Enzymological characteristics of pepsinogens and pepsins purified from lizardfish (Saurida micropectoralis) stomach.

One major pepsinogen, PG-I, and two minor pepsinogens, PG-II and PG-III were purified from lizardfish stomach by ammonium sulfate precipitation and two chromatographic columns. The three purified PGs migrated as single bands in native-PAGE gels with molecular weights (MW) ranging from 36 to 38 kDa. Each PG was converted to pepsin (P) at pH 2.0, and the MW were determined as 32 kDa (for P-I), 31 kDa (for P-II) and 30 kDa (for P-III). The optimum pH and temperature of pepsins were 2.0-3.5, and 40-50 °C. All 3 pepsins were strongly inhibited by pepstatin A. Divalent cations slightly stimulated the pepsin activities, but ATP had no effect on the pepsins. Purified pepsins were effective in the hydrolysis of various proteins. Km and kcat of the three pepsins for hemoglobin hydrolysis were 107.64-276.61 µM and 18.30-32.68 s-1, respectively. The new pepsins have potential for use in protein food procession and modification.

Influence of amino acids on thermal stability and heat-set gelation of bovine serum albumin.

This study investigated the parallels in the influence of amino acid additives on thermal denaturation temperature (Td) and heat-set gelation of bovine serum albumin (BSA). Complete denaturation of BSA occurred only when the gelation temperature (TG) was 14 °C above Td. Under these conditions, the relative effects of various amino acid additives on elevation of Td and gel strength followed a particular order. Further, while zwitterionic amino acids increased both the strength of junction zones and participation of protein in the gel network, sucrose increased the gel strength primarily by strengthening the existing junction zone. The net increase in Td of BSA was linearly correlated with the net increase in gel strength (ΔGS), indicating that the underlying molecular mechanism in both cases might be same. The results suggest that the rheological properties of protein gels can be enhanced by using amino acids, instead of polyols and sugars, as additives.

Hofmeister Order of Anions on Protein Stability Originates from Lifshitz-van der Waals Dispersion Interaction with the Protein Phase.

The mechanism underpinning the Hofmeister order of anions on protein stability and other physical and biological processes has been a mystery since its discovery in 1888. In the present study, we investigated electrostatic and Lifshitz-van der Waals (L-vdW) dispersion (electrodynamic) interactions between Hofmeister salts and four monomeric globular proteins. It is shown that structure-stabilizing salts exerted positive L-vdW pressure, whereas structure-destabilizing salts exerted negative L-vdW pressure on proteins. The relative order of the L-vdW pressure followed the Hofmeister series and it overshadowed the electrostatic pressure at high salt concentrations. The net change in the thermal denaturation temperature (ΔTd) of proteins in 0.8 M Hofmeister salt solutions followed a linear relationship (r2 > 0.8) with the net electrodynamic pressure regardless of the physicochemical differences between proteins. This study also revealed that segregation of anions into structure stabilizers and destabilizers depended on the dielectric susceptibility of the anion in the ultraviolet region: ions having absorbtion spectrum in the ultraviolet region (e.g., Cl-, Br-, I-, and SCN-) exerted a negative electrodynamic pressure, whereas those with absorbtion spectrum only in the infrared region, for example, SO42-, exerted a positive electrodynamic pressure. The lack of ultraviolet absorption of SO42- ions was because of quenching of ultraviolet radiation by water at below 170 nm.

Removal of off-flavour-causing precursors in soy protein by concurrent treatment with phospholipase A2 and cyclodextrins.

A simple mild process to remove phospholipids in soy protein isolate has been developed. The method includes two steps: A 5% soy protein isolate solution is concurrently treated with 0.5-1.5 µkat phospholipase A2/g protein and 10 mmol/l ß-cyclodextrin for 4 h at 43 °C, pH 8.0; secondly, soy protein is separated from the treated solution by precipitating at pH 4.5. The treatment removed more than 92% of the off-flavour precursors in SPI. Comparing α-, ß-, and γ-cyclodextrins, α-cyclodextrin was more effective (>95% removal of precursors) than ß-cyclodextrin, while γ-cyclodextrin essentially had no effect. Under accelerated storage conditions at 45 °C for 90 days, the rate of hexanal production in the treated SPI was 12-times slower than that in the untreated SPI. The treatment lowered the thermal denaturation temperature and enthalpy of denaturation of soy proteins but not its solubility, indicating that the treatment caused some structural changes in soy proteins.

