Dodecyl Sulfate Gel Electrophoresis Research Articles

Cyclic-NDGA Effectively Inhibits Human γ-Synuclein Fibrillation, Forms Nontoxic Off-Pathway Species, and Disintegrates Preformed Mature Fibrils.

Parkinson's disease arises from protein misfolding, aggregation, and fibrillation and is characterized by LB (Lewy body) deposits, which contain the protein α-synuclein (α-syn) as their major component. Another synuclein, γ-synuclein (γ-syn), coexists with α-syn in Lewy bodies and is also implicated in various types of cancers, especially breast cancer. It is known to seed α-syn fibrillation after its oxidation at methionine residue, thereby contributing in synucleinopathy. Despite its involvement in synucleinopathy, the search for small molecule inhibitors and modulators of γ-syn fibrillation remains largely unexplored. This work reveals the modulatory properties of cyclic-nordihydroguaiaretic acid (cNDGA), a natural polyphenol, on the structural and aggregational properties of human γ-syn employing various biophysical and structural tools, namely, thioflavin T (ThT) fluorescence, Rayleigh light scattering, 8-anilinonaphthalene-1-sulfonic acid binding, far-UV circular dichroism (CD), Fourier transform infrared spectroscopy (FTIR) spectroscopy, atomic force microscopy, ITC, molecular docking, and MTT-toxicity assay. cNDGA was observed to modulate the fibrillation of γ-syn to form off-pathway amorphous species that are nontoxic in nature at as low as 75 μM concentration. The modulation is dependent on oxidizing conditions, with cNDGA weakly interacting (Kd ∼10-5 M) with the residues at the N-terminal of γ-syn protein as investigated by isothermal titration calorimetry and molecular docking, respectively. Increasing cNDGA concentration results in an increased recovery of monomeric γ-syn as shown by sodium dodecyl sulfate and native-polyacrylamide gel electrophoresis. The retention of native structural properties of γ-syn in the presence of cNDGA was further confirmed by far-UV CD and FTIR. In addition, cNDGA is most effective in suppression of fibrillation when added at the beginning of the fibrillation kinetics and is also capable of disintegrating the preformed mature fibrils. These findings could, therefore, pave the ways for further exploring cNDGA as a potential therapeutic against γ-synucleinopathies.

Proteome Analysis of Adult Acute Lymphoblastic Leukemia by Two-dimensional Blue Native/Sodium Dodecyl Sulfate Gel Electrophoresis.

Despite the significant progress in the treatment of Acute Lymphoblastic Leukemia (ALL) in children, it still remains as one of the most challenging malignancies in adults. Identification of new biomarkers may improve the management of adult ALL. Proteins expressed on the cell surface can be considered as disease-associated biomarkers with potential for diagnosis and targeted therapies. Thus, membrane proteome studies give essential information about the disease-related biomarkers. We applied 2-dimensional blue-native SDS-PAGE technique followed by MALDI-TOF/TOF-mass spectrometry to study the cell membrane proteome of peripheral blood mononuclear cells of adult B-ALL patients in comparison to that of the healthy controls. Sixty seven differentially expressed protein spots were detected, among them 52 proteins were found to be up-regulated but the other 15 proteins were down-regulated in B-ALL. Five differentially expressed proteins, involved in energy metabolism pathways, were detected in B-ALL patients compared to the healthy control group. Differentially expressed proteins provide an insight into the molecular biology of B-ALL. Further studies must be done to confirm our data to be considered as potential targets for detection and treatment of B-ALL.

Highly efficient protein expression of Plasmodium vivax surface antigen, Pvs25, by silkworm and its biochemical analysis

