Improvement of foaming property of egg white protein by phosphorylation through dry-heating in the presence of pyrophosphate.

Egg white protein (EWP) was phosphorylated by dry-heating in the presence of pyrophosphate at pH 4 and 85 degrees C for 1 d, and the foaming properties of phosphorylated EWP (PP-EWP) were investigated. The phosphorus content of EWP increased to 0.71% as a result of phosphorylation. To estimate the foaming properties of EWP, the foams were prepared by 2 methods: bubbling of the 0.1% (w/v) protein solution and whipping of the 10% (w/w) protein solution with an electric mixer. The foaming power, which was defined as an initial conductivity of foam from 0.1% (w/v) protein solution, was a little higher in PP-EWP than in native EWP (N-EWP), and the foaming stability of PP-EWP was much higher than that of dry-heated EWP (DH-EWP) and N-EWP. The microscopic observation of foams from the 10% (w/w) solution showed that the foams of PP-EWP were finer and more uniform than those of N- and DH-EWP. Although there were no significant differences in the specific gravity and overrun of the foams between PP- and DH-EWP (P < 0.05), the specific gravity and overrun of the foams from PP-EWP were smaller and higher, respectively, than that of the foams from N-EWP. The drainage volume was smaller in the foams from PP-EWP than in those from N- and DH-EWP. These results demonstrated that phosphorylation of EWP by dry-heating in the presence of pyrophosphate improved the foaming properties, and that it was more effective for the foam stability than for the foam formation.

Improvement of functional properties of bovine serum albumin through phosphorylation by dry-heating in the presence of pyrophosphate.

Bovine serum albumin (BSA) was phosphorylated by 2 methods. One is dry-heating in the presence of pyrophosphate, and the other is conjugation with maltopentaose through the Maillard reaction and subsequent dry-heating in the presence of pyrophosphate. The phosphorus content of BSA was increased to approximately 0.45% by dry-heating at pH 4.0 and 85 degrees C for 5 d in the presence of pyrophosphate, and approximately 0.91% by glycation and subsequent phosphorylation. The circular dichroism spectra showed that the change of secondary structure in the BSA molecule by phosphorylation was mild. However, tryptophan fluorescence intensity of BSA decreased by phosphorylation. The differential scanning calorimetry thermograms of BSA showed a disappearing of the 1st peak and a lowering of the 2nd peak denaturation temperature by phosphorylation. These results indicated molten (partially unfolded) conformations of BSA formed by both phosphorylation methods. The functional properties of BSA such as heat stability and calcium phosphate solubilizing ability were improved by phosphorylation alone and further by phosphorylation after glycation. Transparent gels of BSA with relatively high water-holding capacity were obtained by phosphorylation alone, and the immunogenicity of BSA was reduced significantly by glycation and phosphorylation, respectively.

Genetic evidence that antibacterial activity of lysozyme is independent of its catalytic function.

A catalytically inactive mutant of hen egg white lysozyme was constructed by site-directed mutagenesis to elucidate the role of enzymatic activity on its antimicrobial activity against Gram-positive bacteria. The catalytic residue aspartic acid at position 52 of lysozyme was substituted with serine (D52S-Lz) and the mutant cDNA was inserted into a yeast expression vector, pYES-2. Western blot analysis indicated that the mutation did not affect secretion of the D52S-Lz lysozyme into the medium of the expressing Saccharomyces cerevisiae, INVSC1. In addition, circular dichroism and fluorescence spectral analysis revealed no change in the structure of D52S-Lz compared to that of wild-type (Wt-Lz) lysozyme. The mutation (D52S) abolished the catalytic activity of lysozyme. Antimicrobial tests against Staphylococcus aureus and Bacillus subtilis revealed that the catalytically inactive D52S-Lz was as bactericidal as the Wt-Lz lysozyme. Heat treatment leading to enzyme inactivation had no effect on the bactericidal activity of either wild-type or the mutant D52S-Lz lysozyme. The binding affinity of D52S-Lz to the isolated peptidoglycan of S. aureus was unaffected. Our results provide the first demonstration of direct genetic evidence that the antimicrobial activity of lysozyme is operationally independent of its muramidase activity, and strongly suggest the antimicrobial action of lysozyme is due to structural factors.