Nanostructure and functionality of enzymatically repolymerized whey protein hydrolysate.

Whey proteins (WPI) were polymerized with transglutaminase (TGase) before and after partially hydrolyzing the protein with thermolysin to produce protein nanoparticles/polymers. Electrophoresis and atomic force microscopy (AFM) were used to determine the size and structural characteristics of the polymers. The foaming and emulsifying properties of these nanoparticles were studied. The polymerized WPI (WPI-TG) produced more stable foams than the repolymerized WPI hydrolysate (WPIH-TG). In contrast, WPIH-TG produced better emulsions with better storage stability than WPI-TG emulsions. These differences were due to their structure and electrostatic properties: The WPI-TG particles were linear, less than 100 nm in size with lower net negative charge, whereas the WPIH-TG polymers were much larger and were highly negatively charged as judged from zeta potential. This suggested that while protein nanoparticles may provide Pickering stability to both emulsions and foams, strong lateral electrostatic repulsion between nanoparticles within the adsorbed film destabilizes foams but not emulsions.

A two-step enzymatic modification method to reduce immuno-reactivity of milk proteins.

A two-step enzymatic approach to reduce immuno-reactivity of whey protein isolate and casein has been studied. The method involves partial hydrolysis of proteins with proteases, followed by repolymerization with microbial transglutaminase. Whey protein isolate partially hydrolyzed with chymotrypsin, trypsin, or thermolysin retained about 80%, 30%, and 20% of the original immuno-reactivity, respectively. Upon repolymerization the immuno-reactivity decreased to 45%, 35%, and 5%, respectively. The immuno-reactivity of hydrolyzed and repolymerized casein was negligible compared to native casein. The repolymerized products were partially resistant to in vitro digestion. Peptides released during digestion of repolymerized thermolysin-whey protein hydrolysate had less than 5% immuno-reactivity, whereas those of whey protein control exhibited a sinusoidal immuno-reactivity ranging from 5 to 20%. Peptides released during digestion of repolymerized thermolysin-casein hydrolysates had no immuno-reactivity. These results indicated that it is possible to produce hypoallergenic milk protein products using the two-step enzymatic modification method involving thermolysin and transglutaminase.

In vitro digestibility and IgE reactivity of enzymatically cross-linked heterologous protein polymers.

Homologous and heterologous cross-linked polymers of whey protein isolate (WPI), soy protein isolate (SPI) and casein (CN) and their binary mixtures, viz., WPI+SPI, WPI+CN and SPI+CN, were produced using transglutaminase, and their in vitro IgE reactivity and digestibility under simulated gastro-intestinal conditions were studied. The results showed that the IgE reactivity of protein components in heterologous polymers was significantly lower than that in homologous polymers, suggesting that each protein component masked the IgE-reactive epitopes in the other protein component more effectively in heterologous polymers than in homologous polymers. In vitro digestion under simulated gastro-intestinal conditions revealed that both homologous and heterologous polymers were less digestible than untreated proteins, but the peptides released during the time course of digestion were less IgE-reactive. The results of this study indicate that hypoallergenic protein products could be produced by transglutaminase-mediated heterologous polymerization of protein mixtures.

Water at Biological Phase Boundaries: Its Role in Interfacial Activation of Enzymes and Metabolic Pathways.

