• Journal of Oral Medicine and Pain
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• v.39 no.4
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• pp.127-132
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• 2014

Purpose: Many substances in saliva or oral health care products interact with each other. The aim of this study was to investigate interactions between hyaluronic acid (HA), lysozyme, peroxidase, and glucose oxidase (GO) in enzymatic activities at low pH levels. Methods: HA (0.5 mg/mL), hen egg-white lysozyme (HEWL, $30{\mu}g/mL$), bovine lactoperoxidase (bLPO, $25{\mu}g/mL$), and GO ($50{\mu}g/mL$) were used. The influences of HA, bLPO, and GO on HEWL activity were determined by measuring the turbidity of a Micrococcus lysodeikticus suspension. The influences of HA and HEWL on bLPO activity were determined by the NbsSCN assay, measuring the rate of oxidation of 5-thio-2-nitrobenzoic acid (Nbs) to 5,5'-dithiobis(2-nitrobenzoic acid) $(Nbs)_2$. The influences of HA and HEWL on GO activity were determined by measuring oxidized o-dianisidine production. All experiments were performed at pH 4, 5, and 6. Results: HA and GO did not affect the enzymatic activity of HEWL at pH 4, 5, and 6. bLPO enhanced the enzymatic activity of HEWL at pH 5 (p<0.05) and pH 6 (p<0.05) significantly. The enzymatic activity of bLPO was not affected by HA and HEWL at pH 4, 5, and 6. HA and HEWL did not affect the enzymatic activity of the GO at pH 4, 5, and 6. Conclusions: Peroxidase enhances lysozyme activity at low pH, otherwise there were no significant interactions in enzymatic activities between HA, lysozyme, peroxidase, and GO at low pH levels.

• Journal of Oral Medicine and Pain
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• v.43 no.1
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• pp.1-7
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• 2018

Purpose: The purpose of the study was to investigate the influences of hyaluronic acid on the candidacidal activities of lysozyme, the peroxidase system, and the glucose oxidase-mediated peroxidase (GO-PO) system at different concentrations of antimicrobial enzymes. Methods: Hyaluronic acid was used at a final concentration of 0.5 mg/mL. Hen egg-white lysozyme (HEWL) was used at concentrations ranging from 10 to $100{\mu}g/mL$. The peroxidase system included bovine lactoperoxidase (bLPO), potassium thiocyanate (KSCN, 1 mM), and hydrogen peroxide ($100{\mu}M$). The GO-PO system included bLPO, KSCN (1 mM), glucose oxidase (10 units/mL), and glucose ($30{\mu}g/mL$). The final concentration of bLPO in the peroxidase and GO-PO systems ranged from 12.5 to $100{\mu}g/mL$. Candida albicans strains ATCC 10231, 11006, and 18804 were utilized. Candidacidal activities of antimicrobials and the influence of hyaluronic acid on their candidacidal activities were determined based on colony forming units. Results: Candidacidal activities of the peroxidase and GO-PO systems increased with increasing concentrations of bLPO. This tendency was the same in the presence or absence of hyaluronic acid. Candidacidal activity of HEWL was not significantly concentration-dependent. Candidacidal activities of the GO-PO system were higher than those of the corresponding peroxidase system. Candidacidal activity was inhibited in the presence of hyaluronic acid in the following order: HEWL, the peroxidase system, and the GO-PO system. Conclusions: Hyaluronic acid inhibited the candidacidal activities of HEWL, the peroxidase system, and the GO-PO system. The GO-PO system exhibited better candidacidal activity than HEWL and the peroxidase system both in the presence and absence of hyaluronic acid.

• Korean Journal of Food Science and Technology
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• v.31 no.2
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• pp.458-464
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• 1999

The value of lysozyme as a natural food preservative is continuously increased due to its unique antimicrobial activity. To determine the optimum separation concentration among the various hen egg white protein (HEWP) concentrations (0.25, 0.5, 1.0, w/v), protein concentrations, lysozyme concentrations, specific activities (SA), and purification factors of prefiltered solution (PFS) and PM30 permeate solution (PMS) were compared. The purity of lysozyme separated at each step was analyzed and confirmed by gel permeation chromatography and electrophoresis. The fouling deposits on membrane were observed by SEM. The non-enzymatic proteins were removed over 99% by ultrafiltration (UF). The increased feed concentration did not contribute to the increase of SA. SA of PMS was 18 to 31 times higher than that of PFS. The optimum feed concentration was decided as 0.25% based on SA and purification factor. The non-enzymatic region of gel chromatogram was proved to be ovalbumin. The thickness of deposit on the UF membrane was approximately $0.9{\mu}m$ and removed by cleaning with 0.1 N NaOH. Therefore, UF using PM30 membrane was very effective to separate the antimicrobial lysozyme from various HEWPs.

