• BMB Reports
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• 제37권3호
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• pp.275-281
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• 2004

Rhodanese was isolated and purified from the cytosolic fraction of liver tissue homogenate of the fruit bat, Eidolon helvum, by using ammonium sulphate precipitation and CM-Sephadex C-50 ion exchange chromatography. The specific activity was increased 130-fold with a 53% recovery. The $K_m$ values for KCN and $Na_2S_2O_3$ as substrates were $13.5{\pm}2.2\;mM$ and $19.5{\pm}0.7\;mM$, respectively. The apparent molecular weight was estimated by gel filtration on a Sephadex G-100 column to be 36,000 Da. The optimal activity was found at a high pH (pH 9.0) and the temperature optimum was $35^{\circ}C$. An Arrhenius plot of the heat stability data consisted of two linear segments with a break occurring at $35^{\circ}C$. The apparent activation energy values from these slopes were 11.5 kcal/mol and 76.6 kcal/mol. Inhibition studies on the enzyme with a number of cations showed that $Mg^{2+}$, $Mn^{2+}$, $Ca^{2+}$, and $Co^{2+}$ did not affect the activity of the enzyme, but $Hg^{2+}$ and $Ba^{2+}$ inhibited the enzyme.

• Journal of Microbiology
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• 제33권2호
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• pp.115-119
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• 1995

The alkaline protease of Xanthomonas sp. YL-37 has been purified, and the properties of the enzyme investigated. The alkaline protease of Xanthomonas sp. YL-37 was purified form crude enzyme by ammonium sulfate fractionation, CM-cellulose ion exchange chromatography, and Sephadex G-100 gel filtration. Through the series of chromatographies, the enzyme was purified to homogenecity with specific activity of 4.23 fold higher than that of the crude broth. The molecular weight of the purified protease has been estimated to be 62 KDa on SDS-polyacrylamide gel electrophoresis. The optimal pH and temperature for alkaline protease activity were 11.0 and 50.deg.C, respectively. The enzyme was stable between pH 5.0 and 10.0 and up to 50.deg.C. Enzyme activity was lost up to 50% on heating at 70.deg.C for 30 minutes. The activity of alkaline protease was inhibited by Cu$\^$2+/, Zn$\^$2+/, Hg$\^$2+/, PMSF, and activated by Mn$\^$2+/ and Ca$\^$2+/. The $K_{m}$ value for casein as a substrate was 4.0 mg/ml.

• 미생물학회지
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• 제26권2호
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• pp.73-81
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• 1988

$\alpha$-Amylase (EC.3.2.1.1) of Neurospora crassa (ATCC9279) was cloned in E. coli HB101 using shotgun method, and the enzymes isolated from both N. crassa and E. coli were compared. Chromosomal DNA isolated from the spores of N. crassa was partially digested with PstI restriction endonuclease and rejoined to pBR322 which had been digested with the same enzyme. The resulting recombinant DNA were introduced into E. coli HB101 which had competancy by treating with $CaCl_{2}$. As the result, about 8000 colonies which showed tetracycline resistance were selected and two of the colonies which had 13.5Kb recombinant plasmid exhibit starch degrading activity on starch-containing plate when treated with D-cycloserine. $\alpha$-Amylases from both N.crassa and E. coli were isolated by using ammonium sulfate precipitation, DEAE-cellulose ion exchange column chromatography and Bio-Gel P150 gel foltration column. As the result, about 81.3 fold and 5.6 fold purifications in specific activities were obtained respectively, and specific activities of the gel filtrates were 6.1u/mg and 85u/mg respectively. The properties of both enzymes were compared and they showed quite the similar patterns in optimal temperature, optimal pH and had same molecular weight about 100,000 daltons on gel filtration method. Optimal temperatures for both enzymes were $70^{\circ}C$ and optimal pH were about 6 and 10.

