• Journal of Microbiology and Biotechnology
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• 제18권4호
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• pp.616-623
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• 2008

In spite of an increasing interest in fucoidans as biologically active compounds, no convenient commercial sources with fucoidanase activity are yet available. A marine bacterial strain that showed confluent growth on a minimal medium containing fucoidan, prepared from Korean Undaria pinnatifida sporophylls, as the sole carbon source was isolated and identified based on a 16S rDNA sequence analysis as a strain of Sphingomonas paucimobilis, and named Sphingomonas paucimobilis PF-1. The strain depolymerized fucoidan into more than 7 distinct low-molecular-mass fucose-containing oligosaccharides, ranging from 305 to 3,749 Da. The enzyme activity was shown to be associated with the whole cell, suggesting the possibility of a surface display of the enzyme. However, a whole-cell enzyme preparation neither released the monomer L-fucose from the fucoidan nor hydrolyzed the chromogenic substrate p-nitrophenyl-${\alpha}$-L-fucoside, indicating that the enzyme may be an endo-acting fucoidanase rather than an ${\alpha}$-L-fucosidase. Therefore, this would appear to be the first report on fucoidanolytic activity by a Sphingomonas species and also the first report on the enzymatic degradation of the Korean Undaria pinnatifida sporophyll fucoidan. Moreover, this enzyme activity may be very useful for structural analyses of fucose-containing polysaccharides and the production of bioactive fucooligosaccharides.

• Biomolecules & Therapeutics
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• 제16권2호
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• pp.100-105
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• 2008

The sphingolipid metabolites act as lipid mediator for cell proliferation and apoptosis in mammalian cells. In bacteria, sphingolipid metabolism remains unknown. The purpose of this study was to investigate whether sphingolipid metabolism is potential target for fumonisin $B_1$($FB_1$) and desipramine in Sphingomonas chungbukensis, Gram-negative bacteria, by comparing the intracellular contents of bacterial sphingolipids with ones of HIT-T15 ${\beta}$-cells, hamster pancreatic cells. The concentrations of ceramide and dihydroceramide were 18.0 ${\pm}$ 12.0 and 0.025 ${\pm}$ 0.018 nmol/mg protein, respectively, in HIT-T15 cells. However, the concentrations of ceramide and dihydroceramide in the bacterial culture were 2.0 ${\pm}$ 1.2 and 10.6 ${\pm}$ 5.5 nmol/mg protein, respectively. $FB_1$ decreased the level of ceramide from 18.0 to 3.8 nmol/mg protein in HIT-T15 ${\beta}$-cells. However, dihydroceramide content in $FB_1$-treated HIT-T15 cells was slightly decreased compared with the control culture. When S. chungbukensis was treated with either $FB_1$ or desipramine, dihydroceramide level was increased by 5- and 4-fold, respectively, compared with the control bacteria. These results indicate that $FB_1$ and desipramine may act as an activator in bacterial sphingolipid biosynthetic pathway, and bacterial sphingolipid metabolism pathway appears to be different from the pathway of mammalian cells.

• Journal of Microbiology and Biotechnology
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• 제14권6호
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• pp.1134-1141
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• 2004

A bacterial strain capable of hydrolyzing sulfate ester bonds in p-nitrophenyl sulfate and agar was isolated from the Southeast coast of Korea. The isolated strain (AS6330) is aerobic, Gram-negative, rod-shaped, and motile. Octadecanoic acid was the major cellular fatty acid in the isolate. An almost complete 16S rDNA sequence of the isolate was determined and the sequence similarity of the 16S rDNA with those of known Sphingomonas spp. was found to be at most $96.4\%$, implying that the isolate was a new Sphingomonas species. The organism was grown optimally at NaCl concentration of $1.5-3.5\%$. Optimum culture conditions were determined to be $30^{\circ}C$ and pH 7.0 for 48 h fermentation using a laboratory fermentor under constant culture conditions. Partially purified arylsulfatase through Q-Sepharose and phenyl­Sepharose chromatographies catalyzed hydrolysis of sulfate ester bonds in agar, and $97\%$ of sulfates in agar were removed after 4 h reaction at $45^{\circ}C$ and pH 7.0. The arylsulfatase from the isolated bacterium might be useful for the removal of sulfate groups in agar.

• Journal of Microbiology and Biotechnology
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• 제21권5호
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• pp.494-502
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• 2011

Coenzyme $Q_{10}$ ($CoQ_{10}$) is a widely used supplement in heart diseases treatment or antioxidative dietary. The microbial production of $CoQ_{10}$ was enhanced by addition of solanesol and novel precursors recovered from waste tobacco. The novel precursors were separated by silica gel and identified as ${\alpha}$-linolenic acid (LNA) and butylated hydroxytoluene (BHT) based on the effect on $CoQ_{10}$ production and GC-MS. The effects of novel precursors on $CoQ_{10}$ production by Sphingomonas sp. ZUTE03 were further evaluated in a two-phase conversion system. The precursor's combination of solanesol (70 mg/l) with BHT (30 mg/l) showed the best effect on the improvement of $CoQ_{10}$ yield. A maximal $CoQ_{10}$ productivity (9.5 mg $l^{-1}$ $h^{-1}$) was achieved after 8 h conversion, with a molar conversion rate of 92.6% and 92.4% on BHT and solanesol, respectively. The novel precursors, BHT and LNA in crude extracts from waste tobacco leaves, might become potential candidates for application in the industrial production of $CoQ_{10}$ by microbes.

