Ammonium Sulfate Precipitation Research Articles

Characterization and biocontrol mechanism of Streptomyces olivoreticuli as a potential biocontrol agent against Rhizoctonia solani

Rhizoctonia solani is a widespread and devastating plant pathogenic fungus that infects many important crops. This pathogen causes tobacco target spot, a disease that is widespread in many tobacco-growing countries and is destructive to tobacco. To identify antagonistic microorganisms with biocontrol potential against this disease, we isolated Streptomyces strains from forest inter-root soil and screened a promising biocontrol strain, ZZ-21. Based on in vitro antagonism assays, ZZ-21 showed a significant inhibitory effect on R. solani and various other phytopathogens. ZZ-21 was identified as Streptomyces olivoreticuli by its phenotypic, genetic, physiological and biochemical properties. Complete genome sequencing revealed that ZZ-21 harbored numerous antimicrobial biosynthesis gene clusters. ZZ-21 significantly reduced the lesion length in detached inoculated leaf assays and reduced the disease index under greenhouse and field conditions. Based on an in vitro antagonistic assay of ZZ-21 culture, the strain exhibited an antifungal activity against R. solani in a dose-dependent manner. The culture filtrate could impair membrane integrity, possibly through membrane lipid peroxidation. ZZ-21 could secrete multiple extracellular enzymes and siderophores. According to a series of antifungal assays, the extracellular metabolites of ZZ-21 contained antimicrobial bioactive compounds composed of proteins/peptides extracted using ammonium sulfate precipitation, which were stable under stress caused by high temperature and protease K. The EC50 value for ammonium sulfate precipitation was determined to be 21.11 μg/mL in this study. Moreover, the proteins/peptides also exhibited biocontrol ability and were observed to alter the plasma membrane integrity of R. solani which were evaluated by biocontrol efficacy assays on detached tobacco leaves and PI staining. Overall, strain ZZ-21 shows the potential to be developed into a biopesticide against tobacco target spot disease.

The bacterial tubulin homolog FtsZ generates electrical oscillations

FtsZ, a major cytoskeletal protein in all bacteria and archaea, forms a ring that directs cytokinesis. Bacterial FtsZ is considered the ancestral homolog of the eukaryotic microtubule (MT)-forming tubulins, sharing GTPase activity and the ability to assemble into protofilaments, rings, and sheets, but not MTs. Previous studies from our laboratory demonstrated that structures of isolated brain MTs spontaneously generate electrical oscillations and bursts of electrical activity similar to action potentials. No information about whether the prokaryotic tubulins may share similar properties is available. Here, we obtained by ammonium sulfate precipitation an enriched protein fraction of the endogenous FtsZ from wild-type Escherichia coli ATCC 25922 without any transfection or overexpression of the protein. As revealed by electron microscopy, FtsZ was detected by dot blot analysis and immunofluorescence that assembled into filaments and sheets in a polymerization buffer. We used the patch-clamp technique to explore the electrical properties of sheets of FtsZ and bacterial cells. Electrical recordings at various holding potentials ranging from ±200 mV showed a complex oscillatory behavior, with several peak frequencies between 12 and 110 Hz in the power spectra and a linear mean current response. To confirm the oscillatory electrical behavior of FtsZ we also conducted experiments with commercial recombinant FtsZ, with similar results. We also detected, by local field potentials, similar electrical oscillations in K+-depolarized pellets of E. coli cultures. FtsZ oscillations had a wider range of frequency peaks than MT sheets from eukaryotic origin. The findings indicate that the bacterial cytoskeleton generates electrical oscillators that may play a relevant role in cell division and unknown signaling mechanisms in bacterial populations.

Candida albicans chitinase 3 with potential as a vaccine antigen: production, purification, and characterisation.

Chitinases are widely studied enzymes that have already found widespread application. Their continued development and valorisation will be driven by the identification of new and improved variants and/or novel applications bringing benefits to industry and society. We previously identified a novel application for chitinases wherein the Candida albicans cell wall surface chitinase 3 (Cht3) was shown to have potential in vaccine applications as a subunit antigen against fungal infections. In the present study, this enzyme was investigated further, developing production and purification protocols, enriching our understanding of its properties, and advancing its application potential. Cht3 was heterologously expressed in Pichia pastoris and a 4-step purification protocol developed and optimised: this involves activated carbon treatment, hydrophobic interaction chromatography, ammonium sulphate precipitation, and gel filtration chromatography. The recombinant enzyme was shown to be mainly O-glycosylated and to retain the epitopes of the native protein. Functional studies showed it to be highly specific, displaying activity on chitin, chitosan, and chito-oligosaccharides larger than chitotriose only. Furthermore, it was shown to be a stable enzyme, exhibiting activity, and stability over broad pH and temperature ranges. This study represents an important step forward in our understanding of Cht3 and contributes to its development for application.

