• 한국식품과학회지
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• 제21권1호
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• pp.154-163
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• 1989

L-Lysine 생산 균주인 B. flavum ATCC 21528, B. lactofermentum ATCC 21086 및 C. glutamicum 820을 이용하여 L-lysine 생산성이 우수한 균주를 분리할 목적으로 N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) $250{\mu}g/ml$ 농도로 처리한 다음 $300{\mu}g/ml$의 penicillin-G로 변이주를 농축하여 B. flavum $37-2(Hos^-,\;Kan^r,\;AEC^r)$, B. lactofermentum $6-2(Ile^-,\;Val^-,\;Str^r,\;ACE^r)$ 및 C. glutamicum $57-5(Met^-,\;Thr^-,\;Rif^r,\;AEC^r)$ 등의 변이주를 분리하였다. 분리된 변이주의 원형질체 형성은 lysozyme $500{\mu}g/ml$를 함유한 LS 용액으로 6시간 처리시 원형질체의 형성율은 97-99%였으며, 세포벽 재생율은 0.5M sodium succinate를 함유한 RCM에 0.7% osft agar를 중층하였을 때 33-37%를 나타내었다. 또 각각의 원형질체를 동량 혼합후 30% PEG 6,000에서 융합을 시킨 다음 분리된 융합주 BBFL 21, BCFG 37 및 BCLG 59는 $1.25{\times}10^{-6}{\sim}5.83{\times}10^{-7}$의 융합 빈도를 나타내었다. 분리된 융합주 BBFL 21은 LPB에서 $30^{\circ}C$, 72hr 배양하였을때 $411.1ng/ml{\cdot}hr$로 높은 L-lysine 생산성을 나타내었다. 발효 방법을 개선할 목적으로 융합주 BBFL 21를 sodium alginate, polyacrylamide, agar, x-carrageenan등으로 고정화 하여 회분식 발효를 행한 바 $413ng/ml{\cdot}hr$로 sodium alginate로 처리했을 때 가장 좋았다. 고정화 균체를 이용하여 관형 발효기를 제작하여 연속 발효를 행한 바 $416.7ng/ml{\cdot}hr$의 가장 높은 L-lysine 생산성을 나타내어 회분식 보다 높은 수율을 얻었다.

• 환경생물
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• 제37권4호
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• pp.741-748
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• 2019

본 연구에서는 바이오매스 폐기물인 Corynebacterium glutamium을 Alg를 이용한 고정화와 PEI 표면개질 과정을 통하여 유해 미세조류인 Microcystis aeruginosa를 제거할 수 있는 흡착소재인 PEI-AlgBF를 개발하였다. 녹조의 발생단계에 상관없이 PEI-AlgBF는 수계로부터 M. aeruginosa를 성공적으로 제거할 수 있었으며 유해조류 제거과정에서 M. aeruginosa 세포의 파괴를 유발하지 않았다. 흡착소재의 표면적은 M. aeruginosa의 제거효율에 매우 큰 영향을 주는 주요인자로 확인할 수 있었다. PEI-AlgBF를 사용한 M. aeruginosa 흡착/제거 방식은 기존 기술에 비하여 환경영향성이 낮기 때문에 보다 안전하고 안정적인 유해조류의 제어 방식이 될 것이다.

• Journal of Microbiology and Biotechnology
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• 제2권4호
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• pp.243-247
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• 1992

The thr operon of Escherichia coli TF427, an $\alpha$-amino-$\beta$-hydroxyvaleric acid (AHV)-resistant threonine overproducer, was cloned in a pBluescriptII $KS^＋$ plasmid by complementation of E. coli mutants. All clones contained a common 8.8 kb HindIII-generated DNA fragment and complemented the thrA, thrB, and thrC mutants by showing that these clones contained the whole thr operon. This thr operon was subcloned in the plasmid vectors pBR322, pUC18, and pECCG117, an E. coli/Corynebacterium glutamicum shuttle vector, to form recombinant plasmids pBTF11, pUTF25 and pGTF18, respectively. The subcloned thr operon was shown to be present in a 6.0 kb insert. A transformant of E. coli TF125 with pBTF11 showed an 8~11 fold higher aspartokinase I activity, and 15~20 fold higher L-threonine production than TF125, an AHV-sensitive methionine auxotroph. Also, it was found that the aspartokinase I activity of E. coli TF125 harboring pBTF11 was not inhibited by threonine and its synthesis was not repressed by threonine plus isoleucine.

