Patent Application
20230192747
Conjugation of reducing sugars and polysaccharides with proteins to improve their functional properties has been extensively studied. These studies often employ a Maillard reaction, i.e., Schiff base formation between the reducing end of the carbohydrate and the amino groups in proteins, for conjugation. These methods typically involve heating a homogeneously mixed dry powder at 60-80° C. and at 70-80% relative humidity for a few hours to a few days to catalyze the carbonyl-amine reaction. One of the shortcomings of the dry heating method is that the reaction cannot be controlled. It is not possible to limit the reaction to the initial Schiff base formation. The reaction progresses further and produces undesirable advanced Maillard reaction products, which adversely affect the color and flavor of the product. See:
Akhtar, M.; Dickinson, E. Emulsifying properties of whey protein-dextran conjugates at low pH and different salt concentrations. Colloids Surf B: Biointerfaces 2003, 31, 125-132;
Akhtar M.; Dickinson E. Whey protein-maltodextrin conjugates as emulsifying agents: An alternative to gum arabic. Food Hydrocolloids 2007, 21, 607-616;
Aoki, T.; Kitahata, K.; Fukumoto, T.; Sugiomoto, Y.; Ibrahim, H. R.; Kimura, T.; Kato, Y.; T.Matsuda. Improvement of functional properties of β-lactoglobulin by conjugation with glucose-6-phosphate through the Maillard reaction. Food Res. International 1997, 30, 401-406;
Dickinson, E.; Semenova, M. G. Emulsifying properties of covalent protein-dextran hybrids. Colloids Surf. 1992, 64, 299-310;
Diftis D.; Kiosseoglou V. Improvement of emulsifying properties of soybean proteinisolate by conjugation with carboxymethyl cellulose. Food Chemistry 2003, 81, 1-6;
Dunlap, C. A.; Côté, G. L. β-lactoglobulin-dextran conjugates: effect of polysaccharide size on emulsion stability. J. Agric. Food Chem. 2005, 53, 419-423;
Kato A.; Murata K.; Kobayashi K. Preparation and characterization of ovalbumindextranconjugate having excellent emulsifying properties. J. Agric.Food Chem. 1988, 36, 421-425;
Kato A.; Sato T.; Kobayashi K. Emulsifying properties of protein-polysaccharide complexes and hybrids. Agric. Biol. Chem. 1989, 53, 2147-2152;
Kato, A. Industrial applications of Maillard-type protein-polysaccharide conjugates. Food Sci. Technol. Res. 2002, 8, 193-199; and
Neirynck N.; Van der Meeren P.; Gorbe S. B.; Dierckx S.; Dewettinck K. Improved emulsion stabilizing properties of whey protein isolate by conjugation with pectins. Food Hydrocolloids 2004, 18, 949-957.
It has been reported recently that whey protein (WPI)-polysaccharide conjugates could be produced in aqueous solutions under “molecular crowding” conditions by incubating a mixture of concentrated WPI and polysaccharide solution at 60° C., at pH 6.5, for 48 h. Zhu, D.; Damodaran, S.; Lucey, J. A. Formation of whey protein isolate (WPI)-dextran conjugate in aqueous solutions. J. Agric.Food Chem. 2008, 56, 7113-7118. The purified WPI-dextran conjugate so produced was white in color and exhibited better heat stability in the pH range 3 to 7.5, and better emulsifying properties than unmodified WPI. (Zhu, D.; Damodaran, S.; Lucey, J. A. Physicochemical and emulsifying properties of whey protein isolate (WPI)-dextran conjugates produced in aqueous solution. J. Agric.Food Chem. 2010, 58, 2988-2994.) However, a major shortcoming of the above method is that the yield of the protein-polysaccharide conjugate was less than 10% even under molecular crowing conditions. The low yield might be due to the reversible nature of the Schiff base formation in aqueous solutions. Thus, an alternate strategy is needed to produce protein-polysaccharide conjugate to improve the functional properties of underutilized food proteins.
