• BMB Reports
• /
• v.35 no.2
• /
• pp.206-212
• /
• 2002

The NS2B-NS3(pro) polyprotein segment from the dengue virus serotype 2 strain 16681 was purified from overexpressing E. coli by metal chelate affinity chromatography and gel filtration. Enzymatic activity of the refolded NS2B-NS3(pro) protease complex was determined in vitro with dansyl-labeled peptide substrates, based upon native dengue virus type 2 cleavage sites. The 12mer substrate peptides and the cleavage products could be separated by reversed-phase HPLC, and were identified by UV and fluorescence detection. All of the peptide substrates (representing the DEN polyprotein junction sequences at the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 sites) were cleaved by the recombinant protease NS2B-NS3(pro). No cleavage was observed with an enzymatically inactive S135A mutant of the NS3 protein, or with a modified substrate peptide of the NS3/NS4A polyprotein site that contained a K2093A substitution. Enzymatic activity was dependent on the salt concentration. A 50% decrease of activity was observed in the presence of 0.1M sodium chloride. Our results show that the NS3 protease activity of the refolded NS2B-NS3(pro) protein can be assayed in vitro with high specificity by using cleavage-junction derived peptide substrates.

• YAKHAK HOEJI
• /
• v.41 no.4
• /
• pp.421-432
• /
• 1997

A new lectin was partially purified from starfish,Asterina pectinifera by means of physiological saline extraction, salt fractionation, ion exchange chromatography and hy droxyapatite chromatography, and it was named APL. The biochemical properties of the APL were characterized. In addition, its effects on lymphocyte mitogenicity and cancer cell agglutinability were tested. The APL agglutinated nonspecifically human erythrocytes and rabbit blood cells. Agglutinability was decreased to 30% of control activity below pH 5 and above pH 9 and was relatively unstable at increasing temperatures above 60$^{\circ}C$. The activity was reduced by addition of two kinds of metal ions, $Ba^{2+},\;Mn^{2+}$ and chelating agent, EDTA. APL was proved to be glycoproteins containing 9% sugars. For carbohydrate specificity, it was found that the activity of APL was inhibited by D(+)-glucosamine, D(+)-galactosamine, stachyose, N-acetyl-galactosamine and methyl-${\alpha}$-D-galactopyranoside among 35 sugars tested. In amino acid composition, the contents of acidic amino acids such as aspartic acid and glutamic acid were relatively high. This result suggest that the isoelectric point would be in a lower range. APL was found that it promotes the division of human lymphocytes. APL was proved to be a potent agglutinin for cancer cells such as HeLa, L929 and L1210 cells. Significant changes on the HeLa cell surfaces affected by APL were observed under the electron microscope.

• Microbiology and Biotechnology Letters
• /
• v.48 no.4
• /
• pp.491-505
• /
• 2020

A newly isolated strain, Proteus vulgaris OR34, from olive mill waste was found to secrete an alkaline extracellular lipase at 11 U·ml-1 when cultivated on an optimized liquid medium. This lipase was purified 94.64-fold with a total yield of 9.11% and its maximal specific activity was shown to be 3232.58 and 1777.92 U·mg-1 when evaluated using the pH-stat technique at 55℃ and pH 9 and Tributyrin TC4 or olive oil as the substrate. The molecular mass of the pure OR34 lipase was estimated to be around 31 kDa, as revealed by SDS-PAGE and its substrate specificity was investigated using a variety of triglycerides. This assay revealed that OR34 lipase preferred short and medium chain fatty acids. In addition, this lipase was stable in the presence of high concentrations of bile salt (NaDC) and calcium ions appear not to be necessary for its activity. This lipase was inhibited by THL (Orlistat) which confirmed its identity as a serine enzyme. In addition, the immobilization of OR34 lipase by adsorption onto calcium carbonate increased its stability at higher temperatures and within a larger pH range. The immobilized lipase exhibited a high tolerance to organic solvents and retained 60% of its activity after 10 months of storage at 4℃. Finally, the OR34 lipase was applied in biodiesel synthesis via oleic acid mediated esterification of methanol when using hexane as solvent. The best conversion yield (67%) was obtained at 12 h and 40℃ using the immobilized enzyme and this enzyme could be reused for six cycles with the same efficiency.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.38 no.9
• /
• pp.1284-1288
• /
• 2009

