• Journal of Microbiology and Biotechnology
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• v.14 no.1
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• pp.97-104
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• 2004

Water-sludge bacteria were screened to find a lipase enantioselectively hydrolyzing itraconazole precursor, which is well known as the starting material of antifungal drug agents. A bacterial strain was isolated and identified as Acinetobacter junii SY-01. After the strain was cultivated, the enzyme was purified 39.4-fold using ultrafiltration and gel filtration through a Sephadex G-100 chromatographic column and the activity yield was 34.9%. The molecular weight of the enzyme was about 40 kDa, as measured by SDS-PAGE, and the optimum pH was 7.0- 9.0 and stable at pH 6.0- 9.0. The optimum temperature was 45- $5^{\circ}C$, and 73% of the enzymes activity remained after incubation at 70% for 1 h. Enzyme activity was enhanced by gall powder, sodium deoxycholate, a cationic detergent Tween 80, and a non-ionic detergent Triton X-100, but was markedly inhibited by metal ions such as $Hg^{2＋},Cu^{2＋},Ni^{2＋}/,Ca^{2＋}$, and an anionic-surfactant sodium dodecylsulfate. The $K_{m}$ values for (R)- and (S)-enantiomers of the itraconazole precursor were 0.385 and 21.83 mM, respectively, and the $V_{max} values ($\mu$Mㆍmin^{-1}.)$ were 6.73 and 6.49, respectively. The acetyl group among the different acyl moieties of itraconazole precursor showed the highest enantioselectivity for the hydrolysis by the Acinetobacter junii SY-01 lipase, and the lipase from Acinetobacter junii SY-01 displayed better enantioselectivity than that of commercially available lipases and esterases.

• Journal of Microbiology and Biotechnology
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• v.2 no.3
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• pp.189-196
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• 1992

A $\alpha$-l, 4-D-glucan maltohydrolase $(\beta$-amylase), secreted by the mesophilic aerobic bacterium Bacillus polymyxa No.26, was purified and characterized. The enzyme production was increased after a logarithmic phase of bacterial growth and paralleled with the onset of bacterial sporulation. By applying anion exchange chromatography and gel filtration the enzyme was purified 16.7-fold and had a specific activity of 285.7 units/mg. Two enzyme activities were eluted on a column of DEAE-Sephadex chromatography, and they were designated as E-I for a major enzyme peak and E-II for a minor peak. Of them, E-I enzyme peak was further purified by using gel chromatography. The molecular mass of this enzyme was determined to be 64, 000 daltons and consisted of a single subunit, showing an isoelectric point of 8.9. The enzyme was able to attack specifically the $\alpha$-l, 4-glycosidic linkages in soluble starch and caused its complete hydrolysis to maltose and $\beta$-limited dextrin. This amylolytic enzyme displayed a temperature optimum at $45^\circ{C}$ and a pH optimum at 7.0. The amino acid composition of the purified enzyme was quite similar to the other bacterial $\beta$-amylases reported. Surprisingly, the purified enzyme from this aerobe only exhibited hydrolytic activity on soluble starch, not on starch granules. The degradation of from starch by $\beta$-amylase was greatly stimulated by pullulanase addition. These results differentiated from other $\beta$-amylases reported. Based on a previous result that showed the enzyme system involves in effective degradation of raw starch granules, this result strongly suggested that the purified enzyme (E-I) can be a synergistic part of starch granule-digestion and E-II plays a crucial role in digestion of starch granules.

• Archives of Pharmacal Research
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• v.29 no.5
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• pp.418-423
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• 2006

Tabanus anticoagulant protein (TAP) was isolated from the whole body of the tabanus, Tabanus bivittatus, using three purification steps (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and ion exchange chromatography on DEAE Sephadex gel). The purified TAP, with a molecular weight of 65 kDa, was assessed to be homogeneous by SDS-polyacrylamide gel electrophoresis, and an isoelectric point of 7.9 was determined by isoelectric focusing. The internal amino acid sequence of the purified protein was composed of Ser-Leu-Asn-Asn-Gln-Phe-Ala-Ser-Phe-lle-Asp-Lys-Val-Arg. The protein was activated by $Cu^{2+}\;and\;Zn^{2+}$, and the optimal conditions were found to be at pH $3\sim6\;and\;40\sim70^{\circ}C$. Standard coagulation screen assays were used to determine thrombin time and activated partial thromboplastin time. Chromogenic substrate assays were performed for thrombin and factor Xa activity. TAP considerably prolonged human plasma clotting time, especially activated partial thromboplastin time in a dose-dependent manner; it showed potent and specific antithrombin activity in the chromogenic substrate assay. Specific anti-factor Xa activity in TAP was not detected. Overall, this result suggested that TAP has significant anticoagulant activity on blood coagulation system.

