• 한국미생물·생명공학회지
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• 제27권2호
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• pp.124-128
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• 1999

TK6 was able to produce NAD+ dependent cyclohexanol dehydrogenase(CDH). The production of CDH was increased rapidly at the logarithmic phase and maintained constantly after that. In order to investigate the inductive production of CDH by various substrates, the bacteria were grown in the media containing alicyclic hydrocarbons and various alcohols as a sole crabon souce. CDH was induced most actively by cyclohexanol. Cyclohexanone and cyclohexane-1,2-diol also induced remarkable amount of CDH but it was induced weakly by 1-propanol, 1-butanol, 1-pentanol, 1-hexanol, 2-propanol, and 2-methyl-1-propanol. The dehydrogenase of the bacteria grown in the media containing cyclohexanol were weakly active for various alcohols, but the dehydrogenase activity for cyclohexane-1,2-diol was twice as much as that for cyclohexanol. Activity staining on PAGE of the cell free extract of Rhodococcus sp. TK6 grown in the media containing cyclohexanol reveals at least sever isozyme bands of CDH and we nominated the four major activity bands as CDH I, II, III, and IV. CDH I was strongly induced by cyclohexanol, cyclohexane-1,2-diok, but its activity was specific to cyclohexane-1,2-diol and 1-pentanol. CDH IV was strongly induced by cyclohexanol and cyclohexane-1,2-diol, and its activity was very specific to cyclohexane-1,2-diol.

• Journal of Microbiology and Biotechnology
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• 제15권6호
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• pp.1189-1196
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• 2005

The cyclohexanol dehydrogenase (ChnA), produced by Rhodococcus sp. TK6, which is capable of growth on cyclohexanol as the sole carbon source, has been previously purified and characterized. However, the current study cloned the complete gene (chnA) for ChnA and its flanking regions using a combination of a polymerase chain reaction (PCR) based on the N-terminal amino acid sequence of the purified ChnA and plaque hybridization from a phage library of Rhodococcus sp. TK6. A sequence analysis of the 5,965-bp DNA fragment revealed five potential open reading frames (ORFs) designated as partial pte (phosphotriesterase), acs (acyl-CoA synthetase), scd (short chain dehydrogenase), stp (sugar transporter), and chnA (cyclohexanol dehydrogenase), respectively. The deduced amino acid sequence of the chnA gene exhibited a similarity of up to $53\%$ with members of the short-chain dehydrogenase/reductase (SDR) family. The chnA gene was expressed using the pET21 a(+) system in Escherichia coli. The activity of the expressed ChnA was then confirmed (13.6 U/mg of protein) and its properties investigated.

• 한국미생물·생명공학회지
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• 제27권2호
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• pp.107-112
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• 1999

A bacterium, which can utilize cyclohexanol as a sole source of carbon and energy, was isolated from sludge in sewage of Ulsan Industrial Complex for Petrochemicals, Korea and identified as Rhodococcus sp. TK6. The growth conditions of the bacteria were investigated in cyclohexanol containing media. The bacteria utilized cyclohexanol, cyclohexanone, cyclohexane-1,2=diol, cyclopentanol, cyclopentanone, and $\varepsilon$-caprolactone but not cyclohexane, cyclohexane-1,2-dione, and cyclooctanone. The bacteria were able to utilize alcohols such as ethanol, 1-propanol, 1-butanol, 1-pentanol, 1-hexanol, 2-methyl-1-propanol, 3-methyl-1-butanol, 2-propanol, and 2-butanol as well as cyclohexanol, organic acids such as adipate, propionate, butyrate, valerate, n-caproate, and 6-hydroxycaproate, and aromatic compounds such as phenol, salicylate, p-hydroxbenzoate, and benzoate as a sole source of carbon and energy. Cyclohexanone as a degradation product of cyclohexanol by Rhodococcus sp. TK6 was determined with gas chromatography.

• Journal of Microbiology and Biotechnology
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• 제16권4호
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• pp.511-518
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• 2006

A gene cluster for cyclohexanone oxidation was cloned from Rhodococcus sp. TK6, which is capable of growth on cyclohexanone as the sole carbon source. The 9,185-bp DNA sequence analysis revealed seven potential open reading frames (ORFs), designated as ssd-chnR-chnD-chnC-chnB-chnE-partial pcd. The chnBCDE genes encode enzymes for the four-step conversion of cyclohexanone to adipic acid, catalyzed by cyclohexanone monooxygenase (ChnB), $\varepsilon-caprolactone$ hydrolase (ChnC), 6-hydroxyhexanoate dehydrogenase (ChnD), and 6-oxohexanoate dehydrogenase (ChnE). Furthermore, the presence of a regulatory element in the downstream region of the chnD gene supports the notion that chnR is a putative regulatory gene. Among them, the activity of ChnB was confirmed and characterized, following their expression and purification in Escherichia coli harboring the modified chnB gene (chnB gene with 6 successive codons for His at the 3' terminus).

• Journal of Microbiology and Biotechnology
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• 제12권1호
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• pp.39-45
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• 2002

Activity staining on the native polyacrylamide gel electrophoresis (PAGE) of a cell-free extract of Rhodococcus sp. TK6, grown in media containing alcohols as the carbon source, revealed at least seven isozyme bands, which were identified as alcohol dehydrogenases that oxidize cyclohexanol to cyclohexanone. Among the alcohol dehydrogenases, cyclohexanol dehydrogenase II (CDH II), which is the major enzyme involved in the oxidation of cyclohexanol, was purified to homogeneity. The molecular mass of the CDH II was determined to be 60 kDa by gel filtration, while the molecular mass of each subunit was estimated to be 28 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The CDH II was unstable in acidic and basic pHs, and rapidly inactivated at temperatures above $40^{\circ}C$ . The CDH II activity was enhanced by the addition of divalent metal ions, like $Ba^2+\;and\;Mg^{2+}$. The purified enzyme catalyzed the oxidation of a broad range of alcohols, including cyclohexanol, trans-cyclohexane-1,2-diol, trans-cyclopentane-l,2-diol, cyclopentanol, and hexane-1,2-diol. The $K_m$ values of the CDH II for cyclohexanol, trans-cyclohexane-l,2-diol, cyclopentanol, trans-cyclopentane-l,2-diol, and hexane-l,2-diol were 1.7, 2.8, 14.2, 13.7, and 13.5 mM, respectively. The CDH II would appear to be a major alcohol dehydrogenase for the oxidation of cyclohexanol. The N-terminal sequence of the CDH II was determined to be TVAHVTGAARGIGRA. Furthermore, based on a comparison of the determined sequence with other short chain alcohol dehydrogenases, the purified CDH II was suggested to be a new enzyme.