• Food Science of Animal Resources
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• v.35 no.4
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• pp.473-478
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• 2015

The aim of this study was to screen and In vitro characterize the properties of bacteriocin produced by lactic acid bacteria isolated from Vietnamese fermented pork (Nem chua). One hundred and fifty LAB were isolated from ten samples of Nem chua and screened for bacteriocin-producing lactic acid bacteria. Antimicrobial activity of bacteriocin was carried out by spot on lawn method against both gram positive and gram negative bacteria. One isolate, assigned as KL-1, produced bacteriocin and showed inhibitory activity against Lactobacillus sakei, Leuconostoc mesenteroides and Enterococcus faecalis. To characterize the bacteriocin-producing strain, optimum temperature, incubation period for maximum bacteriocin production and identification of bacteriocin-producing strain were determined. It was found that the optimum cultivation temperature of the strain to produce the maximum bacteriocin activity (12,800 AU/mL) was obtained at 30℃. Meanwhile, bacteriocin production at 6,400 AU/mL was found when culturing the strain at 37℃ and 42℃. The isolate KL-1 was identified as L. plantarum. Antimicrobial activity of cell-free supernatant was completely inhibited by proteolytic enzyme of trypsin, alpha-chymotrypsin and proteinase K. Bacteriocin activity was stable at high temperature up to 100℃ for 10 min and at 4℃ storage for 2 d. However, the longer heating at 100℃ and 4℃ storage, its activity was reduced.

• Food Science of Animal Resources
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• v.38 no.5
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• pp.1064-1079
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• 2018

In this study, the antimicrobial activity of a bacteriocin produced by Enterococcus faecalis KT11, isolated from traditional Kargı Tulum cheese, was determined, and bacteriocin KT11 was partially characterized. The results showed that bacteriocin KT11 was antagonistically effective against various Gram-positive and Gram-negative test bacteria, including vancomycin- and/or methicillin-resistant bacteria. The activity of bacteriocin KT11 was completely abolished after treatment with proteolytic enzymes (proteinase K, ${\alpha}$-chymotrypsin, protease and trypsin), which demonstrates the proteinaceous nature of this bacteriocin. Additionally, bacteriocin KT11 remained stable at pH values ranging from 2 to 11 and after autoclaving at $121^{\circ}C$ for 30 min. In addition, the activity of bacteriocin KT11 was stable after treatment with several surfactants (EDTA, SDS, Triton X-100, Tween 80 and urea) and organic solvents (chloroform, propanol, methanol, ethyl alcohol, acetone, hexane and ethyl ether). Cell-free supernatant of E. faecalis KT11 was subjected to ammonium sulfate precipitation and then desalted by using a 3.5-kDa cut-off dialysis membrane. The bacteriocin activity was determined to be 711 AU/mL in the dialysate. After tricine-SDS-PAGE analysis, one peptide band, which had a molecular weight of ~3.5 kDa, exhibited antimicrobial activity. Because the bacteriocin KT11, isolated from E. faecalis KT11, exhibits a broad antimicrobial spectrum, heat stability and stability over a wide pH range, this bacteriocin can be used as a potential bio-preservative in foods. Additionally, bacteriocin KT11 alone or in combination with conventional antibiotics may provide a therapeutic option for the treatment of multidrug-resistant clinical pathogens after further in vivo studies.

• The Korean Journal of Zoology
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• v.31 no.3
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• pp.185-190
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• 1988

To investigate whether malignant behavior of skin tumor correlates with changes in the level of proteolytic activities in skin tumors, rats were treated with 7,12-dimethylbenzanthracene followed by photbor ester. Tumors induced upon the treatrnents exhibited more than 20-fold increase in the activity of plasminogen activator and about 3-fold of plasmin-like activity. as compared to those in treated controls. Furthermore, the former activity was raised to about 6-fold even in the preneoplastic dssues of the skin tissues. On the other hand, the proteolytic activity against casein and insulin decreased to several-fold in the tumor tissues while antitrvpsin activity remained similar in both tumor and controls. Thus, the increase in the activities of plasmInogen activator and plasminlike enzyme appears to occur as a charaderistic to skin cancer and may involve in invasion and metsstasis of the tumor.

