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β-Lactoglobulin is capable of binding fat-soluble compounds including vitamin A palmitate and is suggested to specifically enhance intestinal uptake of retinol. In this study, bioavailability of a vitamin-A-retinyl palmitate complex in skim milk and in water-based liquids was investigated in vitamin-A-depleted rats. First, rats were fed a vitamin-A-free pellet diet for 6 wk and were thereafter gavage-fed with vitamin A in oil, vitamin-A-β-lactoglobulin complex, vitamin A in oil + skim milk, and vitamin-A-β-lactoglobulin + skim milk for 2 wk and 42 wk. Vitamin A repletion, as judged by vitamin A accumulation in serum and liver, occurred in all the treatments. Vitamin-A-β-lactoglobulin complex treatments had statistical equivalence with oil-based vitamin A treatments. In a second experiment, vitamin-A-depleted rats were fed UHT-processed skim milk fortified with either oil-based or freeze-driedβ-lactoglobulin-complexed retinyl palmitate. Liver and serum vitamin A were analyzed by HPLC to indicate vitamin A status in the rats. Results showed no significant difference in bioavailability of retinyl palmitate from milk made with either regular oil-based orβ-lactoglobulin-complexed fortifiers. The vitamin-A-β-lactoglobulin complex, being water soluble, may be useful for fortification of nonfat products.

Milkfat globule membranes (MFGMs) were prepared from bovine cream according to standard procedures. These membranes and peptide hydrolysates, which were generated by proteolysis with immobilized digestive enzymes, were screened for antibacterial activity against Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica Typhimurium, Pseudomonas fluorescens, Bacillus cereus, Lactobacillus acidophilus, and Lactobacillus gasseri. Assays were first performed on beef heart infusion (BHI) plates spotted with test protein-peptide fractions and then seeded with lawns of indicator cells to monitor the zone of growth inhibition. Under these experimental conditions, MFGMs were most active against Salmonella Typhimurium and P. fluorescens. However, antibacterial activity was not seen after plating on Luria-Bertani (LB) medium. We determined that the antimicrobial effects observed on BHI plates were due to the generation of H2O2 by xanthine oxidase, a major protein constituent of the MFGMs, as a result of purine catalysis. This substrate is present in BHI but lacking in LB medium. Evaluation of purified xanthine oxidase alone resulted in analogous data trends. The growth of probiotic Lactobacillus strains were affected only marginally when grown on lactobacilli deMan Rogosa Sharpe plates, suggesting the decreased sensitivity of these bacteria to H2O2. In this study, several MFGM hydrolysates exhibited variable antibacterial activity against test food pathogens on agar plates prepared with M9 minimal media, and this variation was not attributable to xanthine oxidase enzymatic activity. The probiotic microorganisms were mostly resilient to these antibacterial fractions. Bovine MFGM fractions may represent an excellent resource material from which to generate native, naturally occurring biodefensive proteins and/or peptides.

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. Cette etude utilise l'interaction avidine-biotine comme modele pour l'adsorption selective et l'immobilisation de proteines de fusion enzyme-streptavidine recombinantes pour la preparation de bioreacteur. L'application de cette technologie permet de realiser la purification et l'immobilisation dans une seule etape. Cette etude caracterise enfin la trypsine immobilisee par cette methode en comparaison avec celle immobilisee sur des billes de verre.

Extracellular production of keratinase–streptavidin fusion protein (KER–STP) was accomplished by the cloning of Bacillus subtilis with a transforming plasmid carrying the kerA - stp fusion gene. The fusion protein was readily immobilized onto a biotinylated solid matrix by mixing in the culture medium. The properties and reaction kinetics of free and immobilized keratinase (KE) were characterized and compared. Heat stability and pH tolerance were greatly improved by immobilization, but the catalytic efficiency ( k cat / K m ) was reduced by eightfold. The yield of bio-immobilization using bioselective adsorption of the fusion protein was approximately 20%, as estimated from the activity of free KE. HPLC analysis of reaction products demonstrated the hydrolysis of feather keratin, casein, and bovine serum albumin (BSA) by the immobilized KE. The rates of reactions are lower than those of the free enzyme. On the other hand, the stability of the enzyme was greatly improved.

