• 한국식품영양과학회지
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• 제33권2호
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• pp.229-235
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• 2004

콩 단백질을 효소로 가수분해하였을 때 생성되는 항균활성 Peptide를 조사하고 천연 항균제로서의 이용 가능성을 조사하기 위하여 본 실험을 실시하였다. 분리 콩 단백질에 5종의 단백질 가수분해 효소를 작용시켜 생성된 가수분해물의 항균력을 측정하고, membrane filter를 이용해서 한외여과 하여, 분자량별로 분리된 각 fraction의 항균활성을 측정하였으며, 항균활성이 가장 높은 분획을 high peformance liquid chromatography로 분취한 항균성 peptide의 항균활성을 측정하였다 분리 콩 단백질에 5종의 단백질 분해 효소를 작용시켜 제조한 가수분해물 중 Aspergilius saitoi protease로 작용시킨 것이 항균활성이 가장 높았다 Aspergillus saitoi protease로 작용시킨 콩 단백질의 가수분해물을 여과 한계량 10,000, 3,000, 1,000 membrane filter로 cut-off하여 한외여과한 각 fraction의 항균활성을 측정한 결과 분자량 1,000∼3,000인 fraction의 항균활성이 가장 높게 나타났다. Aspergillus saitoi protease로 작용시킨 콩 단백질의 분자량 1,000∼3,000 범위 가수분해물의 MIC는 0.5∼0.8 mg/mL였으며 그람 양성균과 음성균 모두의 증식을 억제하는 경향을 보였다. Aspergillus saitol protease로 작용시킨 콩 단백질의 가수분해물을 121$^{\circ}C$, 10분간 열처리하였을 때도 그 항균활성을 유지하는 것으로 보았을 때 이는 열에 대단히 안정함을 알 수 있었다. 한외여과하여 얻어진 콩 단백질의 분자량 1,000∼3,000범위 가수분해물을 동결건조하여 HPLC의 결과 얻어 진 peak 별로 분획 수집을 반복하여 항균 활성을 측정한 결과 retention time 16.02(IV)의 peak에서 최고 항균활성을 확인하였다.

• Journal of Nutrition and Health
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• 제19권6호
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• pp.363-373
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• 1986

This study was devised to elucidate whether soused shrimp exhibits a digestive action on boiled pork meats. and the mechanism by which sousing with a high concentration of sodium chloride preserves nutrients in foods for a prolonged pe\ulcornerriod. Protease was isolated from soused shrimp using a combination of ammonium sulfate fractionation. DEAE - cellulose ion exchange chromatography and gel filtra\ulcornertion. The isolated protease had specific activity of 1.560 units. 210 purification fo\ulcornerld with an yield of 38%. Its optimum pH and temperature were 8.0 and $43^{\circ}C$ respectively. The molecular weight of the enzyme was 35.000. The Km value of the enzyme for casein was 1.6 x $10^{-6}$ M The e=yme required the presence of cu\ulcornerpric ion to exhibit its full activity. Eighty eight percent of the enzyme activity was in\ulcornerhibited by 3.5M NaCI showing a reversibly linear decrease of the enzyme activity as NaCI concentration increased. The nature of the inhibition by NaCl was rever\ulcornersible and noncompetitive. The protease activity in soused shrimp was well preser\ulcornerved with the elapse of time at least in part due to NaCI induced suppression of autodigestion. The enzyme was denatured by acid easily. i.e. 1% of the original activity remained after staying at pH 2 for 10 minutes. which is within the norm\ulcorneral range of pH of the human stomach. Soused shrimp was observed to be one of those containing the highest protease activity compared with the other soused foo\ulcornerds such as soused oyster. squid. clam. and Pollack intestine with respect to spec\ulcornerific activities of dialized 1:4 whole homogenates(w/v) in 5 mM sodium phospha\ulcornerte - 2.4 mM j3 - mercaptoethanol buffer. pH 8.0. Casein and boiled meats including pork, beef, and chicken appeared to be the good substrates for the protease. Casein was the best. Therefore. the ingestion of boiled meats including pork together with soused sh\ulcornerrimp would help digestion of boiled pork in human not only by increasing appe\ulcornertite also by the direct proteolytic digestion of boiled meats by soused shrimp to\ulcorner some extent. And a high concentration of sodium chloride inhibited the protease activity reversibly in a remarkable degree, which ensued in a significant retardat\ulcornerion of autodigestion of protein in foods by proteases, and hereby contributed to the preservation of foods for an extended period.

