• Asian-Australasian Journal of Animal Sciences
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• 제29권1호
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• pp.126-133
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• 2016

A gene from Actinomyces sp. Korean native goat (KNG) 40 that encodes an endo-${\beta}$-1,4-glucanase, EG1, was cloned and expressed in Escherichia coli (E. coli) $DH5{\alpha}$. Recombinant plasmid DNA from a positive clone with a 3.2 kb insert hydrolyzing carboxyl methyl-cellulose (CMC) was designated as pDS3. The entire nucleotide sequence was determined, and an open-reading frame (ORF) was deduced. The ORF encodes a polypeptide of 684 amino acids. The recombinant EG1 produced in E. coli $DH5{\alpha}$ harboring pDS3 was purified in one step using affinity chromatography on crystalline cellulose and characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis/zymogram analysis of the purified enzyme revealed two protein bands of 57.1 and 54.1 kDa. The amino terminal sequences of these two bands matched those of the deduced ones, starting from residue 166 and 208, respectively. Putative signal sequences, a Shine.Dalgarno-type ribosomal binding site, and promoter sequences related to the consensus sequences were deduced. EG1 has a typical tripartite structure of cellulase, a catalytic domain, a serine-rich linker region, and a cellulose-binding domain. The optimal temperature for the activity of the purified enzyme was $55^{\circ}C$, but it retained over 90% of maximum activity in a broad temperature range ($40^{\circ}C$ to $60^{\circ}C$). The optimal pH for the enzyme activity was 6.0. Kinetic parameters, $K_m$ and $V_{max}$ of rEG1 were 0.39% CMC and 143 U/mg, respectively.

• Journal of Microbiology and Biotechnology
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• 제6권2호
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• pp.86-91
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• 1996

Thermostable $\beta$-mannanase from Bacillus sp. YA-14 was purified by acetone precipitation, CM-cellulose, Sephadex G-100 and hydroxyapatite column chromatography from culture supernatant. The final enzyme preparation appeared to be homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). $\beta$-Mannanase appeared to be a monomeric protein with a molecular weight of 67, 000 daltons. The optimal pH and temperature of the enzyme reaction were pH 6.0 and $75^{\circ}C$ , respectively. The enzyme was stable at a pH range of 6.0 to 9.0 and at temperatures between 45 and $85^{\circ}C$. The kinetic constants of $\beta$-mannanase as determined with a galactomannan (locust bean) as substrate were a Vmax of 25 unit/ml and a Km of 1.1 mg/ml. The enzyme had only limited activity on galactomannan substrate. It was suggested that mg $\beta$-mannanase activity is limited by the number of branched $\alpha$-galactose residues.

• Journal of Life Science
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• 제10권1호
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• pp.1-5
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• 2000

Pectinase was isolated from culture medium of Bacillus sp. BS-214 and purified 105-fold with 3.4% yield by ammonium sulfate precipitation, gel filteration using Sephadex G-75 and DEAE-cellulose followed by gel filteration through Sephadex G-100. The molecular weight of the purified enzyme was estimated to be about 43 kDa on SDS-PAGE and by gel filtration, indicating that the enzyme is a monomer. the optium pH and temperature of the enzyme were 9.0 and 55$^{\circ}C$, respectively. the enzyme was stable at 60$^{\circ}C$ for 30min and in a pH range from 7.5 to 10.5 for 12 h ant 4$^{\circ}C$. The enzyme activity was highly enhanced by Ca2+, and also K+, Li+ and Na+showed a positive effect, while stongly inhibited by Zn2+ and Hg2+.

• 생명과학회지
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• 제12권4호
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• pp.490-495
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• 2002

병원성 미생물인 Vibrio fluvialis로부터 hemolysin을 분리정제 하였다. 즉, hemolysin의 정제는 균 배양액을 황산암모늄, ion exchange chromatography를 행하였으며 SDS-PAGE를 통해 그 단백질의 크기가 약 79kDa임을 알게되었다. 정제된 hemolysin (VFH)는 35$^{\circ}C$에서 최적활성을 나타내었고, 4$0^{\circ}C$에서는 그 활성이 감소되었다. RTG-2 어류유래 세포주에 hemolysin (VFH)의 처리로 세포독성을 측정한 결과 50$\mu\textrm{g}$의 VFH가 약 80%의 cell line을 사멸시켰다. 또한 현미경을 통한 관찰에서도 세포의 형태변화를 관찰할 수 있었다.

