• Journal of Plant Biology
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• 제38권4호
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• pp.349-357
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• 1995

The alkaline invertase ($\beta$-D-fructofuranoside fructohydrolase, EC 3.2.1.26) was isolated and characterized from the hypocotyls of mung bean (Phaseolus radiatus L.). The enzyme was purified by consecutive step using diethylaminoethyl (DEAE)-cellulose anion exchange, 1st Sephadex G-200, DEAE-Sephadex A50 and 2nd Sephadex G-200 chromatography. The overall purification was about 77-fold with a yield of about 6%. The finally purified enzyme exhibited a specific activity of about 48 $\mu$mol of glucose produced mg-1 protein min-1 at pH 7.0 and appeared to be a single protein by nondenaturing polyacrylamide gel electrophoresis (PAGE). The enzyme had the native molecular weight of 450 kD and subunits molecular weight of 63 kD and 38 kD as estimated by Sephadex G-200 chromatography and SDS-PAGE, respectively, suggesting that the enzyme is a heteromultimeric protein composed of two types of subunits. On the other hand, the enzyme appeared to be not a glycoprotein according to the results of Con A chromatography and glycoprotein staining. The enzyme had a Km for sucrose of 19.7 mM at pH 7.0 and maximum activity around pH 7.5. The enzyme was most active with sucrose as substrate, compared to raffinose, cellobiose, maltose and lactose. These results indicate the alkaline invertase is a $\beta$-fructofuranosidase.

• 미생물학회지
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• 제31권1호
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• pp.54-62
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• 1993

Intracellular adenosine deaminase (ADA) from Aspergillus oryzae was purified using ammonium sulfate fractionation, a DEAE-Sephadex A-50 anion exchange chromatography, an ultrafiltration using a PM 10 membrane and two times of Sephadex G-100 gel filtration chromatography. The enzyme was purified 151 fold with a 9% recovery. Purified enzyme gave a single protein band with a molecular weight of 105,000 delton. The enzyme was reasonably stable. The enzyme activity was kept even after 1 hr incubation at 55.deg.C, but decreased significantly at 60.deg.C. The pH optimum was found to be from 6.5 to 7.5. Among tested compounds, the substrate activity was found with adenosine, adenine arainofuranoside, formymcin A, 2'-deoxyadenosine, 3'-deoxyadenosine, 2', 3'-isopropylidene adenosine, 2,6-diaminopurine deoxyriboside, .betha.-nicotinamide adenine dinucleotide (reduced form), 6-chloropurine riboside, 2'-adenine monophosphate (AMP), 3'-AMP and 5'-AMP. The values of Km of adenosine and 2'-deoxyadenosine were calculated to be 500 and .$710\mu$m, respectively. ADA was sensitivite to $Zn^{2+}$, $^Cu{2+}$ and $Fe^{3+}$, p-chloromercuribenzoate and mersalyl acid inactivated the enzyme. The activity of enzyme was not changed when ADA was incubated with dithiothreititol, 2-mercaptoethanol, N-ethylmaleimide, iodoacetic acid and iodoacetamide.

• BMB Reports
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• 제35권2호
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• pp.228-235
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• 2002

Methionine aminopeptidase (MetAP) catalyzes the removal of an amino-terminal methionine from a newly synthesized polypeptide. The enzyme was purified to homogeneity from Bacillus stearothermophilus (KCTC 1752) by a procedure that involves heat precipitation and four sequential chromatographs (including DEAE-Sepharose ion exchange, hydroxylapatite, Ultrogel AcA 54 gel filtration, and Reactive red 120 dye affinity chromatography). The apparent molecular masses of the enzyme were 81,300 Da and 41,000 Da, as determined by gel filtration chromatography and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), respectively. This indicates that the enzyme is comprised of two identical subunits. The MetAP specifically hydrolyzed the N-terminal residue of Met-Ala-Ser that was used as a substrate, and exhibited a strong preference for Met-Ala-Ser over Leu-Gly-Gly, Leu-Ser-Phe, and Leu-Leu-Tyr. The enzyme has an optimal pH at 8.0, an optimal temperature at $80^{\circ}C$, and pI at 4.1. The enzyme was heat-stable, as its activity remained unaltered when incubated at $80^{\circ}C$ for 45 min. The Km and Vmax values of the enzyme were 3.0mM and 1.7 mmol/min/mg, respectively. The B. stearothernmophilus MetAP was completely inactivated by EDTA and required $Co^{2+}$ ion(s) for activation, suggesting the metal dependence of this enzyme.

