• Korean Journal of Microbiology
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• v.47 no.4
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• pp.302-307
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• 2011

Native chitin deacetylase of Aspergillus nidulans was purified to apparent homogeneity by a combination of phenyl-Sepharose and Q-Sepharose column chromatography. In order to analyze the amino acid residues involved in the enzyme activity, the enzyme was chemically modified with chemical agent, which selectively reacted with the specific amino acid residue on the protein. When the enzyme was chemically modified with diethylpyrocarbonate, which specifically reacted with histidine residues on the protein, the activity was eliminated. The chitin deacetylase, chemically modified with 100 ${\mu}M$ modifier at the residue of arginine or tyrosine, has shown to have decreased activities. It was shown that the modification at aspartic acid or glutamic acid did not affect the enzyme activity to a greater extent, which would not implicate that acid amino residues were directly involved in catalytic reaction and would affect on the global structures of the proteins. This results demonstrated that histidine and tyrosine residues of enzyme would participate in an important function of the chitin deacetylase activity.

• Applied Biological Chemistry
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• v.34 no.3
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• pp.249-257
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• 1991

To elucidate enzymatic properties of ${\alpha}-galactosidase$ (EC 3, 2, 1, 22) from germinated soybean, changes in the enzyme activities and oligosaccharide contents during germination of soybean were determined. ${\alpha}-Galactosidase$ from germinated soybean was purified by ammonium sulfate fractionation, ion exchange chromatography and gel filtration. Their chemical and enzymatic properties was investigated. ${\alpha}-galactosidase$ activity of sobeam was maximized when it was germinated at $25^{\circ}C$ for 120 hour. Raffinose and stachyose in soybean were decomposed completely after 96 hours and 120 hours of germination, respectively. Soybean ${\alpha}-galactosidase$ was purified by 6.6 fold by ammonium sulfate fractionation, ion exchange chromatography on DEAE-Cellulose and Sephadex A-50, and gel filtration on Sephadex G-150. Its specific activity was 825 Units/mg protein and the yield was 2.5% of the total activity of crude extracts. The purified ${\alpha}-galactosidase$ of soybean was found to be homogeneous by polyacrylamide gel electrophoresis and by HPLC. Isoelectric point of soybean ${\alpha}-galactosidase$ was determined analytical isoelectric focusing to be pH 4.8. The soybean ${\alpha}-galactosidase$ was monomeric and its molecular weight was estimated to be 30,000 by SDS-PAGE. The optimal temperature and pH for the soybeam ${\alpha}-galactosidase$ activity were $40^{\circ}C$ and pH 6.0 and 75% of its activity was lost by heating at $60^{\circ}C$ for 10 min. The enzyme was appeared to have higher affinity to raffinose than to stachyose. The Km value of soybean enzyme was 5.3 mM for ${\rho}-nitrophenyl-{\alpha}-D-galactopyranoside$ and the activation energy on PNPG was calculated to be 13.02 Kcal per mole.

• Journal of Life Science
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• v.23 no.12
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• pp.1486-1494
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• 2013

A high protease-producing strain was isolated and identified from the digestive tract of octopus vulgaris by detecting a hydrolysis circle of protease and its activity. The strain was identified by morphology observation, biochemical experiments, and 16S rRNA sequence analysis. The protease obtained from the strain was purified by a three-step process involving ammonium sulfate precipitation, carboxy methyl-cellulose (CM-52) cation-exchange chromatography, and DEAE-Sephadex A50 anion-exchange chromatography. The properties of protease were characterized as well. The strain Bacillus sp. QDV-3, which produced the highest activity of protease, was isolated. On the basis of the phenotypic and biochemical characterization and 16S rRNA gene-sequencing studies, the isolate was identified as follows: domain: Bacteria; phylum: Firmicutes; class: Bacilli; order: Bacillales; family: Bacillaceae; and genus: Bacillus. The isolate was shown to have a 99.2% similarity with Bacillus flexus. A high active protease designated as QDV-E, with a molecular weight of 61.6 kDa, was obtained. The enzyme was found to be active in the pH range of 9.0-9.5 and its optimum temperature was $40^{\circ}C$. The protease activity retained more than 96% at the temperature of $50^{\circ}C$ for 60 min. Phenylmethylsulfonyl fluoride (PMSF) inhibited the enzyme activity, thus confirming that this protease isolated from Bacillus sp. QDV-3 is an alkaline serine protease. Metal ions, $Mn^{2+}$ and $Mg^{2+}$, were determined to enhance the protease activity, whereas $Ba^{2+}$, $Zn^{2+}$, and $Cu^{2+}$ were found to inactivate the enzyme.