• Journal of Microbiology and Biotechnology
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• v.15 no.2
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• pp.239-246
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• 2005

Some characteristics of two forms of glucoamylase (glucan 1 A-$\alpha$-glucosidase, EC 3. 2. I. 3) purified from Aspergillus coreanus NR 15-1 were investigated. The enzymes were produced on a solid, uncooked wheat bran medium of A. coreanus NR 15-1 isolated from traditional Korean Nuruk. Two forms of glucoamylase, GA-I and GA-II, were purified to homogenity after 5.8-fold and 9.6-fold purification, respectively, judged by disc- and SDS-polyacrylamide gel electrophoresis. The molecular mass of GA-I and GA-II were estimated to be 62 kDa and 90 kDa by Sephadex G-1OO gel filtration, and 64 kDa and 91 kDa by SDS-polyacrylarnide gel electrophoresis, respectively. The optimum temperatures of GA-I and GA-II were 60$^circ$C and 65$^circ$C, respectively, and the optimum pH was 4.0. The activation energy (Ea value) of GA-I and GA-II was 11.66 kcal/mol and 12.09 kcal/mol, respectively, and the apparent Michaelis constants (K_{m}) of GA-I and GA-II for soluble starch were found to be 3.57 mg/ml and 6.25 mg/ml, respectively. Both enzymes were activated by 1 mM Mn^{2+} and Cu^{2+}, but were completely inhibited by 1 mM N­bromosuccinimide. The GA-II was weakly inhibited by 1 mM p-CMB, dithiothreitol, EDTA, and pyridoxal 5-phosphate, but GA-I was not inhibited by those compounds. Both enzymes had significant ability to digest raw wheat starch and raw rice starch, and hydrolysis rates of raw wheat starch by GA-I and GA-II were 7.8- and 7.3-fold higher than with soluble starch, respectively.

• Korean Journal of Food Science and Technology
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• v.35 no.3
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• pp.499-502
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• 2003

The methanol (MeOH) extracts from the fruits of Rubus coreanum showed antioxidative activity. The antioxidative substance in MeOH extracts was successively purified with solvent fractionation, adsorption chromatography, and gel filtration. The purified active substance was isolated by HPLC and identified as quercetin by EI-MS, and $^1H-NMR$ analyses. The amount of quercetin was 0.25 mg per 100 g in fresh fruits of Rubus coreanum Miquel.

• Journal of the Korean Chemical Society
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• v.24 no.1
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• pp.25-33
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• 1980

Pyrocatechase as a phenolytic dioxygenase was extracted from the benzoate-induced cells of a soil bacterium, a member of Pseudomonadaceae, and purified partially by DEAE-cellulose ion-exchange chromatography and Sephadex G-75 gel filtration. Final preparation of the enzyme yielding 200 fold purification over the crude extracts showed a specific activity of about 40 ${\mu}moles$ per minute per mg protein based on catechol as the substrate. The enzyme showed a very limited substrate specificity towards catechol for its catalytic activity. Based on the inhibition study with the substrate analogues, it was assumed that ortho dihydroxy groups on the aromatic ring may participate in the enzyme-substrate binding. The $K_m$ value for catechol was obtained as $1.9{\times}10^{-6}M$, and the optimum activity of the enzyme was obtained at the pH range of 7∼10 and $35^{\circ}C$. With SH-group blocking agents the enzyme was inhibited seriously. The activity of enzyme was also inhibited by the addition of some heavy metals, $Ag^+$ and $Cu^{2+}$, but was not affected by EDTA. General property of the enzyme was characterized and the possible nature of the enzyme active center was also discussed.

