• Fisheries and Aquatic Sciences
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• 제17권2호
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• pp.181-187
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• 2014

This study was performed to identify the optimum fractionation method and conditions to obtain exopeptidase-active fractions from octopus hepatopancreas (HP) crude extracts (CEs) using four techniques: solid ammonium sulfate fractionation, polyethylene glycol (PEG) fractionation, anion exchange chromatography, and gel filtration chromatography. The fractions with the highest total activity toward L-leucine-p-nitroanilide (Leu-pNA) were fraction IV from the ammonium sulfate and PEG fractionation, and fraction II in ion exchange and gel filtration chromatography. The total exoprotease activity of these fractions was highest in fraction IV (4,050.20 U) of ammonium sulfate fractionation, followed by fraction II (3,600.28 U) from gel filtration chromatography, fraction IV (2,861.30 U) from PEG fractionation, and fraction II (2,576.28 U) from ion exchange chromatography. These results suggest that ammonium sulfate fractionation using 60-80% ammonium sulfate was the most efficient method for separating the exoprotease active fractions from CEs of octopus HP.

• 한국미생물·생명공학회지
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• 제15권5호
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• pp.306-311
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• 1987

황산암모늄 분획, 2차례의 DEAE-Cellulose, DEAE-Sephadex A-50 및 Sephacryl S-200 superfine gel filtration으로 정제한 결과 Streptomyces sp. J-350P의 세포외 adenine deaminase는 0.3%의 수율로서 1764배로 정제되었다. Streptomyces sp. J-350P의 세포외 adenine deaminase는 pH6.5~8.5 사이에서 안정하였고, pH7.0에서 10분간 열처리하였을 때 5$0^{\circ}C$까지는 안정하였다. 효소 활성 최적 pH와 온도는 pH6.5와 35$^{\circ}C$ 였다. Sephacryl S-200 superfine gel filtration 의한 분자량의 측정 결과 본 효소의 분자량은 약 36,000이었다.

• Archives of Pharmacal Research
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• 제28권6호
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• pp.667-674
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• 2005

The leaves of Persimmon (Diospyros kaki L.) has long been used for tea in Korea since it was thought to be effective against hypertension. An anticoagulant fraction was purified through gel filtration G-100, hydrophobic, gel filtration G-150, and FPLC, Phenyl superpose column chromatographies. The purified fraction was homogenous and its Mr was estimated 10,000 Da by gel filtration and SDS-PAGE. The purified fraction was sensitive to treatment of subtilisin B, but not to heat and its activity was not changed after periodate oxidation, indicating that the activity was not due to carbohydrates. It delayed thrombin time (TT), activated partial thromboplastin time (APTT), and prothrombin time (PT) using human plasma. TT was more sensitive than APTT and PT, suggesting that the anticoagulant activity may be caused by a degradation or a defect of fibrin or thrombin. It did not cause the hydrolysis of fibrin after incubation. However, it inhibited thrombin-catalyzed fibrin formation with a competitive inhibition pattern. These results indicate that it may be an antithrombotic agent and that it is bound to fibrinogen binding sites of thrombin.

• BMB Reports
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• 제35권6호
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• pp.576-582
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• 2002

Collagenase from the internal organs of a mackerel was purified using acetone precipitation, ion-exchange chromatography on a DEAE-Sephadex A-50, gel filtration chromatography on a Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel, and gel filtration chromatography on a Sephadex G-75 column. The molecular mass of the purified enzyme was estimated to be 14.8 kDa by gel filtration and SDS-PAGE. The purification and yield were 39.5-fold and 0.1% when compared to those in the starting-crude extract. The optimum pH and temperature for the enzyme activity were around pH 7.5 and $55^{\circ}C$, respectively. The $K_m$ and $V_{max}$ of the enzyme for collagen Type I were approximately 1.1 mM and 2,343 U, respectively. The purified enzyme was strongly inhibited by $Hg^{2+}$, $Zn^{2+}$, PMSF, TLCK, and the soybean-trypsin inhibitor.

• 한국식품위생안전성학회지
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• 제14권3호
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• pp.233-237
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• 1999

Lactobacillus sp. GM7311을 MRS broth에 배양하여 배양 상등액으로부터 n-propanol/acetone처리 , ion-exchange chromatography, gel filtration 및 SDS-PAGE를 이용해 박테이로신을 부분 정제하였다. 정제 과정 중 가장 큰 문제점은 n-propanol 과 acetone등의 유기용매 처리 단계에서의 급격한 활성 감소로서 회수율은 48.0%를 나타내었다. 거러나 이후 CM-cllulose를 통한 ion-exchange chromatography와 Sephacryl HR-100에 의한 gel filtration 과정을 거치면서 활성이 크게 증가하여, 비활성 뿐만 아니라 정제도에서도 493배의 증가를 얻을 수 있었으며 최종 회수율은 8.3%였다. SDS-PAGE로 정제도를 확인했을 때 분자량 약 8,200과 2,500의 2개의 band를 관찰할 수 있었으며, 2개의 band를 각각 나누어 활성을 측정한 결과 두 부분 모두 활성이 있는 것으로 나타나 Lactobacillus sp. GM7311이 2가지 성분의 박테리오신을 생산하는 것으로 추정할 수 있었다.

