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A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein dye binding
... Protein content, activity of enzyme, and specific activity were all evaluated in each experiment. The experiments involving the extraction buffer were conducted in duplicate (6,7). ...
... Treatment included 15 minutes of centrifugation at 10,000 rpm and filtering through paper. The supernatant was evaluated for activity of enzyme, protein content, and specific activity (6,7). ...
... Subsequently, the mixture was subjected to centrifugation at 10,000 revolutions per minute for 15 minutes (19). The filtrate was collected in order to assess activity of the enzymes, the amount of protein, and specific activity (6,7). ...
This study was aimed to test four types of different plants for the purpose of selecting the optimal plant as a source of the amylase enzyme, including wheat, barley, rice, and potatoes. Among these plants, wheat was selected as the best plant source for enzyme extraction because it possessed the highest effectiveness of the enzyme specific activity (3.0 U/mg protein). Furthermore, sodium acetate buffer (0.2 M, pH 6.0) was determined to be the optimal extraction buffer with a 1:5 (w:v) ratio after 75 min, and it provided 68 U/mg protein. The enzyme was concentrated with sucrose before being purified by gel filtration chromatography using Sephadex G-150. The results demonstrated a 1.8-fold increases in final purification folds with a 332% yield of enzymes. At pH 6.0, the purified enzyme showed its highest activity and stability. The activity of the purified enzyme was most effective at 45 °C and remained stable up to 37 °C. At concentrations of 5 and 10 M, various ionic and chemical substances had an impact on amylase. فعاليه اعلى نوعيه ل األميليز نزيم 3.0 ملغم / وحدة الصوديوم خالت محلول اختيار تم كما بروتين). 0.2 M ، pH 6.0) استخالص بنسبه استخالص محلول كأفضل 1 : 5 بعد ح) و: 75 النوعيه الفعاليه بلغت حيث دقيقة، 68 ملغم / وحدة تم بروتين. باستخدام الهالمي الترشيح افيا كروماتوجر اسطة بو النزيم تنقية ج سيفادكس هالم-150 التركيز بعد با لسكروز. في زيادة النتائج أظهرت ات مر عدد بمقدار النهائية التنقية 1.8 مرة مقدا انزيميه بحصيله رها 332 ٪. المنقى النزيم أظهر و الحموضة درجة عند وثبات نشاط أقصى 6.0. و المنقى النزيم لنشاط المثلى ارة الحر درجة كانت 45 ومستقر مئوية درجة عند 37 بتركيز الكيميائية و األيونية المركبات ببعض األميليز تأثر مئوية. درجة 5 و 10 ملم. اميليز، استخالص، مفتاحيه: كلمات ج سيفادكس مثلى، ظروف-150 ثباتيه ،
... After incubation, each flask's culture was centrifuged, and the filtrate's enzyme activity, protein content, and specific activity were all assessed. 13,14 . ...
... isolate was added. To determine enzyme activity, concentration of protein, and specific activity 13,14 , the supernatants were collected after centrifuging the cultures at 10,000 rpm for 10 minutes. ...
... After inoculating the medium with a bacterial culture isolate at a concentration of 3 x 10 11 cells per mL, the medium was incubated for 24 hours at 150 rpm and 37°C in a shaker incubator. Thereafter, measurements were made of the activity of enzyme, protein amount, and specific activity 13,14 . ...
The objective of this study was to isolate and identify the asparaginase-producing bacteria, then purify and characterize the enzyme in order to investigate their properties in the future. Fifteen local bacterial isolates were isolated from various sites in the city of Baghdad, identified by conventional morphological and biochemical procedures, and confirmed using vitek 2 methods, and submitted to primary screening processes for asparaginase production. For secondary screening, eight isolates with the greatest yellow zone ability on a specific solid medium were chosen. Bacillus sp. was reported to have the highest enzyme production (7.5 U/mg proteins). After 24 hours of incubation, submerged fermentation yielded optimal conditions for the production of L-asparaginase (L-ASNase) by the chosen isolate, with medium (2) serving as the optimal medium for production and fructose serving as the optimal source of carbon. In pH 6 at 40°C, Sephadex G-150 gel filtration chromatography was used to purify the enzyme. The final purification folds were increased by 2.5 times, resulting in an enzyme yield of 93.7%. It also showed the highest purified enzyme activity and stability was at 37°C. Also it revealed the highest activity and stability at pH 7.0 and pH 8.0 respectively. Enzyme lost activity when exposed to several metallic ions at concentrations of 1, 5, and 10 mM.
... Protein content, activity of enzyme, and specific activity were all evaluated in each experiment. The experiments involving the extraction buffer were conducted in duplicate (6,7). ...
... Treatment included 15 minutes of centrifugation at 10,000 rpm and filtering through paper. The supernatant was evaluated for activity of enzyme, protein content, and specific activity (6,7). ...
... Subsequently, the mixture was subjected to centrifugation at 10,000 revolutions per minute for 15 minutes (19). The filtrate was collected in order to assess activity of the enzymes, the amount of protein, and specific activity (6,7). ...
This study was aimed to test four types of different plants for the purpose of selecting the optimal plant as a source of the amylase enzyme, including wheat, barley, rice, and potatoes. Among these plants, wheat was selected as the best plant source for enzyme extraction because it possessed the highest effectiveness of the enzyme specific activity (3.0 U/mg protein). Furthermore, sodium acetate buffer (0.2 M, pH 6.0) was determined to be the optimal extraction buffer with a 1:5 (w:v) ratio after 75 min, and it provided 68 U/mg protein. The enzyme was concentrated with sucrose before being purified by gel filtration chromatography using Sephadex G-150. The results demonstrated a 1.8-fold increases in final purification folds with a 332% yield of enzymes. At pH 6.0, the purified enzyme showed its highest activity and stability. The activity of the purified enzyme was most effective at 45 °C and remained stable up to 37 °C. At concentrations of 5 and 10 M, various ionic and chemical substances had an impact on amylase.
... Optimum conditions for laccase extraction: Type of extraction buffer: Three types of buffers were examined to select the best one to be used for further experiments, the experiments were done by mixing the best plant samples obtained from the previous experiments. The experiments were conducted by mixing the plant sample for 15 minutes at room temperature, with 0.02 M sodium acetate (pH 4, 5, and 6), 0.02 M phosphate-buffered saline (pH 7, 7.6, and 8), and 0.02 M tris-base (pH 9, and 10) .The best buffer in terms of enzyme activity, protein content, and specific activity was measured using the same procedures (19,8,17). ...
... The type best buffer was derived from a previous experiment of 0.01, 0.02, 0.05, 0.1, 0.15, 0.2, 0.25, and 0.3 M was experimented to find the optimum concentration. The best concentration buffer in terms of enzyme activity, protein content, and specific activity was measured using the same procedures (19,8,17). ...
... Followed by centrifuging at 10000 rpm for 15 minutes. The measurements of enzymatic activity, amount of protein, and specific activity were conducted (19,8,17). Purification of enzyme with gel filtration chromatography Sephacryl S-300 preparation : A suspension of approximately 20 grams of Sephacryl S-300 was prepared in 500 millilitres of distilled water at a temperature of 90 o C. The suspension was subjected to gentle agitation for a duration of 3 hours to facilitate the swelling of the gel beads. Following this, the mixture was stored overnight in a refrigerator at a temperature of 4 o C, in accordance with the instructions provided by the manufacturer (Pharmacia-Sweden). ...
This study set out to screen 36 common plants which have the greatest level of laccase enzyme activity. It revealed that the enzymatic activity of fenugreek seeds was the highest in comparison with other plants. The optimal enzyme-specific activity was 5340.38 units per milligram protein which were obtained by extracting the enzyme with a sodium phosphate buffer at a concentration of 0.02 M and pH 8.0, at a ratio of 1:40 (weight to volume), and extracting time of 210 minutes. The enzyme yield was 27.6% after extraction and purification by gel filtration using Sephacryl S-3 after 1.01 purification fold. The optimum circumstances for enzymatic activity and stability were found by using 0.1 M sodium acetate as a buffer at pH 5. Also, the maximal activity and stability of purified laccase was obtained at 20 oC for 15 min by using o-tolidine as a substrate. This research sheds light on how to isolate and characterize the laccase enzyme, an important biochemical with numerous biotechnological and technological uses through fenugreek seeds as a source of the laccase.
... Soluble proteins were quantified following the procedure adapted from Bradford (1976) [85]. ...
... Soluble proteins were quantified following the procedure adapted from Bradford (1976) [85]. ...