A helix-loop-helix peptide at the upper lip of the active site cleft of lysozyme confers potent antimicrobial activity with membrane permeabilization action.

Recently, we have found that partially unfolded lysozyme exerts broad spectrum antimicrobial action in vitro against Gram-negative and Gram-positive bacteria independent of its catalytic activity. In parallel, an internal peptide (residues 98-112) of hen egg white lysozyme, obtained after digestion with clostripain, possessed broad spectrum antimicrobial action in vitro. This internal peptide is part of a helix-loop-helix domain (87-114 sequence of hen lysozyme) located at the upper lip of the active site cleft of lysozyme. The helix-loop-helix (HLH) structures are known motifs commonly found in membrane-active and DNA-binding proteins. To evaluate the contribution of the HLH peptide to the antimicrobial properties of lysozyme, the HLH sequence and its secondary structure derivatives of chicken and human lysozyme were synthesized and tested for antimicrobial activity against several bacterial strains. We found that the full HLH peptide of both chicken and human lysozymes was potently microbicidal against both Gram-positive and Gram-negative bacteria and the fungus Candida albicans. The N-terminal helix of HLH was specifically bactericidal to Gram-positive bacteria, whereas the C-terminal helix was bactericidal to all tested strains. Outer and inner membrane permeabilization studies, as well as measurements of transmembrane electrochemical potentials, provided evidence that HLH peptide and its C-terminal helix domain kill Gram-negative bacteria by crossing the outer membrane via self-promoted uptake and causing damage to the inner membrane through channel formation. The results are discussed in terms of proposed mechanisms for the catalytically independent antimicrobial activity of lysozyme that offer a new strategy for the design of potential antimicrobial drugs in the treatment of infectious diseases.

Occurrence of ovalbumin in ovarian yolk of chicken during oogenesis.

Yolk specimens from chicken ovaries during oogenesis gave a positive signal for ovalbumin as analyzed by Western blotting, indicating that the ovarian yolk contains ovalbumin. Northern blotting and reverse transcriptase-polymerase chain reaction gave a negative signal for ovalbumin mRNA in the liver and other organs except oviduct, whereas the laying hen serum was found to indicate immunologically the presence of ovalbumin. It was therefore assumed that ovalbumin synthesized in the oviduct might partly be secreted into the blood circular system, from which it is taken up into the oocyte.

Ovotransferrin antimicrobial peptide (OTAP-92) kills bacteria through a membrane damage mechanism.

Ovotransferrin antimicrobial peptide (OTAP-92) is a cationic fragment of hen ovotransferrin (OTf). OTAP-92 consists of 92 amino acid residues located within the 109-200 sequence of the N-lobe of OTf. This study was aimed to delineate the antimicrobial mechanism of OTAP-92 and to identify its interaction with bacterial membranes. OTAP-92 caused permeation of Escherichia coli outer membrane (OM) to 1-N-phenylnaphthylamine fluorescent probe in a dose-dependent manner. These results suggested that OTAP-92 crossed the bacterial OM by a self-promoted uptake. Cytoplasmic membrane of E. coli was found to be the target for OTAP-92 bactericidal activity, as assayed by the unmasking of cytoplasmic beta-galactosidase due to membrane permeabilization in a kinetic manner. Pretreatment of bacteria with uncoupler, carbonyl cyanide m-chlorophenylhydrazone, markedly enhanced permeation of cytoplasmic membrane, suggesting that the membrane permeation due to OTAP-92 is independent of the transmembrane potential. In an E. coli phospholipid liposome model, it was demonstrated that OTAP-92 has the ability to dissipate the transmembrane electrochemical potential. Intrinsic fluorescence spectra of the two tryptophan residues in OTAP-92, using liposomal membrane, have identified the lipid-binding region as a helix-sheet motif, and suggested an adjacent Ca(2+)-sensitive site within OTAP-92. These data indicated that OTAP-92 possesses a unique structural motif similar to the insect defensins. Further, this cationic antimicrobial peptide is capable of killing Gram-negative bacteria by crossing the OM by a self-promoted uptake and cause damage to the biological function of cytoplasmic membrane.