• Journal of Radiation Protection and Research
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• v.22 no.1
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• pp.23-27
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• 1997

Radiation effects on carbohydrate moiety of chicken ovomucoid, a protease inhibitor as a typical allergenic glycoprotein of egg white, was observed. The trypsin inhibitory activity of chicken ovomucoid decreased exponentially and the inactivation was more significant irradiated in $N_2$ than in $O_2$. From the protein blotting, radiation caused protein degradation in $O_2$ and protein aggregation also in $N_2$. The patterns of carbohydrate blotting were also similar with that of protein blotting. Sugar chains in low molecular weight fraction (MW<5,000) were released by radiation and those in $O_2$ were higher than in $N_2$. From the HPLC patterns of the degradation of sugar chains, all peaks of oligosaccharides have the tendency to decrease with the increase of radiation dose and more remarkable in $O_2$ than in $N_2$. These results suggest that ionising radiation could cause the overall conformational changes of ovomucoid by the degradation and release of oligosaccharides.

• BMB Reports
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• v.40 no.4
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• pp.604-609
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• 2007

An Asp/His catalytic site of 10-formyltetrahydrofolate dehydrogenase (FDH) was suggested to have a similar catalytic topology with the Asp/His catalytic site of serine proteases. Many studies supported the hypothesis that serine protease inhibitors can bind and modulate the activity of serine proteases by binding to the catalytic site of serine proteases. To explore the possibility that soybean trypsin inhibitor (SBTI) can recognize catalytic sites of FDH and can make a stable complex, we carried out an SBTI-affinity column by using rat liver homogenate. Surprisingly, the Rat FDH molecule with two typical liver proteins, carbamoyl-phosphate synthetase 1 (CPS1) and betaine homocysteine S-methyltransferase (BHMT) were co-purified to homogeneity on SBTI-coupled Sepharose and Sephacryl S-200 followed by Superdex 200 FPLC columns. These three liver-specific proteins make a protein complex with 300 kDa molecular mass on the gel-filtration column chromatography in vitro. Immuno-precipitation experiments by using anti-FDH and anti-SBTI antibodies also supported the fact that FDH binds to SBTI in vitro and in vivo. These results demonstrate that the catalytic site of rat FDH has a similar structure with those of serine proteases. Also, the SBTI-affinity column will be useful for the purification of rat liver proteins such as FDH, CPS1 and BHMT.

• BMB Reports
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• v.36 no.3
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• pp.294-298
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• 2003

The pathological effect of the Bacillus thuringiensis Cry $\delta$-endotoxins on susceptible insect larvae had extensive damage on the midgut epithelial cells. In this study, an ex vivo assay was devised for assessing the insecticidal potency of the cloned Cry4B mosquito-larvicidal protein that is expressed in Escherichia coli. Determination of toxicity was carried out by using a cell viability assay on the midguts that were dissected from 5-day old Aedes aegypti mosquito larvae. After incubation with the toxin proteins, the number of viable epithelial cells was determined photometrically by monitoring the quantity of the bioreduced formazan product at 490 nm. The results showed that the 65-kDa trypsin-activated Cry4B toxin exhibited toxic potency ca. 3.5 times higher than the 130-kDa Cry4B protoxin. However, the trypsin-treated products of the non-bioactive Cry4B mutant (R158A) and the lepidopteran-specific Cry1Aa toxin displayed relatively no ex vivo activity on the mosquito-larval midguts. The ex vivo cytotoxicity studies presented here confirms data that was obtained in bioassays.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.21 no.2
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• pp.85-96
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• 1988

Purification and some properties of alkaline proteinases in the pyloric caeca of skipjack, Katsuwonus vagans, were investigated. Four alkaline proteinases, temporarily designated proteinases I, II, III and IV, were identified from the tissue extract of the pyloric caeca by ammonium sulfate fractionation, DEAE-Sephadex A-50 chromatography, and Sephadex G-100 and G-200 gel filtration. Result of disc-polyacrylamide gel electrophoretic analysis showed that the purified proteinases II and III were homogenous with the yields of $1.5\%\;and\;1.2\%$, and those specific activities were increased to 33 to 37 fold over that of the crude enzyme solution, respectively. Molecular weight of the proteinases II and III determined by sephadex G-100 gel filtration were 28,500 and 24,200, respectively. The optimum conditions for the caseinolytic activity of the two enzymes were pH 9.6 and $48^{\circ}C$. The reaction rates of the two alkaline proteinases were constant to the reaction time to 80 min in the reaction mixture of $3.4{\mu}g/ml$ of enzyme concentration and $2\%$ casein solution. The Km values against casein substrate determined by the method of Lineweaver-Burk were $0.56\%$ for proteinase II and $0.30\%$ for proteinase II. The proteinases II and III were inactivated under the presence of $Ag^+,\;Hg^{2+},\;Ni{2+},\;Fe^{2+},\;and\;Cu^{2+}$, and but activated by $Mn^{2+}\;and\;Ca^{2+}$ and markedly inhibited by the soybean trypsin inhibitor and N-p-toluenesulfonyl-L-lysine chloromethyl ketone. Therefore, the proteinases II and III were found to be a group of serine proteases and assured to be trypsin-like proteinases.

