• Journal of Microbiology and Biotechnology
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• 제11권2호
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• pp.242-250
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• 2001

A Gram-positive bacterium (strain JB-13) that was isolated from soil as a producer of cyclodextrin glucanotransferase (CGTase) [EC 2.4.1.19] was identified as Panibacillus sp. JB-13. This CGTase could catalyze the transglucosylation reaction from soluble starch to L-ascorbic acid (AA). A main product formed by this enzyme with ${\alpha}-glucosidase$ was identified as $2-O-{\alpha}-D-glucopyranosyl{\;}_{L}-ascorbic$ acid (AA-2G) by the HPLC profile and the elemental analysis. CGTase was purified to homogeneity using ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Seohadex A-50, and gel chromatography on Sephacryl S-200HR. The molecular weight was determined to be 66,000 by both gel chromatography and SDS-PAGE. The isoelectric point of the purified enzyme was 5.3. The optimum pH and temperature was PH 7.0 and $45^{\circ}C$ respectively. The enzyme was stable in the range of pH 6-9 and at temperatures of $75{\circ}C$ or less in the presence of 15 mM ${CaCl_2}.\;{Hg^2+},\;{Mn^+2},{Ag^+},\;and\;{Cu^2+}$ all strongly inhibited the enzyme's activity.

• Journal of Microbiology and Biotechnology
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• 제14권3호
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• pp.607-611
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• 2004

Glycinin, one of the predominant storage proteins in soybeans, was examined as to whether it could be used as a calcium-binding mediator after chemical phosphorylation and enzymatic hydrolysis. Glycinin is composed of six subunits. One of the proglycinin subunits $(A_{la}B_{lb})$ was overexpressed in E. coli to obtain nonphosphorylated proteins with homogeneity. To investigate the enhanced calcium-binding properties of the phosphopeptides, the proglycinin was purified, phosphorylated, and hydrolyzed with trypsin. The proglycinin expressed in E. coli was purified by ammonium sulfate precipitation, ion-exchange chromatography, and cryoprecipitation. Chemical phosphorylation by sodium trimetaphosphate was performed to obtain phosphorylated proglycinin. After the phosphorylation, one-dimensional isoelectric focusing gel electroanalysis confirmed the phosphorylation of the proglycinin. The phosphorylated peptides were then hydrolyzed with trypsin, followed by a binding reaction with calcium chloride. The calcium-bound phosphopeptides were finally separated using immobilized metal $(Ca^{2+})$ chromatography. Consequently, a limited tryptic hydrolysate of the isolated phosphopeptides exhibited an enhanced calcium-binding ability, suggesting the potential of glycinin phosphopeptides as a calcium-binding mediator with greater availability.

• Journal of Microbiology and Biotechnology
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• 제24권6호
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• pp.788-794
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• 2014

An extracellular ${\beta}$-glucosidase from Aspergillus niger Au0847 was purified to homogeneity by precipitation with ammonium sulfate, anion exchange, and gel filtration. The purified protein was composed of two subunits with molecular masses of 110 and 120 kDa. Au0847 ${\beta}$-glucosidase exhibited relatively high thermostability and pH stability, and its highest activity was obtained at $65^{\circ}C$ and pH 4.6, respectively. As a potential metalloprotein, its enzymatic activity was potently stimulated by manganese ion and DTT. The ${\beta}$-glucosidase displayed avid affinity and high catalytic efficiency for geniposide. Au0847 ${\beta}$-glucosidase has potential value as an industrial enzyme for the hydrolysis of geniposide to genipin.