Quantification of the lung cancer tumor marker CYFRA 21-1 using protein precipitation, immunoaffinity bottom-up LC-MS/MS.

Numerous studies have proven the potential of cytokeratin 19 fragment 21-1 (CYFRA 21-1) detection in the (early) diagnosis and treatment monitoring of non-small cell lung cancer (NSCLC). Conventional immunoassays for CYFRA 21-1 quantification are however prone to interferences and lack diagnostic sensitivity and standardization. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an emerging approach based on a different, often superior, detection principle, which may improve the clinical applicability of CYFRA 21-1 in cancer diagnostics. Therefore, we developed and validated a protein precipitation, immunoaffinity (IA) LC-MS/MS assay for quantitative analysis of serum CYFRA 21-1. Selective sample preparation was performed using ammonium sulfate (AS) precipitation, IA purification, tryptic digestion and LC-MS/MS quantification using a signature peptide and isotopically labeled internal standard. The workflow was optimized and validated according to EMA guidelines and results were compared to a conventional immunoassay. Significant interference effects were seen during IA purification, which were sufficiently solved by performing AS precipitation prior to IA purification. A linear calibration curve was obtained in the range of 1.0-100 ng/mL (R2=0.98). Accuracy and precision were well within acceptance criteria. In sera of patients suspected of lung cancer, the method showed good correlation with the immunoassay. A robust AS precipitation-IA LC-MS/MS assay for the quantification of serum CYFRA 21-1 was developed. With this assay, the clinically added value of LC-MS/MS-based detection over immunoassays can be further explored.

Cassava Root Cortex Peroxidase (CCP) as a Potential Alternative Source of Peroxidase

AbstractCassava is an important starch crop in the world. Starch factories normally generate a huge amount of cassava root residual which may cause pollution to the environment. In order to find some extra uses for the root cortex, cassava root cortex peroxidase (CCP) is found in quantities up to 20 mg kg−1 in deteriorated cortex tissueand demonstrates some applications similar to horseradish peroxidase (HRP). The major native CCP is a 105‐kDa dimeric peroxidase with two 54‐kDa monomers. CCP is found to be tolerant to a broad pH range from 3 to 11 with maximum activity at pH 5.0 and maintains wide temperature range activity from 20 to 70 °C with an optimum at 65 °C. This indicates CCP to be one of the most robust peroxidases. Despite simple purification with ammonium sulfate precipitation, partial purified CCP is capable of determining glucose concentrations with glucose oxidase similar capability to HRP. For application as a reporter enzyme in immunoassays, the lab‐made secondary antibody conjugated with CCPsuccessfully detects the specific antigen in Western blot analysis using achemiluminescent substrate in the same way as HRP. From the properties and results of trial applications, CCP can be equally replaced in all applications of HRP. The enzyme should be found more potential applications to gain the acceptance. Since raw material for CCP is industrially massive, it should emphasize not only optimization of purification and process cost for industry, but also commercializeto the market.

Bioefficacy of engineered Beauveria bassiana with scorpion neurotoxin, LqqIT1 against cotton mealybug, Phenacoccus solenopsis and cowpea aphid, Aphis craccivora.

