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Ammonium Sulphate Precipitation (ammonium + sulphate_precipitation)
Selected AbstractsPurification and characterization of ,-glucosidase in Apis cerana indicaINSECT SCIENCE, Issue 3 2008Chanpen Chanchao Abstract Apis cerana indica foragers were used for the isolation of a full-length ,-glucosidase cDNA, and for purification of the active nascent protein by low salt extraction of bee homogenates, ammonium sulphate precipitation and diethylaminoethyl-cellulose and Superdex 200 chromatographies. The molecular mass of the purified protein was estimated by polyacrylamide gel electrophoresis resolution, and the pH, temperature, incubation, and substrate optima for enzymic activity were determined. Conformation of the purified enzyme as ,-glucosidase was performed by BLAST software homology comparisons between matrix assisted laser desorption ionization time of flight mass spectroscopy analysed partial tryptic peptide digests of the purified protein with the predicted amino acid sequences deduced from the ,-glucosidase cDNA sequence. [source] A heat-stable trypsin inhibitor in adzuki bean (Vigna angularis): effect of extraction media, purification and biochemical characteristicsINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 1 2010Sappasith Klomklao Summary Trypsin inhibitor from adzuki bean (Vigna angularis) seed was isolated and characterised. Extraction of seed with NaCl at the concentration of 0.15 m showed a higher recovery of trypsin inhibitor than other solvents tested (P < 0.05). Optimal extraction time for the recovery trypsin inhibitor from adzuki bean seed was 30 min (P < 0.05). Purification of inhibitor was achieved by heat-treatment at 90 °C for 10 min, followed by ammonium sulphate precipitation with 30,65% saturation and size exclusion chromatography on Sephacryl S-200, presenting a yield and purification of 53.9% and 10.91-fold, respectively. The apparent molecular weight of trypsin inhibitor was estimated to be 14 kDa based on SDS-PAGE and inhibitor activity of zones separated by electrophoresis. The purified inhibitor was stable over a broad pH range and retained high inhibitory activity toward trypsin after incubation at 90 °C for 60 min. NaCl, at 0,3% concentration, did not affect the inhibitory activity of purified trypsin inhibitor, however, the activity was lost when sample was treated with ,-mercaptoethanol prior to electrophoresis. [source] Production, purification and thermal characterisation of invertase from a newly isolated Fusarium sp. under solid-state fermentationINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 7 2008Iram Shaheen Summary Production of invertase employing a newly isolated Fusarium sp. under solid-state fermentation was optimised. Different process parameters were optimised. The maximum enzyme activity under optimum conditions was 47.23 ± 2.12 U gds,1 with nitrogen additives. The enzyme was purified by ammonium sulphate precipitation, diethylaminoethyl cellulose ion-exchange chromatography and Sephadex gel filtration. This protocol gave 20.25-fold purification and 5.53% recovery. The optimum pH and temperature for activity were 5.0 and 50 °C. The Km and Vmax values for the enzyme were 8.33 mm and 21.48 ,mol min,1, respectively. A detailed kinetic study of thermal inactivation has been carried out. Enthalpy of activation (,H*) decreased when entropy (,S*) of activation increased at higher temperatures. Moreover, free energy of denaturation (,G*) increased at higher temperature making the enzyme thermally stable. A possible explanation for the thermal inactivation of invertase at higher temperatures is also discussed. [source] Detection and characterization of the novel bacteriocin entomocin 9, and safety evaluation of its producer, Bacillus thuringiensis ssp. entomocidus HD9JOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2003A. Cherif Abstract Aims: To identify and characterize new bacteriocins from a collection of 41 strains belonging to 27 subspecies of Bacillus thuringiensis, and to evaluate the safety of the producers. Methods and Results:Bacillus thuringiensis ssp. entomocidus HD9 produced in the culture supernatant an antimicrobial activity against Gram-positive bacteria including Listeria monocytogenes, one of four pathogenic Pseudomonas aeruginosa and several fungi. Production of the antibacterial activity, named entomocin 9, started during mid-logarithmic growth reaching its maximum at the early stationary phase. Entomocin 9 retained more than 72% of activity after incubation for 20 min at 121°C. Activity was lost after proteinase K treatment, it was stable in a pH range between 3 and 9, and resistant to lyophilization. After partial purification with ammonium sulphate precipitation followed by gel-filtration and anion-exchange chromatography, an active protein of ca 12·4 kDa was isolated. The mode of action of entomocin 9 was bactericidal and caused cell lysis of growing cells. Despite the presence of a range of virulence related genes, including haemolysin BL, nonhaemolytic enterotoxin, cytotoxin K and several hydrolytic activities, B. thuringiensis HD9 was not toxic against Vero cells. Conclusions: Entomocin 9 is a novel heat-stable, bacteriocin produced by B. thuringiensis HD9. The absence of toxicity against Vero cells suggests the suitability of strain HD9 for a safe application in antimicrobial treatments. Significance and Impact of the Study: New finding on entomocin 9 would make B. thuringiensis attractive in biotechnological applications as an antimicrobial agent in agriculture and food industry. [source] Purification and characterization of a bacteriocin-like compound (Lichenin) produced anaerobically by Bacillus