• Title/Summary/Keyword: ion exchange column chromatography

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Studies of separation and quantitation for selenium species in food (식품중의 셀레늄 화학종의 분리 및 정량연구)

  • Jang, Hee-Young;Min, Hyungsik;Lee, Jonghae;Pak, Yong-Nam
    • Analytical Science and Technology
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    • v.26 no.3
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    • pp.182-189
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    • 2013
  • The purpose of this research is to separate and quantitate selenium species in some food samples with HPLC-ICP-MS. Cation exchange chromatography showed efficient separation only for inorganic Se species while reversed phase ion pair chromatography showed good separation for both inorganic and organic Se species. $C_8$ column ($Symmetryshield^{TM}\;RP_8$, 3.5 ${\mu}m$, $4.6{\times}150$ mm) was used with optimum condition of 5% methanol mobile phase, 0.05% of nonafluorovaleric acid ion pairing reagent. Five standard Se species of Se(IV), Se(VI), SeCys(selenocystein), SeMet(selenomethionine) and Se-M-C(seleno methyl cystein) were separated successfully under the optimum condition (mobile phase; 5% methanol, ion-pairing reagent; 0.05% nonafluorovaleric acid, flow rate; 0.9 mL $min^{-1}$). To extract Se species, microwave assisted and enzyme-assisted extraction methods were studied. In enzyme-assisted extraction method, protease I for garlic, protease I plus trypsin for pork and mackerel, and protease XIV for tuna showed the best extraction efficiency. With the optimum condition for each sample, it was found that mostly inorganic Se, SeCys and SeMet are present in the sample studied ranging from few ${\mu}g$ $g^{-1}$ to few tens of ${\mu}g$ $g^{-1}$.

Isolation and Characterization of L-Ascorbic Acid-Producing Enzyme in Neurospora crassa (Neurospora crassa의 L-Ascorbic Acid 생산효소의 순수 분리 및 이의 특성에 관한 연구)

  • Kim, In-Sil;Lee, Yeon-Hee
    • 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.

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Purification of carbosymethyl cellulase from hybrid between aspergillus niger and penicillium verruculosum

  • Yang, Young-Ki;Lee, Jung-Sup;Park, Hyung-Nam;Moon, Myung-Nim;Kim, Hong-Sub;Kim, Jong-Se;Lim, Chae-Young;Rhee, Young-Ha
    • Journal of Microbiology
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    • v.34 no.1
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    • pp.90-94
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    • 1996
  • The carboxymethyl cellulase (CMCase) was purified from the induced culture filtrate of hybrid TAPW15703 between Aspergillus niger and penicillium verruculosum made by nuclear transfer. The enzyme was purified 80 fold with an overall yield 17% from the culture medium by ammonium sulfate fractionation, Sephadex G-75 gel permeation chromatography, and DEAE-ion exchange column chromatography. The molecular weight of the CMCase has estimated to be 32,000 daltons on SDS-polyacrylamide gel electrophoresis and Sephadex G-150 gel permeation chromatography. The purified enzyme functions optimally at pH 4.0 and 4$0^{\circ}C$ The Km value for carbosymethyl cellulose was 68 mM. The enzyme activity was increased by the presence of $Mg^{2+}$and Mn$^{2+}$.

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Studies on Antibiotic Producers of Korean Soil Microbes (IV) -Isolation and Antibiotic Activity of Streptomyces Strain DMC-42- (한국(韓國) 토양균(土壤菌)중 항생물질(抗生物質) 생성균(生成菌)에 관한 연구(硏究) 제 4 보(第4報) -스트렙토마이세스속(屬) 균주(菌株) DMC-42의 분리(分離) 및 그 항균작용(抗菌作用)-)

  • Kim, Hwa-Ki;Kim, Jung-Woo;Kim, Ha-Won;Choi, Eung-Chil;Kim, Byong-Kak
    • The Korean Journal of Mycology
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    • v.13 no.2
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    • pp.89-97
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    • 1985
  • To find antibacterial strains of the soil microorganisms in Korea, they were isolated from the soil samples of different locations and screened for antibacterial activity against several standard microorganisms. An isolate among them had antibacterial activities against gram-positive and gram-negative bacteria. The examination of its morphological, biochemical, cultural and physiological characteristics according to the International Streptomyces Project methods showed that it belongs to the genus Streptomyces. This strain appears to be a novel strain when it was compared with those species of the genus which have been so far reported. The antibiotic metabolite was produced in the submerged culture of the strain. This metabolite was extracted from the culture filtrate and purified by ion-exchange column chromatography. Physico-chemical properties of the antibacterial metabolite were characterized.