Disclosed herein is a compound (a conjugate) comprising a protein moiety covalently bonded to a polysaccharide moiety via a phosphate bridge. The preferred manner of making the conjugate is by reacting a mixture of a protein with a polysaccharide in the presence of POCl3, for a time, a temperature, and a pH wherein the conjugate is formed. By way of example and not limitation, a conjugate as disclosed herein can be made by reaction whey protein isolate and dextran with POCl3 at pH 10-10.5. This results in the formation of protein-phosphate-polysaccharide (PPP) conjugates cross-linked via a phosphate bridge. Molecules of this genus are referred to herein as “PPP conjugates.” The extent of conjugation increased with the weight ratio of protein to POCl3 used in the reaction. This was confirmed by staining SDS-PAGE gels with Coomassie Blue G-250 for proteins and PAS reagent for carbohydrates. 31P NMR of the PPP conjugates also confirmed the presence of —Npro—PO2−—ODex cross-links in the PPP conjugate. Quantitative analysis of the 31P NMR revealed that about 32% of the phosphorylated lysine residues in the PPP conjugate were involved in the —Npro—PO2−—ODex bond. Fluorescence and CD spectroscopic analysis showed that while the conjugation caused changes in the tertiary structure, it did not significantly affect the secondary structure contents of the protein portion of the conjugate. Incubation of the PPP conjugate at pH 1.5 at 40° C. caused time-dependent hydrolysis of all N-phosphate bonds, including the —Npro—PO2−—ODex cross-links, and release of free amine groups. This indicated that lysine residues cross-linked to the polysaccharide in the PPP conjugate will be biologically available during transit through the gastro-intestinal tract.
It is shown herein that proteins contain hydroxyl groups (serine and threonine residues), ε-amino groups (in lysine residues), and himidazole group (on histidine residues) that can react with POCl3. Polysaccharides contain several hydroxyl groups that can also react with POCl3. In dilute solutions, reaction of POCl3 with polysaccharides and proteins generally produces N- and O-phosphorylated proteins, and O-phosphorylated polysaccharides. (Zhu, D.; Damodaran, S. Chemical Phosphorylation Improves the Moisture Resistance of Soy Flour-Based Wood Adhesive. J. Appl. Polym. Sci. 2014, 131, 40451.) However, in a reaction mixture of protein and polysaccharide, these reactions can be shifted to produce protein-phosphate-polysaccharide (PPP) conjugate, in which POCl3 acts as a bi-valent cross-linking reagent. This is shown schematically in
Thus, disclosed herein is a compound comprising a protein moiety covalently linked to a polysaccharide moiety via a phosphate bridge. The protein moiety may comprise a whey protein. The polysaccharide moiety may comprise glucose residues. The phosphate bridge may be bound to an oxygen atom on the protein moiety, a nitrogen atom on the protein moiety, or both. The polysaccharide moiety may comprise dextran. In all versions of the compound, the protein moiety, the polysaccharide moiety, or both, may be pharmacologically active or nutritionally significant.
Also disclosed herein is a method of making a conjugate having a protein moiety and a saccharide moiety linked by a phosphate bridge, the method comprising contacting, in a solvent, a protein, a polysaccharide, and POCl3 for a time, at a temperature, and at a pH wherein a reaction occurs that yields a protein-phosphate bridge-polysaccharide (“PPP”) conjugate. The solvent may be water. The reaction generally takes place at neutral to alkaline pH, from about pH 8 to about pH 14, or from about pH 8 to about pH 12, or from about pH 9 to about pH 11, or from about pH 10 to about pH 11.
It is generally preferred that in all versions of the method, the protein is present in a concentration of from about 5% (w/v) to about 50% (w/v) with respect to the solvent, and the polysaccharide is present in a concentration of from about 5% (w/v) to about 50% (w/v) with respect to the solvent. However, concentrations above and below these stated ranges are explicitly within the scope of the disclosed method. It is also generally preferred that the POCl3 is present at a protein-to-POCl3 ratio (w/w) of from about 0.5 to about 5. Again, this is a preferred range; concentrations above or below this ratio are explicitly within the scope of this disclosure.
The reaction is preferably conducted at a temperature of from about 10° C. to about 30° C. Temperatures above or below this range is also within the scope of the method.
The conjugates disclosed herein are useful in a number of applications. For example, they can be used as food-grade emulsifying agents. Emulsifying agents are extensively used in commercial food products as foaming agents, texture modifiers, etc. They are also useful to improve the solubility and stability of proteins in aqueous solutions—e.g., in protein drinks.
