A. niger produced alpha-glucosidase, alpha-amylase and two forms of glucoamylase when grown in a liquid medium containing raw tapioca starch as the carbon source. The glucoamylases, which formed the dominant components of amylolytic activity manifested by the organism, were purified to homogeneity by ammonium sulfate precipitation, ion-exchange and two cycles of gel filtration chromatography. The purified enzymes, designated GA1 and GA2, a raw starch digesting glucoamylase, were found to have molar masses of 74 and 96 kDa and isoelectric points of 3.8 and 3.95, respectively. The enzymes were found to have pH optimum of 4.2 and 4.5 for GA1 and GA2, respectively, and were both stable in a pH range of 3.5-9.0. Both enzymes were thermophilic in nature with temperature optimum of 60 and 65 degrees C, respectively, and were stable for 1 h at temperatures of up to 60 degrees C. The kinetic parameters Km and V showed that with both enzymes the branched substrates, starch and amylopectin, were more efficiently hydrolyzed compared to amylose. GA2, the more active of the two glucoamylases produced, was approximately six to thirteen times more active towards raw starches compared to GA1.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
The traditional use of papaya to treat many diseases, especially skin conditions and its prohibition for consumption during pregnancy has prompted us to determine whether papaya extracts both from green and ripe fruits improve wound healing and also produce foetal toxicity. Aqueous extracts of green papaya epicarp (GPE) and ripe papaya epicarp (RPE) were applied on induced wounds on mice. GPE treatment induced complete healing in shorter periods (13 days) than that required while using RPE (17 days), sterile water (18 days) and Solcoseryl ointment (21 days). Extracts were administered orally (1 mg/g body weight/day) to pregnant mice from day 10 and onwards after conception. 3 (n=7) mice and 1 (n=6) mice given RPE and misoprostol, an abortive drug, respectively experienced embryonic resorption while this effect was observed in none of the mice given GPE (n=5) and water (n=5). The average body weight of live pups delivered by mice given GPE (1.12+/-0.04 g) was significantly lower than those delivered by mice given water (1.38+/-0.02 g). In SDS-PAGE, proteins were distributed in three bands (Mr range approximately 8-29 kDa). Band intensity at Mr approximately 28-29 kDa was higher in GPE than in RPE. In contrast, band intensity at low Mr (approximately 8 kDa) was found to be higher in RPE than in GPE. Notably, the band corresponding to Mr approximately 23-25 kDa was absent in RPE. These differences in composition may have contributed to the different wound healing and abortive effects of green and ripe papaya.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
An amylase enzyme from pitaya peel was purified 234.2-folds with 72.1% recovery using ammonium sulphate precipitation, gel filtration and ion exchange chromatography. Gel filtration chromatography and SDS-PAGE revealed that the enzyme is monomeric with a molecular weight of 42.1kDa. The apparent Km and Vmax of the amylase were 2.7 mg/ml and 34.30 u/min/mg of protein, respectively. The enzyme was highly active and stable over a wide pH range from pH 3 to pH 11.0, with optimum activity being observed at pH 5.0. The enzyme was highly selective for soluble starch, amylopectin, glycogen and pulullan. The purified amylase did not require calcium and displayed extreme stability with regard to surfactants and oxidising agents. EDTA, a powerful chelating agent, did not have any significant effect on the stability of the enzyme. Such characteristics have not been previously reported for this type of enzyme from fruit peel. This enzyme, which possesses unique properties, could be widely used in different types of industries, especially in food and biotechnological applications.
The study was aimed to differentiate between porcine and bovine gelatines in adulterated samples by utilising sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) combined with principal component analysis (PCA). The distinct polypeptide patterns of 6 porcine type A and 6 bovine type B gelatines at molecular weight ranged from 50 to 220 kDa were studied. Experimental samples of raw gelatine were prepared by adding porcine gelatine in a proportion ranging from 5% to 50% (v/v) to bovine gelatine and vice versa. The method used was able to detect 5% porcine gelatine added to the bovine gelatine. There were no differences in the electrophoretic profiles of the jelly samples when the proteins were extracted with an acetone precipitation method. The simple approach employing SDS-PAGE and PCA reported in this paper may provide a useful tool for food authenticity issues concerning gelatine.
