• Journal of Microbiology
• /
• 제34권1호
• /
• pp.82-89
• /
• 1996

Purine nucleoside phosphorylase (PNP) was purified in Micrococcus luteus (M. luteus) using streptomycin sulfate and amomonium sulfate fractionation, three times by a Sephadex G-100 gel filtration and a DEAE-Sephadex A-50 ion exchange chromatography. The enzyme was purified 72 folds with a 11% recovery and showed a single band in a nondenaturing gel electrophoresis. The M. W. of PNP turned out to be 1.35 * 10$^{5}$ delton in G-150 gel filtration chromatography. The stability of the enzyme was increased by treatment with both substrates, MgCI$_{2}$ or CaCI$_{2}$, but not significantly kcal/mol. M. luteus PNP catalyzed the phosphorolysis of inosine, deoxyinosine, guanosine and deoxyguanosine with the Km value of 1.5 * 10$^{-3}$ M, 3.0 * 10$^{-3}$ M, 5.0 * 10$^{-4}$ M, respectively. The enzyme was reacted with adenosine, 1-methylnosine and 1-methylguanosine as substrates, which were shown to be poor substrates for mammalian enzyme.

• Journal of Microbiology and Biotechnology
• /
• 제3권3호
• /
• pp.188-193
• /
• 1993

Superoxide dismutase (SOD) was purified from Pseudomonas polycolor to an electrophoretically homogeneous state and partially characterized. SOD was purified by ammonium sulfate fractionation, column chromatography on DEAE-Sephadex A-50, phenyl-Toyopearl 650 M, and gel filtration on Sephadex G-100. The molecular weight and subunit molecular weight of the purified enzyme were estimated to be 40, 000 and 20, 000, respectively. The purified enzyme remained stable at pH 9.0~11.0, $25^{\circ}C$ for 40 hr, but rapidly became inactive below 9.0. SOD was stable up to $45^{\circ}C$ at pH 9.0 with about 80% relative activity, but rapidly became inactive at temperature above that. The enzyme was insensitive to cyanide and fluoride, and sensitive to hydrogen peroxide and azide. The results suggest that the enzyme be an iron-containing SOD.

• 생약학회지
• /
• 제19권1호
• /
• pp.19-27
• /
• 1988

The hexane and ether extracts from the roots of Angelica dahurica caused a significant inhibition of hepatic drug-metabolizing enzyme (DME) activity. Through systematic fractionation by $SiO_2\;column$ and vacuum liquid chromatography monitoring by bioassays, three furanocoumarins, phellopterin, byakangelicin and tert-O-methylbyakangelicin were isolated as active principles. These components have biphasic responses, both inhibitory and inducing effects on DME system. Tert-O-methyl byakangelicin was found to have the strongest enzyme inhibitory potency.

• Archives of Pharmacal Research
• /
• 제21권6호
• /
• pp.692-697
• /
• 1998

GTP cyclohydrolase I catalyzing the first reaction in the biosynthesis of pterin moiety of folic acid in bacteria, was purified from Streptomyces tubercidicus by at least 203-fold with a yield of 32% to apparent homogeneity, using ammonium sulfate fractionation, DEAE-cellulose, Sepharose CL-6B, and hydroxylapatite column chromatography. The molecular weight of the native enzyme was estimated to be 230,000 daltons by gel permeation chromatography. The purified enzyme gave a single band on sodium dodesyl sulfate-polyacrylamide gel electrophoresis and its molecular weight was apparently 58,000 daltons. These results indicate that the enzyme consists of four subunits with the same molecular weight. The $K_m$ and $V_{max}$ values for GTP of the purified enzyme were determined to be 80${\mu}$M and 90nmol/min (mg protein), respectively. The optimum pH and temperature for the enzyme reaction were pH 7.5-8.5 and $40-42^{\circ}C$, respectively. Coenzyme or metal ion was not required for the enzyme activity. The enzyme activity was inhibited by most divalent cations, while it was slightly activated by potassium ion. In case of nucleotides, CTP, GMP, GDP, and UTP inhibited enzyme activity, among which GDP exhibited the strongest inhibitory effect.

• 미생물학회지
• /
• 제27권3호
• /
• pp.245-249
• /
• 1989

Bacillus licheniforntis에서 분비된 단백질 추출물에서 ammonium sulfate fractionation과 Sephadex G-75젤 여과 크로마토그라피를 사용하여 부분 정제된 2개의 서로 다른 활성을 갖는 단백질 분해 효소를 얻었다. 단백질 분해 효소 type l은 카제인에 대해 비교적 높은 활성을 보이며 organotluoride와 EOTA에 의한 억제에 매우 민감했다 이 효소의 최적 pH는 7.5였으며 $75^{\circ}C$에서 빠르게 변성되었다. 단백질 분해효소 type II는 type I에 비해 낮은 카제인 분해 활성을 보이며 ImM PMSF와 10mM EOTA용액과 함께 $37^{\circ}C$에서 30분 간 반응시키면 역사 활성이 억제되었다 한편 이 효소 는 pH 8.0에서 최대 활성을 나타내고 $71^{\circ}C$ 에서 상대적으로 늦게 불환성화 되었다.

