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It has been common to do fractionation (for example using ammonium sulphate as a precipitating agent) before doing a more sophisticated method for purification of a protein. The logic behind this is easy to understand, but in fact, the precipitation step often causes severe loss in yield and activity of the protein, making the whole purification effort too costly. In this work we evaluated the specific activity (thus, purification factor) and total activity (yield) during the purification process of papain from a crude extract using ion exchange chromatography (IEC), with and without prior fractionation using ammonium sulphate. Detail assays in each step were recorded and SDS-PAGE was also done to reveal the protein profile of the purification products. [ABSTRACT FROM AUTHOR]

This paper describes the purification and characterization of a novel acid stable and pH tolerant α-amylase from a Aspergillusoryzae JGI 21 isolated from Mangalore. The enzyme displayed a molecular weight of 22 kDa and it was stable over a broad range of acidic and alkaline pH with maximum activity and stability at 6.5. The optimum temperature of enzyme stability was found to be around 24+/-2°C. The purification of α-amylase by ammonium sulphate precipitation and ion-exchange chromatography resulted in 23.56 fold increase in its activity (100.38 U/mg protein). Considering its promising properties, this enzyme can find potential applications in the food industry as well as in laundry detergents. [ABSTRACT FROM AUTHOR]

Abstract: Screening of crustacean proteins in relation to molting cycle revealed a 48kDa protein which is cross reactive to Gas7 antibody and expressed predominantly in the intermolt stage where the cells remain quiescent. This 48kDa protein of Penaeus monodon has been purified to homogeneity using ammonium sulphate precipitation, ion-exchange chromatography and electro elution. MALDI-TOF analysis of the purified protein revealed the presence of ANK repeat and LEM domain regions homologous to those of mouse. RT-PCR revealed 684bp in all molting stages with varied expression. Bioinformatic analysis showed that the ankyrin repeat domain in the 48kDa protein of P. monodon is highly conserved among Homo sapiens, Mus musculus, Rattus rattus, Danio rerio and Drosophila mojavensis. Secondary structure prediction revealed a commonly conserved region between mouse Gas7 and the ANK repeat region of P. monodon. Thus, we concluded that the isolated 48kDa protein from P. monodon is a novel, Gas7-like protein with ANK repeat and LEM domain involved in molting. [Copyright &y& Elsevier]

Objective: Some properties of cellulase purified from the culture supernatant of Bacillus coagulans Co4, isolated from cocoa pod dumpsite were investigated for possible biotechnological applications. Methods: The crude cellulase was purified to apparent homogeneity using a combination of acetone precipitation, CM Sepharose CL-6B ion exchange chromatography and gel filtration on Sephadex G-100. The molecular and thermodynamic properties of the purified enzyme were studied following standard procedures. Results: The specific activity of the purified cellulase rose from 0.10 to 47 units/mg of protein, at the end of purification. The molecular weight was found to be 14.5 kDa; and an apparent Km value of 0.18±0.06 mg/ml of carboxylmethylcellulose. The optimum pH and temperature were 7.5 and 60°C respectively. The activation energy for carboxylmethylcellulose hydrolysis (Ea) was 16.5 kJ/mol. Na+ and K+ had no effects on its activity at concentrations up to 200 mM, whereas Ca2+ and Mg2+ served as inhibitors at concentrations above 25 and 40 mM respectively. The cellulase retained 40% residual activity when heated at 60°C for 40 minutes. Conclusison: On the basis of these properties, it is concluded that the purified cellulase is moderately thermostable and may have applications in the bioconversion of agricultural wastes into economically useful products. [ABSTRACT FROM AUTHOR]

Abstract: Aspergillus niger RNase belonging to RNase T2 family is purified to homogeneity using ammonium sulfate precipitation and anion-exchange chromatography from A. niger. The enzyme is glycosylated monomer with molecule mass of 28.5kDa. Optimum activity of the enzyme is achieved at pH 3.5 and temperature 55°C. The enzyme shows broad substrate specificity with poly (A), poly (C), poly (G), poly (U) and yeast RNA. The enzyme is highly stable against various metal ions and organic solvents, thus dictates its applicability in various industries. Two peptide sequences, PGGTLLQTQFWDYDP and TLDSYTALSDAGITPSEDATYK obtained by MS/MS analysis and confirmed by BLAST search, show substantial sequence homology with Aspergillus phoenicis, Aspergillus clavatus and P. merneffei. Multiple sequence analysis of the peptide sequences of purified enzyme shows 97%, 75% and 72% identity with A. phoenicis, A. clavatus and P. merneffei, respectively using Clustal W tool. Spectroscopic studies by absorbance, fluorescence, and circular dichroism reveal that the enzyme has α+β type secondary structure with approximately 29% α-helix, 24% β-sheet and 47% random coil by K2D software. [ABSTRACT FROM AUTHOR]