• KSBB Journal
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• 제21권6호
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• pp.428-432
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• 2006

본 연구는 미생물이 오염물 분해를 위해 황산염이 필요한 동전기 생물학적복원에서 황산염원의 종류가 제거효율에 미치는 영향을 조사하였다. 대표오염물과 적용 미생물은 각각 phenanthrene과 Sphingomonas sp. 3Y였다. Magnesium sulfate를 이용했을 때 Mg이온은 음극에서 전기적으로 생성되는 수산화이온과 결합하여 전해질 pH를 크게 감소시키고 미생물활성을 저해하는 현상이 관찰되었다. 따라서 ammonium sulfate와 disodium sulfate를 이용했을 때 전해질 및 토양 pH가 중성영역으로 유지되었으며 미생물 활성도 높게 유지되었다. 하지만 전자의 경우 ammonium이 미생물의 오염물 분해를 지연시켜 제거효율이 오히려 magnesium sulfate의 21.8%보다 12.0%로 더 낮게 나타났으며, 반면 후자의 경우 27.2%로 제거효율이 증가된 것을 관찰할 수 있었다. 이와 같은 제거효율의 차이는 장기간의 처리에서는 더욱 두드러질 것으로 예상된다.

• Journal of Microbiology and Biotechnology
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• 제23권4호
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• pp.473-482
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• 2013

The desulfurization ability of Sphingomonas subarctica T7b was evaluated using resting and immobilized cells with dibenzothiophene (DBT), alkyl DBTs, and commercial light gas oil (LGO) as the substrates. The resting cells of S. subarctica T7b degraded 239.2 mg of the initial 250 mg of DBT/l (1.36 mM) within 24 h at $27^{\circ}C$, while 127.5 mg of 2-hydroxybiphenyl (2-HBP)/l (0.75 mM) was formed, representing a 55% conversion of the DBT. The DBT desulfurization activity was significantly affected by the aqueous-to-oil phase ratio. In addition, the resting cells of S. subarctica T7b were able to desulfurize alkyl DBTs with long alkyl chains, although the desulfurization rate decreased with an increase in the total carbon number of the alkylated DBTs. LGO with a total sulfur content of 280 mg/l was desulfurized to 152 mg/l after 24 h of reaction. Cells immobilized by entrapment with polyvinyl alcohol (PVA) exhibited a high DBT desulfurization activity, including repeated use for more than 8 batch cycles without loss of biodesulfurization activity. The stability of the immobilized cells was better than that of the resting cells at different initial pHs, higher temperatures, and for DBT biodesulfurization in successive degradation cycles. The immobilized cells were also easily separated from the oil and water phases, giving this method great potential for oil biodesulfurization.

• Bulletin of the Korean Chemical Society
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• 제30권6호
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• pp.1360-1364
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• 2009

The bacterium Sphingomonas chungbukensis DJ77 produces the extracellular polysaccharide gellan in high yield. Gellan produced by this bacterium is widely used as a gelling agent, and the enzyme UDP-glucose pyrophosphorylase (UGP) is thought to play a key role in the gellan biosynthetic pathway. The UGP gene has been successfully cloned and over-expressed in E. coli. The expressed enzyme was purified with a molecular weight of approximately 32 kDa, as determined by a SDS-polyacrylamide gel, but the enzyme appears as ca. 63 kDa on a native gel, suggesting that the enzyme is present in a homodimer. Kinetic analysis of UDP-glucose for UGP indicates $K_m$ = 1.14 mM and $V_{max}$ = 10.09 mM/min/mg at pH 8.0, which was determined to be the optimal pH for UGP catalytic activity. Amino acid sequence alignment against other bacteria suggests that the UGP contains two conserved domains: An activator binding site and a glucose-1-phosphate binding site. Site-directed mutagenesis of Lys194, located within the glucose-1-phosphate binding site, indicates that substitution of the charge-reversible residue Asp for Lys194 dramatically impairs the UGP activity, supporting the hypothesis that Lys194 plays a critical role in the catalysis.