• Journal of Microbiology and Biotechnology
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• v.10 no.2
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• pp.251-257
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• 2000

Chitin synthases are identified as key enzymes of chitin biosynthesis in most of the fungi. Among them, chitin synthase II has been reported to be and essential enzyme in chitin biosynthesis, and exists as a membrane-bound form. To search and screen new antifungal agents from natural resources to inhibit chitin synthase II, the assay conditions were established using the enzyme isolated from Saccharomyces cerevisiae ECY38-38A(pAS6) that overproduces only chitin synthase II. This enzyme was activated only by partial proteolysis with trypsin. Its actibity reached the maximum at $80{\;}\mu\textrm{g}/ml$ of trypsin and was strongly stimulated by 2.0 mM $Co^{2+}$, 1.0 nM UDP-[$^{14}C$]-GicNAc, and 32 mM free-GlcNAc. Under these assay conditions, the highest chitin synthase II activity was observed by incubation at $30^{\circ}C$ for 90 min. However, and extremely narrow range of organic solvents up to as much as 25% of DMSO and 25% of MeOH was useful for determining optimal assay conditions. After a search or potent inhibitors of chitin synthase II from natural resources, prodigiosin was isolated from Serratia marcescens and purified by solvent extration and silica gel column chromatographies. The structure of prodigiosin was determined by UV, IR, Mass spectral, and NMR spectral analyses. Its molecular weight and formula were found to be 323 and $C_{20}H_{25}N_{3}O$, respectively. Prodigiosin ingibited chitin synthase II by 50% at the concentration of $115{\;}\mu\textrm{g}/ml$.

• Bulletin of the Korean Chemical Society
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• v.31 no.9
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• pp.2509-2513
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• 2010

In order to increase protease stability of a novel Trp-rich model antimicrobial peptide, $K_6L_2W_3$ (KLWKKWKKWLK-$NH_2$)and investigate the effect of L-amino acid to peptoid residue conversion on biological functions, we synthesized its antimicrobial peptoid, $k_6l_2w_3$. Peptoid $k_6l_2w_3$ had similar bacterial selectivity compared to peptide $k_66L_2W_3$. The bactericidal rate of $k_6l_2w_3$ was somewhat slower than that of $K_6L_2W_3$. Peptoid $k_6l_2w_3$ exhibited very little dye leakage from bacterial outer-membrane mimicking PE/PG liposomes, as observed in $K_6L_2W_3$, indicating that the major target site of $K_6L_2W_3$ and $k_6l_2w_3$ may be not the cell membrane but the cytoplasm of bacteria. Trypsin treatment of $K_6L_2W_3$ completely abolished antimicrobial activities against Escherichia coli and Staphylococcus aureus. In contrast, the antimicrobial activity of $k_6l_2w_3$ was completely preserved after trypsin treatment. Taken together, our results suggested that antimicrobial peptoid $k_6l_2w_3$ can potentially serves as a promising therapeutic agent for the treatment of microbial infection.

• Natural Product Sciences
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• v.20 no.2
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• pp.113-118
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• 2014

Ribes fasciculatum which belongs to Saxifragaceae has been widely used as a traditional medicine for the treatment of symptoms associated with lacquer poison. However, pharmacological studies on the R. fasciculatum are extremely limited until now. Thus, in this study, we evaluated the possible anti-inflammatory effects of ethyl acetate fraction of R. fasciculatum (ERF) using IFN-${\gamma}$/LPS-stimulated peritoneal macrophage model. We investigated the change in nitrite level in the absence or presence of ERF after LPS stimulation, and we found that ERF effectively attenuates the NO production in a dose dependent manner without notable toxicity. To determine the mechanism of the inhibitory action of ERF on NO production, we performed iNOS enzyme activity assay and Western blotting. Here we showed that both of iNOS enzyme activities and iNOS expressions were significantly down-regulated by ERF, indicating that these dual activities of ERF are responsible for ERF-mediated NO suppression. In addition, ERF inhibitied the expression of cyclooxygenase-2 (COX-2), an another key enzyme in inflammation through suppression of NF-${\kappa}B$ activation. We also tested anti-inflammatory properties of ERF not only in vitro, but in vivo using trypsin-induced paw edema model in mice. Our results revealed that the increased paw volume in response to trypsin injection was recovered by ERF supplement dose dependently.