• BMB Reports
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• 제41권11호
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• pp.790-795
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• 2008

An inducible lysine 6-dehydrogenase (Lys 6-DH), which catalyzes the oxidative deamination of the 6-amino group of L-lysine in the presence of $NAD^+$, was purified to homogeneity from Achromobacter denitrificans, yielding a homodimeric protein of 80 kDa. The enzyme was specific for the substrate L-lysine and $NAD^+$ served as a cofactor. The dimeric enzyme associated into a hexamer in the presence of 10 mM L-lysine. The $K_m$ values for L-lysine and $NAD^+$ were 5.0 and 0.09 mM, respectively. The lys 6-dh gene was cloned and overexpressed in E. coli. The open reading frame was 1,107 nucleotides long and encoded a peptide containing 368 amino acids with 39,355 Da. The recombinant enzyme was purified to homogeneity and characterized. Enzyme activities and kinetic properties of the recombinant enzyme were almost the same as those of the endogenous enzyme obtained from A. denitrificans. Crystals of the enzyme were obtained using the hanging drop method.

• Journal of Microbiology and Biotechnology
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• 제9권6호
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• pp.854-860
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• 1999

Superoxide dismutase (SOD) was purified to homogeneity from fruiting bodies of edible mushroom, Lentinus edodes, by ammonium sulfate precipitation, diethylaminoethyl (DEAE)-Sepharose FF ion-exchange chromatography, Sephacryl S-200 gel filtration chromatography, and preparative PAGE. The molecular weight of the purified enzyme was estimated to be approximately 54 kDa by gel filtration chromatography, and the enzyme was shown to be consisted of two identical subunits of molecular weight 27 kDa by SDS-PAGE. The isoelectric point of the enzyme was 4.9 as determined by isoelectric focusing. The enzyme had optimal pH and temperature of pH 8.0 and $20^{\circ}C$, respectively. The activity of the enzyme was inhibited by hydrogen peroxide, but inhibited less by cyanide and azide. The native enzyme was found to contain 0.89g-atom of iron, 0.75g-atom of zinc, and 0.46g-atom of copper per mol of enzyme. Analysis of amino acids composition revealed that the SOD from L. edodes contained a relatively large amount of glutamic acid/glutamine, proline, cysteine, isoleucine, and leucine, but only a small amount of aspartic acid/asparagine, tyrosine, and tryptophan when compared to the other iron-containing SODs.

• Journal of Microbiology and Biotechnology
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• 제7권2호
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• pp.121-126
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• 1997

A bacterial strain LC-413, producing an extracellular inulin fructotransferase (depolymerizing) which converts inulin into di-D-fructofuranose dianhydride (DFAIII), was isolated from soil. Inulin fructotransferase from the isolate identified as a strain Flabobacterium sp. was purified from the culture broth by ammonium sulfate precipitation, followed by column chromatograpies on DEAE-Toyopearl 650 M and phenyl-Toyopearl 650 M. The purified enzyme gave a single band on an electrophoretic disc-gel. The molecular weight of the enzyme was estimated to be 44, 000 Da by SDS-polyacrylamide gel electrophoresis, and 45, 000 Da by gel filtration, suggesting the monomeric state of the enzyme. The isoelectric point of the enzyme was about pH 4.5. The optimal pH and temperature for the enzyme reaction were 6.0 and $50^{\circ}C$, respectively. The purified enzyme digested inulin into di-D-fructofuranose-l, 2': 2, 3'-dianhydride, confirming the enzyme was an inulin fructotransferase (inulinase II).