Specifically disclosed and claimed herein are:
1. A compound comprising a protein moiety covalently linked to a polysaccharide moiety via a phosphate bridge.
2. The compound of claim 1, wherein the polysaccharide moiety comprises a saccharide residue selected from the group consisting of ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose, talose, ribulose, xylulose, psicose, fructose, sorbose and tagatose.
3. The compound of claim 1, wherein the phosphate bridge is bound to an oxygen atom on the protein moiety.
4. The compound of claim 1, wherein the phosphate bridge is bound to a nitrogen atom on the protein moiety.
5. The compound of claim 1, wherein the polysaccharide moiety is dextran.
6. The compound of claim 1, wherein the protein moiety comprises a whey protein.
7. The compound of claim 6, wherein the polysaccharide moiety comprises a saccharide residue selected from the group consisting of ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose, talose, ribulose, xylulose, psicose, fructose, sorbose and tagatose.
8. The compound of claim 6, wherein the phosphate bridge is bound to an oxygen atom on the protein moiety.
9. The compound of claim 6, wherein the phosphate bridge is bound to a nitrogen atom on the protein moiety.
10. The compound of claim 6, wherein the polysaccharide moiety is dextran.
11. A method of making a conjugate having a protein moiety and a saccharide moiety linked by a phosphate bridge, the method comprising contacting, in a solvent, a protein, a polysaccharide, and POCl3 for a time, at a temperature, and at a pH wherein a reaction occurs that yields a protein-phosphate bridge-polysaccharide (“PPP”) conjugate.
12. The method of claim 11, wherein the solvent is water.
13. The method of claim 11, wherein the pH is selected from a range of from about pH 8 to about pH 14, or from about pH 8 to about pH 12, or from about pH 9 to about pH 11, or from about pH 10 to about pH 11.
14. The method of claim 11, wherein the protein is present in a concentration of from about 5% (w/v) to about 50% (w/v) with respect to the solvent, and the polysaccharide is present in a concentration of from about 5% (w/v) to about 50% (w/v) with respect to the solvent.
15. The method of claim 11, wherein POCl3 is present at a protein-to-POCl3 ratio (w/w) of from about 0.5 to about 5.
16. The method of claim 11, wherein the temperature is from about 10° C. to about 30° C.
Numerical ranges as used herein are intended to include every number and subset of numbers contained within that range, whether specifically disclosed or not. Further, these numerical ranges should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 2 to 8, from 3 to 7, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.
All references to singular characteristics or limitations as used herein shall include the corresponding plural characteristic or limitation, and vice-versa, unless otherwise specified or clearly implied to the contrary by the context in which the reference is made. That is, unless specifically stated to the contrary, “a” and “an” mean “one or more.” The phrase “one or more” is readily understood by one of skill in the art, particularly when read in context of its usage. For example, “one or more” substituents on a phenyl ring designates one to five substituents.
All combinations of method or process steps as used herein can be performed in any order, unless otherwise specified or clearly implied to the contrary by the context in which the referenced combination is made.
The methods of this disclosure can comprise, consist of, or consist essentially of the essential elements and limitations of the method described herein, as well as any additional or optional ingredients, components, or limitations described herein or otherwise useful in synthetic organic chemistry.
The term “about” is used herein generally to mean a value±5% of a given numerical value. Thus, “about 60%” refers to a value of 60±5% of 60 (i.e., between 57 and 63).
The term “contacting” refers to the act of touching, making contact, or of bringing to immediate or close proximity, including at the molecular level, for example, to bring about a chemical reaction, or a physical change, e.g., in a solution or in a reaction mixture.
An “effective amount” refers to an amount of a chemical or reagent effective to facilitate a chemical reaction between two or more reaction components, and/or to bring about a recited effect. Thus, an “effective amount” generally means an amount that provides the desired effect.
As used herein, the terms “phosphodiester bond” and “phosphate bridge” are synonymous and refer to a divalent linkage having the structure R—P(O2)−—R′, wherein R and R′ can be the same or different.
