• Applied Biological Chemistry
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• v.15 no.2
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• pp.111-142
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• 1972

An experimental Chung-Kook-Jang was prepared using the strain Bacillus subtilis sp. isolated by the author Samples were taken in 12 hrs interval during the fermentation and the oligopeptides were separated by the method of molecular sieving using the ion exchange resin column of Dowex-50. Only the X-16 fraction of oligopeptides was taken and the components of oligopeptides were developed in two dimensional thin layer chromatograms. The each peptide spot was eluted and each peptide was isolated. The pattern and kinds of amino acids, and N and C-terminal amino acids were studied. Fourteen different oligopeptides could be detected by the two dimensional thin layer chromatography, all of which were consisted of $4{\sim}9$ kinds of amino acids. No dipeptides and no tripeptides could be found. The N and C-terminal amino acids and the residual component amino acids of all these 14 peptides could be summarized as the follows. [P]-I. Pro (Cys Ala Asp Trp Ile Val) Glu [P]-II. Val (His Arg Glu Thr Ala Met) Asp [P]-III. Glu (Cys Lys Asp Thr Met) Ala [P]-IV. Glu(His Ser Ala) Met) [P]-V. Ile (Cys Asp Arg Gly Pro T.p Phe) His [P]-VI. Gly(Asp ser) Lys [P]-VII. Thr(Pro Tyr Phe) Asp [P]-VIII. Phe(Tyr Leu Ile) Val [P]-IX. Trp (Phelle) Thr [P]-X. Ile (Arg Leu) Phe [P]-XI. Asp (Lys His Ser Gly Glu Pro) Ala [P]-XII. Glu (Cys Asp Gly) Ser [P]-XIII. Ala (Arg Tyr) Glu [P]-XIV. Met (Glu Ala) His It appears that the protease of the Bacillus subtilis K-27 syrain has rather wider range of specificity than proteases of Aspergoillus soya, pepsin, chymotrypsin, and trypsin.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.24 no.5
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• pp.273-288
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• 1991

To elucidate the physiological and biochemical differences between chondrichthyes and osteichthyes, the properties of the specific digestive enzymes in cat-shark, Cephaloscyllium umbratile, and mackerel, Scomber japonicus, were studied. Homogenous trypsin proved through the disc-electrophoresis, SDS-PAG electrophoresis and gel filtration was obtained from the pancreas of cat-shark by $50-70\%$ saturated ammonium sulphate fractionation, DEAE-Sephadex A-50 column chromatography, benzamidine-Sepharose 6B affinity chromatography and Sephadex G-75-120 gel filtration. Two types of trypsins were also obtained from the pyloric caeca of mackerel by $30-70\%$ saturated ammonium sulphate fractionation and the slightly modified procedure from the method adopted in the purification of cat-shark trypsin. The two trypsins, designated trypsin A and B, were proved their homogeneity by disc- and SDS-PAG electrophoresis and gel filtration. The molecular weights of the trypsins were estimated to be 31,700 for cat-shark trypsin, 30,000 for mackerel trypsin A and 29,000 for mackerel trypsin B by SDS-PAG electrophoresis, but those were estimated to be 21,500 for cat-shark trypsin, 23,700 for mackerel trypsin A and 21,500 for mackerel trypsin B by gel filtration. The trypsins exhibited their optimum conditions at pH 9.0 and on temperature ranged from $45^{\circ}C\;to\;50^{\circ}C$ for cat-shark, and at pH 8.0 and a temperature of $50^{\circ}C$ for mackerel trypsin A and B, respectively. The cat-shark trypsin was stable at pH 10.0 and the temperature below $10^{\circ}C$, whereas the mackerel trypsin A and B, were stable in the range over pH 7.0 to pH 9.0 below $10^{\circ}C$ and at pH 8.0 below $35^{\circ}C$, respectively. The mackerel trypsins were severely inhibited by some heavy metal ions such as $Ag^{2+},\;Cu^{2+}\;and\;Hg^{2+}$ compared to cat-shark trypsin. All of the enzymes were also inhibited by antipain, leupeptin, TLCK(tosyllysine chloromethyl ketone) and SBTI(soybean trypsin inhibitor) remarkably. The inhibitory effects of PMSF(phenylmethane sulphonylfluoride), DFP(diisopropyl fluorophosphate) and benzamidine were indicated that these enzymes belong to serine-proteases.