Cotton mealybug, Phenacoccus solenopsis (Tinsley) and cowpea aphid Aphis craccivora (Koch) are notorious polyphagous, hemipteran sap sucking insect pests. A recombinant toxin gene 'LqqIT1' from the scorpion Leiurus quinquestriatus quinquestriatus (Ehrenberg) was cloned in the pAL1 fungal expression vector and then expressed in the entomopathogenic fungus Beauveria bassiana (Balasmo) using genetic modification techniques. The genetically transformed B. bassiana strain (BbLqqIT1-3) and its un-transformed parent strain (Bb-C) were screened to infect the third instar nymphs of P. solenopsis and first instar nymph of A. craccivora through leaf treatment and topical application (spray) method at 1 * 107 spores per ml concentration. The recombinant strain BbLqqIT1-3 was highly pathogenic against A. craccivora but non pathogenic to P. solenopsis. BbLqqIT1-3 induced 72 and 43.33% mortality in A. craccivora nymphs 96 h after leaf treatment and topical application, respectively. The nymphs of A. craccivora infected with BbLqqIT1-3 displayed classical neurotoxic symptoms such as sluggishness, solublize and liquification of the body. Crude soluble toxin protein, BbLqqIT1a-CSE and Bb-WT-CSE was extracted from the BbLqqIT1-3 and Bb-C, respectively using ammonium sulphate precipitation method, and their oral toxicity was analyzed at 5 µg/ml concentration. The survival of the studied insects was negatively affected by the crude soluble toxin extracts. The LT50 values of BbLqqIT1a-CSE against P. solenopsis and A. craccivora were 22.18 and 17.69 h, respectively. Exposure to crude soluble toxin extracts also accounted for the imbalance of ionic concentrations in the hemolymph of treated insects such as hyperpotassemia (3.53-8.18 meq/ml) in the P. solenopsis and hypopotassemia (7.52-0.47 meq/ml) in A. craccivora. The transformed fungus BbLqqIT1-3 strain exhibited promising results in invitro study.

L-Arginase from P. aeruginosa Isolated from Sewage as Anti-Biofilm Formation by Pathogenic Bacteria

Background: The enzyme arginase plays a significant role in the growth of many organisms as important biological enzyme. Objective: The study was designed to isolate L-arginase from P. aeruginosa that isolated from sewages and to evaluate the influence of arginase on biofilm-generating bacteria. Materials and Methods: The current study includes 28(54.8%) isolates from soil with 24 (46.2%) from sewage water out of 52 isolated from soil and sewage for the purpose of obtaining Pseudomonas aeruginosa. Using standard laboratory methods to isolate Pseudomonas aeruginosa, 33 isolates were isolated, of which 15 (45.45%) were from soil and 18 (54.54%) from sewage. The study also included 177 different eyes (urinary fluid, excrement, blood, wounds, sputum, and cerebrospinal fluid) of patients suffering from various injuries. Results: The statistical analysis of the isolates indicated that there is a high significant (P≤0.01) for the growth of some types of bacteria, such as P. aeruginosa 30(16.89%), Klebsiella, Serratia 33(18.88%) and Acinetobacter 33(18.88%). While the results showed that Lactobacillus is the lowest number of isolates 7(3.9%). Optimum condition of arginase activity and stability comprising pH level of eight degrees, the temperature of 37℃ is the best conditions for the production of arginine, concentrations of L-arginine substrate (0.1-2.5)g/l on the activity of arginase enzyme for arginine production, The finding also shows that the arginase enzyme exhibited the highest activity after 96 hours of the incubation process L- arginase Purification Assay including Ammonium Sulphate precipitation in (60-80)%, Dialysis, Ion exchange chromatography, Gel filtration. Conclusions: This study concluded that the enzyme arginase had a significant effect on the strong biofilm of pathogenic bacteria.

Purification and characterization of the low molecular weight xylanase from Bacillus cereus L-1.

Xylanase is widely used in various industries such as food processing, paper, textiles, and leather tanning. In this study, Bacillus cereus L-1 strain was isolated and identified as capable of producing low molecular weight xylanase through 16s rRNA sequencing. Maximum xylanase yield of 15.51 ± 2.08 U/mL was achieved under optimal fermentation conditions (5% inoculum, 20g/L xylan, pH 6.0, for 24h). After purification via ammonium sulfate precipitation and High-S ion exchange chromatography, electrophoretic purity xylanase was obtained with a 28-fold purification and specific activity of 244.97 U/mg. Xylanase had an optimal pH of 6.5 and temperature of 60°C and displayed thermostability at 30°C and 40°C with 48.56% and 45.97% remaining activity after 180min, respectively. The xylanase retained more than 82.97% of its activity after incubation for 24h at pH 5.0 and was sensitive to metal ions, especially Mg2+ and Li+. Purified xylanase showed a molecular weight of 23kDa on SDS-PAGE, and partial peptide sequencing revealed homology to the endo-1,4-beta-xylanase with a molecular weight of 23.3kDa through LC/MS-MS (liquid chromatography-tandem mass spectrometry). This study suggests that the purified xylanase is easier to purify and enriches low molecular weight xylanases from bacteria source.

