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Purification and Characterization of the D-xylulokinase from Candida sp. L-16 (Candida sp. L-16이 생산하는 D-Xylulokinase의 정제 및 특성)

  • 이종수;주길재
    • Food Science and Preservation
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    • v.9 no.4
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    • pp.429-433
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    • 2002
  • The D-xylulokinase from Candida sp. L-16 was purified through a sequence of ammonium sulfate fractionation, DEAE-cellulose chromatography, Sephadex G-100 and Sephadex G-200 gel filtration. The specific activity of the purified Dxylulokinase was increased to 23.2 fold and the yield was 11.2%. The enzyme was showed to be a single protein band by SDS-PAGE. The molecular weight of the enzyme was 150,000 dalton, this enzyme was identified to be a dimer with two subunits. The optimum conditions of the enzyme were pH 8.0 and 40$\^{C}$, respectively. The enzyme was relatively stable between pH 7.0 to pH 9.0, but it was unstable over 30$\^{C}$. The enzyme showed substrate specificity on D-xylulose, D-arabinose and D-ribose, Km value and Vmax for D-xylulose were 0.042 mM and 117 units/ml, respectively. The activation energy of the enzyme was 4.75 Kcal/mol. The one was inhibited by metabolic intermediates such as 6-phosphogluconic acid, 2-keto-gluconic acid. The enzyme was activated by EDTA and thiol compounds such as cysteine-HCI, DTT and glutathione.

Purification and Characterization of Protease from Bacillus subtilis PANH765 (Bacillus subtilis PANH765가 생산하는 Protease의 정제 및 특성)

  • 이창호;우철주;베동호;김관필
    • Food Science and Preservation
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    • v.10 no.2
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    • pp.246-251
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    • 2003
  • Pretense produced by Bacillus subtilis PANH765 was purified from culture supernatant by using ammonium sulfate fractionation DEAE-cellulose ion exchange chromatography, and gel filtration with Sephacryl S 200 HR and Sepharose CL-6B. DEAE-cellulose ion exchange column chromatography, separated the pretense into one fraction. This fraction was further purified using Sephacryl S 200 HR and Sepharose CL-6B gel titration. The molecular mass of pretense was estimated to be 35.0 kDa by the SDS-PAGE and gel filtration using Sepharose CL-6B. The results indicated that the purified pretense are monomeric proteins. Specific activity and purification folds of pretense were 657 U/mg and 4.35, respectively. The optimum temperature, optimum pit stable at a temperature range and pH ranges for the purified protease were 65$^{\circ}C$, 7.05, 50 ∼ 75$^{\circ}C$ and 6.0 ∼ 7.5, respectively. The pretense activity was decreased by the presence of PMSF and DFP, which the protease activity was increased by the presence of Na$\^$+/, K$\^$+/, Mg$\^$2+/ and NH$_4$$\^$+/ ions.

Purification of a Protease Produced by Bacillus subtilis PCA 20-3 Isolated from Korean Traditional Meju (전통 메주로부터 분리한 Bacillus subtilis PCA 20-3 유래 Protease 의 정제)

  • Lim, Seong-Il;Yoo, Jin-Young
    • Korean Journal of Food Science and Technology
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    • v.31 no.6
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    • pp.1635-1641
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    • 1999
  • Bacillus subtilis PCA20-3 was isolated from meju and was found to produce a protease. The strain produced the maximum amount of enzyme in the medium containing soytone (0.2%), soluble starch (2%), $(NH_4)_2SO_4\;(0.1%),\;CaCl_2(0.1%),\;yeast\;extract\;(0.01%),\;K_2HPO_4\;(0.1%),\;and\;KH_2PO_4\;(0.1%)$. Protease was first concentrated by ammonium sulfate (80% saturation, w/v) precipitation of culture supernatant. Then the enzyme was purified by column chromatography using CM Sephadex C-50. The collected proteins were rechromatographed using Sephadex G-100 gel filtration column. The fraction with protease active from Sephadex G-100 gel chromatography was found to be pure when examined by SDS-polyacrylamide gel electrophoresis and YMC-pak reverse phase chromatography. Specific activity, yield and purity were 76 U/mg. 2.7%, and 7.6 fold, respectively. The molecular weight of the enzyme was estimated to be 31.5 kDa by SDS-PAGE. The number of amino acids calculated from molecular weight was evaluated about 321 residues. N-terminal sequence of the enzyme was $Val^1-Pro^2-Tyr^3-Gly^4-Val^5-Ser^6-Gln^7-Gly^8-Lys^9-Ala^{10}$.