Almond processing generates a high quantity of by-products, presenting the untapped potential for alternative applications and improved sustainability in production. This study aimed to evaluate whether the incorporation of almond by-products (hulls/shells) can improve the biochemical characteristics of green bean pods when used as an alternative to traditional growing media in green bean plants. Four substrates were prepared: the Control substrate (C): 70% peat + 30% perlite; substrate (AS): 70% peat + 30% shells; substrate (AH): 70% peat + 30% perlite + 1 cm hulls as mulch; substrate (MIX): 70% peat + 15% shells + 15% hulls. Plants were grown in each of these substrates and subjected to two irrigation levels, 100% and 50% of their water-holding capacity. Biochemical parameters (photosynthetic pigments, total phenolics, flavonoids, ortho-diphenols, soluble proteins, antioxidant capacity) and color were evaluated in the harvested pods. Results showed that pods from plants growing in AH substrate presented statistically significant higher values in their total phenolic content, while AS and MIX substrates did not reveal significant benefits. Summarily, this study highlights the potential of almond hulls as a promising medium for green bean cultivation, particularly when employed as mulch. Further research is recommended to gain a more comprehensive understanding of the application of almond by-products as natural fertilizers/mulch.
... The total protein was estimated in the homogenates using Bradford assay with bovine serum albumin (BSA) as a standard. 25 ...
... 7,[14][15][16] Based on these results, the present study sought to investigate the therapeutic potential of the S1PR1 agonist SEW2871 in a PHBresistant PTZ kindling model of epilepsy, specifically focusing on its effects on seizure frequency, neuroprotection and inflammatory markers. 20,48 Our findings indicate that the initial administration of PHB at 25 observed at a dose of 0.75 mg/kg. These findings align with previous research on an another S1P analogue, fingolimod. ...
Epilepsy is a prevalent neurological disorder characterized by neuronal hypersynchronous discharge in the brain, leading to central nervous system (CNS) dysfunction. Despite the availability of anti‐epileptic drugs (AEDs), resistance to AEDs is the greatest challenge in treating epilepsy. The role of sphingosine‐1‐phosphate‐receptor 1 (S1PR1) in drug‐resistant epilepsy is unexplored. This study investigated the effects of SEW2871, a potent S1PR1 agonist, on a phenobarbitone (PHB)‐resistant pentylenetetrazol (PTZ)‐kindled Wistar rat model. We measured the messenger ribonucleic acid (mRNA) expression of multi‐drug resistance 1 (MDR1) and multi‐drug resistance protein 5 (MRP5) as indicators for drug resistance. Rats received PHB + PTZ for 62 days to develop a drug‐resistant epilepsy model. From day 48, SEW2871 (0.25, 0.5, 0.75 mg/kg, intraperitoneally [i.p.]) was administered for 14 days. Seizure scoring, behaviour, oxidative markers like reduced glutathione, catalase, superoxide dismutase, inflammatory markers like interleukin 1 beta tumour necrosis factor alpha, interferon gamma and mRNA expression (MDR1 and MRP5) were assessed, and histopathological assessments were conducted. SEW2871 demonstrated dose‐dependent improvements in seizure scoring and neurobehavioral parameters with a reduction in oxidative and inflammation‐induced neuronal damage. The S1PR1 agonist also downregulated MDR1 and MRP5 gene expression and significantly decreased the number of dark‐stained pyknotic nuclei and increased cell density with neuronal rearrangement in the rat brain hippocampus. These findings suggest that SEW2871 might ameliorate epileptic symptoms by modulating drug resistance through downregulation of MDR1 and MRP5 gene expression.
... Two distinct forms of mucins are secreted by human salivary glands: oligomeric mucin (MG1), which has a molecular weight exceeding 1000 k Da, and monomeric mucin (MG2), which has a molecular weight between 200 and 250 k Da. Mucin-coated bacteria may be unable to connect to the surface, which plays a function in oral bacterial adhesion [14]. The current study aims to the optimizing the conditions and purification of extracellular protease, which was produced from E.coli AJ55 isolated from UTI patients, and mucin protein cleavage by this protease enzyme. ...
... To determine the proteolysis activity of the protease, the absorbance at 280 nm was measured. To calculate and ascertain the protein concentration in samples, the Bradford method was used [14]. ...
Proteases have various applications in the food, pharmaceutical, medicine, pathogenicity of some pathogenic bacteria, and detergent sectors as well as meeting the needs of approximately 60% of the global enzyme industry, whereas they catalyze the breakdown of protein molecules into peptides and amino acids. Production and purification of protease enzyme by the isolate Escherichia coli AJ55 was scrutinized in the present study. Cultivation optimum conditions, were various complex medium, carbon source, nitrogen source, temperature, pH of the medium, and time of incubation were optimized to enhance the total protease production in shake flask culture of E.coli AJ55. The nutrient broth supplemented with 2% glucose and 2% yeast extract, with a pH of 7.0 and incubated at 37 °C for 24 hours, better conditioned for producing the maximum production of protease. Escherichia coli AJ55 proteolytic enzyme was separated and purified using ion-exchange chromatography on a DEAE-Cellulose column and Sephadex G-150 gel after being precipitated with 0-70% saturated ammonium sulfate. Protease that had been partially purified had a yield of 34%, a purification fold of 13.4, an activity of 12.16 U/ml, a protein concentration of 0.005 mg/ml, and a specific activity of 2432 U/mg. By using gel filtration chromatography on a Sephadex G-150 gel, partially purified protease was examined for its ability to cleave the mucin protein. The findings of mucin biodegradation showed that the five fractions of the small peptides were produced after treatment of mucin with partially purified protease.
... The protein concentration in samples was assessed and determined using the Bradford technique [12]. ...
... By incubating the selected isolates at various time intervals (1,2,3,4,5,6,7,8,9,10,11,12, and 13 days), the keratinase production parameter was first optimized. Conical flasks were incubated for various time durations of spaced one day apart in order to find the optimal incubation time for keratinase synthesis. ...
Keratin is a fibrous, insoluble structural protein that is highly cross-linked with hydrophobic, hydrogen, and disulfide bonds. Keratinases are enzymes that belong to the category of serine hydrolases that are capable of breaking down keratin. The results of the determination of the better fermentation system showed that the production of keratinase from local A.terreus A13 isolate by submerged fermentation (SmF) system was the best system to give the highest specific activity (113.4 U/mg) of keratinase compared with solid-state fermentation (SSF). The optimum conditions for keratinase production by SmF, were determined via cultivation conditions, including carbon source, nitrogen source, temperature, pH of the medium, and time of incubation were optimized to enhance the production of total keratinase production in a culture of A.terreus A13 with incubator shaker. The highest product of total keratinase was achieved in feather broth with 2 % sucrose, and 0.5 % soya bean, with a pH of 5.5 at 28 °C for 8 days. Separation and purification of keratinase from a local isolate of A.terreus A13 was done by precipitating with 0-75 % saturated ammonium sulfate, then by ion-exchange chromatography on DEAE-Cellulose column and sephadex G-150 gel. Partially purified keratinase gave an activity of 5.1 U/ml, protein concentration of 0.004 mg/ml, and specific activity of 1275 U/mg with purification fold of 4.96 and 49 % as yield. The aim of the present study was to optimize the production of keratinase from A. terreus A13, cultivated using optimum conditions, and its use for the biodegradation of feathers.
... Bovine serum albumin (BSA) was used as a standard for protein quanti cation. To determine enzyme activity, protein concentration, and speci c activity, the protein concentration was measured using the Bradford technique (Bradford, M. 1976). A standard curve for the Bradford assay was constructed using BSA. ...
... The residual activity was incubated at room temperature in several buffers with different pH values ranging from pH (3)(4)(5)(6)(7)(8)(9) to investigate the pH value and stability of WCP. The partial peroxide was stable throughout a wide pH range (3-7) according to the results of the pH stability testing as shows in (Fig. 4b). ...