In vitro α-Amylase and α-Glucosidase Inhibitory Activity of Dioscorin from Dioscorea alata

Dioscorea alata is a species of yam found in many countries in Southeast Asia. It contains many bioactive compounds consisting of steroidal saponins, phenolic compounds flavonoids, polysaccharides, and proteins. Eighty to eighty-five of the soluble proteins in the tuber of D. alata are dioscorin. Dioscorin (31 kDa) is the major storage protein of yam tuber, exhibiting biochemical and immunomodulatory properties, such as antioxidant activity, reducing blood pressure and angiotensin I converting enzyme (ACE) inhibitors activity. The aims of this research focused on extraction of the total soluble protein from D. alata tuber, purification of dioscorin, and study of in vitro inhibitory activity of α-amylase and α-glucosidase of dioscorin extracts. The results showed that a maximum yield of total soluble protein was obtained from the buffers extraction using Tris-HCl buffer at pH 9.0 resulting in the protein concentration of 15.10 mg/g with a protein extraction yield of 31.56 ± 2.36%. The crude protein was further recovered using ammonium sulfate precipitation and dialysis. SDS-PAGE analysis showed that dioscorin from crude extracts was precipitated at the concentration of ammonium sulfate of 60% saturation. The purified dioscorin was obtained from anion exchange chromatography (DEAE-Sephacel) and its molecular weight of 31 kDa was estimated on SDS-PAGE. Furthermore, the α-amylase inhibitory activities of crude extract (1 mg/mL), purified dioscorin obtain from gastric digestion and purified dioscorin obtained from intestinal digestion were at 14.44% 2.21% and 78.13%, respectively. Moreover, the results showed that the α-glucosidase inhibitory activity of the crude extract, and purified dioscorin obtained from gastric digestion were at 10.56% and 2.61%, respectively.

L-Asparaginase from an acrylamide degrader, Cupriavidus oxalaticus ICTDB921: Production, kinetic modelling, purification and characterization

In the present study, a novel soil isolate and previously reported acrylamide degrader, Cupriavidus oxalaticus ICTDB921, was found to have better and significantly higher l-asparaginase (LAase) activity than other bacterial strains that were screened for the same. The medium and fermentation conditions were optimized for maximizing LAase production followed by its purification and biochemical characterization. The optimum conditions of pH (7), temperature (30 °C) and agitation (180 rpm), showed the highest biomass accumulation (DCW 3.15 g/L) and LAase activity (22.94 IU/mL) after 12 h at 160 mM l-asparagine. Kinetic modelling of optimized batch data showed the biomass accumulation, LAase formation and l-asparagine utilization to best fit with the logistic model, Leudeking-Piret model and exponential decay model, respectively. The crude LAase from this isolate showed high potency at pH 7 to 9, and temperatures from 30 to 40 °C with reasonable pH (6–9) and thermal (up to 50 °C) stability. The LAase was stable against most amino acids and metal ions, and it could degrade aliphatic amides like urea and formamide. A sequential purification by ammonium sulphate precipitation, thermal treatment and chromatographic separation achieved approximately 8-fold purity. The purified subunit showed a molecular weight of 78 kDa, and km and Vmax of 24.1 mM and 40 μmol/min, respectively. The study demonstrates the potential of LAase from Cupriavidus oxalaticus in the mitigation of asparagine in food as well as for therapeutic applications.

Optimization and purification of laccase activity from Mammaliicoccus sciuri isolated from the soils of Similipal, Odisha, India: a kinetics study of crystal violet dye decolorization

Four laccase-producing bacteria were found in soil samples from the Similipal Biosphere Reserve in Odisha, according to the current study. The isolates (SLCB1 to SLCB4) were evaluated for their laccase-producing ability in LB broth supplemented with guaiacol. The ABTS assay was performed to assess the laccase activity. The bacterium Mammaliicoccus sciuri shows the highest laccase activity i.e., 0.5125 U/L at the optimized conditions of pH 5.5, temperature 32.5 °C, ABTS concentration of 0.75 μl with an incubation time of 9 d. Laccase activity of M. sciuri grown in Sawdust was significantly increased in comparison to that in other agro wastes. The partially purified laccase enzyme after ammonium sulfate precipitation and dialysis showed a molecular weight of ∼58.5 kDa as determined by SDS-PAGE. A decolorization efficiency of 66.67% was recorded for the dye crystal violet after 1 h treatment with dialyzed laccase enzyme compared with phenol red, brilliant blue, and methylene blue.

