버섯 중 Aminotransferase 의 동질효소 분리정제 및 그 특성에 관한 연구(II) -표고버섯(Lentinus edodes)중의 Branched Chain Amino Acid Aminotransferase(Enzyme II)의 정제 및 특성-

민태진,배강규,Min, Tae-Jin,Bae, Kang-Gyu 생화학분자생물학회 1992 한국생화학회지 Vol.25 No.6

Enzyme I and II of branched chain amino acid aminotransferase (BCAT) in Lentinus edodes (Berk.) Sing was purified by ammonium sulfate fractionation, DEAF-cellulose column chromatography and sephadex G-150 gel filtration. We have been studied molecular weight, N-terminal amino acid, composition of amino acids, optimum pH, optimum temperature, $K_m$ value and effect of organic compounds and metal ions about enzyme II, to identify isozyme patterns between enzyme I and II. Apparent molecular weight of enzyme II was estimated 72,000 dalton, N-terminal amino acid was determined glycine. The enzyme II was existed as negatively charged amino acids rather than positively charged ones by more than 3.67%. Optimum pH and temperature were found to be 8.2 and $38^{\circ}C$, respectively. It was stable for 30 min under $20^{\circ}C$. This enzyme showed the activity toward L-leucine, as a substrate. It was inhibited by both organic compounds of hydroxylamine, N-ethylmaleimide and p-chlromercuribenzoate and metal ions of $Cu^{2+}$, $Hg^{2+}$ and $Fe^{2+}$. The $K_m$ values were determined to be 3.39 mM for L-leucine as a amino group donor, 0.28 mM for $\alpha$-ketoglutarate as a amino group acceptor. Although their molecular weight, N-terminal amino acid, composition of amino acids, optimum pH and temperature and $K_m$ values had been different between enzyme I and II, but they showed same substrate specificity for L-leucine as a substrate. 표고버섯, Lentinus edodes (Berk.) Sing 중의 가지달린 아미노산 aminotransferase(BCAT)의 enzyme I과 II를 황산암모늄 분별침전, DEAE-cellulose column 크로마토그래피 및 Sephadex G-150 겔 여과하여 각각 분리정제한 후, enzyme I과 II의 동질효소 여부를 구명하기 위하여 enzyme II의 분자량, N-말단, 아미노산 조성, 최적 pH, 최적 온도, $K_m$값, 유기물 및 금속이온 효과 등을 실험하였다. 이 효소의 겉보기 분자량은 72,000 dalton 이었고, N-말단 아미노산은 L-leucine이었으며, 음성잔기를 가진 아미노산이 양성잔기를 가진 것보다 3.67% 더 많이 존재하였다. 최적 pH 및 최적 온도는 각각 8.2, $38^{\circ}C$ 였으며, $20^{\circ}C$이하에서 30분 동안 안정하였다. 이 효소는 기질인 가지달린 아미노산 중 L-leucine에 대해서만 활성을 보였으며, hydroxylamine, N-ethylmaleimide 및 p-chloromercuribenzoate 등의 유기물과 $Cu^{2+}$, $Hg^{2+}$ 및 $Fe^{3+}$ 등의 금속이온에 의하여 활성이 억제 되었다. 아미노기 공여체인 L-leucine과 아미노기 수용체인 $\alpha$-ketoglutarate에 대하여 활성도 측정시의 조건하에서 측정된 $K_m$값은 각각 3.39 및 0.28 mM이었다. 이로써 enzyme II와 I은 분자량, N-말단 아미노산, 아미노산 조성, 최적 pH 및 최적 온도 그리고 $K_m$값 등은 서로 다르지만, L-leucine 기질에 대하여 같은 기질 특이성을 보이는 효소였다.