• Journal of Microbiology
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• 제33권2호
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• pp.115-119
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• 1995

The alkaline protease of Xanthomonas sp. YL-37 has been purified, and the properties of the enzyme investigated. The alkaline protease of Xanthomonas sp. YL-37 was purified form crude enzyme by ammonium sulfate fractionation, CM-cellulose ion exchange chromatography, and Sephadex G-100 gel filtration. Through the series of chromatographies, the enzyme was purified to homogenecity with specific activity of 4.23 fold higher than that of the crude broth. The molecular weight of the purified protease has been estimated to be 62 KDa on SDS-polyacrylamide gel electrophoresis. The optimal pH and temperature for alkaline protease activity were 11.0 and 50.deg.C, respectively. The enzyme was stable between pH 5.0 and 10.0 and up to 50.deg.C. Enzyme activity was lost up to 50% on heating at 70.deg.C for 30 minutes. The activity of alkaline protease was inhibited by Cu$\^$2+/, Zn$\^$2+/, Hg$\^$2+/, PMSF, and activated by Mn$\^$2+/ and Ca$\^$2+/. The $K_{m}$ value for casein as a substrate was 4.0 mg/ml.

• 미생물학회지
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• 제26권2호
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• pp.73-81
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• 1988

$\alpha$-Amylase (EC.3.2.1.1) of Neurospora crassa (ATCC9279) was cloned in E. coli HB101 using shotgun method, and the enzymes isolated from both N. crassa and E. coli were compared. Chromosomal DNA isolated from the spores of N. crassa was partially digested with PstI restriction endonuclease and rejoined to pBR322 which had been digested with the same enzyme. The resulting recombinant DNA were introduced into E. coli HB101 which had competancy by treating with $CaCl_{2}$. As the result, about 8000 colonies which showed tetracycline resistance were selected and two of the colonies which had 13.5Kb recombinant plasmid exhibit starch degrading activity on starch-containing plate when treated with D-cycloserine. $\alpha$-Amylases from both N.crassa and E. coli were isolated by using ammonium sulfate precipitation, DEAE-cellulose ion exchange column chromatography and Bio-Gel P150 gel foltration column. As the result, about 81.3 fold and 5.6 fold purifications in specific activities were obtained respectively, and specific activities of the gel filtrates were 6.1u/mg and 85u/mg respectively. The properties of both enzymes were compared and they showed quite the similar patterns in optimal temperature, optimal pH and had same molecular weight about 100,000 daltons on gel filtration method. Optimal temperatures for both enzymes were $70^{\circ}C$ and optimal pH were about 6 and 10.

• BMB Reports
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• 제29권2호
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• pp.116-121
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• 1996

In earlier studies, the genes encoding Escherichia coli thioredoxin and Corynebacterium nephridii thioredoxin C-3 were fused via a common restriction site in the nucleotide sequence coding for the active site of the proteins to generate two chimeric thioredoxins, designated E-C3 (N to C-terminal) and C3-E. The hybrid thioredoxins were overexpressed in E. coli from the cloned chimeric thioredoxin genes by a T7 promoter/polymerase system. To investigate the structure-function relationship of thioredoxin, we purified the E-C3 hybrid thioredoxin through ammonium sulfate fractionation, DEAE-cellulose chromatography, and Sephadex G-50 gel filtration. Its purity was examined on SDS-polyacrylamide gel electrophoresis and the molecular weight of the purified E-C3 hybrid thioredoxin was estimated to be 12,000. On native polyacrylamide gels, the purified E-C3 hybrid thioredoxin shows a much lower mobility than E. coli thioredoxin. E-C3 hybrid thioredoxin exhibits a 40-fold lower catalytic efficiency with E. coli thioredoxin reductase than E. coli thioredoxin. It was shown to catalyze the reduction of insulin disulfide by dithiothreitol. The purified E-C3 hybrid thioredoxin was also characterized in other aspects.