Plasmodium vivax ookinete surface protein, Pvs25, is a candidate for a transmission-blocking vaccine (TBV) for malaria. Pvs25 has four EGF-like domains containing 22 cysteine residues forming 11 intramolecular disulfide bonds, a structural feature that makes its recombinant protein expression difficult. In this study, we report the high expression of recombinant Pvs25 as a soluble form in silkworm, Bombyx mori. The Pvs25 protein was purified from hemolymphs of larvae and pupae by affinity chromatography. In the Pvs25 expressed by silkworm, no isoforms with inappropriate disulfide bonds were found, requiring no further purification step, which is necessary in the case of Pichia pastoris-based expression systems. The Pvs25 from silkworm was confirmed to be molecularly uniform by sodium dodecyl sulfate gel electrophoresis and size-exclusion chromatography. To examine the immunogenicity, the Pvs25 from B. mori was administered to BALB/c mice subcutaneously with oil adjuvant. The Pvs25 produced by silkworm induced potent and robust immune responses, and the induced antisera correctly recognized P. vivax ookinetes in vitro, demonstrating the potency of Pvs25 from silkworm as a candidate for a malaria TBV. To the best of our knowledge, this is the first study to construct a system for mass-producing malaria TBV antigens using silkworm.

Statistical Approach to Enhance α-Amylase Production from Bacillus licheniformis and Purification of the Enzyme

Background: The most widely used thermostable enzymes are the amylases in the starch industry. These are among the most important enzymes and are of great significance in present day biotechnology. Objective: The main objective of the present study was to enhance α-amylase production from Bacillus licheniformis using Response Surface Methodology (RSM) and the purification of the enzyme to homogeneity. Method: Bacterial culture producing α-amylase isolated from hot spring (Himachal Pradesh) was identified as Bacillus licheniformis using 16S rDNA gene sequencing (NCBI Accession No.: KR340466). Medium components and physical culture parameters viz. pH, temperature, inoculum size, peptone concentration and starch concentration were optimized using RSM. Among these five factors, three factors (starch concentration, peptone concentration and inoculum size) had a positive effect on amylase production. A 4.09-fold increase in the production of α-amylase from B. licheniformis was achieved using RSM as compared to One Factor At a Time. The enzyme was purified by using Diethylaminoethyl Cellulose column chromatography and subsequently by Sephadex G-75 gel filtration chromatography. Result: A purification fold of 23.39 and a yield of 12.12% were observed. A single band of 33 kDa was obtained using Sodium Dodecyl Sulphate (SDS) and native-Poly Acrylamide Gel Electrophoresis (PAGE), which indicated that the enzyme was purified to homogeneity and was a monomer. The enzyme showed stability at 50 and 65°C temperatures and at alkaline pH. Conclusion: The stability of purified enzyme at high temperatures and alkaline pH suggested its wide application in textile, detergent and paper industries.

Capillary Sodium Dodecyl Sulfate Agarose Gel Electrophoresis of Proteins

Capillary sodium dodecyl sulfate gel electrophoresis has long been used for the analysis of proteins, mostly either with entangled polymer networks or translationally cross-linked gels. In this paper capillary agarose gel electrophoresis is introduced for the separation of low molecular weight immunoglobulin subunits. The light (LC~24 kDa) and heavy (HC~50 kDa) chain fragments of a monoclonal antibody therapeutic drug were used to optimize the sieving matrix composition of the agarose/Tris-borate-EDTA (TBE) systems. The agarose and boric acid contents were systematically varied between 0.2–1.0% and 320–640 mM, respectively. The influence of several physical parameters such as viscosity and electroosmotic flow were also investigated, the latter to shed light on its effect on the electrokinetic injection bias. Three dimensional Ferguson plots were utilized to better understand the sieving performance of the various agarose/TBE ratio gels, especially relying on their slope (retardation coefficient, KR) value differences. The best resolution between the LC and non-glycosylated HC IgG subunits was obtained by utilizing the molecular sieving effect of the 1% agarose/320 mM boric acid composition (ΔKR = 0.035). On the other hand, the 0.8% agarose/640 mM boric acid gel showed the highest separation power between the similar molecular weight, but different surface charge density non-glycosylated HC and HC fragments (ΔKR = 0.005). It is important to note that the agarose-based gel-buffer systems did not require any capillary regeneration steps between runs other than simple replenishment of the sieving matrix, significantly speeding up analysis cycle time.