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Effects of Extraction Methods on Histidine-containing Low-molecular Weight Peptides and Pro-oxidants Contents in Tuna Thunnus Extracts (다랑어(Thunnus) 추출물 중의 Histidine 함유 저분자 펩타이드 및 산화촉진물질 함량에 미치는 추출방법의 영향)

  • Kim, Hong-Kil;Song, Ho-Su
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.50 no.6
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    • pp.684-693
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    • 2017
  • We investigated methods for extracting histidine-containing low-molecular-weight (LMW) peptides such as anserine, carnosine and histidine from the edible meat of tuna byproducts. Extracts were treated by several methods including heat treatment ($80^{\circ}C$, 10 min), DOWEX ion exchange (IEC), ultrafiltration (UF), and carboxymethyl (CM)-cellulose column chromatography (IEC+CMC); then the levels of protein, total iron, histidine, carnosine, and anserine were measured. Extracts treated with IEC+CMC using CM-cellulose were analyzed for total iron, protein, histidine, and anserine content, which were $6.27{\pm}0.26mg/mL$, $5.20{\pm}0.21{\mu}g/mL$, 0.80 mg/mL, 0.208 mg/mL, and 4.40 mg/mL, respectively, in yellowfin tuna; and $9.05{\pm}0.82mg/mL$, $4.06{\pm}0.20{\mu}g/mL$, 1.62 mg/mL, 0.012 mg/mL, and 7.28 mg/mL in bigeye tuna. By comparison in IEC-UF treated extracts, protein, total iron, and histidine content decreased by 43%, 73%, and 27% in yellowfin and 0.4%, 54%, and 23% in bigeye tuna, wheres carnosine and anserine content increased by 22% and 17%, respectively. Freeze-dried (FD) extracts exhibited similar trends as non-dried extracts, i.e., dipeptide content increased with purification steps, whereas pro-oxidant (total iron and protein) content decreased. IEC+CMC treated FD extracts had the highest anserine, content, and the greatest reductuion in pro-oxidants.

Characterization and Expression of Antibacterial Protein Gene, Nuecin (곤충세포주에서 누에신 단백질의 발현 및 성상구명)

  • 윤은영;구태원;황재삼;김상현;강석우;김근영;진병래
    • Journal of Sericultural and Entomological Science
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    • v.44 no.2
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    • pp.64-68
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    • 2002
  • The antibacterial protein gene, nuecin was expressed in Sf9 cells using baculovirus expression vector system (BEVS). The antibactetial activity of mature nuecin against Pectobacterium carotovorum subsp. carotovorum, Ralstonia solanacearum and Pseudomonas tolaasii was significantly high, demonstrating that nuecin had a wider antibacterial spectrum on gram negative and positive bacteria. The result appears to be superior to other antibacterial peptide, attacin. The nuecin was purified by SP-sepharose and Mono Q HR ion-exchange chromatography, and then by Superdex 200 HR 10/30 column. The purified nuecin is quite stable at 80$\^{C}$ and 100$\^{C}$ for several hours of incubation and in a wide pH range (pH 2-12).

Purification and Biological Activities of Bombesin Like Immunoreactivity from Skin of the Frog, Bombina orientalis in Korea (한국산 무당개구리 피부에 존재하는 Bombesin 유사면역 반응물질의 순수정제 및 생물학적 활성)

  • Kwon, Hyeok-Yil;Kim, Yil;Park, Hyoung-Jin
    • The Korean Journal of Physiology
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    • v.24 no.2
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    • pp.363-375
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    • 1990
  • The present investigation was performed to purify bombesin-like immunoreactivity (BBS-LI) from the skin of frogs, B. orientalis inhabiting Korea. For extraction of BBS-LI, the fresh skin of 360 g from frogs was immersed in 1,800 ml of 100% methanol and then kept at $4^{\circ}C$ for 5 days. BBS-LI was partially purified by liquid chromatography using an alkaline alumina column followed by a Sephadex G-10 column. BBS-LI was further purified by using sequential HPLC of reversed phase C18 preparation, gel permeation, SP-ion exchange and reversed phase C18 analysis. BBS-LI in fractions of each step was monitored by radioimmunoassay for which bombesin antiserum with a titer of 1 : 188,800 was raised in a guinea pig. Eventually, two different BBS-LI were successfully purified and each BBS-LI showed the following character. 1) BBS-LI was well separated into two peaks in SP-ion exchange HPLC. One (BBS-LI-K1) bound to the column while the other (BBS-LI-K2) did not. 2) BBS-LI-K1, 73.8% of total BBS-LI, was not differentiated from synthetic bombesin in reversed phase C18 analytical and gel permeation HPLC. 3) BBS-LI-K2, 26.2% of total BBS-LI, eluted later than synthetic bombesin in reversed phase C18 analytical HPLC, but it eluted with a retention time identical to that of synthetic bombesin in gel permeation HPLC. 4) The two forms of BBS-LI and synthetic bombesin identically stimulated gastrin release and pancreatic exocrine secretion including volume, protein output and amylase output in anesthetized rats. It is concluded from the above results that the skin of B. orientalis contains two different forms of BBS-LI which are very identical to bombesin immunologically and biologically. In comparison with synthetic bombesin containing 14 amino acid residues, the major form shows quite similar pattern in all HPLC used in the present study, but the minor form exhibits quite different pattern in SP-ion exchange and reversed phase C18 analytical HPLG.