• 한국균학회지
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• 제14권2호
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• pp.101-107
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• 1986

The properties of carboxyl proteinase which was contained in Poria cocos (Schw.) Wolf were investigated by means of the purification with 0.65 ammonium sulfate saturation, DEAE cellulose and Sephadex G-75 gel filtration. This enzyme was found to hydrolyze only peptide bond between glutamyl-L-tyrosine of carbobenzoxy-L-glutamyl-L-tyrosine among the synthetic substrates of carbobenzoxy-L-glutamyl-L-tyrosine, hippuryl- L-phenylalanine and hippuryl-L-arginine. This enzyme was inhibited by $Zn^{+2},\;Fe^{+2},\;Ca^{+2},\;CN^{-1},\;P_2O_7^{-4}$ ions, but stimulated by $Hg^{+2}$ ion. Also, this enzyme was inhibited by organic compounds such as L-lysine, L-phenylalanine, hippuryl-L-phenylalanine, diazoacetyl-DL-norleucine methyl ester (DAN) and 1,2-epoxy-3-(P-nitrophenoxy)propane(EPNP). In particular, the activity was inhibited by L-lysine till 20 minutes of preincubation time rapidly, and by DAN in the presence of $Cu^{+2}$ ion more rapidly after 30 minutes than DAN in the absence of $Cu^{+2}$ ion. L-Lysine was found to be a competitive inhibitor and its $K_i$ value was determined to be 0.12 mmole by Dixon plot.

• Journal of Pharmaceutical Investigation
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• 제31권2호
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• pp.101-106
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• 2001

Alginate-chitosan (anion-cationic polymeric complex) was prepared to control the release rate of silver sulfadiazine (AgSD). Na-alginate (2%) solution containing AgSD was gelled in $CaCl_2$ solution. The gel beads formed were immediately encapsulated with chitosan (CS). The gel matrix and membrane were then reinforced with chondroitin-6-sulfate (Ch6S). Release rate of AgSD from the gel matrix was investigated by placing alginate beads in the sac of cellulose membrane simmered in HEPES-buffer solution. The concentration of AgSD released was analyzed by UV at 264 nm. Incorporation capacity of AgSD in Ca-alginate gel was more than 90%. Alginate-Ch6S-CS could control the release rate of AgSD. The amount of AgSD release was dependent on the AgSD loading dose. Incorporation of tripolyphosphate (polyanionic crosslinker) onto the alginate-Ch6S-CS bead increased the release rate of AgSD. Collagen-coating had no influence on the AgSD release rate. Alginate-Ch6S-CS beads with a sufficiently high AgSD encapsulation were capable of controlling the release of the drug over 10 days. In summary, alginate-Ch6S-CS beads could be used as a sustained delivery for AgSD and provide local targeting with low silver toxicity and patient discomfort.

• Journal of Nutrition and Health
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• 제16권2호
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• pp.115-124
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• 1983

The biochemical nature of the protein constituents of six year old fresh Panax ginseng root was studied. Total protein constituents were extracted with phosphate buffer of pH 7.4, ionic strength of 0.1 and fractionated by ultrafiltration using four different membranes which cut down the materials of molecular weight of 500, 1,000, 5,000 and 10,000, respectively. Each fraction was subjected to ion exchange chromatography using DEAE - cellulose to isolate component proteins. The protein fraction larger than molecular weight of 10,000 was refractionated by the method of ammonium sulfate precipitation. The electrophoresis of the refractionated protein constituents was performed. The amino acid composition of the protein constituents was determined by gas- liquid chromatography. From the results, it could be summarized that eleven different protein constituents smaller than molecular weight of 10,000 were isolated from the fresh Panax ginseng root. At least eleven different protein constituents larger than molecular weight of 10,000 were identified from the electrophoretic patterns. These protein costituents seem to be compounded of all or some of five different subunits.