• Journal of Microbiology and Biotechnology
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• 제23권10호
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• pp.1365-1371
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• 2013

The Streptomyces coelicolor wblA gene is known to play a negative role in both antibiotic biosynthesis and the expression of genes responding to oxidative stress. Recently, WhcA, a WblA ortholog protein, was confirmed to interact with dioxygenase-encoding SpiA ($\underline{s}$tress $\underline{p}$rotein $\underline{i}$nteracting with Whc$\underline{A}$) in Corynebacterium glutamicum. We describe here the identification of a SpiA ortholog SCO2553 protein ($SpiA_{sc}$) that interacts with WblA in S. coelicolor. Using heterologous expression in E. coli and in vitro pull-down assays, we show that WblA specifically binds $SpiA_{sc}$, and is influenced by oxidants such as diamide. These data indicate that the interaction between WblA and $SpiA_{sc}$ is not only specific but also modulated by the redox status of the cell. Moreover, a $spiA_{sc}$-disruption mutant exhibited a less sensitive response to the oxidative stress induced by diamide present in solid plate culture. Real-time RT-PCR analysis also showed that transcription levels of oxidative stress response genes (sodF, sodF2, and trxB) were higher in the $spiA_{sc}$-deletion mutant than in wild-type S. coelicolor. These results show that $SpiA_{sc}$ negatively regulates WblA during oxidative stress responses in S. coelicolor.

• 한국임상수의학회지
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• 제31권1호
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• pp.36-39
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• 2014

Many bacteria colonized in the horse semen affect quality of the sperm and some may cause infection in the mare reproductive tract and infertility of susceptible mare. This study was initiated to determine the prevalence of bacteria in testes of Jeju horses by determining rRNA sequence. The samples were swabed from the testes of nine Jeju horses (aged from 8 to 12 months after birth). Bacteria isolated from testes were identified by 16S rDNA sequencing. 1.6-kbp PCR products for 16S rRNA coding region were obtained using the universal primers. The PCR products were further purified and sequenced. Maximum similar species were found by BLAST search in the GenBank DNA database. BLAST results showed that the sequences were similar to those of Acinetobacter sp (A. schindleri, A. ursingii)., Bacillus cereus, Corynebacterium glutamicum, Escherichia coli, Gamma proteobacterium, Micrococcus luteus, Pseudomonas mendocina, Shigella sonnei, Sphingomonas sp., Staphylococcus sp (S. cohnii, S. saprophyticus, S. xylosus)., and Stenotrophomonas maltophilia. DNA sequences for 16S rRNA is provided useful informations for species identification of pathogenic microorganisms for the reproductive organs in horses.

• Journal of Microbiology and Biotechnology
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• 제8권3호
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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.

• Journal of Microbiology and Biotechnology
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• 제23권12호
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• pp.1726-1736
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• 2013

Biosynthesis of indole-3-acetic acid (IAA) via the indole-3-pyruvic acid pathway involves three kinds of enzymes; aminotransferase encoded by aspC, indole-3-pyruvic acid decarboxylase encoded by ipdC, and indole-3-acetic acid dehydrogenase encoded by iad1. The ipdC from Enterobacter cloacae ATCC 13047, aspC from Escherichia coli, and iad1 from Ustilago maydis were cloned and expressed under the control of the tac and sod promoters in E. coli. According to SDS-PAGE and enzyme activity, IpdC and Iad1 showed good expression under the control of $P_{tac}$, whereas AspC was efficiently expressed by $P_{sod}$ originating from Corynebacterium glutamicum. The activities of IpdC, AspC, and Iad1 from the crude extracts of recombinant E. coli Top 10 were 215.6, 5.7, and 272.1 nmol/min/mg-protein, respectively. The recombinant E. coli $DH5{\alpha}$ expressing IpdC, AspC, and Iad1 produced about 1.1 g/l of IAA and 0.13 g/l of tryptophol (TOL) after 48 h of cultivation in LB medium with 2 g/l tryptophan. To improve IAA production, a tnaA gene mediating indole formation from tryptophan was deleted. As a result, E. coli IAA68 with expression of the three genes produced 1.8 g/l of IAA, which is a 1.6-fold increase compared with wild-type $DH5{\alpha}$ harboring the same plasmids. Moreover, the complete conversion of tryptophan to IAA was achieved by E. coli IAA68. Finally, E. coli IAA68 produced 3.0 g/l of IAA after 24 h cultivation in LB medium supplemented with 4 g/l of tryptophan.