In this work, the peroxidase enzyme was extracted from cabbage legs obtained from restaurant waste. Additionally, a comparison was made between crude and pure enzymes to evaluate their efficacy as catalysts for the biodegradation of common contaminants, textile dyes, and bisphenol A. The concentration of protein, bioactivity of peroxidase, and specific activity of both crude and pure enzymes were experimentally determined. The extracted peroxidase enzyme displayed optimal bioactivity at pH 6 and a temperature of 40°C. Moreover, it exhibited good stability across a wide pH range (3–7), retaining 65% of its original bioactivity at 60°C. The addition of FeSO 4 and ZnCl 2 enhanced enzyme activity by 153% and 120%, respectively, whereas exposure to CuCl 2 , AgNo 3 , and HgCl 2 ions reduced enzyme activity by 60%, 53%, and 25%, respectively. The crude enzyme exhibited remarkable efficiency in decolorizing the synthetic dyes, with percentage decolorization of 86% and 78% for reactive blue 49 (RB) and reactive green 19 (RG), respectively, after a 10-hour incubation at the laboratory scale. Similarly, the pure enzyme exhibited a decolorization percentage of 79% and 75% for RB and RG, respectively, under the same conditions. The crude enzyme showed a high degradation efficiency of 92.8% for bisphenol A degradation in aqueous solution after 5 h.
... In order to prepare NH 4 Cl standard curve for the urease assay, serial concentrations (100-500 µM) from a stock solution of NH 4 Cl (0.5 mM) were prepared in triplicate. The standard curve of NH 4 Cl was plotted between the ammonium chloride concentration (µM) and the corresponding absorbance of standard ammonium chloride at 625 nm, as shown in (Fig. 1). ...
... : is the Concentration of ammonia, T: is the Time of reaction, 60 min. C: is the Constant, (14) Protein concentration, was measured according to the method described by Bradford (4). Determination of o4ptimum condition for urease extraction Type of plant material: Chickpea, Tomato, soybean, Mustard, Baker, Lebbeck, bean, Sesame, Male Iraqi berries, Female Iraqi berries, Indian berries, Potato, Radish, Iegumes, Peas, Watermelon, and Phaseolus were extracted by using 0.02 M of phosphate buffer pH 7.0. ...
In the current study, Seventeen types of plants commonly used namely (Chickpeas, Tomato, Soybean, Mustard, Baker, Lebbeck, Bean, Sesame, Male Iraqi berries, Female Iraqi berries, Indian berries, Potato, Radish, Legumes, peas, Watermelon, and Phaseolus were obtained and screened for urease activity, among these plants, Sesame was chosen with maximum enzymatic activity, and it had the highest productivity of urease enzyme (1.623 U/mg protein). The optimum extraction ratio represented by 1:10 (W: V) after 90 minutes and 0.8414U/mg protein. Sodium phosphate buffer (0.1 M, pH 7.0) was chosen as the best extraction buffer with specific activity 0.9004U/mg protein.
... In this assay, the enzyme activity (one unit, IU) is defined as the enzyme amount that releases one micromole of glucose per milliliter per minute. Using the Bradford method, the protein concentration was determined by employing bovine serum albumin for preparing a standard curve (Bradford, 1976). Glucose concentration was estimated using the phenol-sulfuric acid method with standard glucose (Lam et al., 2021), and the resulting glucose concentration was expressed as micrograms per gram of wheat straw depending on the provided equation: ...
Glucose is considered as one of the most important monosaccharides, consisting of six carbon atoms. Glucose can be bound with other sugars or with other glucose to form complex compounds or polysaccharides such as cellulose. Therefore, it is possible to biodegrade cellulose to produce glucose using the cellulase enzyme produced by microorganisms. One of the significant filamentous fungal isolates like Aspergillus terreus can be used for this purpose. Aspergillus terreus AJ3 was activated via culturing on potato dextrose agar media, then the optimum conditions were determined for cellulase and glucose production by using this isolate. The better parameters after investigation were wheat straw, corn step liquor as nitrogen source, moisten at ratio 1:1 (v:w) with mineral salts solution at pH 6.0, and were incubated at 30°C for 6 days. The cellulase purification date demonstrated that, following precipitation by ammonium sulfate (0-75%), gel filtration (Sephadex G-150) was an effective procedure for enzyme purification, with specific activity of around 1433.25 U/mg, yield of approximately 49% and 2.45 as purification fold. The findings of enzyme characterization demonstrated that the molecular weight of cellulase was 26 kDa, and the best pH for cellulase activity was 4.5 and the pH stability was ranged from 4.0-8.5. Additionally, the better temperature for cellulase activity was 40°C, while the thermal constantly of enzyme was ranged from 20-50°C. The Thin Layer Chromatography outcomes for glucose detection showed that the wheat straw and cellulose were hydrolyzed to glucose, depended on retention factor (Rf) values of the standard glucose and the test samples (0.36).
... The protein content of cellulase in the initial solution before immobilization and in the supernatant after immobilization was estimated using the Bradford method [17], with bovine serum albumin as the standard protein. The loading efficiency of cellulase protein is calculated as follows: ...
This study investigates the immobilization of cellulase on zeolitic imidazolate frameworks (ZIFs) by physical adsorption, specifically the ZIF-8-NH2 and Fe3O4@ZIF-8-NH2, to enhance enzymatic hydrolysis efficiency. The immobilization process was thoroughly analyzed, including optimization of conditions and characterization of ZIF carriers and immobilized enzymes. The impacts on the catalytic activity of cellulase under various temperatures, pH levels, and storage conditions were examined. Additionally, the reusability of the immobilized enzyme was assessed. Results showed the cellulase immobilized on Fe3O4@ZIF-8-NH2 exhibited a high loading capacity of 339.64 mg/g, surpassing previous studies. Its relative enzymatic activity was found to be 71.39%. Additionally, this immobilized enzyme system demonstrates robust reusability, retaining 68.42% of its initial activity even after 10 cycles. These findings underscore the potential of Fe3O4@ZIF-8-NH2 as a highly efficient platform for cellulase immobilization, with promising implications for lignocellulosic biorefinery.
... The harvesting buffer was supplemented with aprotinin (10 μg/mL), and the cells were subsequently resuspended and sonicated. The nuclear and cell debris was separated by centrifuging the mixture again at 850 g for five minutes at 4 • C. The resultant supernatant was then mixed with 7.5 % glycerol and kept at − 80 • C. Protein concentration was determined using the Bradford microplate assay, with bovine serum albumin serving as the reference protein [27]. ...
... Optical density was measured using an ELISA reader at a wavelength of 450 nm. Total protein was measured by the Bradford method using bovine serum albumin as the standard [26]. Data were expressed in picograms per milligram (pg/mg) of protein. ...
Objectives
Despite the fact that fibromyalgia, a widespread disease of the musculoskeletal system, has no specific treatment, patients have shown improvement after pharmacological intervention. Pregabalin has demonstrated efficacy; however, its adverse effects may reduce treatment adherence. In this context, neuromodulatory techniques such as transcranial direct current stimulation (tDCS) may be employed as a complementary pain-relieving method. Consequently, the purpose of this study was to evaluate the effect of pregabalin and tDCS treatments on the behavioral and biomarker parameters of rats submitted to a fibromyalgia-like model.
Methods
Forty adult male Wistar rats were divided into two groups: control and reserpine. Five days after the end of the administration of reserpine (1 mg/kg/3 days) to induce a fibromyalgia-like model, rats were randomly assigned to receive either vehicle or pregabalin (30 mg/kg) along with sham or active- tDCS treatments. The evaluated behavioral parameters included mechanical allodynia by von Frey test and anxiety-like behaviors by elevated plus-maze test (time spent in opened and closed arms, number of entries in opened and closed arms, protected head-dipping, unprotected head-dipping [NPHD], grooming, rearing, fecal boluses). The biomarker analysis (brain-derived neurotrophic factor [BDNF] and tumor necrosis factor-α [TNF-α]) was performed in brainstem and cerebral cortex and in serum.
Results
tDCS reversed the reduction in the mechanical nociceptive threshold and the decrease in the serum BDNF levels induced by the model of fibromyalgia; however, there was no effect of pregabalin in the mechanical threshold. There were no effects of pregabalin or tDCS found in TNF-α levels. The pain model induced an increase in grooming time and a decrease in NPHD and rearing; while tDCS reversed the increase in grooming, pregabalin reversed the decrease in NPHD.
Conclusions
tDCS was more effective than pregabalin in controlling nociception and anxiety-like behavior in a rat model-like fibromyalgia. Considering the translational aspect, our findings suggest that tDCS could be a potential non-pharmacological treatment for fibromyalgia.
... The protein content of cellulase in the initial solution before immobilization and in the supernatant after immobilization was estimated using the Bradford method [11], with bovine serum albumin as the standard protein. The loading e ciency of cellulase protein is calculated as follows: ...
