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Purification Factor (purification + factor)
Selected AbstractsCharacterization of a new tyrosinase from Pycnoporus species with high potential for food technological applicationsJOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2005S. Halaouli Abstract Aims:, Tyrosinase production by Pycnoporus cinnabarinus and Pycnoporus sanguineus was screened among 20 strains originating from various geographical areas, particularly from tropical environments. The tyrosinase from the most efficient strain was purified and characterized and tested for food additive applications. Methods and Results:, Monophenolase and diphenolase activities of tyrosinase were measured from cell lysate from the 20 Pycnoporus strains, for 8,10 days of cultivation. The strain P. sanguineus CBS 614.73 showed the highest productivity (45·4 and 163·6 U g,1 protein per day for monophenolase and diphenolase respectively). P. sanguineus CBS 614.73 tyrosinase was purified from concentrated cell lysate, anion-exchange, size-exclusion and hydroxyapatite chromatography, with a final yield of 2% and a purification factor of 35,38. The pure enzyme was a monomere with a molecular mass of 45 kDa and it showed four isoforms or isoenzymes with pI between 4·5,5. No N-glycosylation was found. The N-terminal amino acid sequence was IVTGPVGGQTEGAPAPNR. The enzyme was shown to be almost fully active in a pH range of 6,7, in a large temperature range (30,70°C), and was stable below 60°C. The main kinetic constants were determined. The tyrosinase was able to convert p -tyrosol and p -coumaric acid into hydroxytyrosol and caffeic acid, respectively, and it could also catalyse the cross-linking formation of a model protein. Conclusions:, Among the genus Pycnoporus, known for the production of laccase, the strain P. sanguineus CBS 614.73 was shown to produce one other phenoloxidase, a new monomeric tyrosinase with a specific activity of 30 and 84 U mg,1 protein for monophenolase and diphenolase respectively. Significance and Impact of the Study:, This study identified P. sanguineus CBS 614.73 as a potential producer of a tyrosinase which demonstrated effectiveness in the synthesis of antioxidant molecules and in protein cross-linking. [source] Purification and Characterization of Thermostable ,-Amylase from a Newly Isolated Thermophilic Bacillus stearothermophilus GRE 1ASIA-PACIFIC JOURNAL OF CHEMICAL ENGINEERING, Issue 5-6 2005S.M. Zakir Hossain A thermophilic bacterium, Bacillus stearothermophilus GRE 1, isolated from an Ethiopian hyperthermal spring produced a thermostable ,-amylase. Enzyme production in shake flask experiments while using optimum nutrient supplements and environmental conditions was 2.35 U/ml. Under optimized conditions in a bioreactor, 5-6 fold higher enzyme activity was obtained than that of a shake flask. Gel filtration chromatography yielded a purification factor of 33.62-fold and a recovery of 46.52%. The optimum temperature for activity was determined to be 60-70d,C and optimum pH was in the range of 5.5-6.0. The enzyme maintained 50% of its original activity after 45 minutes of incubation at 80d,C, and is stable at pH values of 5.0-9.0. Enzyme activity was strongly inhibited by Cu2+, Zn2+ and Fe2+. The enzyme is calcium independent and 94% and 86% relative activities were displayed with low concentrations of Co2+ and Mg2+, respectively. [source] Microbial bio-production of a recombinant stimuli-responsive biosurfactantBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2009W. Kaar Abstract Biosurfactants have been the subject of recent interest as sustainable alternatives to petroleum-derived compounds in areas ranging from soil remediation to personal and health care. The production of naturally occurring biosurfactants depends on the presence of complex feed sources during microbial growth and requires multicomponent enzymes for synthesis within the cells. Conversely, designed peptide surfactants can be produced recombinantly in microbial systems, enabling the generation of improved variants by simple genetic manipulation. However, inefficient downstream processing is still an obstacle for the biological production of small peptides. We present the production of the peptide biosurfactant GAM1 in recombinant E. coli. Expression was performed in fusion to maltose binding protein using chemically defined minimal medium, followed by a single-step affinity capture and enzymatic cleavage using tobacco etch virus protease. Different approaches to the isolation of peptide after cleavage were investigated, with special emphasis on rapid and simple procedures. Solvent-, acid-, and heat-mediated precipitation of impurities were successfully applied as alternatives to post-cleavage chromatographic peptide purification, and gave peptide purities exceeding 90%. Acid precipitation was the method of choice, due to its simplicity and the high purification factor and recovery rate achieved here. The functionality of the bio-produced peptide was tested to ensure that the resulting peptide biosurfactant was both surface active and able to be triggered to switch between foam-stabilizing and foam-destabilizing states. Biotechnol. Bioeng. 