• Bulletin of the Korean Chemical Society
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• 제30권7호
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• pp.1547-1552
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• 2009

Sphingomonas chungbukensis DJ77 has the ability to produce large quantities of an extracellular polysaccharide that can be used as a gelling agent in the food and pharmaceutical industries. We identified, cloned and expressed the UDP-glucose dehydrogenase gene of S. chungbukensis DJ77, and characterized the resulting protein. The purified UDP-glucose dehydrogenase (UGDH), which catalyzes the reversible conversion of UDP-glucose to UDPglucuronic acid, formed a homodimer and the mass of the monomer was estimated to be 46 kDa. Kinetic analysis at the optimal pH of 8.5 indicated that the $K_m\;and\;V_{max}$ for UDP-glucose were 0.18 mM and 1.59 mM/min/mg, respectively. Inhibition assays showed that UDP-glucuronic acid strongly inhibits UGDH. Site-directed mutagenesis was performed on Gly9, Gly12 Thr127, Cys264, and Lys267. Substitutions of Cys264 with Ala and of Lys267 with Asp resulted in complete loss of enzymatic activity, suggesting that Cys264 and Lys267 are essential for the catalytic activity of UGDH.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XIII)
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• pp.775-779
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• 2003

충남 태안소재 순천향대학교 해양연구소의 해양수산연구소에서 probiotics 개발을 궁극적인 목표로 하여 대하양식장에서 대하의 효율적 생산을 위한 생육환경을 조사하였다. 대하의 생장에 영향을 미치는 용존산소, 수체온도, pH, 염도, $NH_4-N$, $NO_2-N$, $NO_3-N$, 클로로필 a를 포함하는 몇가지 환경요인들을 조사하였다. 수 표본에 용존되어 있는 $NH_4-N$, $NO_2-N$, 그리고 $NO_3-N$의 농도는 각각 0.024-0.034 mg/L, 0.03-0.02, 0.004-0.009 mg/L였다. 클로로필 a는 0.002-0.018 $ug/m^3$ 범위에 있었다. 양식장에서 채취한 수 표본에서 다른 세균들의 분포를 알아보기 위하여 marine agar plates에서 분리 계수하였다. 총세균수는 대략 $6.5\;{\times}\;10^4$까지 증가하였다. BIOLOG 시험들 통하여 13가지의 분리 세균들은 Corynebacterium nirilophilus, Clavibacter agropyri, Sphingomonas adhaesiva, Brevundimonas vesicularis, Vibrio parahaemolyticus, Pseudomonas bathycetes, Vibrio tubiashii, Sphingomonas macrogoltabidus, Rhodococcus, rhodochrous, Burkholderia glumae, Corynebacterium urealyticus, Rhodococcus fascians, Psychrobacter immobilis로 각각 동정되었다. 향후 연구는 대하의 효율적인 생산을 위한 정보를 제공하기 위한 병독성이나 환경과 probiotics의 연관성을 규명에 집중 될 것이다.

• 미생물학회지
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• 제41권3호
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• pp.195-200
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• 2005

Comamonas sp. strain DJ-12의 pmcABCDEFT 유전자군은 protocatechuate (PCA)의 분해과정에 관여하는 PCA 4,5-dioxygenase, 4-carboxy-2hydroxymuconic semialdehyde (CHMS) dehydrogenase, 2-pyrone04,5-dicarboxylate(PDC) hydrolase, 4-oxalomesaconate (OMA) hydratase, 그리고 4-oxalocitramalate (OCM) aldolase 등의 효소들을 생산하는 유전자들과 transporter의 역학을 하는 유전자로 각각 확인되었다. 이 유전자군은 Comamonas sp. strain DJ-12의 chromosomal DNA로부터 얻은 PCR 산물들을 T-vector에 ligation하여 재조합 플라스미드 pMT1, pMT2, pMT3, pMT4, pMT5, pMT6, pMT7, pMT8, pMT9, pMT10을 제조하였다. 이들 재조합 플라스미드의 염기서열을 분석한 결과 PCA 4,5-dioxygenase 유전자는 alpha(pmcA)와 beta(pmcB) 두 개의 subunit으로 구성 되어있으며, 각각 450 bp와 870 bp이었다. CHMS dehydrogenase 유전자(pmcC)는 960 bp, PDC hydrolase 유전자(pmcD)는 918 bp이였으며, OMA hydratase 유전자(pmcE)는 1029 bp, OCM aldolase 유전자 (pmcF)는 689 bp, 그리고 transporter 유전자(pmcT)는 1,398 bp이였다. 이들 pmc 유전자들은 pmcT-pmcE-pmcF-pmcD-pmcA-pmcB-pmcC의 순서로 배열되어 있었다. Comamonas sp. strain DJ-12의 pmcABCDEFT 유전자산물의 아미노산 서열을 분석한 결과, Comamonas testosteroni BR6020 및 Psedomonas ochraceae NG.J1와 $94{\~}98\%$의 높은 유사성을 보였고, 그 유전자들의 배열 순서도 동일하였다. 그러나 Sphingomonas paucimobilis SYK-6, Sphingomonas sp. LB126, 그리고 Arthrobacter keyser 12B와는 아미노산 서열이 $52{\~}74\%$의 유사성을 보였고, 그 유전자의 배열 구조도 상이하였다.