Preparation and characterization of milk calcium salts by using casein phosphopeptide.

Milk calcium salt solution was prepared by the following procedures using casein phosphopeptides (CPP). To CPP solution, 1 M citric acid, 1 M CaCl2 and 1 M K2HPO4 were added with stirring, while adjusting the pH to 6.7. The prepared solution was left for 12 hr at 25 degrees C and then used for subsequent experiments, or lyophilized. The concentrations of organic phosphate of CPP, calcium, inorganic phosphate, and citrate in the typical milk salt solution were 7, 30, 22, and 10 mM, respectively, which were close to those in bovine milk. The lyophilized sample was easily dissolved in water. No crystal structure of hydroxyapatite was shown in the lyophilized milk calcium salts by X-ray diffraction analysis, although the pattern of KCl crystal was observed. The X-ray diffraction pattern of commercial whey mineral, which was prepared by precipitation at alkaline pH from rennet whey, was similar to that of hydroxyapatite. It was confirmed by high-performance gel chromatographic analysis that the form of calcium phosphate in the milk calcium salts was similar to that of casein micelles.

Ovalbumin in developing chicken eggs migrates from egg white to embryonic organs while changing its conformation and thermal stability.

Ovalbumin was detected in developing chicken eggs. The large majority of these ovalbumin molecules was found to be in a heat-stable form reminiscent of S-ovalbumin. About 83 and 90% of the ovalbumin population was in a heat-stable form in day 14 or stage 40 amniotic fluid and day 18 or stage 44 egg yolk, respectively, whereas ovalbumin in newly deposited eggs was in the heat-unstable, native form. Purified preparations of stable ovalbumin from egg white and amniotic fluid showed a less ordered configuration than native ovalbumin, as analyzed by circular dichroism and differential scanning calorimetry. In addition, mass spectrometric analysis exhibited distinct size microheterogeneity between the stable and native forms of ovalbumin. Immunohisotochemical study revealed that ovalbumin was present in the central nervous system and other embryonic organs. These results indicated that egg white ovalbumin migrates into the developing embryo while changing its higher order structure.

Enhanced secretion of hydrophobic peptide fused lysozyme by the introduction of N-glycosylation signal and the disruption of calnexin gene in Saccharomyces cerevisiae.

The insertion of a hydrophobic pentapeptide (Phe-Phe-Val-Ala-Pro) into the C-terminus in hen egg white lysozyme by genetic modification resulted in an unstable structure which caused little secretion in a yeast expression system, although this modification is useful to enhance bactericidal action to gram-negative bacteria [Ibrahim et al. (1994) J. Biol. Chem. 269, 5059-5063]. To enhance the secretion of the unstable hydrophobic pentapeptide fused lysozymes (H5-Lz), we attempted to introduce the signal sequence (Asn-X-Ser/Thr) of N-linked glycosylation into lysozyme and to suppress the quality control of the unstable mutant in the yeast expression system. The polymannosyl hydrophobic fused lysozyme (H5/G49N-Lz) having the N-glycosylation signal sequence was expressed in the medium at 3.4 times that of unglycosylated lysozyme. Further, the secretion of the unstable mutant lysozyme was done in the Saccharomyces cerevisiae disrupted calnexin gene to avoid the degradation of the unstable mutant by the quality control. Although disruption of the calnexin gene did not lead to gross effects on the levels of growth of S. cerevisiae (W303-1b), the secretion amount of H5/G49N-Lz in calnexin disrupted S. cerevisiae was 2.5 times larger than that in wild type S. cerevisiae. These results suggest that the secretion of unstable glycosylated lysozyme (H5/G49N) was suppressed by the quality control function of calnexin and that the disruption of calnexin is effective to increase the secretion of unstable glycosylated protein.