This study investigates the immobilization of cellulase on zeolitic imidazolate frameworks (ZIFs) by physical adsoption, specifically ZIF-8-NH 2 and Fe 3 O 4 @ZIF-8-NH 2 , to enhance enzymatic hydrolysis efficiency. The immobilization process was thoroughly analyzed, including optimization of conditions and characterization of ZIF carriers and immobilized enzymes. The impacts on the catalytic activity of cellulase under various temperatures, pH levels, and storage conditions were examined. Additionally, the reusability of the immobilized enzyme was assessed. Results showed the cellulase immobilized on Fe 3 O 4 @ZIF-8-NH 2 exhibited a high loading capacity of 339.64 mg/g, surpassing previous studies. Its relative enzymatic activity was found to be 71.39 %. Additionally, this immobilized enzyme system demonstrates robust reusability, retaining 68.42 % of its initial activity even after 10 cycles. These findings underscore the potential of Fe 3 O 4 @ZIF-8-NH 2 as a highly efficient platform for cellulase immobilization, with promising implications for lignocellulosic biorefinery.
... 0.005 N H 2 SO 4 was used as the mobile phase as a flow rate of 0.6 ml/min and the column temperature was maintained at 60 °C. Enzyme concentration was measured by the Bradford protein assay using bovine serum albumin (BSA) as standard [9]. ...
Ultrafiltration was used to clarify glucose from cellulose hydrolysate using polyethersulfone (PES) membrane. The flux behavior of PES membrane was studied in concentrating glucose from cellulose hydrolysate during dead end ultrafiltration in different pH of solutions and Kumar’s model was applied to analyse the fouling mechanism. The permeation of glucose achieved more than 93% for all the different pH solution. The permeate flux decreased over time as a result of membrane fouling. The minimum fouling was obtained at pH solution above the IEP due to protein-protein and membrane-protein repulsions alleviating aggregation and fouling. Cake formation blocking was identified as the dominant mechanism for flux decline. Keywords: glucose, enzyme hydrolysis, lignocellulosic biomass, ultrafiltration, polyethersulfone
... Using the phenol-sulfuric acid method, the neutral sugar content of polysaccharides derived from H. coralloides was determined (Dubois, Gilles, Hamilton, Robers & Smith, 1956). The protein content was analysed by the Coomassie brilliant blue G-250 assay (Bradford, 1976). The sulfuric acid-carbazole method was applied to assess the uronic acid contents (Blumenkrantz & Asboe-Hansen, 1973). ...
... Protein was determined using the dye-binding method of Bradford, (1976). Commassie Brilliant Blue G250 (100mg) was dissolved in 500 ml of 95% ethanol. ...
Thirty bacterial isolates belonging to Bacillus spp. obtained from different local sources in Kirkuk-Iraq were screened for their capabilities for producing Amylase. It was found that the isolates designated (5M, 8M, 9M, 12M, 13M, 17M, 18M, 24M, 27M, 30M) have higher capability for producing Amylase. All isolates subjected to secondary screening. The Isolate (13M) was found to be the highest producers with enzyme activity of (6.329U/ml). The identification tests of this isolate were carried out by studying the morphological, microscopic characteristics and biochemical test using Vitek2 compact system. The tests results were confirmed by identification of 16S rRNA gene using polymerase chain reaction (PCR) and its nitrogen base sequencing. The results revealed that this isolate belongs to Bacillus subtilis. Recorded in gene bank under the ACCESSION: MN744663.
... The amount of enzyme that, under experimental conditions, releases 1 µmole of ammonia per minute was designated as one unit of L-ASNase. Based on the Bradford method, protein content was determined [18]. ...
Acrylamide is a toxic chemical that is created when foods are heated; it is also available in foods containing different additives. The purpose of the study was to determine whether Bacillus spp. isolates could reduce the concentration of acrylamide in food, as well as to compare the different treatments of crude and pure L-asparaginase produced from the same bacteria in acrylamide reduction in potato slices. Our findings reveal that this bacterium could degrade acrylamide and reduce its concentration. Furthermore, the acrylamide content of potato slices reduced dramatically with increasing enzymatic treatment time, reaching the under detection limit (UDL) after 30 minutes of treatment with 84 U/ml of crude and purified L-asparaginase. In addition, the purified enzyme is more active in removing acrylamide from potato slices than the crude enzyme at all three times. The removal efficiency rises as the enzyme concentration increases. After bleaching the potato slices for 20 minutes at 80 °C with 84 U/ml of pure enzyme, the acrylamide level was totally decreased.
... Lipid Analysis: Samples were analyzed by gas chromatography model DANI 2015 in Ministry of Industry and Minerals / IBN SINA state company. Determination of Protein and Carbohydrate: The protein determined according to Bradford, (1976) method and the carbohydrate according to Dubois et al., 1956(Azeez, 2001Al-Rekabi, 2003) Statistical Analyses: The result express as mean ± sd. Data were analyzed by one way analysis of variance (ANOVA) followed by Fisher's test for multiple comparison, using stat view version 5.0. ...
Two local microalgae are used in this study: Chlorella vulgaris as model of green algae and Chroococcus minor as model of blue green algae which used to test their ability to produce lipids as a source of biodiesel through starvation of some nutrients (-N) 100, (-N) 50%, (-P) 50%, (-N) 50 %+(-P) 50% and (-Fe) 50%, the study showed that the accumulation of lipids in C.vulgaris more efficient than C.minor , in C.vulgaris The highest lipid content was 41% from DW in treatment (-N) 100% while the lowest lipid content was 8% at control, carbohydrate content increased from 18% at control to 34% at (-N) 100% while the highest protein was 51% of DW in the control. In C.minor the highest lipid content was 31% in (-N) 100% while the lowest lipid content was 5% at control. The highest carbohydrate content was 28% of DW (-N) 100%, while the highest protein was 40% of DW in the control.
... The protein determined according to Bradford, (1976)method [28] and the carbohydrate according to [29]. ...
Two locally isolated microalgae (green algae Chlorella vulgaris and blue green algae Chroococcus minor) were used in the current study to test their ability to production biodiesel through stimulated in different pH levels treatments (pH 5,pH 9) and effect of pH level on the quantity of protein ,carbohydrate. Showed that the accumulation of lipids in C.vulgaris more efficient than C.minor, The treatment pH 9 was recorded C. vulgaris the highest lipid content from 8% at control to 32% as well as highest carbohydrate content from 18% at control to 25% but showed decreased protein content from 51% to 31%.The treatment pH 9 was recorded in C. minor was recorded the highest lipid content from 5% at control to 12% as well as highest carbohydrate content from 15% at control to 18% but showed decreased protein content from 40 % to 30% .The results revealed that Stearic acid and Oleic acid content increased content for both algae at pH 9 levels.
... We employed the Bradford method to ascertain the protein content in the samples [33]. ...
Background
Cholesterol (Cho) is an essential lipophilic molecule in cells; however, both its decrease and its increase may favor the development of neurological diseases such as Alzheimer’s disease (AD). Although copper (Cu) is an essential trace metal for cells, the increased plasma concentration of its free form has been linked with AD development and severity. AD affects aged people, but its prevalence and severity are higher in women than in men. We have previously shown that Cu promotes Cho de novo synthesis in immature neurons as well as increased Cho in membrane rafts and Aβ levels in culture medium, but there are no results yet regarding sex differences in the effects of sublethal Cu exposure on Cho de novo synthesis.
Methods
We examined the potential sex-specific impact of sublethal Cu concentrations on de novo Cho synthesis in primary cultures of male and female astrocytes. We also explored whether this had any correlation with variations in Cho and APP levels within neuronal membrane rafts.
Results
Flow cytometry analysis demonstrated that Cu treatment leads to a greater increase in ROS levels in female astrocytes than in males. Furthermore, through RT-PCR analysis, we observed an upregulation of SREBP-2 and HMGCR. Consistently, we observed an increase in de novo Cho synthesis. Finally, western blot analysis indicated that the levels of ABCA1 increase after Cu treatment, accompanied by a higher release of radiolabeled Cho and an elevation in Cho and APP levels in neuronal membrane rafts. Importantly, all these results were significantly more pronounced in female astrocytes than in males.
Conclusions
Our findings confirm that Cu stimulates Cho synthesis in astrocytes, both in a ROS-dependent and -independent manner. Moreover, female astrocytes displayed elevated levels of HMGCR, and de novo Cho synthesis compared to males following TBH and Cu treatments. This corresponds with higher levels of Cho released into the culture medium and a more significant Cho and APP rise within neuronal rafts. We consider that the increased risk of AD in females partly arises from sex-specific responses to metals and/or exogenous substances, impacting key enzyme regulation in various biochemical pathways, including HMGCR.