• 한국가축번식학회지
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• 제12권3호
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• pp.141-147
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• 1988

These experiments were carried out to develop new techniques for in vitro separation of x-and Y-bearing spermatozoa. The results obtained in these experiments were summarized as follows: 1. Following centrifugation of discontinuous percoll density gradient, populatin of spermatozoa increased progressively from low to high density. The highest concentration of spermatozoa was observed at the 4th fraction which included 36.6% of spermatozoa. 2. As increasing percoll concentration, the higher motility index was obtained and the highest motility index(74.2) was obtained at the 5th fraction. 3. The percentage of B-body bearing spermatozoa following percoll density gradient centrifugation was decreased from 39.7% to 25.6%. 4. The sperm population following chromatography by sephadex gel and percoll density gradient centrifugation was decreased in 1st, 5th and 6th fractions but the reverse was turn for 2nd, 3rd and 7th fractions, and the highest sperm concentration was observed at the 7th fraction which included 37.4% of spermatozoa. 5. Motility index of spermatozoa was increased from 77.6 to 79.4 after the sephadex gel filtration, however it was decreased at all fractions after percoll density gradient centrifugation. The lowest motility index(33.2) was obtained from the 7th fraction. 6. The rate of B-body bearing spermatozoa was shown the trend to decrease by the sephadex gel filtration and the trend was accelerated by the percoll density gradient centrifugation. The lowest percentage of B-body bearing spermatozoa, 12.0% was obtained from the 5th fraction.

• 한국응용곤충학회지
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• 제34권2호
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• pp.140-146
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• 1995

연두금파리의 난세포성숙에 따른 단백질의 변화와 난특이성단백질의 특성을 확인하기 위하여 gel filtration, 전기영동 및 분자량측정, 아미노산과 지방산함량을 측정하여 얻은 결과는 다음과같다. 연두금파리 암컷성충의 난소단백질은 단백질원을 섭식시킨 후 72시간 이후 빠르게 증가하였고, 완전한 성숙이 일어나는 96시간에 최고의 함량을 나타냈다. DEAE-cellulose와 Sephacryl 5-200으로 gel filtration하고 7.5% native polyacrylamide gel에서 전기영동한 결과 난소에서 분리된 특이단백질은 ${R}_{f}$ 0.4에서 혈림프 및 난소와 다른 밴드가 확인되었으며, 분자량은 110,000 dalton이였다. 분리된 난특이성단백질내 아미노산 조성은 asparagine 외 모두 13종이 검출되었으며, asparagine, glutamic acid와 함께 tyrosine이 특이하게 높게 나타났다. 지방산은 난소와 함께 난특이성단백질에서 palmitic acid의 4종이 분리되었다. 따라서, 연두금파리의 난에는 지방체에서 합성, 분비된 난황단백질이외에 난소에만 존재하는 특이단백질이 있음을 알 수 있다.

• Journal of Microbiology and Biotechnology
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• 제6권6호
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• pp.402-406
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• 1996

Inulin fructotransferase (depolymerizing) (EC 2.4.1.93) was purified 34-fold from the culture broth of Arthrobacter sp. A-6 by using a combination of ammonium sulfate fractionation, DEAE-Sepharose CL-6B chromatography and Sephacryl S-200 gel filtration. The purified enzyme converts inulin into di-D-fructofuranose dianhydride III(DFA III) and small quantities of fructo-oligosaccharides. The temperature and pH optima of the enzyme were $70^{\circ}C$ and 6.0, respectively. Molecular weight of the enzyme was determined to be 49 kDa by 12$%$ SDS-polyacrylamide gel electrophoresis, and 145 kDa by Sephacryl S-200gel filtration. This indicates that the functional inulin fructotransferase of Arthrobacter sp. A-6 has a homomeric trimer structure. The enzyme had an isoelectric point of pH 4.6. The N-terminal amino acid sequence of the purified enzyme subunit was Ala-Asp-Asn-Pro-Asp-Gly(\ulcorner)-Ser-Asn-Met(or Glu)-Tyr-Asp-Val.

• 한국식물병리학회지
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• 제11권4호
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• pp.312-317
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• 1995

The polygalacturonase (PG) production in rotten apples by Botryosphaeria dothidea was purified by using gel filtration and ion exchange column chromatography, and the biochemical characters of PG were investigated. The purified PG appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with approximate molecular weight of 49 kilodalton (kDa). The molecular weight was equal to the native molecular weight estimated by gel filtration. The Km and Vmax values of PG were 0.51 mg/ml and 90.9 $\mu$M/min/ml, respectively. Optimum pH was 4.0~5.0, and the PG activity was stable from pH 5.0~10.0. Optimum temperature of the enzyme activity was 4$0^{\circ}C$. The PG activity was relatively stable at 2$0^{\circ}C$, but it was reduced 45% at 4$0^{\circ}C$ and completely inactivated at 8$0^{\circ}C$. The PG activity was considerably inhibited by Cu2+, Zn2+, SDS and EDTA, whereas it was not effected by Ca2+, K+, Mg2+ or Na+ ions.

• 한국동물학회지
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• 제36권2호
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• pp.238-244
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• 1993

We identified juvenile hormone binding protein (JHBP) from last instar larval hemollvnph of Hvphantria cunea using gel filtration and non-SDS PAGE. Two kinds of JHBP in hemollnnph were found at two peaks by gel filtration (Sephadex G-100) and also at Rm values of 0.13 and 0.57 by non-SDS PAGE. JHBP was partially purified using anion exchange chromatosraphv, preparative gel filteration, and preparative PAGE. Dextrin coated charcoal (DCC) binding assay was employed to monitor the location of JHBP in chromatographic profile during the purification process. Purity of JHBP was checked by silver staining of 1091 SDS-Polyacrvlamide.