Plant polyphenols applied as natural antioxidant ingredients, are known to bind to cysteine residues on meat proteins. The aim of this study was to examine the effect of light exposure on the formation of cysteine-phenol adduct in meat added 4-methylcatechol (4MC), a model polyphenol, during storage through quantitative LC-MS/MS-based analysis. Cysteine-4-methylcatechol adduct (Cys-4MC) formation in meat added 1500 ppm 4-MC increased significantly (by 50%) when stored under light in oxygen at 4 °C for 7 days as compared to storage in the dark. This was reflected by a significant decrease in thiol concentrations in the same sample. Gel electrophoresis showed loss in myosin heavy chain (MHC), and a resulting increase in cross-linked MHC (CL-MHC) and larger protein polymers in samples added 4MC. Protein blots stained with nitroblue tetrazolium (NBT) showed intensive protein-polyphenol binding in the meat samples added 4MC, but no major differences between storage conditions.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Myosin heavy chain (MHC) isoforms in goat muscles and their possible relationships with meat quality have not been fully elucidated. This study characterized the MHC isoforms in different caprine muscles using sodium dodecyl sulphate glycerol gel electrophoresis (SDS-GGE). The relationships between MHC isoforms, calpain systems and meat quality characteristics of different muscles in goats were examined. Four muscles, namely infraspinatus (IF), longissimus dorsi (LD), psoas major (PM) and supraspinatus (SS) were obtained from ten Boer crossbred bucks (7-10 months old; 26.5 ± 3.5 kg, BW). The percentages of MHC I, MHC IIa and MHC IIx in SS, IF, PM and LD were 47.2, 38.3, 32.1, 11.9; 28.0, 42.1, 33.0, 36.4; and 24.8, 19.6, 34.9 and 51.7, respectively. IF and SS had higher levels of calpastatin, total collagen and insoluble collagen contents than did PM and LD. PM had longer sarcomere length than did other muscles. LD had higher collagen solubility, troponin-T degradation products and glycogen content than did other muscles. These results infer that variable fiber-type composition could account partially for the differences in the physicochemical properties of goat muscles.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Purification and characterization of polyphenol oxidase (PPO) from Chinese parsley (Coriandrum sativum) were achieved. Crude PPO exhibited an enzyme activity of 1,952.24 EU/mL. PPO was partially purified up to 6.52x with a 10.89% yield using gel filtration chromatography. Maximal PPO activity was found at 35°C, pH 8.0 for 4-methylcatechol and at 40°C, pH 7.0 for catechol. PPO showed a higher affinity towards 4-methylcatechol, but a higher thermal stability when reacting with catechol. LCysteine was a better inhibitor than citric acid for reducing PPO activity at concentrations of 1 and 3mM in the presence of either substrate. Two 46 kDa isoenzymes were identified using SDS-PAGE. Isolation and characterization of Chinese parsley serves as a guideline for prediction of enzyme behavior leading to effective prevention of enzymatic browning during processing and storage, including inhibition and inactivation of PPO.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Electrophoretically detectable variation in the fungus Neurospora intermedia has been surveyed among isolates from natural populations in Malaya, Papua, Australia and Florida. The principal result is a pattern of genetic variation within and between populations that is qualitatively no different than the well documented patterns for Drosophila and humans. In particular, there is a high level of genetic variation, the majority of which occurs at the level of local populations. Evidence is presented which argues that N. intermedia has a population structure analogous to that of an annual vascular plant with a high level of vegetative reproduction. Sexual reproduction appears to be a regular feature in the biology of the species. Substantial heterokaryon function seems unlikely in natural populations of N. intermedia. Theoretical considerations concerning the mechanisms underlying the observed pattern of variation most likely should be consistent with haploid selection theory. The implications of this constraint upon the theory are discussed in detail, leading to the presentation of a model based upon the concept of environmental heterogenicity. The essence of the model, which is equally applicable to haploid and diploid situations, is a shifting distribution of multiple adaptive niches among local populations such that a given population has a small net selective pressure in favor of one allele or another, depending upon its particular distribution of niches. Gene flow among neighboring populations with differing net selective pressures is postulated as the principal factor underlying intrapopulational allozyme variation.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
MPT64 is a specific protein that is secreted by Mycobacterium tuberculosis complex (MTBC). The objective of this study was to obtain optimum culture conditions for MPT64 synthetic gene expression in Escherichia coli BL21 (DE3) by response surface methodology (RSM). The RSM was undertaken to optimize the culture conditions under different cultivation conditions (medium concentration, induction time and inducer concentration), designed by the factorial Box-Bhenken using Minitab 17 statistical software. From the randomized combination, 15 treatments and three center point repetitions were obtained. Furthermore, expression methods were carried out in the flask scale fermentation in accordance with the predetermined design. Then, the MPT64 protein in the cytoplasm of E. coli cell was isolated and characterized using sodium dodecyl sulfate polyacrilamide electrophoresis (SDS-PAGE) then quantified using the ImageJ program. The optimum conditions were two-fold medium concentration (tryptone 20 mg/mL, yeast extract 10 mg/mL, and sodium chloride 20 mg/mL), 5 h of induction time and 4 mM rhamnose. The average concentration of recombinant MPT64 at optimum conditions was 0.0392 mg/mL, higher than the predicted concentration of 0.0311 mg/mL. In conclusion, the relationship between the selected optimization parameters strongly influenced the level of MPT64 gene expression in E. coli BL21 (DE3).