• Korean Journal of Food Science and Technology
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• v.34 no.4
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• pp.552-558
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• 2002

Polyphenol oxidase from Flammulina velutipes was purified and characterized. Purification of polyphenol oxidase was achieved by ammonium sulfate precipitation, Superdex G-200 gel filtration chromatography, Phenyl superose affinity chromatography, Mono-Q anion exchange chromatography and Superdex S-200 gel filtration chromatography on FPLC. After these purification steps specific activity of purified polyphenol oxidase increased to 199.1 units/mg. Polyphenol oxidase from F. velutipes was composed of a single polypeptide with molecular weight of about 40 kDa. Optimum pH and temperature for the enzyme reaction were found to be 6.0 and $25^{\circ}C$, respectively. The activity of the enzyme gradually decreased at acidic pH between 3 and 5, and the enzyme lost its activity at alkaline pH between 8 and 10. This enzyme exhibited high substrate specificity to o-diphenols. Km-values for L-DOPA and caffeic acid were found to be 3.97 mM and 1.78 mM, respectively. 2-mercaptoethanol, L-ascorbic acid, sodium bisulfite, EDTA and $Mg^{2+}$ inhibited the activity of pholyphenol oxidase and $Cu^{2+}$, $Fe^{2+}$, $Zn^{2+}$ and $Ni^{2+}$ increased enzyme activity. The activity of enzyme was well maintained at $-70^{\circ}C$ for over 4 months, and at $-20^{\circ}C$ for 1 months.

• Korean Journal of Food Science and Technology
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• v.36 no.2
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• pp.361-364
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• 2004

EPA and DHA extracted from methyl esterified squid oil were purified by silver exchanged resin, silver nitrate-impregnated silica gel, silver exchanged zeolite, and silica gel column chromatography, among which column chromatography using mixture of silver exchanged resin and silica gel (10% by weight) showed the best result. By this simple purification method, EPA and DHA were concentrated from 12.5 to 27.9% (yield, 86,0%) and from 21.7 to 49.5% (yield, 87.3%), respectively. Silver exchanged resin had additional advantages of outstanding reusability and simple recovery of silver.

• Analytical Science and Technology
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• v.12 no.2
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• pp.99-104
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• 1999

A stepwise gradient elution with two wavelengths detection was performed for the separation and determination of some anions in saline water. The eight anions such as iodate, bromate, nitrite, bromide, nitrate, chromate, iodide and thiocyanate were successfully separated using AS-7 column and sodium chloride/sodium phosphate buffer solution as an eluant within 40 min. The separation behaviors of anions were studied at various sodium chloride concentrations. The peak shapes of anions of bromate, nitrite, bromide and nitrate gradually broadened as the concentration of sodium chloride increased until 1.0 M in the sample solutions. However, no effect was observed in the peak shapes of chromate, iodide and thiocynate. A good linearity was obtained at the range of ppm(mg/L). The detection limit was proved to be $10-720{\mu}g/L$ for the eight anions with $50{\mu}L$ injection volume. This method was applied to the determination of $Br^-$, ${NO_3}^-$ and $I^-$ in sea water.

• Korean Journal of Microbiology
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• v.30 no.2
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• pp.101-107
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• 1992

Cytochrome c oxida5e from chemotrophically grown R p , geliitinosu was purified by cytochrome c affinity chromatography and DEAE-Sephacel ion exchange chromatography. The molecular weight of the cytochrome c oxidase was approximately 110.000 Da by sephacryl s-300 gel chromatography and approximately 52, 000 Da by SDS-gel electrophoresis, respectively. Therefore. cytochrolne c oxidase of Rps. gehtinosu seems to be dimer. The cytochrome c oxidasc was very sensitive to temperature. It's Km and Vmax were 20 pM and 44 unitlmg protein for horsc heart cytochrome c as a substrate. respectively, and its optimum pH and temperature were 6.4 and 25$^{\circ}$C. respectively. The absorption peaks of the reduced cytochrome c oxidase showed at 554 nm, 523 nm. and 422 nm. The activiiy of cytochrome c oxidase was inhibited by KCN, and NaN3, but not by CO, antimycir~ A. and myxothiazol. The cytochrome c-551 was produced either in phototrophically or chemotrophically grown Rps. gelaiinosci. The rcduced cytochrome c-551 was oxidized by b-type cytochrome c oxidase from Rp.v. gc.lrtino.sc~. Km and Vmax of cytochrome c oxidase was 26 pM and 31 unitlnlg protein For cytochrome c-551 as a substrate. respectively. Thercfore. thc electron transfer chain of chemotrophically grown Rps. glatinosa seems lo be ubiquinol cytochrome bc, complex -'cytochrome c-55lMb-type cytochrome c oxidase+02.