Identification of various inorganic compound crystals contained in solar salts, which are produced from 12 areas of Jeonnam, was firstly made by the X-ray diffraction (XRD) technique. The analysis of the XRD spectra was carried out on the basis of Joint Committee on Powder Diffraction Standards (JCPDS) data and the results of Energy Dispersive X-ray Spectrometer (EDX) measurements. In particular, the analysis of the XRD spectra supported that each solar salt contains $Na_2S$ (Shinan Jeungdo and Sinui), $KMgCl_3$ (Shinan Bigeum), $Ca(ClO_3)_2$ (Shinan Docho), $CaAl_4O_7$ (Haenam Songji), $CaSiO_3$ and $CaCl_2$ (Goheung) as inorganic compound crystals, which have not been reported for the solar salts. Also, the XRD results indicated that the solar salts maintain a cubic NaCl crystal structure without any change of lattice parameters etc. However, it was shown in the Field Emission Scanning Electron Microscope (FE-SEM) images that an external form of the solar salts has a lamination layer shape of a cubic structure, which is different from a simple cubic form for the purified salts and the reagent NaCl.

• Korean Journal of Microbiology
• /
• v.45 no.2
• /
• pp.193-199
• /
• 2009

A bacterium producing the halotolerant extracellular protease was isolated from squid jeotgal, and was identified as Bacillus sp. SJ-10 based on morphological, physiological and biochemical characteristics, as well as phylogenetic analysis using 16S rRNA gene sequence. The strain grew at $20^{\circ}C\sim55^{\circ}C$, pH 5~8, and 0%~14% NaCl and optimal growth conditions were $35{\pm}5^{\circ}C$, pH 7, and 5% NaCl. The major cellular fatty acids were anteiso-$C_{15:0}$, anteiso-$C_{17:0}$, and $C_{16:0}$ DNA G+C content was 50.58 mol% and menaquinone consisted of MK-7 Phylogenic analysis based on the 16S rRNA gene sequence indicated that SJ-10T belongs to the genus Bacillus. About 40 kDa of the salt-tolerant protease was purified by 40% ammonium sulfate saturation and Mono Q column chromatography. The optimal activity of the protease was pH 8 and stable at pH 5~10. The optimum temperature and NaCl concentration were $35{\pm}5^{\circ}C$ and $5{\pm}1%$, respectively.

• Korean Journal of Medicinal Crop Science
• /
• v.20 no.2
• /
• pp.124-128
• /
• 2012

When ginseng seedlings are cultured in paddy fields, quality degradation and yield reduction are induced by severe plant loss with chlorosis on leaves occurred physiological disorder by excessive salt and poor drainage, rusty-root occurrence, and root rot etc. Accordingly, in order to solve these problems, this study was performed to investigate the treatment method, concentrations and time of activated ion calcium as environment-friendly agricultural materials. Activated ion calcium is an enriched and purified water-soluble mineral calcium component for absorbing quickly into plant as a highly functional calcium and it is an alkaline calcium of 37% (370 $m{\ell}$/1 ${\ell}$) concentration with pH 13. Treatment method was that ginseng seeds were sown after removing water in the shade after seed immersion for 1 minute with active ion calcium of 20-fold diluted solution, and then irrigated $4{\ell}$ per 3.3 $m^2$ with 200-fold, 400-fold, and 600-fold diluted solution before emergence on late March, and supplied 1 time on leaves with 500-fold diluted solution in June and July respectively. The disease rate by treatment of activated ion calcium was that on the treatment of soil irrigated with 200-fold diluted solution compared to non-treated soil, damping-off was 33%, anthracnose was 100% reduced and the occurrence rate of rusty-root was 30% reduced. In addition, when active ion calcium of 200-fold diluted solution were soil irrigated, first and second grade ginseng were respectively 26% and 22% produced more, compared with control.