• 한국식품영양과학회지
• /
• 제26권2호
• /
• pp.193-197
• /
• 1997

볶은 들깨박에 존재하는 항암효소계 유도물질을 분리하기 위해 용매분획과 preparative TLC를 실시하여 이들에 대한 암예방지표효소인 quinone reductase와 AHH 유도활성을 조사하였다. 볶은 들깨박의 메탄올 추출물을 용매분획하여 QR을 측정한 결과 chloroform층에서 가장 높은 활성이 나타났다. 들깨박 메탄올 추출물을 TLC로 분리한 분획가운데 QR과 AHH유도활성은 F1$(R_{f}=0.8)$에서 가장 높았으며, 항산화능은 F1$(R_{f}=0.8)$과 F2$(R_{f}=0.7)$에서 가장 강한 것으로 나타나 QR유도성분과 항산화성분이 동일성분일 가능성이 높은 것으로 사료 된다.

• 한국미생물·생명공학회지
• /
• 제14권6호
• /
• pp.467-471
• /
• 1986

Fusarium moniliforme으로부터 순모세포벽 분해효소를 생산하고 분리, 정제하여 효소특성 및 protoplast 제조실험을 하였다. Ammonium nitrate를 0.2％ 첨가한 Baker's yeast 배지에서 7일간 진탕배양으로 효소를 생산한 후 Ammonium sulfate로 분획하고 Sephadex(G-100) column chromatography하여 세개의 peak를 얻었다 첫 번째 peak는 proteolytic, lytic activity 및 laminarin 분해력가를 보였으며, 두 번째 Peak는 lytic activity와 laminarin 분해력가를 동시에 가지고 있었으며, 세 번째 peak는 lytic activity만을 가지고 있었다. 분리된 세개의 peak를 혼합하였을때 개개의 peak보다 훨씬 높은 역가을 나타내어 상승효과를 보였고 또한 환원제에 의한 효소력가의 상승효과도 있었다. protoplast 수율은 99.2％정도였다

• 한국미생물·생명공학회지
• /
• 제23권5호
• /
• pp.573-579
• /
• 1995

A Bacillus strain capable of producing an extracellular malto-oligosaccharides forming $\alpha $-amylase was isolated from soil and designated as Bacillus sp. SUH4-2. The enzyme was purified by ammonium sulfate fractionation, DEAE-Toyopearl and Mono-Q HR 5/5 column chromatographies using a FPLC system. The specific activity of the enzyme was increased by 16.1-fold and the yield was 13.5%. The optimum temperature for the activity of $\alpha $-amylase was 60-65$\circ$C and more than 50% of initial activity was retained after the enzyme was incubated at 60$\circ$C for 40 min. The enzyme was stable over a broad pH range of 5.0-8.0 and the optimum pH was 5.0-6.0. The molecular weight of the enzyme was determined to be about 63.6 kD and isoelectric point was around 5.8. The enzyme activity was strongly inhibited by Mn$^{2+}$, Ni$^{2+}$, and Cu$^{2+}$ ; slightly by Ca$^{2+}$. The purified enzyme produced starch hydrolyzates containing mainly maltose and maltotriose from soluble starch. The starch hydrolyzates were composed of 11% glucose, 59% maltose, 25% maltotriose and 5% maltotetraose.

• 미생물학회지
• /
• 제23권1호
• /
• pp.25-33
• /
• 1985

Cellulomonas flavigena KIST 321이 생산하는 균체외 cellulose 분해효소를 gel filtration법과 ion-exchange chromatography법으로 3종의 다른 효소를 분리하여 이들을 A, B, 및 C 효소로 명명하였다. 분리된 각 효소를 결정적 기질에 처리하여 일어나는 기질의 구조변화를 적회전 분광법과 X-ray crystallography법으로 측정하여 다음 결론을 얻었다. B효소는 결정성 cellulosem이 구성단위인 glucopyranose의 불안정화로 그 결정도를 감소시키는 $C_1$형의 효소이며 A 및 C 효소는 $C_x$형의 효소로 B 효소의 반응생성물에 작용하여 glucose를 생산하였다. 이들 각 효소의 작용에서 본균의 cellulase의 작용기작을 고찰하였다.

• BMB Reports
• /
• 제29권5호
• /
• pp.455-461
• /
• 1996

A serine proteinase was purified from Acanthamoeba culbertsoni by 41~80% ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography and gel filtration chromatography. The molecular weight of the purified enzyme was estimated to be 108.0 kDa by gel filtration chromatography and 54.0 kDa by SDS-PAGE. Therefore, the purified enzyme seemed to be a dimer. Isoelectric point was 4.5. The enzyme activity was highly inhibited by the serine proteinase inhibitors diisopropyl fluorophosphate (OFP) and phenylmethyl sulfonylfluoride (PMSF). It had a narrow pH optimum of 6.5~7.5 with a maximum at pH 7.0. These data suggested that the purified enzyme was a neutral serine proteinase. Optimal temperature was $37^{\circ}C$. It was stable for at least 16 h at $4^{\circ}C$ and $37^{\circ}C$, but it was rapidly inactivated at $65^{\circ}C$ The activity of the purified enzyme was not influenced significantly by $Mg^{2+}$, $Mn^{2+}$, $Zn^{2+}$ or $Ca^{2+}$. However, the enzyme activity was highly inhibited by $Hg^{2+}$ The enzyme degraded type I collagen and fibronectin, but not BSA, hemoglobin, lysozyme, immunoglobulin A or immunoglobulin G.