Abstract: Crude water-soluble polysaccharides were isolated from gel juice, skin juice and flowers of Aloe arborescens by ethanol precipitation, Sevag deproteinization. Result indicates that skin juice contained 2.4 times the level of polysaccharides than gel juice from one plant, suggesting the potential industrial application of A. arborescens skin rather than discarded. After anion-exchange chromatography, neutral polysaccharides accounted for 59.1% and 79.8% of the total recovered neutral and acidic polysaccharide preparations from gel juice and skin juice, respectively, whereas the crude flower polysaccharides were largely composed of weakly acidic polysaccharides (85.2%). Sugar analysis of the polysaccharides after gel permeation chromatography revealed that glucose and galactose were the most abundant monosaccharide in the neutral polysaccharides from gel juice and skin juice, respectively. The acidic polysaccharides from the two juices consisted of glucuronic acid, galactose, glucose, mannose and xylose with variable proportions. Except glucuronic acid (16.4%) in flower acidic polysaccharide, the flower neutral and acidic polysaccharides contained galactose, glucose and mannose as the main sugar components. Glucuronic acid was as the major uronic acid in all acidic polysaccharides from different tissues. [Copyright &y& Elsevier]

Abstract: A procedure based on BIA Separations CIM DEAE anion-exchange chromatography was developed to separate double-stranded (ds) RNA of hypovirus infecting phytopathogenic fungus Cryphonectria parasitica. Using a linear gradient of 25mM 4-morpholinepropanesulfonic acid (MOPS), pH 7.0 as a binding buffer, and 25mM MOPS, 1.5M NaCl, 0.1mM EDTA, 15% isopropanol (v/v), pH 7.0 as an elution buffer, hypoviral dsRNA was additionally purified from nucleic acid species present in preparations partially purified by standard CF-11 cellulose chromatography. Moreover, crude phenol/chloroform extracts of the fungal tissue were also applied to monolithic supports and CIM DEAE chromatograms revealed clear evidence for hypoviral presence without CF-11 chromatography, nucleic acid precipitation, and electrophoresis. [Copyright &y& Elsevier]

Abstract: The present work deals with the purification and drying of bromelain, one of the vegetal proteases. The purification of the enzyme from fruit Ananas comosus was carried out by precipitation and by chromatography. Further, the drying of chromatographically purified fraction was carried out in freeze dryer and spray dryer. The activity was expressed in terms of casein digesting units (CDU). The bromelain extract of higher purity was obtained from the fruit using ion exchange chromatography. The elution efficiency in chromatographic purification was obtained in the range of 80–90%. The fold purity obtained by chromatography was 3.3 times as compared to that obtained by precipitation. Bromelain thus obtained was subjected to spray and freeze drying. It was observed that the yield obtained in spray dryer at an outlet temperature of 40–50°C was almost 50–70% and in case of freeze dryer the yield obtained was 96%. The resulting residual activity and the specific activity was compared between spray-dried and freeze-dried powder. The purity of enzyme obtained in the present study was found to be 2.8 times more than that of a commercial sample. [Copyright &y& Elsevier]

Papain, a proteolytic enzyme was purified from latex of Carica papaya by a variety of techniques such as two-step salt precipitation, organic solvent precipitation (ethanol) and ion exchange chromatography. Fold purity obtained using ion exchange chromatography was in the range of 4.5 to 5 times as compared to the precipitation techniques. The purified papain was entrapped in ionotropically cross-linked alginate beads, in order to develop formulation for site-specific delivery of papain in intestine. The entrapment efficiency for papain in alginate beads was found to be in excess of 86%. Drying at low temperature and low air velocity results into maximum retention of papain activity; however, drying time increases significantly. The dissolution study shows the possible use of alginate beads for site-specific intestinal delivery of papain. The shelf-life of papain can be improved significantly by entrapment in alginate matrix, which was confirmed by an FTIR study. [ABSTRACT FROM AUTHOR]