Mammary fluids, colostrum and milk, deliver nature's first host defense systems upon birth, and these essential liquids are critical for survival of the neonate. The identification and characterization of anti-infectious proteins were among the early scientific discoveries and this group of proteins has long been recognized for promoting health benefits in both newborns and adults. Among the more widely studied are the immunoglobulins, lactoperoxidase, lysozyme, and lactoferrin. Recently, it was shown that alpha--lactalbumin may also function in a protective capacity dependent upon its folding state. Some of these, especially lactoferrin, also display an immunomodulatory role in which case a totally separate cascade of host defense responses is initiated. It was noted that the mechanism of action for this cluster of sentry proteins does vary; thus, this protective strategy provides for a broad range of responsive reactions to infection. Presently, there is a major focus on the discovery of novel peptides that can be generated from existing milk proteins via proteolytic reactions. To date, this substrate list includes alpha--lactalbumin, beta-lactoglobulin, all casein fractions, and lactoferrin. Again, the immunoregulatory effects prompted as a result of the appearance of these peptides are currently being defined. Herein, we review the principal biological properties associated with each of these contributing milk components with a special emphasis on the role of biodefensive milk peptides. We envision future contributions emerging from this research field as an opportunity to develop effective new therapies to be used in treating infectious diseases and promoting health benefits in vivo.

Native soybean lectins (SBL) could potentially have deleterious effects on young animals. The objectives of this study were to determine the optimum processing temperature and time at which SBL is inactivated and to investigate the possibility of using urease activity (UA) to predict residual lectin levels in soybean meal (SBM). Raw defatted SBM was steam-heated at incremental temperatures between 90 and 120 degrees C for 5 to 20 min in an autoclave. The processed meals were subjected to native-PAGE and measurement of total carbohydrate-binding lectin (TCBL), agglutinating lectin (AL), UA, and trypsin inhibitor (TI). Processing severity was evaluated by determining protein solubility in 0.2% potassium hydroxide. Results indicated that levels of all antinutrients (TCBL, AL, UA, and TI) decreased with increasing processing temperature (P0.05). The intensity of the lectin band on the electrophoresis gel was considerably reduced when meal was heated at 100 degrees C for 5 min. This result implied that lectin inactivation occurred at 100 degrees C. More than 90% of all the original antinutrient levels in the raw meal were destroyed when meals were heated at 100 degrees C for 5 min. Meals processed at 100 degrees C for 5 to 20 min had protein solubility values (80 to 85%) indicative of adequate processing. The denaturation pattern of UA was highly correlated with that of SBL (ror = 0.73), indicating that UA could be used for monitoring lectin levels in commercial meals. We concluded that UA of 0.03 to 0.09 units of pH change are indicative of adequately processed meals that contain negligible lectin levels.

Immobilized keratinase can improve stability while retaining its proteolytic and keratinolytic properties. Conventional purification followed by chemical immobilization is a laborious and costly process. A new genetic construct was developed to produce the keratinase-streptavidin fusion protein. Consequently, the purification and immobilization of the fusion protein onto a biotinylated matrix can be accomplished in a single step. The method was tested in both the Bacillus subtilis and Escherichia coli systems. In B. subtilis, the fusion protein was produced extracellularly and readily immobilized from the medium. In E. coli, the fusion protein was produced intracellularly in inclusion bodies; additional separation and renaturation processes were required prior to immobilization from the cell extract. The overall efficiencies were approximately the same, 24-28%, using both systems.

Expression of fusion protein trypsin-streptavidin (TRYPSA) in Escherichia coli was evaluated and the protein purified. Protein expression was induced by 1 mM isopropylthio-beta-D-galactoside (IPTG), and the enzyme activity was measured by the hydrolysis rate of p-toluenesulfonyl-L-arginine methyl ester (TAME). Expression of the fusion protein in the cell-free extract decreased with increased induction time; correspondingly, that in the inclusion bodies increased. The total expression in Luria-Bertani broth (LB) and Terrific Broth (TB) media reached the highest levels in 2 hr at 30 degrees C. The optimum expression level was 35 and 48 U/L in LB and TB, respectively. Expression of the fusion protein was verified by Western Blot analysis using streptavidin antiserum, and the fusion protein was purified using a benzamidine Sepharose 6B affinity column at room temperature. The molecular size of the soluble purified fusion protein was determined by size-exclusion chromatography using Superose 12 FPLC. A molecular weight of 39-40 kDa was obtained, indicating that the soluble protein exists as a monomer; thus, the presence of the trypsin domain must prevent the streptavidin domain from tetramer formation.

