The Proteinase Distributed in the Intestinal Organs of Fish 1. Purification of the Three Alkaline Proteinases from the Pyloric Caeca of Mackerel, Scomber japonicus

변재형,김형낙,PYEUN Jae-Hyeung,KIM Hyeung-Rak The Korean Society of Fisheries and Aquatic Scienc 1986 한국수산과학회지 Vol.19 No.6

고등어 유문수조직중에 분포하는 알칼리성단백질분해효소의 정제조건을 검토하고 염석, DEAE-Sephadex A-50 칼럼 크로마토그래피 및 Sephadex G-100 겔 여과에 의해 3종류의 알칼리성단백질분해효소의 분리${\cdot}$정제방법을 확립하였다. 유문수조직중에 분포하는 알칼리성단백질분해효소의 추출용매는 $1\%$ NaCl 용액이 가장 효과적이었다. 정제된 가칭 Enz, A, B 및 C의 각 알칼리성단백질분해효소는 전기영동적으로 균질함이 증명되었으며, 조효소에 비해 고유활성이 Enz. A는 34배, B는 53배 그리고 C는 37배로 각각 증가하였다. 각 효소의 수율은 조효소에 대해 $1.6\%,\;2.1\%$ 및 $1.5\%$였고, 총수율은 $5.2\%$였다. In the previous paper(Kim et al, 1986), the alkaline proteinase from the pyloric caeca of mackerel was shown relatively strong activity in the alkaline pH range. Therefore purification of the enzyme has been undertaken to identify the proteolytic enzyme and three alkaline proteinases were isolated by ammonium sulfate fractionation, DEAE-Sephadex A-50 column chromatography and Sephadex G-100 gel filtration. One percent sodium chloride solution was the most effective for the extraction of alkaline proteinase from the pyloric caeca of mackerel. Three alkaline proteinases temporarily designated Enz. A, B and C were isolated from the pyloric caeca of mackerel, and identified to be homogeneous with electrophoresis. The specific activity of the purified Enz. A, B and C was increased to 34, 53 and 37-fold over the crude enzyme solution, respectively. Yield of them was 1.6, 2.1 and $1.5\%$, respectively, and a combined yield was $5.2\%$.

유전자 조작 , 균주분리 고활성 단백질분해효소 생산균주의 개발을 위한 Aspergillus oryzae 의 원형질체 융합에 의한 변이

변재형 ( Jae Hyeung Pyeun ) 한국미생물생명공학회 1998 한국미생물·생명공학회지 Vol.26 No.6

혈합육어(멸치, 고등어, 황다랭이 및 날개다랭이)의 Trypsin - 1. 정제와 반응조건

변재형(Jae-Hyeung Pyeun),조득문(Deuk-Moon Cho),허민수(Min-Soo Heu) 한국식품영양과학회 1993 한국식품영양과학회지 Vol.22 No.4

