탄닌 흡착 크로마토그래피에 의한 Penicillium citrinum Nuclease P1의 부분정제

엠써네쓰,전문진,서원철,임번삼 생화학분자생물학회 1992 BMB Reports Vol.14 No.2

As a new approach for protein purification, affinity characteristics of tannin for enzyme protein and its applicability to adsorption chromatography were investigated. Crude nuclease P₁ was first purified by fractionation of ammonium sulfate followed by tannin adsorption chromatography. Result showed that adsorption capacity of nuclease P₁ onto one gram of immobilized tannin was 40 ㎎ and recovery yield of the enzyme was 90%. Specific activity of the purified nuclease P₁ increased to 15.6 fold higher than of crude extract, recovering 55.396 of enzyme yield.

Penicillium citrinum 의 Nuclease P1 고정화와 생체 반응기에 의한 누클레오티드의 생산

엠써네쓰,전문진,서원철,임번삼 생화학분자생물학회 1992 BMB Reports Vol.14 No.2

Nuclease P₁ from Penicillium citrinum was immobilized on cellulose by covalent binding method. Their general properties and applicational possibilities were investigated. Optimum reaction temperature of immobilized nuclease P₁ was 70℃ with broader activity range than that of native enzyme, while optimum pH was shifted to pH 5.0 from pH 5.5 for the native enzyme. Km values of native and immobilized nuclease P₁ on RNA as a substrate were 0.92% and 1.48%, respectively. Finally, continuous production of 5'-nucleotides by enzymatic hydrolysis of RNA was tried using packed-bed reactor and/or continuous-flow stirred-tank reactor.

Analysis of Reaction Kinetics in Continuous Microbial Bioreactor

Nam Soon Oh,Manfred Sernetz 한국미생물생명공학회(구 한국산업미생물학회) 1993 한국미생물생명공학회 학술발표초록집 Vol.1993 No.1

다공성 섬유소 입자에 대한 α - Amylase 와 Glucoamylase 의 고정화

지영민,전문진,엠써네쓰 ( Y . M . Chi,M . Chun,M . Sernetz ) 생화학분자생물학회 1984 BMB Reports Vol.17 No.1

For the immobilization of α-amylase and glucoamylase effectively on porous cellulose beads, the optimal activation methods were studied. And their enzymatic properties were investigated. The results obtained were as follows; 1) The most effective method of enzyme immobilization was obtained when the 3% gallotannin, pH 4, was used as activation reagent after 0.1M p-benzoquinone, pH 12, activation in case of α-amylase and when the 3% gallotannin, pH 9, was used as activation reagent after 0.1M p-benzoquinone, pH 12, activation in case of glucoamylase. 2) The immobilized enzymes activities were increased by the addition of CaCl₂ to immobilizing mixture. The immobilized a-amylase activity was increased from 69.17 μ/g to 77.18μ/g by addition of 5×10^(-4)M CaCl₂ to the immobilizing mixture. The immobilized glucoamylase activity was increased from 157.37μ/g to 189. 31μ/g by addition of 5×10^(-3)M CaCl₂ to the immobilizing mixture. The maximum immobilization rate of a-amylase was 5.07 and glucoamylase was 47.33%. 3) The optimum pH of α-amylase was 4.5-5 for native enzyme and also 4.5-5 for immobilized enzyme. The optimum pH of glucoamylase was 4.5 in both cases. 4) The optimum temperatures for the native and immobilized α-amylase were both 55℃. The optimum temperature was 55℃ for the native glucoamylase and 50℃ for the immobilized glucoamylase. 5) The Km value of native a-amylase was 0.48% and that of immobilized α-amylase was 1.43% while Vmax (1.52㎎ hydrolyzed soluble starch/min) was unaltered. The Km value of native glucoamylase was 0.83% and that of immobilized glucoamylase was 2.0%.

β - Galactosidase 의 Tannin 활성화 섬유소 입자에의 고정화

홍영수,권석태,전문진,엠써네쓰 한국농화학회 1983 Applied Biological Chemistry (Appl Biol Chem) Vol.26 No.4

β-Galactosidase(β-D-galactoside galactohydrolase, E.C. 3. 2. 1. 23) from E. coli K-12 CSH 36 was immobilized on porous cellulose beads which were previously activated with tannin and p-benzoquinone. Their general properties and applicational possibities were investigated. The most effective, enzyme immobilization was obtained when tannin and p-benzoquinone, pH 11.0, were used together as activation reagents and a period of 6 hours of activation. The optimum pH of β-galactosidase was 5.5 for free enzyme and 6. 0 for the immobilized enzyme, the optimum temperatures for native and immobilized enzyme were both 50℃. Kms of native β-galactosidase and immobilized enzyme for ONPG(o-nitrophenyl galactopyranoside) were about 4.0×10^(-4)M and 7.5×10^(-4)M, respectively. In the case of tannin : p-benzoquinone activated cellulose beads, the immobilized enzyme retained over 80% of the initial enzyme activity after 20 runs, which is very promising result far a possible industrial application.