Bioactive food foams stabilized by licorice extract/whey protein isolate/sodium alginate ternary complexes

Interactions among multi-component food matrices play a key role in modulating the foam properties. In this study, foams containing mixtures of sodium alginate (ALG; 0.5, 1.0, and 1.5% w/v), whey protein isolate (WPI; 0.2 and 0.6% w/v), and licorice extract (LE; 0.1, 0.3, and 0.5% w/v) were formed and characterized. The interactions between the components were confirmed by turbidity, ζ-potential, particle size, Fourier transforms infrared spectroscopy, and sodium dodecyl-sulphate gel electrophoresis. The pre-foam dispersions adopted a shear-thinning behavior with high apparent viscosity as a function of ALG level. Surface tension results indicated that ALG and LE were the main components at the air/water interface, and the pre-foam dispersions possessed a good antioxidant activity (up to 56.53%). The ALG/WPI/LE foamed systems had up to 446.03% overrun, up to 81.69% air-volume fraction, and up to 40.80 h physical stability. Foams with higher overrun and air-volume fraction displayed greater firmness and cohesiveness, as well. Additionally, the elastic properties (Gʹ) were superior to the viscous ones (Gʹʹ) at all frequency range which indicated the solid-like behavior of the foams. A remarkable free radical scavenging activity (up to 49.66%) was also observed in the foams. Therefore, the bioactive foams with high foamability, stability, and viscoelasticity could be used to design novel healthy aerated foods with low calorie content.

Preparation of Staphylococcal Protein A Imprinted Supermacroporous Cryogel Beads.

Protein A is the most commonly used ligand in IgG purification due to its specific binding to the Fc receptor of most immunoglobulins, making it commercially important. Molecular imprinting is a method based on the selective recognition of various molecules. Molecular imprinted polymers are materials that are easy to prepare, durable, cheap and have molecular recognition capability. Cryogels are prepared by radical polymerization in a partially frozen environment. The unique structure of cryogels combined with osmotic, chemical and mechanical stability make them attractive chromatography matrices for a variety of biological compounds/specimens (plasmids, pathogens, cells). In this protocol, protein A imprinted supermacroporous poly(2-hydroxyethyl methacrylate) cryogels were prepared in spherical form for protein A purification. The characterization of the prepared cryogels were made by swelling test, scanning electron microscopy (SEM), Fourier transform infrared spectrophotometer (FTIR), and Brunauer-Emmett-Teller (BET) surface area analysis. After characterization, optimum conditions for protein A adsorption were determined in the batch system. The maximum protein A adsorption capacity was determined after optimization of the imprinted cryogels. Protein A relative selectivity coefficients of imprinted cryogels were examined for both Fc and protein G. Protein A was isolated from the bacterial cell wall using fast performance liquid chromatography (FPLC). The separated protein A was determined by sodium dodecyl sulfate gel electrophoresis (SDS-PAGE). In the last stage, the reusability of the cryogel was examined.

Isolation and Purification of Thrombolytic Enzyme Extracted from Earthworm Punjab, Pakistan

The Cardiovascular disease due to thrombus (clot) formation is the major factor of death throughout the world. Earthworms being the eco engineers has thrombolytic enzyme that can be used for thrombolysis. The thrombolytic enzyme was isolated and purified from supernatant of earthworm Apporectodea longa by column chromatography. Six Strain BKT 11, BKT 15, BKT 17, BKT 26, BKT 27 and BKT 28 shows the thrombolytic activity 791.64 U/mg, 1362.39 U/mg, 1205.4 U/mg, 710.63 U/mg, 529.66 U/mg and 625.00 U/mg respectively. Thrombolytic activity was confirmed by blood clot lysis method. Different concentrations 50 ?l,100 ?l, 150 ?l, 200 ?l and 250 ?l of extracted enzyme were applied on 25mg of wet blood clot along with control where distill water used. These fractions of extracted enzymes represent the dissolution of clot (thrombolysis). The molecular weight 32 KDa was determined by sodium dodecyl sulphate gel electrophoresis (SDS-PAGE). Results show that extracted elute have potential of fibrinolytic activity in this specie of earthworm and it can serve as a suitable therapeutic agent. Keywords: Thrombolytic activity, Casein plate assay, Blood clot lysis, spectrophotometry, Gel electrophoresis.