CD36, a common milk fat globule membrane glycoprotein, was isolated from skim milk by methods similar to those previously utilized for the isolation of sulfhydryl oxidase. Two separate methods that were employed, gave similar purity as observed by electrophoresis. The first was based on differential centrifugation and size-exclusion chromatography, whereas the second combined ultrafiltration and affinity chromatography. After significant purification, the protein was identified by Western blotting and sequence analysis. Deglycosylation decreased the apparent molecular mass from approximately 85 to 57 kDa. These results suggested tissue-specific glycosylation. The purified fractions also exhibited low levels of sulfhydryl oxidase activity, the significance of which will require further study.

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.

α-Lactalbumin is a whey protein with high digestibility and low potential for causing allergic problems in infants, making it a strong candidate for use in infant formulas. The development of an efficient and scalable process for isolation of α-lactalbumin is necessary to allow its use on a large scale. Affinity chromatography using short peptides as ligands is a promising technique because it allows the recovery of specific proteins without the use of harsh chemicals or problems due to ligand release. In the present paper we describe the identification of the hexapeptide WHWRKR, obtained from a combinatorial library, that shows affinity for α-lactalbumin.

A trypsin-streptavidin (TRYPSA) fusion protein was designed and its expression in Escherichia coli was evaluated. The streptavidin gene was PCR modified and cloned into the pET expression vector. The trypsin gene was subsequently inserted into this plasmid, thus generating a colinear fusion of trypsin and streptavidin genes (pTRYPSA). This engineering strategy was verified, and TRYPSA was expressed after IPTG induction using the E. coli strains, BL21(DE3) and BL21(DE3)pLysS. Standard protein fractions of the cell lysate were prepared and trypsin activity was primarily detected in the periplasmic and inclusion body fractions. Immunoblotting showed a single Western-positive band exhibiting a molecular weight of 39,000 Da. A biotinylated porous glass affinity matrix was prepared and selective adsorption resulted in a one-step purification and immobilization of TRYPSA from crude cell lysate. Trypsin activity was verified using a synthetic substrate. This enzyme bioreactor should serve as an excellent prototype for future studies that will examine the effect of limited proteolysis on functional characteristics of milk proteins, including gelling, emulsifying and foaming properties.

Whey protein polymers (WP-polymers) were prepared by heating whey protein isolate below the critical concentration for gelation at neutral pH and low salt conditions. The effects of WP-polymers and salt types (CaCl2 or NaCl) on rheological properties (large-strain and small-strain analysis), water holding properties, turbidity and microstructure of heat-induced whey protein isolate gels were investigated. Replacement of native whey protein isolate with WP-polymers increased fracture stress, fracture modulus, held water, and the translucency of gels. With both salt types, the addition of WP-polymers changed the gel structure from particulate to fine-stranded. However, the effect of WP-polymers on rheological properties was salt specific. Addition of 20‐100% WP-polymers in the presence of 10 mM CaCl2 caused a continued increase in fracture stress. In contrast, protein dispersions containing 30 mM NaCl did not form self-supporting gels when $60% WP-polymers were added. Dispersions containing 200 mM NaCl formed self supporting gels at all levels of WP-polymer addition but fracture stresses for gels containing 20‐100% WP were similar. Dispersions containing 80% WP-polymers and 200 mM NaCl had lower gel points (time and temperature) than dispersions with 80% WP-polymers and 10 mM CaCl2. It appeared that CaCl2 was more effective in increasing gel fracture stress while NaCl was more effective in decreasing gelation time. Different gel properties may be prepared by altering the amount of WP-polymers and salt types. q 2001 Elsevier Science Ltd. All rights reserved.