무흡광 색소 생물의 감광수용체 개발 연구 (4) - 표고버섯 중의 광감응성 Mitochondrial ATP synthase 의 유기물 및 금속이온 유입효과 -

민태진,이완기,김재웅,민태익 ( Tae Jin Min,Wan Gie Lee,Jae Woong Kim,Tae Ick Mheen ) 한국균학회 1989 韓國菌學會誌 Vol.17 No.2

흰느타리버섯 ( Pleurotus cornucopiae ( Per . ) Rolland ) 중의 Protease 의 분리정제 및 성질에 관한 연구 ( 2 )

민태진,이수용,김재웅 ( Tae Jin Min,Soo Young Lee,Jae Woong Kim ) 생화학분자생물학회 1985 BMB Reports Vol.18 No.2

The enzymic properties of protease in the Pleurotus cornucopiae(Per.) Rolland were investigated. The Km value was 0.45 mM when hippuryl-L-phenylalanine was used as a substrate. This enzyme showed substrate specificity hydrolyzing the peptide bond of glycyl-L-phenylalanine. This enzyme was inhibited by L-lysine, diazoacetyl-DL-norleucine methyl ester(DAN), 1,2-epoxy-(p-nitrophenoxy)propane(EPNP) and EDTA, also was inhibited by anion as SO₃^(-2), SO₄^(-2) and HP₄^(-2) but increased with NO₂^(-2) ion. The L-cysteine was found to be competitive inhibitor and its K_i value was determined to be 2.4 mM by Dixon plot.

흰느타리버섯(Pleurotus cornucopiae (per.) Rolland)중의 Protease의 분리정제 및 성질에 과한 연구 (II)

민태진,이수용,김재웅,Min, Tae-Jin,Lee, Soo-Young,Kim, Jae-Woong 생화학분자생물학회 1985 한국생화학회지 Vol.18 No.2

흰느타리버섯(Pleurotus cornucopiae (Per.) Rolland)의 protease를 분리 정제하여 그 성질을 조사하였다. Hippuryl-L-phenylalanine 기질에 대한 이 효소의 $K_m$ 값은 0.45 mM 이었고, glycyl-L-phenylalanine의 펩티드 결합을 분해시키는 특이성을 보였다. L-Cysteine, L-Lysine, DAN, EPNP, EDTA, ${SO_3}^{-2}$, ${SO_4}^{-2}$, 및 ${HPO_4}^{-2}$은 효소활성을 저해하였고, ${NO_2}^{-1}$은 활성을 증가시켰다. L-Cysteine은 경쟁적 방해제로 작용하였고, 그것의 $K_i$값은 2.4 mM 이었다. $pK_a$값은 4.4였다. The enzymic properties of protease in the Pleurotus cornucopiae(Per.) Rolland were investigated. The $K_m$ value was 0.45 mM when hippuryl-L-phenylalanine was used as a substrate. This enzyme showed substrate specificity hydrolyzing the peptide bond of glycyl-L-phenylalanine. This enzyme was inhibited by L-lysine, diazoacetyl-DL-norleucine methyl ester(DAN), 1,2-epoxy-(p-nitrophenoxy) propane(EPNP) and EDTA, also was inhibited by anion as ${SO_3}^{-2}$, ${SO_4}^{-2}$ and ${HPO_4}^{-2}$ but increased with ${NO_2}^{-1}$ ion. The L-cysteine was found to be competitive inhibitor and its $K_i$ value was determined to be 2.4 mM by Dixon plot.

누에(Bombyx mori L.)의 요소첨식 효과에 관한 연구

민태진,김택영,Min, Tae-Jin,Kim, Taik-Yung 생화학분자생물학회 1978 한국생화학회지 Vol.11 No.3

누에(수원 $108{\times}107$호 교잡종)에 3령기에서 5령기까지 요소를 농도별(1,000ppm~2,000ppm)로 첨식시켜 성장도, 고치량의 변화, 체내 Urease의 활성도, 핵산, 단백질의 함량 변화 및 Acrylamide gel올 이용한 disc electrophoresis를 통하여 단백질의 이동상의 변화를 관찰하였다. 요소 1600ppm($U_4$) 처리구에서, 성장도 및 고치량의 증가율은 각각17.43%, 20.11%로 최고치였다. Urease의 활성도는 3령기에서 5령기로 갈수록 크게 감소하였고, 요소의 농도 증가에 따라 증가하나 $U_4$ 이상에서는 감소하였다. 그러나 요소처리구는 대조구에 비하여 감소하였다. 핵산량은 3령기에서 5령기로 갈수록 크게 감소하고, 요소처리구는 대조구에 비하여 감소하지만 요소 농도 증가에 따라 증가하였다. 5령기 끝날, 누에의 체내 단백질의 전기이동상은 대조구(C)에서 6개, $U_4$구에서는 5개의 이동상을 확인하였다. The change of the growth rate, the weight of cocoon, and the change of Urease activity, nucleic acid, protein contents, and the changes in disc electrophoretic patterns of the protein in the silkworm (Suwon No $108{\times}107$ confused species) from the 3rd instar stage to the 5th instar stage by supplemental feeding of different concentrations of Urea has been studied. On the experiment group, $U_4$ which was treated with 1,600ppm Urea, the rate of growth increased to 17.43% and the rate of cocoon's weight increased 20.11% more than the control groups. The activity of Urease was increased with the increasing of the concentration of Urea treatment, but not more than the control groups. Beyond $U_4$ activity of Urease was decreased gradually with the increasing of the concentration of Urea. The contents of nucleic acid were decreased sharply at first in the Urea treatments but gradually increased according to the Urea concentration. In the 5th instar stage, disc electrophoretic patterns of protein in. the silkworms were found to be five in the $U_4$ group compared to six in the control group.