• Journal of Microbiology and Biotechnology
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• 제14권4호
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• pp.829-835
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• 2004

A fibrinolytic enzyme has been found in several bacteria isolated from fermented food. This study was carried out to investigate the purification and characteristics of the fibrinolytic enzyme produced by Bacillus subtilis KCK-7 originated from Chungkookjang. The fibrinolytic enzyme was purified to homogeneity from the culture supernatant using ammonium sulfate fractionation and chromatographies on DEAE-cellulose and on Sephadex G-100. The final specific activity of the purified enzyme increased 11.0-fold, and the protein amount in the purified enzyme was about 16% of that in the culture supernatant. The molecular weight of the purified enzyme was estimated to be about 45,000 by SDS-PAGE. The optimum pH and temperature for the enzyme activity were pH 7.0 and $60^{\circ}C$, respectively. The enzyme activity was relatively stable up to $60^{\circ}C$ over the pH range of 7.0-10.0. The fibrinolytic enzyme activity increased by $Ca^{2+}$ and $Cu^{2+}$, whereas it was inhibited by $Hg^{2+}$ and $Ba^{2+}$. In addition, it was severely inhibited by PMSF and DFT. It is suggested that the purified enzyme was a serine protease for the fibrinolysis. The purified enzyme could completely hydrolyze fibrin in vitro within 8 h. Hence, it is suggested that the purified enzyme can be put into practice as an effective thrombolytic agent.

• 생명과학회지
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• 제8권3호
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• pp.326-332
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• 1998

Cyclodextrin glucanotransferase from B. stearothermophilus KJ16 that can produce both cyclodextrin glucanotransferase and cyclodextrinase was purified by ammonium sulfate precipitation, DEAE-cellulose chromatography, Sephadex G-100 chromatography, and FPLC. The molecular weight of the purifice enzyme was about 65,000 dalton by SDS-PAGE. The optimal pH and temperature were 6.0 and $60^{\circ}C$, respectively. The enzyme was stable at $50^{\circ}C$ for 1 hr and in the pH range of 5.5 and 8.5. Mercaptoethanol and dithiothreitol inhibited the enzyme activity strongly. The enzyme produced 60% cyclodextrin(CD) from 5% soluble starch with the $^{\alpha}$, $^{\beta}$, $^{\gamma}$-CD ratio of 42:46:12. Amylopectin was the most suitable substrate with 67% conversion to CD.

• 한국미생물·생명공학회지
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• 제25권1호
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• pp.44-50
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• 1997

$\beta $-Glucosidase (EC 3.2.1.21) was purified from Aspergillus niger SFN-416 by a sequential process of ammonium sulfate precipitation, Sepadex G-100 and DEAE-Sephacel column chromatography. Molecular weight of the enzyme was 46, 000 daltons. The K$_{m}$ and V$_{max}$ values for PNPG were 0.67 mM and 25 moles/ml $\cdot $min., respectively. The optimum pH and temperature of the enzyme activity were 3.5 and 58$\circ $C, respectively. The enzyme activity was decreased by addition of metal ions, and increased by addition of metanol, ethanol, isopropanol and 1-butanol at a concentration of 10% (v/v). Stability of the enzyme was increased by addition of isopropanol and 1-butanol at a concentration of 10% (v/v).

• 환경위생공학
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• 제5권1호
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• pp.49-64
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• 1990

Glucoamylase from the culture filtrate of Aspergillus nicer was purified by ammonium sulfate precipitation, aceton precipitation, DEAE-cellulose ion exchange chromatography and Sephadex G-50 gel fillration. Glucoamylase was secreted into the medium upon growth on glucose, sucrose or a variety of other hexose sugars or hexose sugar polymers and little or no glucoamylase activity was found when glycerol or xylose was used as the carbon source. The optimum pH and temperature (or the maximum enzyme activity were found to be 5.0 and $50^{\circ}C$, respectively. The enzyme was considerably thermostable, for no loss of activity was observed when the enzyme was preincubated at $60^{\circ}C$ for 30 min. The enzyme activity was inhibited by 20 mM of $Hg^{2+}$, $Fe^{2+}$. The km value for starch was 0.045%.