The unfolding equilibrium of beta-trypsin induced by thermal and chemical denaturation was thermodynamically characterized. Thermal unfolding equilibria were monitored using UV absorption and both far- and near-UV CD spectroscopy, while fluorescence was used to monitor urea-induced transitions. Thermal and urea transition curves are reversible and cooperative and both sets of data can be reasonably fitted using a two-state model for the unfolding of this protein. Plots of the fraction denatured, calculated from thermal denaturation curves at different wavelengths, versus temperature are coincident. In addition, the ratio of the enthalpy of denaturation obtained by scanning calorimetry to the van't Hoff enthalpy is close to unity, which supports the two-state model. Considering the differences in experimental approaches, the value for the stability of beta-trypsin estimated from spectroscopic data (deltaGu = 6.0 +/- 0.2 kcal/mol) is in reasonable agreement with the value calculated from urea titration curves (deltaGUH2O = 5.5 +/- 0.3 kcal/mol) at pH 2.8 and 300 degrees K.

Bioactive peptides have been identified within the amino acid sequences of native milk proteins. Hydrolytic reactions, such as those catalyzed by digestive enzymes, result in their release. These peptides directly influence numerous biological processes evoking behavioral, gastrointestinal, hormonal, immunological, neurological, and nutritional responses. The specific bioreactions associated with each physiological class have been well characterized. Herein, we review the scientific literature and attempt to stimulate consideration of the continued use of bioactive peptides and their expanded development as a commercial product. Several applications have already evolved. For example, phosphopeptides derived from casein fractions are currently used as both dietary and pharmaceutical supplements. Potentially, the addition of bioactive peptides to food products could improve consumer safety as a result of their antimicrobial properties. Lastly, bioactive peptides may function as health care products, providing therapeutic value for either treatment of infection or prevention of disease.

The isolation of individual proteins from whey would allow production of more consistent and reliable products by the food industry and possibly would also increase their use in the pharmaceutical industry. α-Lactalbumin is the second most prevalent protein in bovine milk whey and has many uses including serving as an excellent protein source in infant formulas, power drinks and other beverages that require soluble, nutritional protein. In this study, we describe two methods for production of α-lactalbumin from whey protein isolate using bioselective adsorption. The use of a peptide ligand (WHWRKR) attached to a resin allowed production of an α-lactalbumin-rich fraction with a purity of 90.6% and a recovery of 47.9%, while also producing other fractions of commercial interest. The combined use of an amino resin followed by the WHWRKR resin produce a highly purified α-lactalbumin (100%) with a yield of 35.2%.

Whey protein isolate (WPI) was subjected to limited tryptic hydrolysis and the effect of the limited hydrolysis on the rheological properties of WPI was examined and compared with those of untreated WPI. At 10% concentration (w/v in 50 mM TES buffer, pH 7.0, containing 50 mM NaCI), both WPI and the enzyme-treated WPI (EWPI) formed heat-induced viscoelastic gels. However, EWPI formed weaker gels (lower storage modulus) than WPI gels. Moreover, a lower gelation point (77 °C) was obtained for EWPI as compared with that of WPI which gelled at 80 °C only after holding 1.4 min. Thermal analysis and aggregation studies indicated that limited proteolysis resulted in changes in the denaturation and aggregation properties. As a consequenece, EWPI formed particulated gels, while WPI formed fine-stranded gels. In keeping with the formation ofa particulate gel, Texture Profile Analysis (TPA) ofthe heat-induced gels (at 80 °C for 30 min) revealed that EWPI gels possessed significantly higher (p < 0.05) cohesiveness, hardness, gumminess, and chewiness but did not fracture at 75% deformation. The results suggest that the domain peptides, especially β-lactoglobulin domains released by the limited proteolysis, were responsible for the altered gelation properties.

Control of microorganisms such as Bacillus cereus spores is critical to ensure the safety and a long shelf life of foods. A bifunctional single chain antibody has been developed for detection and binding of B. cereus T spores. The genes that encode B. cereus T spore single-chain antibody and streptavidin were connected for use in immunoassays and immobilization of the recombinant antibodies. A truncated streptavidin, which is smaller than but has biotin binding ability similar to that of streptavidin, was used as the affinity domain because of its high and specific affinity with biotin. The fusion protein gene was expressed in Escherichia coli BL21 (DE3) with the T7 RNA polymerase-T7 promoter expression system. Immunoblotting revealed an antigen specificity similar to that of its parent native monoclonal antibody. The single-chain antibody-streptavidin fusion protein can be used in an immunoassay of B. cereus spores by applying a biotinylated enzyme detection system. The recombinant antibodies were immobilized on biotinylated magnetic beads by taking advantage of the strong biotin-streptavidin affinity. Various liquids were artificially contaminated with 5 × 10 4 B. cereus spores per ml. Greater than 90% of the B. cereus spores in phosphate buffer or 37% of the spores in whole milk were tightly bound and removed from the liquid phase by the immunomagnetic beads.