• 한국식품저장유통학회지
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• 제13권1호
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• pp.88-94
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• 2006

단백 효소 가수분해물의 천연 항균제로서의 응용성을 검토하기 위하여 casein에 5종의 단백질 가수분해 효소를 작용시켜 얻어진 가수 분해물의 항균활성을 측정하고 활성이 가장 높은 가수분해물을 부분 정제하여 그 응용성을 검토하였다. Casein에 5종 단백질 분해효소를 작용시켜 얻은 가수분해물의 항균활성은 Aspergillus oryzae protease에 의한 것이 가장 높았다. 효소처리에 의하여 얻어진 가수분해 물을 30,000, 10,000, 3,000 membrane filter로 한외여과 하였을 때 항균활성은 3,000이하 분획물에 대부분 함유되어 있었으며 공시균주에 대한 최소저해농도는 $1.0\~1.5\;mg/mL$이었다. Aspergillus oryzae pretense로 작용시킨 casein 가수분해물은 $121^{\circ}C$에서 10분간 가열했을 때도 그 항균 활성을 유지하는 것으로 보아 열에 안정하였다. 가수분해물을 HPLC로 220 nm 280 nm에서 검출된 peak 별로 수집하여 항균활성을 측정 한 결과 retention time 12.6, 13.2 분에서 분취된 peptide가 활성이 있었다. 가수분해 동결건조물을 된장에 첨가하였을 때 미생물의 생육이 저해되었으며 실용성이 있었다.

• 동의생리병리학회지
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• 제18권3호
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• pp.759-766
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• 2004

In the present study, we investigated the effects of aqueous extract of the healthful decoction utilizing Phellinus linteus (HDPL) on the cell growth of human lung carcinoma tumor cell line A549. Exposure of A549 cells to HDPL resulted in growth inhibition and induction of apoptosis in a dose-dependent manner as measured by hemocytometer counts, fluorescence microscopy and flow cytometric analysis. This increase in apoptosis was associated with inhibition and/or degradation of apoptotic target proteins such as poly(ADP-ribose) polymerase (PARP), b-catenin and phospholipase C- 1 (PLC- 1) protein. HDPL treatment induced the down-regulation of anti-apoptotic Bcl-2 expression, an anti-apoptotic gene, however, the level of Bax. a pro-apoptotic gene, was increased by HDPL treatment. In addition, HDPL-induced apoptotis of A549 cells was connected with activation of caspase-3 and caspase-9 protease in a dose-dependent manner, however, the levels of inhibitor of apoptosis proteins family were remained unchanged. Taken together, these results indicated that the anti-proliferative effects of HDPL were associated with the induction of apoptotic cell death through regulation of several major growth regulatory gene products such as Bcl-2 family expression and caspase protease activity, and HDPL may have therapeutic potential in human lung cancer.

