• 한국미생물·생명공학회지
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• 제21권6호
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• pp.577-581
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• 1993

The amino acid threonine was produced from glycine and ethanol in a reaction mixture using cell free extract of the methylotrophic bacterium isolated from soil and identified as mellthylo-bacterium sp. KJ29. Although the isolate could grow on carbon source other than methanol, only the cell free extract from the cells grown on methanol produced threonine. Methanol dehydrogenase (MDH) activity was present only in the cells grown on methanol when compared to the cells grown on heterotrophic substrates.

• 약학회지
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• 제36권4호
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• pp.315-320
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• 1992

The amino acid threonine was produced from glycine and ethanol in a reaction mixture using resting cells of a newly isolated gram-negative methylotrophic bacterium, capable of growth on methanol. The isolate could utilize $C_1$ compounds and a variety of multicarbon substrates as sole carbon and energy source. To obtain cells of isolate with high threonine producing activity, we investigated optimum cultural conditions. Optimal growth was at the initial concentration of 0.5%(v/v) methanol, at $30^{\circ}C$ and pH 7.0. The growth was not affected by antibiotics inhibiting cell wall synthesis, but was completely suppressed by those inhibiting protein synthesis. The optimum reaction conditions from threonine production by resting cells of this strain were found.

• BMB Reports
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• 제38권6호
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• pp.695-702
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• 2005

The groESL bicistronic operon of a restricted facultative methylotrophic bacterium Methylovorus sp. strain SS1 DSM 11726 was cloned and characterized. It was found to consist of two ORFs encoding proteins with molecular masses of 11,395 and 57,396 daltons, which showed a high degree of homology to other bacterial GroES and GroEL proteins. The genes were clustered in the transcription order groES-groEL. Northern blot analyses suggested that the groESL operon is transcribed as a bicistronic 2.2-kb mRNA, the steady-state level of which was markedly increased by temperature elevation. Primer extension analysis demonstrated one potential transcription start site preceding the groESL operon, which is located 100bp upstream of the groES start codon. The transcription start site was preceded by a putative promoter region highly homologous to the consensus sequences of Escherichia coli ${\sigma}^{32}$-type heat shock promoter, which functioned under both normal and heat shock conditions in E. coli. Heat shock mRNA was maximally produced by Methylovorus sp. strain SS1 approximately 10min after increasing the temperature from 30 to $42^{\circ}C$. The groESL operon was also induced by hydrogen peroxide or salt shock.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2008년도 International Meeting of the Microbiological Society of Korea
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• pp.163-163
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• 2008

Methylophaga aminisulfidivorans $MP^T$, a restricted facultative marine methylotrophic bacterium, was able to utilize methanol as a sole carbon and energy source, and possessed a methanol dehydrogenase (MDH) that is a key enzyme in the process of methanol oxidation. During purification of MDH, three types of MDH (MDH I, II, and III) were obtained in the cell free extracts from $MP^T$ cells grown on methanol. When analyzed by SDS-PAGE and ESI-FT ICR MS, MDH I was confirmed to consist of two subunits and with molecular masses of ~66 and ~10 kDa, respectively, in a form of ${\alpha}_2{\beta}_2$. While MDH II and MDH III contained an additional ~30 kDa protein, designated ${\gamma}$, in a form of ${\alpha}_2{\beta}_2{\gamma}$ and ${\alpha}_2{\beta}_2{\gamma}_2$, respectively. MDH III showed 1.5.2.0 times higher activity than MDH II, while MDH I remained the lowest activity. Based on these observations and experimental data, it seems that the original MDH conformation is ${\alpha}_2{\beta}_2{\gamma}2$ within $MP^T$ growing on methanol, and subunit ${\gamma}$ keeps MDH in an active form, and/or makes MDH easily bind to the substrate, methanol.