Identification of Goats’ and Cows’ Milk Protein Profile in Banyumas Regency by Sodium Dedocyl Sulphate Gel Electrophoresis (Sds-Page)

Protein is one of the nutrient components in milk that is related to product quality. The components of milk protein are divided into casein alpha-s1, beta, alpha-s2, kappa, and whey fractions such as alpha lactalbumin and beta lactoglobulin. There are no existing data of milk protein fraction in dairy cow and goats in Banyumas Regency. This study aimed to determine the profile in form of protein fractions of cow and goat milk in Banyumas. Milk sample from fifty cows and thirty dairy goats was taken by random sampling in some areas. The milk protein profile was identified by the technique of sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) and protein quantity prediction by software. The data obtained were analyzed statistically by Mann-Whitney between cows and goats. The results were significantly different (p<0.05) between cows and goats in molecular weight of protein alpha-S1 casein (29.66 vs 33.37 kDa), alpha-S2 (27.76 vs 29.49 kDa), beta (24 , 48 vs 25.59 kDa) and beta lactoglobulin (15.75 vs 15.97 kDa). The quantity of casein alpha-S1 (7.88 vs 4.16 g/l), alpha-S2 (1.31 vs. 4.02 g/l), beta (8.74 vs 14.24 g/l), kappa (2.41 vs. 4.28 g/l) and alpha lactalbumin (0.91 vs 0.7 g / l) was significantly different (p <0.05) between cow's and goat's milk, respectively. In conclusion, milk protein profile of cows and goats in Banyumas Regency is different.

Purification of a Thermostable β-mannanase from Paenibacillus Thiaminolyticus - characterization and its Potential Use as a Detergent Additive

Endo-1, 4- β- D-mannanase (EC 3.2.1.78) is a glycoside hydrolase involved in random cleavage of β-1, 4- D-manno-pyranosyl linkages within mannans and heteromannans and generates branched and linear oligosaccharides. A β-mannanase was purified from a thermotolerant bacterium Paenibacillus thiaminolyticus isolated from a soil sample. Enzyme was purified to homogeneity with specific activity of 8812 U/mg protein. Sodium dodecyl sulfate (SDS) and native poly-acryl amide gel electrophoresis indicated that the purified mannanase is a monomeric protein with a molecular mass of 38 kDa. The purified enzyme was found to be maximally active at temperature and pH of 60°C and 7.0, respectively. It was stable at 55°C for 24 h and maintained more than 50 % activity up to 3 h at 60°C. The enzyme was very stable in the pH range of 5.0-9.0. Purified β-mannanase demonstrated high stability after 1 h of pre-incubation with most of the tested organic solvents. Enzyme retained significant stability in the presence of various detergent additives, commercially available detergents and dish washing liquids. The high compatibility and substantial stability in the presence of nonionic detergents and dishwashing liquids confirmed its utility as an additive to dish washing liquids and laundry detergents. Enzyme exhibited efficacious de-staining of heteromannan based stains of chocolate ice cream and salad dressing in the wash performance test for detergent application. It also exhibited anti-soil redeposition effect on cotton swatches treated with tennis court clay and heteromannans.

Analysis of protein denaturation, aggregation and post-translational modification by agarose native gel electrophoresis

Agarose native gel electrophoresis has been developed to separate proteins and protein complexes in the native state. Here, we applied this technology to analyze proteins that undergo degradation, post-translational modification or chemical/physical changes. Antibodies showed aggregation/association upon acid or heat treatment. Limited reduction of disulfide bonds resulted in non-covalent aggregation of bovine serum albumin and cleavage of only inter-chain linkages of an antibody that had no effects on its overall structure. Native agarose gel analysis showed changes in mobility of human transferrin upon Fe3+ binding. Analysis of a commercial glycated human hemoglobin A1c showed no difference in electrophoretic pattern from un-modified hemoglobin. Native agarose gel showed aggregation of a virus upon acid or heat treatment. We have extracted bands of bovine serum albumin from the agarose native gel for sodium dodecylsulfate gel electrophoresis analysis, showing degradation of aged sample. Lastly, we analyzed phosphorylation of Zap70 kinase by native gel and Western blotting. These applications should expand the utility of this native gel electrophoresis technology.

Renal Expression of Light Chain Binding Proteins.