• Applied Microscopy
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• v.34 no.1
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• pp.31-42
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• 2004

Acute respiratory distress syndrome (ARDS) is a kind of acute lung injury characterized by inflammatory disruption of alveolar-capillary barrier and notorious for its high mortality. Neutrophils cause cell damage through the production of free radicals, inflammatory mediators, and proteases in ARDS. $PLA_2$ might serve a primary regulatory role in the activation of neutrophils. This present study was performed to elucidate the effect of rutin known as $PLA_2$ inhibitor on ARDS induced by endotoxin. Endotoxin had increased lung myeloperoxidase (MPO) activity, BAL (bronchoalveolar lavage) protein content, numbers of neutrophils in BALF (bronchoalveolar lavage fluid) compared with those of control rat (p<0.001). In addition, histological evidence of lung injury was correlated with neutrophil influx into alveolar space and cerrous perhydroxide granules were found in lining of endothelial cell, alveolar type I, II cells. In contrast, pretreated group of rutin had significantly decreased all of the parameters (p<0.001). These data suggest that inhibition of $PLA_2$ is one step approach that block the process of ARDS. Accordingly, we conclude that rutin can be used as the prophylactic agent for ARDS on the bases of these experimental results.

• Proceedings of the Korean Biophysical Society Conference
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• 2003.06a
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• pp.80-80
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• 2003

${\beta}$-lapachone(${\beta}$-Lap), a natural o-naphthoquinone, presents in the bark of the Lapacho tree. ${\beta}$-Lap is cytotoxic against a variety of human cancer cells and it potentiates the anti-tumor effect of Taxol. In addition, ${\beta}$-Lap has been reported to radiosensitize cancer cells by inhibiting the repair of radiation-induced DNA damage.In the present study, we investigated the cytotoxicity of ${\beta}$-Lap against RKO human colorectal cancer cells as well as the combined effect of ${\beta}$-LaP and ionizing radiation. An incubation of RKO cells with 5 ${\mu}$M of ${\beta}$-Lap for 4 h killed almost 90％ of the clonogenic cells. An incubation of RKO cells with 5 ${\mu}$M of ${\beta}$-Lap for 4 h or longer also caused massive apoptosis. Unlike other cytotoxic agents, ${\beta}$-Lap did not increase the expression of p53 and p21 and it suppressed the NFkB expression. The expression of Caspase 9 and 3 was minimally altered by ${\beta}$-Lap. Radiation and ${\beta}$-Lap acted synergistically in inducing clonogenic cell death and apoptosis in RKO cells when ${\beta}$-Lap treatment was applied after but not before the radiation exposure of the cells. Interestingly, a 4 h treatment with 5 ${\mu}$M of ${\beta}$-Lap starting 5 h after irradiation was as effective as that starting immediately after irradiation. The mechanisms of ${\beta}$-Lap-induced cell killing is controversial but a recent hypothesis is that ${\beta}$-Lap is activated by NAD(P)H: quinone-onidoreductase (NQO1) in the cells followed by an elevation of cytosolic Ca$\^$2+/ level and activation of proteases leading to apoptosis. It has been reported that NQO1 level in cells is markedly up-regulated for longer than 10 h after irradiation. Indeed, using immunological staining of NQO1, we observed a significant elevation of NQO1 expression in RKO cells 5h after 2-4 Gy irradiation. Such a prolonged elevation of NQO1 level after irradiation may be the reasons why the ${\beta}$-Lap treatment applied S h after irradiation was as effective as that applied immediately after irradiation in killing the cells. In view of the fact that the repair of radiation-induced damage is usually completed within 1-2 h after irradiation, it is highly likely that the ${\beta}$-Lap treahment applied 5 h after irradiation could not inhibit the repair of radiation-induced damage. For in vivo study, RKO cells were injected S.C. into the hind-leg of Nu/Nu mice, and allowed to grow to 130 mm3 tumor. The mice were i.p. injected with ${\beta}$-lapachone or saline 2 h after irradiation of tumors with 10 Gy of X-rays. The radiation induced growth delay was increased by 2.4 $\mu\textrm{g}$/g of ${\beta}$-lapachone. Taken together, we may conclude that the synergistic interaction of radiation and ${\beta}$-Lap in killing cancer cells is not due to radiosensitization by ${\beta}$-Lap but to an enhancement of ${\beta}$-Lap cytotoxicity by radiation through an upregulation of NQO1. The fact that NQO1 is elevated in tumors and that radiation causes prolonged increase of the NQO1 expression may be exploited to preferentially kill tumor cells using ${\beta}$-Lap in combination with radiotherapy.

