• Journal of Life Science
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• v.19 no.3
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• pp.397-402
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• 2009

To isolate bacteria secreting protease, which can dissolve fibrin efficiently, we prepared chunggukjang using rice straw and isolated, preliminarily, approximately 100 bacterial stains. Their capabilities to dissolve milk protein as well as fibrin included in media were then examined and finally, five strains named J1 - J5 were selected. Among them, J-4, which is close to bacillus subtilis, showed highest activity for fibrin dissolution. Proteases secreted from the J-4 strain were partially purified from culture supernatant using DEAE-sepharose column chromatography and identified with SDS-polyacrylamide gel electrophoresis. Three proteins were subjected to analysis with MALDI-TOF and PMF (Peptide Mass Fingerprinting). 41.9 kDa protein was identified as a neutral protease. On the other hand, 45 kDa protein turned out to be bacillopeptidase F, with a molecular mass of 91.7 kDa, indicating that partially purified peptide is a degradation product.

• Journal of Microbiology
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• v.45 no.5
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• pp.402-408
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• 2007

A strain producing a potent protease was isolated from turban shell. The strain was identified as Bacillus sp. S17110 based on phylogenetic analysis. The enzyme was purified from culture supernatant of Bacillus sp. S17110 to homogeneity by ammonium sulfate precipitation, SP-Sepharose, and DEAE-Sepharose anion exchange chromatography. Protease activity of the purified protein against casein was found to be stable at pH 7 to pH 10 and around $50^{\circ}C$. Approximately 70% of proteolytic activity of the enzyme was detected either in the presence of 100 mM SDS or Tween 20. The enzyme activity was enhanced in the presence of $Ca^{2+},\;Zn^{2+},\;Mg^{2+}$, but was inhibited by EDTA, indicating that it requires metal for its activity. The purified enzyme was found to be a monomeric protein with a molecular mass of 75 kDa, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography. The purified enzyme was analyzed through peptide fingerprint mass spectra generated from matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) and a BLAST search, and identified as immune inhibitor A (inhA) deduced from nucleotide sequence of B. cereus G9241. Since InhA was identified as protease that cleave antibacterial proteins found in insect, inhA-like protease purified from Bacillus sp. S17110 might be pathogenic to sea invertebrates.

• Journal of the Korean Society of Food Science and Nutrition
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• v.20 no.6
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• pp.551-558
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• 1991

We investigated the physicochemical and functinal properties of rapeseed(Brassica napus var. Youngsan) protein prepared by combining various soluvent and purification procedures, such as ultrafiltration (UF) concentration and acid-washin. The lightness value(YCIE) of each protein was greadully improved and its hydrophobicity increased by the degree of purification. The analysis of each protein by sodium dodecyl sulfate-plyacrylamide gel electrophoresis (SDS-PAGE) had nine bands revealed without difference, the considerable portion of which were of $1.96~1.59{\times}10^4$ dalton molecular weight. The content of amino acid increased a little more in the other processed proteins than in the control, and decreased considerably in the proteins extracted by the mixed solvent of 1% sodium hexametaphosphate(SHMP) and 0.25M ethlene diamine tetraacetic acid (EDTA). The better proteins were purified, the lower the kinematic viscosities were in their values. The water absorption and foaming properties were scarcely different according to the processes. The oil absorption and the emulsion activity index normally increased according to the degree of purification. The properties of heat coagulation revealed high values only in the proteins processed by EDTA while they showed considerably low values the other proteins.

• Microbiology and Biotechnology Letters
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• v.22 no.3
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• pp.240-245
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• 1994