The terms “saccharide” and “monosaccharide” refer to compounds of the molecular formula Cn,H2nOn, wherein “n” is an integer of from 3 to 7, and all isomers thereof, without limitation. Saccharides include aldoses and ketoses. A non-limiting list of examples includes ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose, talose, ribulose, xylulose, psicose, fructose, sorbose and tagatose. The term “polysaccharide refers to an oligomer or polymer of saccharide residues, and all isomers, anomers, and epimers thereof.
The term “solvent” refers to any liquid that can dissolve a compound to form a solution. Solvents include water and various organic solvents, such as hydrocarbon solvents, for example, alkanes and aryl solvents, as well as halo-alkane solvents. Examples include hexanes, benzene, toluene, xylenes, chloroform, methylene chloride, dichloroethane, and alcoholic solvents such as methanol, ethanol, propanol, isopropanol, and linear or branched (sec or tert) butanol, and the like. Aprotic solvents that can be used in the method include, but are not limited to perfluorohexane, α,α,α-trifluorotoluene, pentane, hexane, cyclohexane, methylcyclohexane, decalin, dioxane, carbon tetrachloride, freon-11, benzene, toluene, triethyl amine, carbon disulfide, diisopropyl ether, diethyl ether, t-butyl methyl ether (MTBE), chloroform, ethyl acetate, 1,2-dimethoxyethane (glyme), 2-methoxyethyl ether (diglyme), tetrahydrofuran (THF), methylene chloride, pyridine, 2-butanone (MEK), acetone, hexamethylphosphoramide, N-methylpyrrolidinone (NMP), nitromethane, dimethylformamide (DMF), acetonitrile, sulfolane, dimethyl sulfoxide (DMSO), propylene carbonate, and the like.
CD=circular dichroism. NMR=nuclear magnetic resonance. PAS=periodic acid (H5IO6/HIO4)-Schiff reagent (or stain). PPP conjugates=a molecule comprising a protein moiety covalently linked to a polysaccharide moiety via a phosphate bridge. SDS PAGE=sodium dodecyl sulfate polyacrylamide gel electrophoresis. TNBS=trinitrobenzenesulfonic acid. WPI=whey protein isolate.
Materials: Whey protein isolate (WPI) samples were provided by Agropur Ingredients (Minneapolis, Minn., USA). The sample contained 97.9% protein and 4.7% moisture. The various molecular weight dextrans (6,000, and 10,000 Da) from Leuconostoc spp, were purchased from Alfa Aesar (Tewksbury, Mass., USA). 2,4,6-Trinitrobenzenesulfonic acid (TNBS), Pierce glycoprotein staining kit for carbohydrate staining, and pre-stained molecular weight markers (EZ-run™) were purchased from Thermo Fisher Scientific (Waltham, Mass., USA). POCl3 (CAS No. 10025-87-3) was purchased from Millipore Sigma (Burlington, Mass. USA).
Synthesis of WPI-Dextran Conjugates: Molecular crowding conditions were created by dissolving 10% (w/v) WPI and 20% (w/v) dextran in water. The solution was stirred on a magnetic stirrer at room temperature until the solution was homogeneous. The pH of the solution was adjusted to 10.5. The protein and the polysaccharide were cross-linked via a phosphate bridge by reacting with POCl3. A calculated amount of POCl3 was added in small (μL) aliquots to the protein-polysaccharide mixture solution over a period of up to one hour with vigorous stirring. The pH of the solution was continuously maintained between about 10 and about 10.5 during the reaction by adding small amounts of concentrated base (10 M NaOH). The extent of conjugation was varied by varying the protein-to-POCl3 ratio (w/w) from 0 to 3. Once the reaction was completed, the solution was stirred for an additional 15 min at about pH 10 to about pH 10.5 and then the pH was adjusted to pH 7. Because the final volumes of the reacted solutions were different at different protein:POCl3 ratios (1:0.5, 1:1, 1:2, and 1:3), all the solutions were made up to a same final volume to avoid variations in protein and dextran concentrations. Small aliquots (2 mL) of these samples were withdrawn and stored frozen for subsequent chemical and electrophoretic analyses. The rest of the samples were dialyzed using either 6-8 kDa molecular weight cut-off membrane for 72 h at 4° C. to remove salts (NaCl and Na3PO4) formed during the reaction. The samples were then lyophilized and stored at 4° C. for future use.