• The Journal of the Korean Society for Microbiology
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• v.12 no.1
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• pp.19-32
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• 1977

The specificity of the N- and C-terminal antigenic determinant($P_{17}$: sequence $Lys^1-{cys-}^6-Asn^{27},\;{Trp^{12}}_2-Cys^{127}-Leu^{129}$) of hen egg-white lysozyme(HL) was studied in more detail. In a Scatchard plot of the binding of $^{14}C$-acetyl HL with guinea pig purified anti-$P_{17}$ antibody experimental values bent sharply aear r=1. This suggests of two antibody populations with different affinities for HL or possible steric hindrance in the binding of a second HL molecule to the second binding site of the antibody molecule. The antigenic activities of various peptides were tested by measuring their inhibition of the binding of $^{14}C-acetyl-P_{17}$ with the antibody, Only $P_{17}$ and $P_{17}t$(sequence $Lys^1-cys^6-Homoser^{12},\;Trp^{123}-Cys^{127}-Leu^{128})$) were inhibitory, with $K_1$ values of $2.0{\times}10^4$ and $8.1{\times}10^3$, respectively. These results indicate that the direct binding site of $P_{17}$ to anti-$P_{17}$ antibody may be located in the terminal portion of $P_{17}$ (sequence $Lys^1-Cys^6-Homoser^{12},\;Trp^{123}-Cys^{127}-Leu^{129})$) while the rest of $P_{17}$ may be important in maintaining the conformation of this determinant. The single disulphide bond involved in this determinant is essential for manifestation of immunological activity.

• Journal of Life Science
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• v.28 no.3
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• pp.300-306
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• 2018

Lysozymes are innate immune factors that play a critical role in the defense against pathogens in various invertebrate animals including spoon worms. In this study, an invertebrate-type lysozyme was isolated from the body wall of spoon worm, Urechis unicinctus. The acidified body wall extract was partially separated using a Sep-Pak C18 cartridge. Among the fractions, the materials that were eluted with 60% methanol/0.1% trifluoroacetic acid showed the most potent antimicrobial activity against Bacillus subtilis KCTC 1021. A series of high performance liquid chromatography (HPLC) steps were then utilized to isolate a single antimicrobial absorbance peak. The molecular weight of the antimicrobial peak was approximated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which was approximately 13 to 14 kDa. The partial primary structure of this antimicrobial protein that was analyzed, using LC-MS/MS, was CTGGRPPTCEDYAK (1611.69 Da). Homology search of these fourteen residues, using the National Center for Biotechnology Information Basic Local Alignment Search Tool (NCBI BLAST), revealed that the isolated protein was similar to the invertebrate-type lysozymes described in other animals. Then, the antimicrobial and lysozyme enzymatic (muramidase) activities of this protein were assessed. The isolated protein possessed antimicrobial activity and potent muramidase activity, which were comparable to those of hen egg white lysozyme. Therefore, the isolated protein was designated as Urechis unicinctus invertebrate-type lysozyme from the body wall, Uu-iLysb.

• Journal of Oral Medicine and Pain
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• v.34 no.3
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• pp.227-235
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• 2009