• 해양환경안전학회:학술대회논문집
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• 해양환경안전학회 2004년도 춘계학술발표회
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• pp.81-84
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• 2004

Control of Sediment is very important in prawn farm due to the eruption of toxic material such as W1ionized H2S, NH3 and NO2-. In this study, column test study, column with filter media such as activated carbon, zeolite, oyster shell and iron chloride to evaluate the reduction of toxicity from sediment ammonia-N(NH3) was effectively removed by Zeolite and oyster shell. It was indicated that ammonium ion(NH4+) was removed by ion exchange of zeolite. And the ammonia in the column of oyster shell was existed as the form of NH4+, which is not toxic for prawn because oyster shell was stably kept around pH 8. Therefore, some of ammonia(NH3) was reduced by oyster shell. Hydrogen sulfide and COD were effectively removed by adsorption of activated carbon and a partial removal of hydrogen sulfide was accomplished by Oyster shell. Phosphorous was removed by activated carbon, oyster shell and iron chloride. In prawn farm, the concentration of ammonia was increased with increase of pH by algae photosynthesis in the column of activated carbon, zeolite and iron chloride, but it was revealed that pH was stably kept in the column of oyster shell.

• Fisheries and Aquatic Sciences
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• 제7권3호
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• pp.99-108
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• 2004

Vitellogenin (VTG) was purified from serum of $estradiol-l7{\beta}-treated rockfish$(Sebastes schlegeli) by precipitation with $EDTA-Mg^{2+}$ and ammonium sulfate and two step chromatography (anion exchange chromatography and gel permeation chromatography) was performed on FPLC system. Rockfish VTG (rfVTG) was characterized and its properties were determined. The monomers have apparent, molecular mass of about 188 kDa as indicated by SDS-PAGE. Amino acid composition analysis of rfVTG was similar to VTG from other oviparous teleosts. Cysteine and lysine were present at relatively high level. Leucine was present at relatively lower level than in other species. The N-terminal amino acid sequence was evaluated to identify rfVTG. Western blot analysis using an antibody against the purified VTG showed that the antibody reacted with both plasma of $estradiol-l7{\beta}-treated rockfish$ treated male and purified VTG, whereas there was no reaction with male serum of the control. An ELISA was developed using monoclonal and polyclonal antibodies against rfVTG. The assay range was 3.2 ng/mL and 1,000 ng/mL and the value of the intra and inter assay variations were within $9.7{\%}$ and $11.2{\%}$, respectively. Recovery rate was $96.8{\%}$. The sandwich ELISA could be useful for the detection of VTG and could be good for screening of estrogenic compounds.

• Journal of Microbiology and Biotechnology
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• 제24권10호
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• pp.1337-1345
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• 2014

A novel antifungal protein produced by the fungal strain Penicillium citrinum W1, which was isolated from a Southwest Indian Ocean sediment sample, was purified and characterized. The culture supernatant of P. citrinum W1 inhibited the mycelial growth of some plant pathogenic fungi. After saturation of P. citrinum W1 culture supernatants with ammonium sulfate and ion-exchange chromatography, an antifungal protein (PcPAF) was purified. The N-terminal amino acid sequence analysis showed that PcPAF might be an unknown antifungal protein. PcPAF displayed antifungal activity against Trichoderma viride, Fusarium oxysporum, Paecilomyces variotii, and Alternaria longipes at minimum inhibitory concentrations of 1.52, 6.08, 3.04, and $6.08{\mu}g/disc$, respectively. PcPAF possessed high thermostability and had a certain extent of protease and metal ion resistance. The results suggested that PcPAF may represent a novel antifungal protein with potential application in controlling plant pathogenic fungal infection.

• Journal of Microbiology and Biotechnology
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• 제23권11호
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• pp.1554-1559
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• 2013

A protease produced by Bacillus polyfermenticus SCD was purified and characterized as a new detergent material. The protease was purified from supernatant produced by B. polyfermenticus SCD, by ammonium sulfate precipitation, ion-exchange chromatography on a DEAE-Sephadex A-50, and finally gel filtration chromatography on Sephadex G-50. The molecular mass of this enzyme was 44 kDa based on SDS-PAGE. The optimum temperature and pH were $50^{\circ}C$ and pH 8.0. The ranges of its stability to the pH and temperature were 7.0 to 9.0 and under $40^{\circ}C$, respectively. The enzyme was highly stable in the presence of the surfactants like Triton X-100 (0.1%), showing a 2-fold increase in its proteolytic activity. However, the enzyme was slightly inhibited by the chelating agent EDTA (1 mM). The enzyme has a maximum activity at $50^{\circ}C$ and the activity can be increased by surfactants such as Triton X-100 and Tween 80.