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Apolipoprotein(a) [apo(a)] exhibits a genetic size polymorphism explaining about 40% of the variability in lipoprotein(a) [Lp(a)] concentration in Tyroleans. Lp(a) concentrations and apo(a) phenotypes were determined in 7 ethnic groups (Tyrolean, Icelandic, Hungarian, Malay, Chinese, Indian, Black Sudanese) and the effects of the apo(a) size polymorphism on Lp(a) levels were estimated in each group. Average Lp(a) concentrations were highly significantly different among these populations, with the Chinese (7.0 mg/dl) having the lowest and the Sudanese (46 mg/dl) the highest levels. Apo(a) phenotype and derived apo(a) allele frequencies were also significantly different among the populations. Apo(a) isoform effects on Lp(a) levels were not significantly different among populations. Lp(a) levels were however roughly twice as high in the same phenotypes in the Indians, and several times as high in the Sudanese, compared with Caucasians. The size variation of apo(a) explains from 0.77 (Malays) to only 0.19 (Sudanese) of the total variability in Lp(a) levels. Together these data show (I) that there is considerable heterogeneity of the Lp(a) polymorphism among populations, (II) that differences in apo(a) allele frequencies alone do not explain the differences in Lp(a) levels among populations and (III) that in some populations, e.g. Sudanese Blacks, Lp(a) levels are mainly determined by factors that are different from the apo(a) size polymorphism.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Three human saliva genetic markers, namely, salivary peroxidase (SAPX), Pm, and Ph proteins, were investigated in the three major ethnic groups of Malaysia: Malays, Chinese, and Indians. For Pm, the allelic frequencies of Pm+ for Malays, Chinese, and Indians are 0.385 +/- 0.030, 0.282 +/- 0.026, and 0.289 +/- 0.026 respectively. For Ph, the allelic frequencies of Ph+ are 0.082 +/- 0.016 for Malays, 0.109 +/- 0.017 for Chinese, and 0.062 +/- 0.013 for Indians. For SAPX, the allelic frequencies of SAPX1 in Malays, Chinese, and Indians are 0.762 +/- 0.027, 0.755 +/- 0.027, and 0.723 +/- 0.026 respectively.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Hybridoma clone C3A8, which is a fusion product between splenic lymphocytes of Balb/c mice immunized with MCF7 breast carcinoma cells and SP2/0 myelomas, was produced and characterized. A stable clone that secreted IgM monoclonal antibody (MAb) with kappa light chain was obtained through limiting dilutions. Cell-ELISA screening, flow cytometry analysis, and immunofluorescence staining revealed that the MAb C3A8 had bound specifically and strongly to MCF7 and HT29 but cross reacted weakly or not on HeLa cell line. The MAb C3A8 reacted positively with paraffin-embedded tissues of human breast and colon cancers but there were no positive reactions on normal tissues. Western blot analysis showed the MAb recognized a 55 kDa protein, which was present in the extract of MCF7 and HT29 cell lines. Our results demonstrated that MAb C3A8 could be used for basic and clinical research of breast and colon cancers.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Three bacterial species of Clostridium (septicum, tertium and sporogenes) were identified to produce extracellular proteases cleaving IgA to Fab and Fc fragments, as demonstrated by SDS-PAGE and immunoelectrophoretic procedures. These enzymes acted on monometric IgA1 paraproteins and normal serum IgA1 but had no activity on IgA2 paraproteins and intact secretory IgA1 from human colostrum. Their action on polyclonal serum IgA1 suggested the absence of neutralizing anti-clostridial IgA protease activity. Although the enzymes were shown not to act on secretory IgA1, they were, however, able to digest free alpha-heavy chains of the dimeric IgA molecules. Susceptibility of the alpha-heavy chain to the proteases was more likely due to the change to a more accessible conformation than because of the absence of neutralizing anti-enzymic activity.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