... After extraction, the extract was centrifuged at 14,000 × g for 30 min at 4ºC. Protein content was quantified according to Bradford [9]. Soluble protein content was estimated from a standard curve using bovine serum albumin (BSA), and results were expressed as μg protein g − 1 DM. ...
This study tests the hypothesis that a more salt-excluder rootstock can attenuate salt stress in grapevine plants by enhancing photosynthesis and providing ionic and oxidative protection. Plants of ‘BRS Vitória’ variety, grafted with the rootstocks IAC 313 (salt-excluder) and SO4, were subjected to salinity by adding NaCl (0, 50, and 100 mM) for 30 days. Plants with SO4 showed more severe salt toxicity symptoms in leaves and lower chlorophyll content under salinity. Conversely, plants with IAC 313 showed improved photosynthesis and stomatal conductance, along with higher carboxylation efficiency under salt compared to SO4. Under salinity, plants with SO4 showed higher losses of K+ in stems, roots, and petioles, as well as increased accumulation of Na+ in these organs, relative to IAC 313. Furthermore, plants with IAC 313 had lower leaf Na+ content under salinity and reduced leaf Cl− content at 50 mM NaCl, a response associated with a higher Na+ allocation in petioles of IAC 313. At 50 mM, IAC 313 exhibited better photochemical activity, as indicated by electron transport rate and non-photochemical quenching. However, at 100 mM, both rootstocks showed similar trends, suggesting that the photosynthetic restriction was primarily due to stomatal disturbances. Plants with IAC 313 showed better APX activity and ascorbate balance under salinity. IAC 313 showed more salt-resistance traits than SO4, although the growth was similarly affected in both rootstocks. This response could be due to the reduced time of salt treatment (30 days). In summary, our data indicate that IAC 313 rootstock possesses better salt tolerance traits than SO4.
... Alanine amino transferees (ALT) and Aspartate amino transferees (AST) were determined calorimetrically according to the method of Reitman and Frankle, (1957), and alkaline phosphatase (ALP) activity was determined according to the method of Weisshaar, (1975). While total protein was determined according the method of Bradford, (1976) and albumin content was determined by the method of (Doumas et al., 1971). ...
... The protein concentration was determined using the Bradford method [36]. This method uses Coomassie blue (Quick start/Bradford; Catalogue No. 500-0205; Bio-Rad) to determine the total concentration of proteins in each extract sample. ...
... Algae samples were centrifuged by cooling centrifuge model Rotina 380 R. for 5000 r/min for 30 min, 4 C º . The supernatant was collected and the protein determined according to Bradford (1976) method and the carbohydrate according to Dubois et al., (1956). (Kochert, 1978;Azeez,2001;Al-Rekabi,2003). ...
... Protein concentration was determined according to the method described by Bradford. [14] Optimum carbon and nitrogen source for cytosine deaminase production ...
This study was aimed to isolate a higher cytosine deaminase producer Escherichia coli and studying the optimum condition for its production, a total of twenty-five urine samples were collected for the isolation of E. coli bacterium from Al-Imamein AlKadhumain medical city hospital in Baghdad. From these samples, a total of 10 bacterial isolates were obtained when subjected to morphological and microscopical tests. Cytosine deaminase enzyme activity was determined using cytosine as substrate, results indicated that 10 isolates of them are cytosine deaminase producer with different specific activity ranged between (0.122-0.35) U/mg and the isolate Escherichia coli E9 was the most efficient in the production of cytosine deaminase with specific activity of 0.35 U/mg protein. Therefore, it was chosen to determine the optimum conditions for cytosine deaminase production. Maximum cytosine deaminase production was achieved after supplementation of the minimal salt medium (pH 8.5) with 0.1% citric acid, 0.1% peptone and incubated at 37 o C for 24h. Under these conditions, the specific activity of cytosine deaminase produced in culture supernatant was sharply increased to 0.7 U/mg protein. KEYWORDS: This study was aimed Al-Imamein AlKadhumain to 0.7 U/mg protein.
... Protein concentration was determined according to the method described by Bradford, (16) ...
This study was aimed to isolate a locally higher cytosine deaminase producer Escherichia coli. Ten E. coli bacterial isolates were obtained from twenty-five urine samples collected from Al-Imamein AlKadhumain medical city hospital in Baghdad. These isolates were identified by cultural, microscopical and biochemical tests, identifications also done by vitek 2 system. Cytosine deaminase activity was assayed for these isolates and it was found that all isolates were enzyme producer with variable activities and E. coli E9 was the most efficient with specific activity of (0.350 U/mg).
... After stirring at 25°C for 2 h, the supernatant was obtained by centrifugation (8000 g, 4°C, 20 min). The protein content in the supernatant was determined by Bradford method (Bradford, 1976). The protein solubility was expressed as a percentage of the total protein content. ...
The Moringa seed protein (MOS) is a new protein resource, the development and utilisation of MOS is still in its early stages due to the late introduction of its varieties. To improve the range of available MOS protein, this study investigated the effects of different pH levels on the ultrasonic modification of MOS protein (UMOWP), exploring the feasibility of UMOWP emulsion systems in food. The zeta potential analysis revealed that the isoelectric point (pI) of UMOWP was 5.8. The secondary structures of UMOWP could be regulated by pH. Moreover, different secondary structures led to different tertiary structures and chemical bonds in UMOWP. Compared with pH values near pI, surface hydrophobicity significantly increased but the total S–S group and particle size decreased away from pI. Furthermore, UMOWP partially dissociated at pH 3, and the degree of dissociation was weaker at pH 9 and pH 11. These structural changes led to significant improvements in the emulsification properties of UMOWP at pH 11, resulting in better environmental stability of the emulsion. These findings provide a foundation for the potential application of MOS proteins in the food industry.
... Superoxide dismutase (SOD) activity was measured according to spechtrometric method described by Giannopolitis and Ries (1977). The soluble protein content was determined conform Bradford (1976). ...
The use of silicon in plant cultivation is one of the strategies to mitigate the negative effects of salinity. This study aimed to evaluate the effect of silicate fertilization on the morphophysiological, biochemical and nutritional characteristics of Jatropha curcas L. plants under saline stress. The work was carried out in a greenhouse at the State University of Goiás. The experiment was set up in a completely randomized design in a 5 × 2 factorial arrangement (plants irrigated with salt water with sodium chloride (NaCl) and electrical conductivities equal to 0 dS m-1; 2 dS m-1; 4 dS m-1; 6 dS m-1 and 8 dS m-1 applied at 80 days after emergence (DAE) and absence or presence of silica fertilization of 1 g L-1 with Si applied at 80 and 95 DAE by volume of 30 mL of the solution with the aid of a spray manual), five replicates and experimental plot of one plant per pot. The evaluations were carried out at 130 DAEs. The absence of differences in the concentrations of photosynthetic pigments and visible damages is indicative of the absence of severe toxic effects caused by salinity. The tolerance of Jatropha curcas L. plants to salinity is independent of silicon. The Jatropha curcas L. plant tolerates salinity by minimizing transpiration and remaining hydrated through the water stored in the succulent stem. In addition, the plants control sodium uptake and eliminate toxic compounds through increases in Calcium concentration and antioxidative metabolism respectively.
... The homogenate was centrifuged at 12,000×g for 10 min at 4°C, and the supernatant was immediately collected for SOD, CAT, and GPX activity assays. The protein content of the goldfish livers was determined using the Bradford method [27] with bovine serum albumin as the standard. SOD activity was determined based on the ability of the enzyme to inhibit the reduction of NBT [28]. ...
NaCl has beneficial effects in preventing fish disease. However, the effects of NaCl in regulating fish growth and survival at different food supply levels under hypoxic conditions are unknown. Herein, the effects of NaCl on body weight, survival rate (SR), H2O2 content, lipid peroxidation, protein carbonylation, antioxidant (superoxide dismutase [SOD], catalase [CAT], and glutathione peroxidase [GPX]) enzyme activities, and the glutathione (GSH) content were investigated in goldfish (Carassius auratus) livers at three food supply levels (50, 200, and 400 mg day⁻¹ fish⁻¹) under hypoxic conditions. The highest and lowest SRs were detected in the 200 and 400 mg food groups, respectively. Interestingly, 50 mM NaCl markedly elevated survival, but not the body weight of goldfish. Enhanced H2O2 content; SOD, CAT, and GPX activities; GSH content; and reduced lipid peroxidation and protein carbonylation were detected in goldfish livers after NaCl treatment compared with those in the control. However, these effects of NaCl were dramatically attenuated by 50 µM hydroxychloroquine, an inhibitor of autophagy. This showed that nutrition stress reduced goldfish survival, which could be improved by NaCl via regulation of the antioxidant system and autophagy under hypoxic conditions.
