• Bioinformatics and Biosystems
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• v.2 no.1
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• pp.28-32
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• 2007

The protein phosphorylation is one of the important processes in the cell signaling pathway. A variety of protein kinase families are involved in this process, and each kinase family phosphorylates different kinds of substrate proteins. Many methods to predict the kinase-specific phosphoryrated sites or different types of phosphorylated residues (Serine/Threonine or Tyrosin) have been developed. We employed Supprot Vector Machine (SVM) to attempt the prediction of protein kinase specific phosphorylation sites. 10 different kinds of protein kinase families (PKA, PKC, CK2, CDK, CaM-KII, PKB, MAPK, EGFR) were considered in this study. We defined 9 residues around a phosphorylated residue as a deterministic instance from which protein kinases determine whether they act on. The subsets of PSI-BALST profile was converted to the numerical vectors to represent positive or negative instances. When SVM training, We took advantage of multiple SVMs because of the unbalanced training sets. Representative negative instances were drawn multiple times, and generated new traing sets with the same positive instances in the original traing set. When testing, the final decisions were made by the votes of those multiple SVMs. Generally, RBF kernel was used for the SVMs, and several parameters such as gamma and cost factor were tested. Our approach achieved more than 90% specificity throughout the protein kinase families, while the sensitivities recorded 60% on average.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2001.06a
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• pp.163-164
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• 2001

A number of bacterial species produce extracellular lipases. Among them, many lipase genes have been cloned and sequenced. A comparison of primary sequences revealed only very limited sequence homology among them. Based on the sequence homologies and molecular sizes (Mr), bacterial lipases were classified into four discrete groups. From soil samples taken around Taejon, five different lipase-producing bacteria were isolated; Proteus vulgaris K80, Bacillus stearothermophilus Ll, B. pumilus B26, Staphylococcus haemolyticus L62, S. aureus B56. Nucleotide sequence analysis showed that Staphylococcus lipase genes (L62 and B56) composed of pre-pro-mature parts, Bacillus lipase genes (Ll and B26) pre-mature parts, and Proteus lipase gene (K80) mature part only. In addition, the molecular sizes of their mature parts were quite different from 19,000 to 45,000. Finally, they had very little homology (less than 20％) in their amino acid sequences. Judging from the above results, lipase K80 belonged to bacterial lipase Group I, lipase L1 and lipase B26 Group III, and lipase L62 and lipase B56 Group IV. This diversity in their primary structures was also reflected in their enzymatic properties; temperature effects, pH effects, substrate specificity, detergent effects, and so on.

• Journal of Microbiology and Biotechnology
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• v.9 no.3
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• pp.249-254
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• 1999

A catechol 2,3-dioxygenase (C23O) was purified to apparent homogeneity from Pseudomonas putida SU10 through several purification steps consisting of ammonium sulfate precipitation and chromatographies on DEAE 5PW, Superdex S-200, and Resource-Q. Gel filtration indicated a molecular mass under nondenaturing conditions of about 130 kDa. The enzyme has a subunit of 34 kDa as was determined by SDS-PAGE. These results suggest that the native enzyme is composed of four identical subunits. The N-terminal amino acid sequence (30 residues) of the enzyme has been determined and exhibits high identity with other extradiol dioxygenases. The reactivity of this enzyme towards catechol and methyl-substituted catechols is somewhat different from that seen for other catechol 2,3-dioxygenases, with 4-methylcatechol cleaved at a higher rate than catechol or 3-methylcatechol. $K_m$ values of the enzyme for these substrates are between 3.5 and 5.7 M.

• Journal of Microbiology and Biotechnology
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• v.15 no.3
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• pp.519-524
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• 2005

An ${\alpha}$-L-rhamnosidase (EC 3.2.1.40.), which transforms quercitrin to quercetin, was purified from Fusobacterium K-60, a human intestinal anaerobic bacterium. The specific activity of the purified ${\alpha}$-L-rhamnosidase was 2.89 mol/min/mg protein. ${\alpha}$-L-Rhamnosidase, whose molecular size was 170 kDa by gel filtration, was composed of four subunits ($M_r$ 41,000 Da) with pI and optimal pH values of 5.2 and 5.5-7.0, respectively. The apparent $K_m$ and $V_{max}$ values for p-nitrophenyl-${\alpha}$-L-rhamnopyranoside and quercitrin were determined to be 0.057 mM and 3.4 mol/min/mg, and 0.077 mM and 5.0 mol/min/mg, respectively. This enzyme was strongly inhibited by $Cu^{2+},\;Mn^{2+}$, L-rhamnose, and p-chlormercuriphenylsulfonic acid. These findings suggest that the biochemical properties and substrate specificity of the purified enzyme are different from those of the previously purified ${\alpha}$-L-rhamnosidase. This is the first reported purification of quercitrin-hydrolyzing ${\alpha}$-L-rhamnosidase from intestinal bacteria.

• Journal of Microbiology and Biotechnology
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• v.15 no.3
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• pp.491-496
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• 2005

The biosynthetic gene cluster for the aminoglycoside antibiotic kasugamycin was isolated and characterized from the kasugamycin producing strain, Streptomyces kasugaensis KACC 20262. By screening a fosmid library using kasA, the gene encoding aminotransferase, we isolated a 22 kb DNA fragment. The fragment contained seventeen complete open reading frames (ORFs); one of these ORFs, kasD, was identified as the gene for dNDP-glucose 4,6-dehydratase, which catalyzes the conversion of dNDP-glucose to 4-keto-6-deoxy-dNDP-glucose. The enzyme showed a broad spectrum of substrate specificity. In addition, ksR was overexpressed in E. coli BL21 and proved to be a self-resistance gene against kasugamycin. These findings suggest that the isolated gene cluster is highly likely responsible for the biosynthesis of kasugamycin.

