Hydrolysis Of Β-lactoglobulin Research Articles

Effect of high hydrostatic pressure on the steady-state kinetics of tryptic hydrolysis of β-lactoglobulin

The rate of hydrolysis of β-lactoglobulin (genetic variant B) by trypsin in aqueous solution at pH of 7.6 and ionic strength 0.08 M (NaC1) at 25.0 °C increased with increasing hydrostatic pressure. At constant pressure (ambient, 100, 150 and 250 MPa studied), the initial rate of disappearance of β-lactoglobulin B could be analyzed using Michaelis–Menten kinetics (five initial substrate concentrations ranging from 0.11 to 0.88 mM used at each pressure). The over-all catalytic efficiency (ratio between catalytic constant kc and Michaelis constant Km) increased by a factor of 103 at 250 MPa compared with ambient pressure. While kc increased only for pressures up to 100 MPa, corresponding to an initial pressure-melted state (factor of 102 up to 100 MPa) with no further increase at higher pressure, Km continuously decreased up to 250 MPa, corresponding to a binding volume of −37 ml mol−1 of β-lactoglobulin to trypsin. Thus, the enhanced digestibility of β-lactoglobulin at elevated pressure is the result of a volume decrease during the association process and a volume decrease during the catalytic step. Pressure treatment of whey concentrates should be considered as an alternative processing technology for reduction of allergenicity of whey products.

A functional comparison of ovine and porcine trypsins

Trypsin was isolated from ovine and porcine pancreas using affinity chromatography on immobilized p-aminobenzamidine. Molecular masses of the two proteins were 23 900 and 23 435 Da, determined by matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) mass spectrometry. The purified trypsins were compared using the kinetic properties K m and k cat which were determined at pH 8.0 and between 25 and 55 °C. Comparison of the Michaelis constants for ovine and porcine trypsins toward N-α -benzoyl-arginine- p-nitroanilide (Ba pNA) indicated that ovine trypsin had higher affinity for this substrate than the porcine enzyme. The rates of the reactions catalysed by the two enzymes correlated strongly over the range of temperatures and substrate concentrations tested, as did the k cat values. The specific activity of ovine trypsin for Ba pNA was, on average, approximately 10% higher than that of the porcine enzyme over the range of conditions tested. Porcine trypsin was less susceptible to denaturation at low pH or high temperature than was ovine trypsin. Porcine and ovine trypsin produced seven identically sized fragments from auto-catalytic hydrolysis. Proposed regions of identity between ovine and porcine trypsins were I 54–K 77, L 98–R 107, S 134–K 178 and N 209–K 116. Hydrolysis of β-lactoglobulin, egg white lysozyme or casein by ovine or porcine trypsin yielded virtually identical patterns of fragments although the rate at which fragments were produced, in the case of β-lactoglobulin, differed between the two enzymes. On balance the two enzymes appear to be functionally identical in their action.

Effect of limited hydrolysis on the interfacial rheology and foaming properties of β-lactoglobulin A

Hydrolysis of β-lactoglobulin (β-Lg), genetic variant A, using a serine protease specific for glutamic and aspartic acid residues from Bacillus licheniformis (BLP), resulted in improved foam overrun and foam stability. Limited hydrolysis (19–26% hydrolysed β-Lg) led to a more rapid increase in the viscoelastic properties of air/water interfacial films and a concomitant increase in foam overrun compared with intact β-Lg, presumably due to increased exposure of hydrophobic areas. The increased exposure did not, however, cause formation of an interfacial layer with increased viscoelastic properties. More extended hydrolysis (86% hydrolysed β-Lg) resulted in a higher initial overrun than the unhydrolysed sample and the best foam stability. The interfacial elasticity and viscosity, though, was the lowest observed. Thus, high maximum values of these interfacial properties are not necessary prerequisites for formation of a voluminous and stable foam.