흰느타리버섯(Pleurotsu cornucopiae(pers) Rolland)중의 Protease의 분리정제 및 그 성질에 관한 연구(I)

민태진,홍성일,김재웅,Min, Tae-Jin,Hong, Sung-Il,Kim, Jae-Woong 생화학분자생물학회 1983 한국생화학회지 Vol.16 No.1

흰느타리버섯(Pleurotus cormkopiae(pers.) Rolland)중의 protease를 DEAE-sephadex A-50, CM-cellulose, DEAE-cellulose 및 Sephadex G-75를 사용하여 분리정제하고 그 성질을 조사하였다. 이 버섯중에는 bovine serum albumin을 기질로 하였을 때 2종류의 활성분획을 함유하고 있었고, protease(분획 I)의 최적 pH는 4.0, 최적온도는 $50^{\circ}C$, pH 안정도는 pH 3.0~4.0 그리고 열안정도는 $40^{\circ}C$ 이하에서 안정하였다. 또한$Ca^{++}$, $Cu^{++}$, $Zn^{++}$, $Fe^{++}$ 및 $Mg^{++}$ 이온은 이 효소의 활성도를 저해하였으나, $Co^{++}$ 이온은 효소의 활성을 증가시켰다. 이 효소의 subunit는 1개 였고, 걷보기 분자량은 56,000 daltons이었으며 아미노산 조성은 17종이었다. The enzyme properties of protease of the Pleurotus Cornucopiae (Pers.) Rolland were investigated by purification with gel filteration using DEAE-sephadex A-50, CM-cellulose, DEAE-cellulose and Sephadex G-75. Two kinds of active fractions were isolated from this mushroom study. The active fraction showed protease activity for the bovine serum albumin substrate, and its optimum pH, optimum temperature, pH stability and thermal stability were 4.0, $50^{\circ}C$, 3.0~4.0 and $20{\sim}40^{\circ}C$, respectively. The activity of the enzyme was inhibited by $Ca^{++}$, $Cu^{++}$, $Zn^{++}$, $Fe^{++}$ and $Mg^{++}$, but increased with $Co^{++}$. The enzyme had one subunits composed of 17 amino acids. The apparent molecular weight was about 56,000 daltons.

Purification and Properties of Carboxyl Proteinase from the Poria cocos (SchW.) Wolf (I)

민태진,정광식,김재웅,Min, Tae-Jin,Chung, Kwang-Sik,Kim, Jae-Woong 생화학분자생물학회 1983 한국생화학회지 Vol.16 No.3

복령[Poria cocos (Schw.) Wolf.]중의 carboxyl proteinase을 황산 암모늄 65% 포화용액, DEAE-cellulose 및 Sephadex G-75을 이용한 gel-filtration 방법으로 분리 정제하여 그 성질을 조사하였다. 이 버섯중에서 활성분획분 I과 II을 얻었으며, 활성분획분 II에 대하여 bovine hemoglobin을 기질로 사용하여 얻은 carboxyl proteinase의 활성도의 최적 pH는 2.0, 최적온도는 $70^{\circ}C$, pH 안정성은 1.2~2.5 및 열안정성은 $25^{\circ}C{\sim}75^{\circ}C$였다. 이 효소의 subunit는 1개였고, 18종의 아미노산으로 구성된 단백질이였으며, 겉보기 분자량은 25,500(PAGE)~23,000 (HPLC)였다. 이 효소는 carbobenzoxy-L-glutamyl-L-tyrosine (CBZ-glu-tyr.)의 glutaml-L-tyrosine간의 부위를 절단하는 특이성을 가졌었다. CBZ-glu-tyr.을 사용하여 측정한 Km 값은 0.29 mM 이였다. The enzymic properties of carboxyl proteinase isolated from the Poria cacos (Schw.) Wolf. were investigated. Two kinds of active fraction I and II were isolated from this mushroom. The active fraction II showed carboxyl proteinase activity to bovine hemoglobin substrate, and its optimum conditions were the followings; temperature, $70^{\circ}C$, pH stability, 1.2~2.5 and thermal stability, $25^{\circ}C{\sim}75^{\circ}C$. The enzyme appeared to be one subunit, and consisted of 18 kinds of amino acids. The apparent molecular weight were 25,500 (PAGE) and 23,000(HPLC), respectively. The enzyme appeared to hydrolyze peptide bond between glutaml-L-tyrosine. The Km value of this enzyme was 0.29mM when carbobenzoxy-L-tyrosine were used as a subtrate.