• Journal of Applied Biological Chemistry
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• v.43 no.3
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• pp.136-140
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• 2000

A novel 6-deoxyhexose biosynthetic gene cluster different from the one for the biosynthesis of streptomycin was isolated from Streptomyces griseus using specifically designed PCR primers to compare the sequence of known dTDP-glucose synthase genes. We cloned a 5.8-kb DNA from Streptomyces griseus ATCC10137, which contained the 4-ketoreductase homologue (grsB), dTDP-glucose synthase (grsD), and dTDP-glucose 4, 6-dehydratase (grsE) genes. Escherichia coli cultures containing plasmid of the PCR product which encoded the grsE region under the controUed T7 promoter were able to catalyze the formation of dTDP-4-keto-6-deoxy-D-glucose from TDP-glucose. The enzyme showed high substrate specificity, being specific to only dTDP-glucose that is known to be incorporated into secondary metabolites such as antibiotics.

• Journal of Microbiology and Biotechnology
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• v.23 no.2
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• pp.144-148
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• 2013

Lycopene cyclase converts lycopene to ${\beta}$-carotene by catalyzing the formation of two beta-rings at each end of the linear carotene structure. This reaction takes place as a two-step reaction in which both sides of of the lycopene molecule are cyclized into ${\beta}$-carotene rings via the monocyclic ${\gamma}$-carotene as an intermediate. The crtY gene coding for lycopene cyclase from Paracoccus haeundaensis consists of 1,158 base pairs encoding 386 amino acids residues. An expression plasmid containing the crtY gene (pET44a-CrtY) was constructed and expressed in Escherichia coli, and produced a recombinant protein of approximately 43 kDa, corresponding to the molecular mass of lycopene cyclase. The expressed protein was purified to homogeneity by His-tag affinity chromatography and showed enzymatic activity corresponding to lycopene cyclase. We also determined the lycopene substrate specificity and NADPH cofactor requirements of the purified protein. The $K_m$ values for lycopene and NADPH were 3.5 ${\mu}M$ and 2 mM, respectively. The results obtained from this study will provide a wider base of knowledge on the enzyme characterization of lycopene cyclase at the molecular level.

• Fisheries and Aquatic Sciences
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• v.13 no.1
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• pp.49-55
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• 2010

A bacterial strain with phytase and glucose-1-phosphatase activity was isolated from seawater. The colony was identified as an Enterobacter cloacae strain and named E. cloacae B11. A gene, agpEnB11, coding for an intracellular acid glucose phosphatase was cloned from the strain and sequenced. It comprised 1,242 nucleotides and encoded a polypeptide of 413 amino acids. Recombinant glucose-1-phosphatase (AgpEn) was overexpressed in Escherichia coli and purified using Ni-NTA column under native conditions. Purified protein displayed a single band of 47 kDa on SDS-PAGE. AgpEn hydrolyzed a wide variety of phosphorylated compounds, with high activity for glucose-1-phosphate and glucose-6-phosphate. Optimum pH and temperature for enzyme activity were pH 5.0 and $50^{\circ}C$, respectively. Enzyme activity was stimulated by $Ca^{2+}$ and $Co^{2+}$, and inhibited by $Cu^{2+}$.

• Korean Journal of Veterinary Service
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• v.34 no.4
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• pp.397-401
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• 2011

Aspiration of lytic bone lesions is an excellent diagnostic test in the initial evaluation of primary bone tumor. However, cytologically, it can be difficult to differentiate osteosarcoma (OSA) from other bone neoplasms, including fibrosarcoma, chondrosarcoma, synovial cell sarcoma, malignant fibrous histiocytoma and malignant peripheral nerve sheath tumor. The purpose of this study is to introduce alkaline phosphatase (ALP) staining to differentiate OSA from other mesenchymal tumors. Tumors actively producing bone are specifically positive for ALP staining. Unstained, cytologic specimens were incubated for 10 minutes with nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate toluidine salt-phosphatase substrate. Among 20 cases of cytology specimen, 14 were positive for ALP staining and histopathology, 6 were negative for ALP staining and histopathology. ALP staining was 100% sensitive and specificity for the diagnosis of OSA. Aspirate cytology with ALP staining was a simple, fast, safe and accurate diagnostic test for the evaluation of suspected OSA lesions in dogs.

• The Korean Journal of Zoology
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• v.39 no.2
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• pp.215-222
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• 1996

In the previous studies, we have demonstrated that prorenin converting enzyme (PRECE) was identical to the epidermal grouch factor-binding protein (EGF-BP) type B, which was a member of the mouse glandular kallikrein family, To examine whether or not EGF-BP type A and C are involved in the processing of prorenin, we have cloned the CDNAS of the EGF-BP type h and C from a library of male ICR mouse submandibular gland (SMGI. And then CHO cells were transfected with the EGF-BP expression plasmids. and stable cell lines expressing a high level of the EGF-BPS precursor were obtained. The conditioned medium was then treated with trypsin, which has been knotvn to effectively convert the EGF-BP type A and C precursor to the active forms. 수ubsequentlv, the prorenin converting activity of the trypsin-treated or untreated medium was examined. PRECE converted exactly prorenin to renin, but the prorenin converting activities of EGF-BP type A and C were not detected. From these results, it seems that only type B of these EGF-BPs is involved in processing Ren 2 prorenin in mouse SMG.