Identification of peptides in aggregates formed during hydrolysis of beta-lactoglobulin B with a Glu and Asp specific microbial protease.

The purpose of the present study was to identify the peptides responsible for aggregate formation during hydrolysis of beta-lactoglobulin by BLP at neutral pH. Hydrolysates taken at various stages of aggregate formation were separated into a precipitate and a soluble phase and each was analyzed by CE and mass spectrometry. The aggregates consisted of six to seven major peptides of which four were tentatively identified. The peptides were positively charged at neutral pH and had a high charge-to-mass ratio at low pH. The fragment f135-158 seemed to be the initiator of aggregation, since it was present at high concentration in the aggregates at all stages, and the concentration of this peptide remained low in the supernatant. F135-158 contains several basic and acid amino acids alternating with hydrophobic amino acids, which is in accordance with formation of noncovalently linked aggregates, as previously shown.

Beta-lactoglobulin hydrolysis. 2. Peptide identification, SH/SS exchange, and functional properties of hydrolysate fractions formed by the action of plasmin.

beta-Lactoglobulin (betaLg) was hydrolyzed by plasmin to a degree of hydrolysis of 4%. The hydrolysate was fractionated by ion-exchange chromatography and subsequent hydrophobic-interaction chromatography. The betaLg peptide fraction consisting of smaller peptides (mostly <2 kDa) had poor foam- and emulsion-forming and -stabilizing properties. Most of the betaLg peptides were identified (in either the nonreduced or reduced form) by mass spectrometry on the basis of the known primary structure of the intact protein and the specificity of the enzyme. The peptides formed during betaLg/plasmin-hydrolysis were (1) peptides lacking a cysteyl residue, (2) peptides composed of a single amino acid chain containing intramolecular disulfide bonds, and (3) peptides composed of two amino acid chains linked by an intermolecular disulfide bond. It appeared that significant SH/SS-exchange had taken place during hydrolysis. Many of the peptides present in the peptide fraction that exhibited good functional properties were disulfide-linked fragments.

Secondary structure changes and peptic hydrolysis of β-lactoglobulin induced by diols

Secondary structure changes and peptic hydrolysis of β-lactoglobulin induced by diols

Conformation of β-Lactoglobulin at an Oil/Water Interface as Determined from Proteolysis and Spectroscopic Methods

The rates of appearance of tryptic peptides following the hydrolysis of β-lactoglobulin in solution or adsorbed at the oil/water interface of an emulsion were investigated as a function of time. It was also shown using hydrophobic labeling that the region 15–40 of β-lactoglobulin was in the oil phase. The fluorescence results suggested that the conformation of β-lactoglobulin was modified upon adsorption at the oil/water interface and that at least one tryptophan in adsorbed β-lactoglobulin was in a more hydrophobic environment. The data obtained by circular dichroism in the peptidic region indicated that the adsorbed β-lactoglobulin was characterized by a higher content in α-helix than the protein in solution.

Effect of High-pressure Treatment on the Tryptic Hydrolysis of Bovine β-Lactoglobulin AB

Abstract The effects of high-pressure treatment on the tryptic hydrolysis of bovine β-lactoglobulin AB were studied. Isostatic pressure (100–800 MPa, 15 min) was applied to the protein substrate prior to its hydrolysis, as well as following its digestion at atmospheric pressure. Hydrolysis (100 min at 40°C) was also conducted under high pressure (100–400 MPa). To characterize the hydrolysates on a molecular level, peptide mapping by RP-HPLC was coupled on-line to detection by electrospray ionisation mass spectrometry. Under pressures up to 400 MPa, tryptic hydrolysis of β-lactoglobulin was increased with an optimum at 300 MPa. Analysis of the digests showed that the enhanced hydrolysis was due to accelerated breakdown of large intermediate hydrolysis products into final tryptic peptides. Among others, it was found that under these conditions the Tyr20–Ser21 peptide bond splitting was complete, resulting to entire conversion of Val15–Arg40 intermediate into Val15–Tyr20 and Ser21–Arg40 end-products. Increasing pressures applied during pre-pressurization (100–800 MPa) resulted in small but progressive reduction in residual intact β-lactoglobulin, but no change in the peptide profile of the hydrolysates obtained was observed. Similar to this, post-pressurization of the tryptic hydrolysate had no measurable effect on the peptide profiles.

