• Journal of Microbiology and Biotechnology
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• v.14 no.6
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• pp.1249-1255
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• 2004

This study has developed Corynebacterium ammoniagenes (c. ammoniagenes) purine gene transcriptional reporters (purF-lacZ and purE-lacZ) that function in Escherichia coli (E. coli) DH5a. After transformation of a C. ammoniagenes gDNA library into E. coli cells harboring either purF-lacZ or purE-lacZ, C. ammoniagenes clones were obtained that repress purF-lacZ and purE-lacZ gene expression. The potential purE and purF regulatory genes are homologous to the genes encoding transcription regulators, the regulatory subunit of RNA polymerase, and genes for purine nucleotide biosynthesis of various bacteria. The C. ammoniagenes purE-lacZ and purF-lacZ reporters were repressed by adenine and guanine within E. coli, indicating similarity in the regulatory mechanism of purine biosynthesis in C. ammoniagenes and E. coli. Gene regulation of pur-lacZ by adenine and guanine was partly abolished in cells expressing potential purine regulatory genes, indicating functionality of the purine gene regulators in repression of purE-lacZ and purF-lacZ. The purE-lacZ and purF-lacZ reporters can be used for the screening of genes involved in the regulation of the de novo synthesis of the purine nucleotides.

• Journal of Microbiology and Biotechnology
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• v.18 no.4
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• pp.639-647
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• 2008

The heat-inducible expression vectors for Corynebacterium glutamicum and C. ammoniagenes were constructed by using the ${\lambda}O_L1$ and the cryptic promoters, CJ1 and CJ4 that express genes constitutively in C. ammoniagenes. Although the promoters were isolated from C. ammoniagenes, CJ1 and CJ4 were also active in C. glutamicum. To construct vectors, the $O_L1$ from the ${\lambda}P_L$ promoter was isolated and fused to the CJ1 and CJ4 promoters by recombinant PCR. The resulting artificial promoters, CJ1O and CJ4O, which have one ${\lambda}O_L1$, and CJ1OX2, which has two successive ${\lambda}O_L1$, were fused to the green fluorescent protein (GFP) gene followed by subcloning into pCES208. The expression of GFP in the corynebacteria harboring the vectors was regulated successfully by the temperature-sensitive cI857 repressor. Among them, C. ammoniagenes harboring plasmid pCJ1OX2G containing GFP fused to CJ1OX2 showed more GFP than the other ones and the expression was tightly regulated by the repressor. To construct the generally applicable expression vector using the plasmid pCJ1OX2G, the His-tag, enterokinase (EK) moiety, and the MCS were inserted in front of the GFP gene. Using the vector, the expression of pyrR from C. glutamicum was tried by temperature shift-up. The results indicated that the constructed vectors (pCeHEMG) can be successfully used in the expression and regulation of foreign genes in corynebacteria.

• The Korean Journal of Food And Nutrition
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• v.6 no.3
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• pp.158-168
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• 1993

For the conversion of WAD to NADP, Immobilized Brevibacterium ammoniagenes cells with NAD kinase was coupled with ATP-generating system by acetate kinase. The membrane permeability of B. ammoniagenes was improved by toluene treatment of cells. The toluene treated B. ammoniagenes cells were immobilized for stable enzyme activity. Partially purified acetate kinase was used in the reaction system. The optimum conditions for the efficient conversion of UAD to WADP by energy-coupled system were investigated. B. ammoniagenes cells treated with toluene for the Improvement of membrane permeability showed 4.5 fold improved permeability in the conversion of NAD to NADP compared with Intact cells. 3% k-carrageenan as the immobilization matrix of B. ammoniagenes showed the best efficiency for the conversion of NAD to NADP The optimum conditions for the WAR to WARP conversion reaction coupled nth ATP-generating system were 10mM acetylphosphate, 5mM ADP 200mM inorganic phosphate, 10mM MgCl2, 250mg/ml Immobilized cells, 49.3mUnit/ml acetate kinase, pH 7.5 and 37$^{\circ}C$. Under the optimum conditions, 72% of 5mM(340mg/ml ) NAD was converted to UADP In 12 hours.

