• 한국생물공학회:학술대회논문집
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• 2002.04a
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• pp.445-448
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• 2002

The Pseudomonas fluorescens KCTC 1767, a selected and identified as potential candidate for stereo-specific resolution of rac-ketoprofen ethyl ester, was systematically investigated in order to induce the high level expression and detailed characterization of the expressing enzyme esterase. We cloned the esterase gene from chromosomal DNA of Pseudomonas fluorescens KCTC 1767 by PCR with two synthetic primers that desinged for simple purification. The recombinant esterase from Pseudomonas fluorescens KCTC 1767 exibited a high conversion rate and enantioselectivity to the (S)-ketoprofen ethyl ester as expected. The enzyme was easily purified to homogeniety by using a metal chelating affinity chromatography as a protein with poly histidine taq, and thus obtained 0.6 mg of protein from a 100 mL culture broth in a single step. The purified enzyme was steadily stable at the pH range from 7.0 to 10. The activity was also retained to be about 70% after the preincubation at $40^{\circ}C$ but over $50^{\circ}C$ lost the activity completely. The molecular mass of the esterase was estimated to be about 43 kDa on SDS-PAGE, and an identical result was also shown in gel filteration chromatography. The specific activity was calculated 27 mM/mg-protein/min by using the rac-ketoprofen ethly ester as a substrate.

• Journal of Microbiology and Biotechnology
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• v.22 no.3
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• pp.339-342
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• 2012

This study describes the characterization of a new angiotensin I-converting enzyme (ACE) inhibitory peptide from a Korean traditional rice wine. After purification of the ACE inhibitor peptides with ultrafiltration, Sephadex G-25 column chromatography, and successively $C_{18}$ and SCX solid-phase extraction, reverse-phase HPLC, and size exculsion chromatography, two types of the purified ACE inhibitors with $IC_{50}$ values of 0.34 mg/ml and 1.23 mg/ml were finally obtained. The two purified ACE inhibitors (F-1 and F-2) were found to have two kinds of novel oligopeptides, showing very little similarity to other ACE inhibitory peptide sequences. The amino acid sequences of the two purified oligopeptides were found to be Gln-Phe-Tyr-Ala-Val (F-1) and Ala-Gly-Pro-Val-Leu-Leu (F-2), and their molecular masses were estimated to be 468.7 Da (F-1) and 357.7 Da (F-2), respectively. They all showed a clear antihypertensive effect on spontaneously hypertensive rats at a dosage of 500 mg/kg.

• Journal of Microbiology and Biotechnology
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• v.20 no.12
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• pp.1711-1716
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• 2010

A gene encoding the ${\beta}$-xylosidase/${\alpha}$-arabinofuranosidase (XylC) of Paenibacillus woosongensis was cloned into Escherichia coli. This xylC gene consisted of 1,425 nucleotides, encoding a polypeptide of 474 amino acid residues. The deduced amino acid sequence exhibited an 80% similarity with those of both Clostridium stercorarium ${\beta}$-xylosidase/${\alpha}$-N-arabinosidase and Bacillus cellulosilyticus ${\alpha}$-arabinofuranosidase, belonging to the glycosyl hydrolase family 43. The structural gene was subcloned with a C-terminal His-tag into a pET23a(+) expression vector. The His-tagged XylC, purified from a cell-free extract of a recombinant E. coli BL21(DE3) Codon Plus carrying a xylC gene by affinity chromatography, was active on para-nitrophenyl-${\alpha}$-arabinofuranoside (pNPA) as well as para-nitrophenyl-${\beta}$-xylopyranoside (pNPX). However, the enzymatic activities for the substrates were somewhat incongruously influenced by reaction pHs and temperatures. The enzyme was also affected by various chemicals at different levels. SDS (5 mM) inhibited the enzymatic activity for pNPX, while enhancing the enzymatic activity for pNPA. Enzyme activity was also found to be inhibited by addition of pentose or hexose. The Michaelis constant and maximum velocity of the purified enzyme were determined for hydrolysis of pNPX and pNPA, respectively.

• Journal of Microbiology and Biotechnology
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• v.24 no.11
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• pp.1525-1535
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• 2014

The xynC gene, which encodes high molecular weight xylanase from Paenibacillus sp. DG-22, was cloned and expressed in Escherichia coli, and its nucleotide sequence was determined. The xynC gene comprised a 4,419bp open reading frame encoding 1,472 amino acid residues, including a 27 amino acid signal sequence. Sequence analysis indicated that XynC is a multidomain enzyme composed of two family 4_9 carbohydrate-binding modules (CBMs), a catalytic domain of family 10 glycosyl hydrolases, a family 9 CBM, and three S-layer homologous domains. Recombinant XynC was purified to homogeneity by heat treatment, followed by Avicel affinity chromatography. SDS-PAGE and zymogram analysis of the purified enzyme identified three active truncated xylanase species. Protein sequencing of these truncated proteins showed that all had identical N-terminal sequences. In the protein characterization, recombinant XynC exhibited optimal activity at pH 6.5 and $65^{\circ}C$ and remained stable at neutral to alkaline pH (pH 6.0-10.0). The xylanase activity of recombinant XynC was strongly inhibited by 1 mM $Cu^{2+}$ and $Hg^{2+}$, whereas it was noticeably enhanced by 10 mM dithiothreitol. The enzyme exhibited strong activity towards xylans, including beechwood xylan and arabinoxylan, whereas it showed no cellulase activity. The hydrolyzed product patterns of birchwood xylan and xylooligosaccharides by thin-layer chromatography confirmed XynC as an endoxylanase.