Many life-sustaining activities in living cells occur at the membrane-water interface. The pertinent questions that we need to ask are, what are the evolutionary reasons in biology for choosing the membrane-water interface as the site for performing and/or controlling crucial biological reactions, and what is the key physical principle that is very singular to the membrane-water interface that biology exploits for regulating metabolic processes in cells? In this chapter, a hypothesis is developed, which espouses that cells control activities of membrane-bound enzymes through manipulation of the thermodynamic activity of water in the lipid-water interfacial region. The hypothesis is based on the fact that the surface pressure of a lipid monolayer is a direct measure of the thermodynamic activity of water at the lipid-water interface. Accordingly, the surface pressure-dependent activation or inactivation of interfacial enzymes is directly related to changes in the thermodynamic activity of interfacial water. Extension of this argument suggests that cells may manipulate conformations (and activities) of membrane-bound enzymes by manipulating the (re)activity of interfacial water at various locations in the membrane by localized compression or expansion of the interface. In this respect, cells may use the membrane-bound hormone receptors, lipid phase transition, and local variations in membrane lipid composition as effectors of local compression and/or expansion of membrane, and thereby local water activity. Several experimental data in the literature will be reexamined in the light of this hypothesis.

Beyond the hydrophobic effect: Critical function of water at biological phase boundaries--A hypothesis.

Many life-sustaining processes in living cells occur at the membrane-water interface. The pertinent questions that need to be asked are what is the evolutionary reason for biology to choose the membrane-water interface as the site for performing and/or controlling crucial biological reactions and what is the key physical principle that is singular to the membrane-water interface that biology exploits for regulating metabolic processes in cells? In this review, a hypothesis is developed, which espouses that cells control activities of membrane-bound enzymes and receptor activated processes via manipulating the thermodynamic activity of water at the membrane-water interfacial region. In support of this hypothesis, first we establish that the surface pressure of a lipid monolayer is a direct measure of a reduction in the thermodynamic activity of interfacial water. Second, we show that the surface pressure-dependent activation/inactivation of interfacial enzymes is fundamentally related to their dependence on interfacial water activity. We extend this argument to infer that cells might manipulate activities of membrane-associated biological processes via manipulating the activity of interfacial water via localized compression or expansion of the interface. In this paper, we critically analyze literature data on mechano-activation of large pore ion channels in Escherichia coli spheroplasts and G-proteins in reconstituted lipid vesicles, and show that these pressure-induced activation processes are fundamentally and quantitatively related to changes in the thermodynamic state of interfacial water, caused by mechanical stretching of the bilayer.

Electrodynamic pressure modulation of protein stability in cosolvents.

Cosolvents affect structural stability of proteins in aqueous solutions. A clear understanding of the mechanism by which cosolvents impact protein stability is critical to understanding protein folding in a biological milieu. In this study, we investigated the Lifshitz-van der Waals dispersion interaction of seven different solutes with nine globular proteins and report that in an aqueous medium the structure-stabilizing solutes exert a positive electrodynamic pressure, whereas the structure-destabilizing solutes exert a negative electrodynamic pressure on the proteins. The net increase in the thermal denaturation temperature (ΔTd) of a protein in 1 M solution of various solutes was linearly related to the electrodynamic pressure (PvdW) between the solutes and the protein. The slope of the PvdW versus ΔTd plots was protein-dependent. However, we find a positive linear relationship (r(2) = 0.79) between the slope (i.e., d(ΔTd)/dPvdW) and the adiabatic compressibility (ßs) of the proteins. Together, these results clearly indicate that the Lifshitz's dispersion forces are inextricably involved in solute-induced stabilization/destabilization of globular proteins. The positive and/or negative electrodynamic pressure generated by the solute-protein interaction across the water medium seems to be the fundamental mechanism by which solutes affect protein stability. This is at variance with the existing preferential hydration concept. The implication of these results is significant in the sense that, in addition to the hydrophobic effect that drives protein folding, the electrodynamic forces between the proteins and solutes in the biological milieu also might play a role in the folding process as well as in the stability of the folded state.

Off-flavor precursors in soy protein isolate and novel strategies for their removal.

