• 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.

• Applied Biological Chemistry
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• 제29권3호
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• pp.304-310
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• 1986

CL 배지에서 자란 A. calcoaceticus C10은 glucose dehydrogenase(GDH)와 cyclohexanol dehydrogenase(CDH)를 모두 생산하였다. A. calcoaceticus C10에 의한 사이클로헥사놀의 산화가 비특이적인 GDH에 의한 것인지를 알아보기 위하여 GDH와 CDH의 차이를 조사한 결과 GDH는 $NAD^+$와 $NADP^+$를 모두 조효소로 이 용하였으나 CDH는 $NAD^+$만을 조효소로 이용하였으며 $NADP^+$를 이용하지 못하였다. GDH는 LB 배지와 0.2%의 포도당 또는 사이클로헥사놀을 첨가한 LB 배지 및 CL 배지에서 모두 생산되었으나 CDH는 사이클로헥사놀을 첨가한 배지에서만 생산되었으며 7.5% polyacrylamide 젤 전기영동 결과 GDH와 CDH는 서로 다른 활성 밴드를 나타내었다. 이로써 GDH와 CDH는 서로 다른 것이며 사이클로헥사놀의 산화는 비특이적인 GDH에 의한 것이 아님을 확인하였다. LB 배지에서 A. calcoaceticus C10을 4시간 배양 후 사이클로헥사놀을 첨가할 경우 배양 24시간에 LB 배지에서보다 약 8배의 CDH 활성을 나타내었으며 생육도는 약 2배의 증가현상을 나타내었다. CDH는 사이클로헥사놀, 사이클로헥사논 cyclohexan-1,2-diol 및 cyclohexene oxide에 의해 유도되었으나 ${\varepsilon}-caprolactone$과 adipate에 의해서는 유도되지 않았다.

• 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.

• 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.

• Current Research on Agriculture and Life Sciences
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• 제5권
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• pp.68-74
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• 1987

A. calcoaceticus C10은 CL 배지에서 ${\varepsilon}$-caprolactone 및 succinate에 의해 생육에 아무 영향도 받지 않았으나 adipate와 xylose에 의해서 균의 생육도가 증가하였다. 특히 이 균이 탄소원으로 이용할 수 없는 포도당에 의해서도 생육도가 증가하였으며 6시간 배양 후 0.2%의 포도당을 첨가한 경우에는 16시간 후 CL 배지에서 생육한 것보다 약 2배의 생육도를 냐타내었다. A. calcoacelicus C10이 생산하는 cyclohexanol dehydrogenase(CDH)는 ${\varepsilon}$-caprolactone, succinate, xylose 및 포도당에 의해 이화물 억제를 받지 않았으나 사이클로헥사놀 대사경로의 최종생산물인 adipate에 의하여 생합성 억제를 받았다. CL 배지에 0.1%의 adipate를 첨가한 배지에서는 CDH가 다소 유도되었으나 0.2%의 adipate를 첨가한 배지에서는 CDH가 거의 유도되지 않아 0.5%의 adipate를 첨가한 것과 비슷한 억제효과를 나타내었다.

• 한국미생물·생명공학회지
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• 제13권1호
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• pp.71-77
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• 1985

오니(汚泥)로부터 사이클로헥사놀 이용능이 우수한 균(菌)을 분리하여 Acinetobacter calcoaceticus C-15로 동정하였다. A. calcoaceticus C-15의 배양을 위한 최적배지의 조성은 0.2% 사이클로헥사놀, 0.11% $NH_4Cl$, 0.05% $KH_2PO_4$, 0.2% $K_2HPO_4$, 0.02% $MgSO_4{\cdot}7H_2O$ 및 0.05% yeast extract이었다. 이 균(菌)의 생육최적(生育最適) pH는 7.2, 온도는 $33^{\circ}C$ 부근이었다. 사이클로헥사놀 및 사이클로헥사논을 기질로 했을 때 $33^{\circ}C$에서 본(本) 균(菌)의 증식속도는 각각 $0.27hr^{-1}$ 및 $0.15hr^{-1}$이었다. 통기배양에서 사이클로헥사놀에 대한 증식수율은 1.0이었다. 본(本) 균(菌)은 사이클로헥사놀과 사이클로헥사논 이외에 유기산으로 benzoate, adipate, acetate, citrate를 잘 이용하나 salicylate, phthalate, ${\beta}$-hydroxybenzoate, gluconate는 이용할 수 없었다. 알코올로는 에탄올, 1-부탄올, 1-펜탄올을 잘 이용하나 메탄올, 1-헥산올, m-크레졸, 글리세롤은 잘 이용하지 못했다. 또 본(本) 균(菌)은 크실로스 이외의 당류는 잘 이용할 수 없었다. 본(本) 균(菌)의 무세포추출액(無細胞抽出液)으로부터 사이클로헥사놀을 사이클로헥사논으로 전환시키는 사이클로헥사놀 dehydrogenase활성을 검출하였으며, 이 효소의 조효소는 $NAD^+$ 이었다.