The variable-region genes of monoclonal antibody against Bacillus cereus spores were cloned from mouse hybridoma cells by reverse transcription-PCR. The heavy- and light-chain variable-region genes were connected by a 45-base linker DNA to allow folding of the fusion protein into a functional tertiary structure. For detection of protein expression, a 10-amino-acid strep tag (biotin-like peptide) was attached to the C terminus of recombinant antibody as the reporter peptide. The single-chain antibody construct was inserted into the expression vector and expressed in Escherichia coli under the control of the T7 RNA polymerase-T7 promoter expression system. The expressed single-chain antibody was detected on Western blots by using a streptavidin-conjugated enzyme system. This small recombinant antibody fragment (ca. 28,000 Da by calculation) had B. cereus spore binding ability and antigen specificity similar to those of its parent native monoclonal antibody.

A gene for a streptavidin-lipase fusion protein was constructed by cloning a PCR-modified Pseudomonas fluorescens B52 lipase gene into a streptavidin chimeric protein expression vector, pStp4. The plasmids, pSTLP1 or pSTLP2, were used to transform Escherichia coli NM522 or DH5α. Plasmid pSTLP2, which contains a lacl q gene upstream from the promoter, gave better expression of the bifunctional fusion protein when the cells were induced. Correct insertion of the fused gene was confirmed by DNA sequencing of the linker region and the lipase gene, by identification of a protein band of the correct molecular size (66 kDa) on SDS-PAGE, and by Western blot analysis using antistreptavidin antibody. Streptavidin-lipase fusion protein was purified and immobilized in a single step from recombinant cell lysates by bioselective adsorption on 6-(biotinamido)hexanamidopropyl-controlled pore glass. The hydrolytic activity of the resulting immobilized enzyme exhibited a pH optimum between 7 and 8 and a preference for short-chain fatty acids. Comparison of the hydrolytic activity of immobilized B52 lipase with that of commercial SAM-2 lipase from P. fluorescens suggests that about 30% of the activity was retained upon immobilization. Transesterification of tricaprylin with oleic acid in hexane by immobilized lipase indicated that 11% of the reaction products were derived from transesterification.

The binding of palmitate to β -lactoglobulin at protein concentrations ranging from 1 to 200 μM was determined using an ultrafiltration method with [ 14 C]palmitate. Fit of the data to theoretical models required the assumption of two independent sets of binding sites; however, binding characteristics were dependent on the protein concentration. A model assuming one set of sites on the protein monomer and another on the dimer was consistent with the data. The analysis suggests that 2mol of palmitate are bound/mol of dimer and that the binding constant is of the order of 10 5 M –1 ; a larger number of palmitate molecules are bound per mole of monomer with a smaller binding constant of the order of 10 4 M –1 . Apparently, formation of the dimer, by hydrophobic interactions at the monomer contact site, eliminated palmitate binding sites on the monomer but formed a higher affinity pocket for binding to the dimer.

A series of simple and phenol-free silica-based protocols for isolating and cleaning RNA or DNA fragments from different sources were developed. Cytoplasmic RNA isolated from hybridoma cells by this method was used in RT-PCR. DNA fragments obtained using this protocol were suitable for further subcloning, gene transformation and DNA sequencing. © Rapid Science Ltd. 1998

Bioselective adsorption was evaluated as a possible technology for food processing to enhance safety usingBacillus cereusin milk as a model system. Cataphote™ Microbeads class −400 were derivatized by attaching 3-aminopropyl groups (2.7 nm2/molecule) onto the surface of the bead. Carbohydrates on the Fc region of monoclonal antibody 183 againstB. cereusT spores were oxidized with potassium meta-periodate to allow for an oriented antibody immobilization (270 nm2/molecule). The adsorption matrix was characterized for its ability to bindB. cereusspores in comparison to a control matrix containing immobilized bovine serum albumin. When 2.5 × 106spores in skim milk were added to 1 mL of each matrix, the IgG-matrix was capable of removing 96% of that amount, 70% of which were bound with high affinity and were only eluted with 0.1 mol/L acetic acid. In contrast, the control matrix removed 90% of the spores added but only 7% were retained after washing the matrix. In addition, the IgG-matrix showed an excellent regeneration ability; the binding level did not decrease significantly after 28 trials with buffer or milk. Calculations determined that the bioadsorbant was capable of removing 8 × 106spores/m2. Thus, bioselective adsorption has promise as a technology to enhance safety of liquid foods or to improve analytical methodology.

