• 한국수산과학회지
• /
• 제48권2호
• /
• pp.178-186
• /
• 2015

Protease inhibitors (PI) of trypsin and papain as target proteases from the roe of bastard halibut Paralichthys olivaceus were fractionated out using ammonium sulfate precipitation (A), DEAE 650M anion exchange chromatography (D), and Sephacryl S-300 gel filtration (S). The recovery percentages of the fractions with the strongest inhibitory activity for each fractionation method were 13% for the A4 fraction, 21.2% for the D3 fraction, and 21.3% for the S2 fraction, with specific inhibitory activities of the fractions toward trypsin and casein of 168, 139, and 218 U/mg, respectively, while no inhibition of papain was observed. The $IC_{50}$ for the trypsin-specific substrate $N{\alpha}$-benzoyl-$\small{L}$-arginine-p-nitroanilide (BAPNA) was 0.65, 1.55, 2.26, and 2.85 mg/mL for the A4, S2, A3, and D3 fractions, respectively. These results suggest that chromatographic fractionation methods (D and S) based on the molecular mass and charge of the protein were more effective at fractionating PI than was ammonium sulfate precipitation based on protein solubility, and that the bastard halibut roe extract acts as a serine protease inhibitor. Therefore, the PI fraction from fish roe might be useful for inhibiting proteases in foodstuffs, and could constitute an alternative food-grade inhibitor for the surimi industry.

• Journal of Ginseng Research
• /
• 제13권2호
• /
• pp.254-259
• /
• 1989

A radioprotective ginseng protein fraction was obtained from Korean white ginseng powder by the following isolation and purification procedures: Tris-HCI buffer extraction, 70% ammonium sulfate fractionation, CM-rellulosr column chromatography, heat inactivation and Sephadex G-75 column chromatography. This fraction was further purified by Sepharose 4B and Sephadex G-150 column chromatographies. Three fractions obtained were subjected to Native-PAGE and SDS-PAGE using gradient gels and the silver staining method. Molecular weights of the native proteins and their subunits were estimated.

• KSBB Journal
• /
• 제16권5호
• /
• pp.435-441
• /
• 2001

In spite of the powerfulness for the simultaneous study of proteome expression and post-translational modification, 2-D PAGE has inevitable limitation on detect low aboundant proteins. Since many of the low abundant proteins are likely to have very important regulatiory functions in cells, separation and analysis of low copy number proteins is an important issue in proteome studies and challenge for 2-D techniques. Among various methods developed to detect low abundant proteins, electrophoretic protein prefractionation, chromatographic protein prefractionation, and subcellular fractionation are explained in this paper. Their practical strengths and weaknesses are also explained with current research trends.

• 한국농업기계학회:학술대회논문집
• /
• 한국농업기계학회 2000년도 THE THIRD INTERNATIONAL CONFERENCE ON AGRICULTURAL MACHINERY ENGINEERING. V.III
• /
• pp.792-802
• /
• 2000

The coagulation of Chinese cabbage juice can be accomplished by applying the combine method of the formic acid with rate of 3% and in four hours the propionic acid with rate of 1 % in the juice. The separation of coagulation into the protein paste and the brown juice completed in 6.5 hours by set up method in special storage. The protein paste can be stored safely for 30 days in anaerobic condition.

• 한국식품영양과학회지
• /
• 제18권4호
• /
• pp.423-429
• /
• 1989

선화녹두(sunhwa-nogdu ; SH)와 재래종 녹두(conventional variety ; C) 2품종의 분리녹두단백(mungbean protein isolate : MPI)을 제조하여, 이들의 아미노산 조성 그리고 용해도에 의한 단백질의 분획 및 분획된 단백질의 전기영동 패턴을 연구하여 다음과 같은 결과를 얻었다. 즉, MPI의 아미노산조성을 보면 MPI-SH, MPI-C 둘 다 glutamic acid, aspartic acid의 함량이 높게 나타났으며, 함황아미노산의 함량은 낮게 나타났다. 한편 MPI의 아미노산조성은 원시료인 MBF와 거의 유사함을 나타내었고, 두 품종간에도 큰 차이가 없었다. 그리고 MPI를 용해도에 의하여 분별정량 했을 때 $87.6{\sim}92.9%$가 수용성 성분이었으며, prolamin, glutelin, residue는 MPI-C에서 0.25, 1.13, 10.74%로 MPI-SH의 0.25, 0.91, 5.63%에 비해 각각 높게 나타났다. 또한, 전분획의 전기영동 패턴을 살펴본 결과 $6{\sim}10$개의 band를 나타내며 $43,000{\sim}68,000\;dalton$ 범위의 분자량에 집중하는 경향을 나타내었다.

• 한국식품과학회지
• /
• 제22권1호
• /
• pp.56-60
• /
• 1990

역류분배 방법을 이용한 단백질의 분리정제 가능성을 높이기 위하여 4가지 모델단백질에 대하여 수용성 액체 2상계에서의 분획계수를 pH 변화에 따라 조사하였다. 또한 역류분배를 이용한 다중분배추출방식으로 모델 단백질의 혼합물을 수준의 pH(4.5-12.0)에서 분획하였다. 그 결과 pH에 따라 단백질들의 등전점 차이로 인한 분획계수(K)값이 다르게 나타서, 다중분배를 행할 때 각 단백질 분획들이 위치하는 튜브번호가 다르게 되므로 역류분배 방법을 통한 단백질의 분리 가능성을 보여주었다.