• 대한의생명과학회지
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• 제11권4호
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• pp.509-515
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• 2005

This study was conducted to determine the kinetics of cyclohexane metabolites (the biomarker on cyclohexane exposure), the changes of hepatic cyclohexane metabolizing enzyme activities and the metabolites of cyclohexane in urine or serum. The rats were sacrificed at 2, 4, 8, 12 and 24 hr after administration of one dose of cyclohexane (1.56 g/kg body weight, i.p.). The metabolites of cyclohexane in urine were identified as cyclohexanol, cyclohexanone, trans-l,2-cyclohexanediol and 1,4-cyclohexanediol with cyclohexane metabolite being 124.00, 0.78, 23.28 and 2.75 (g/g of creatinine, $1\times10^{-3}$). Most of the cyclohexanol and trans-l,2-cyclohexanediol were determined to be in the form of $\beta-glucuronide$ conjugates, whereas cyclohexanone and 1 ,4-cyclohexanediol were found as free forms. In toxicokinetics of serum cyclohexane metabolites, cyclohexanol showed a rapid increase, reaching the plateau at 4 hr, after this time rapidly decreased throughout 24 hr. Changes of cyclohexanone also showed the similar pattern with cyclohexanol except somewhat lower concentration. Trans-l,2-cyclohexanediol, however, showed a gradual increase until 12 hr with the continued same levels throughout 24 hr. On the other hand, 1,4-cyclohexanediol was detected as trace levels at 4 and 12 hr, respectively. The administration of cyclohexane led to a significant increase of hepatic aniline hydroxylase activity from 2 to 8 hr. The activity of hepatic alcohol dehydrogenase showed a significant increase at 4 hr and then were recovered to the level of the control at 24 hr. On the other hand, there were no differences in liver weightlbody weight between the control and cyclohexane-treated animals. However, there were the changes of aniline hydroxylase and alcohol dehydrogenase activities on time-dependent pattern after cyclohexane treatment, which influence on the degree of cyclohexane metabolites both in blood and urine. These results suggest that differential determination of cyclohexane metabolites in urine and serum may be able to be as a biomarker of cyclohexane-exposure in the body. But in this fields further study is needed.

• 대한의생명과학회지
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• 제12권4호
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• pp.361-370
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• 2006

To evaluate an effect of pathological liver damage on the conjugation of cyclohexane metabolites, rats were pretreated with 50% $CCl_4$ dissolved in olive oil (0.1 ml/100 g body weight) 10 or 17 times intraperitoneally at intervals of every other day. On the basis of liver function, the animals pretreated with $CCl_4$ 10 times were identified as acutely liver damaged ones and the animals pretreated with $CCl_4$ 17 times were identified as severly liver damaged ones. To these liver damaged animals, cyclohexane (a single dose of 1.56 g/kg body weight, i.p.) was administered at 48 hr after the last injection of $CCl_4$. The rats were sacrificed at 4 or 8 hr after injection of cyclohexane. The cyclohexane metabolites, cyclohexanol (CH-ol), cyclohexane-1,2-diol (CH-1,2-diol), cyclohexane-1,4-diol (CH-1,4-diol), and their glucuronyl conjugates and cyclohexanone were detected in the urine of cyclohexane treated rats. The urinary concentration of cyclohexane metabolites was generally more increased in liver damaged animals than normal ones, and the increasing rate was higher in $CCl_4$ 17 times injected rats than 10 times injected ones. And liver damaged.ats, especially $CCl_4$ 17 times treated ones, had an enhanced ability of glucuronyl conjugation to CH-ol analogues compared with normal group. Futhermore, CH-1,2 and 1,4-diol were all conjugated with glucuronic acid in $CCl_4$ 17 times injected animals. On the other hand, the increasing rate of activities of hepatic cytochrome P450 dependent aniline hydroxylase, alcohol dehydrogenase and urine diphosphate glucuronyl transferase was higher in 17 times $CCl_4$-treated rats compared with normal and $CCl_4$ 10 times injected animals. Taken all together, it is assumed that an increased urinary excretion amount of cyclohexane metabolites in liver damaged rats might be caused by an increase in the activities of cyclohexane metabolizing enzymes. And enhanced conjugating ability of CH-ol in liver damaged animals and novel finding of conjugating form of CH-1,2 and 1,4-diol might be caused by increase in the activity of hepatic diphosphouridine glucuronyltransferase.