Relation the phage defense mechanism of phage resistant Lactococcus lactis subsp. cremoris ATCC 11602-A1 to its cell wall components was investigated. To determine whether teichoic acid which is known to be one of the phage receptor site present on the cell wall, phage adsorption was examined after treatment 5% TCA(60%$\CIRC $C) and concanavalin A to the cell wall of A1 and parent strain. However, the adsorption rate of two strains did not change. Total amount of phosphate after TCA treatment did not change in both strains, but a difference between the two strains was observed. Ribitol and glycerol, components of teichoic acid, could not be detected in the cell walls of two strains by GC analysis. These results suggest that although teichoic acid was not present in the cell walls of both strains, the composition of cell wall of two strains was not identical. Measurement of amount of protein and SDS-polyacryamide gel electrophoresis were carried out to examine the involvement of cell wall protein in phage resistance, showing that protein is nothing to do with phage adsorption of parent strain, but phage resistance of A1 is related to protein. Cell wall carbohydrates of A1 contained rhamnose, glucose, and galactose. Total amount of carbohydrate of 1% SDS-treated A1 cell wall was reduced to the level of parent strain. The results suggest that phage resistance of A1 was due to the presence of a higher level of carbohydrates then parent strain, and to interaction of carbohydrate and protein.

• Journal of Microbiology and Biotechnology
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• v.22 no.7
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• pp.930-938
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• 2012

High levels of xylanase activity (143.98 IU/ml) produced by the newly isolated Paenibacillus campinasensis G1-1 were detected when it was cultivated in a synthetic medium. A thermostable xylanase, designated XynG1-1, from P. campinasensis G1-1 was purified to homogeneity by Octyl-Sepharose hydrophobic-interaction chromatography, Sephadex G75 gel-filter chromatography, and Q-Sepharose ion-exchange chromatography, consecutively. By multistep purification, the specific activity of XynG1-1 was up to 1,865.5 IU/mg with a 9.1-fold purification. The molecular mass of purified XynG1-1 was about 41.3 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Sequence analysis revealed that XynG1-1 containing 377 amino acids encoded by 1,134 bp genomic sequences of P. campinasensis G1-1 shared 96% homology with XylX from Paenibacillus campinasensis BL11 and 77%~78% homology with xylanases from Bacillus sp. YA-335 and Bacillus sp. 41M-1, respectively. The activity of XynG1-1 was stimulated by $Ca^{2+}$, $Ba^{2+}$, DTT, and ${\beta}$-mercaptoethanol, but was inhibited by $Ni^{2+}$, $Fe^{2+}$, $Fe^{3+}$, $Zn^{2+}$, SDS, and EDTA. The purified XynG1-1 displayed a greater affinity for birchwood xylan, with an optimal temperature of $60^{\circ}C$ and an optimal pH of 7.5. The fact that XynG1-1 is cellulose-free, thermostable (stability at high temperature of $70^{\circ}C{\sim}80^{\circ}C$), and active over a wide pH range (pH 5.0~9.0) suggests that the enzyme is potentially valuable for various industrial applications, especially for pulp bleaching pretreatment.

• Applied Biological Chemistry
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• v.41 no.7
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• pp.522-526
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• 1998

Ferritin from germinated soybean seeds was purified by ammonium sulfate precipitation (0.55 saturation), ion-exchange chromatography on DEAE-cellulose, gel filtration chromatography on Sephacryl S-300, and HPLC with Bio-Scale Q2 column. SDS-PAGE analysis showed that the purified ferritin is composed of subunit with an apparent M, 21,000. The molecular mass of the native soybean ferritin estimated by gel filtration on Sephacryl S-300 and non-denaturing polyacrylamide gel electrophoresis appeared to be $510{\sim}560\;kDa$. Soybean ferritin contained 833 mol Fe/mol protein, which is 31-fold more iron than pumpkin ferritin and stained positive for iron on non-denaturing gel. Soybean ferritin cross-reacted with anti-soybean rabbit ferritin antiserum.

• Food Science of Animal Resources
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• v.43 no.2
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• pp.305-318
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• 2023

This study investigated the protein digestibility of chicken breast and thigh in an in vitro digestion model to determine the better protein sources for the elderly in terms of bioavailability. For this purpose, the biochemical traits of raw muscles and the structural properties of myofibrillar proteins were monitored. The thigh had higher pH, 10% trichloroacetic acid-soluble α-amino groups, and protein carbonyl content than the breast (p<0.05). In the proximate composition, the thigh had higher crude fat and lower crude protein content than the breast (p<0.05). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of myofibrillar proteins showed noticeable differences in the band intensities of tropomyosin α-chain and myosin light chain-3 between the thigh and breast. The intrinsic tryptophan fluorescence intensity of myosin was lower in the thigh than in the breast (p<0.05). Moreover, circular dichroism spectroscopy of myosin revealed that the thigh had higher α-helical and lower β-sheet structures than the breast (p<0.05). The cooked muscles were then chopped and digested in the elderly digestion model. The thigh had more α-amino groups than the breast after both gastric and gastrointestinal digestion (p<0.05). SDS-PAGE analysis of the gastric digesta showed that more bands remained in the digesta of the breast than that of the thigh. The content of proteins less than 3 kDa in the gastrointestinal digesta was also higher in the thigh than in the breast (p<0.05). These results reveal that chicken thigh with higher in vitro protein digestibility is a more appropriate protein source for the elderly than chicken breast.