• 한국식품과학회지
• /
• 제16권3호
• /
• pp.322-328
• /
• 1984

Pizopus oryzae가 생산(生産)하는 glucoamylase를 유안(硫安)및 acetone 분획(分劃)과 이온교환수지의 column chromatography에 의하여 정제(精製)하였다. 즉(卽) 조효소액(粗酵素液)을 유안분획(硫安分劃) acetone 분획(分劃), DEAE-cellulose column chromatography, CM-cellulose column chromatography에 의(依)하여 두가지형(型)의 glucoamylase를 분리정제(分離精製)하였으며 이들을 각각(各各) glucoamylase I과 II라고 하였다. Glucoamylase I과 II의 Specific activity는 각각(各各) 157.6U/mg protein (조효소액(粗酵素液)의 37.5배(倍)) 164.7U/mg protein (조효소액(粗酵素液)의 39.2배(倍)) 이었고 수율(收率)은 각각(各各) 4.3%, 3.8%이었다. Glucoamylase I과 II는 polyacrylamide disc gel electrophoresis와 SDS-polyacrylamide gel electrophoresis에 의(依)하여 각각(各各) 단일(單一)한 band를 나타내었고 이 단백질(蛋白質) band는 옥도염색(沃度染色)에 의(依)해 glucoamylase 활성(活性)을, PAS염색(染色)에 의(依)해 glycoprotein임을 확인(確認)하였다.

• 한국수산과학회지
• /
• 제46권2호
• /
• pp.119-128
• /
• 2013

A protease inhibitor was fractionated from fish eggs using methods based on protein solubility. Fractionation efficiency was evaluated with regard to percent recovery and total inhibitory activity (U). The fractionation of protease inhibitor (PI) from egg extracts of skipjack tuna (ST, Katsuwonus pelamis), yellowfin tuna (YT, Thunnus albacores), and Alaska pollock (AP, Theragra chalcogramma) was performed by precipitation with cold acetone or ammonium sulfate (AS). Fractions exhibiting the strongest inhibitory activity contained 20-40% (v/v) cold acetone or 40-60% saturated AS fractions. AS fractionation was more effective in isolating PI than was precipitation with acetone. The total inhibitory activity and percent recovery of fraction obtained with AS 40-60% toward trypsin and $N{\alpha}$-benzoyl-L-arginine-p-nitroanilide (BAPNA) were 4,976 U and 24.2% for ST, 3,331 U and 38.1% for YT, and 4,750 U and 43.8% for AP, respectively. In comparisons against six commercial proteases, 40-80% AS fractions, made by combining the 40-60% and 60-80% AS fractions from fish egg extract, exhibited the strongest inhibition of trypsin when using a casein substrate. These results suggest that fish eggs act as serine protease inhibitors and may be useful for protease inhibition in foodstuffs.

• BMB Reports
• /
• 제37권1호
• /
• pp.59-74
• /
• 2004

The study of whole patterns of changes in protein expression and their modifications, or proteomics, presents both technological advances as well as formidable challenges to biological researchers. Nutrition research and the food sciences in general will be strongly influenced by the new knowledge generated by the proteomics approach. This review examines the different aspects of proteomics technologies, while emphasizing the value of consideration of "traditional" aspects of protein separation. These include the choice of the cell, the subcellular fraction, and the isolation and purification of the relevant protein fraction (if known) by protein chromatographic procedures. Qualitative and quantitative analyses of proteins and their peptides formed by proteolytic hydrolysis have been substantially enhanced by the development of mass spectrometry technologies in combination with nanoscale fluidics analysis. These are described, as are the pros and cons of each method in current use.

• BMB Reports
• /
• 제33권6호
• /
• pp.448-453
• /
• 2000

In yeast the key gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBPase), is selectively targeted from the cytosol to the lysosome (vacuole) for degradation when glucose starved cells are replenished with glucose. The pathway for glucose induced FBPase degradation is unknown. To identify the receptor-mediated degradation pathway of FBPase, we investigated the presence of the FBPase receptor on the vacuolar membrane by cell fractionation experiments and binding assay using vid mutant (vacuolar import and degradation), which is defective in the glucose-induced degradation of FBPase. FBPase sedimented in the pellets from vid24-1 mutant after centrifugation at $15,000{\times}g$ for 15 min, suggesting that FBPase is associated with subcellular structures. Cell fractionation experiments revealed that FBPase is preferentially associated with the vacuole, but not with other organelles in vid24-1. FBPase enriched fractions that cofractionated with the vacuole were sensitive to proteinase K digestion, indicating that FBPase is peripherally associated with the vacuole. We developed an assay for the binding of FBPase to the vacuole. The assay revealed that FBPase bound to the vacuole with a Kd of $2.3{\times}10^6M$. The binding was saturable and specific. These results suggest that a receptor for FBPase degradation exists on the vacuolar membrane. It implies the existence of the receptor-mediated degradation pathway of FBPase by the lysosome.