Immobilization of $\alpha$-Amylase and Glucoamylase on Porous Cellulose Beads

지영민,전문진,엠써네쓰,Chi, Y.M.,Chun, M.,Sernetz, M. 생화학분자생물학회 1984 한국생화학회지 Vol.17 No.1

다공성 섬유소 업자를 제조하고, $\alpha$-amylase와 glucoamylase를 각각 효과적으로 고정화 시키기 위하여 섬유소 담체를 활성화 시키고 그 최적 조건을 조사하였으며 그것의 효소학적 성질에 관하여 연구하였다. 가장 효과적 인 $\alpha$-amylase와 glucoamylase의 고정화는 다공성 섬유소 담체를 0.1M p-benzoquinone으로 활성화 시킨 후 3% gallotannin으로 재활성화 시켰을 때 잔여 활성도가 가장 높았으며 이때 $CaCl_2$를 첨가함으로써 고정화율이 증가되었다. 유리의 $\alpha$-amylase와 고정화 $\alpha$-amylase의 반응 최적 pH는 차이가 없었으며, 반응 최적 온도는 모두 $55^{\circ}C$이었다. 유리의 glucoamylase와 고정화 glucoamylase의 반응 최적 pH에도 차이가 없었으나 반응 최적 온도는 유리의 glucoamylase는 $55^{\circ}C$, 고정화 glucoamylase는 $50^{\circ}C$로 나타났다. Starch에 대한 $\alpha$-amylase와 gucoamylase의 Km 값은 고정화시킴으로써 증가되었고 저장성 또한 증가되었다. For the immobilization of $\alpha$-amylase and glucoamylase effectively on porous cellulose beads, the optimal activation methods were studied. And their enzymatic properties were investigated. The results obtained were as follows; 1) The most effective method of enzyme immobilization was obtained when the 3% gallotannin, pH 4, was used as activation reagent after 0.1M p-benzoquinone. pH 12, activation in case of $\alpha$-amylase and when the 3% gallotannin, pH 9, was used as activation reagent after 0.1M p-benzoquinone, pH 12, activation in case of glucoamylase. 2) The immobilized enzymes activities were increased by the addition of $CaCl_2$ to immobilizing mixture. The immobilized $\alpha$-amylase activity was increased from 69.17u/g to 77.18u/g by addition of $5{\times}10^{-4}M$ $CaCl_2$ to the immobilizing mixture. The immobilized glucoamylase activity was increased from 157.37u/g to 189.31u/g by addition of $5{\times}10^{-3}M$ $CaCl_2$ to the immobilizing mixture. The maximum immobilization rate of $\alpha$-amylase was 5.07% and glucoamylase was 47.33%. 3) The optimum pH of $\alpha$-amylase was 4.5-5 for native enzyme and also 4. 5-5 for immobilized enzyme. The optimum pH of glucoamylase was 4.5 in both cases. 4) The optimum temperatures for the native and immobilized $\alpha$-amylase were both $55^{\circ}C$. The optimum temperature was $55^{\circ}C$ for the native glucoamylase and $50^{\circ}C$ for the immobilized glucoamylase. 5) The Km value of native $\alpha$-amylase was 0.48% and that of immobilized $\alpha$-amylase was 1.43% while Vmax (1.52mg hydrolyzed soluble starch/min) was unaltered. The Km value of native glucoamylase was 0.83% and that of immobilized glucoamylase was 2.0%.

Immobilization of Nuclease $P_1$ from Penicillium citrinum and Production of 5'-nucleotides by Bioreactor

서원철,임번삼,전문진,엠 써네쓰,Suh, W.C.,Lim, B.S.,Chun, M.,Sernetz, M. Korean Society for Biochemistry and Molecular Biol 1987 한국생화학회지 Vol.20 No.1

Penicillum citrinum 의 nuclease $P_1$을 탄닌 및 p-benzoquinone을 사용하여 셀룰로오스에 고정화하였다. 고정화 nuclease P1의 최적 반응온도는 $70^{\circ}C$로서 수용성 효소와 별 차이가 없었으며 최적pH는 5.5에서 5.0으로 산성쪽으로 약간 이동하였다. 또한 기질인 RNA에 대해 수용성 효소와 고정화 효소의 Km값은 각각 0.92% 및 1. 48%로 나타났다. 최종적으로 Packed-Bed Reactor와 Continuous-Flow Stirred-Tank Reactor에 의한 5'- 누클레오티드의 연속적 생산을 각각 비교 검토하여 보았다. Nuclease $P_1$ from Penicillium citrinum was immobilized on cellulose by covalent binding method. Their general properties and applicational possibilities were investigated. Optimum reaction temperature of immobilized nuclease $P_1$ was $70^{\circ}C$ with broader activity range than that of native enzyme, while optimum pH was shifted to pH 5.0 from pH 5.5 for the native enzyme. Km values of native and immobilized nuclease $P_1$ on RNA as a substrate were 0.92% and 1.48%, respectively. Finally, continuous production of 5'-nucleotides by enzymatic hydrolysis of RNA was tried using packed-bed reactor and/or continuous-flow stirred-tank reactor.

