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  • immobilized digestive enzyme assay (IDEA)  (1)
  • streptavidin  (1)
  • β-lactoglobulin  (1)
  • 1
    ISSN: 1573-4943
    Keywords: β-Structural Domains ; β-lactoglobulin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Incubation of β-lactoglobulin with immobilized trypsin at 5–10°C results in a time-dependent release of several fragments of the core domain in yields approaching 15%. Digests were fractionated by ion-exchange chromatography with a Mono Q HR5/5 column and analyzed after disulfide reduction by polyacrylamide gel electrophoresis in sodium dodecylsulfate. Three fragments with approximate molecular weights of 13.8, 9.6, and 6.7 kD were identified. The fraction from ion-exchange chromatography yielding the 6.7 kD fraction after disulfide reduction was further characterized because it was most homogeneous and gave the highest yield. The C-terminal cleavage site of the 6.7 kD core fragment appeared to be Lys100 or Lys101 as determined by C-terminal amino acid analysis. The exact masses, after reduction with dithiothreitol, are 6195 and 6926 as determined by laser desorption mass spectrometry, corresponding to residues 48–101 and 41–100. Prior to reduction, β-lactoglobulin C-terminal residues 149–162 are connected to these core domain fragments as shown by C-terminal analysis and mass spectrometry. Structural studies indicate that these 7.9 and 8.6 kD core domain fragments released by immobilized trypsin retain much of their native structure. CD spectra indicate the presence of antiparallel β-sheet structure similar to the native protein but the α-helix is lost. Spectra in the aromatic region indicate the existence of tertiary structure. Moreover, structural transitions in urea are completely reversible as measured by CD spectra, although the extrapolated ΔG D H20 and the urea concentration at the transition midpoint are lower than for the native protein. The core domain fragments also display apH-dependent binding to immobilizedtrans-retinal as does intact protein. A single endotherm is obtained for both core domain fragments and native protein upon differential scanning calorimetry, but again, the domain is less stable as indicated by a transition peak maxima of 56.9°C as compared with 81.1°C for native protein.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1573-9104
    Keywords: immobilized digestive enzyme assay (IDEA) ; protein digestibility
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract The digestibility of the protein in various foods or food components was analyzed using an immobilized digestive enzyme assay (IDEA) system. The assay consists of two bioreactors, one containing pepsin and the other containing trypsin, chymotrypsin and intestinal mucosa peptidases. The fraction of the peptide bonds hydrolyzed during an extent of hydrolysis assay was correlated with independentin vivo determinations of the digestibilities. A correlation coefficient of 0.80 was obtained. The derived linear regression equation can be used to predict digestibility. The method is sensitive to structural modification of protein, as for example, those caused by effects of heat treatment.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 44 (1994), S. 1348-1354 
    ISSN: 0006-3592
    Keywords: streptavidin ; galactosidase ; fusion proteins ; plasmid vector ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Covalently immobilized biotin was used as a biospecific adsorbant to investigate the application of streptavidin as an affinity domain for simultaneous purification and immobilization of recombinant proteins. A streptavidin-β-galactosidase fusion protein was constructed and tested as a model system. The gene for streptavidin from Streptomyces avidinii was modified by polymerase chain reaction to mutate the stop codon and to facilitate cloning into an Escherichia coli expression vector yielding a versatile plasmid with 37 unique restriction enzyme sites at the 3' end. E. coli β-galactosidase was cloned in-frame to the streptavidin gene. Analysis of lysates of induced recombinant E. coli cells by SDS-PAGE and Western blots indicated that the 133.6-kDa fusion protein was expressed. Sulfosuccinimidyl-6-(biotinamido) hexanoate was covalently immobilized on 3-aminopropyl-controled-pore glass beads. Exposure of recombinant cell lysates to this support indicated that streptavidin-β-galactosidase was bioselectively adsorbed. The resulting biocatalyst contained 300 mg protein per gram of beads and exhibited a specific activity of 306 βmol/min per milligram protein with o-nitrophenyl-β-D-galactopyranoside as substrate corresponding to approximately 50% of that observed for commercially pure E. coli β-galactosidase. © 1994 John Wiley & Sons, Inc.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
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