• Journal of Microbiology and Biotechnology
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• 제12권5호
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• pp.819-825
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• 2002

Methanol dehydrogenase (MDH) is a key enzyme in the process of methanol oxidation in methylotrophic bacteria. However, information on MDH genes from genus Methylobacillus is limited. In this study, a 6.5-kb HindIII DNA fragment of Methylobacillus sp. SK-5 chromosomal DNA was isolated from the genomic library of the strain by using a degenerate oligonucleotide probe that was designed based on JV-terminal amino acid sequence of the MDH $\alpha$ subunit purified from the strain. Molecular analysis of the fragment revealed four tightly clustered genes (mxaFJGI) involved in the methanol oxidation. The first and fourth genes were very similar to mxaF (77% identity for nucleotides an 78% identity for amino acids) and mxaF (67% Identity for nucleotides and 68% Identity for amino acids) genes, respectively, from Methylovorus sp. SSI. Genes mxaF and mxaI encode $\alpha$ and $\beta$ subunits of MDH, respectively. The two subunits were identified from purified MDH from Methylobacillus sp. SK-5. A dendrogram constructed by comparison of amino acid sequences of MDH u subunits suggests that MxaF from Methylobacillus sp. SK-5 belongs to a subfamily cluster of MDH u subunits from $\beta$-subgroup Proteobacteria. The subfamily cluster is separated from the other subfamily that consists of $\beta$- and $\gamma$-subgroup Proteobacteria. This study provided information on mn genes from a methylotrophic bacterium in $\beta$-subgroup Proteobacteria, which would aid to better develop a gene probe to detect one-carbon metabolizing bacteria.

• 미생물학회지
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• 제34권4호
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• pp.212-218
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• 1998

세포외 다당류를 분비하는 새로운 methylotrophic bacteria를 분리하여 세균의 특징과 그 세균이 생산하는 세포외 다당류의 물리 화학적 특성을 조사하였다. 분리균주는 그람음성의 간균으로 편모가 없는 비운동성 세균이며, DNA의 G+C 함량은 53-56%이었고, plasmid를 가지고 있지 않았다. 메탄올과 메틸아민만을 기질로 이용하였으며, 탄소동화경로로는 ribulose monophosphate pathway를 이용하는 절대 methylotrophic bacteria이었다. 성장을 위한 최적온도와 pH는 각각 $35^{\circ}C$와 6.5이었고, 0.5%(v/v)의 메탄올이 포함된 배지에서 가장 빨리 성장하였다(세대시간=2.4시간). 분리균주는 절대 호기성 세균으로 질소원과 산소가 결핍된 조건에서 다량의 세포외 다당류를 분비하였다. 다당류 생산을 위한 최적온도와 pH는 각각 $30^{\circ}C$와 6.5이었고, 1.0%(v/v)의 메탄올이 포함된 배지에서 배지내의 탄소 대질소비가 57.4일 때 가장 많이 생산되었다. 정제된 다당류는 포도당과 galactose로 이루어져 있었다. 에탄올 처리전의 다당류는 낮은 pH에서 더 높은 점도를 보였고, 온도와 염류농도의 변화에도 비교적 안정하였다. 에탄올 처리후의 다당류는 xanthan gum보다 높은 점도를 나타내었고, pH, 온도, 염류농도의 변화에 대해 점도변화가 크지 않았다. 냉동건조된 다당류를 전자현미경을 이용하여 관찰하였을 때, 에탄올 처리전의 다당류는 얇은 막이 겹친 구조를 하고 있었고, 에탄올 처리후의 다당류는 굵은 섬유상의 모습을 띠고 있었다.

• Journal of Microbiology and Biotechnology
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• 제12권3호
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• pp.476-482
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• 2002

Methanol dehydrogenase (MDH) and c-type cytochromes from marine methanol-oxidizing bacterium, Methylophaga sp. MP, were purified and characterized. The native MDH had a molecular mass of 148 kDa and its isoelectric point was 5.5. Two c-type cytochromes, $c_L\;and\;c_H$, were found, and their isoelectric points were 3.4 and 8.0, respectively. The purified MDH had higher thermal stability than that of the other soil methylotrophic bacteria. The electron flow rate from MDH to cytochrome $c_L$was higher than that from MDH to cytochrome $c_H$, indicating that the physiological primary electron acceptor for MDH is cytochrome $c_L$. The electron transfer from MDH to phenazine ethosulfate (PES, artificial electron acceptor) in the two dye (PES/DCPIP)-linked assay system was not inhibited by NaCl, whereas the electron flow from MDH to cytochrome $c_L$ in the cytochrome/DCPIP-linked assay system was suppressed significantly by NaCl. Metal chelating agents such as EDTA showed the same effects on the MDH activity.