Overproduction of human light chains (LCs) and immunoglobulins can result in various forms of renal disease such as cast nephropathy, monoclonal immunoglobulin deposition disease, LC proximal tubulopathy, AL amyloidosis, and crystal storing histiocytosis. This is caused by cellular uptake of LCs and overwhelmed intracellular transport and degradation in patients with high urine LC concentrations. LC kappa and lambda purification was evaluated by sodium dodecyl sulfate gel electrophoresis. LC and myeloma protein binding to immobilized renal proteins was measured by enzyme-linked immunosorbent assay (ELISA). The human protein microarray (HuProt™) was screened with purified kappa and lambda LC. Identified LC partners were subsequently analyzed in silico for renal expression sites using protein databases, Human Protein Atlas, UniProt, and Bgee. Binding of urinary LCs and immunoglobulins to immobilized whole renal proteins from 22 patients with myeloma or plasma cell dyscrasia was shown by ELISA. Forty lambda and 23 kappa interaction partners were identified from HuProt™ array screens, of which 21 were shared interactors. Among the total of 42 interactors, 12 represented cell surface proteins. Lambda binding signals were approximately 40% higher than kappa signals. LC interaction with renal cells and disease-causing pathologies are more complex than previously thought. It involves an extended spectrum of proteins expressed throughout the nephron, and their identification has been enabled by recently developed methods of protein analysis such as protein microarray screening. Further biochemical studies on interacting proteins are warranted to elucidate their clinical relevance.

Proteomic Analysisof Food Allergens by MALDI TOF/TOF Mass Spectrometry.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is largely recognized as an important tool in the analysis of many biomolecules such as proteins and peptides. The MS analysis of digested peptides to identify a protein or some of its modifications is a key step in proteomics. MALDI-MS is well suited for the peptide mass fingerprinting (PMF) technique, as well as selected fragmentation of various precursors using collisional-induced dissociation (CID) or post-source decay (PSD).In the last few years, MALDI-MS has played a significant role in food chemistry, especially in the detection of food adulterations, characterization of food allergens, and investigation of protein structural modifications induced by various industrial processes that could be an issue in terms of food quality and safety.Here, we present simple extraction protocols of allergenic proteins in food commodities such as milk, egg, hazelnut , and lupin seeds. Classic bottom-up approaches based on Sodium Dodecyl Sulphate(SDS) gel electrophoresis separation followed by in-gel digestion or direct in-solution digestion of whole samples are described. MALDI-MS and MS /MS analyses are discussed along with a comparison of data obtained by using the most widespread matrices for proteomic studies, namely, α-cyano-4-hydroxy-cinnamic acid (CHCA) and α-cyano-4-chloro-cinnamic acid (CClCA). The choice of the most suitable MALDI matrix is fundamental for high-throughput screening of putative food allergens.

Myofascial pain syndrome in female patients with chronic nonspecific back pain: diagnosis and treatment

Objective: to elucidate the frequency and pathogenesis of myofascial pain syndrome (MFPS) in chronic nonspecific lower back pain (CNLBP) and to optimize the diagnosis and treatment of MFPS in CNLBP. Patients and methods. The investigation covered 121 patients with CNLBP. The patients' mean age was 42.1±10.5 years; the pain duration was 7.9±4.3 months. The possible causes of CNLBP were determined: these were facet joints (FJs); sacroiliac joints (SIJs); skeletal muscles with the development of MFPS; MFPS concurrent with FJs; MFPS concurrent with SIJs. Twenty patients had MFPS only (its mean duration was 5.3±2.2 months; the mean pain intensity scores were 6.5±1.1 on a numerical rating scale). Six patients underwent examinations of open biopsy specimens of the muscle straightening the spinal column; a comparison group consisted of 3 healthy women matched for age and gender. The patients were prescribed therapy with aceclofenac 200 mg/day in combination with tolperisone 450 mg/day and nondrug therapy (cognitive behavioral therapy and kinesio- and ergotherapy). When the treatment was insufficiently effective, ultrasonography of the muscle straightening the spinal column was additionally performed; a local anesthetic was injected into myofascial trigger points (MTPs). Results and discussion. MFPS was a cause of pain syndrome in 63 (52%) patients, while MFPS this was an isolated cause of pain in 20 (16.5%) cases and was concurrent with FJ osteoarthritis in 23 (19%), and with SIJ dysfunction in 20 (16.5%). Muscle ultrasonography in patients with MFPS revealed MTPs, whereas examinations of biopsy specimens of the muscle straightening the spinal column showed no evidence of necrosis, fibrosis, or inflammatory infiltration in the presence of transformation of the myosin phenotype, by increasing the proportion of rapidly fatigued type II muscle fibers. The results of sodium dodecyl sulfate (SDS) gel electrophoresis indicated a decrease in the content of titin and nebulin, the sarcomeric cytoskeletal proteins involved in maintaining muscle contractility. A two-week cycle of therapy with aceclofenac and tolperisone reversed pain syndrome in 5 (25%) of the 20 patients and reduced the intensity of back pain in 15 (75%), but the pain increased during physical exercise and impeded active rehabilitation. The additional administration of anesthetics into MTPs and the continuous intake of aceclofenac and tolperisone in combination with kinesiotherapy could relieve pain syndrome and enhance motor activity. Conclusion. More than half of the patients with CNLBP had MFPS only or concurrent with joint pathology (FS and FJs). The changes found in the back muscle biopsy specimens of patients with CNLBP are potentially reversible and can be reversed during kinesiotherapy.

