복상어와 고등어의 Trypsin 에 관한 비교효소학적 연구 : 2 . 정제 Trypsin 의 효소적 성질 2 . Enzymatic Properties of the Trypsins

변재형,조득문,허민수 한국수산학회 1992 한국수산과학회지 Vol.25 No.5

전보(Pyeun 등, 1991)에 이어 복상어의 췌장에서 정제한 trypsin과 고등어 유문수에서 정제한 trypsin A 및 trypsin B에 대하여 그 효소적 특성을 비교 검토한 내용을 요약하면 다음과 같다. 1. 30℃, 45℃, 55℃ 및 60℃의 각 온도 조건에서 효소의 안정성을 검토한 결과, 복상어의 trypsin이 고등어의 두 trypsin보다 안정하였으며, 고등어 trypsin A는 가장 불안정하였다. 55℃에서의 변성속도정수는 복상어 trypin이 10.68×10^(-4) sec^(-1), 고등어 trypsin A는 47.18×10^(-4) sec^(-1), 그리고 고등어 trypsin B는 34.06×10^(-4) sec^(-1)로서 고등어 trypsin A의 변성속도가 가장 빨랐다. 활성화에너지는 복상어 trypsin이 4.07㎉/mole, 고등어 trypsin A는 11.61㎉/mole, 그리고 고등어 trypsin B는 8.43㎉/mole이었다. 2. Km정수는 복상어 trypsin이 248.59μM, 고등어 trypsin A는 53.7μM, 그리고 고등어 trypsin B는 96.5μM이었다. 3. Dixon식에서 구한 저해제 TLCK에 대한 저해제 정수는 복상어 trypsin 1.50×10^(-6) M, 고등어 trypsin A는 2.86×10^(-6) M, 그리고 고등어 trypsin B는 3.90×10^(-6) M였으며, 저해제 DFP에 대한 저해제 정수는 복상어 trypsin이 9.28×10^(-6) M, 고등어 trypsin A는 2.11×10^(-4) M, 그리고 고등어 trypsin B는 1.60×10^(-4) M이었다. 4. 복상어와 고등어 trypsin의 효소적 특성의 두드러진 차이는 pH와 온도에 대해서 복상어의 trypsin은 고등어의 두 trypsin보다 알칼리성측과 고온에 대해 안정하였으며, 저해제에 의하여서는 복상어 trypsin이 보다 예민하게 나타났다. 또한 BA-p-NA 합성기질에 대한 친화성에 있어서는 고등어의 두 trypsin이 복상어의 trypsin보다 높은 것을 알 수 있었다. 5. 아미노산 조성은 세 효소 모두 비슷하였으나, 고등어 trypsin B의 경우 arginine의 함량이 높은 특징을 보였다. 산성아미노산과 염기성아미노산의 비는 복상어 trypsin이 3.57, 고등어 trypsin A는 3.36, 그리고 고등어 trypsin B는 2.23이었다. A comparative study of enzymatic properties between the trypsin from the cat-shark Cephaloscyllium umbratile(C-T) and the two trypsins from the mackerel Scomber japonicus (M-T_A and M-T_B) was carried out following after the previous paper(Pyeun et al., 1991). Trypsin from cat-shark(C-T) showed the higher heat stability compared to the others (M-T_A and M-T_B) and its denaturation constant(K_D) was 10.68×10^(-4) sec^(-1) at 55℃ with BA-p-NA substrate. The activation energies(Ea) of the trypsins measured at a temperature range from 30℃ to 50℃ were estimated to be 4.07㎉/mole for C-T, 11.61㎉/mole for M-T_A, and 8.43㎉/mole for M-T_B, respectively. The Km values were 24.9×10^(-5) M for C-T, 5.37×10^(-5) M for M-T_A, and 9.65×10^(-5) M for M-T_B. On the other hand, the Ki values for TLCK and DFP determined by Dixon plot were 1.50×10^(-6) M and 9.28×10^(-6) M for C-T, 2.86×10^(-6) M and 2.11×10^(-4) M for M-T_A, and 3.90×10^(-6) M and 1.60×10^(-4) M for M-T_B. Similar amino acid profiles were showed between three trypsins each other, with few exceptions of M-T_B containing higher amount of arginine, and the smaller amount of tryptophan in C-T than the others.

Comparative Biochemical Properties of Proteinases from the Hepatopancreas of Shrimp. : 1. Purification of Protease from the Hepatopancreas of Penaeus japonicus

Pyeun, Jae Hyeung,KIM, Hyeung Rak,KIM, Doo Sang,AHN, Chang Bum,Choi, Sung Mi,Oh, Eun Sil,Cho, Deuk Moon 한국수산학회 1998 Fisheries and Aquatic Sciences Vol.1 No.2

A protease, which had no tryptic and chymotryptic activity, was purified from the hepatopancreas of shrimp, P. japonicus, through ammonium sulfate fractionation, Q-Sepharose ionic exchange, benzamidine Sepharose 6B affinity, and Sephacryl S-100 gel chromatography. Molecular weight (M.W.) of the protease was estimated to be 24 kDa by gel filtration and. showed a single peptide band by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protease had a low ratio of acidic to basic amino acids, which is different with proteases from marine animals. The enzyme was partially inhibited by benzamidine, tosyl-L-lysine chloromethyl ketone (TLCK), phenylmethylsulfonyl fluoride (PMSF), soybean trypsin inhibitor (SBTI), and pepstatin. The enzyme did not have any activity against benzoyl-D, L-arginine p-nitroanilide (BAPNA) or benzoyl-L-tyrosine ethyl ester (BTEE) which is a specific substrate of trypsin and chymotrypsin, respectively. However, the enzyme showed activity forward N-CBZ-L-tyrosine p-nitrophenyl ester (CBZ-Tyr-pNE), N-CBZ-L-tryptophan p-nitrophenyl ester (CBZ-Trp-pNE), and N-CBZ-L-proline p-nitrophenyl ester (CBZ-Pro-pNE). The protease did not showed tryptic and chymotryptic activity, which was not reported in shrimp hepatopancreas.