혈합육어 중 연근해 온대산 멸치의 내장과 고등어의 유문수, 그리고 원양 열대산 황다랭이 및 날개다랭이의 유문수를 각각 시료로 하여 trypsin을 분리 정제하였으며, 그 분자량과 아미노산 조성 및 효소의 반응 최적조건에 관하여 비교분석하였다. 실험결과를 요약하면 다음과 같다. 1. 멸치의 내장과 고등어, 황다랭이 및 날개다랭이의 유문수에서 각각 황산암모늄염석, benzamidine-Sepharose 6B 친화성 크로마토그래피, DEAE-Sephadex A-50 크로마토그래피, Sephadex G-75 겔 여과 크로마토그래피 등의 방법을 혼용하여 고등어부터는 2종의 trypsin(고등어 trypsin A와 고등어 trypsin B로 명명함)을, 그리고 다른 어류로부터는 각각 1종의 trypsin을 분리 정제하였다. 2. 멸치 trypsin과 고등어 trypsin B는 고등어 trypsin A와 황다랭이 trypsin 및 날개다랭이 trypsin에 비하여 그 고유활성이 월등히 높았다. 3. 이들 혈합육어 trypsin의 분자량은 22kDa~26kDa 범위였다. 4. 이들 trypsin은 공통적으로 glycine, serine, aspartic acid를 많이 함유하고 있었으며, tryptophan, methionine, lysine, tyrosine의 함유량은 적었다. 5. BA-p-NA기질에 대한 반응 최적조건은 멸치 trypsin은 PH 8.0에서 45℃, 고등어 trypsin A와 B는 PH 8.0에서 50℃, 그리고 황다랭이 trypsin은 PH 9.0에서 55℃, 날개다랭이 trypsin은 PH 9.0에서 50℃였다. 6. 위에 든 실험 결과들은 혈합육 어류의 서식온도가 이들 어류의 trypsin의 반응 최적 온도와는 어느정도 관계가 있을 것으로 추측되었다. Deterioration of fish muscle is known to occur more quickly in the dark fleshed fish than in the white fleshed fish, causing by their high intestinal proteolytic activity. Muscle degradation which suffer post-mortem autoproteolysis is affected by trypsin with its unique activation function towards other enzymes. To compare physicochemical and enzymatic properties for the trypsins of the dark fleshed fish, trypsins from the viscera of anchovy (Engraulis japonica), and the pyloric caeca of mackerel(Scomber japonicus), yellowfin tuna(Thunnus albacores) and albacore (Thunnus alalunga) were purified through ammonium sulfate fractionation, benzamidine-Sepharose 6B, DEAE-Sephadex A-50, and Sephadex G-75 chromatography. Two trypsins from mackerel (designated mackerel trypsin A and mackerel trypsin B), and one each from anchovy, yellowfin tuna and albacore were isolated as electrophoretical homogeneity. The purities of anchovy trypsin, mackerel trypsin A and H, yellowfin tuna trypsin, and albacore trypsin increased to 78.1, 4.8, 9.3, 120, and 16o-fold, respectively, compared to crude enzyme solutions. Molecular weights of the trypsins from the dark fleshed fish estimated by SDS-polyacrylamide electrophoresis were ranged from 22kDa to 26kDa. The trypsins contained higher amount of glycine, serine and aspartic acid, and less amount of tryptophan, methionine, lysine and tyrosine. Optimal conditions for amidolytic reactions of the enzymes were pH 8.0 and 45℃ for anchovy trypsin, pH 8.0 and 50℃ for mackerel trypsin A and B, pH 9.0 and 55℃ for yellowfin tuna trypsin, and pH 9.0 and 50℃ for albacore trypsin. It was supposed that the habitat temperature of the dark fleshed fish is slightly connected with the optimal reaction temperature of the trypsins of the fish.

海産腹足類의 Paramyosin의 分離 및 그 特性에 關한 硏究

변재형(Jae-Hyeung Pyeun) 한국식품영양과학회 1973 한국식품영양과학회지 Vol.2 No.1

The muscle of abalone, Notohaliotis discus (REEVE), and top-shell, Turbo cornutus Solander, were examined for protein composition.<br/> Then paramyosins which are known as one of the important structural protein of the muscle fibrils were isolated from the both muscle and their physico-chemical properties such as solubility, salting-out behaviour, intrinsic viscosity, ATPase activity, etc. involving amino acid composition and N-terminal amino acid residues were investigated to elucidate phylogenie characteristics more intensively from the viewpoint of comparative biochemistry.<br/> The analysis of protein composition resulted in the following estimations: abalone muscle; water-soluble protein of 22 %, salt-soluble protein, 34%, alkali-soluble protein, 20%, and stroma protein, 24%, and top-shell muscle; water-soluble protein of 16%, salt-soluble protein, 30%, alkali-soluble protein, 29%, and stroma protein, 25%, respectively.<br/> It is demonstrated in sedimentation analysis that paramyosin and myosin-actomyosin account for approximately 65% and 35% of the salt-soluble protein of abalone, and that the composition of both sediments in top-shell was approximately 70% and 30%, respectively.<br/> The ultracentrifugally homogenous paramyosins isolated essentially according to Bailey's ethanol-dried method from both of the muscle showed a S°_(20,w) of 3. 14s for abalone and a S°_(20,w) of 3.50s for top-shell.<br/> The both paramyosins were commonly rich in arginine, aspartic acid, and glutamic acid, while scarcely contained proline and tryptophan, in rough accord with the other paramyosins thus far reported.<br/> It is clear that these gastropod paramyosins showed of having the characteristic N-terminal amino acid residues such as N-aspartic acid, N-valine, N-serine, and N-threonine in common.<br/> The abalone paramyosin completely salted in with KCl beyond 0.35μ and the top-shell paramyosin beyond 0.30μ. The abalone paramyosin was salted-out between 18% and 30% saturation of ammonium sulphate and the top-shell paramyosin between 22% and 29% saturation.<br/> The intrinsic viscosities at abalone and top~shell paramyosins at 25℃ were estimated respectively to be 3.1 dl/g and 2.6 dl/g showing somewhat higher than the values for some other paramyosins from lamellibranchs.<br/> In regard with the ATPase activity, the para myosin specimens did not exhibit any significant activity over through the pH conditions of 5 to 9.5. irrespective of the presence of Ca^(++) or Mg^(++). So was the case with the abalone paramyosin prepared by a slightly modified Bailey's wet-extraction method.