Phosphorylated WPI and dextran controls also were prepared in a similar manner. Briefly, 10% (w/v) WPI and 20% (w/v) dextran solutions were phosphorylated separately using same amounts of POCl3 as were used in the above WPI-polysaccharide mixture reactions. After the reaction, the volumes of these phosphorylated control solutions were also made up to a final volume as the above samples; small (2 mL) aliquots these samples were withdrawn and stored frozen for subsequent chemical and electrophoretic analyses and the rest of the solutions were dialyzed as above and freeze dried and stored at 4° C. for future use.
Determination of Protein and Dextran Contents: Protein estimation using the Biuret method was not possible with protein-dextran samples as the dextran precipitated upon the addition of the Biuret reagent. Therefore, the protein content of the samples was determined using the 205/280 nm absorbance method using the following equation:
where A205 is the absorbance at 205 nm and A280 is the absorbance at 280 nm. See Scopes, R. K. Measurement of protein by spectrophotometry at 205 nm, Analytical Biochemistry 1974, 59, 277-252 and Whitaker, J. R. and Granum, P. E. An Absolute method for protein determination based on difference in absorbance at 235 and 280 nm. Analytical Biochemistry 1980, 109, 156-159. Because dextran absorbs at 205 nm (but not at 280 nm), the final protein content was determined by quantitatively subtracting the A205 contribution from dextran. The phenol-sulfuric acid method (Dubois et al., 1956) was used to determine the concentration of dextran in samples. Dubois, M. et al. Colorimetric Method for determination of sugars and related substances. Analytical Chemistry 1956, 28, 350-356.
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE): Gel electrophoresis was done on a “Mini-Protean”®-brand device (Biorad Laboratories, Mass., USA) using a 4% stacking and 12% resolving gel under reducing conditions according to the method of Laemmli (1970). Two gels were run simultaneously at 200 mV for 55 minutes. After electrophoresis, one of the gels was stained with Coomassie blue G-250 and the other was stained with PAS glycoprotein staining kit. See Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227, 680-685.
Lysine Estimation: Initial experiments to determine the lysine content of protein-dextran conjugates using the method of Hall et al (1956) was unsuccessful due to caramelization of dextran during acid hydrolysis at 100° C. Therefore, the method of Habeeb (1966), with modifications, was used to determine the lysine content of protein-dextran conjugates. Briefly, to 1 mL of sample solution containing 200 μg protein was added 1 mL of 1M NaHCO3 (pH 8.5) and 1 mL of 0.1% TNBS. The modification involved use of 1M NaHCO3, as opposed to 4% NaHCO3 originally proposed (Habeeb (1966)), to ensure that the pH was maintained at 8.5 throughout the reaction. The contents were incubated at 40° C. for 2 h. The reaction mixture was then mixed with 3.5 mL of concentrated HCl and the absorbance was measured at 415 nm. The lysine estimation was done in triplicates. See Hall, R. J. et al. Observations on the Use of 2,4,6-trinitrobenzenesulphonic acid for the determination of available lysine in animal protein concentrates. Analyst, 1973, 98, 673-686 and Habeeb, A. F. S. A. Determination of free amino groups in proteins by trinitrobenzenesulfonic acid. Analytical Biochemistry 1966, 14, 328-336.
Dephosphorylation: The time course of dephosphorylation of the protein-dextran conjugates under acidic condition was studied as follows: The pH of protein-dextran conjugate solution (20 mL) containing 0.2 mg/mL protein concentration was adjusted to 1.5 and incubated at 40° C. Aliquots (1 mL) were withdrawn at 15 min intervals for the first 2 h and at 30 min intervals for the next 2 h and were subjected to lysine determination, as described above. A control consisting of unmodified WPI at the same protein concentration as in the protein-dextran conjugate sample was treated in the same manner and its lysine content as a function of incubation time at pH 1.5 and 40° C. was determined. These measurements were done in duplicates.
Fluorescence Measurements: Fluorescence spectroscopic measurements were done using Perkin Elmer LS-5B luminescence spectrometer (Perkin Elmer, Billerica, Mass., USA) to study conformational changes in the protein upon conjugation with polysaccharide.
