in the presence of organic solvents could suggest this enzyme as a good candidate for peptide synthesis industry.
Abbas Zamani, Mohammad Goli,
Volume 10, Issue 4 (12-2021)
Abstract
The present study was conducted to evaluate the effect of fish meal replacement with yellow meal worm (Tenebrio molitor) (TM) on growth performance and proteolytic enzymes activity of trypsin and alkaline protease in koi (Cyprinus rubrofuscus) juvenile (weight: 0.95 ± 0.15 g) for 8 weeks. Five experimental diets were prepared with replacement levels of 0 (control), with 25% (D1), 50% (D2), 75% (D3) and 100% (D4) of fish meal with TM as isonitrogenous and isoenergetic in triplicate. Parameters of body weight gain, specific growth rate and condition factor in control, D1 and D2 diets were not shown a significant difference, while these indices had a decreased amount in D3 and D4 diets significantly (p < 0.05). The highest amount of feed conversion ratio was indicated in control and D1 diets with a significant difference than those from other treatments (p < 0.05). The survival rate was 100 % in the diets. The lipid efficiency ratio and the protein efficiency ratio in diets control, D1 and D2 were significantly the higher than other treatments (p < 0.05). The highest activity of trypsin and alkaline protease enzymes from intestine was observed in control, D1 and D2 groups which showed a significant difference compared to D3 and D4 groups (p < 0.05). The findings of growth performance and protease enzymes activity were revealed that the diets containing up to 50% TM could be appropriate for koi growth.
Volume 11, Issue 1 (1-2022)
Abstract
The Sunn pest, Eurygaster integriceps (Puton) (Hem.: Scutelleridae), is an economic pest of wheat that reduces the quantitative and qualitative properties of wheat products. We investigated the effect of E. integriceps feeding on six different wheat cultivars (Ghermez (Red), Noodle, Antanius, Sabalan, Azar 2 (with two types of cultivation), and Sardari) on the protein content in the adult’s gut and fat body and their digestive enzymatic activity. All qualified values of the insect feeding on wheat cultivars differed significantly. The least amount of adult weight and protein content of gut and fat body were observed in the insects fed on Ghermez (Red) cultivar, and the highest amount belonged to the Sardari cultivar. The same results for protein content were obtained from SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Also, the lowest and highest gut α-amylase, pectinase, and protease activities were in Ghermez and Sardari cultivars, respectively. Therefore, it was concluded that the type of wheat cultivar affects the food preference of this insect and, thereby, physiological parameters of the insect gastrointestinal tract. Planting a wheat cultivar like “Ghermez,” which may be resistant, can be a suitable and cost-effective method to decrease the chemicals applied against this pest.
Volume 14, Issue 4 (3-2012)
Abstract
Objective: The use of antiretroviral drugs has proven remarkably effective in controlling the progression of human immunodeficiency virus (HIV) disease, but these benefits can be compromised by the development of drug resistance. This study aims to assess the drug resistance profile of the Pr gene in highly active antiretroviral therapy (HAART)-treated and naïve HIV-1 infected patients.
Methods: A total of 30 samples from naïve and 16 samples of highly active antiretroviral therapy (HAART)-treated patients were collected and divided into two groups. After RNA extraction, RT nested PCR was performed. The final products were sequenced and then analyzed for drug-resistant mutations and subtypes.
Results: No drug resistant mutations were noted in group one that have never used drug, but 40% of group two samples which are under treatment contained drug resistant mutations. According to the results, the following subtypes were seen among patients: A (50%), B (40.6%), D (6.2%), and C (3.2%).
Conclusion: Transmission of drug-resistant viruses and their detection are very important epidemiologically. However our data and other studies suggest that other PIs should be replaced by LPV in the HAART regime.
Volume 14, Issue 62 (4-2017)
Abstract
The effect of addition of a commercial enzyme derived from Bacillus Polymyxa type IX on Iranian UF-Feta cheese during a 60- day ripening period on chemical composition, pH and proteolysis of cheese samples was investigated. No Significant differences were observed in the chemical composition between experimental and control cheeses. In experimental cheese, pH values were significantly (P< 0.05) higher throughout whole ripening period and at 60d it was 4.6 and 4.77 in control and experimental cheeses, respectively. Soluble nitrogen in pH=4.6 was significantly (P< 0.05) higher in experimental cheese at 45 and 60d. At 60d, this index was 6.38 and 7.73% in the control and experimental treatments, respectively. Significant difference in the level of trichloroacetic acid (TCA) 12% was also observed at 45 and 60d. Urea- polyacrylamide gel electrophoresis of the pH 4.6-insoluble fractions showed that αs1-casein was hydrolysed faster than the β-casein. Intact αs1-casein values from 100 % on the first day reached to 82.93 % and 71.24 % and in β- casein reached to 90.28 and 90.56% at 60d in control and experimental cheeses, respectively. The concentration of total free amino acids in whole ripening period was significantly (P< 0.05) higher in experimental cheese and at the end of the ripening period was 0.68 versus 0.54 in control cheese (mg Glycine/g cheese). Levels of individual free amino acids were also different between treatments.