Off-flavors remain a major hurdle in expanding the use of soy protein isolate (SPI) in mainstream food applications. The complexity in solving this problem arises from the presence of protein-bound precursors in SPI. Among the most predominant sources of off-flavors in SPI is the residual amount of phospholipids that contain polyunsaturated fatty acids (PUFAs). Autoxidation of PUFAs generates several classes of volatile compounds that contribute to the beany, grassy, or green odor of SPI. In addition, several polyphenolic compounds, such as isoflavones, saponins, phenolic acids, etc., impart bitter and astringent tastes to SPI. Traditional methods for removing protein-bound precursors from SPI and their limitations are reviewed. The most notable trade-off of conventional methods is the loss of protein functionality to some degree. Therefore, pursuit of gentler treatments to overcome SPI off-flavor has been the focus of industry and academia alike. Novel approaches that employ ß-cyclodextrin to remove both SPI-bound precursors and volatile compounds are described.

On the molecular mechanism of stabilization of proteins by cosolvents: role of Lifshitz electrodynamic forces.

Several ionic and nonionic additives are known to affect structural stability of proteins in aqueous solutions. At a fundamental level, the mechanism of stabilization or destabilization of proteins by cosolvents must be related to three-body interactions between the protein, additive, and the water medium. In this study, the role of the Lifshitz-van der Waals electrodynamic interaction between various additives (sucrose, glycerol, urea, poly(ethylene glycol)-200, betaine, taurine, proline, and valine) and bovine serum albumin (BSA) in water medium was examined. The electrodynamic interaction energy was attractive for all of the additives studied here when both far ultraviolet and infrared relaxations of the additives were included in their dielectric susceptibility representations. However, when only the infrared contribution was included for structure stabilizers and both far ultraviolet and infrared contributions for the structure destabilizers, the resulting electrodynamic interaction energy (E/kT) followed the structure stabilizing and/or destabilizing behavior of the additives; that is, the interaction was attractive for urea and PEG200 (structure destabilizers), whereas it was repulsive for sucrose, glycerol, betaine, taurine, alanine, valine, and proline (structure stabilizers). The electrodynamic interaction energy E/kT at any given surface-to-surface separation distance between the additives and BSA was positively correlated (r(2) = 0.92) with the experimental thermal denaturation temperature (T(d)) of BSA in 1 M solutions of the additives. These analyses provided a mechanistic basis for the experimental observations of exclusion of the structure-stabilizing additives from the protein-water interface and binding of the structure-destabilizing additives to the protein surface. The role of water structure in the three-body electrodynamic interaction is discussed. It is hypothesized that in the case of additives that enhance water structure the hydration shells formed around the additives effectively dampen the contribution of ultraviolet frequencies to the dielectric susceptibility of the additives and thus impart repulsive electrodyanamic interaction between the additive and the protein, whereas the opposite occurs in the case of additives that breakdown the hydrogen-bonded structure of water.

Straightforward process for removal of milk fat globule membranes and production of fat-free whey protein concentrate from cheese whey.

A straightforward method for the separation of milk fat globule membrane (MFGM) and production of fat-free whey protein concentrate/isolate from cheese whey has been developed. Lowering of the conductivity of the whey from its initial value of about 5600 µS cm(-1) to about 2000-500 µS cm(-1) via diafiltration with water caused selective precipitation of MFGM when incubated for 30 min at pH 4.2 and 35 °C. The whey proteins remained soluble in the supernatant under these conditions. Experimental evidence suggested that precipitation of MFGM at pH 4.2 was not due to a nonspecific effect of lowering of the conductivity of the whey but due to the specific effect of removal of Ca2+ from the whey. The lipid content of whey protein isolate obtained by this process was <0.2%, and the protein loss was <14%. The method provides an industrially feasible process for the production of fat-free whey protein concentrate/isolate. The MFGM, which is reported to contain bioactive/nutraceutical lipids and proteins, is a valuable byproduct of the process.

Composition, thermotropic properties, and oxidative stability of freeze-dried and spray-dried milk fat globule membrane isolated from cheese whey.

