• Mycobiology
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• v.37 no.2
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• pp.121-127
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• 2009

Purification and characterization of intracellular cellulase produced by A. oryzae ITCC-4857.01 are reported. The enzyme was purified by ion-exchange chromatography using DEAE-cellulose followed by Gel filtration. The purification achieved was 41 fold from the crude extract with yield of 27%. The purified enzyme showed single band on poly acrylamide gel. The molecular weight as determined by SDS-PAGE and gel filtration was 38 KDa and 38.6 KDa respectively and contained only one subunit. The enzyme is glycoprotien as nature and contained 0.67% neutral sugar. The apparent Km value of the enzyme against cellulose was 0.83%. The enzyme showed the highest relative ativities on CMC followed by avicel, salicin and filter paper. The optimum pH of activity was 5.5 and very slight activity was observed at or above pH 7.5 as well as bellow pH 3.5. The optimum tempreture of the activity was $45^{\circ}C$ and the highest activity was exhibited in 35 to $45^{\circ}C$. The enzyme lost their activities almost completely (95${\sim}$100%) at $80^{\circ}C$ or above and as well as bellow $25^{\circ}C$.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.10 no.3
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• pp.620-624
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• 2009

Apolipophorin-III (apoLp-III) was isolated and purified from the last instar larval hemolymph of Galleria mellonella by gel chromatography (Sephadex G-100) and ion exchange chromatography (CM-52). In the present study, I wanted to show that apoLp-III is taken up into the adult ovary in Galleria mellonella. Adult ovary tissues were incubated at room temperature for 30 min with fluorescein isothiocyanate (FITC)-labeled apoLp-III. Fluorescence microscopy and sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) revealed that adult ovary tissues internalize fluorescence-labeled apoLp-III. The results suggest that apoLp-III is taken up by the adult ovary.

• Journal of the Korean Society of Food Science and Nutrition
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• v.27 no.5
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• pp.808-812
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• 1998

Melania snail(Semisulcopira bensoni) is used as ingredient in Korean traditional soup and nutritional foods. Generally, lipoxygenase in several food products may produce off-flavors during their processing and storage. Therefore, the inactivation of lipoxygenase is required to make the better extracts from Melania sanil. Also, the quality on freshness of Melania snail may be evaluated by lipoxygenase activity. The lipoxygenae activity was the highest at 40~60% saturation among several concentrations in salting-ouot saturated solution of ammonium sulfate. The partial purification of lipoxygenase was successfully obtained by Sephacryl S-200 gel chromatography. The first peak among three peaks for protein determination showed the highest activity of lipoxygenase in 13~16 fractions among 100 fractions. The highest peak of lipoxygenase activity by ion exchange chromatography was shown at 0.1M NaCl. In the purification step, the specific activity was 20.8U/mg and activity yield was 19.8%. The optimum pH and temperature were pH6.0~8.0 and 3$0^{\circ}C$, respectively. Molecular weight of the lipoxygenase was estimated about 35kDa by SDS-PAGE.

• Journal of the Korean Society of Food Science and Nutrition
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• v.21 no.1
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• pp.76-83
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• 1992

Enterotoxigenic E. coli is one of the major causative agents of the infantile diarrhea and traveler's diarrhea. The heat-stable enterotoxin (ST) is thought to be a virulence factor in the pathogenesis of the diarrhea and to be a maker for identification of the enterotoxigenic E. coli from non pathogenic E. coli. ST producing E. coli KM-7 strain was isolated from the swine and molecular cloning of ST gene of KM-7 strain. Transformant eKT-53 $(ST^+,\;LT^-)$ was selected by infant mouse assay (IMA). The culture supernatant of eKT-53 strain was performed purification by multipled steps. The culture supernatant (crude ST) was purified by sequentially applying batch adsorption chromatography on Amberlite XAD-2 resin, ion exchange chromatography on DEAE-Sephacel anion exchanger, gel filtration chromatography on Bio-Gel P-6 and preparative polyacrylamide slab gel electrophoresis. About 113-fold purification was achieved with a yield of about 11% of crude ST and the minimum effective dose(MED) of this purified ST was about 2.8ng in IMA. Homogeneity of purified ST was demonstrated by showing a single band in analytical SDS polyacrylamide disc gel electrophoresis.

• Korean Journal of Environmental Biology
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• v.29 no.4
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• pp.305-311
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• 2011

The distribution of flocculating activity of culture broth was examined and the major constituent with flocculating activity was identified. Most of flocculating activity was found in culture broth without cells. As the activity was maintained by the digestion with pronase, it suggests that the activity is due to the polysaccharide. The bioflocculant obtained from $Lactobacillus$ $jensenii$ YW-33 was precipitated by 60~80% EtOH fractionation (LJ-80). LJ-80 was separated by ion-exchange chromatography using DEAE-Toyopearl 650C and LJ-80-II showed more potent flocculating activity than those of other fractions. The major activity fraction LJ-80-II was further purified on the gel permeation using Sepharose CL-6B to LJ-80-II-1. GPC (Sepharose CL-6B) and HPLC were used to determine whether LJ-80-II-1 has a homogenecity. The molecular weight of purified LJ-80-II-1 was estimated over 800,000 dalton by gel permeation chromatography. Purified LJ-80-II-1 contained 98.4% total sugar, 0.6% protein. Main sugar of purified LJ-80-II-1 was composed of mannose : galactose : glucose with a molar ratio of 1.61 : 0.25 : 1.00.