A 5% protein suspension (4 mL) was whipped in a modified 50-mL centrifuge tube using a tissumizer equipped with a flat-bottom generator. Drainage time at 50% liquid weight and the weight of the foam formed/ unit volume were used for calculating foam stability and foam overrun, respectively. The foaming properties of a variety of milk proteins were determined using this method. This method distinguished differences in foaming properties among the proteins. Values for overrun confirmed published results. Compared with standard methods, this method required much less sample (about 1/20) and less measuring time (about 1/5 to 1/10).

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

Trypsin was immobilized on 2 Celite™ derivatives and the kinetic properties of trypsin immobilized on these derivatives were determined and compared. Celite™ was derivatized with organosilane to give aminopropyl-Celite (APC) and a portion of this derivative was then succinylated to give succinamidopropyl-Celite (SAPC). Trypsin was covalently immobilized on APC using glutaraldehyde to activate amino groups and on SAPC using water-soluble carbodiimide to activate surface carboxyl groups. Enzyme loadings were 13.9 and 17.8 mg ml −1 of beads on APC and SAPC, respectively. Using p -tosyl- l -arginine methyl ester as substrate, the catalyst specific activity, K M app and k cat / K M app were 17.8 U ml −1 of beads, 3.60 and 21.0 mM −1 min −1 , respectively, for trypsin-APC as compared with 24.5 U ml −1 of beads, 3.77 and 20.3 mM −1 min −1 , respectively, for trypsin-SAPC. With β -lactoglobulin as substrate, K M app and k cat / K M app were 0.36 and 1.62 mM −1 min −1 for trypsin-APC and 0.54 and 1.39 mM −1 min −1 for trypsin-SAPC, respectively. The pH range for optimal activity was much larger for both immobilized forms as compared with the soluble enzyme. The optimal temperature ranges were 40–50°C for trypsin-APC and 50–60°C for trypsin-SAPC. The two methods of immobilization on Celite™ gave biocataysts with similar kinetic properties but immobilization on SAPC yielded slightly higher loadings and higher specific activities.

A streptavidin-β-galactosidase fusion protein was expressed in Escherichia coli and bioselectively adsorbed from crude cell lysates to biotin covalently immobilized on controlled pore glass. Michaelis constants for the immobilized enzyme with o -nitrophenyl-β- d -galactopyranoside and lactose were 0.28 and 0.4 m m , respectively. Very similar values were obtained with a commercial preparation of soluble enzyme, indicating that neither folding of the fusion protein nor interaction of the streptavidin domain with immobilized biotin altered the structure of the substrate binding site. As compared to soluble enzyme, the apparent optimum pH for activity was shifted 0.5 units to the acid region and the optimum temperature was 5°C lower. Similar bioreactor activities were regenerated five times by desorption of the fusion enzyme protein with 6 m guanidinium chloride followed by readsorption from cell lysates.

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...

A rapid and sensitive spectrophotometric assay was developed for the measurement of biotin-binding sites of immobilized avidin. The method is based on the reaction of avidin with excess biotin followed by assay of the unbound biotin using the HABA (2-[4′-hydroxyazobenzene] benzoic acid) method. Three solids possessing variable amounts of monomeric avidin were examined; viz., succinamidopropyl-controlled-pore glass (CPG-500), crosslinked 6% beaded agarose (Sepharose-CL-6B**), and crosslinked bis-acrylamide/azlactone (3M Emphaze Biosupport Medium AB1. Results indicate that the total biotin-binding sites of monomeric avidin immobilized on CPG-500, Sepharose-CL-6B, and 3M Emphaze are 0.229, 0.093, and 0.218 µmol biotin per mL beads, respectively. Assays for exchangeable biotinbinding sites gave values greater than 90% of the total sites. The spectrophotometric HABA method described is an alternative to assays based on tracers, thus the handling of radioactive material is avoided.