Variability of Current and Sea Level Difference in the Western Channel of the Korea Strait in Winter 1995 ~ 96

Pyeun, Jae Hyeung,Kim, Jeong Sook,Yoon, Hong Joo,Lee, Sang Ryong,Byun, Sang Kyoung,Park, Moon Jin 한국수산학회 1998 Fisheries and Aquatic Sciences Vol.1 No.2

As a part of the long-term ADCP mooring program to measure the mass flux through the Korea Strait, current velocity data were obtained for 39 days in the deepest point of the strait. Near-surface velocity of this abservation was compared with Izuhara-Pusan sea level difference (SLD) to investigate the geostrophic relationship. Principal direction of the Tsushima Current at the mooring station is 44.6 degrees to the north from the east. Variability of the tidal current is greater than the nontidal current by a factor of two. Correlation coefficient of tidal current a against SLD is 0.46 but the nontidal current is not correlated. The current velocity (U in ㎝/s) can be estimated from the demeaned SLD (in. ㎝) by the relation U=23.63+0.64SLD where the maximum range of SLD is 52.9 ㎝. Current is coherent with SLD at semidiurnal, diurnal and 42.7-hour periods. A dominant nontidal variability with about 5-day period is not coherent with SLD.

Purification and Characterization of Trypsins Affecting on the Autolysis of Shrimp , Penaeus japonicus

Pyeun, Jae Hyeung,KIM, Hyeung Rak,KIM, Doo Sang,AHN, Chang Bum 한국수산학회 1996 한국수산과학회지 Vol.29 No.6

Two trypsins were purified from shrimp hepatopancreas through ammonium sulfate fractionation, Q-Sepharose ionic exchange, benzamidine Sepharose-6B affinity, and Sephacryl S-300 gel chromatography. Both enzymes had a single polypeptide chain with a molecular weight(M.W.) of 32kDa by sodium dodecylsulfate polyacrylamide gel electrophoresis(SDS-PAGE), although trypsin A and B were estimated to be a molecular weight of 27.2 and 22.8kDa, respectively, using Sephacryl S-300 gel filtration. Both trypsins had similar amino acid compositions and rich in glycine, valine, alanine, aspartic acid, and glutamic acid, but low in methionine and basic amino acids. Both enzymes were completely inactivated by soybean trypsin inhibitor(SBTI), phenylmethylsulfonyl fluorid(PMSF), tosyl-L-lysine chloromethyl ketone(TLCK), benzamidine, leupeptin, however, not affected by tosyl-L-phenylalanine chloromethyl ketone(TPCK) and pepstatin.

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.

방어 Myosin과 갈색띠 매물고둥 Paramyosin 의 물리화학적 성질

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

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.

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.

멸치 육과 내장으로부터 분리한 Cathepsin L, Chymotrypsin 및 Trypsin의 단백질분해 특성

번재형 ( Jae Hyeung Pyeun ),허민수 ( Min Soo Heu ),조득문 ( Deuk Moon Cho ),김형락 ( Hyeung Rak Kim ) 한국수산학회 1995 한국수산과학회지 Vol.28 No.5

어류의 사후 초기의 변화를 육 및 장기조직중에 분포하는 단백질분해효소의 작용과 관련하여 검토할 목적으로 멸치의 육 및 장기에서 분리한 cathepsin L과 chymotrypsin 및 trypsin의 단백질 기질에 대한 특성과 근원섬유단백질에 대한 분해능을 전기영동적으로 분석하여 다음의 결론을 얻었다. 이들 세 효소의 casein에 대한 친화도는 유사하였고, 근원섬유단백질에 대한 친화도는 casein에 대한 친화도보다 높았다. 멸치와 방어의 근원섬유단백질에 대한 cathepsin L과 chymotrypsin의 활성은 trypsin보다 훨씬 높게 나타났다. 0~25%까지의 식염농도에서 세 효소의 단백질분해활성은 식염의 농도에 반비례하였으며, 식염의 공존상태에서 세 효소는 casein 보다 근원섬유단백질에 대하여 높은 활성을 나타내었다. 근원섬유단백질의 효소 분해시에 cathepsin L은 chymotrypsin과 trypsin에 비하여 염농도와 온도에 의한 영향이 적었다. 따라서, 멸치의 사후변화와 젓갈 숙성중의 자가소화는 trypsin보다는 cathepsin L과 chymotrypsin의 단백질분해활성이 더욱 깊이 관여할 것으로 판단된다. Proteolytic properties of enzymes from the muscle and viscera of anchovy have been examined. Cathepsin L, chymotrypsin, and trypsin showed similar Km values for casein. However, they had higher Km values for myofibrillar proteins than those for casein. The k(cat) of cathepsin L and chymotrypsin for myofibrillar proteins were higher than that of trypsin, and also cathepsin L and chymotrypsin caused higher hydrolysis in myofibrillar proteins of anchovy and yellowtail. In the presence of sodium chloride (0~25%), proteolytic activity for myofibrillar proteins from yellowtail was higher than that for casein. Proteolytic activity was decreased with the increase of sodium chloride concentration. Cathepsin L had been less affected by NaCl concentration and temperature on the hydrolysis of myofibrillar proteins than chymotrypsin and trypsin. Cathepsin L and chymotrypsin were more responsible to the autolysis of muscle proteins from fish than trypsin.

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