Volume 15, Issue 1 (3-2023)
Abstract
Hepatitis C virus (HCV) NS3/4A Serine protease is an important drug target for treating patients with hepatitis C virus. However, its amino acid mutations, particularly A156G, commonly lead to the rapid emergence of drug resistance. Bosiprevir, simiprevir, and viniprevir drugs approved by the FDA show distinct resistance profiles against the HCV NS3/4A protease. In order to show the behavior of each of these drugs in the interaction with the protease in the wild type and A156G mutant, molecular dynamics simulation and binding free energy calculations were performed. MMPBSA-based binding free energy calculations showed that the binding affinity of each of the drugs in the interaction with NS3/4A protease in the wild type is significantly more than the interaction with the protease in the A156G mutant state. Free energy landscape (FEL) calculations revealed that in the presence of each of the drugs, more basins of conformations are formed. We hope that our data can provide useful insights for the design of a new effective inhibitory drug for the treatment of patients with the hepatitis C virus.
Volume 15, Issue 1 (3-2023)
Abstract
The COVID-19 pandemic has created a global health crisis, and developing effective treatments is essential to prevent the spread of the disease and save millions of lives. One of the key proteins involved in the replication cycle of SARS-CoV-2, the virus that causes COVID-19, is the main protease enzyme, 3CLpro. Due to its high importance, this enzyme is the subject of molecular, structural, and clinical investigations, and efforts have been made to develop drugs that can inhibit its activity. One such drug is the chemical compound N3, which has been found to have a high inhibitory effect against 3CLpro. However, traditional medicine perspectives on this issue have been less explored. In this research, molecular docking interaction simulation and all-atom molecular dynamics (MD) simulation were conducted to study the potential inhibitory capability of generally available 21 plant-extracted compounds against the 3CLpro enzyme. Three compounds with the highest inhibition probability were selected from the molecular docking results and subjected to 100 ns of MD simulation to investigate their stability and structural-dynamic-energetic features. Beside the complexes stability, the results from the simulation demonstrated that, all our selected three compounds induce N3 comparable structural-dynamics characteristics to 3CLpro and, therefore, are expected to have a similar inhibitory ability against this enzyme. Compound number 5 was found to have the most favorable binding energy and was proposed as the best plant substitute for N3. The results from this research can be directly used to design experimental research for 3CLpro enzyme inhibition, saving the time-financial cost.
Volume 16, Issue 5 (9-2014)
Abstract
The date palm fruit stalk borer is one of the most important pests of date palm in the world. Biochemical properties of digestive proteases in Oryctes elegans Prell larvae were investigated in this research and optimal total proteolytic and trypsin activities were obtained at pH 9.0 and 11.0, respectively. Activity staining of protease on SDS-PAGE showed one isoform. Also, zymogram pattern of trypsin using nitro-cellulose membrane revealed two isoforms. The inhibitory effect of PMSF, TLCK, TPCK, EDTA, iodoacetate and iodoacetamide were determined on O. elegans proteolytic activity. The iodoacetamide showed the highest inhibition on total proteolytic activity. Therefore, cysteine protease accounted for the major proteases in the gut of O. elegans. Total proteolytic activity was inhibited 22.3 and 12.15% by inhibitors extracted from Vicia faba and Lathyrus sativus, respectively. However, the inhibitors extracted from seeds of Prosopis farcta, Panecum miliaceum, and Alhagi maurorum showed negligible inhibitory effects on proteolytic activities. Trypsin activity was inhibited 91.5 and 82.3% by inhibitors extracted from V. faba and L. sativus, respectively. Electrophoretic analysis showed that inhibitors extracted from V. faba reduced the intensity of total proteolytic and trypsin activities. The inhibitor from V. faba was purified by ammonium sulfate precipitation and gel-filtration, also the molecular mass of inhibitor was determined 35 kDa. This purified inhibitor was able to inhibit trypsin activity by 72.7%. In addition, the highest inhibition of trypsin activity by inhibitor from V. faba occurred at pH 11.0. Also, the stability of inhibitor from V. faba was evaluated at different pHs and temperatures. This inhibitor was stable at pH 11.0 and 30 °C.