방어 Myosin과 갈색띠 매물고둥 Paramyosin 의 물리화학적 성질

변재형(Jae-Hyeung Pyeun),최영준(Yeung-Joon Choi) 한국식품과학회 1987 한국식품과학회지 Vol.19 No.1

The Proteinase Distributed in the Intestinal Organs of Fish 2. Characterization of the Three Alkaline Proteinases from the Pyloric Caeca of Mackerel, Scomber japonicus

김형낙,변재형,KIM Hyeung-Rak,PYEUN Jae-Hyeung The Korean Society of Fisheries and Aquatic Scienc 1986 한국수산과학회지 Vol.19 No.6

전보(Pyeun and Kim, 1986)에서와 같이 고등어 유문수조직에서 정제한 3종의 알칼리성단백질분해효소에 대하여 활성최적조건, 열안정성. 기질친화도, 화학약제에 대한 영향 및 분자량 등을 규명하였다. 각 정제효소의 반응최적조건을 기질별로 검토한 결과, casein에 대하여 Enz. A는 pH 9.4, Enz. B와 Enz. C는 pH 9.8이었으며, Hb에 대하여 Enz. A는 pH 9.2, Enz. B는 pH 10.2, 그리고 Enz. C는 pH 9.8이었고, 최적반응온도는 공히 $45^{\circ}C$였다. 효소농도 $2{\mu}g/ml,\;2\%$ casein 기질의 반응조건에서 반응시간(x)에 대한 활성도 (y)의 관계를 분석한 결과, Enz. A는 60분. Enz. B는 40분, 그리고 Enz. C는 50분까지 1차 반응의 관계가 성립하였으며, 이때의 반응속도식은 Enz. A는 y=3.6x, Enz. B는 Y=6.0x, 그리고 Enz. C는 y=4.2x였다. 열안정성을 검토하기 위하여 $50^{\circ}C$에서 5분간 가열했을때, Enz, A는 $90\%$, Enz. B는 $33\%$, 그리고 Enz. C는 $37\%$가 각각 불활성화하였다. Lineweaver-Burk의 도식에 의한 효소의 기질친화도를 측정한 결과, casein기질에 대하여 Enz. A는 Km이 $5.0{\times}10^{-3}\%$, Enz. B는 Km이 $1.0{\times}10^{-3}\%$, 그리고 Enz. C는 Km이 $3.6{\times}10^{-3}\%$였다. 금속 ion에 의한 영향을 검토한 결과, $Ag^+,\;Hg^{2+}$는 효소활성을 저하시켰으나, $Mn^{2+},\;Sn^{2+}$ 및 $Pb^{2+}$ 이온은 활성을 증가시켰다. Enz. B와 Enz. C는 soybean trypsin inhibitor에 의해 상당히 저해되었다. 따라서 효소 B와 C는 serine 계 단백질분해효소로 판단되었다. SDS-PAG 전기영동과 Sephadex G-100 겔 여과법에 의하여 각 정제효소의 분자량을 측정한 결과, Enz. A는 $27,500{\pm}2.500$, Enz. B는 $20,500{\pm}1,500$, 그리고 Enz. C는 $15,250{\pm}250$이었다. The characteristics of the three alkaline proteinases, Enz. A, B and C, from the pyloric caeca of mackerel have been investigated. The optimum condition for the activity of the Enz. A, B and C was pH 9.4, 9.8 and 9.8 at $45^{\circ}C$ for $2\%$ casein solution, and was pH 9.2 10.2 and 9.8 at $45^{\circ}C$ for $5\%$ hemoglobin denatured by urea, respectively. Enz. A, B and C by heat treatment at $50^{\circ}C$ for 5 min were inactivated 90, 33 and $37\%$, respectively, over the original activity. The reaction rate of the three alkaline proteinases was constant to the reaction time to 40 min in the reaction condition of $2{\mu}g/ml$ of enzyme concentration and $2\%$ casein solution. The reaction rate equation and Km value against casein substrate determined by the method of Lineweaver and Burk were: Enz. A, Y=3.6X and $Km=5.0{\times}10^{-3}\%$; Enz. B, Y=6.0X and $Km=1.0{\times}10^{-3}\%$; Enz. C, Y=4.2X and $Km=3.6{\times}10^{-3}\%$. The three alkaline proteinases were inactivated by $Ag^+$ and $Hg^{2+}$, but activated by $Mn^{2+},\;Sn^{2+}\;and\;Pb^{2+}$, Enz. B and C were remarkably inhibited by the soybean trypsin inhibitor. Molecular weight of Enz. A, B and C determined by SDS-polyacrylamide gel electrophoresis and Sephadex G-100 gel filtration was in the range of $27,500{\pm}2,500,\;20,500{\pm}1,500\;and\;15,250{\pm}250$, respectively.