IN VITRO ANTIBACTERIAL AND ANTIBIOFILM ACTIVITIES OF ULTRAFILTRATED-BROMELAIN AGAINST DENTAL CARIES PATHOGENS

Dental plaque on tooth surfaces significantly contributes to dental caries leading to acidic demineralization of the tooth enamel and dentin. This is mainly caused by the formation of pathogenic bacterial biofilm. Dental caries affects approximately 3.5 million people around the world. Compared to synthetic anticariogenic agents such as chlorohexidine, plant-based compounds were found to have fewer side effects and higher economic value. Pineapple (Ananas comosus) is one of the largely commercialized plants in Malaysia with beneficial attributes such as its Bromelain enzyme. This study aimed to purify Bromelain enzyme from MD2 pineapple core and to evaluate their antibacterial properties against dental caries-associate pathogens in vitro. This study evaluated the effect of Bromelain enzyme isolated from MD2 pineapple core using 50% Ammonium Sulphate precipitation incorporated with centrifugal ultrafiltration against Streptococcus mutans, Staphylococcus aureus, Lactobacillus acidophilus, and Lactobacillus casei. Bromelain enzyme showed effectivity of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) for all cariogenic organisms. Ultrafiltrate Bromelain (UFB) enzyme had significant antibacterial activity against S.mutans with a MIC value of 250 μg/ml and complete susceptibility at an MBC value of 1000 μg/ml. Biofilm inhibition against S.mutans increased from 45.98% to 57.48% with ultrafiltration. A 3.25 purification fold increase was observed in the ultrafiltrate sample. In conclusion, the ultrafiltration technique is efficient in purifying Bromelain enzyme and effectively inhibits S. mutans biofilm formation and thus possesses a potential inhibitory effect on dental caries in vitro.

Extraction and Purification of Bromelain Enzyme from Fruits and its Therapeutic Application Study

Food processing industries release a lot of fruit wastes mainly of seed, skin, rind, pomace, crown etc. containing potential bioactive compounds. Bromelain is a crude protein extracted from certain fruits. Bromelain is a sub-type of cysteine protease enzyme extensively investigated for its therapeutic properties such as anti-microbial and anti-oxidant activity with good ferric reducing power. The present study deals with isolating and purifying bromelain from kiwi and avocado fruits and assessing their therapeutic application. Fruit samples were taken from a juice shop in VIT, Vellore, for performing all the experimental analysis. Different parts such as stem, pulp, peel and leaves of the avocado and kiwi fruits were collected for the bromelain extraction process. The extraction of protein was done by the ammonium sulfate precipitation method. Estimation of protein content was performed and the concentration of crude enzyme was found to be 970μg/ml and 900μg/ml for avocado peel and kiwi pulp respectively. Avocado peel showed good activity in ferric reducing power and also a potent radical scavenging activity of 40% at 200μg concentration. Crude bromelain extracted from both fruits showed a broad spectrum of antimicrobial activity against Escherichia coli and Staphylococcus aureus. Hence it can be concluded that bromelain can be used as an effective health supplement.

Purification and characterization of extracellular lipase from a thermotolerant strain: Bacillus subtilis TTP-06.

In current study, lipase from a thermotolerant Bacillus subtilis TTP-06 was purified in a stepwise manner by using ammonium sulfate precipitation and column chromatography. Thenceforth, it was subjected to sodium dodecyl sulfate- and native-polyacrylamide gel electrophoresis to check the homogeneity of the purified enzyme. The ideal substrate concentration, pH, temperature, reaction duration and lipase specificity were identified. With a yield of 11.02%, purified lipase displayed activity of 8.51 U/mg. Thenceforward, the homogeneously purified enzyme was considered to be a homo-dimer of 30kDa subunits. Enzyme had Km and Vmax value of 9.498mM and 19.92molmg-1min-1, respectively. Additionally, the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) method was used to investigate the purified lipase and estimate its 3-D structure, which revealed a catalytic triad of serine, aspartate and histidine.