• Journal of Microbiology and Biotechnology
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• v.22 no.12
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• pp.1692-1697
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• 2012

We report a glycoside hydrolase (GH)-118 ${\beta}$-agarase from a strain of Agarivorans, in which we previously reported recombinant expression and characterization of the GH-50 ${\beta}$-agarase. The GH comprised an open reading frame of 1,437 base pairs, which encoded a protein of 52,580 daltons consisting of 478 amino acid residues. Assessment of the entire sequence showed that the enzyme had 97% nucleotide and 99% amino acid sequence similarities to those of GH-118 ${\beta}$-agarase from Pseudoalteromonas sp. CY24, which belongs to a different order within the same class. The gene corresponding to a mature protein of 440 amino acids was inserted, recombinantly expressed in Escherichia coli, and purified to homogeneity with affinity chromatography. It had maximal activity at $35^{\circ}C$ and pH 7.0 and had 208.1 units/mg in the presence of 300 mM NaCl and 1 mM $CaCl_2$. More than 80% activity was maintained after 2 h exposure to $35^{\circ}C$; however, < 40% activity remained at $45^{\circ}C$. The enzyme hydrolyzed agarose to yield neoagarooctaose as the main product. This enzyme could be useful for industrial production of functional neoagarooligosaccharides.

• Microbiology and Biotechnology Letters
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• v.22 no.1
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• pp.65-72
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• 1994

Purification and characterization of pectate lyase from Bacillus sp HSA-925. Bacillus sp. HSA-925 isolated from soil produced constitutively an extracellular pectate lyase when cultivated in LB broth. The pectate lyase(EC 4.2.2.2) was purified from the cuylture broth by preciptation with ammonium sulfate, followed by column chromatography on CM-cellulose C-50 and repeated gel filtration on Sephadex G-75G. The enzyme had a molecular weight of 32-33 kDa. The activity was mazimum at pH 9.5 AND 45$\CIRC $C. The enzume activity was stable at 55$\circ $C for 15 min and between pH7-12. The activation energy, Km and V$_{max}$ for the pectate lyase were 5.8779 kcal/mol, 6.33$\times $10$^{-2}$ mol/ml and 2.09$\times $10$^{2}$ $\mu $mol/min respectively. The enzyme was activated by Ca$^{2+}$, Cu$^{2+}$ and inhibited by Li$^{+}$, Hg$^{2+}$, EDTA.

• Preventive Nutrition and Food Science
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• v.7 no.4
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• pp.421-426
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• 2002

In order to obtain basal data for industrial application of inulinase from Bacillus sphaeicus 188-1, its intracellular inulinase was purified by ammonium sulfate fractionation and column chromatography on DEAE-Sephadex A-50 and Sephadex G-100. The enzyme was homogeneous as judged by SDS-polyacrylamide gel electrophoresis, with an apparent molecular weight of 29 kDa. Inulinase activity was optimal at pH 6.5 and 4$0^{\circ}C$. The enzyme activity was significantly inhibited by Cu$^{2＋}$, Cd$^{2＋}$ and Hg$^{2＋}$. The inulinase exhibited an apparent Km value of 0.014% for inulin.

• Journal of Plant Biotechnology
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• v.36 no.4
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• pp.397-402
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• 2009

Ornithinine carbamoyltransferase (OTC) is an enzyme that catalyzes the key step in arginine biosynthesis in bacteria and plants. OTC is also involved in the urea cycle and deficiency of the enzyme in human leads to disease. The argF gene encoding OTC has been reported in many bacteria and few plants. Here we report the characterization of a gene encoding OTC from rice (OsOTC). Analysis of a cDNA sequence from rice revealed that the full-length open reading frame of OsOTC consisted of 367 amino acids, corresponding to a protein of approximately 39.7 kDa. The predicted amino acid sequence of OsOTC harbor distinct five OTC signature sites and is highly homologous to that of enzymes of plants, animals and many bacterial OTCs. Expression of OsOTC in argF mutants of Escherichia coli showed that the gene was able to functionally complement to the mutant. These results suggest that the OsOTC encode a protein for ornithine carbamoyltransferase in rice.

• Korean Journal of Veterinary Research
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• v.44 no.3
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• pp.421-426
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• 2004

This study was performed to report the outbreaks of canine intestinal spirochetosis and to characterize the canine isolates. Three canine isolates were weakly beta-hemolytic and had sharp end shape with 5 flagella. Isolates didn't produce indole but fermented fructose. In $API-ZYM^{(R)}$ study, isolates were alpha-glucosidase negative and alpha-galactosidase positive, which is the typical characteristics of B. pilosicoli. In multilocus enzyme electrophoresis(MEE) study, isolates were divided into 2 electrophoretic types. Isolates showed different properties with B. hyodysenteriae but closely related with pathogenic canine intestinal spirochete(B. pilosicoli). This is the first report of the isolation and characterization of canine intestinal spirochete in Korean dogs.

• Journal of Microbiology and Biotechnology
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• v.26 no.2
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• pp.255-262
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• 2016

The carrageenan-degrading marine bacterium Vibrio sp. strain NJ-2 was isolated from rotten red algae, and κ-carrageenase with high activity was purified from the culture supernatant. The purified enzyme with molecular mass of 33 kDa showed the maximal activity of 937 U/mg at 40℃ and pH 8.0. It maintained 80% of total activity below 40℃ and between pH 6.0 and 10.0. The kinetics experiment showed the K_m and V_max values were 2.54 g/ml and 138.89 mmol/min/mg, respectively. The thin layer chromatography and ESI-MS analysis of hydrolysates indicated that the enzyme can endolytically depolymerize the κ-carrageenan into oligosaccharides with degrees of depolymerization of 2-8. Owing to its high activity, it could be a valuable tool to produce κ-carrageenan oligosaccharides with various biological activities.