• 한국식품저장유통학회지
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• 제7권2호
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• pp.201-206
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• 2000

${\beta}$-Galactosidase was extracted and purified from strawberry. The purified ${\beta}$-Galactosidase from strawberry was investigated their physicochemical characteristics. ${\beta}$-Galactosidase was purified 25.74 fold from strawberry. The purification procedure include ammonium sulfate fraction, acetone powder treatment and gel and ion exchange chromatography. Yield of the enzyme purification was 18.11%. The purified enzyme has native molecular weight of 116,000 dalton. Vmax value and Km value of ${\beta}$-Galactosidase were 0.077 mM ONPG/ml/15mim and 1.75x10-2mM, respectively. The optimum temperature and pH of ${\beta}$-Galactosidase were 43$^{\circ}$C and pH 4.0, respectively. The ${\beta}$-Galactosidase activity was stable below 50$^{\circ}$C and at pH 4.0 to pH 6.0. Among the metal ions Ca and Mg were did not affect, whereas K, Cu and Zn show a little effect on the enzyme activity. The ${\beta}$-Galactosidase activities were inhibited by treatment with EDTA and SDS.

• BMB Reports
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• 제30권4호
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• pp.274-279
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• 1997

Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of branchedchain amino acids, valine, leucine, and isoleucine. ALS is the target site for several structually diverse classes of herbicides including sulfonylureas, imidazolinones. and triazolopyrimidines. We have purified ALS from etiolated barley shoots to homogeneity. The five major purification steps are ammonium sulfate fractionation, DEAE anion exchange, hydroxylapatite, Bio-Gel A gel filtration, and low pressure Mono-Q chrornatoqraphy. Approximately 170-fold purification was achieved and the yield was 0.45% of initial activity in the crude extract. Both SDS-PAGE and Western blot analysis showed a single polypeptide of ALS with an apparent molecular mass of 64 kDa. The result of nondenaturing gel electrophoresis with activity staining indicated that the molecular mass of its native form is approximately 225 to 250 kDa. The values of $K_m$ for pyruvate. pl. and optimum pH of ALS were determined to be 2.0 mM, 5.2. and 7.0. respectively Feedback inhibition studies showed that ALS is more susceptible to leucine than valine. And $IC_{50}$ value of Cadre, a class of irnidazolinones, is about $1.5\mu{M}$ for ALS.

• 생명과학회지
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• 제6권4호
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• pp.250-263
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• 1996

In this study Carotenoprotein from Styela clava were extracted, and purified by ammonium sulfate fraction, Sephadex G- 200 gel chromatography and DEAE-cellulose ion exchange chromarography. The purified carotenoprotein from styela clava had absorption maxima of 487nm, 463nm and 280nm, and the carotenoid liberated from carotenoprotein had 478nm and 452nm with inflexion. One miligram of caroteno-protein contained 0.35 $\mu$g of carotenoid. The carotenoprotein had an approximate molecular weight of 398 kDa (gel filtration). SDS-PAGE showed only a single polypeptide chain with a molecular weight of 62.4 kDa. The amino acid composition of the carotenoprotein were mainly glutamic acid(11.48%), valine(10.75%), leucine (10.45%), aspartic acid(9.94%), while cysteine and tryptophan were not found. The carotenoprotein contained lipid as structure units. In the carotenoprotein, the major fatty acids were oleic acid, plamitoleic acid and palmiunsaturated fatty acids 33.6%, saturated fatty acids 18.6%. In addition, the levels of higher unsaturated fatty acids were high as much as 30.8% of the total fatty acids. Carotenoid was extracted from the carotenoprotein by acetone. Thin layer chromatography showed only carotenoid to be present. Its chemical reactivities and spectroscopic properties were studied and elucidated as astaxanthin.