licheniformis isolated from water buffaloJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2001P. Pattnaik Aims:,To characterize a bacteriocin-like factor from Bacillus licheniformis 26 L-10/3RA isolated from buffalo rumen. Methods and Results:,The culture supernatant exhibited the antibacterial activity against a number of indicator organisms in a cut-well agar assay under anaerobic conditions. The inhibitory component was purified by following ammonium sulphate precipitation, gel filtration and ion exchange chromatography and confirmed to be a single peptide. A single band on tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis confirmed that the peptide was purified to homogeneity and having an estimated molecular mass of approximately 1400 dalton. Complete amino acid sequence of the peptide yielded 12 amino acids from the N-terminal end (ISLEICXIFHDN). No homology with previously reported bacteriocins was observed and has been designated as Lichenin. Lichenin was found to be hydrophobic, sensitive to atmospheric oxygen, retained biological activity even after boiling for 10 min and was active over a pH range of 4·0,9·0. Conclusions:,The Lichenin represents the first anaerobiosis specific expression of bacteriocin-like compound isolated from Bacillus licheniformis 26 L-10/3RA of buffalo rumen origin. Significance and Impact of the Study:,Lichenin could be a potential condidate for manipulating the rumen function at molecular level intended for improving the productivity of the ruminant. [source] Characterization of a bacteriocin produced by Prevotella nigrescens ATCC 25261LETTERS IN APPLIED MICROBIOLOGY, Issue 5 2004J. Kaewsrichan Abstract Aims:, To characterize the antimicrobial activity produced by Prevotella nigrescens ATCC 25261, and to evaluate its safety on cultured gingival fibroblasts. Methods and Results:, An antimicrobial activity was obtained from purifying the culture supernatant of Pr. nigrescens ATCC 25261. Purification of the active compound was achieved with ammonium sulphate precipitation followed by anion-exchange and gel filtration chromatography. As revealed by SDS-PAGE, the active fraction was relatively homogeneous, showing a protein with an approximate molecular weight of 41 kDa. The antimicrobial compound, named nigrescin, exhibited a bactericidal mode of action against Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythensis, and Actinomyces spp. Nigrescin was stable in a pH range between 6·5 and 9·5, at 100°C for 10 min, and resistant to lyophilization. But its activity was lost after proteinase K treatment. Despite at very high concentrations beyond the minimum inhibitory concentration (MIC), nigrescin was not toxic to the gingival fibroblasts. Conclusion:, Nigrescin is a novel bacteriocin produced by Pr. nigrescens ATCC 25261. It exhibits antimicrobial activity against species that are implicated in periodontal diseases. The absence of toxicity on the gingival fibroblasts suggests the possibility in using of nigrescin for an application in periodontal treatment. Significance and Impact of the Study:, Novel evidence on nigrescin would make Pr. nigrescens ATCC 25261 attractive in biotechnological applications as an antimicrobial agent in clinical dentistry. [source] Solvent extraction of bacteriocins from liquid culturesLETTERS IN APPLIED MICROBIOLOGY, Issue 3 2000L.L. Burianek A solvent extraction method was developed to concentrate lacidin from the culture of Lactobacillus acidophilus OSU133. The new method concentrates the bacteriocin at the interface between chloroform and the aqueous culture of the producing bacterium. Compared with other extraction procedures, the new method effectively recovers higher bacteriocin yield and results in relatively clean preparations. Recovery of lacidin by the chloroform extraction procedure, compared with ammonium sulphate precipitation and cell acidification methods, was >10-fold and about 100-fold greater, respectively. The new extraction procedure saves time and is easy to perform. This method is also effective in recovering subtilin, bacillicin, pediocin and nisin from cultures of Bacillus subtilis ATCC 6633, B. subtilis OSY1115/C, Pediococcus acidilactici PO2 and Lactococcus lactis ATCC 11454, respectively. [source] Purification and characterization of a subtilisin-like serine protease induced during the senescence of wheat leavesPHYSIOLOGIA PLANTARUM, Issue 4 2003Irma N. Roberts A senescence-specific protease accounting for almost 70% of the total peptide hydrolytic activity of protein extracts, was isolated from detached wheat leaves induced to senescence by incubation in the dark for 72 h. Purification to apparent homogeneity was performed by ammonium sulphate precipitation, ion exchange chromatography and gel filtration chromatography. The enzymatic activity was followed by its ability to hydrolyse the synthetic peptide Suc-AAPF-pNA. SDS/PAGE and gel filtration analysis indicated that the enzyme was a dimer composed of two identical subunits of 59 kDa. The apparent Km and Vmax for the peptide were 1.18 mm and 2.27 mmol pNA mg,1 h,1, respectively. The enzyme was active at pH values above 8.0 and remained active after heat treatment at 60°C for 10 min. It was inhibited by chymostatin, indicating that the enzyme possesses a chymotrypsin-like activity. Rubisco was readily hydrolysed by the purified protease. A sequenced internal fragment of 17 amino acids showed a high level of similarity (65,75% identity) with a highly conserved region of several plant subtilisin-like serine proteases. The absence of this enzymatic activity in fractionated extracts from non-senescent tissues suggests that it might play a role in the senescing process. [source] | ||||||||||