• Journal of Microbiology and Biotechnology
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• v.8 no.3
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• pp.214-221
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• 1998

A Brevibacterium ammoniagenes gene coding for glucose/mannose-specific enzyme II ($EII^{Glc}$) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) was cloned by complementing an Escherichia coli mutation affecting a ptsG gene, and the complete DNA nucleotide sequence was determined. The cloned gene was identified to be a ptsG, which enables the E. coli transportment to use glucose more efficiently than mannose as the sole carbon source in an M9 minimal medium. The ptsG gene of B. ammoniagenes consists of an open reading frame of 1,983 nucleotides putatively encoding a polypeptide of 661 amino acid residues and a TAA stop codon. The deduced amino acid sequence of the B. ammoniagenes $EII^{Glc}$ shows, at $46\%$, the highest degree of sequence similarity with the Corynebacterium glutamicum EII specific for both glucose and mannose. In addition, the $EII^{Glc}$ shares approximately $30\%$ sequence similarities with sucrose-specific and ${\beta}$-glucoside-specific EIIs of the several bacteria belonging to the glucose-PTS class. The 161-amino-acid C-terminal sequence of $EII^{Glc}$ is also similar to that of E. coli enzyme $IIA^{Glc}$, specific for glucose ($EIIA^{Glc}$). The B. ammoniagenes $EII^{Glc}$ consists of three domains; a hydrophobic region (EIIC) and two hydrophilic regions (EIIA, EIIB). The arrangement of structural domains, IIBCA, of the $EII^{Glc}$ is identical to those of EIIs specific for sucrose or ${\beta}$-glucoside. While the domain IIA was removed from the B. ammoniagenes $EII^{Glc}$ the remaining domains IIBC were found to restore the glucose and mannose-utilizing capacity of E. coli mutant lacking $EII^{Glc}$ activity with $EIIA^{Glc}$ of the E. coli mutant. $EII^{Glc}$ contains a histidine residue and a cysteine residue which are putative phosphorylation sites for the protein.

• Korean Journal of Microbiology
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• v.45 no.3
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• pp.233-238
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• 2009

The expression of Corynebacterium ammoniagenes purF was analyzed by utilizing a plasmid carrying a cat gene fused to the purF promoter region. Adenine and guanine repressed the expression of the purF gene by 20~30% but hypoxanthine did not exert such repressive effect. The expression purF was maximal at the late log phase and remained constant throughout the stationary phase. Promoter $P_{180}$ which was developed in C. glutamicum was also functional in C. ammoniagenes, achieving maximal activity at the late log phase. The promoter outperformed Escherichia coli $P_{tac}$ promoter by 40~50% level. DNA-affinity purification identified a protein which could bind to the promoter region of the purF gene. The protein showed high similarity to the CRP-family transcriptional regulator encoded by NCgl0120 in C. glutamicum. The size of the screened protein agreed with the expected protein size from the ORF NCgl0120. The corresponding gene in C. ammoniagenes encoded a 42 kDa polypeptide composed of 400 amino acids with expected pI of 4.9. The encoded protein showed 14.1% and 15.8% identity with E. coli and Bacillus subtilis PurR, respectively, suggesting that the isolated protein might be a novel type of regulatory protein involved in the regulation of purine metabolism.

• Microbiology and Biotechnology Letters
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• v.28 no.3
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• pp.147-151
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• 2000

School of Food Biotechnology, W0050ng University, San 7-6, Jayang~dong. Dong-ku1 Taejon 300-100, Korea - The nucleotide sequence of approximately 2.4 kb immediately adjacent to ptsG gene coding for the glucose permease of Brevibacterium ammoniagenes was detennined. A putative open reading frame (ORP) of 1.467 nucleotides encoding a polypeptide of 489 amino acid residues and a TAA stop codon was identified. The deduced amino acid sequence of the ORF product has a high homology with the 30S ribosomal protein S 1 of Mycohacteriwn tuberculosis (83 % ). M leprae (74%), Streptomyces coelicola (77%), and Escherichia coli (40%). suggesting that the predicted product of ORF is a ribosomal protein S 1. The ORF is located at a distance of 266 nucleotides upstream from ptsC gene with a same translational direction.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.24 no.2
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• pp.111-116
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• 1991

The effective p.eduction of 5'-GMP(5'-Guanylic acid) by enzymatic conversion of 5'-XMP(5'-Xanthyic acid) was investigated. The Iyophilized Brevibacterium ammoniagenes ATCC 19216 which were used as the XHP aminase source, was immobilized by entrapping in K-carrageenan, agar, polyacrylamide or Ca-alginate. $3\%$ K-carrageenan was selected as the most suitable matrix. In the production of 5'-GMP using the free cells of 3. ammoniagenes ATCC 19216, the optimum conditions were $42^{\circ}C$, PH 7.0, 100mg/ml glucose, 120mg/ml cell ,8mg/ml $MgSO_4\cdot7H_2O$, 5mg/ml POESA, 5mg/ml phytic acid. Under the conditions, $94.5\%$ of 5'-GMP was converted within 8 hours. In the production of 5'-GMP using the immobilized whole cells of B. ammoniagenes ATCC 19216, the optimum conditions were $37^{\circ}C$, pH 7.5, 50mg/ml glucose, 1mg/ml $KH_2PO_4$, 10mg/ml phytic acid, 60mg/ml cell, 8mg/ml $MgSO_4\;\cdot\;7H_2O$, 5mg/ml POESA. Under the conditions, $64.7\%$ of 5'-GMP was converted within 40 hours.