The milk fat globule membrane (MFGM) was isolated from cheese whey using a recently developed novel method. The cheese-derived MFGM contained about 17-19% lipids and 65-70% protein on a dry weight basis. About 50% of the lipids in MFGM were phospholipids. Compositional analysis of the cheese whey-derived MFGM showed that it is a rich source of phosphatidylserine, sphingomyelin, and bioactive proteins CD36, butyrophilin, xanthine oxidase, and mucin 1. Utilization of MFGM in foods as a source of nutraceutical lipids depends on its oxidative stability. In this context, the impact of drying methods, namely, freeze-drying versus spray-drying, on the storage stability of MFGM was studied. Freeze-dried (FD) and spray-dried (SD) MFGM samples were morphologically very different when examined by light microscope: The thermotropic phase transition temperature (T(m)) of lipids in the FD-MFGM was 37.8 °C, and it was 48 °C in SD-MFGM. This 10 °C difference in T(m) indicated that the drying method altered the thermodynamic state of phospholipids in MFGM. At all storage temperatures studied, the zero-order rate constant of lipid oxidation, as measured by hexanal production, was 1-2 orders of magnitude greater in the spray-dried than in the freeze-dried MFGM. The results clearly indicated that the choice of drying method affects morphological characteristics, the T(m) and oxidative stability of phospholipids in MFGM.

Chemical modification and structural analysis of protein isolates to produce hydrogel using Whitemouth croaker (Micropogonias furnieri) wastes.

Recovery and alteration of fish protein from wastes and its use has been regarded as a promising alternative to develop useful products once polymer gels have a high capacity of water uptake. This study aims to produce hydrogel, a super absorbent biopolymer from modified fish protein, in order to evaluate the protein structure. In the modified proteins, analyses of the extent of modification of the lysine residues, electrophoresis, and electrometric titration were performed. In the hydrogels were realized assays of swelling water. The proteins with more modifications were shown as 63.5% and 75.9% of lysine residues, from fish protein isolate obtained with alkaline and acid solubilization, respectively. The modified protein in that same rate presented 332.0 and 311.4 carboxyl groups. Accordingly, the hydrogel produced from alkaline and acid isolates reached a maximum water uptake in 24 h of 79.42 and 103.25 g(water)/g(dry gel), respectively.

Removal of soy protein-bound phospholipids by a combination of sonication, ß-cyclodextrin, and phospholipase A2 treatments.

One of the contributing factors to generation of off-flavours in soy protein isolate (SPI) during storage is autoxidation of residual amounts of phospholipids present in SPI. Thus, removal of phospholipids from SPI is a likely first step to improve its flavour stability and enhanced utilisation of SPI in food products. We describe a ß-cyclodextrin-based (ßCD) process to remove protein-bound phospholipids and free fatty acids in SPI. Treating SPI solution (8%) with 10mM ßCD alone at pH 8.0 decreased the phospholipid content of SPI by about 36%. A greater than 99% removal of phospholipids and free fatty acids was achieved by using a combination of treatments involving sonication of the SPI solution for 5min at 50°C followed by treatment with phospholipase A2 and ßCD. SPI prepared by this method was white in colour. The results presented here offer a process for removing residual off-flavour causing phospholipids from soy protein.

Zinc-induced precipitation of milk fat globule membranes: a simple method for the preparation of fat-free whey protein isolate.

A simple method to isolate milk fat globule membrane (MFGM) from cheese whey has been developed. The method was based on the premise that divalent cations should be able to form insoluble complexes with MFGM under certain solution conditions; this ability, however, is dependent on their coordination chemistry. Incubation of cheese whey with 0-50 mm CaCl2 or MgCl2 at 30 °C did not cause precipitation of MFGM, whereas incubation with Zn(Ac)2 under similar conditions induced selective precipitation of MFGM in a concentration-dependent manner at pH 5.2, with complete precipitation occurring above 20 mm Zn(Ac)2. The whey proteins remained soluble in the supernatant under these conditions. The ability or inability of a cation to induce precipitation of MFGM is related to the ionic radius as well as its coordination geometry. Calcium and magnesium ions have a strong tendency to form hexa-coordinated (n = 6) complexes in a regular octahedral geometry, whereas zinc prefers to form a tetra-coordinated complex in a tetrahedral geometry with MFGM phosphate groups. It is proposed that the tetrahedral geometry of zinc coordination in the zinc-MFGM complex permits hydrophobic interaction between MFGM particles, resulting in precipitation at 30 °C. Further processing of the supernatant using membrane ultrafiltration/diafiltration resulted in a fat-free whey protein isolate.