... Then, the homogenates were centrifuged at 11,000 rpm for 40min at 4°C. The total protein concentration was determined by the Bradford method (Bradford 1976). Samples containing 100µL of total extract from each experimental group were incubated for 5 min at 80°C with concentrated 4x Laemmli buffer (0.250 mM Tris-HCl buffer pH 6.8, 0.5% bromophenol blue, 50% glycerol, 10% sodium dodecyl sulfate, and 500 mM dithiothreitol) (4:1, v/v). ...
Abstract To evaluate the effects in adults rats submitted of a low-protein, high-carbohydrate (LPHC; 6% protein, 74% carbohydrate) diet and reversion (R) to a balanced diet introduced after weaning. Research methods & procedures: Male rats weigting approximately 100g (30 to 32 d old) were treated with control (C; 17% protein, 63% carbohydrate) or LPHC diets for 120 days. The reverse group (R) was treated with the LPHC diet for 15 days, and changed to C diet for another 105 days. Results: The LPHC group showed an increase in serum fasting triglycerides (TAG). Serum adiponectin was increased only in the LPHC group. Lipoprotein lipase (LPL) activity was decreased in the extensor digitorum longus (EDL) and cardiac muscles. The adiponectin receptor 1 content is the same among groups in the cardiac muscle, but it is lower in the EDL muscle in the LPHC group. In animals from the R group, these parameters are the same as the LPHC group. Thus, the LPHC diet administered for a long period, it promotes an increase in TAG. It is possible that there is adiponectin resistance in the EDL muscle, due to the lower LPL activity. The reversal of the LPHC diet did not normalize these parameters.
... Through the FOX method (Jiang et al., 1992), we determined lipoperoxidation (LPO). The quantification of the total protein concentration used bovine serum albumin as a standard according to the Bradford method (Bradford, 1976) for calculating the enzymes' specific activity. ...
Ciprofloxacin (CIP) is an antibiotic commonly used in human and veterinary medicine. It is present in the aquatic environment, but we still know very little about its effect on non-targeted organisms. This study aimed to evaluate the effects of long-term exposure to environmental CIP concentrations (1, 10, and 100 μg.L-1) in males and females of Rhamdia quelen. After 28 days of exposure, we collected the blood for the analysis of hematological and genotoxic biomarkers. Additionally, we measured 17 β-estradiol and 11 keto-testosterone levels. After the euthanasia, we collected the brain and the hypothalamus to analyze acetylcholinesterase (AChE) activity and neurotransmitters, respectively. The liver and gonads were assessed for biochemical, genotoxic, and histopathological biomarkers. At 100 μg.L-1 CIP, we observed genotoxicity in the blood, nuclear morphological changes, apoptosis, leukopenia, and a reduction of AChE in the brain. In the liver was observed oxidative stress and apoptosis. At 10 μg.L-1 CIP, leukopenia, morphological changes, and apoptosis were presented in the blood and a reduction of AChE in the brain. Apoptosis, leukocyte infiltration, steatosis, and necrosis occurred in the liver. Even at the lowest concentration (1 μg.L-1), adverse effects such as erythrocyte and liver genotoxicity, hepatocyte apoptosis, oxidative stress, and a decrease in somatic indexes were observed. The results showed the importance of monitoring CIP concentrations in the aquatic environment that cause sublethal effects on fish.
... The 200 mg of fresh leaf tissue was homogenized in an ice-cold extraction buffer and centrifuged at 15,000 × g for 20 min at 4 °C. The resultant supernatant was used to assess enzyme activity, with protein content determined by the method of Bradford [33]. The activity of SOD was quantified by using bovine serum albumin as a standard method [34,35]. ...
Heat stress poses a threat to plants in arid and semiarid regions, leading to soil salinization and plant mortality. Researchers are exploring remedies to alleviate these effects, including using gibberellic acid (GA3) to regulate plant enzymes and antioxidants. Additionally, sodium nitroprusside (SNP) is gaining attention, but its combined effect with GA3 requires further research. To address this gap, we investigated the effects of GA3 and SNP on plants under heat stress conditions. For that, wheat plants were cultivated under 40 °C for 6 h per day (15 days). Sodium nitroprusside (donor of NO and SNP) and gibberellic acid (GA3), respectively, with 100 µM and 5 µg/ml concentrations, were applied as foliar sprays at 10 days after sowing (DAS). Results showed that SNP + GA3 treatment had the highest plant height (4.48% increase), plant fresh weight (29.7%), plant dry weight (87%), photosynthetic rate (39.76%) and stomatal conductance (38.10%), and Rubisco (54.2%) compared to the control. Our findings indicate a significant increase in NO, H2O2, TBARS, SOD, POD, APX, proline, GR, and GB that greatly scavenged reactive oxygen species (ROS) for decreasing the adverse effect of stress. Such findings confirmed the efficacy of the combined treatment of SNP + GA3 under high-temperature stress compared to the solitary application of GA3, SNP, and control. In conclusion, using SNP + GA3 is a better strategy for mitigating heat stress in wheat than individual applications. Further research is recommended to validate the effectiveness of SNP + GA3 in other cereal crops.
Graphical Abstract
... Protein was assayed using Bradford method (Bradford, 1976). The standard curve was produced using bovine serum albumin (#A2153; ...
Some d ‐amino acid functions for food production are widely known: d ‐alanine improves sensory evaluations of sake , beer, and fermented foods. Therefore, for the application of d ‐amino acids, alanine racemase (ALRase) in Lactobacillus sakei ZH‐2, which has strong racemization, was analyzed using molecular biological methods. It had been hypothesized that ALRase coding DNA, alr , in ZH‐2 strain differs from those of other Lactobacillus sakei strains. However, complete genome sequencing by the National Center for Biotechnology (NCBI) revealed the amino acid sequence of alr in ZH‐2 strain to have homology of 99.4% similarity with the alr in Lactobacillus sakei 23K strain. However, it is considered that the sequence of alr was a unique amino acid sequence in the lactic acid bacteria group. DNA “ alr ” of ZH‐2 strain has a 1140 bp DNA base with 41 kDa molecular mass. Its molecular mass was inferred as approximately 38.0 kDa using SDS‐PAGE. Its optimum conditions are pH 9.0 at 30–40°C, showing stability at pH 9.0–10.0 and 4–40°C. Its cofactor is pyridoxal phosphate. Its activity is activated more by copper and zinc ions than by the lack of a metal ion. Additionally, its K m is 1.32 × 10 ⁻³ (mol), with V max of 4.27 × 10 ⁻⁵ (μmol ⁻¹ min ⁻¹ ). ALRase reacted against alanine most strongly in other substrates such as amino acids. The enzyme against serine was found to have 40% activity against alanine. The enzyme converted up to 54.5% of d ‐alanine from l ‐alanine ZH‐2 strain.
... Enzyme activity = Where : Reaction time: 20 minutes Volume of the enzymatic solution: 0.2 ml Amount of increase in absorption per enzymatic unit: 0.001 Protein determination Protein content was calculated according to [6]. ...
This study was conducted in the laboratories of Biology Department, faculty of Science, which deal with isolation and purification of protease by Bacillus subtilis which carried out for enhanced production of protease using casein(10%) as the substrate of enzyme , the production was carried out by submerged fermentation , the best conditions were the isolated of protease in synthetic medium , it gave high titer of protease activity , the ammonium sulfate as nitrogen source , incubation period 48 h , the casein as carbon source, incubation temperature 37°C and pH= 7, the protease was purified using precipitation by ammonium sulphate (60%), dialysis against sucrose and ultra filtration, analyses of the protease for molecular weight was carried out by SDS-PAGE electrophoresis which revealed 32 KDa, the refined protease had a maximum activity at pH = 9 and in temperature 30 °C.
... One unit of the enzyme activity (U) was defined as the amount of enzyme required to liberate 1 μmol of reducing sugar per minute. The amount of enzyme was quantified by the Bradford protein assay, using bovine serum albumin (BSA) as a standard (Bradford 1976). ...