• Journal of the Korean Society of Food Science and Nutrition
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• v.46 no.4
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• pp.435-441
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• 2017

The present study was carried out to evaluate the applicability of Tenebrio molitor larvae (mealworm) as a health functional food material in order to contribute to the development of the domestic insect industry and health functional food industry. Protein hydrolysates were prepared from mealworm powder by enzymatic hydrolysis using five different proteases (alcalase, bromelain, flavourzyme, neutrase, and papain), and the hydrolysates were then tested for their antioxidant activities. Based on available amino group contents and sodium dodecyl sulphate-polyacrylamide gel electrophoresis analyses, mealworms treated with alcalase ($4,781.39{\mu}g/mL$), flavourzyme ($5,429.35{\mu}g/mL$), or neutrase ($3,155.55{\mu}g/mL$) for 24 h showed high degree of hydrolysis (HD) value, whereas HD values of bromelain ($1,800{\mu}g/mL$) and papain-treated ($1,782.61{\mu}g/mL$) mealworms were much lower. Protein hydrolysates showing high HD values were further separated into > 3 kDa and ${\leq}3kDa$ fractions by a centrifugal filter system and then lyophilized, and the production yields of the low molecular weight protein hydrolysates (${\leq}3kDa$) by alcalase, flavourzyme, and neutrase were 42.05%, 26.27%, and 30.01%, respectively. According to the RC_{50} values of the protein hydrolysates (${\leq}3kDa$) obtained from three different antioxidant analyses, all three hydrolysates showed similar antioxidant activities. Thus, alcalase hydrolysates showing the highest production yield of low molecular weight protein hydrolysates were further tested for their inhibitory effects on peroxidation of linoleic acid by measuring thiobarbituric acid values, and the results show that peroxidation of untreated linoleic acid increased dramatically during 6 days of incubation. However, pretreatment with the hydrolysates ($100{\sim}800{\mu}g/mL$) significantly inhibited linoleic acid peroxidation in a dose-dependent manner over 6 days.

• Journal of the Korean Society of Food Science and Nutrition
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• v.46 no.10
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• pp.1164-1170
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• 2017

Protaetia brevitarsis larvae (PBL) has recently been registered as a temporary food in Korea, and this study evaluated the application potential of PBL proteins as health functional food materials. Protein hydrolysates were prepared from PBL powder by enzymatic hydrolysis using five different proteases (alcalase, bromelain, flavourzyme, neutrase, and papain), and based on the results from the peptide content and SDS-PAGE analyses, PBL treated with alcalase or flavourzyme showed a high degree of hydrolysis (HD) value, whereas the HD value of those treated with neutrase, bromelain, or papain was minimal. The protein hydrolysates showing a high HD value were separated further into the fractions of >3 kDa and <3 kDa by a centrifugal filter system and then lyophilized, and according to the $RC_{50}$ values of the protein hydrolysates (<3 kDa) obtained from three different antioxidant analyses; the alcalase hydrolysates showed the highest antioxidant activity. Therefore, the alcalase hydrolysates were tested further for their inhibitory effects on the peroxidation of linoleic acid by measuring the thiobarbituric acid values. The results showed that the peroxidation of untreated linoleic acid increased dramatically during 6 days of incubation, but a pretreatment with the hydrolysates ($100{\sim}800{\mu}g/mL$) significantly inhibited the linoleic acid peroxidation in a dose-dependent manner for 6 days. Our current studies are focused on the identification of active peptide sequences from alcalase hydrolysates.

• Journal of Aquaculture
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• v.22 no.1
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• pp.105-111
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• 2009

In this study, we carried out an experiment for estimation the larval digestibility in aspects which digestive enzymatic activities and nutrition of the rotifers, Brachionus rotundiformis. Thus we enhanced the digestive enzymatic activity through the addition of starch for the increase of digestibility of rotifer (starch-rotifer), and compared with the feed efficiency through rearing of the olive flounder, Paralichthys olivaceus used rotifer lipid-enriched with Algamac $2000^{(R)}$ (CE-rotifer). The digestive enzyme activities (except for TG-lipase), total protein contents, total essential amino acid, essential amino acids (methionin and phenylalanine) of starch-rotifer (the rotifer used a starch as additive, and enriched not) was assayed significantly higher than CE-rotifer (P<0.05). And total lipid, lipid classes (except for sterol) and fatty acids as DHA and EPA showed higher in CE-rotifer than starch-rotifer (P<0.05). But, sterol contents and ST/TG ratio were shown significantly higher in starch-rotifer (P<0.05). The flounder larvae supplied the two rotifers showed standard length and body weight that not significantly differed with ranges $3.72{\sim}3.79\;mm$ and $32.9{\sim}37.8\;mg$/larva on 6 days after hatching (DAH), respectively (P>0.05). However, these of 12 DAH showed the values of significantly higher to $5.94{\pm}0.249\;mm$, $144.0{\pm}23.86\;mg$/larva and $26.2{\pm}12.13%$ in standard length, body weight and survival in CE-flounder than that of starch-flounder (P<0.05). The hydrolytic enzymatic activities of flounder larvae severally supplied the two rotifers showed the significantly higher activities in acidic -amylase, neutral -amylase, TG-lipase, lysozyme and acidic phosphatase in starch-flounder on 5 DAH (P<0.05). But neutral $\alpha$-amylase, three proteases and two phosphatases of CE-flounder on 11 DAH showed the significantly higher activities than that of starch-flounder (P<0.05). Therefore, for the flounder, Paralichthys olivaceus larvae just depleted yolk was more beneficial to supply the feed, rotifer, enhanced the digestibility than to supply the feed lipid-enriched for aspect of larval digestibility up to 6 DAH, thereafter nutrition of absorption due to the development of digestive organs suggested that enrichment effect appeared with larval somatic growth. Consequently, investigation more detailed about the larval digestive physiological and nutritional requirement variations after 6 DAH will be necessary, thereafter.