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

Size-exclusion chromatography was used to investigate the interaction of sucrose ester P-1670, composed of 70% pure palmitic acid monoester, with native milk proteins as they are present in skim milk. Addition of sucrose ester to fresh raw skim milk or to casein micelles that had been isolated by chromatography of fresh raw skim milk without cooling resulted in a small decrease in the elution volume and an increase in the light scattering of the micelle fraction, indicating an increase in micelle size. In the skim milk sample, the elution volume of whey proteins also decreased slightly. Similar resolution of casein micelles and whey proteins was obtained with tandem glycerylpropyl controlled-pore glass columns containing beads of mean pore diameters of 52 and 286 nm and with Sephacryl S-400. Analysis of micellar fractions by SDS-PAGE showed that the distribution of individual caseins in the micelles was not affected by sucrose ester. Quantitative determination of sucrose ester using the periodate reaction confirmed the binding of the surfactant by casein micelles.

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

The interaction of sucrose ester, P-1670 (70% pure palmitic acid monoester), with milk proteins was examined by affinity chromatography. The ester was covalently bound to 3-aminopropyl controlled-pore glass by periodate oxidation of the carbohydrate to aldehyde, formation of the Schiff base, followed by reduction to secondary amine. In phosphate buffer, pH 7, α -LA did not appear to interact with sucrose ester, but β -LG exhibited binding. However, casein micelles were very tightly bound, and caseins could only be eluted following addition of urea. On application of skim milk, the strengths of the interactions, as reflected by their elution volumes, decreased in the order: α s -CN, κ -CN, β -CN, β -LG, and α -LA. Micelles dissociate after addition of urea, allowing individual caseins to interact with sucrose ester moieties in the presence of increasing urea concentrations. Apparent dissociation constant was 2.04 μM for the binary complex using solutions of pure β -LG in agreement with the value for soluble complexes measured by other methods. Hydrophobic interaction appears to be the major force stabilizing complex formation, even though the affinities of the individual caseins do not follow the order of their overall hydrophobicities. Tight binding of micelles to the immobilized sucrose esters suggests that the alkyl chains are able to insert into hydrophobic regions of surface submicelles.

SummaryA laboratory scale bioreactor was used for continuous acidification and inoculation of milk with a proteinase-negative, lactose-fermenting strain,Lactococcus lactissubsp.lactisC2S. Calcium alginate-entrapped cells were immobilized on a spiral stainless steel mesh incorporated into a column bioreactor and used to acidify and inoculate reconstituted skim milk. Characteristics of the immobilized cell bioreactor (ICB) were compared with those of a free cell bioreactor (FCB) during challenge with a virulent phage. Steady state biomass and lactate productivities were respectively 25-fold and 12-fold larger with the ICB than with the FCB. The ICB and the FCB were inoculated with the prolate phage c2 at multiplicities of infection of 0·25 and 0·02 respectively. Within 90 min of the infection, the FCB viable cell concentration dropped by five orders of magnitude and never recovered, while the plaque forming units/ml increased dramatically. In the ICB, released cells decreased immediately after infection, but subsequently increased, while the plaque forming units/ml steadily declined, indicating that phage were being washed out of the bioreactor. Productivity of FCB decreased to zero, whereas productivity of the ICB only decreased ∼ 60% and subsequently recovered to its initial steady state value.

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

Mixtures of lignin peroxidase isozymes were immobilized onto controlled-pore glass (CPG) beads through three different functional groups (free amino groups, free carboxylate groups, or the aldehyde groups of the metaperiodate-oxidized carbohydrate portions of the proteins) and catalytic properties of the immobilized proteins were determined. Some loss of activity occurred in all immobilized samples, but relatively high activities were maintained with enzymes immobilized via the modified carbohydrate moieties. The effectiveness of using the immobilized enzymes to treat anthracene and pulp mill bleach plant effluent was tested. Bleach plant effluent precipitated onto the aminopropyl-CPG beads inactivating the enzymes, probably due to reactions with free amino groups. In contrast anthracene was rapidly and completely degraded by the immobilized lignin peroxidases.