A novel chitinase gene of 888 bp from Streptomyces bacillaris was cloned and expressed in Escherichia coli BL21. The purified recombinant enzyme (SbChiAJ103) was identified as the first microbial-derived family 19 endochitinase that showed exochitinase activity. SbChiAJ103 exhibited the substrate preference for N-acetylchitooligosaccharides with even degrees of polymerization and the capability to specifically hydrolyze colloidal chitin into (GlcNAc)2. Mono-methyl adipate was employed as a novel linker for the efficient covalent immobilization of chitinase on magnetic nanoparticles (MNPs). The immobilized SbChiAJ103, SbChiAJ103@MNPs, exhibited superior pH tolerance, temperature stability, and storage stability than free SbChiAJ103. Even after incubation at 45 °C for 24 h, SbChiAJ103@MNPs could retain more than 60.0% initial activity. As a result, the enzymatic hydrolysis yield of SbChiAJ103@MNPs increased to 1.58 times that of free SbChiAJ103. Moreover, SbChiAJ103@MNPs could be reused by convenient magnetic separation. After 10 recycles, SbChiAJ103@MNPs could retain almost 80.0% of its initial activity. The immobilization of the novel chitinase SbChiAJ103 paves the way to the efficient and eco-friendly commercial production of (GlcNAc)2.
Key points
• The first microbial GH19 endochitinase with exochitinase activity was reported.
• Mono-methyl adipate was first employed to immobilize chitinase.
• SbChiAJ103@MNPs showed excellent pH stability, thermal stability, and reusability.
Graphical Abstract
Sesbania virgata (Cav.) Pers. seeds are protein sources with health and environmental benefits. In this research, proteins with lectin activity were identified in a protein fraction from S. virgata seeds (PFLA), as well its antioxidant and antimicrobial potentials, in addition to cytotoxic effects. To obtain PFLA, seed flour was homogenized in Glycine-NaOH (100 mM; pH 9.0; NaCl 150 mM) and precipitated in ammonium sulfate. PFLA concentrates bioactive lectins (32 HU/mL, 480 HU/gFa, 18.862 HU/mgP) and essential amino acids (13.36 g/100g protein). PFLA exerts antioxidant activity, acting as a promising metal chelating agent (~77% of activity). Analyzes of cell culture assay results suggest that antioxidant activity of PFLA may be associated with the recruitment of essential molecules to prevent the metabolic impairment of cells exposed to oxidative stress. PFLA (256-512 µg/mL) also exhibits antifungal activity, inhibiting the growth of Aspergillus flavus, Candida albicans, Candida tropicalis and Penicillium citrinum. Cytotoxic analysis indicates a tendency of low interference in the proliferation of 3T3 and HepG2 cells in the range of PFLA concentrations with biological activity. These findings support the notion that PFLA is a promising adjuvant to be applied in current policies on the management of metal ion chelation and fungal infections.
Biostimulants are chemical or biological components adopted to improve nutrient uptake/efficiency and tolerance to abiotic stresses in crops. We studied three biostimulants (Stimulate®, tryptophol and Bacillus subtilis C-3102) associated to two sanitizers (sodium hypochlorite or thymol), on initial growth of propagules of Arracacia xanthorrhiza. Sodium hypochlorite associated to B. subtilis improve the leaf gas exchange, furthermore this treatment showed greater root volume. The interaction among sodium hypochlorite and tryptophol improves the plant branching; in addition this association showed better results for root dry mass. Different biostimulants improve differently the arracacha organs development, hence it is necessary to evaluate the plant morphophysiological competence to apply the correct biostimulant and sanitizer.
Keywords:
Arracacia xanthorrhiza; phenolic compound; NaClO; bacteria; plant growth; plant metabolism
This study was aimed at inhibition of purified protease produced by C. albicans using plant seeds extract. One hundred two local C. albicans isolates that were isolated and identified by microscopic and biochemical tests were submitted to primary and secondary screening techniques in order to select the qualified C. albicans isolate for protease synthesis. Among these isolates, nineteen isolates with the highest hydrolysis zone on skim milk media (primary screening) were chosen for secondary screening. It has been found that C. albicansV56 had the highest productivity of the enzyme (41.8U/mg protein). The optimum conditions of protease production by selected isolate using solid state fermentation by using wheat bran as best substrate , temperature 28 °C and pH 7, after 5 days of incubation . The enzyme was purified by concentration with sucrose, then used gel filtration chromatography using Sephadex G-75. The results show two peaks. The first peak has a purification folds 6.5 time with an enzyme yield of 13.08 %, while peak 2 has a purification folds 3.3 time with an enzyme yield of 20%. The purified enzyme exhibited maximal activity at pH 6.0 for peak 1 and 3.0 for peak 2, whereas the maximum stability was 7.0 for peak 1 and 8 for peak 2. The optimal temperature for purified enzyme activity was 40 for peak 1 and 30 for peak 2, and it was stable until 45°C. Protease activity was inhibited with local plant seed extracts. Lathyrus sativus extract inhibits approximately 50 % of protease activity.
Methyl-Coenzyme M reductase (Mcr) plays an important role in the regulation of the global carbon cycle. The enzyme catalyzes the reversible conversion of methyl-coenzyme M and coenzyme B to methane and the corresponding heterodisulfide CoM-S-S-CoB, which constitutes the first step of the anaerobic oxidation of methane. Mcr homologs of varies methanogenic archaea also catalyze the anaerobic oxidation of extended alkanes. Fully active, purified enzyme is exclusively achievable via strictly anaerobic purification from Methanothermobacter marburgensis . This microbe expresses two isoenzymes and most studies were performed with isoenzyme I that exhibits a limited substrate-promiscuity (c.a. 0.5%) towards the homologous substrate ethyl-coenzyme M. Here, cell-free lysates from the different species such as Methanosarcina mazei, Methanococcus maripaludis, Methanothermococcus okinawansis , and Mt. marburgensis were screened for Mcr activity and substrate promiscuity. An assay relying on titanium (III) citrate and cobalamin to regenerate coenzyme B and coenzyme M was used to test the ability of different cell extracts for the catalytic activity towards the C2-substrate ethyl-coenzyme M. Cell extracts from M. mazei showed a ratio of ethane-to methane-production of ca. 8% at 37 °C, and about 14% at 49 °C. The level of substrate-promiscuity towards ethyl-coenzyme M for M. marburgensis cell extracts under our assay conditions were much higher than previously reported for purified isoenzyme I, indicating that isoenzyme II is much more promiscuous for ethane formation than isoenzyme I. Our experiments demonstrate that Mcr activity can be quickly and conveniently studied via cell-free lysates, and that substrate promiscuity towards ethane formation is generally larger than anticipated.
Xanthomonas euvesicatoria is a major cause of bacterial spot disease in various crops. The present study was
focused on the pathosystem pepper (Capsicum annuum L.) - X. euvesicatoria 269p (wild strain). The infectious
process was studied using several different modes of in vivo inoculation under controlled conditions. The
spread of the pathogen in different parts of the plants was monitored by a new qPCR procedure developed for
the detection of X. euvesicatoria, as well as by re-isolation of viable bacterial cells. Photosynthesis, the number
of viable pathogens, oxidative stress markers, activities of the main antioxidant enzymes, and levels of nonenzymatic
antioxidants in the novel single-leaf model system were studied. The most important observation is
that the invasion of the pathogen causes local infection and the dissemination of bacteria to the healthy parts of
the host is blocked. The plants limit bacterial colonization around the entry points. Oxidative burst and
alterations in antioxidant defenses are detected in infectious leaf lesions. Localized ROS overproduction
resembles a hypersensitive response, but several differences can be observed. We assumed that pepper plants
are more likely to manifest an intermediate phenotype, similar to lesions simulating disease or leaf flecking. By
localizing the infection, possibly involving oxidative stress, the plant survives. However, the same applies to
bacteria. The pathogen multiplies at the infection spots and is transmitted to other plants. Our conclusion is that
the intermediate phenotype in the studied pathosystem is an example of long and successful co-evolution for
both species.
The objective of this study was to evaluate the β-glucosidase activity in the non-conventional yeasts under cellulose, glucose and sucrose substrates. The participation of the enzyme β-glucosidase and its contribution to the enzymatic degradation of tannins is known. Within the classification of tannins are ellagitannins, molecules of gallic acid and ellagic acid, which are considered as nutraceutical compounds due to the properties that they present and that they can be used in the design of food and new drugs, synthesis of materials with antimicrobial capacity. The extracellular β-glucosidase activity was mainly presented in the Candida and Pichia strains, being the glucose and sucrose media the most capable for inducing the activity that showed maximum values with P. pastoris in glucose (0.1682±0.00 µmol/min mg protein), and C. utilis in cellulose (0.1129±0.1349 µmol/min mg of protein), and sucrose (0.0657±0.0214 µmol/min mg protein). Additionally, I. terricola and P. kluyvery stood out in a qualitative cellulose degradation approach measured by Congo red method (9.60±0.04 mm and 9.20±0.05 mm respectively). These indicate that P. pastoris and C. utilis have potential as β-glucosidase producers, especially when growing under complex carbon sources for biomass conversion, new biofuels production and polyphenol degradation with more manageable bioreactor process.