패류 가공에 관한 연구 1. 개량조개 Mactra Sulcataria REEVE의 토사 배출에 대하여

이응호,변재형,허종화,LEE Eung-Ho,PYEUN Jae-Hyeung,HUR Jong-Wha 한국수산과학회 1970 한국수산과학회지 Vol.3 No.1

살아 있는 개량조개 속에 들어 있는 토사의 배출에 대하여 실험한 결과를 요약하면 다음과 같다. 1) 토사는 주로 입수관 및 출수관 주변, 아가미 주변, mantle주변 그리고 중장선을 위시한 소화 기관 속에 존재하였다. 그리고 소화기관 속에 들어 있는 모래의 크기는 $180\times10\mu\~550\times200\mu$정도의 극히 작은 것들이었다. 2) 시료를 밑바닥에 깔아 놓은 것과 중간층에 수하시킨 것 그리고 단층으로 깔든지 또는 수하시킨 것과 두껍게 깔든지 또는 수하시킨 것 사이에 토사의 배출에 있어 뚜렷한 차이는 찾아 볼 수 없었다. 3) 개량조개는 처음 1시간 정화시키면 약 $50\%$의 토사를 배출하며, $22\~26$ 시간만에 배출물의 양이 거의 일정한 값에 도달하지만, 배출물 속에 토사가 배출되지 않을 때까지는 약 42시간 소요되었다. 4) 토사의 배출에 미치는 PH의 영향을 보면 개량조개가 살고 있는 환경 해수보다 약 1정도 높은 쪽(pH 8.75)이 빨랐다. 5) 토사의 배출에 미치는 온도의 영향은 개량조개가 살고 있는 환경 해수의 온도 때가 온도를 더 높였을 때보다 토사의 배출이 빨랐다. 6) 토사의 배출에 미치는 염분 농도의 영향은 개량조개가 살고 있는 환경 해수의 염분 농도($30.61\%_{\circ}$)보다 약 배로 묽은 쪽($15.14\%_{\circ}$)이 배출이 빨랐다. A study on the purification of the surf clam Mactra Sulcataria REEVE for the remove of sand and fecal piles was conducted in the laboratory. The sand was mostly distributed around the inhalent and exhalent siphon, mantle and gill, and were also distributed in the digestive tract including the mid-gut gland. The sand particles in the digestive tract were extremely small and their sizes were about $180\times10\mu\;to\;550\times200\mu$. It could be seen that there was little, if any, difference in the rate of sand removal when either the hanging Purification method was used or the method of laying the surf clams in thick or thin layers on the bottom of the experimental vessel was used. The surf clam removed about $50\%$ of its sand during the first hour of purification. The weight of the removed sand and fecal piles on a dry basis reached a constant value after 22 to 26 hours of purification. After 42 hours of Purification, no sand could be seen in the fecal piles. The surf clam removed the sand in its body more rapidly when it was in sea water with the pH of 8.75 than when it was in natural sea water. Also high temperature had a much greater depressing effect upon the removal of sand In the surf clam than did natural sea water. Also low salinity had greater accelerating effect upon the removal of sand in the body than did natural sea water. However the surf clam died when the salinity was $8.19\%_{\circ}$.