• 한국식품영양과학회지
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• 제24권5호
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• pp.779-784
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• 1995

5'-GMP(5'-Guanylic acid) 생산 융합균조 fusant RC102를 고정화하여 5'-GMP을 연속적으로 생산하고자 하였다. 균체 고정화용 담체로서 k-carrageenan, polyacrylamide, Ca-alginate 및 agar 등 4가지 담체에 고정화한 결과 최적 담체로서 3% k-carrageenan을 선정하였으며, 이담체에 고정화한 균체를 이용하여 5'-GMP 연속적으로 생산하였다. 고정화 균체에 의한 5'-GMP 생산의 최적 온도 및 pH은 $32^{\circ}C$, pH 8.0, 미량온소로서 $Mn^{2+}과\;Zn^{2+}$의 농도는 각각 $30\mu\textrm{g}/L,\;1{\times}10^{-6}%$이었다. $Mn^{2+}$이 풍부하고 값싼 CSL(Corn Steep Liquor)을 배지로서 사용하기 위해서 penicillin G와 D-cycloserine 및 POSEA(polyoxyethylene stearylamine) 농도 최적 농도는 각각 0.8unit/ml, 0.8unit/ml, 그리고 5mg/ml으로 나타났으며, 항생물질 보다 계면활성제가 효과적이었다. 이상의 최적 조건에서 고정화 fusant RC102는 15일간 안정되게 5'-GMP를 생산하였다.

• Journal of Microbiology and Biotechnology
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• 제9권5호
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• pp.582-588
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• 1999

A Brevibacterium lactofermentum gene coding for a glucose-specific permease of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) was cloned, by complementing an Escherichia coli mutation affecting a ptsG gene with the B. lactofermentum genomic library, and completely sequenced. The gene was identified as a ptsG, which enables an E. coli transformant to transport non-metabolizable glucose analogue 2-deoxyglucose (2DG). The ptsG gene of B. lactofermentum consists of an open reading frame of 2,025 nucleotides encoding a polypeptide of 674 amino acid residues and a TAA stop codon. The 3' flanking region contains two stem-loop structures which may be involved in transcriptional termination. The deduced amino acid sequence of the B. lactofermentum enzyme $II^{GIe}$ specific to glucose ($EII^{GIe}$) has a high homology with the Corynebacterium glutamicum enzyme $II^{Man}$ specific to glucose and mannose ($EII^{Man}$), and the Brevibacterium ammoniagenes enzyme $II^{GIc}$ specific to glucose ($EII^{GIc}$). The 171-amino-acid C-terminal sequence of the $EII^{Glc}$ is also similar to the Escherichia coli enzyme $IIA^{GIc}$ specific to glucose ($IIA^{GIc}$). It is interesting that the arrangement of the structural domains, IIBCA, of the B. lactofermentum $EII^{GIc}$ protein is identical to that of EIIs specific to sucrose or $\beta$-glucoside. Several in vivo complementation studies indicated that the B. lactofermentum $EII^{Glc}$ protein could replace both $EII^{ Glc}$ and $EIIA^{Glc}$ in an E. coli ptsG mutant or crr mutant, respectively.

• Journal of Dairy Science and Biotechnology
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• 제29권2호
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• pp.81-85
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• 2011

Recently, antimicrobial resistance of microorganisms has been a major concern of its relation to food safety and national health, therefore, customer's needs to organic animal food is still increasing. In this study, we reviewed the usage of antimicrobials in animal farms and antimicrobial resistance of microorganisms isolated from organic dairy farm environments. The isolates from dairy farms were Acinetobacter sp., A. lwoffi, A. johnsonii, A. towneri, Aerococcus viridans, Aeromonas media, A. veronii, Bacillus pseudofirmus, B. pumilus, B. licheniformis, Corynebacterium glutamicum, Escherichia coli, Enterococcus faecium, Lysinibacillus fusiformis, Paenibacillus illinoisensis, Staphylococcus epidermidis, S. hominis, Streptococcus equinus, S. lutetiensis, and Saccharomyces cerevisiae. Indicator microorganisms isolated from organic dairy farms were tested for susceptibility to 20 types of antimicrobials. E. coli (ATCC 25922) isolated from dairy farm fence showed resistance to 8 types of antimicrobials, such as oxacillin, penicillin, vancomycin, etc., and E. faecium isolated from feces showed resistance to 9 types of antimicrobials, such as cephalothin, oxacillin, streptomycin, etc., respectively. However, these results showed less antimicrobial resistance compare with customary dairy farm.