• BMB Reports
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• 제32권6호
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• pp.579-584
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• 1999

Mantis egg fibrolase (MEF-3) was purified from the egg cases of Tenodera sinensis using ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, DEAE Affi-Gel blue gel affinity chromatogragphy, and MONO-Q anion-exchange chromatography. This protease had a molecular weight of 35,600 Da as determined by SDS-polyacrylamide gel electrophoresis under reducing conditions and its isoelectric point was 6.0. The N-terminal amino acids sequence was Ala-Thr-Gln-Asp-Asp-Ala-Pro-Pro-Gly-Leu-Ala-Arg-Arg. This sequence was 80% homologous to the serine protease from Tritirachium album. MEF-3 readily digested the ${\alpha}$-and ${\beta}$-chains of fibrinogen and more slowly the ${\gamma}$-chains. It showed strong proteolytic and fibrinolytic activities. Phenylmethanesulfonyl fluoride and chymostatin inhibited its proteolytic activity, while EDTA, EGTA, cysteine, ${\beta}$-mercaptoethanol, elastinal, tosyl-lysine chloromethylketone, and tosyl-amido-2-phenylethyl chloromethyl ketone did not affect its proteolytic activity. Among the chromogenic protease substrates, the most sensitive one to the hydrolysis of MEF-3 was benzoyl-Phe-Val-Arg-p-nitroanilide. Based on these experimental results, we speculated that MEF-3 is a serine protease with a strong fibrin(ogen)olytic activity.

• BMB Reports
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• 제28권2호
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• pp.170-176
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• 1995

Rhodanese from mouse liver was purified to near homogeneity by ammonium sulfate precipitation, CM-Sephadex ion exchange, hydroxyapatite and Sephacryl S-200-HR gel filtration chromatographies with a purification of 776 folds. The molecular weight was determined by Sephadex G-150 gel filtration and found to be 34.8 KDa. SOS-PAGE showed molecular weight 34 KDa and two identical subunits splitting by aging for 3 weeks at $-70^{\circ}C$ the molecular weight of which was 17 KDa. The optimal pH of enzyme activity was 9.4 and the pI value of the enzyme was 6.6. Rhodanese showed the optimal reaction temperature of $25^{\circ}C$ and near linear increasing pattern until 10 min. incubation. $K_m$ values of rhodanese for KCN and $Na_{2}S_{2}O_{3}$ as substrates were 12.5 mM and 8.3 mM, respectively. Rhodanese activity was inhibited by more than 70% at a concentration of 100 ${\mu}M$ of $Ni^{2+}$, $Zn^{2+}$, $Cd^{2+}$, $Hg^{2+}$ and $Cu^{2+}$. Other metal ions, such as $Mn^{2+}$, $Mg^{2-}$, $Ca^{2+}$, and $Fe^{2+}$ showed no effect on rhodanese activity.

• BMB Reports
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• 제28권2호
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• pp.100-106
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• 1995

S-adenosylmethionine (SAM) synthetase was purified to homogeneity from soybean (Glycine max) axes. The enzyme was purified 216-fold with a 1.5% yield by ammonium sulfate fractionation, acetone fractionation, ion exchange chromatography with DEAE-sephacel, gel filtration with Sephacryl S-300, and afffinity chromatography with ATP-agarose. The enzyme activity reached a maximum 3 days after germination. SAM synthetase had a subunit molecular weight of 57,000 daltons from a silver stained single band on SDS-PAGE. The molecular weight of the enzyme was 110,000 daltons from Sephacryl S-300 gel filtration. The enzyme was composed of two identical subunits. The $K_m$ values of the enzyme for L-methionine and ATP were 1.81 and 1.53 mM, respectively. The enzymatic activity was not affected by polyamines, agmatine, or SAM analogues, but was inhibited by SAM. The inhibition pattern was showed non-competitive for L-methionine and uncompetitive for ATP. The activity of SAM synthetase was inhibited by thiol-blocking reagents. The enzyme was induced by treatment with $10^{-3}$ M putrescine at germination. Experimental data revealed a possible novel regulation mechanism of polyamine biosynthesis through several endogenous intermediates.