Physicochemical and emulsifying properties of whey protein isolate (WPI)-dextran conjugates produced in aqueous solution.

The physicochemical and emulsifying properties of protein and polysaccharide conjugates prepared under mild conditions were investigated. The covalently linked conjugates of whey protein isolate (WPI) and dextran (DX, 440 kDa) were produced by incubating aqueous solutions containing 10% WPI and 30% DX at pH 6.5 and 60 degrees C for 48 h. After purification by anion-exchange chromatography and affinity chromatography, the conjugate had a weight-average molecular weight (M(w)) of 531 kDa and a radius of gyration (R(g)) of 30 nm as determined by size exclusion chromatography-multiangle laser light scattering (SEC-MALLS); the molar binding ratio of WPI to DX was calculated to be approximately 1:1. The purified conjugate had significantly improved heat stability when subjected to 80 degrees C for 30 min and remained soluble over a range of pH from 3.2 to 7.5 and ionic strengths from 0.05 to 0.2 M in contrast to native WPI. The emulsifying ability and emulsion stability made with WPI-DX conjugate were also improved compared to WPI and gum arabic (an emulsifier containing naturally derived glycoproteins).

pH-stability and thermal properties of microbial transglutaminase-treated whey protein isolate.

Whey protein isolate (WPI) was treated to various extents using microbial transglutaminase (MTGase) and changes in pH-stability and thermal stability of its protein components were investigated. The MTGase treatment significantly increased the denaturation temperature (T(d)) of beta-lactoglobulin in WPI, from 71.84 degrees C in the untreated sample to 78.50 degrees C after 30 h of incubation with MTGase. The enthalpy change of denaturation of WPI did not change upon cross-linking, indicating that the increase in T(d) was primarily due to covalent cross-linking and not due to an increase in nonpolar interactions within the protein. The surface hydrophobicity (S(o)) of the protein decreased upon cross-linking; however, this decrease was not due to burial of the surface hydrophobic cavities in the protein interior, but due to occlusion of the hydrophobic cavities to the fluorescent probes. Fluorescence emission and circular dichroism spectroscopic analyses revealed no major changes in the secondary and tertiary conformations as a result of cross-linking. However, unlike native WPI, the cross-linked protein exhibited a U-shaped pH-stability profile with maximum turbidity at pH 4.0-4.5. The study revealed that even though enzymatic cross-linking significantly improved the T(d) of beta-lactoglobulin in WPI without causing major structural changes, a reduction in the hydrophilic-hydrophobic balance of the protein surface as a result of elimination of the positive charge on lysyl residues caused precipitation at pH 4.0-4.5.

Ice-structuring peptides derived from bovine collagen.

Antifreeze proteins belonging to structurally diverse families of genetically coded proteins from several living organisms have been isolated and characterized in the past. This paper reports that collagen peptides of a certain molecular size range derived from Alcalase hydrolysis of bovine gelatin are able to inhibit recrystallization of ice in frozen ice cream mix as well as in frozen sucrose solutions in a manner similar to natural antifreeze proteins. The optimum conditions for producing such ice-structuring peptides (ISP) were hydrolysis at pH 9.0 for 30 min at 45 degrees C and an Alcalase-to-gelatin ratio of 0.176 unit per gram of gelatin. The collagen peptides were fractionated on size exclusion (Sephadex G-50) and ion exchange (sulfopropyl-Sephadex C-25) columns, and the molecular mass distribution of the ice-structuring peptide fractions was determined by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. The collagen peptide fractions in the molecular mass range of 600-2700 Da inhibited ice recrystallization in a supercooled ice cream mix and in concentrated sucrose solutions. The cationic collagen peptides within this fraction with molecular mass in the range of 1600-2400 Da were more effective than the anionic peptides in inhibiting ice crystal growth.