• The Korean Journal of Zoology
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• v.27 no.4
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• pp.199-212
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• 1984

A cytoplasmic endoprotease, named protease Ci, has been partially purified by classical chromatographic procedures. This enzyme degrades insulin, glucagon and bovine growth hormone to trichloroacetic acid-soluble materials, but shows little or no hydrolysis of bovine serum albumin, casein or globin. It has a molecular weight of about 120,000 as determined by gel filtration on Sephadex G200, and it appears to be consisted of two identical subunits having molecular weight of 54,000 when estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Protease Ci has an optimum pH of 7.5, and has an isoelectric point of 5.5. This enzyme is a metalloprotease, since it is inhibited by o-phenanthroline and can be activated by the addition of divalent metal cations, such as $Mn^++$ and $Co^++$. Protease ci is inhibited by p-hydroxymercuribenzoic acid, but not by either of leupeptin or Ep475 which are specific inhibitors of sulfhydryl protease. It is distinct from protease Pi, a perplasmic insulin degrading enzyme, since protease Ci is localized to the cytoplasm. The physiological function of protease Ci is presently unknown.

• Korean Journal of Microbiology
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• v.30 no.2
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• pp.71-77
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• 1992

A 50 KD metalloprotease of Serratia marcesrens ATCC 21074 was purified by ammoniumsulfate precipitation. DEAE-cellulose ion exchange chromatography, and sephadex ti-100gel filtration. Optimal pH and temperature of enzyme were pH 8.0 and 37"C, respectively.This enzyme was stable in the ranges of 10-37$^{\circ}$C and pH 5.0--11.0. Thermal denaturationwas investigated by differential scanning calorimetry. Onset temperature of denaturationand endothermic peak temperature were 376$^{\circ}$C and 43.2"C. re:,pectively. The denaturationenthalpy was -8.4mJimg. The purified metalloprotease was ri.sistant to autodigestion for24 hr at 30$^{\circ}$C. Metalloprotease in culture supernatant was also resistant to autodigestionin this conditions. Heat-denatured enzyme. however. was rapidly digested by the nativeenzyme. The metalloprotease was stable to proteolytic digestion by mammalian proteasessuch as trypsin. a-chymotrypsin, and elastase. But the enzyme was easily digested bybacterial protease. thermolysin.bacterial protease. thermolysin.

• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.2
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• pp.144-149
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• 2001

For the production and purification of a single chain human insulin precursor, four types of fusion peptides $\beta$-galactosidase (LacZ), maltose binding protein (MBP), glutathione-S-transferase (GST), and (His)(sub)6-tagged sequence (HTS) were investigated. Recombinant E. coli harboring hybrid genes was cultivated at 37$\^{C}$ for 1h, and gene induction occurred when 0.2mM of isopropyl-D-thiogalactoside (IPTG) was added to the culture broth, except for E. coli BL21 (DE3) pLysS harboring a pET-BA cultivation with 1.0mM IPTG, followed by a longer than 4h batch fermentation respectively. DEAE-Sphacel and Sephadex G-200 gel filtration chromatography, amylose affinity chromatography, glutathione-sepharose 4B affinity chromatography, and a nickel chelating affinity chromatography system as a kind of immobilized metal ion affinity chromatography (IMAC) were all employed for the purification of a single chain human insulin precursor. The recovery yields of the HTS-fused, GST-fused, MBP-fused, and LacZ-fused single chain human insulin precursors resulted in 47%, 20%, 20%, and 18% as the total protein amounts respectively. These results show that a higher recovery yield of the finally purified recombinant peptides was achieved when affinity column chromatography was employed and when the fused peptide had a smaller molecular weight. In addition the pET expression system gave the highest productivity of a fused insulin precursor due to a two-step regulation of the gene expression, and the HTS-fused system provided the highest recovery of a fused insulin precursor based on a simple and specific separation using the IMAC technique.

• Journal of the Korean Chemical Society
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• v.29 no.3
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• pp.295-303
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• 1985

Carboxypeptidase B from Salmo Salar eggs was purified by CM-cellulose, 0.5 ammonium sulfate saturation, DEAE-cellulose, and Sephadex G-75 gel filtration and its enzymatic properties were investigated. Optimum temperature was 55$^{\circ}C$, pH optima were 4.0 and 7.0 at 37$^{\circ}C$, and the enzyme was stable at pH 2.0∼3.0 and 5.5∼7.0 for 1.5h. This enzyme showed substrate specificity hydrolyzing the peptide bond of glycyl-L-arginine. Its K$_m$ values was 0.21mM, and the enzyme activity was stimulated by Cu$^{2+}$ and Fe$^{3+}$, while inhibited by Zn$^{2+}$. The lysine was found to be competitive inhibitor and its K$_i$ value was determined to be 4.3mM. Molecular weight of this enzyme was determined to be 36,400 daltons by SDS-PAGE and the enzyme was monomeric protein composed of 19 kinds of amino acid residues.