Insight into the thrombolytic ability of an extracellular fibrinolytic enzyme from Bacillus amyloliquefaciens GXU-1 isolated from Sipunculus nudus

Bacterial fibrinolytic enzymes have an important role in thrombolytic therapy due to their ability to dissolve fibrin. Therefore, purification, characterization and activity determination are of prime importance for bacterial fibrinolytic enzymes. In the current study, marine Bacillus amyloliquefaciens was first screened from Sipunculus nudus living in the Beibu Gulf of China and denoted as Guangxi University-1 (GXU-1). Then, an extracellular fibrinolytic enzyme (FEB-1) was purified from GXU-1 using ammonium sulfate precipitation, hydrophobic chromatography and gel filtration chromatography. The specific activity of FEB-1 was determined to be as high as 6789.74 U/mg. The relative molecular weight of FEB-1 was measured as 30 kDa through SDS‒PAGE. The optimum in vitro fibrinolytic activity of FEB-1 was identified at 37 °C and pH = 8. Furthermore, the activity of FEB-1 can be well preserved at 20–45 °C and pH = 6.0 to 9.0. The combination analysis of SDS‒PAGE and the molecular docking calculation revealed that FEB-1 can cleave more Aα- and Bβ-chains of fibrinogen than γ-chain. It is noteworthy that FEB-1 is comparatively stable under human-body environmental conditions, indicating its potential application in thrombosis therapy.

Production of laccase enzyme from Curvularia lunata MY3: purification and characterization

Laccase-producing fungus (MY3) was successfully isolated from soil samples collected from Mansoura Governorate, Egypt. This fungal isolate has shown a high laccase production level over other isolated fungi. The identity of this isolate was determined by the molecular technique 18SrRNA as Curvularia lunata MY3. The enzyme purification was performed using ammonium sulfate precipitation followed by Sephacryl S-200 and DEAE-Sepharose column chromatography. The denatured enzyme using SDS-PAGE had a molar mass of 65 kDa. The purified laccase had an optimum temperature at 40 °C for enzyme activity with 57.3 kJ/mol activation energy for 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) oxidation. The enzyme had an optimum pH of 5.0, and it has shown a high stability at the acidic range (4.5 to 5.5). Mn2+ and Mg2+ ions enhanced the enzyme activity, while most of the enzyme activity was inhibited by Hg2+. Some compounds such as 2-mercaptoethanol, L-cysteine, and sodium azide at a concentration of 10 mmol/L had shown a high suppression effect on the enzyme activity. The enzyme strongly oxidized ABTS and syringaldazine and moderately oxidized DMP and guaiacol. The antimicrobial activity of the purified enzyme towards three pathogenic strains (Escherichia coli ATCC-25922, Staphylococcus aureus NRRLB-767, and Candida albicans ATCC-10231) was evaluated for the potential use as an antimicrobial therapeutic enzyme.

Extraction, purification and characterization of Lipoxygenase from African oil bean (Pentaclethra macrophylla Benth.) seed

Lipoxygenase (linoleate:oxygen oxidoreductase, LOX) was extracted from African oil bean seed and purified in three purification steps using ammonium sulphate precipitation, ion exchange chromatography on DEAE-Sephadex A50 column and gel filtration chromatography on Sephadex G 200. The optimum pH and temperature were investigated while the pH and temperature stability were also examined. The effect of metal ions on LOX was also investigated. The molecular weight and kinetic parameters were determined. The single protein band on SDS-PAGE suggested that the enzyme was homogenous. The molecular weight was found to be 77.54 kDa. A purification fold of 32.7 and 10.7% yield of the enzyme was achieved. The optimum temperature of the purified lipoxygenase was 40 °C and the enzyme was thermally stable at 40 °C and rapidly lost activity at 50 °C and above. The purified enzyme had an optimum pH of 6.0 and was stable at pH 5.0 and 6.0. The kinetic parameters Km and Vmax of the purified lipoxygenase were 0.1 mM and 94.40 μmol/min, respectively. The activity of the enzyme was enhanced in the presence of Ca2+ and Mg2+ while Zn2+, Cu2+ and Fe2+ significantly reduced the enzyme activity. Total inactivation of the lipoxygenase activity was observed at alkaline environment. This study confirmed lipoxygenase activity in African oil bean seed and showed properties that could be explored in improving its utilization in industrial food processing.

Expression of Untagged Arrestins in E. coli and Their Purification.