• Natural Product Sciences
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• 제26권3호
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• pp.207-213
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• 2020

Characterization and in vitro inhibition studies of protease inhibitor from the mushroom Pleurotus floridanus (PfTI) towards the pest Papilio demoleus is studied. The addition of 1 mM Mn²⁺, Na²⁺, Ba²⁺ and Ni ²⁺ enhanced the PfTI activity. The ICP-atomic emission spectrum showed the presence of Ca²⁺, Mg²⁺ and Zn²⁺ in the PfTI. Surfactants SDS and CTAB at a concentration of 1% reduced the PfTI activity whereas, the nonionic detergents Triton X and Tween 80 increased the activity. The inhibitory activity gradually decreased with increase in concentration of DMSO and H₂O₂. The activity was increased by dithiothreitol up to a concentration of 80 μM and inactivated at 140 μM. The activity of PMSF modified PfTI was drastically reduced to 0.234 U/mL at 4 mM concentration and similar results were obtained for modification of cysteine by N-Ethylmaleimide at slightly higher concentrations. The complex of trypsin and PfTI showed complete loss in fluorescence intensity at 343 nm compared with control. In vitro inhibition studies of PfTI with midgut proteases isolated from citrus pest P. demoleus with protease activity of 1.236 U was decreased to 0.613 U by 50 μL (0.1 mg/mL) of the inhibitor. Inhibitor was stable up to 0.04 M concentration of HCl.

• Parasites, Hosts and Diseases
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• 제60권1호
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• pp.1-6
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• 2022

The encystation of Acanthamoeba leads to the development of metabolically inactive and dormant cysts from vegetative trophozoites under unfavorable conditions. These cysts are highly resistant to anti-Acanthamoeba drugs and biocides. Therefore, the inhibition of encystation would be more effective in treating Acanthamoeba infection. In our previous study, a sirtuin family protein-Acanthamoeba silent-information regulator 2-like protein (AcSir2)-was identified, and its expression was discovered to be critical for Acanthamoeba castellanii proliferation and encystation. In this study, to develop Acanthamoeba sirtuin inhibitors, we examine the effects of sirtinol, a sirtuin inhibitor, on trophozoite growth and encystation. Sirtinol inhibited A. castellanii trophozoites proliferation (IC50=61.24 µM). The encystation rate of cells treated with sirtinol significantly decreased to 39.8% (200 µM sirtinol) after 24 hr of incubation compared to controls. In AcSir2-overexpressing cells, the transcriptional level of cyst-specific cysteine protease (CSCP), an Acanthamoeba cysteine protease involved in the encysting process, was 11.6- and 88.6-fold higher at 48 and 72 hr after induction of encystation compared to control. However, sirtinol suppresses CSCP transcription, resulting that the undegraded organelles and large molecules remained in sirtinol-treated cells during encystation. These results indicated that sirtinol sufficiently inhibited trophozoite proliferation and encystation, and can be used to treat Acanthamoeba infections.

• KSBB Journal
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• 제19권4호
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• pp.291-294
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• 2004

박테리오신의 일종인 leucocin A로 형질전환된 효모가 보이는, B. subtilis에 대한 항균 활성을 파악하기 위하여 형질 전환되지 않은 효모와 형질전환된 효모를 배양하면서 시간별로 채취하여 광학밀도, 건조중량, 총단백질량, 단백질 분해효소 그리고 항균활성을 측정하였다. Leucocin A의 항균활성은 성장양상과 비례하였다. 배양초기에 비해 12시간 배양 후에 항균활성이 3배 이상 증가하는 것으로 나타났고, 이때 B. subtilis의 성장이 70.57% 감소하는 것으로 나타났다. 정지기 이후에는 항균활성이 급속히 감소하였는데, 이는 항균활성을 보이는 leucocin A가 단백질이고, 단백질 분해효소가 정지기 이후 증가한 것에 의한 것으로 사료되었다. 이 연구는 향후 식품산업 등에 사용할 수 있는 bacteriocin의 산업적 생산에 필요한 기초자료를 제공할 수 있을 것이다.

