• Journal of Microbiology and Biotechnology
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• v.4 no.2
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• pp.102-107
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• 1994

The $\beta$-1,4-endoglucanase gene from Actinomyces sp. 40 was cloned into Escherichia coli DH5$\alpha$ with pUC19. Chromosomal DNA from Actinomyces sp. 40 was cleaved with the restriction enzyme Sau3AI and ligated into pUC19 for the transformation of Escherichia coli DH5$\alpha$. Positive clones of $\beta$-1,4-endoglucanase gene were detected as the clear zones on a medium supplemented with carboxymethylcellulose (CMC). This transformant possessed a single plasmid, designated pDS1, which contained the vector DNA and a 3.5 kilobase (kb) Sau3AI insertion fragment encoding endoglucanase. The size of the cloned fragment was reduced to 2.0 kb. The endoglucanase activity produced by the E. coli DH5$\alpha$ (pDS6) was higher than that of Actinomyces sp. 40 strain. The optimum pH and temperature of the cloned enzyme were pH 4.0$\sim$5.0 and 55$^{\circ}C$, respectively. The cloned enzyme was stable at 55$^{\circ}C$ or below and in buffer ranging from pH 4.0 to 7.0. The enzyme degraded CMC but did not degrade xylan, cellobiose, and methyl-umbelliferylcellobiopyranoside (MUC).

• Asian-Australasian Journal of Animal Sciences
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• v.7 no.3
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• pp.373-382
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• 1994

A bacterial strain No. 40, which produced extracellular endoglucanase, was isolated from the rumen of Korean native goals and identified to be a genus of Actinomyces sp. The optimum conditions for endoglucanase production in PY-CMC medium were initial pH of 7.0 and 4 days of cultivation at $39^{\circ}C$. When localization of endoglucanase activity of Actinomyces sp. was determined, 68% of the enzyme activity was found in the extracellular fraction, 11% of the activity was detected in the periplasmic space and the remaining activity was in the intracellular and cell-bound fractions. The maximal endoglucanase activity was observed at pH 5.0 and it was most s table at pH 5.0. The optimum temperature of this enzyme activity was $55^{\circ}C$, but enzyme activity was gradually lost at temperature above $60^{\circ}C$. The crude enzyme was activated by addition of 10 mM cysteine and 10 mM DTT. But it was inhibited by addition of 10 mM $Cu^{{+}{+}}$ and $Fe^{{+}{+}}$. This crude enzyme could digest carboxymethylcellulose (CMC), and degrade xylan, avicel, pNPG, and pNPC to a less extent.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.1
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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.

• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.202-205
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• 2000

A gene coding $endo-{\beta}$,-1, 4 glucanase from Actinomyces sp. KNG40 and phytase from Hansenula polymorpha were cloned into Esherichia coli JM101 by using E. coli/Lactobacillus shuttle vector pNZ3004 and pNZ123. The plasmid p3PS(1-4) and p123(1-4) have slpA promoter and slpA signal sequence. So, I constructed expression vectors, p3PS(1-4)Endo, phy and p123(1-4)Endo, phy. These constructed vector was transformed in target host Lactobacillus gasseri and reutri. These transformed host expressed endoglucanase and phytase as extracellular fraction. In the enzyme activity of the same vector, host L, gasseri was higher activity than L. reuteri. This indicates that L. gasseri recongnize promoter and signal sequence very well.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.32 no.5
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• pp.436-446
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• 2006

The purpose of this study was to isolate and identify the bacteria in chronic maxillary sinusitis (CMS) lesions from 3 patients and to determine the antimicrobial susceptibility of them against 10 antibiotics. One of them was odontogenic origin and the others were non-odontogenic origin. Pus samples were collected by needle aspiration from the lesions and examined by culture method. Bacterial culture was performed in three culture systems (anaerobic, CO2, and aerobic incubator). Identification of the bacteria was performed by 16S rRNA gene (16S rDNA) nucleotide sequencing method. To test the sensitivity of the bacteria isolated from the maxillary sinusitis lesions against seven antibiotics, penicillin G, amoxicillin, tetracycline, ciprofloxacin, cefuroxime, erythromycin, clindamycin, and vancomycin, minimum inhibitory concentration (MIC) was performed using broth dilution assay. Our data showed that enterobacteria such as Enterobacter aerogenes (30%), Klebsiella pneumoniae (25%), and Serratia marcescens (15%) were predominately isolated from the lesion of non-odontogenic CMS of senile patient (70 year old). Streptococcus spp. (40.3%), Actinomyces spp. (27.4%), P. nigrescens, M. micros, and P. anaerobius strains were isolated in the lesion of odontogenic CMS. In the lesion of non-odontogenic CMS, Streptococcus spp. (68.4%), Rothia spp. (13.2%), and Actinomyces sp. (10.5%) were isolated. The susceptibility pattern of 10 antibiotics was determined according to the host of the bacteria strains ratter than the kinds of bacterial species. Even though the number of CMS was limited as three, these results indicate that antibiotic susceptibility test must be accompanied with treatment of CMS. The combined treatment of two or more antibiotics is better than single antibiotic treatment in the presence of multidrug-resistant bacteria in the CMS lesions.