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Analysis of Anions by Indirect Photometric Detection (I) (간접 분광 검출법에 의한 음이온의 분석(I))

  • Park, Man-Ki;Kim, Bak-Kwang;Park, Jeong-Hill;Kim, Kyoung-Ho;Lee, Mi-Yung;Jung, Jae-Eun
    • YAKHAK HOEJI
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    • v.34 no.3
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    • pp.215-218
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    • 1990
  • An ion chromatographic method based on indirect photometric detection of UV transparent anions was developed. Separation of anion was accomplished on strong anion exchange column (Waters SAX) using UV detector at 254 nm. Among examined UV-active additives (Dns-H, Dns-glu, Dnsser, Dns-val), Dns-glu showed the highest sensitivity. Studies on the effects of the pH and ionic strength of eluent revealed that the increase of pH and ionic strength of the eluent decreased capacity factor. The best eluent for the separation of acetate, fluoride, chloride, nitrate and bromide was $1\;{\times}\;10^{-4}M$ Dns-glu in 5 mM phosphate buffer (pH 6.30). The detection limit of chloride ion was 2.1 ng in this condition.

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Separation Characteristics of Alkali and Alkaline Earth Metal Ions Using Novel HDBPDA Ion Exchanger (새로운 HDBPDA 이온교환체를 사용한 알칼리 및 알칼리 토금속 이온들의 분리특성)

  • Kim, Dong Won;Kim, Chang Suk;Choi, Ki Young;Jeon, Young Shin;Hong, Choon Pyo
    • Analytical Science and Technology
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    • v.6 no.5
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    • pp.471-477
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    • 1993
  • The novel macrocylic ligand {(4, 5):(13, 14)-dibenzo-6, 9, 12-trioxa-3, 15, 21, -triazabicyclo [15. 3. 1]heneicosa-(1, 17, 19)(18, 20, 21) triene: HDBPDA} was synthesized, and protonable constants of the ligand and the complex stability constants with alkali metals alkaline earth metals were determined. We evaluated the resolution factor(${\Delta}$) from equation that inducing from stability constants(pK). Also, this ligand was grafted on chloromethylated styrene-divinyl benzene(Merrifield resin) for HDBPDA, ion exchanger. Alkali and alkaline earth metal ions were separated using water by the column chromatography with this ion exchanger. Selectivity(${\alpha}$) and resolution(Rs) of alkali and alkaline earth metal ions were measured from the elution curves chromatogram. The selectivity and resolution values of the various ions calculated from the elution curves were compared with those abtained from pK values. The results were in a good agreement between tow methods. Ion exchange capacity of the resin were determined using the alkali and alkaline earth metal ions and pH dependence of capacity was also discussed.

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Trypsin Inhibitor from Streptomyces sp. ( Part 1) Isolation of microorganism and purification of the inhibitor (Streptomyces 속 균주가 생성하는 Trypsin Inhibitor (제1보) 균의 분리 및 저해물질의 정제)

  • Yi, Dong-Heui;Seu, Jung-Hwn
    • Microbiology and Biotechnology Letters
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    • v.10 no.4
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    • pp.275-281
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    • 1982
  • One strain of Streptomyces sp. (AS-707) isolated from soil was found to produce a biologically active substance that showed a strong inhibitory activity against proteolytic enzymes viz. trypsin, papain, $\alpha$-chymotrypsin, Azotobacter protease, and Bacillus pretense. The substance was separated from culture filtrate by ion exchange column chromatography using Amberlite IRC-50 and CM-cellulose column chromatography. It was found that the recovery yield was 26% as activity basis. The substance was stable in wide pH range from 2.0 to 12.0 at 37$^{\circ}C$, but it was unstable in alkaline pH values at 6$0^{\circ}C$. The activity was thermostable to give 90% activity compared to the intact sample when it was treated at pH5.6 at 10$0^{\circ}C$ for 2 hours.

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