복령 ( Poria cocos ( Schw . ) Wolf ) 중의 Carboxyl Proteinase 의 분리정제 및 그 성질에 관한 연구 ( 1 )

민태진,정광식,김재웅 ( Tae Jin Min,Kwang Sik Chung,Jae Woong Kim ) 생화학분자생물학회 1983 BMB Reports Vol.16 No.3

The enzymic properties of carboxyl proteinase isolated from the Poria cacos (Schw.) Wolf. were investigated. Two kinds of active fraction I and II were isolated from this mushroom. The active fraction II showed carboxyl proteinase activity to bovine hemoglobin substrate, and its optimum conditions were the followings: temperature, 70℃, pH stability, 1.2∼2.5 and thermal stability, 20°∼75 ℃. The enzyme appeared to be one subunit, and consisted of 18 kinds of amino acids. The apparent molecular weight were 25,500 (PAGE) and 23,000(HPLC), respectively. The enzyme appeared to hydrolyze peptide bond between glutaml-L-tyrosine. The Km value of this enzyme was 0.29 mM when carbobenzoxy-L-tyrosine were used as a subtrate.

흰느타리버섯 ( Pleurotsu cornucopiae ( pers ) Rolland ) 중의 Protease 의 분리정제 및 그 성질에 관한 연구 ( 1 )

민태진,홍성일,김재웅 ( Tae Jin Min,Sung Il Hong,Jae Woong Kim ) 생화학분자생물학회 1983 BMB Reports Vol.16 No.1

The enzyme properties of protease of the Pleurotus Cornucopiae (Pers.) Rolland were investigated by purification with gel filteration using DEAE-sephadex A-50, CM-cellulose, DEAE-cellulose and Sephadex G-75. Two kinds of active fractions were isolated from this mushroom study. The active fraction showed protease activity for the bovine serum albumin substrate, and its optimum pH, optimum temperature, pH stability and thermal stability were 4.0, 50℃, 3.0∼4.0 and 20∼40℃, respectively. The activity of the enzyme was inhibited by Ca^(++), Cu^(++), Zn^(++), Fe^(++) and Mg^(++), but increased with Co^(++). The enzyme had one subunits composed of 17 amino acids. The apparent molecular weight was about 56,000 daltons.

누에 ( Bombyx mori L . ) 의 요소첨식 효과에 관한 연구

민태진,김택영 ( Tae Jin Min,Taik Yung Kim ) 생화학분자생물학회 1978 BMB Reports Vol.11 No.3

The change of the growth rate, the weight of cocoon, and the change of Urease activity, nucleic acid, protein contents, and the changes in disc electrophoretic patterns of the protein in the silkworm (Suwon No 108×107 confused species) from the 3rd instar stage to the 5th instar stage by supplemental feeding of different concentrations of Urea has been studied. On the experiment group, U₄ which was treated with 1,600ppm Urea, the rate of growth increased to 17. 43% and the rate of cocoon`s weight increased 20.11% more than the control groups. The activity of Urease was increased with the increasing of the concentration of Urea treatment, but not more than the control groups. Beyond U₄ activity of Urease was decreased gradually with the increasing of the concentration of Urea. The contents of nucleic acid were decreased sharply at first in the Urea treatments but gradually increased according to the Urea concentration. In the 5th instar stage, disc electrophoretic patterns of protein in. the silkworms were found to be five in the U₄ group compared to six in the control group.