Aggregate Formation during Hydrolysis of β-Lactoglobulin with a Glu and Asp Specific Protease from Bacillus licheniformis

The hydrolysis of isolated β-lactoglobulin (9 and 70−200 mg/mL) by a Bacillus licheniformis protease was followed to assess whether aggregates and gels, respectively, were formed during hydrolysis. Changes during hydrolysis were monitored by electrophoresis, dynamic light scattering, and fluorescence and circular dichroism spectroscopy. Gelation was monitored by dynamic oscillation rheology. Upon hydrolysis of a β-lactoglobulin preparation with the B. licheniformis protease aggregates were formed and a soft gel resulted from only 70 mg/mL of β-lactoglobulin. The aggregates consisted of a number of peptides with molecular weight ranging from 2000 to 6000 and pI from 5 to 8. As the aggregates were solubilized in either SDS or urea or at extreme pH values, it is proposed that noncovalent interactions, mainly electrostatic and hydrophobic, are major interacting forces. These kinds of aggregates are thought to be important in protease-induced gelation of whey protein isolate solutions. Keywords: β-Lactoglobulin; proteolysis; aggregation; fluorescence; circular dichroism

Hydrolysis of β-lactoglobulin by four different proteinases monitored by capillary electrophoresis and high performance liquid chromatography

Abstract The hydrolysis of β-lactoglobulin (β-lg) by four enzymes [porcine trypsin (PT), Fusarium oxysporum trypsin (FOT), Bacillus licheniformis proteinase (BLP) and Bacillus subtilis proteinase (Neutrase®)] was characterised by size exclusion—and reversed phase high performance liquid chromatography (SE-HPLC and RP-HPLC) and by capillary electrophoresis (CE). After 24 h of hydrolysis, all β-lg had been degraded by PT and BLP, but a large part of the protein was still intact after hydrolysis by FOT or Neutrase®. The main fraction of peptides was found to have MWs ranging from 1.0 to 3.0kDa. The hydrolysis catalysed by each enzyme resulted in different peptide profiles by RP-HPLC and CE. Hydrolysates produced by PT or FOT are resolved into 18 peaks, while BLP hydrolysates were resolved into 25 peaks, corresponding well to the numbers of possible cleavage sites in β-lg. Neutrase®, with a broad specificity, produced the largest number of peptides. Seven peptides from PT hydrolysis, eight from FOT hydrolysis and one from BLP hydrolysis were identified by mass spectrometry (MS) and Edman degradation.

HOW TO INCREASE ?-LACTOGLOBULIN SUSCEPTIBILITY TO PEPTIC HYDROLYSIS

Since β-lactoglobulin is resistant to peptic hydrolysis in physiological conditions, the increase of its digestibility by this enzyme was sought by the destabilization of its folding using methods that do not influence the biological value of protein, such as high pressure, medium polarity changes (alcohol addition), and esterification (ethylation). For example, the rate of hydrolysis of β-lactoglobulin by pepsin (negligible at 0.1 MPa) increased considerably with pressure up to 300 MPa. The susceptibility of all potential β-lactoglobulin proteolytic sites to peptic cleavage remained constant over the pressure range that was studied. The addition of alcohols decreases the bulk dielectric constant of the medium and, according to CD measurements, increases significantly the proportion of helical structure in β-lactoglobulin while increasing susceptibility to peptic hydrolysis. In the presence of alcohols (ethanol, ethylene glycol), β-lactoglobulin hydrolysis by pepsin was initiated when its secondary structure began to change and diversified peptic peptide populations were obtained. The chemical modification of β-lactoglobulin by mild esterification yields a 40%-ethylated β-lactoglobulin derivative that is rapidly hydrolyzed by pepsin. As compared with peptic hydrolysis of β-lactoglobulin in aqueous ethanol, 22 new sites of pepsin cleavage were induced by esterification of the protein.