Purified arrestin proteins are necessary for biochemical, biophysical, and structural studies of these versatile regulators of cell signaling. Described herein is a basic protocol for arrestin expression in Escherichia coli and purification of tag-free wild-type and mutant arrestins. The method includes ammonium sulfate precipitation of arrestins from cell lysates, followed by Heparin-Sepharose chromatography. Depending on the arrestin type and/or mutations, the next step is Q-Sepharose or SP-Sepharose chromatography. In many cases, the nonbinding column is used as a filter to bind contaminants without retaining arrestin. In some cases, both chromatographic steps must be performed sequentially to achieve high purity. Purified arrestins can be concentrated up to 10 mg/ml, remain fully functional, and withstand several cycles of freezing and thawing, provided that the overall salt concentration is maintained at or above physiological levels. © 2023 Wiley Periodicals LLC. Basic Protocol: Large-scale expression and purification of arrestins Alternate Protocol: Purification of arrestin-3 and truncated form of arrestin-1-(1-378) Support Protocol: Small-scale test expression of wild-type and mutant arrestins in E. coli.

Characterization of Thermostable Cellulase from Bacillus licheniformis PANG L Isolated from the Himalayan Soil.

This study aimed to isolate, purify, and characterize a potential thermophilic cellulase-producing bacterium from the Himalayan soil. Eleven thermophilic bacteria were isolated, and the strain PANG L was found to be the most potent cellulolytic producer. Morphological, physiological, biochemical, and molecular characterization identified PANG L as Bacillus licheniformis. This is the first study on the isolation of thermostable cellulase-producing Bacillus licheniformis from the Himalayan soil. This bacterium was processed for the production of cellulase enzyme. The optimum conditions for cellulase production were achieved at 45°C after 48 h of incubation at pH 6.5 in media-containing carboxymethyl cellulose (CMC) and yeast extract as carbon and nitrogen sources, respectively, in a thermo-shaker at 100 rpm. The enzyme was partially purified by 80% ammonium sulphate precipitation followed by dialysis, resulting in a 1.52-fold purification. The optimal activity of partially purified cellulase was observed at a temperature of 60°C and pH 5. The cellulase enzyme was stable within the pH ranges of 3-5 and retained 67% of activity even at 55°C. Cellulase activity was found to be enhanced in the presence of metal ions such as Cd2+, Pb2+, and Ba2+. The enzyme showed the highest activity when CMC was used as a substrate, followed by cellobiose. The Km and Vmax values of the enzyme were 1.8 mg/ml and 10.92 μg/ml/min, respectively. The cellulase enzyme obtained from Bacillus licheniformis PANG L had suitable catalytic properties for use in industrial applications.

Purification and fractionation of phycobiliproteins from Arthrospira platensis and Corallina officinalis with evaluating their biological activities

Phycobiliproteins (PBPs) are a class of water-soluble pigments with a variety of biological functions that are present in red macroalgae and cyanobacterial species. The crude forms of phycocyanin (C-PC) from the blue green alga Arthrospira platensis and allophycocyanin (APC) from the red macroalga Corallina officinalis were extracted and purified by ammonium sulphate precipitation, anion exchange chromatography, and size exclusion chromatography methods, respectively. The obtained C-PC and APC from A. platensis and C. officinalis were 0.31 mg/mL and 0.08 mg/mL, respectively, with molecular masses of “17.0 KDa and 19.0 KDa” and “15.0 KDa and 17.0 KDa” corresponding to α and β subunits, respectively. FT-IR was used to characterize the purified APC and C-PC in order to look into their structures. Highly purified extracts (A620/A280 > 4.0) were obtained from subtractions’ PC3 and PC4 that were tested for their biological activities. APC and C-PC crude extracts plus their fractions exhibited potent anti-oxidant in different ratios by using three techniques. PC1 showed high anti-inflammatory (75.99 and 74.55%) and anti-arthritic (78.89 and 76.92%) activities for C. officinalis and A. platensis, respectively compared with standard drugs (72.02 and 71.5%). The methanolic and water extracts of both species showed greater antibacterial efficacy against Gram +ve than Gram −ve marine bacteria. Our study shed light on the potential medical uses of C-PC and APC extracted from the tested species as natural substances in a variety of foods and drugs. Further investigations are required to explore the diverse chemical natures of distinct PBPs from different cyanobacteria and red algae because their amino acid sequences vary among different algal species.