Microbial proteases are one of the most demanding enzymes for various industries with diverse applications in food, pharmaceutics, and textile industries to name the few. An extracellular alkaline metalloprotease was produced and purified from moderate halophilic bacterial strain, Bacillus cereus TS2, with some unique characteristics required for various industrial applications. The protease was produced in basal medium supplemented with casein and was partially purified by ion exchange chromatography followed by ammonium sulphate precipitation. The alkaline metalloprotease has molecular weight of 35 kDa with specific activity of 535.4 µM/min/mg. It can work at wide range of pH from 3 to 12, while showing optimum activity at pH 10. Similarly, the alkaline metalloprotease is stable till the temperature of 80 °C and works at wide range of temperature from 20 to 90 °C with optimum activity at 60 °C. The turnover rate increases in the presence of NaCl and Co+2 with k cat/KM of 1.42 × 103 and 1.27 × 103 s-1.M-1 respectively, while without NaCl and Co+2 it has a value of 7.58× 102. The alkaline metalloprotease was relatively resistant to thermal and solvent mediated denaturation. Applications revealed that the metalloprotease was efficient to remove hair from goat skin, remove blood stains and degrade milk, thus can be a potential candidate for leather, detergent, and food industry.
The in silico enzyme digestion and peptide function annotation of pili nut (Canarium ovatum Engl.) 11S globulin protein sequence revealed the presence of bioactive peptides with antihypertensive and antioxidative activities. Molecular docking simulations showed that the proline-histidine (PH) ligand can better inhibit angiotensin I-converting enzyme (ACE) activity than captopril. This result was verified through biochemical assays. Pili kernel protein was extracted with 0.5 M NaCl buffer using the modified Osborne fractionation method, which yielded 100 mg of protein per gram of defatted pili kernel determined via Bradford assay. SDS-PAGE suggested that the protein extract had a major legumin-like (11S) globulin structure with an approximately 57 kDa molecular weight. Enzymatic hydrolysis using pepsin, thermolysin, α-chymotrypsin, and trypsin was used to release the potential antihypertensive and antioxidative peptides with an average rate of 0.03869 mm 2 h-1 , estimated from band intensity. The hydrolysates were tested for ACE inhibition and free radical scavenging. Captopril and enalapril had %ACE inhibitions of 74.02% and 83.57%, respectively, whereas the 6 h hydrolysates had a peak %ACE inhibition of 99.73%. These same hydrolysates, however, only had 51.32% 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity compared to ascorbic acid, which had 75.59%. These results show the potential of pili nut as a functional food, especially for preventing high blood pressure and other cardiovascular diseases.
Abstract Fifteen polar extracts from leaf, seed, pod, stem, flower and root of Crotalaria spectabilis were prepared using aqueous systems, based on the principles of green chemistry, and showed different protease inhibitor (PI) activities on trypsin, papain, pepsin and the extracellular L. amazonensis serine protease (LSPIII). The most pronounced inhibitory effect on LSPIII was observed in leaf (CS-P), root, stem, flower (CS-FPVPP) and pod (CS-VA) extracts. Crotalaria extracts exhibited low cytotoxicity on macrophages; however, they decreased the viability of L. amazonensis promastigotes and amastigotes, as observed in leaf (CS-AE, CS-P, CS-T and CS-PVPP), seed (CS-ST), flower and root (CS-RA) extracts. CS-P was chosen to study PI and secondary metabolites and a 10-12 kDa protein, analyzed by mass spectrometry, was identified as a serine PI homologous with papaya latex serine PI. Glycosylated flavonoids, such as quercetins, vitexin and tricin were the major secondary metabolites of CS-P. The presence of PIs in C. spectabilis is a new finding, especially in other organs than seeds since PIs have been reported only in seed legumes. Besides, this is the first report of antileishmanial activity of C. spectabilis extracts and the identification of serine polypeptide PI and glycosylated flavonoids from leaf.
The present study aimed to investigate the response of soybean cultivars with different susceptibility levels to the root-knot nematode Meloidogyne javanica at varied time intervals by analyzing the initial plant-nematode interaction using antioxidant enzymes as oxidative stress markers. A 4 × 4 × 2 factorial method with 5 repetitions was used to analyze 4 soybean cultivars at 4 different collection times-6, 12, 24, and 48 h-with and without M. javanica inoculation. The parameters evaluated were the activities of antioxidant enzymes phenol peroxidase (POX) and ascorbate peroxidase (APX); the concentrations of hydrogen peroxide (H 2 O 2) and malondialdehyde (MDA); and the number of M. javanica juveniles penetrated into each plant. H 2 O 2 concentration varied among the cultivars with and without inoculation and at different collection times as indicated by MDA concentration and POX and APX activities, demonstrating a rapid response of the host to an infection by M. javanica. Oxidative stress caused by M. javanica did not vary among the soybean cultivars regardless of their susceptibility level; however, the antioxidant enzymes POX and APX responded according to the susceptibility level of the cultivars.
This study evaluates using different levels of the white button mushroom powder (WBMP) on some mucosal innate immune parameters (lysozyme, protease, esterase, alkaline phosphatase activities, and total immunoglobulin levels), and the relative expression of some principal immune-relevant genes (lysozyme, TNF-α, and IL-1β) in the zebra danio intestine. Zebrafish specimens (1.75 ± 0.25 g) were divided into experimental units based on the additives to a diet including 5, 10, and 20 g of WBMP per kg of food weight, alone or in conjunction with the antibiotic (10 mg/kg BW), and the AGRIMOS (1 g/kg food weight). Following the 11-day experimental duration, the skin mucus and intestine were sampled. To assess the immune gene expression, the real-time PCR detection system was conducted according to the ΔΔCt method using the IQ5 software (Bio-RAD). Results showed that all groups had a significant increase in terms of mucosal lysozyme activity compared to the control group. Examination of total immunoglobulin, protease, esterase, and ALP activity in fish under experimental treatment showed that there was no significant difference between the trial groups and the control groups. The most expression of the lysozyme gene was related to the group that was separately taken the lower concentration (5 g per kg of FW) of WBMP. In conclusion, the amount of 1% mushroom powder in the diet can improve its immune function. Our recommendation is that given the positive effects that mushroom powder added on the diet alone, avoid taking antibiotics for this purpose.
Background
Psoriasis is a chronic inflammatory disease in which there is hyperproliferation and abnormal differentiation of keratinocytes. Since high levels of KLK7, an enzyme inhibited by zinc (Zn²⁺) ions, are present in psoriatic lesions, we have studied the effect of zinc ions in the viability of keratinocytes, as well as in the activity of KLK5 and KLK7 and in the expression of epidermal markers.
Methods and Results
The cells were cultured in the absence or presence of Zn²⁺ ions (5.0, 10 and 25 µM). Cell viability was evaluated by the MTT method after during 14 days. Cell death was evaluated by flow cytometry using propidium iodide. The activity of the KLK was evaluated on the hydrolysis of synthetic substrates. Expression of involucrin, filaggrin, cytokeratins (CK) 5, 10 and 14 was evaluated by quantitative PCR. Cell incubation with Zn²⁺ ions did not result in significant changes in cell viability. By MTT assay, it was observed that the cultures incubated with 10 and 25 µM Zn²⁺ ions showed a decrease in the number of viable cells in comparison to the control. Cells cultured for 1 day in the presence of 25 µM Zn²⁺ ions displayed a decrease in KLK7 activity. In the presence of Zn²⁺ ions, it was shown an increase in the expression of CK5, 10 and 14, involucrin and filaggrin.
Conclusions
These results have shown that zinc ions can affect the differentiation of HaCat cells, contributing for future therapeutic trials related to psoriasis based on the modulation of KLK activity.
Aquatic Environment Monitoring Report No. 57 collects together work carried out in 2002-03 by CEFAS
scientists in support of our monitoring and surveillance duties (see overleaf for the background to the work). The
information presented covers both environmental surveillance at offshore and coastal sites and site-specific work carried out in support of risk assessments and regulatory procedures. Some of the science reported here forms part of wider efforts to integrate data from Departments and Agencies in the UK to provide a comprehensive picture of the quality of the marine environment via the UK National Marine Monitoring Programme (NMMP). Other components are unique to CEFAS due to our requirement to understand ecosystem response resulting from potential pressures from deposit, extraction and discharge activities.
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