Protein expression patterns in hek-Blue™ - Cells treated with Clinacanthus nutans extracts

Background: Clinacanthus nutans (CN) is a small shrub native to tropical Asia known for their anti-oxidant, anti-inflammation, anti-cancer, and anti-viral activities. Objectives: This study aimed to investigate the effect of CN extract on human embryonic kidney cell line (HEK-Blue™-4) in a proteomic perspective. Materials and Methods: Comparative proteomic profiling through two-dimensional sodium dodecyl sulfate gel electrophoresis was performed on HEK-Blue™-4 treated with CN leaf polar extract. Results: We successfully identified seven upregulated proteins, of which five promoted the growth of the HEK-Blue™-4 cells. Interestingly, a potent antioxidant enzyme which neutralizes reactive oxygen or nitrogen species, peroxiredoxin-1 was also upregulated in HEK-Blue™-4 cell lines after treatment with CN leaf polar extract. Conclusion: CN leaf polar extract promotes the growth of HEK-Blue™-4 cells and induced the expression of peroxiredoxin-1, which protects the cells from reactive oxygen species during the inflammation process.

Variations in Amino Acid and Protein Profiles in White versus Brown Teff (Eragrostis Tef) Seeds, and Effect of Extraction Methods on Protein Yields.

Data on variations in amino acid compositions and protein profiles among white and brown teff, a grain of growing interest, is either limited or contradicting at the moment. In this study, three white (Addis-W, Mekel-W and Debre-W) and three brown (Addis-B, Mekel-B and Debre-B) teff seed samples were used for whole flour amino acid analysis and protein fractionation with three different methods. White and brown seed types showed different physical changes during protein extraction. Brown teff displayed higher essential amino acid content than white with lysine present in high concentration in both seed types. Extraction with tert-butanol increased prolamin yields in teff compared to ethanol. The major protein fraction in teff was glutelin with white teff containing higher glutelin proportion than brown. Sodium Dodecyl Sulfate Gel Electrophoresis (SDS-PAGE) analysis revealed clear genetic variability between white and brown teff seed types.

Immunogenicity assay of KatG protein from Mycobacterium tuberculosis in mice: preliminary screening of TB vaccine

the tuberculosis (tB) disease is still widely found even though BCg vaccine given to many people. Ineffectiveness of the BCG vaccine is one of causes that make the difficulties in preventing TB transmission. objective of the research was to determine the immunogenicity of katg protein of M. tuberculosis clinical iso-late L19 in mice. the katg protein as antigen was prepared by expression of the katg gene of M. tuberculosis clinical isolate L19 in Escherichia coli BL21 using pColdII-DNA vector. After purification by affinity chromatography, the katg was vaccinated to mice to detect its immunogenicity. the expression of katg in e.coli BL21 could result in katg protein with molecular weight 80 kda in sodium dodecyl sulfate gel electrophoresis (SDS-PAGE). The pure KatG protein could significantly stimulate the immune response of mice by triggering the antibodies production of Igg1, Igg2a, Igg2b, Igg2c, Igg3, and IgM. the highest antibody level was obtained when the mice were vaccinated by KatG L19 with the dose of 45 μg/ml. Of the antibodies, the IgG2c isotype was dominantly produced in the blood serum. the katg protein exhibited a high immunogenici ty in mice, so it is possible to develop as a vaccine candidate for tB. a clinical test should be performed in a future to ensure its safety as a therapeutic protein.