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

A laboratory-scale bioreactor with lactococci immobilized in calcium alginate gel was developed for continuous acidification and inoculation of milk. Cells were entrapped in a calcium alginate film coating a spiral mesh and placed in a column through which milk was recirculated from a reservoir. Steady-state conditions were achieved by addition of fresh milk using a pH controller to maintain the pH at 5.7 and acidified milk was continuously removed during operation periods up to 5 d. Immobilized and free cell bioreactors were compared using both proteinase-positive and proteinase-negative strains of Lactococcus lactis ssp. lactis C2. Productivities were 1.5- to 3.5-fold larger with immobilized cell bioreactors than with free cell bioreactors because of higher cell densities, although specific productivities were lower for immobilized cells. Productivity increase was larger for proteinase-negative cells, which do not grow as well as free cells in milk. However, high densities can be immobilized, resulting in productivities of immobilized proteinase-negative cells that were similar to those of proteinase-positive cells. Free proteinase-negative cells responded to amino acid and peptide supplementation by increasing productivity (5-fold), but the immobilized cells did not respond proportionally, suggesting that free cell activity was limited by substrate availability but that immobilized cells were limited by product inhibition.

Incubation of beta-lactoglobulin with immobilized trypsin at 5-10 degrees 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 HR 5/5 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, beta-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 beta-sheet structure similar to the native protein but the alpha-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 delta GDH20 and the urea concentration at the transition midpoint are lower than for the native protein. The core domain fragments also display a pH-dependent binding to immobilized trans-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 degrees C as compared with 81.1 degrees C for native protein.

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

A chemically prepared conjugate of avidin and E. coli β-galactosidase was adsorbed to biotinylated controlled-pore glass beads and used in a fluidized-bed bioreactor to assess the feasibility of bioselective adsorption immobilization technology. Biotinylated 200 nm pore diameter porous glass beads were prepared by reaction of 3-aminopropyl-glass beads with sulfosuccinimidyl-6-(biotinamido) hexanoate. Avidin and biotinylated β-galactosidase were sequentially adsorbed to the matrix. The fluidized-bed bioreactor was characterized with respect to β-galactosidase activity using both a lactose solution and o-nitrophenyl β-D-galactopyranoside (ONPG) as substrates. A lactose solution (4.5%, pH 7) was assayed for lactose hydrolysis at various flow rates. The bioreactor was operated for three months at 65–75% lactose hydrolysis with no loss in enzyme activity. The biocatalyst was characterized by amino acid analysis to determine the amount of each of the two proteins adsorbed. Results indicated 162 μ protein/mg beads of which 36% was avidin and 64% was β-galactosidase corresponding to 1 mole of avidin per mole of β-galactosidase monomer. Biocatalyst activity using ONPG as the substrate was 430 μmoles/min/mg protein, yielding a specific activity of 672 μmoles/min/mg β-galactosidase. These results lead to the conclusion that biospecific adsorption of the β-galactosidase conjugate onto biotinylated glass beads via avidin results in a biocatalyst that is stable and retains a high specific activity.

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 rapid, sensitive, and convenient spectrophotometric assay was developed for the measurement of amino groups on solid supports. This method is based on the reaction of amino groups of solids with an excess ofo-phthaldialdehyde (OPA) and subsequent quantitative determination of unreacted OPA by reaction with glycine. Four solids possessing variable quantities of amino groups were examined. Results indicate that about 70% of the total surface amino concentration (determined by the microKjeldahl method) are available for ligand attachment. Unlike the spectrophotometric 2,4,6-trinitrobenzenesulfonic acid method, the OPA spectrophotometric assay is more rapid, sensitive, and convenient, and unlike the spectrofluorimetric OPA, it does not require sophisticated instrumentation.

The effects of various food components on the in vitro digestibility of proteins, measured by the immobilized digestive enzyme assay (IDEA) system, were investigated. The digestibility of unprocessed and processed sodium caseinate and soybean protein was examined. Varying concentrations of sucrose (0, 5, 10 and 20%) or starch (0, 0.5, 1 and 2%) had no significant effects on digestibility of protein. Similarly, the presence of emulsified (1% lecithin) vegetable oil (0, 0.5, 1 and 2%) did not affect digestibility. Therefore, these common ingredients of foods did not affect the extent of hydrolysis of the protein samples under the conditions of the IDEA method, suggesting that this assay is suitable for use with complex foods.