• Journal of Microbiology and Biotechnology
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• 제30권8호
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• pp.1261-1271
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• 2020

Cytochrome c_L (Cytc_L) is an essential protein in the process of methanol oxidation in methylotrophs. It receives an electron from the pyrroloquinoline quinone (PQQ) cofactor of methanol dehydrogenase (MDH) to produce formaldehyde. The direct electron transfer mechanism between Cytc_L and MDH remains unknown due to the lack of structural information. To help gain a better understanding of the mechanism, we determined the first crystal structure of heme c containing Cytc_L from the aquatic methylotrophic bacterium Methylophaga aminisulfidivorans MP^T at 2.13 Å resolution. The crystal structure of Ma-Cytc_L revealed its unique features compared to those of the terrestrial homologues. Apart from Fe in heme, three additional metal ion binding sites for Na⁺, Ca⁺, and Fe²⁺ were found, wherein the ions mostly formed coordination bonds with the amino acid residues on the loop (G93-Y111) that interacts with heme. Therefore, these ions seemed to enhance the stability of heme insertion by increasing the loop's steadiness. The basic N-terminal end, together with helix α4 and loop (G126 to Y136), contributed positive charge to the region. In contrast, the acidic C-terminal end provided a negatively charged surface, yielding several electrostatic contact points with partner proteins for electron transfer. These exceptional features of Ma-Cytc_L, along with the structural information of MDH, led us to hypothesize the need for an adapter protein bridging MDH to Cytc_L within appropriate proximity for electron transfer. With this knowledge in mind, the methanol oxidation complex reconstitution in vitro could be utilized to produce metabolic intermediates at the industry level.

• 한국미생물·생명공학회지
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• 제18권3호
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• pp.273-279
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• 1990

PHB 생산을 위하여 메탄올을 기질로 한 선별배지에서 토양, 하천수, 퇴비 등으로부터 분홍색 색소를 가지는 PHB 축적 facultative methylotroph를 분리하여, 균주의 특성을 검토하였다. 분리균주의 최적 생육조건과 PHB 축적을 위한 배양조건을 조사한 결과 균체의 생육은 메탄올 농도 1.0(v/v), 질소원인$ NH_4C$ 농도 1.0g/l, 즉 C/N ratio 13.2 일때 그리고 pH 7.0과'$30^{\circ}C$에서 가장 좋았으며, PHB는 C/N ratio가 50.8, 즉 메탄올 농도 1.0(v/v )$NH_4CL$ 0.26g/l 일때, 그리고 pH 6.0일 때 건조중량의 약 40까지 축적되었다. 고농도 메탄올에 의한 생육저해를 극복하기 위하여 기질을 간헐적으로 계속 공급해주는 fed-batch 배양을 시도한 결과 균체량은 14g/l, PHB 축척량은 5.5g/l까지 증가시킬 수 있었다. 생산된 PHB를 분리.정제하여 IR과 $^I H-NMR$로 구조를 분석한 결과 3-hydroxybutyric acid 의 homopolymer임이 확인되었다. 또한 균주의 pink-pigment를 추출하여 absorption spectrum를 조사하여 그 특성을 규명하였다.

• 미생물학회지
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• 제27권1호
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• pp.63-69
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• 1989

흙으로부터 에탄올을 이용하여 성장하는 분홍색 통성 에단올 자화세율을 분리하여 Methylobacteriu~η sp. strain SY 1이라 명명하였다. 이 세균은 그람음성세균으호 약간 굽은 간균이고 한쪽 끝에 달련 한개의 편모로 운동하였다. 세균의 군략은 매끈 하고 밝은 분홍맺을 띄우며 정성이 높았다. 이 세균이 지니는 DNA의 G+C 함량은 약 66%로 나타났다. 이 제균은 칠대호가 성으호 catalase와 oxidase 활성을 나타내었고, carotenoid 색소와 poly-$\beta$-hydroxy butyrate를 가지고 있었다. 이 세균은 또 한 세가지 종류의 큰 plasmid DNA (45,000, 38,500, 23,000)을 가졌으며, $30^{\circ}C$와 pH7.0에서 0.5%(v/v)의 에탄올을 이용 하여 빼른 성장을 하였다($t_{d}$=6.5시간), 이 세균은 메탄올은 울온 여러까지 종듀의 당, 유기산, 아미노산, 아민 및 알코올을 이용하여 성장할 수 있었고, 메탄올은 serine pathway를 통하여 이 세균의 세포 구성물질로 전환됨을 알 수 있었다.