Zeta Potential Measurements: Zeta potential of protein-polysaccharide conjugate were determined at various pHs using a 90 Plus-brand particle analyzer (Brookhaven Instruments Corp., N.Y., USA).
pH-Turbidity Profile: Solutions containing 0.8 mg/mL protein-polysaccharide conjugate were heat denatured by incubating in a boiling water bath for 10 min and then cooled to room temperature by immersing in a cold-water bath. Aliquots (2 mL) of the solution were then adjusted to various pHs and were allowed to stand at room temperature for 15 minutes, after which the turbidity of the solution was measured at 600 nm in a UV-visible spectrophotometer (Shimadzu UV-P1601 PC, Shimadzu Corp., Kyoto, Japan). A control solution containing a physical mixture of WPI and polysaccharide at same concentrations as found in the protein-polysaccharide conjugates was subjected to same treatment as the samples and its pH-turbidity profile was determined at 600 nm. These measurements were made in triplicates.
31P NMR spectroscopy: Proton-decoupled 31P NMR was acquired on a Bruker Avance III 600 NMR spectrometer (240 MHz for 31P) equipped with a 5 mm cryogenic probe at the National NMR Facility at the University of Wisconsin-Madison, Madison, Wis., USA. The spectra were acquired with 32,768 scans over a period of 24 h at 298 K. The Bruker pulse sequence of zgpg30 was used. Phosphoric acid was used as the internal standard.
Circular Dichroism (CD) Spectroscopy: The secondary structure of the protein was studied using a Biorad Proteon XPR36 model circular dichroism spectrometer (Biorad, Mass., USA). Measurements were done at 1 mm path length with a 0.2% protein concentration. The raw data (in mdeg) were converted to the mean residual ellipticity (deg.cm2,dmol−1) using the following equation (Damodaran, 1989):
[θ]=100dM/C
where d is rotation in degrees/cm path length, M is the average molecular weight of amino acid residues in the protein (=115 Da), and C is protein concentration in mg/mL. (Damodaran, S. Influence of protein conformation on its adaptability under chaotropic conditions. Int. J. Biol. Macromol, 1989, 11, 2-8.) Secondary structure estimates from CD spectra were made using the CDESTIMA program. (Chang, C. T.; Wu, C. S. C.; Yang, J. T. Circular dichroic analysis of protein conformation: inclusion of the beta-turns. Anal. Biochem. 1978, 91, 13-31.)
Extent of Conjugation/Phosphorylation: WPI and dextran contain several reactive groups that can react with POCl3 at pH 10.0 to 10.5. These include the amine groups of lysine and histidine, the hydroxyl groups of serine and threonine residues in WPI, and the hydroxyl groups in dextran (located at positions 2, 3, and 4 of the glucose monomers). Thus, the phosphorylation/cross-linking reaction between protein and polysaccharide can occur between any set of these reactive groups.
The extent of phosphorylation/conjugation in WPI (10%)+dextran (20%) mixtures as a function of the weight ratio of POCl3 to WPI used in the reaction is shown in
SDS-PAGE of WPI-Dextran Conjugates: The formation of WPI-dextran conjugates through a phosphate bridge (a type of PPP conjugate) in the above samples was analyzed by SDS-PAGE under reducing conditions. See
31P NMR: To obtain further corroborative evidence that protein—PO4−—polysaccharide cross-links are formed during the reaction with POCl3, the proton-decoupled 31P NMR spectra of the PPP conjugate were studied. Phosphorylated WPI and phosphorylated dextran were used as controls to identify the chemical shift attributable to protein—PO4−—polysaccharide cross-links.