• BMB Reports
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• 제28권3호
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• pp.197-203
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• 1995

Famesyl protein transferase involved in the first step of post-translational modification of $p21^{ras}$ proteins transfers the famesyl moiety from famesyl pyrophosphate to a cysteine residue in $p21^{ras}$ proteins. The enzyme was first purified 30,000-fold from bovine testis by use of 30~50% ammonium sulfate fractionation, DEAE-Sephacel ion exchange chromatography, Sephacryl S-300 gel filtration chromatography, Sephacryl S-200 gel filtration chromatography, and hexapeptide (Lys-Lys-Cys-Val-Ile-Met) affinity chromatography. The molecular weight of the purified enzyme was estimated to be ~100 kDa by gel filtration and SDS-polyacrylamide gels showed two closely spaced bands of ~50 kDa protein. These indicate that the enzyme consists of two nonidentical subunits, a and 13, which have slightly different molecular weights. The enzyme was inhibited by hexapeptide (Lys-Lys-Cys-Val-Ile-Met), which acted as an alternative substrate that competed for famesylation. Kinetic analysis by measuring initial velocities showed that famesyl protein transferase is a very slow enzyme. EDTA-treated famesyl protein transferase showed little activity with $Mg^{2+}$ or $Zn^{2+}$ alone, but required both $Mg^{2+}$ and $Zn^{2+}$ for the catalytic activity.

• Journal of Nutrition and Health
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• 제16권2호
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• pp.115-124
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• 1983

The biochemical nature of the protein constituents of six year old fresh Panax ginseng root was studied. Total protein constituents were extracted with phosphate buffer of pH 7.4, ionic strength of 0.1 and fractionated by ultrafiltration using four different membranes which cut down the materials of molecular weight of 500, 1,000, 5,000 and 10,000, respectively. Each fraction was subjected to ion exchange chromatography using DEAE - cellulose to isolate component proteins. The protein fraction larger than molecular weight of 10,000 was refractionated by the method of ammonium sulfate precipitation. The electrophoresis of the refractionated protein constituents was performed. The amino acid composition of the protein constituents was determined by gas- liquid chromatography. From the results, it could be summarized that eleven different protein constituents smaller than molecular weight of 10,000 were isolated from the fresh Panax ginseng root. At least eleven different protein constituents larger than molecular weight of 10,000 were identified from the electrophoretic patterns. These protein costituents seem to be compounded of all or some of five different subunits.

• BMB Reports
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• 제30권1호
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• pp.60-65
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• 1997

Expression of human Cu.Zn-superoxide dismutase (SOD) with activity comparable to human erythrocyte enzyme was achieved in E. coli B21(DE3) by using the pET-17b expression vector containing a T7 promoter. Recombinant human SOD was found in the cytosol of disrupted bacterial cells and represented > 25% of the total bacterial proteins. The protein produced by the E. coli cells was purified using a combination of ammonium sulfate precipitation, Sephacryl S-100 gel filtration and DEAE-Sephacel ion exchange chromatography. The recombinant Cu,Zn-SOD and human erythrocyte enzyme were compared using dismutation activity, SDS-PAGE and immunoblotting analysis. The mass of the subunits was determined to be 15,809 by using a electrospray mass spectrometer. The copper specific chelator. diethyldithiocarbamate (DOC) reacted with the recombinant Cu,Zn-SOD. At $50{\mu}M$ and $100{\mu}M$ concentrations of DOC, the dismutation activity was not inhibited for one hour but gradually reduced after one hour. This result suggests that the reaction of DOC with the enzyme occurred in two distinct phases (phase I and phase II). During phase I of this reaction, one DOC reacted with the copper center, with retention of the dismutation activity while the second DOC displaced the copper, with a loss of activity in phase II.

• Journal of Microbiology and Biotechnology
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• 제19권8호
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• pp.818-822
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• 2009

A ${\beta}-1$,3-1,4-glucanase from the fungus Laetiporus sulphureus var. miniatus was purified as a single 26 kDa band by ammonium sulfate precipitation, HiTrap Q HP, and UNO Q ion-exchange chromatography, with a specific activity of 29 U/mg. The molecular mass of the native enzyme was 52 kDa as a dimer by gel filtration. ${\beta}-1$,3-1,4-Glucanase showed optimum activity at pH 4.0 and $75^{\circ}C$. The half-lives of the enzyme at $70^{\circ}C$ and $75^{\circ}C$ were 152 h and 22 h, respectively. The enzyme showed the highest activity for barley ${\beta}$-glucan as ${\beta}-1$,3-1,4-glucan among the tested polysaccharides and p-nitrophenyl-${\beta}$-D-glycosides with a $K_m$, of 0.67 mg/ml, a $k_{cat}$ of 13.5 $S^{-1}$ and a $k_{cat}/K_m$ of 20 mg/ml/s.