PURIFICATION OF REACTIVE SULFHYDRYL ENZYMES BY BIOSELECTIVE ADSORPTION ON MONOMERIC AVIDIN: PURIFICATION OF SULFHYDRYL OXIDASE1

ABSTRACT A biotinylation-bioselective adsorption procedure was developed for single-step purification of bovine sulfhydryl oxidase from solubilized skim milk membrane vesicles. Sulfhydryl oxidase was specifically biotinylated by reaction of its chemically reactive sulfhydryl group with a disulfide-containing biotinylation reagent giving a mixed disulfide between the enzyme and the biotinyl moiety. The biotinylated enzyme was then selectively adsorbed on a monomeric avidin matrix. After washing all nonspecifically bound proteins from the matrix, the enzyme was released by reduction of the disulfide with dithiothreitol. Both the biotinylation and the isolation are performed at pH 7.0 under extremely mild conditions. The resulting enzyme preparation appeared to be homogeneous by gel electrophoresis and the specific activity increased over 3000 fold in comparison to whey. The monomeric avidin matrix can be regenerated and used indefinitely. This purification procedure should be generally applicable to proteins with a chemically reactive sulfhydryl group.     

A RESEARCH NOTE: SENSORY QUALITY CHANGES OF HIGH MOISTURE PINEAPPLE BITS DURING STORAGE

Minimally processed high moisture pineapple bits prepared in sucrose syrups, in order to decrease water activity to 0.92, 0.95 and 0.97, were stored at 30, 35 and 40C and evaluated by selected and trained sensory panelists using the Karlsrühe Quality Test. Color, flavor and texture changes were significantly lower for the 0.92 a_w product stored at 30C. It can be predicted by shelf‐life estimation that this product will maintain a satisfactory sensorial and microbiological quality for up to 50 days.     

TRYPSIN IMMOBILIZATION ON DERIVATIZED CELLULOSE BEADS BY BIOSPECIFIC AVIDIN‐BIOTIN INTERACTION AND CHARACTERIZATION OF THE IMMOBILIZED ACTIVITY1

Trypsin was immobilized on cellulose beads using the biospecific and high affinity avidin‐biotin interaction. Trypsin and cellulose beads were biotinylated with sulfosuccinimidyl‐6‐(biotinamido) hexanoate (NHS‐LC‐biotin). Avidin and biotinylated trypsin were sequentially adsorbed to the biotinylated cellulose beads. A similar procedure was carried out using controlled‐pore glass (CPG) beads. The properties of the two trypsin bioreactors were examined and compared. The substrate used for the assay of trypsin activity was p‐tosyl‐L‐arginine methyl ester and the extent of biotinylation of biotinylated trypsin and of immobilized biotin on cellulose beads and on CPG beads were determined using the 2‐[4′‐hydroxyazobenzene]benzoic acid dye‐binding method. Biotinylated trypsin in solution retained about 82% of the specific activity of native trypsin. Cellulose beads contained 0.184 μmol/mL (1.15 μmol/g) biotin and CPG beads, 0.329 μmol/mL (0.987 μmol/g). After regeneration, the biotin contents became slightly lower, namely, 0,159 μmol/mL for cellulose beads and 0.315 μmol/mL for CPG beads. The specific activities of trypsin immobilized on cellulose beads and CPG beads were 32 U/mL (202 U/g) and 43 U/mL (130 U/g), respectively. These studies indicate that cellulose beads can be biotinylated for use as bioselective support.     

Ascorbic acid loss and sensory changes in intermediate moisture pineapple during storage at 30–40°C

Kinetics of ascorbic acid loss as a function of storage temperature (30, 35 and 40°C) and water activity of intermediate moisture pineapple slices at a _W of 0.92, 0.95 and 0.97 was modelled. Loss was faster at higher a _W but storage temperatures of 30 - 40°C had little effect at the same a _w. Colour changes were larger than flavour changes. Spoilage was only a problem at high a _w.     

Influence of hydrophobic characteristic of organo-modified precursor on wettability of silica film

The objective of this study is to design new hybrid silica materials as templates with hydrophobic properties, prepared at room temperature by a base catalyzed sol–gel process. As silica sources, organoalkoxysilanes functionalized with short hydrophobic chains were used: tetraethylorthosilicate (TEOS), methyltriethoxysilane (MTES), vinyltriethoxysilane (VTES), octyltriethoxysilane (OTES) and isobutyltriethoxysilane (iTES). It was shown that hydrophobicity of the functionalized silica nanoparticles increased as a function of length of the aliphatic chains (MTES < iTES < OTES) or when, instead of a hydrophobic alkyl chains (substituting group of silica precursors), a monounsaturated group was used (VTES). It was observed that the samples responded in a specific way to each type (hydrophilic or hydrophobic) of the dropped liquid. Even though the experiments were limited to short hydrocarbon chains, they showed that there is a threshold to reach high hydrophobicity of the hybrid surface.