On the novel catalytically-independent antimicrobial function of hen egg-white lysozyme: a conformation-dependent activity.

Dependency of the antimicrobial activity on the conformation of lysozme was examined by the means of gradual thermal inactivation at neutral pH and different temperatures. We found that heating of lysozyme at increasing temperatures for 20 min in pH 6.0 results in progressive loss of enzyme activity, while greatly promotes its antimicrobial action to the Gram-negative bacteria without a detrimental effect on the inherent bactericidal activity to Gram-positive ones, suggesting action independent of catalytic function. The most potent bactericidal conformation of lysozyme to either Gram-negative or -positie bacteria was that retaining approximately 50% of the native enzyme activity (HL80/6). HL80/6 showed several fold increase in surface hydrophobicity, with exposed two thiol groups, and 17% deamidation. Spectrophotometric analysis of HL80/6 revealed slight changes in its secondary structures, but considerable global conformational changes as a result of the formation of beta conformation, via cyclic imide, at the three aspartylglycyl sequences of lysozyme molecule. Direct damage to the bacertial membranes by HL80/6, was demonstrated by using ELISA and liposomal membrane model. Furthermore, the antimicrobial activity of HL80/6 was inhibited by the divalent cations Ca2+ and Mg2+ suggesting that HL80/6 interacts at a divalent cation binding site on the bacterial membrane and subsequently permeabilize it. The results introduce an interesting structure-antimicrobial relationship that the antimicrobial action of lysozyme is independent of its catalytic function. In addition, it is worth emphasizing that the naturally-occurring conformational transition of lysozyme at physiological temperatures can be a biologically relevant event to switch its antimicrobial specificity to include the food-borne pathogens and heralding fascinating opportunities for application in formulated food systems.

Identification of a distinct antibacterial domain within the N-lobe of ovotransferrin.

We have evaluated the bactericidal activity of hen ovotransferrin (OTf), which was found to operate regardless of its iron-deprivation properties, with the objective of isolating the bactericidal domain. The amino-terminal half-molecule (N-lobe, residues 1-332) of OTf, isolated by trypsin-nicking, retained the bactericidal activity independently of iron-deprivation, but not the carboxyl-terminal half-molecule (C-lobe, residue 342-686), suggesting the presence of a bactericidal domain within the N-lobe of the molecule. Specific cleavage at the aspartyl residues of OTf, by diluted-acid procedure, yielded fairly large peptides, whereas proteolysis for 150 min produced the strongest bactericidal peptides mixture. The bactericidal domain was purified from the active hydrolysate by gel filtration and reversed-phase HPLC and showed activity against S. aureus as well as E. coli K-12. Electrophoretic analysis on tricine-SDS-PAGE revealed a bactericidal peptide with an average M(r) of 9900 Da under non-reducing conditions. In combination with the specificity of cleavage (Asp-X) and the molecular mass, its N-terminal microsequencing corresponded to a cationic peptide consisting of 92 residues located within the 109-200 sequence of the N-lobe of OTf, containing three intrachain disulfide bridges, featuring a common structural motif occurs in the N-lobes of transferrins for which the sequence is available. Two of the disulfides (C160-C174 and C171-C182) form surface exposed cringle bridges lying on the opposite side of the iron-binding site from the interdomain cleft and showing marked sequence homology to insect defensins, which are blockers of the voltage-dependent K+ channels. The peptide lost antibacterial activity when its disulfide bonds were reduced, indicating the importance of its tertiary structure for the exertion of antibiotic activity.

Bactericidal action of lysozymes attached with various sizes of hydrophobic peptides to the C-terminal using genetic modification.