• Korean Chemical Engineering Research
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• v.49 no.2
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• pp.129-136
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• 2011

Simulated Moving Bed(SMB) process consists of multiple chromatographic columns, which are usually partitioned into four zones. Such a process characteristic allows a continuous binary separations those are impracticable in conventional batch chromatographic processes. Compared with batch chromatography, SMB has advantages of continuity, high purity and productivity. Various researches have been reported for the integration of reaction and recovery during process operation on the purpose of economics and effectiveness. Simulated Moving Bed Reactor(SMBR) is introduced to combine SMB as a continuous separation process and reactor. Several cases of SMBR have been reported for diverse reactions with catalytic, enzymatic and chemical reaction on ion exchange resin as main streams. With an early type of fixed bed using catalyst, SMBR has been developed as SMB using fluidized enzyme, SMB with immobilized enzyme and SMB with discrete reaction region. For simple modeling and optimization of SMBR, a method considering convection only is possible. A complex method considering axial dispersion and mass transfer resistance is needed to explain the real behavior of solutes in SMBR. By combining reaction and separation, SMBR has benefits of lower installation cost by minimizing equipment use, higher purity and yield by avoiding the equilibrium restriction in case of reversible reaction.

• Microbiology and Biotechnology Letters
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• v.26 no.4
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• pp.297-302
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• 1998

$\beta$-Xylosidase B was produced by Escherichia coli HB101/pKMG12 carrying the xylB gene of Bacillus stearothermophilus No.236 on its recombinant plasmid. The $\beta$-xylosidase B produced was purified by ammonium sulfate fractionation, DEAE-Sepharose CL-6B, Sephacryl S-200 and Superdex 200 HR gel filtration. The purified enzyme showed the highest activity at pH 6.5 and 5$0^{\circ}C$. But, the enzyme was observed to be very sensitive to the pH and temperature of the reaction mixture. The enzyme was activated about 35% of its original activity in the presence of 1 mM of $Mn^{2+}$ but it was completely inhibited by $Ag^{+}$, $Cu^{2+}$and $Hg^{2+}$ions. In contrast with the $\beta$-xylosidase A, the B enzyme was found to have $\alpha$-arabinofuranosidase activity though the activity was fairly low compared with the $\alpha$-arabinofuranosidase produced from the arfI gene of the same Bacillus stearothermophilus. Therefore, $\beta$-xylosidase B is considered to be more suitable than $\beta$-xylosidase A at least for the biodegradation of arabinoxylan. The $K_{m}$ and V$_{max}$ values of the $\beta$-xylosidase B for o-nitrophenyl-$\alpha$-D-xylopyranoside were 6.43 mM and 1.45 $\mu$mole/min, respectively. Molecular mass of the enzyme was determind to be about 54 kDa by SDS-PAGE and 160 kDa by Superdex 200HR gel filtration, indicating that the functional $\beta$-xylosidase B was composed of three identical subunits.s.

• Journal of Food Hygiene and Safety
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• v.20 no.4
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• pp.211-216
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• 2005

The bacteriocin produced by E. faecium MJ-14 was precipitated with $50\%$ saturated ammonium sulfate in MRS broth and then the precipitated protein was dissolved in 20 mM sodium phosphate buffer (pH 6.0). The crude bacteriocin was purified by CM-sepharose CL 6B and Sephacry S-100 column chormatograhy. In this case, the purification fold of the bacteriocin was 114, therefore, its activity was 127,293 BU/mg of specific activity. Result from SDS-PAGE of the purified bacteriocin, it was obtained two protein bands of 4.3 kDa and 5.8 kDa having antilisterial activity.