가열조리가 복어 추출물 함질소화합물의 조성에 미치는 영향

양영,한영실,변재형,Yang, Yeong,Han, Yeong-Sil,Pyeun, Jae-Hyeung 한국식품조리과학회 1990 한국식품조리과학회지 Vol.6 No.2

Globefish, Fugu xanthopterus, known to have a severe toxin, is one of the favorite food in Korea and Japan when the toxic part is removed. In this paper, the effect of cooking on the composition of nitrogenous components in the extractives from globefish cooked investigated and the changes of the taste compounds originated from the nitrogenous components in the extractives were discussed. When the sample fish was analysed for general composition, drip amount and pH by the different methods of thawing, the method effective method was the running water thawing. Total nitrogen content in raw globefish and the frozen globefish was not different, and the nitrogen content was increased with the heat treatment. It seemed that the nitrogen content was higher in the extract from the boiled globefish than that of the steamed globefish. Taurine, lysine, glycine and alanine were occupied about 70% of the total free amino acids. Total free amino acid content was higher in the extracts from the frozen sample than those from the raw sample. The amount of free amino acids was increased when the globefish soup cooked under the direct-heat cooking than in the microwave oven-heat cooking. Among nucleotides in the extracts from the thawed and cooked fishes, IMP and inosine contents were increased, and the both components were decreased with the heating time and by the heating method. Tne content of total creatinine-nitrogen were 50% of the total nitrogen content of the extracts, but the concentration of glycinebetaine, TMA and TMAO were only a few amounts. It could be concluded that total creatinine components, including free amino acids such as taurine, lysine, glycine and alanine, and IMP might be the important components contributing to the taste of the cooked globefish.

가열 변성에 따른 방어 Myosin과 갈색띠 매물고둥 Paramyosin의 소수성, 용해도, SH기 및 단백질간 상호작용의 변화

최영준(Yeung-Joon Choi),변재형(Jae-Hyeung Pyeun) 한국식품과학회 1987 한국식품과학회지 Vol.19 No.2

식용해조류중의 미량요소와 특수기능성 당질 - 1 산지와 채취시기별 일반성분의 조성과 무기원소의 분포

조득문,김두상,이동수,김형락,변재형 ( Deuk Moon Cho,Doo Sang Kim,Dong Soo Lee,Hyeung Rak Kim,Jae Hyeung Pyeun ) 한국수산과학회 1995 한국수산과학회지 Vol.28 No.1

Nine species of edible seaweed [green layer(Monostroma nitidium) and sea staghorn(Codium fragile) of green algae; sea mustard(Undaria pinnatifida), seaweed fusiforme (Hizikia fusiforme), gulf weed (Sargassum fulvellium), and sea tangle(Laminaria japonica) of brown algae; seaweed dilatata(Halimeniopsis dilatata), seaweed furcata(Gloiopeltis furcata), and layer(Porphyra tenera) of red algae] collected from Kijang, Chungmu, and Yosu in Korea, were examined and compared on their chemical and mineral composition depending on their harvesting season. Crude protein(N×6.25) showed about 45% on moisture free basis(the contents of every components described below are shown as moisture free basis) for layer and 30% for green layer collected from every seasons and sites. Sea tangle showed the highest content in crude lipid(10%) among brown seaweeds and green layer had 6% of crude lipid regardless their habitats. Ash and carbohydrate in sea staghorn hold about 90% of total solids but those levels were only 50% in layer. The relationship between ash and carbohydrate content showed a tendency with reverse correlation. Mineral compositions were examined on green layer, sea mustard, and seaweed furcata collected from Kijang. Sodium was eminent element(1,798-7,334㎎/100g) followed by potassium. Magnesium and calcium content were low level compared with sodium and potassium. As a micro-element, iron was appraised the highest status(165-330㎎/100g) in green layer, however, iron in sea mustard and seaweed furcata was comparatively low amount (2.7-47.4㎎/100g). The level of zinc was also comparatively high and that was varied on habitats. In conclusion, chemical compositions of these algae were distinctively varied on species and habitats, and mineral compositions were notably changed by the harvesting season.