The genetic modification of lysozyme was attempted to improve the bactericidal activity against Gram-negative bacteria E. coli. The different lengths of hydrophobic peptides were attached to the C-terminus of the hen egg white lysozyme to investigate the most effective length of the hydrophobic peptides for killing bacteria. The oligonucleotides encoding Phe-Val-Pro (H3), Phe-Phe-Val-Ala-Pro (H5) and Phe-Phe-Val-Ala-Ile-Ile-Pro (H7) were fused to the C-terminus Leu 129 of lysozyme cDNA. The reconstructed cDNAs were inserted into the yeast expression vector. The hydrophobic peptide-fused lysozymes were secreted in the yeast carrying the reconstructed cDNA. Although the hydrophobic peptide-fused lysozymes retained 75 80% lytic activity of the wild-type protein, the bactericidal action to E. coli was greatly increased with the length of hydrophobic peptides. These results suggest that the hydrophobic peptides play an important role in killing Gram-negative bacteria. To elucidate the role of catalytic domain in bactericidal action of the hydrophobic fusion lysozyme (H5-Lz), the mutant hydrophobic lysozyme (H5/E35A-Lz) whose glutamic acid was substituted with alanine at the position 35 was constructed to diminish the catalytic activity. The mutant hydrophobic lysozyme (H5/E35A-Lz) was greatly lost the bactericidal action to E. coli, suggesting that not only the length of hydrophobic peptide fused to C-terminus but also the catalytic domain is important for the bactericidal action of the hydrophobic peptide-fused lysozyme.

Occurence of a sialylglycopeptide and free sialylglycans in hen's egg yolk.

Free sialylglycans (FSGs) and a sialylglycopeptide (SGP) as components of hen's egg yolk were found and their chemical structures were determined. SGP and FSGs were isolated from fresh egg yolk by treatment with phenol, gel filtration and successive chromatographies on columns of anion- and cation-exchangers. They were localized in the yolk plasma. The glycan moiety of SGP, which was liberated by PNGase digestion, was studied for the chemical structure by HPLC mapping with p-aminobenzoic ethylester-derivatization, sugar composition analysis, 1H nuclear magnetic resonance and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and the glycomoiety was found to be an N-linked disialyl-biantennary glycan. The amino acid sequence of the peptide moiety of SGP was determined to consist of Lys-Val-Ala-Asn-Lys-Thr, the Asn of which is modified with the disialylglycan moiety. FSGs were determined to be two free disialyl-biantennary glycans whose reducing end was either Man beta1-4GlcNAc (FSG-I) or Man beta1-4GlcNAc beta1-4GlcNAc (FSG-II). Since the molar value of SGP present in one egg yolk (2.8 micromol) is comparable to those of well-known major yolk proteins, low density lipoprotein, lipovitellins and phosvitin, it can be considered that SGP is one of the major components in hen's egg yolk.

Enhanced bactericidal action of lysozyme to Escherichia coli by inserting a hydrophobic pentapeptide into its C terminus.

The mechanism of the enhanced bactericidal action to Escherichia coli of the lysozyme having a hydrophobic pentapeptide (Phe-Phe-Val-Ala-Pro) at its C terminus was investigated. The modified lysozyme, hydrophobic pentapeptide-fused lysozyme (HLz), was secreted in the culture medium from yeast harboring the expression plasmid, in which a synthetic DNA fragment encoding a hydrophobic pentapeptide was introduced to the 3'-end of the coding region of the lysozyme cDNA. Although CD analysis showed that HLZ was considerably different from wild-type lysozyme (WLz) in the secondary and tertiary structures, it retained 76% of the lytic activity of WLz. When E. coli cells were exposed to the WLz or HLz, the survival cells were significantly reduced only in the case of HLz. Periplasmic proteins from the HLz-treated cells were released to an extent similar to that from the WLz-treated cells, indicating that HLz has nearly the same action as WLz with respect to the disruption of the outer membrane and peptidoglycan. Experiments with E. coli phospholipid liposomes revealed that HLz dissipated the valinomycin-induced transmembrane electrochemical potential, but WLz did not. These results suggest that the enhanced bactericidal action of HLz to E. coli is due to disruption of the electrochemical potential of the inner membrane in cooperation with the inherent function of the lysozyme to the outer membrane and peptidoglycan.