• Asian-Australasian Journal of Animal Sciences
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• 제17권6호
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• pp.863-868
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• 2004

Lectin activities and chemical characteristics of Escherichia coli, Lactobacillus spp. and Bifidobacterium spp. originating from the porcine cecal mucosal layer were studied based on hemagglutination assay (HA) and hemagglutination inhibition assay (HIA). Although all the bacterial strains were able to agglutinate erythrocytes of porcine or rabbit origin, much higher HA titers were consistently observed for Lactobacillus spp. than for E. coli or for Bifidobacterium spp. A remarkable reduction in HA titers occurred by the treatment of E. coli and Lactobacillus spp. with protease or trypsin and of Bifidobacterium spp. with protease, trypsin or periodate. There were no significant effects on the HA titers of the three groups of bacteria after the treatment with lipase. Hemagglutination of E. coli was strongly inhibited by D (+)-mannose and D (+)-galactose; Lactobacillus spp. by $\alpha$-L-rhamnose and methyl-$\beta$-galactopyranoside; Bifidobacterium spp. by D (+)-alactose, $\alpha$-L-rhamnose, $\alpha$-L-fucose, L (+)-arabinose, D (+)-mannose, D (-)-fructose at a relatively low concentration (1.43 to 3.75 mg/ml). These results, combined with the enhanced HA activities of the three bacterial strains by modification of rabbit erythrocytes with neuraminidase and abolished HA activity of E. coli after treatment with $\beta$-galactosidase, indicate that it might be the glycoproteinous substances surrounding the surface of the bacterial cells that are responsible for the adhesions of these microorganisms by recognizing the specific receptors on the red blood cell.

• Journal of Microbiology
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• 제43권3호
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• pp.237-243
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• 2005

The protease purified from Bacillus cereus JH108 has the function of leucine specific endopeptidase. When measured by hydrolysis of synthetic substrate (N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide), the enzyme activity exhibited optimal activity at pH 9.0, $60^{\circ}C$. The endopeptidase activity was stimulated by $Ca^{++},\;Co^{++},\;Mn^{++},\;Mg^{++},\;and\;Ni^{++}$, and was inhibited by metal chelating agents such as EDTA, 1,10-phenanthroline, and EGTA. Addition of serine protease inhibitor, PMSF, resulted in the elimination of the activity. The endopeptidase activity was fully recovered from the inhibition of EDTA by the addition of 1 mM $Ca^{++}$, and was partially restored by $Co^{++}\;and\;Mn^{++}$, indicating that the enzyme was stabilized and activated by divalent cations and has a serine residue at the active site. Addition of $Ca^{++}$ increased the pH and heat stability of endopeptidase activity. These results show that endopeptidase requires calcium ions for activity and/or stability. A Lineweaver-Burk plot analysis indicated that the $K_m$ value of endopeptidase is 0.315 mM and $V_{max}$ is 0.222 ) is $0.222\;{\mu}mol$ of N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide per min. Bestatin was shown to act as a competitive inhibitor to the endopeptidase activity.

• Journal of Microbiology and Biotechnology
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• 제20권5호
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• pp.950-957
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• 2010

Serratiopeptidase (SRP), a 50 kDa metalloprotease produced from Serratia marcescens species, is a drug with potent anti-inflammatory property. In this study, a powerful statistical design, evolutionary operation (EVOP), was applied to optimize the media composition for SRP production in shake-flask culture of Serratia marcescens NRRL B-23112. Initially, factors such as inoculum size, initial pH, carbon source, and organic nitrogen source were optimized using one factor at a time. The most significant medium components affecting the production of SRP were identified as maltose, soybean meal, and $K_2HPO_4$. The SRP so produced was not found to be dependent on whey protein, but rather was notably induced by most of the organic nitrogen sources used in the study and free from other concomitant protease contaminant, as revealed by protease inhibition study. In addition, experiments were performed using different sets of EVOP design with each factor varied at three levels. The experimental data were analyzed with a standard set of statistical formula. The EVOP-optimized medium, with maltose 4.5%, soybean meal 6.5%, $K_2HPO_4$ 0.8%, and NaCl 0.5% (w/v), gave a SRP production of 7,333 EU/ml, which was 17-fold higher than the unoptimized media. The application of EVOP resulted in significant enhancement of SRP production.