Direct recovery of Bacillus subtilis xylanase from fermentation broth with an alcohol/salt aqueous biphasic system

Xylanase enzyme degrades linear polysaccharide β-1,4 xylan and the hemicellulose of the plant cell wall. There is a growing demand in finding a cost-effective alternative for industrial scale production of xylanase with high purity for pharmaceutical applications. In this study, an alcohol/salt aqueous biphasic system (ABS) was adopted to recover xylanase from the Bacillus subtilis fermentation broth. The effects of several ABS parameters such as types and concentrations of alcohols and salts (i.e., sulphate, phosphate, and citrate), amount of crude loading and pH of the system on the recovery of xylanase were investigated. Partition coefficient of xylanase (KE), selectivity (S) and yield (YT) of xylanase in top phase of the ABS were measured. Highest KE (6.58±0.05) and selectivity (4.84±0.33) were recorded in an ABS of pH 8 composed of 26% (w/w) 1-propanol, 18% (w/w) ammonium sulphate. High YT of 71.88%±0.15 and a purification fold (PFT) of 5.74±0.33 were recorded with this optimum recovery of xylanase using alcohol/salt ABS. The purity of xylanase recovered was then qualitatively verified with sodium dodecyl sulphate (SDS) gel electrophoresis. The SDS profile revealed the purified xylanase was successfully obtained in the top phase of the one-step 1-propanol/sulphate ABS with a distinct single band.

Amylase Zymography.

Amylase zymography was carried out for the detection of amylases produced by a Geobacillus stearothermophilus strain isolated from the Thermal Center "Las Trincheras" in Venezuela. Zymography is an electrophoretic technique used to study hydrolases by means of thin gels containing copolymerized-specific substrates, under nonreducing conditions. In this study, 0.1% starch was incorporated into the gel as substrate. The formation of clear zones against a dark background in the gel stained with iodine indicated the presence of amylolytic activity. The thermophilic bacteria released several extracellular amylases to a selective growth medium supplemented with 1% soluble starch at 55 °C after 40 h incubation. The amylolytic enzymes showed an optimum temperature of 60 °C and an optimum pH at 6.0. The amylases were partially purified by cold acetone precipitation followed by two chromatographic techniques. These purified amylases showed different molecular masses which were determined by sodium dodecyl sulfate gel electrophoresis and confirmed by zymography.

Aplikasi Metode SDS-PAGE (Sodium Dodecyl Sulphate Poly Acrylamide Gel Electrophoresis) untuk Mengidentifikasi Sumber Asal Gelatin pada Kapsul Keras

Gelatin as the main ingredient of capsules is still a problem for a moslem. Most of gelatin production remains largely derived from non-halal materials. One of gelatin source is came from collagen of the skin and bones of bovine or pork. The main of study is determine the source of gelatin used in hard capsules by using SDS-PAGE (Sodium Dodecyl Sulphate Gel electrophoresis Poly Acrylamide) method. In the early stages, optimization of standards bovine and pork gelatin were hydrolyzed by pepsin at pH 4.5 and 60°C for 1 hour, 2 hours, and 3 hours. Gelatin hydrolyzateswere analyzed by SDS-PAGE to determine the optimal hydrolysis time. Identification of gelatin hydrolyzate fragments were carried by molecular weight. Hydrolysis time optimization throught applied to identify the source of hard gelatin capsules in the samples obtained from market and compared with the simulation of hard gelatin capsules. The results showed there were of specific bands of bovine gelatin with a molecular weight of 11,4 kDa; 34 kDa; 47kDa and specific bands of pork gelatin with a molecular weight of 24.7 kDa; 28 kDa; and 60 kDa. Similar results were obtained on a sample of hard capsules with bands of protein fragments that were identical to bovine gelatinstandard. Based on the results,each of the samples were tested contain of bovine gelatin respectively. DOI :http://dx.doi.org/10.15408/jkv.v0i0.3150