Effect of high hydrostatic pressure on the enzymic hydrolysis of beta-lactoglobulin B by trypsin, thermolysin and pepsin.

Hydrolysis of beta-lactoglobulin B (beta-lg B) by pepsin, a process slow at ambient conditions, is facilitated at a moderately high hydrostatic pressure such as 300 MPa, corresponding to an apparent volume of activation delta V# = -63 ml mol-1 at pH 2.5, 30 degrees C and gamma/2 = 0.16. Digestion of beta-lg by trypsin and thermolysin is likewise enhanced by pressure, and the pressure effect has been traced to pressure denaturation of beta-lg B, which by high-pressure fluorescence spectroscopy has been shown to have a large negative volume of reaction, delta V(o) = -98 ml mol-1, at pH 6.7, 30 degrees C and gamma/2 = 0.16. Pressure denaturation is only slowly reversed following release of pressure and the enhanced digestibility is maintained at ambient pressure for several hours.

Enzymatic Hydrolysis of Milk Proteins Used for Emulsion Formation. 1. Kinetics of Protein Breakdown and Storage Stability of the Emulsions

Hydrolysis of β-lactoglobulin and sodium caseinate in solution or while adsorbed at oil-water interfaces in emulsions formed from unmodified proteins was carried out with trypsin. The hydrolysis was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and dynamic light scattering. The stability during storage of the emulsions containing hydrolyzed proteins was also investigated by small-angle laser light scattering. The order of the hydrolysis of the individual caseins at the oil surface was β > αs >>> κ compared with αs > β > κ in solution. The overall rate of hydrolysis of sodium caseinate was the same whether adsorbed to the interface or in solution. Conversely, the rate of hydrolysis of β-lactoglobulin was much higher on the surface of emulsion than in solution. Emulsions formed from hydrolyzed protein solutions were less stable during storage than those formed from unmodified proteins and subsequently hydrolyzed or those formed with unmodified protein solutions. Keywords: Emulsions; casein; β-lactoglobulin; trypsin; peptides; emulsion stability

Pancreatic elastases I and II in β-lactoglobulin hydrolysis: preliminary results

Pancreatic elastases I and II in β-lactoglobulin hydrolysis: preliminary results

Proteolysis of β-lactoglobulin and β-casein by pepsin in ethanolic media

Abstract Limited proteolysis of β-lactoglobulin and β-casein by pepsin was performed in the presence of varying concentrations of ethanol. β-Lactoglobulin started to be cleaved by pepsin only in ethanol concentrations greater than 20%, when its secondary structure began to change. In 25% ethanol, the rate of hydrolysis of β-lactoglobulin was slow (40% remained intact after 40 h of hydrolysis) and many short and hydrophilic peptides were observed. The rate of hydrolysis of β-lactoglobulin reached its maximum in 30 and 35% ethanol (80% of β-lactoglobulin was hydrolysed after 10 h), and a mixed population of hydrophilic and hydrophobic peptides of different lengths was observed. Large hydrophobic peptides appeared first, then some shorter products. The rate of hydrolysis of β-lactoglobulin decreased at ethanol concentrations equal to or higher than 40%, when only a few long, hydrophobic peptides were produced. As seen by circular dichroism, the addition of ethanol to β-casein induced α-helix formation and reduced the rate of casein hydrolysis without changing the peptide profile. The only exception was the yield of a single peptide (Pro81 − Met93).