A rapid antibody detection test is very useful for the detection of lymphatic filariasis, especially for certification and surveillance of post-mass drug administration. panLF Rapid kit is suitable for this purpose since it can detect all species of lymphatic filaria. It is based on the detection of anti-filarial IgG4 antibodies that react with recombinant B. malayi antigens, BmR1 and BmSXP. There is an increase demand for the test due to its attributes of being rapid, sensitive and specific results, as well as its field-applicability. The main aim of this paper is to obtain high recovery and purity of recombinant antigen BmSXP via a modified method of immobilized metal affinity chromatography (IMAC). The highest product yield of 11.82 mg/g dry cell weight (DCW) was obtained when IMAC was performed using the optimized protocol of 10 mM imidazole concentration in lysis buffer, 30 mM imidazole concentration in wash buffer, and 10 column volume wash buffer containing 300 mM salt concentration. This gave a 54% protein recovery improvement over the manufacturer's protocol which recorded a product yield of only 7.68 mg/g DCW. The recovered BmSXP recombinant antigen showed good western blot reactivity, high sensitivity (31/32, 97%) and specificity (32/32, 100%) in ELISA, thus attesting to its good purity and quality.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Extracts from ‘kesinai’ (Streblus asper) leaves were investigated as a potential source of enzymes that can serve as an alternative to calf rennet in cheese making. Different types of extraction buffers were investigated namely sodium acetate buffer (pH 4.2-5.0), phosphate buffer (pH 6.0-7.0) and Tris-HCl buffer (pH 7.0-9.0). Finally, the milk-clotting enzyme was extracted using 100 mM Tris-HCl buffer (pH 7.4) with and without 5.0 mg/mL polyvinylpyrrolidone, 0.015 mL/mL Triton X-100 and 2 mM sodium metabisulphite. Purification was carried out using acetone precipitation, and ion-exchange and size-exclusion chromatographic techniques. Results showed that 100 mM Tris-HCl buffer (pH 7.4) was the most efficient extraction buffer among the buffers used in the extraction study. After the final purification step of size-exclusion chromatography, the enzyme was purified 3.3-fold with 42.3% of recovery. The enzyme showed an optimum temperature and pH at 60°C and pH 7.4, respectively. The enzyme was stable up to 70°C for one hour and the partially purified enzyme retained 83% and 96% of its original activity at pH 6.0 and 8.0, respectively. The molecular weight of the partially enzyme was estimated to be 75.8 kDa on SDS-PAGE. The milk-clotting activity of ‘kesinai’ enzyme was found to be lower than that of commercial Mucor rennet.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
The study examined the protein profile of Pectoralis major muscle in broiler chickens subjected to different freezing and thawing methods. Pectoralis major muscle was excised from the carcasses of twenty broiler chickens and split into left and right halves. The left half was subjected to slow freezing (-20oC) while the right half was rapidly frozen (-80oC). The samples were stored at their respective temperature for 2 weeks and assigned to either of tap water (27oC, 30 min), room temperature (26oC, 60 min), microwave (750W, 10 min) or chiller (4oC, 6 h) thawing. Changes in myofibrillar proteins following the thawing methods were monitored through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoretic profile indicated differences (p < 0.05) in intensities of the components of myofibrillar proteins among the thawing methods in both slow and rapidly frozen samples. Chiller thawing had significantly higher (p < 0.05) protein concentration than other methods in rapidly frozen samples. However, in slow freezing, there were no significant differences in protein concentration among the thawing methods. In rapidly frozen samples, the protein optical densities at molecular weight of 21, 27, 55 and 151kDa in tap water, chiller and room temperature thawing did not differ (p < 0.05). Similarly, in slowly frozen samples, protein optical densities at molecular weight of 21, 27, 85 and 151 kDa were not significantly different among chill, tap water and room temperature thawing. Microwave thawing consistently caused higher protein degradation resulting in significantly lower (p < 0.05) protein quality and quantity in both freezing methods.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
This study was carried out to extract and compare the characteristic ability of globulins from cottonseed, alfalfa seed, pea seed, mung bean and French bean with cocoa seeds to produce cocoa-specific aroma precursors. The extracted globulins were compared through SDS PAGE, amino acid and oligopeptide profiles. A very low recovery was obtained during globulin extraction from different seeds ranging from 0.5% to 2.7%. Cottonseed produced the highest total protein (13.90 mg/g), followed by cocoa seed (11.91 mg/g), whereas alfalfa seed, mung bean, pea seed and French bean produced 7.86, 4.77, 4.59 and 3.89 mg/g respectively. Two distinctive bands of 51.1 and 33.0 kDa were observed for cocoa vicilin-class globulin (VCG) from SDS PAGE. More than three bands were shown for other seed globulins. Comparative HPLC analyses of the obtained peptide mixtures revealed different and complex patterns of predominantly hydrophobic peptides. A similar high content of amides (glutamic acids-glutamine, aspartic acid- asparagine and arginine) and low concentrations of lysine were observed in all seeds globulin.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Threadfin bream (Nemipterus japonicas) muscle was hydrolysed using protease extracted from