2009;102: 176,187. © 2008 Wiley Periodicals, Inc. [source] A novel purification strategy for retrovirus gene therapy vectors using heparin affinity chromatographyBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2005Marķa de las Mercedes Segura Abstract Membrane separation and chromatographic technologies are regarded as an attractive alternative to conventional academic small-scale ultracentrifugation procedures used for retrovirus purification. However, despite the increasing demands for purified retroviral vector preparations, new chromatography adsorbents with high specificity for the virus have not been reported. Heparin affinity chromatography is presented here as a novel convenient tool for retrovirus purification. The ability of bioactive retroviral particles to specifically bind to heparin ligands immobilized on a chromatographic gel is shown. A purification factor of 63 with a recovery of 61% of functional retroparticles was achieved using this single step. Tentacle heparin affinity supports captured retroviral particles more efficiently than conventional heparin affinity chromatography supports with which a lower recovery was obtained (18%). Intact, infective retroviral particles were recovered by elution with low salt concentrations (350 mM NaCl). Mild conditions for retrovirus elution from chromatographic columns are required to preserve virus infectivity. VSV-G pseudotyped retroviruses have shown to be very sensitive to high ionic strength, losing 50% of their activity and showing membrane damage after a short exposure to 1M NaCl. We also report a complete scaleable downstream processing scheme for the purification of MoMLV-derived vectors that involves sequential microfiltration and ultra/diafiltration steps for virus clarification and concentration respectively, followed by fractionation by heparin affinity chromatography and final polishing by size-exclusion chromatography. Overall, by using this strategy, a 38% yield of infective particles can be achieved with a final purification factor of 2,000. © 2005 Wiley Periodicals, Inc. [source] Single step purification of lactoperoxidase from whey involving reverse micelles-assisted extraction and its comparison with reverse micellar extractionBIOTECHNOLOGY PROGRESS, Issue 3 2010K.E. Nandini Abstract The extraction of lactoperoxidase (EC 1.11.1.7) from whey was studied using single step reverse micelles-assisted extraction and compared with reverse micellar extraction. The reverse micelles-assisted extraction resulted in extraction of contaminating proteins and recovery of lactoperoxidase in the aqueous phase leading to its purification. Reverse micellar extraction at the optimized condition after forward and backward steps resulted in activity recovery of lactoperoxidase and purification factor of the order of 86.60% and 3.25-fold, respectively. Whereas reverse micelles-assisted extraction resulted in higher activity recovery of lactoperoxidase (127.35%) and purification factor (3.39-fold). The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) profiles also evidenced that higher purification was obtained in reverse micelles-assisted extraction as compared of reverse micellar extracted lactoperoxidase. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Purification and Concentration of Alkaline Phosphatase by Selective Permeabilization of Escherichia coli Using Reverse Micellar SolutionsBIOTECHNOLOGY PROGRESS, Issue 6 2003Ritu Bansal-Mutalik Recovery of alkaline phosphatase (AP) from the periplasm of Escherichia coli using reverse micellar solutions (RMSs) of sodium dioctyl sulfosuccinate (AOT) in aliphatic hydrocarbons has been attempted. A variety of surface-active agents, solvents, and reverse micellar conditions were screened, and an excellent recovery of the enzyme in a concentrated form, with a high purification factor, was obtained in a single-step process. The permeabilization process strongly depended on the water content of the RMS as well as on the amount of water coating the microbial cell surface. The product was almost free from nucleic acids. In addition, because of the low affinity of AOT and the organic solvent for the aqueous phase, contamination by the permeabilizing agents would also be negligible. [source] Quantifying Process Tradeoffs in the Operation of Chromatographic SequencesBIOTECHNOLOGY PROGRESS, Issue 4 2003Sheau-Huey Ngiam A method for the rapid representation of key process tradeoffs that need to be made during the analysis of chromatographic sequences has been proposed. It involves the construction of fractionation and maximum purification factor versus yield diagrams, which can be completed easily on the basis of chromatographic data. The output of the framework developed reflects the degree of tradeoff between levels of yield and purity and provides a fast and precise prediction of the sample fraction collection strategy needed to meet a desired process specification. The usefulness of this approach for the purposes of product purification and contaminant removal in a single chromatographic step has been successfully demonstrated in an earlier paper and it is now extended by application to a chromatographic sequence: the separation of a hypothetical three-component protein system by hydrophobic interaction chromatography (HIC) followed by size exclusion chromatography (SEC). The HIC operation has a strong impact upon the subsequent SEC step. The studies show how the analysis of performance in such a chromatographic sequence can be carried out easily and in a straightforward fashion using the fractionation diagram approach. The methodology proposed serves as a useful tool for identifying the process tradeoffs that must be made during operation of a sequence of chromatographic steps and indicates the impact on further processing of the cut-point decisions that are made. [source] | ||||||||||