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Double Salt Precipitation Behavior of Rare Earth by Sodium Sulfate in Sulfuric Liquor of Waste Permanent Magnet Scrap (폐 영구자석 스크랩 황산침출용액으로부터 황산나트륨에 의한 희토류 원소 복염침전 거동 고찰)

  • Yoon, Ho-Sung;Kim, Chul-Joo;Chung, Kyeong Woo;Kim, Ji-Hye;Lee, Eun-Ji;Yoo, Seung-Joon
    • Resources Recycling
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    • v.26 no.5
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    • pp.39-47
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    • 2017
  • In this study, the precipitation of rare earth-sodium sulfate with sodium sulfate was conducted in order to separate rare earth from Fe in rare earth sulfate solution. Neodymium (Nd) was easily precipitated as Nd-sulfate salt with sodium sulfate, on the other hand, excessive sodium sulfate was needed for the precipitation of Dy-sulfate salt. Also neodymium not only promoted the precipitation of dysprosium sulfate salt but also increased recovery of dysprosium sulfate salt in sulfuric acid solution. At the condition of $60^{\circ}C$ precipitation temperature, 3 h reaction time, 7 equivalents sodium sulfate, the recovery of neodymium and dysprosium sulfate salt was 99.7% and 94.3% respectively from the sulfuric acid solution containing Nd of 23.39 mg/ml and Dy of 8.67 mg/ml. Lastly, from the results of separation of Dy to Nd by the method of sulfate double salt, the effect of salting out with NaCl is important to increase the grade of Dy, and 98.7% of Dy grade could be obtained in this study.

Studies on the Hydrolysis of Holocellulose with Trichoderma viride Cellulase. (III). Effects of the Optimum Treated Conditions and Reactivation of Residue of Digested Substrates (Cellulase에 의한 목재당화(木材糖化)에 관(関)한 연구(硏究) - (III) 최적(最適) 처리조건(処理條件)과 효소처리(酵素処理) 잔사(殘渣)의 재기질화(再基質化) 효과(效果) -)

  • Min, Du Sik
    • Journal of Korean Society of Forest Science
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    • v.49 no.1
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    • pp.1-5
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    • 1980
  • In this study, enzymatic saccharification of substrates from Alnus hirsuta Ruper (8-14 years). Quercus acutissima Carruthers, Betula platyphylla var. japonica Nera, Populus euramericana Guiner and Platanus orientalis L. were investigated using crude cellulase preparations of Trichoderma viride Pers. ex. Fr. SANK 16374, and conduced on the optimum treated conditions of the cellulase sacchrification and reactivation of residue of digested substrates. The Trichoderma viride cellulase was produced by the submerged culture process and produced in the culture fluid was salted out quantitatively by the use of ammonium sulfate. The method of dilignification from wood (5 species) was treated by the peracetic acid(PA) method. The reducing sugar was determined by the dinitrosalicylic acid (DNS) method. 1. The results of tests carried out for 96 hr. (Figure 1), show conclusively the initial substrates from 5 species ($S_3$) which has been rendered highly reactive form and the mean rate of reducing sugar was 28.3 %. 2. The results of tests carried out for 96 hr., the reactivation of residue of digested substrates (improvement in the quality of the substrate through preheating in air at $190^{\circ}C$. for 45 min. followed by milling was (60 mesh size) at the same substrate level, increased concentrations of cellulase at the same substrate level, and increased concentrations of cellulase increases the rate of hydrolysis considerably. 3. Figure 1. shows conclusively that the residue of digested substrates ($S_1$ dried at $60^{\circ}C$) which has been rendered extremly resistant to cellulase action can be reactivated into a highly reactive form ($S_2$), almost comparable to that of the initial substrates ($S_3$). And the reducing sugar formation did not show statistically significent differences at 5% levels by initial substrates and the residue of digested substrates (preheating in air at $190^{\circ}C$. for 45 min. fallowed by milling was (60 mesh size).

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Purification of Vibrio anguillarum Growth Inhibition Factor Produced by Bacillus amyloliquefaciens H41. (Bacillus amyloliquefaciens H41이 생산하는 Vibrio anguillarum 생육 저해인자의 정제)

  • Shin, Hyun-Chul;Chung, Kyung-Tae;Kim, Kwang-Hyun;Kim, Byung-Woo;Kwon, Hyun-Ju;Lee, Eun-Woo;Yum, Jong-Hwa;Rhu, Eun-Ju;Jeong, Yu-Jeong;Kim, Young-Hee
    • Journal of Life Science
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    • v.18 no.6
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    • pp.789-795
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    • 2008
  • To study the possible use of probiotics in fish farming, we evaluated antagonism of antibacterial strain Bacillus amyloliquefaciens H41 against the fish pathogenic bacterium Vibrio anguillarum NCMB1. The purification of growth inhibition factor produced by B. amyloliquefaciens H41 was achieved by obtaining supernatant of this bacterium. The growth inhibition factor was purified to homogeneity by 70% ammonium sulfate precipitation, DEAE-sephadex A-50 ion exchange chromatography, sephadex G-200 gel filtration column chromatography, and sephadex G-50 gel filtration column chromatography with 40.8 fold of purification and 2.9% yield. The molecular weight of the purified growth inhibition factor was 48 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH and temperature for the growth inhibition factor were pH 7.5 and $30^{\circ}C$, respectively. The activity of growth inhibition factor was enhanced slightly by some metal ions, such as $Mg^{+2}$, $Mn^{+2}$, but was inhibited by the addition of $Co^{+2}$, $Hg^{+2}$, $Zn^{+2}$ and $Ag^{+2}$. NaCl stability of the growth inhibition factor was observed with 50% residual activity at 3% NaCl concentration. Toxicity test showed that the purified B. amyloliquefaciens H41 growth inhibition factor did not affect the live of Japanese flounder (Paralichthys olivaceus) and the effectiveness was 78% of residual lethality compared to commercial antibacterial agents.