Partial Purification of Nuclease $P_1$ from Penicillium citrinum by Tannin Adsorption Chromatography

서원철,임번산,전문진,엠 써네쓰,Suh, W.C.,Lim, B.S.,Chun, M.,Sernetz, M. Korean Society for Biochemistry and Molecular Biol 1987 한국생화학회지 Vol.20 No.1

효소 단백질에 대한 탄닌의 특이적 흡착성질과 흡착 크로마토그래피의 응용성에 대하여 검토하였다. 고정화 탄닌 1 g당 nuclease $P_1$의 흡착량은 40 mg 이었으며, 흡착된 nuclease $P_1$을 90%의 높은 수율로 회수할수 있었다. 황산 암모니움 염석과 단백질 정제의 새로운 시도방법으로서 탄닌 흡착 크로마토그래피에 의하여 nuclease $P_1$의 부분정제를 시도한 결과 nuclease $P_1$의 비활성은 15.6 배 향상되었으며, 이때 수율은 55.3% 였다. As a new approach for protein purification, affinity characteristics of tannin for enzyme protein and its applicability to adsorption chromatography were investigated. Crude nuclease $P_1$ was first purified by fractionation of ammonium sulfate followed by tannin adsorption chromatography. Result showed that adsorption capacity of nuclease $P_1$ onto one gram of immobilized tannin was 40 mg and recovery yield of the enzyme was 90%. Specific activity of the purified nuclease $P_1$ increased to 15.6 fold higher than that of crude extract, recovering 55.3% of enzyme yield.

Immobilization of $\beta$-Galactosidase from E. coli K-12 CHS36 Using Tannin - Activated Cellulose Beads

홍영수,권석태,전문진,엠써네쓰,Hong, Y.S.,Kwon, S.T.,Chun, M.J.,Sernetz, M. 한국응용생명화학회 1983 Applied Biological Chemistry (Appl Biol Chem) Vol.26 No.4

Tannin 및 p-benzoquinone으로 활성화시킨 다공성 섬유소 입자를 사용하여 대장균 변이주(E. coli K-12 CSH36)로부터 추출한 $\beta$-galactosidase를 고정화시켜 효소학적 성질과 그 응용성에 대하여 연구하였다. $\beta$-galactosidase는 pH 11.0으로 맞춘 tannin과 p-benzoquinone을 사용하여 6시간 동안 활성화시킨 섬유소 입자담체에 고정화 시켰을 때 그 잔여 활성도가 가장 높았다. $\beta$-galactosidase의 최적작용 pH는 5.5였으나 고정화 시켰을 때 pH 6.0으로 변화하였으며 최적작용 온도는 고정화 전이나 후에도 일정하였다. $\beta$-galactosidase의 Km 값은 $4.0{\times}10^(-4)M$으로 나타났으며 고정화 효소의 경우에는 $7.5{\times}10^(-4)M$이었다. 고정화 효소의 재사용성을 검토한 결과, 담체 활성화 물질로 tannin과 p-benzoquinone 용액을 사용하였을 때 20히 사용 후에 초기 효소 활성도의 80% 이상이 유지되는 결과를 나타내었다. $\beta$-Galactosidase($\beta$-D-galactoside galactohydrolase, E.C. 3. 2. 1. 23) from E. coli K-12 CSH 36 was immobilized on porous cellulose beads which were previously activated with tannin and p-benzoquinone. Their general properties and applicational possibities were investigated. The most effective, enzyme immobilization was obtained when tannin and p-benzoquinone, pH 11.0, were used together as activation reagents and a period of 6 hours of activation. The optimum pH of $\beta$-galactosidase was 5.5 for free enzyme and 6. 0 for the immobilized enzyme, the optimum temperatures for native and immobilized enzyme were both $50^{\circ}C$. Kms of native $\beta$-galactosidase and immobilized enzyme for ONPG(o-nitrophenyl galactopyranoside) were about $4.0{\times}10^(-4)M$ and $7.5{\times}10^(-4)M$, respectively. In the case of tannin : p-benzoquinone activated cellulose beads, the immobilized enzyme retained over 80% of the initial enzyme activity after 20 runs, which is very promising result far a possible industrial application.

탄닌 흡착 크로마토그래피에 의한 Penicillium citrinum Nuclease P1의 부분정제

서원철,임번삼,전문진,엠 써네쓰 ( W . C . Suh,B . S . Lim,M . Chun M . Sernetz ) 생화학분자생물학회 1987 BMB Reports Vol.20 No.1

As a new approach for protein purification, affinity characteristics of tannin for enzyme protein and its applicability to adsorption chromatography were investigated. Crude nuclease P₁ was first purified by fractionation of ammonium sulfate followed by tannin adsorption chromatography. Result showed that adsorption capacity of nuclease P₁ onto one gram of immobilized tannin was 40 ㎎ and recovery yield of the enzyme was 90%. Specific activity of the purified nuclease P₁ increased to 15.6 fold higher than of crude extract, recovering 55.396 of enzyme yield.