Many of the protective functions of saliva can be attributed to the biological, physical, structural, and rheological characteristics of salivary glycoproteins. Therefore, the development of ideal saliva substitutes requires understanding of the rheological as well as biological properties of human saliva. In the present study, we investigated the changes of salivary enzymatic activities by saliva substitutes and compared viscosity of saliva substitutes with human saliva. Five kinds of saliva substitutes such as Moi-Stir, Stoppers4, MouthKote, Saliva Orthana, and SNU were used. Lysozyme activity was determined by the turbidimetric method. Peroxidase activity was determined with an NbsSCN assay. $\alpha$-Amylase activity was determined using a chromogenic substrate, 2-chloro-p-nitrophenol linked with maltotriose. The pH values of saliva substitutes were measured and their viscosity values were measured with a cone-and-plate digital viscometer at six different shear rates. Various types of saliva substitutes affected the activities of salivary enzymes in different ways. Stoppers4 enhanced the enzymatic activities of hen egg-white lysozyme, bovine lactoperoxidase (bLP), and $\alpha$-amylase. Saliva Orthana and SNU inhibited bLP activity and enhanced $\alpha$-amylase activity. MouthKote inhibited $\alpha$-amylase activity. Moi-Stir inhibited the enzymatic activities of bLP and $\alpha$-amylase. The pH values were very different according to the types of saliva substitutes. Stoppers4, MouthKote, and Saliva Orthana showed lower values of viscosity at low shear rates and higher values of viscosity at high shear rates compared with unstimulated and stimulated whole saliva. Moi-Stir and SNU displayed much higher values of viscosity than those of natural whole saliva. Collectively, our results indicate that each saliva substitute has its own biological and rheological characteristics. Each saliva substitute affects the enzymatic activity of salivary enzyme and finally oral health in different ways.

• Journal of the Korean Crystal Growth and Crystal Technology
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• v.23 no.1
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• pp.1-7
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• 2013

The three-dimensional structure of the proteins, including the location of the hydrogen atoms can be analyzed by using neutron diffraction. In order to grow single crystals for neutron diffraction experiments, we developed a simple magnetic field device with the commercial magnets and succeeded in growing hen egg-white lysozyme single crystals of more than 1 $mm^3$ in volume using this device. The crystals grown with a magnetic field were larger and had perfect transparency, whereas the crystals grown without a magnetic field had micro-cracks. The result of the X-ray measurement showed a good resolution and small mosaicity for the crystals grown in the presence of a magnetic field.

• Preventive Nutrition and Food Science
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• v.7 no.2
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• pp.139-145
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• 2002

Hen egg-white lysozyme, ovalbumin, egg-yolk phosvitin, acid-precipitated soy protein and $\alpha$$_{sl}$ milk casein were covalently linked with galactomannan through a controlled dry-heating at 6$0^{\circ}C$ under 79% relative humidity without any chemical reagent. Neoglycosylation by the covalent binding of polysaccharide chains brought a significant improvement into the surface functionalities of food proteins. Excellent emulsifying properties and foaming properties were observed in all protein-galactomannan conjugates. Bacterial mutagenesis tests and animal dose test were done to evaluate the food safety of the protein-galactomannan conjugates. The neo-glycoproteins were negative for Ames test using Salmonella typhimurium TA100 (hisG46) and TA98 (hisD3052) strains, and rec-assay using Bacillus subtilis Hl7 (rec) and M45 (re $c^{+}$) strains. All substances were also nontoxic for oral administration to rats. L $D_{50}$ 's of these substances were all more than 7.5 g/kg body-weight of rat. No effect was also observed in the weight increases and the concentrations of total cholesterol, triglyceride and phospholipids in blood serum of the administrated rats with 7.5 g/kg conjugates. Thus, Maillard-type protein-polysaccharide conjugates prepared by covalent attachment of galactomannan to food proteins were proposed to be useful as a safe functional biopolymer in this study.y.

• Molecules and Cells
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• v.44 no.7
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• pp.517-528
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• 2021

A recent genetic study with Brucella abortus revealed the secretion activator gene A (SagA) as an autolysin component creating pores in the peptidoglycan (PGN) layer for the type IV secretion system (T4SS) and peptidoglycan hydrolase inhibitor A (PhiA) as an inhibitor of SagA. In this study, we determined the crystal structures of both SagA and PhiA. Notably, the SagA structure contained a PGN fragment in a space between the N- and C-terminal domains, showing the substrate-dependent hinge motion of the domains. The purified SagA fully hydrolyzed the meso-diaminopimelic acid (DAP)-type PGN, showing a higher activity than hen egg-white lysozyme. The PhiA protein exhibiting tetrameric assembly failed to inhibit SagA activity in our experiments. Our findings provide implications for the molecular basis of the SagA-PhiA system of B. abortus. The development of inhibitors of SagA would further contribute to controlling brucellosis by attenuating the function of T4SS, the major virulence factor of Brucella.