Characterization of Ferritin Isolated from Dog Spleen (개의 비장에서 분리한 페리틴의 특성)

  • Park Jae-Hag;Jun Do Youn;Kim Young Ho
    • Journal of Life Science
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    • v.15 no.3 s.70
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    • pp.439-446
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    • 2005
  • Ferritin is known to be the principle iron-storage protein in a wide variety of rganisms. The electro­phoretic mobility and immunological cross-reactivity of dog splenic ferritin were compared with those of horse, bovine, and pig splenic ferritin after isolation using heat treatment, salting out, column chromatography, and ultrafiltration. These isolation methods allowed the recovery of $\~84{\mu}g$ of the ferritin per g of spleen. The iron content in the dog ferritin was $22.7\%$, which appeared to be higher than those in the other mammalian ferritins tested. The electrophoretic mobility of the dog ferritin under nondenaturing conditions was similar to its bovine counterpart, whereas it was more identical to pig and horse ferritins on an SDS-polyacrylamide gel. The molecular weight of the dog ferritin subunit was 19.5 kDa on an SDS-polyacrylarnide gel, and the subunit was unable to bind with iron. The polyclonal anti-dog ferritin raised in rats was able to cross-react with the pig, bovine, and horse ferritins, upon Ouchterlony double immunodiffusiion. A Western blot analysis also revealed that the anti-dog ferritin, which specifically bound with the dog ferritin subunit, could also recognize the horse, bovine, and pig ferritin subunits and the maximum cross-reactivity was exhibited with the pig ferritin subunit, indicating that the dog ferritin is immunochemically more similar to the pig ferritin than its other mammalian counterparts. Accordingly, these results elucidate the biochemical and immunochemical characteristics of dog ferritin that might have a potential to be applied as an oral iron supplement to treat iron deficiency anemia.

Pediatric Urolithiasis: Our 22-year Experience at a Single Center (소아에서의 요로결석: 단일기관에서 22년간의 경험)

  • Kim, Su-Yon;Kim, Min-Jee;Lee, Joo Hoon;Kim, Kun Suk;Park, Young Seo
    • Childhood Kidney Diseases
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    • v.17 no.2
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    • pp.101-109
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    • 2013
  • Purpose: Pediatric urolithiasis is uncommon in children but is a cause of significant morbidity and damage to the kidney. Although much information on adult urolithiasis is available in the literature, large studies on the pediatric population are still scarce. In this report, we review our experience with pediatric urolithiasis over 22 years at a tertiary referral center. Method: We retrospectively reviewed the records of children with newly diagnosed urolithiasis between January 1991 and May 2013. We assessed the age, sex, family history, initial symptoms, location of stones, underlying cause, stone analysis, treatment, and recurrence among the patients. Results: In total, 137 patients (96 male, 41 female) were assessed. The age range was 0-17 years (mean age, 6.0 years). Forty-three (31%) children were aged <1 year, and 37% (16/43) had a history of intensive care unit (ICU) admission. Thirteen patients (9.5%) had a family history of stones. The most common symptoms at presentation among the patients were gross hematuria (56/137, 41%) and flank or abdominal pain (46/137, 34%). The stones were located in the kidney (85/137, 62%), ureter (29/137, 21%), bladder (2/137, 1.4%), and multiple locations (20/137, 15 %). Congenital abnormalities of the genitourinary (G-U) tract, with or without metabolic abnormality, or urinary tract infection (UTI) was detected in 26 children (19%). Ninety-one patients (66%) underwent metabolic examination, and 38% of these patients exhibited an abnormality. UTI, with or without abnormalities of the G-U tract, or metabolic abnormality was detected in 26 children (19%). Of the 35 stones analyzed, the majority were calcium stones (20/35, 57%), followed by infected stones (5/35, 14%), uric acid stones (4/35, 11%), carbonate apatite stones (3/35, 7%), cystine stones (2/35, 6%), and phosphate stones (1/35, 3%). Five patients (4%) required open procedures, with or without non-open procedures, whereas 77 patients (56%) were managed conservatively; the remaining 55 patients (40%) received some other form of intervention. Eighteen patients (13%) had stone recurrence during the follow-up period. Conclusions: Pediatric urolithiasis is commonly associated with abnormalities of the G-U tract and/or metabolic disorders and/or UTI. Half of the patients will pass their stones spontaneously, and all the techniques of minimally invasive surgery are applicable in the treatment of children with stones. As the recurrence rates are high among this population, long-term follow-up is recommended and the complete clearance of stones is important.