• Food Science and Preservation
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• v.22 no.5
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• pp.727-734
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• 2015

In this study, three GRAS (generally recognized as safety) strain was isolated from Doenjang and Cheonggukjang and identified as a protease-producing microorganism, following the appearance of a clear zone around its colony when cultured on a medium containing skim milk. Based on an analysis of the nucleotide sequence of 16S ribosomal RNA, the strains wereas identified as Bacillus amyloliquefaciens and wereas therefore named Bacillus amyloliquefaciens CDD5, Bacillus amyloliquefaciens CPD4, and Bacillus amyloliquefaciens CGD3. Here, we analyzed the protease and ${\alpha}$-glucosidase inhibitory activities of the three B. amyloliquefaciens strains. Among the isolated strains, B. amyloliquefaciens CGD3 exhibited the highest protease activity (9.21 U/mL, 24 hr). The protease activities of B. amyloliquefaciens CDD5 and B. amyloliquefaciens CPD4 reached 1.14 U/mL and 8.02 U/mL, respectively, at 48 hr. The proteases from the three B. amyloliquefaciens strains showed the highest activities within a pH range of 8.0-9.0 at $50^{\circ}C$, and casein was found to be the preferred substrate on evaluating enzyme activity in the substrate specificity assay. The B. amyloliquefaciens strains exhibited maximal growth when the nutrient broth medium had an initial pH within the range of 5.0-10.0, 6-9% sodium chloride (NaCl), and 5% glucose. B. amyloliquefaciens CDD5 exhibited a low ${\alpha}$-glucosidase inhibition rate (5.32%), whereas B. amyloliquefaciens CPD4 and B. amyloliquefaciens CGD3 exhibited relatively higher inhibition rates of 96.89% and 97.55%, respectively.

• Journal of the Korean Society of Food Science and Nutrition
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• v.35 no.9
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• pp.1139-1145
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• 2006

In this study, antioxidant activities of enzymatic and methanolic extracts from E. cava stem and leave were evaluated by measuring the scavenging activities on 1,1 diphenyl 2 picrylhydrazyl (DPPH), hydroxyl radical, hydrogen peroxide and the inhibitory effects on DNA damage induced by oxidative stress of cells. Enzymatic extracts were prepared by enzymatic hydrolysis of both stem and leave using food grade five different carbohydrases (Viscozyme, Celluclast, AMG, Termamyl, Ultraflo) and five proteases (Protamex, Kojizyme, Neutrase, Flavourzyme, Alcalase). The enzymatic extracts were lower than methanolic extracts in polyphenol contents, but higher in extraction yield by approximately 30%. The enzymatic extracts were superior to methanolic extracts in DPPH and H2O2 scavenging activities and DNA damage protective effect. There were no significant antioxidant activity difference between stem and leave, but the extracts of leave were relatively better than those of stem. In this study it is suggested that E. cava stem as well as its leave would be a good raw materials for antioxidants compound extraction and enzymatic hydrolysis would be a good strategy to prepare antioxidant extracts from seaweeds.

• Journal of Life Science
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• v.33 no.10
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• pp.808-819
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• 2023

This study was conducted to develop a selection marker for the identification of the Bacillus licheniformis K12 strain in microbial communities. The strain not only demonstrates good growth at moderate temperatures but also contains enzymes that catalyze the decomposition of various polymer materials, such as proteases, amylases, cellulases, lipases, and xylanases. To identify molecular markers appropriate for use in a microbial community, a search was conducted to identify variable gene regions that show considerable genetic mutations, such as recombinase, integration, and transposase sites, as well as phase-related genes. As a result, five areas were identified that have potential as selection markers. The candidate markers were two recombinase sites (BLK1 and BLK2), two integration sites (BLK3 and BLK4), and one phase-related site (BLK5). A PCR analysis performed with different Bacillus species (e.g., B. licheniformis, Bacillus velezensis, Bacillus subtilis, and Bacillus cereus) confirmed that PCR products appeared at specific locations in B. licheniformis: BLK1 in recombinase, BLK2 in recombinase family protein, and BLK3 and BLK4 as site-specific integrations. In addition, BLK1 and BLK3 were identified as good candidate markers via a PCR analysis performed on subspecies of standard B. licheniformis strains. Therefore, the findings suggest that BLK1 can be used as a selection marker for B. licheniformis species and subspecies in the microbiome.