bilimbi (Averrhoa bilimbi L.) fruit. This study was performed in order to compare the efficiency of bilimbi protease in producing threadfin bream protein hydrolysate with the commercial protease; alcalase 2.4 L. Initially, protease was extracted and then purified using 40% ammonium sulfate precipitation method. The proteolytic activity of the crude extract and purified protease was determined. Precipitation using 40% ammonium sulfate resulted in bilimbi protease specific activity of 2.36 U/mg and 23.13% recovery. Threadfin bream hydrolysate was prepared based on the pH-stat method by hydrolysis for 2 hrs. Hydrolysis using bilimbi protease produced 34.76% degree of hydrolysis (DH) and 3.75% yield while hydrolysis using alcalase resulted in 86.6% DH with 22.78% yield. Alcalase hydrolysate showed higher solubility than bilimbi protease hydrolysate at pH 7 with 70.87 and 32.16% solubility, respectively. Results also showed that protein content of threadfin bream hydrolysate produced using alcalase was higher (86.86%) than those produced using bilimbi protease (22.12%). However, both hydrolysates showed low moisture content between 3.93 to 7.00%. The molecular weight distribution analysis using SDS–PAGE indicated the distribution of smaller peptides especially in alcalase hydrolysate. Overall, the results showed that alcalase is more efficient enzyme choice than bilimbi protease for preparing threadfin bream hydrolysates. However, both hydrolysates could play an important role thus contribute to the food industry.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Gelatin is widely used in food and pharmaceutical products. However, the addition of gelatin especially in food products becomes a controversial issue among Muslims due to its animal origin. Thus, the present study was aimed to detect and differentiate the origin of gelatin added in processed foods using a combination method of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Principal Component Analysis (PCA). Porcine gelatin had exhibited 11 prominent polypeptides compared to bovine gelatin with 2 prominent polypeptides. Polypeptides of both gelatin sources at molecular weight ranged from 53 to 220 kDa can be used to differentiate between porcine and bovine gelatins using PCA. The efficiency in extracting gelatin from processed foods by different solutions was also evaluated. Extraction of gelatin in processed foods by cold acetone and deionised water had exhibited a similar polypeptide patterns, suggesting both solutions are suitable. The study indicated that approach of a simple gelatin extraction combined with SDS-PAGE and PCA, may provide robust information for gelatin species differentiation of processed foods.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
It is crucial to determine several protein-related parameters at the initial stages of proteomic analysis of any biological samples. In this study, crude protein content, total soluble protein, total phenolic content and the SDS-PAGE profile of fifteen varieties of seaweed from Semporna, Sabah, Malaysia were analysed. The crude protein, total soluble protein and total phenolic content of all seaweed samples were in the range of 3.99 to 13.18 % of dry weight, 0.52 to 1.45 mg/mL in acetone dried powder samples and 8.59 to 48.98 mg PGE/g dry weight, respectively. In general, the differences (crude protein, total soluble protein and total phenolic content) among all fifteen varieties of seaweeds were significant (p< 0.05). There was also a strong positive correlation between crude protein and total soluble protein concentration (Pearson’s Correlation Coefficient (r)=0.923; p=0.01) in these fifteen varieties of seaweed. A distinctive protein pattern was observed in the SDS-PAGE gels between three different seaweed classes of green, red and brown colours. All of these results are important in sample preparations (extractions) before furthering proteomic analysis in order to identify and characterize seaweed proteomes.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel