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The present work investigates the mechanism of binding of alpha-lactalbumin to the peptide ligand WHWRKR and its variants HWRKR and acetylated WHWRKR immobilized on a polymethacrylate chromatographic resin. The presence of two temperature-dependent binding mechanisms and one temperature-independent mechanism was demonstrated. Injections of different forms of alpha-lactalbumin (apo-alpha-lactalbumin, D87A mutant alpha-lactalbumin) displayed similar behaviors when compared to native alpha-lactalbumin, while lysozyme showed little or no binding to the WHWRKR and AcWHWRKR resins. An alternative process for isolation of alpha-lactalbumin from WPI was shown, using consecutive injections of WPI with limited elution.

Lipase from Candida cylindracea was immobilized on glass beads using the biospecific and high-affinity avidin−biotin interaction. Biotinylated lipase and glass beads were prepared by reactions of lipase and 3-aminopropyl glass beads with sulfosuccinimidyl-6-(biotinamido)hexanoate (NHS-LC-biotin). Avidin and biotinylated lipase were sequentially adsorbed to the biotinylated glass beads. Biotinylated lipase in solution retained about 63% of the hydrolytic specific activity of native lipase when an average 3 mol of biotin was incorporated/mol of lipase. Nonporous glass beads contained more biotin and protein (avidin and lipase) per unit of surface area, followed by 302 nm mean pore diameter controlled-pore glass beads (CPG-3000) and 198 nm mean pore diameter controlled-pore glass beads (CPG-2000). The hydrolytic specific activity of lipase immobilized on CPG-3000 and on nonporous beads was essentially the same as that for the biotinylated free enzyme, whereas that immobilized on CPG-2000 was about 50% less. ...

A bifunctional fusion protein was constructed by fusion of the streptavidin gene from Streptomyces avidinii to the lipase gene from Pseudomonas fluorescens, and the resulting streptavidin−lipase was expressed in Escherichia coli. Immobilized streptavidin−lipase was prepared by direct bioselective adsorption from crude cell lysates on biotinylated controlled-pore glass and used to catalyze interesterification of anhydrous butteroil. Changes in the triacylglycerol composition indicated that those with equivalent carbon numbers (ECN) ranging from 36 to 42 decreased, while those with ECN values from 48 to 50 increased following interesterification for 120 h in hexane at 42 °C. Both the melting temperatures and the solid fat content at various temperatures were lower as compared to those of the unmodified butteroil. Addition of unsaturated fatty acids, linoleic and linolenic, yielded modified butteroils with lower melting points and solid fat content, whereas addition of saturated fatty acids, palmitic and ste...

The goals of this study were to express bovine lysozyme in tobacco and to purify the protein with a scaleable process to >90% homogeneity while retaining antimicrobial characteristics. Results showed that the enzyme was expressed at levels equivalent to 1−1.5% of total fraction 2 protein in each of five different transformant groups. The enzyme was subsequently purified to 93% homogeneity using an easily scaleable process while retaining high activity. It was concluded that tobacco was an excellent choice for expression of the recombinant protein and that the purification process developed in this study demonstrates methodology for isolation of high-value enzymes from tobacco and other crop plants. Keywords: Transgenic; lysozyme; recombinant protein; antimicrobial; Nicotiana tabacum

A bioaffinity column of all-trans-retinal immobilized on Celite was capable of isolating high-purity (94.5%) β-lactoglobulin from bovine acid whey. Conditions for producing a potentially hypoallergenic reduced β-lactoglobulin whey were investigated. Reapplication of pH 5.1 eluate to the column resulted in a final purity of 87% α-lactalbumin. The purity of β-lactoglobulin was slightly lower upon elution with buffers containing

The mechanism for stabilization of actomyosin (AM) by sodium lactate (SL) was studied by comparing physical properties (viscosity, density, surface tension, and water activity) of SL and sucrose solutions. Relatively high intrinsic viscosity [η] and Huggins factor (k‘) values showed SL is an effective water structure-maker. Thermal aggregation studies showed that AM stability increased up to 15% SL; higher concentrations of SL destabilized the AM. Surface tension and water activity measurements indicated that the transition from stabilization to destabilization and from increasing to decreasing surface tension coincided with a change in molecular species in the SL solutions. The decrease in surface tension (and AM stability) with increasing SL concentration above the optimum is presented to be due to formation of the lactate dimer lactoyl lactate. This molecule is amphiphilic and would be expected to preferentially absorb at an interface (with for example air or protein) and thus lower surface tension. Ke...

Fragments of the β-barrel domain of β-lactoglobulin were obtained by membrane fractionation of a limited proteolysate prepared with an immobilized trypsin bioreactor. Analysis of this fraction by size-exclusion chromatography under physiological conditions indicated that the fraction contained a predominant peptide (50%) with a size of 8400 Da and several other peptides with sizes ranging from 2000 to 30 700 Da. Analysis of reductively denatured peptides by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol indicated the presence of a major peptide with a size of 6400 Da, suggesting that a small peptide was linked to the 8400-Da peptide by a disulfide bond. Comparison of the surface and emulsifying properties of the peptide fraction with those of intact β-lactoglobulin indicated that the domain peptides have a lower surface hydrophobicity and a slightly higher surface and interfacial tension. Furthermore, the emulsifying activity index for the domain peptides wa...

Binding of hydrogen ions by N-retinyl-Celite indicates an approximate pK of 5.6 for the secondary amino group which is consistent with the previously observed pH dependence of β-lactoglobulin binding by this affinity matrix. N-Retinyl-3-(triethoxysilyl)-n-propylamine was synthesized as a soluble model compound for the affinity ligand. 13C NMR and 1H NMR spectra of this compound and its precursors, (3-aminopropyl)triethoxysilane, all-trans-retinal, and the Schiff base, confirmed formation of the Schiff base and its selective reduction with NaCNBH3. Furthermore, 1H NMR spectra confirmed that protonation of the secondary amine in N-retinyl-3-(triethoxysilyl)-n-propylamine occurred upon treatment at pH 5.1. Keywords: N-Retinyl-Celite; N-retinyl triethoxysilyl n-propylamine; 13C NMR spectra; 1H NMR spectra; hydrogen ion binding

Transglutaminase was iminobilized on a porous glass support by biotinylation followed by adsorption to avidin that had been immobilized by adsorption to the biotinylated aminopropyl glass. Thus, avidin served as a protein spacer between the support and the enzyme. Both the biotinylation of enzyme amino groups and the association of the biotinylated enzyme with soluble avidin caused some loss of enzyme activity. This loss could account for the 3-fold reduction in the specific activity of the immobilized enzyme with carbobenzoxyglutaminylglycine and hydroxylamine as substrates. However, with α s -casein as a substrate, a 24-fold reduction in the k cat value was observed, implying that the rate of reaction with this large substrate molecule was limited by mass transfer. The pH optima and temperature dependences of enzyme catalysis were similar for both the soluble and immobilized enzyme, although the slight differences observed for the immobilized form were also indicative of mass transfer effects. A bimodal pH activity profile with optima at pH 6.5 and 7.5 was obtained with both enzyme forms. Treatment of α s -casein with immobilized enzyme caused a rapid disappearance of the monomeric protein with concomitant appearance of dimers and higher cross-linked polymers

Proteolytic susceptibility of β-lactoglobulin (β-Lg) genetic variants A and B was determined by subjection to immobilized trypsin. Products of limited proteolysis, performed in 50 mM Tris-HCl buffer (pH 8.0) at 4 o C, were characterized by fractionation with anion-exchange chromatography and by denaturing gel electrophoresis of the isolated fractions. Both the rate of appearance of products and the peptides formed were different for the two variants. Two major peptides, with estimated molecular weights of 7500 and 8200 were obtained in homogeneous form by ion-exchange fractionation of β-Lg A hydrolysates. Similar fractionation of β-Lg B hydrolysates yielded fractions that were heterogeneous, containing larger peptides that were not present in β-Lg A hydrolysates

β-Lactoglobulin (β-Lg) genetic variants A and B were purified from milks of individual cows that were homozygous for each variant, and their structural stabilities were examined by susceptibility to proteolysis by immobilized trypsin and by differential scanning calorimetry (DSC). A 15% increase in the value for V M /K M was observed with proteolysis of β-Lg A as compared with β-Lg B. This observation is substantiated by a more rapid disappearance of intact β-Lg A during proteolysis of equimolar mixtures of the two variants. Lower structural stability of variant A is also indicated by a 5 o C lower thermal denaturation temperature observed for β-Lg A by DSC

The gelation of β-lactoglobulin genetic variants A and B (β-Lg A and β-L8 B) was examined with 7% protein solutions at pH 7. Both variants formed viscoelastic solid gels exhibiting low phase angles (

The gelation of β-lactoglobulin treated by limited proteolysis with immobilized trypsin was studied by dynamic rheometry. Gelation conditions were (1) 80 o C for 3 h with 7% protein (w/v) and (2) 60 o C for 12 h with 7% (or 15%) protein (w/v). Thermal transition temperatures of the different components in the limited proteolysis mixture were determined by differential scanning calorimetry

Transglutaminase (EC 2.3.2.13) was covalently immobilized on poly(lysyl)-α s -casein which was covalently attached to 3-aminopropyl porous glass. Attachment of polylysine to the protein spacer, using soluble transglutaminase as a catalyst, greatly increased the concentration of immobilization sites, resulting in a 74% yield of immobilized protein. Catalytic efficiencies, k cat /K m for the immobilized form were 12-13% of that for the soluble enzyme with either α s -casein or β-lactoglobulin as substrate. Also, the catalytic efficiency was 5-fold greater with α s -casein than with β-lactoglobulin using either soluble or immobilized enzyme

Modification of the functionality of whey proteins using microbial transglutaminase (TGase) has been the subject of recent studies. However, changes in rheological properties of whey proteins as affected by extensive cross-linking with TGase are not well studied. The factors affecting cross-linking of whey protein isolate (WPI) using both soluble and immobilized TGase were examined, and the rheological properties of the modified proteins were characterized. The enzyme was immobilized on aminopropyl glass beads (CPG-3000) by selective adsorption of the biotinylated enzyme on avidin that had been previously immobilized. WPI (4 and 8% w/w) in deionized water, pH 7.5, containing 10 mM dithiothreitol was cross-linked using enzyme/substrate ratios of 0.12-10 units of activity/g WPI. The reaction was carried out in a jacketed bioreactor for 8 h at 40 degrees C with continuous circulation. The gel point temperature of WPI solutions treated with 0.12 unit of immobilized TGase/g was slightly decreased, but the gel strength was unaffected. However, increasing the enzyme/substrate ratio resulted in extensive cross-linking of WPI that was manifested by increases in apparent viscosity and changes in the gelation properties. For example, using 10 units of soluble TGase/g resulted in extensive cross-linking of alpha-lactalbumin and beta-lactoglobulin in WPI, as evidenced by SDS-PAGE and Western blotting results. Interestingly, the gelling point of WPI solutions increased from 68 to 94 degrees C after a 4-h reaction, and the gel strength was drastically decreased (lower storage modulus, G'). Thus, extensive intra- and interchain cross-linking probably caused formation of polymers that were too large for effective network development. These results suggest that a process could be developed to produce heat-stable whey proteins for various food applications.

Availability of gram quantities of purified soybean lectin (SBL) to scientists will foster discovery of novel biomedical applications of the lectin and provide the opportunity to investigate the antinutritional effects of SBL in soybean-consuming food animals and poultry. Therefore, a semi-pilot-scale procedure for isolating and purifying SBL was designed. Defatted soyflour was extracted overnight with 0.9% NaCl at 4 degrees C. The extract obtained was filtered (0.45 microm membrane) and subjected to affinity chromatography using a column containing N-acetyl-D-galactosamine resin that is specific for SBL. Bound SBL was eluted off the column with 0.14 M galactose solution. The eluent was ultrafiltered (30 kDa), and the resulting solution (SBL and water) was freeze-dried. Electrophoretic analysis and hemagglutination assay revealed that the freeze-dried SBL was similar to Sigma-grade SBL in purity and activity (35 and 33 HU/mg protein, respectively). The procedure yielded 141 mg of SBL/100 g of soyflour.

A process was developed in which calcium-independent, microbial transglutaminase (mTgase) was immobilized to controlled-pore glass. Avidin was adsorbed to glass beads that had been derivatized and biotinylated. The enzyme was biotinylated and adsorbed to the avidin affinity matrix. Solutions of 8% whey protein isolate (WPI) were then incubated with the mTgase beads, resulting in limited cross-linking of whey proteins. As incubation time increased, intrinsic viscosity increased, gelation temperature decreased, and stronger, more brittle gels were formed upon heating. This process allowed for recycling of the enzyme, eliminated the requirement for a downstream inactivation step, and permitted control over the extent of cross-linking. The functional properties of several batches of WPI were modified using

A method was developed for the production of a hydrolyzed/polymerized whey protein derivative with altered solution and gelation properties using a combination of recombinant DNA and immobilized enzyme technologies. The recombinant fusion proteins trypsin-streptavidin (TrypSA) and streptavidin-transglutaminase (cSAcTG) were produced in Escherichia coli, extracted, and then immobilized by selective adsorption on biotinylated controlled-pore glass. Recirculation through a TrypSA reactor induced limited proteolysis of whey proteins. Hydrolysates were then recirculated through a cSAcTG reactor for incremental periods of time to arrive at increasing degrees of polymerization. The polymers were subsequently analyzed for viscosity/flow behavior, gelation properties, and fracture properties using shear rate ramps/intrinsic viscosity, small-strain oscillatory rheology, and vane viscometry, respectively. By combining limited proteolysis with controlled cross-linking, it was possible to create derivatives of whey proteins with enhanced functional properties. Increases in the degree of whey protein modification were correlated with greater apparent viscosity and intrinsic viscosity, lowered gel point temperatures, and stronger, more brittle gels. This method allowed for recycling of the enzyme, eliminated the requirement for a downstream inactivation step, and permitted control over the extent of modification. Utilization of a similar process may allow for the production of designer proteins engineered with specific functionalities.

The effect of processing on the digestibility of various food proteins was examined by using the immobilized digestive enzyme assay (IDEA) system. The values obtained were compared to true digestibilities determined by rat bioassay. Untreated proteins were also analyzed. Regression analysis of data for all samples resulted in a correlation coefficient (r) of 0.83 (p

Mise en oeuvre d'un systeme comprenant deux bioreacteurs, un contenant de la pepsine, le second de la trypsine, de la chymotrypsine et des peptidases intestinales pour evaluer le degre de racemisation des acides amines et la formation de lysinoalanine (traitement alcalin), ainsi que les consequences de la reaction de Maillard (traitement thermique) sur la digestibilite de proteines d'origine vegetale ou animale

Developpement d'un systeme polyenzymatique immobilise sur lit de verre poreux et comprenant pepsine, trypsine, chymotrypsine et peptidase intestinale de porc. Application a l'evaluation de la valeur nutritive des proteines alimentaires