• Applied Biological Chemistry
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• v.24 no.2
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• pp.105-111
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• 1981

Growth responses of Brevibadterium ammoniagenes BA 17-2, which had been obtained by the treatment of several mutagens in our previous report, were investigated to select the preliminary optimal concentrations of phosphate, $Mg^{++}$, $Mn^{++}$ and thiamine for the production of 5'-XMP aminase. In this experiment it was shown that the concentration of phosphate in the medium has an important effect on the growth of microorganism. Using the medium containing 0.2% of $MgSO_4{\cdot}7H_2O$, 3mg/l of $MnSO_4{\cdot}H_2O$and $1\;mg/l$ of thiamine-HCl, the maximum cell mass was obtained at the concentration of 0.4% of $KH_2PO_4$ and $K_2HPO_4$, respectively. Above the concentration of these phosphates, cell growth was inhibited as the phosphate concentration increased to 1%, but the inhibition was overcome by the addition of 1% of $MgSO_4{\cdot}7H_2O$ and 3mg/l of thiamine-HCl. The 5'-XMP aminase activity was also influenced by the concentration of phosphate, $Mg^{++}$, $Mn^{++}$, and thiamine. In addition, the optimal culture pH and temperature for the enzyme activity were found to be 6.8 and $32^{\circ}C$, respectively.

• Korean Journal of Food Science and Technology
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• v.6 no.1
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• pp.6-11
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• 1974

Four new compounds; 2,2'-methylene-bis [3, 4, 6-trichloro ${{\beta}-(o-nitroanilino)$ propionoxy} benzene]; m.p. $200{\sim}202^{\circ}C,\;C_{31}H_{22}O_8N_4Cl_6$ 2,2'-methylene-bis [3, 4, 6-trichloro ${{\beta}-(p-nitroanilino)$ propionoxy} benzene]; m.p. $168-170^{\circ}C,\;C_{31}H_{22}O_8N_4Cl_6$ : 2,2'-Methylene-bis [3, 4, 6-trichloro ${{\beta}-(o-chloro-p-nitroanilino)$ propionoxy} benzene]; m.p. $170.5-172.5^{\circ}C,\;C_{31}H_{20}O_8N_4Cl_8$ : 2,2'-Methylene-bis [3, 4, 6-trichloro ${{\beta}-(c-methyl-p-nitroanilino)$ propionoxy} benzene]; m.p. $163-164^{\circ}C,\;C_{33}H_{26}O_8N_4Cl_6$-were synthesized by Mannich reaction from 2,2'-Methylene-bis (3, 4, 6-trichloroacetoxy benzene) and their antimicrobial activities against the microorganisms Staphylococcus aureus, Escherichia coli, Bacillus subtilis Natto, Brevibacterium ammoniagenes, Candida tropicalis, Rhodotorula glutinis, Pseudomonas ovalis, Aspergillus candidus Link, Aspergillus awamori Nakazawa. Aspergillus niger var. Tieghem, Aspergillus usami Sakakuchi, Penicillium notatum-were tested. 2,2'-methylene-bis [3, 4, 6-trichloro ${{\beta}-(o-nitroanilino)$ propionoxy} benzene] showed a strong antimicrobial activity against Bacilus subtilis Natto and Brevibacterium ammoniagenes.

• Korean Journal of Food Science and Technology
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• v.4 no.2
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• pp.72-76
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• 1972

Seven new hydroxyamine derivatives of 2,2'-methylene bis(3,4,6-trichloroacetoxy benzene) were synthesized by the Mannich reactions. 13 strains of microorganisms were tested for sensitivity to these derivatives by both paper disk method and tube dilution method. Of these compounds, -NHOH compound displays the most effective antimicrobial activity in vitro against Brevibacterium ammoniagenes, Staphylococcus aureus and Bacillus subtilis. Its minimal inhibitory concentration is $1.6{\mu}g/ml$ for Brevibacterium ammoniagenes, and $5{\mu}g/ml$ for Staphylococcus aureus and Bacillus subtilis.