The 31P NMR of phosphorylated dextran (6 kDa) showed chemical shifts at four different regions, namely, at 18.2493 to 14.9510 ppm, 5.8847 to 2.1904 ppm, -7.6650 ppm, and −21.6512 (
The 31P NMR of phosphorylated WPI showed four distinct chemical shift regions, at 2.48, −0.5886, −6.2587 to −7.0903, and −20.6378 to −21.645 ppm (
The 31P NMR of the PPP conjugate showed peaks corresponding to those present in phosphorylated dextran and phosphorylated WPI, and three additional peaks arising from PPP conjugation (
31P NMR peak assignments for phosphorylated WPI, phosphorylated
De-Phosphorylation: Lysine is an essential amino acid. Both phosphorylation and conjugation of lysine residues in a protein to dextran might affect its biological availability unless it is dephosphorylated and the PPP conjugate is cleaved during transit through the gastro-intestinal tract. N-phosphate linkages are known to be prone to acid hydrolysis. Because the acidity in the human stomach ranges from roughly pH 1.5 to 4.0, it is possible that the —Npro—PO32− and —Npro—PO2−—ODex— linkages in the PPP conjugate might be cleaved during transit through the stomach.
The time course of release of free amine groups in the PPP (10 kDa) conjugate during incubation at pH 1.5 and 40° C. is shown in
To identify which phosphate bonds in the PPP conjugate were broken during acid hydrolysis at pH 1.5, the 31P NMR of dephosphorylated PPP conjugate (400 min sample) was investigated. See
The minor peak at 0.95 ppm in the dephosphorylated PPP conjugate might belong to either a minor fraction of the —Npro—PO32− specie resistant to hydrolysis, presumably because of restricted accessibility, or it could be —O—PO32− of serine residues.
Conformational Changes in WPI-Dextran Conjugates: The fluorescence spectra of PPP conjugates of 6 kDa dextran at various extent of conjugation/phosphorylation is shown in
The changes in the secondary structure of the protein were studied using CD spectroscopy. The CD spectra of native WPI, phosphorylated WPI, and PPP conjugate are shown in
aThe WPI:POCl3 ratio used in the preparation of the conjugate was 1:1.
pH Solubility Profile of WPI-Dextran Conjugates: The pH solubility profile was studied for the native WPI, phosphorylated WPI, WPI-dextran conjugates (6 kDa, 10 kDa, and 20 kDa) between pH 2.5 and 7.5. Compared to the phosphorylated WPI and the WPI-dextran conjugates, the native WPI had a higher turbidity. i.e., decreased solubility at 600 nm. See
The visual appearances of the turbidity of control WPI and WPI-dextran conjugates at pH 4.56 before and after centrifugation at 11,000 g for 2 min are shown in
Reacting a mixture of WPI and dextran with POCl3 at pH 10 -10.5 under “molecular crowding” conditions resulted in the formation of protein-polysaccharide conjugates cross-linked via a phosphate bridge. The extent of conjugation increased with the weight ratio of protein to POCl3 used in the reaction. This was confirmed by SDS-PAGE gels with Coomassie Blue G-250 staining for proteins and PAS reagent for staining carbohydrates. 31P NMR of the PPP conjugate also confirmed the presence of —Npro—PO2−—ODex cross-link in the PPP conjugate. Quantitative analysis of the 31P NMR revealed that about 32% of the phosphorylated lysine residues in the PPP conjugate were involved in the —Npro—PO2−—ODex bond. Incubation of PPP conjugate at pH 1.5 at 40° C. caused hydrolysis of all N-phosphates, including the —Npro—PO2−—ODex cross-link, and release of the amine groups in a time-dependent manner. This indicated that lysine residues cross-linked to the polysaccharide in the PPP conjugate could become biologically available during transit through the gastro-intestinal tract.
Zeta potential as a function of pH for various WPI-dextran (6 kDa) conjugates at various extents of phosphorylation were determined. The results are shown in
It should be noted that di-starch phosphate, which is widely used as modified starch in various food products (e.g., bread and bakery products, breakfast cereals, pastas, and snacks), is phosphate cross-linked amylose. In a recent ‘scientific opinion’ report, the European Food Safety Authority (2010) has declared that phosphated di-starch phosphate is safe for human consumption. This likely is also true of protein-phosphate-polysaccharide (PPP) conjugate as well. Furthermore, post-translational phosphorylation of basic amino acid residues (lysine, histidine, and arginine) in proteins is very common in many biologically important proteins (Ciesla, 2010). Thus, the PPP conjugate disclosed herein is likely biologically safe for human consumption.
This invention was made with government support under 2018-67017-27561 awarded by the USDA/NIFA. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/045515 | 8/11/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63064489 | Aug 2020 | US |