• Microbiology and Biotechnology Letters
• /
• v.16 no.5
• /
• pp.335-342
• /
• 1988

An anion exchange chromatography was employed for the purification of mouse monoclonal antibodies from ascitic fluid and in vitro cultivation media. After cultivation of hybridomas, Alps 25-3, HCGK, A4W, and KW, producing IgG1, the culture supernatants were harvested by centrifugation, precipitated with 50-60％ ammonium sulfate, and dialyzed against 0.025 M Tris-HCI buffer (pH 8.2). Then the dialyzed samples were loaded into a DEAE-Trisacryl M anion exchange column. Monoclonal antibodies bound to the DEAE-Trisacryl M were eluted with 0.025 M Tris-HCI buffer (pH 8.2) containing 30-40 mM NaCl. In ammonium sulfate precipitation, the recovery of the monoclonal antibody was shown to be 90％ and 84％ from in vitro culture media containing 10％ and 2％ fetal bovine serum, respectively. On the other hand, the pretreatment by ultrafiltration enhanced the yield up to 91％ whereas the purity was lower than that by ammonium sulfate treatment. Subsequently, in the DEAE-Trisacryl M chromatographic separation, the purities and recoveries of all the monoclonal antibodies from both the in vitro culture supernatants and ascitic fluids were 70-80％ and 65％ respectively. The monoclonal antibody, Alps 25-3 could be further purified with a purity of 95％ through an immunoadsorbent chromatography.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.12 no.3
• /
• pp.207-211
• /
• 1983

An attempt was made to find out the possibility of utilizing water-melon seed as resources of food fats and protein. The water-melon seed contained 40.40% of crude fat and 28.36% of crude protein. The lipid fraction obtained by silicic acid column chromatography was composed of about 97.35% neutral lipid, and the main components of neutral lipid by thin layer chromatography were triglyceride(50.40%), diglyceride(21.84%) and sterol(11.48%). The predominant fatty acids of total and major lipid classes were linoleic acid(55.30-67.85%), palmitic acid(12.07-28.12%) and oleic acid(9.06-16.40%), whereas stearic acid and linolenic acid were detected as small amounts. The salt soluble protein of watermelon seed was highly dispersible in 0.02M sodium phosphate buffer containing about 0.7M $MgSO_4$, and the extractability of seed protein was about 27%. Glutamic acid and arginine were major amino acids, and the essential amino acids such as lysine, threonine, valine, methionine, isoleucine, leucine and phenylalanine were also detected. The electrophoretic analysis showed 6 bands in water-melon seed protein, and the collection rate of the main protein fraction purified by sephadex G-100 and G-200 was 52.4%. The amino acids of the main fraction protein were also mainly composed of glutamic acid and arginine. The molecular weight for the main protein of the water-melon seed was estimated to be 120,000.

• Journal of Korean Society of Forest Science
• /
• v.35 no.1
• /
• pp.24-32
• /
• 1977

According to the chlorous salt method, most of holocellulose whose lignin was removed, was obtained. In extracting xylan from holocellulose by the different densities of alkali, 5% KOH was extracted three times but still there remained part of xylan in it and another composite of hemicellulose and cellulose was obtained. The extraction of 10% and 20% KOH showed a desirable result. Rather than the ordinary method to use a large quantity of ethanol in the precipitation isolation of xylan, the method to use a small quantity of ethanol in adopting the dialysis with cellophane-membrane by condensing density to one tenth, made il possible to extract a high purity xylan in a high retrieving rate. In isolating glucomannan, the residue of 5% KOH extraction contained a large quantity of xylan, the residue of 10% and 24% KOH extraction, also showed the same result and the comparison between glucose and mannose was approximately 1 : 1. The purification of Fehling solution made it possible to obtain comparatively pure xylan but the process of oxidation dissolution was complicated and the retrieving rate was low. This was not a good method. The ethanol titration purified a high purity xylan in a high retrieving rate and was a very excellent purifying method, considering its simple and easy process. These two purifying methods, however, could not completely remove the residue of arabinose. This will be examined and reported later.

• Korean Journal of Food Science and Technology
• /
• v.16 no.2
• /
• pp.235-241
• /
• 1984

Two fractions of pectinesterase from Chinese cabbage were isolated by ammonium sulfate fractionation, ion exchange chromatography on DEAE-cellulose and Sephadex G-150 gel filtration. The fraction F-A and F-B were purified approximately 340- and 10-fold. The similar salt effects and pH optima (pH 7.5-8.0) were obtained for the two pectinesterase fractions. The maximum activity of both two. fractions were obtained at 20-50mM of divalent rations and at 250mM of monovalent rations. The apparent Michaelis constant of the F-A was 0.01% for citrus pectin. The temperature optima for F-A and F-B were $48^{\circ}$ and $55^{\circ}C$, respectively and both fractions were stable in the region of pH 5.0-8.0 at room temperature. The thermal inactivation of the two fractions followed the first order reaction kinetics. From D and Z-values obtained the thermal resistance of the two fractions were characterized.