• Journal of Microbiology and Biotechnology
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• v.26 no.10
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• pp.1781-1789
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• 2016

Glargine insulin is a long-acting insulin analog that helps blood glucose maintenance in patients with diabetes. We constructed the pPT-GI vector to express prepeptide glargine insulin when transformed into Escherichia coli JM109. The transformed E. coli cells were cultured by fed-batch fermentation. The final dry cell mass was 18 g/l. The prepeptide glargine insulin was 38.52% of the total protein. It was expressed as an inclusion body and then refolded to recover the biological activity. To convert the prepeptide into glargine insulin, citraconylation and trypsin cleavage were performed. Using citraconylation, the yield of enzymatic conversion for glargine insulin increased by 3.2-fold compared with that without citraconylation. After the enzyme reaction, active glargine insulin was purified by two types of chromatography (ion-exchange chromatography and reverse-phase chromatography). We obtained recombinant human glargine insulin at 98.11% purity and verified that it is equal to the standard of human glargine insulin, based on High-performance liquid chromatography analysis and Matrix-assisted laser desorption/ionization Time-of-Flight Mass Spectrometry. We thus established a production process for high-purity recombinant human glargine insulin and a method to block Arg (B31)-insulin formation. This established process for recombinant human glargine insulin may be a model process for the production of other human insulin analogs.

• Microbiology and Biotechnology Letters
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• v.11 no.1
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• pp.47-52
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• 1983

The extracellular inulase produced by Aspergillus sp. C-58 was isolated by pH and charcoal treatment, precipitation with ammonium sulfate from the crude extract, and separated into 3 fractions (P-I, II, III) by DEAE-cellulose column chromatography in the ratio of 31.1:1.7:1 with respect to the activity. The ratio of inulase activity to sucrase activity of P-I, P-II and P-III fraction was 0.23, 0.24 and 1.1 respectively. The enzyme P-I fraction was purified 482 fold with a 22.8% yield by DEAE-Sephadex A-50, Sephadex G-75, Sephadex G-100 (1st and 2nd) column chromatography, and appeared homogeneous on polyacrylamide disc gel electrophoresis and ultracentrifugation.

• Korean Journal of Food Science and Technology
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• v.28 no.5
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• pp.859-864
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• 1996

The rhamnan sulfat extracted from green algae seaweed, Monostroma nitidum was characterized on sugars, sulfate compositions and molecular structure. Rhamnan sulfate was extracted with boiling water, and purified with two steps of cetylpyridinium chloride and ion exchange chromatography. The yield of crude rhamnan sulfate was about 2% from raw material. Rhamnan sulfate fraction, F-4 was composed of 30% rhamnose, 0.9% arabinose, 2.5% xylose, 2% glucose and 32.6% sulfate. Rhamnan sulfate F-4-3 obtained from F-4 fraction was composed of 36.8% rhamnose, 3.6% xylose, 2.7% glucose, 1.4% galactose and 30.8% sulfate. The molecular weight of F-4-3 fraction was estimated as 10,000-10,300 dalton with Sephacryl S-200 gel filtration chromatography.

• Reproductive and Developmental Biology
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• v.34 no.4
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• pp.275-281
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• 2010

The aim of this study was to investigate what protein(s) of porcine epididymal fluid (pEF) are able to enhance the nuclear maturation of porcine germinal vesicle (GV) oocytes in vitro. Proteins of pEF were fractionated by affinity, ion exchange, and gel filtration chromatography. Porcine cumulus-oocytes complexes (COC) from follicles were cultured in tissue culture medium (TCM 199) containing various fractions obtained by chromatography. Porcine COCs were also cultured in TCM 199 containing various meiosis inhibitors and pEF. After 24 or 48 h culture, oocytes were examined for evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II. When porcine COCs were cultured in the medium with meiosis inhibitor such as, dibutyryl cAMP (dbcAMP) and forskolin (Fo), more than 80% of oocytes were unable to resume meiosis. However, porcine COCs supplemented with pEF were able to overcome the inhibitory effect of dbcAMP and Fo. Maturation rate of oocytes was significantly (p<0.05) increased in the media supplemented with cationic protein(s) during in vitro maturation than in those with anionic protein(s) (44.1% vs 20.0%). When oocytes were cultured in the TCM 199 with fractions obtained by gel filtration, the maturation rate of oocytes was significantly (p<0.05) higher in fraction 11 containing 18 kDa than other fractions. The present study suggests that 1) dbcAMP and Fo prevent the spontaneous maturation of oocyte after isolation from follicles, and that pEF contain a substance(s) that improves meiosis resumption in vitro of porcine COCs, 2) cationic 18 kDa protein(s) are responsible for promotion of Mil stage.

• Korean Journal of Microbiology
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• v.32 no.2
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• pp.132-138
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• 1994

L-Ascorbic acid-producing enzyme in Neurospora crassa was found to exist in mitochondria and the activity of this enzyme was increased by the addition of D-fluconno-${\gamma}$-lactone or L-gulono-${\gamma}$-lactone in the media. L-Ascorbic acid-producin enzyme in N. crassa has been purified with ammonium sulfate precipitation. DEAE Sepharose CL-6B ion exchange chromatography. Sephacryl S-200 gel filtration chromatography and Reactive yellow 3-agarose dye affinity column chromatography. The specific activity of this enzyme was increased to 239.6 fold and the yield was 2.1%. The molecular weight of the native enzyme was 150.000 dalton when it was estimated with Sephacryl S-200 gel filtration chromatography. Its molecular weight appeared as 75.000 dalton by SDS-polyacrylamide gel electrophoresis. which suggested that this enzyme was consisted with two identical subunits. The optimal pH for this enzyme was 9.0 and the $K_m$ value for D-galactono-${\gamma}$-lactone was 0.073 mM.