Immobilized Derivatives of Leucine Aminopeptidase and Aminopeptidase M: APPLICATIONS IN PROTEIN CHEMISTRY

Abstract Leucine aminopeptidase and aminopeptidase M have been covalently bound to an arylamine derivative of porous glass. The bound forms of both enzymes retain 100% of their activities at saturating levels of substrate (leucine p-nitroanilide). For the hydrolysis at pH 7.3 and 25° κcat values for bound and free leucine aminopeptidase are 46 ± 5 s-1 and 46 ± 2 s-1, respectively; for aminopeptidase M (pH 7.5 and 25°) κcat values are 23 ± 2 s-1 and 21 ± 0.4 s-1, respectively. Although the Michaelis constants for both enzymes increase on binding, the pH and temperature dependencies of the bound enzymes remain unchanged. These data suggest that the environments and conformations of the enzymes are not significantly changed after coupling to the solid support. The apparent decrease in the binding of substrate could be explained by a decrease in the effective diffusion coefficient of the substrate. Both insoluble enzymes are active against polypeptide substrates. After treatment for removal of contaminating endopeptidases, the immobilized derivatives of leucine aminopeptidase and aminopeptidase M were used successfully in NH2-terminal sequence determination. The bound aminopeptidase M appears to be the better of the two for this purpose. Both bound enzymes will catalyze the hydrolysis of the aminoethylated A and B chains of insulin nearly to completion (≥87% recovery of free amino acids in all cases). These digests are carried out at pH values near neutrality in a volatile buffer with no activating metal. Immobilized pronase (Royer, G. P., and Green, G. M. (1971) Biochem. Biophys. Res. Commun. 44, 426) was used in concert with bound leucine aminopeptidase and bound aminopeptidase M for the hydrolysis of β-lactoglobulin. In both cases the recovery of free amino acids was 93%. These bound enzymes should be quite useful in amino acid composition determinations when acid-labile residues such as tryptophan, glutamine, asparagine, or certain affinity labeled side chains are present.

In vitro synthesis of α-lactalbumin and β-lactoglobulin by microsomes and bound polyribosomes from the mammary gland of lactating sheep

1. Synthesis of α-lactalbumin and β-lactoglobulin has been carried out in vitro by cell-free systems consisting of microsomes or bound polyribosomes derived from the mammary gland of the lactating ewe. 2. The two proteins labelled with 14C and synthesized in vitro have been identified by; (a) their behaviour on chromatography with CM-cellulose and Sephadex G-100 columns and on electrophoresis in polyacrylamide gel: (b) their antigenic properties: (c) their molecular weights, determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate; (d) the number of peptides produced by tryptic hydrolysis of β-[ 14C]lactoglobulin and their chromatographic and electrophoretic properties. 3. The distribution of the radioactivity between the peptides resulting from tryptic hydrolysis of β-lactoglobulin and the determination of the radioactivity of the N-terminal amino acid suggest that most of the radioactive amino acids incorporated in vitro into β-lactoglobulin correspond to the completion of peptide chains which began in vivo.

Étude de l'inhibition de la protéolyse trypsique de la β-lactoglobuline a par le saccharose et quelques autres sucres. Mise en évidence d'un changement de conformation de la β-lactoglobuline a par liaison avec le saccharose