• Analytical Science and Technology
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• v.22 no.3
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• pp.228-234
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• 2009

To gain further insight into herbicide detoxification of plant, we purified a glutathione S-transferase from Brassica oleracea (BoGST) and studied its substrate specificity towards several xenobiotic compounds. The BoGST was purified to electrophoretic homogeneity with approximately 10% activity yield by DEAE-Sephacel and GSHSepharose column chromatography. The molecular weight of the BoGST was determined to be approximately 23,000 by SDS-polyacrylamide gel electrophoresis and 48,000 by gel chromatography, indicating a homodimeric structure. The activity of the BoGST was significantly inhibited by S-hexyl-GSH and S-(2,4-dinitrophenyl)GSH. The substrate specificity of the BoGST displayed high activities towards CDNB, a general GST substrate and ethacrynic acid. It also exhibited GSH peroxidase activity toward cumene hydroperoxide.

• KSBB Journal
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• v.10 no.1
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• pp.63-70
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• 1995

The effect of operating conditions on separation of soybean proteins in a home-made free-flow electrophoresis apparatus was investigated. Measurement of the pH, conductivity, and UV-absorbance(280 nm) were carried out at each run and the purity of the sample was tested with SDS-PAGE analysis. The soybean extract pretreated with Tris and boric acid was mixed with the amino acids composed of glutamic acid, histidine, arginine, glycine(1 mM each) with glycyl-glycine(2mM) and KCl(1mM). When the cellulose acetate was used as a compartment between the electrode and the buffer solution in the cell, pH distribution in the separation cell varied from 3.0 at the anodic side to 8.0 at the cathodic side and had two inflection point. The applied voltage was from 300V to 1000V and the separation was better at a higher voltage but the voltage was limited by the capability of the cooling system due to Joule heat. The proteins focused near the middle of the channel. From the change of pH and conductivity it was found that the ions in the channel moved out to the electrodes through the membrane. In the case when the concentration of the buffer solution was increased 5 times, proteins were focused at 300V. We could not increase up to the ten times of the concentration since the temperature difference between inlet and outlet was more than $25^{\circ}C$ and denaturation of proteins was expected. When ion-exchange membranes were used U-type pH distribution was set up due to the ionic polarization near the membrane. The commercial ampholytes, instead of the mixed amino acids showed not much improvements in purity of the separated sample.

• Journal of Life Science
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• v.8 no.5
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• pp.614-621
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• 1998

An extracellular inulinase from Penicillium sp. which isolated from soil sample was purified to a single protein th-rough ammonium sulfate fractionation, DEAE-Sephacel ion exchange chromatography and Toyopearl HW 65 F gel filtration. The temperature and pH for the enzyme reaction were around 6$0^{\circ}C$ and 4.0, respectively. The enzyme was stable at 3$0^{\circ}C$-5$0^{\circ}C$ and in the pH range of 4 to 5. $CuCl_2$, $HgCl_2$ and EDTA inhibited the enzyme activity strongly. By contrast, $MnCl_2$ and $CaCl_2$ activated the enzyme activity. The molecular weight of the purified enzyme was esti-mated to be 77,000 dalton by SDS-polyacrylamide gel electrophoresis. The Km values of the enzyme for inulin were calculated to be $2.2\times10^{-3}$M. TLC analysis suggested that purified enzyme is exo-type inulinase. The NH2-terminal amino acid sequences of the purified enzyme was determined to be $NH_2$-X-Glu-Ser-Tyr-Thr-Glu-Lys/Leu-Tyr-Arg-Pro.