We were able to show that the inhibition caused by sucrose on tryptic (EC 3.4.4.4) hydrolysis of β-lactoglobulin A is due to the fixation of sucrose on this protein and to the existence of strong stabilizing interactions. The kinetic analysis, based on the steady-state approximation assuming a two step scheme, shows that the total non-competitive inhibition occurs only with native protein substrate. Two molecules of sucrose are involved in the process, and the binding sites on the 1 2 molecule are not identical nor independent. As the presence of sucrose modifies only that part of the β-lactoglobulin molecule that is active (total inhibition), we may say that changes in reaction rate are in inverse proportion to the saturation of the substrate with the inhibitor. Therefore we plot ( υ − υ i) υ i in place of log Y (1− Y) , ( υ i , υ = reaction rate with or without inhibition, Y = “fractional saturation” of the substrate with the inhibitor, i.e. concentration of inhibitor bound per mole and per site of substrate). The n parameter of the so-called Hill plot, in terms of log (υ − υ i) υ i versus log [I] ( I = concentration of inhibitor), is found to be 2.7. Therefore there must be some inter-molecular interactions associated with strong intra-molecular stabilizing interactions. The β-lactoglobulin A dimer is known to dissociate between pH 6 and pH 9 into two monomers (molecular weight 17 500). The calculation for the dimer dissociation linked to the binding of two moles of sucrose per mole of protein gives the same 2.7 value for n, assuming a wide separation of the individual equilibrium curves of the dimer and the monomer with the inhibitor. Empirical and theoretical results are thus in good agreement. These kinetic results are confirmed by a thermodynamic study. The increase of the activation free energy for the 50%-inhibited enzymatic reaction is calculated to be 400 cal, whereas the experimentally determined minimal value of the total free energy of interaction, realised in completely saturating the macromolecule by the ligand, is 760 cal. The thermodynamic study indicates, on the other hand, that the forces involved are of the hydrophobic type and accompanied by a negative variation of entropy. This suggests the occurrence of a conformational change which is all the more plausible since interactions in a macromolecule often go with changes in the tertiary and quaternary structure. Several recent studies show further that changes in conformation of proteins results from reversible combination with non-polar substances. The conformational change of β-lactoglobulin A induced by sucrose is made apparent, in the absence of any enzymatic reaction, by titration and difference spectroscopy curves. It is closely related to the inhibition of the proteolysis of β-lactoglobulin A, since the value of the sucrose concentration which gives the half transformation is the same (0.7 M) as for the 50% inhibition. The observed phenomenon is not due to dissociation of the macromolecule alone and corresponds to a more compact structure of the protein. It is noteworthy that sucrose, which is so largely employed in studies on the native structure of proteins, is not inert and causes a transconformation of β-lactoglobulin A.

Proteinase production by a species of Cephalosporium.

An unidentified Cephalosporium species produced an extracellular proteinase when grown in a variety of fermentation media under submerged culture conditions. Maximal enzyme yields were obtained in a medium containing 2% corn meal, 1% soybean meal, and 0.5% CaCO(3) in tap water. Optimal proteinase production in this medium occurred within a 72- to 96-hr growth period. High enzyme yields were also attained with media in which cottonseed meal, Fermatein, Pharmamedia, or soybean-alpha-protein was substituted for the soybean meal. The substitution of these ingredients for the corn meal resulted in significantly decreased proteinase yields. The addition of minerals or vitamins to the corn meal-soybean meal fermentation medium failed to enhance proteinase production. The enzyme was most active in an alkaline environment; maximal caseinolysis occurred at pH 7.5, whereas pH 8.5 was optimal for either hemoglobin or beta-lactoglobulin hydrolysis. Enzymatic activity was also noted with either bovine albumin fraction V or soybean-alpha-protein substrates, whereas ovalbumin was not susceptible to enzymatic attack. The enzyme was stable within the pH range of 3.0 to 9.5 at 25 C for 2 hr, and at 5 C for 24 hr. The proteinase was stable upon heating for 10 min at 35 to 45 C, but it was totally inactivated at 70 C. The proteinase was unaffected by soybean inhibitor, partially inactivated by lima bean inhibitor, and completely inactivated by ovomucoid inhibitor.

Contribution à l'étude cinétique de la protéolyse de la β-lactoglobuline par la trypsine

The hydrolysis of β-lactoglobulin by trypsin and chymotrypsin is followed to about 90% degradation. It is demonstrated that a simple Henri-Michaelis mechanism ( i. e. E + S ⇄ ES → E + P) cannot explain the experimental results. Another mechanism is suggested and discussed.