• Journal of Microbiology and Biotechnology
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• 제26권12호
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• pp.2076-2086
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• 2016

Fungal cytochrome P450 (CYP) enzymes catalyze versatile monooxygenase reactions and play a major role in fungal adaptations owing to their essential roles in the production avoid metabolites critical for pathogenesis, detoxification of xenobiotics, and exploitation avoid substrates. Although fungal CYP-dependent biotransformation for the selective oxidation avoid organic compounds in yeast system is advantageous, it often suffers from a shortage avoid intracellular NADPH. In this study, we aimed to investigate the use of bacterial glucose dehydrogenase (GDH) for the intracellular electron regeneration of fungal CYP monooxygenase in a yeast reconstituted system. The benzoate hydroxylase FoCYP53A19 and its homologous redox partner FoCPR from Fusarium oxysporum were co-expressed with the BsGDH from Bacillus subtilis in Saccharomyces cerevisiae for heterologous expression and biotransformations. We attempted to optimize several bottlenecks concerning the efficiency of fungal CYP-mediated whole-cell-biotransformation to enhance the conversion. The catalytic performance of the intracellular NADPH regeneration system facilitated the hydroxylation of benzoic acid to 4-hydroxybenzoic acid with high conversion in the resting-cell reaction. The FoCYP53A19+FoCPR+BsGDH reconstituted system produced 0.47 mM 4-hydroxybenzoic acid (94% conversion) in the resting-cell biotransformations performed in 50 mM phosphate buffer (pH 6.0) containing 0.5 mM benzoic acid and 0.25% glucose for 24 h at $30^{\circ}C$. The "coupled-enzyme" system can certainly improve the overall performance of NADPH-dependent whole-cell biotransformations in a yeast system.

• 생명과학회지
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• 제8권6호
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• pp.695-701
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• 1998

한국산 꽃뱀(Natrix tigrina Lateralis)의 간 마이크로좀에 존재하는 mixed function oxidase system 구성 성분들의 함량과 P45O 의존성 monooxygenase의 활성도를 조사하고 이들을 흰쥐(Sp. D)의 것과 상호 비교하였다. 꽃뱀에서의 P45O, b5 함량 및 NADPH-cytochrome c reductase 활성도는 흰쥐에서 보다 낮았으며, 7-ethoxycoumarin O-deethylase와 benzphetamine N-demethylase 활성도 역시 흰쥐에서 보다 상당히 낮았다. 그러나 aryl hydrocarbon hydroxylase와 testosterone hydroxylase 활성도는 흰쥐와 비교할 때 거의 비슷하거나 오히려 높았다. Testosterone의 수산화 반응에 대한 선택특이성을 조사한 결과, 꽃 뱀은 7$\alpha$ 위치에서, 흰쥐는 6$\beta$ 위치에서 가장 높은 수산화 반응물을 생성했다. 그러나 testosterone의 C2와 C3 위치에서의 수산화 반응에 대한 선택특이성은 꽃뱀과 흰쥐에서 비슷하였다. Radioimmunoassay (RlA)를 실시하여 5종 (CYP2B, CYP1A1, CYP1A2, CYP3A 및 CYP2El)의 P45O 동위효소의 구성비를 비교한 결과, 꽃뱀에서는 CYP1A1/1A2가, 흰쥐에서는 CYP2El이 각각 비교적 많이 존재하였다. 부분정제한 P45O을 SDS-PAGE와 RIA로 분석한 결과, 꽃뱀의 간 마이크로좀에 존재하는 P45O중에는 흰쥐와는 다른 종류의 P45O 동위효소가 존재함을 시사하였다.

• 한국산업보건학회지
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• 제19권1호
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• pp.73-79
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• 2009

This study was undertaken to investigate the effects of single and combined exposure of toluene (T) and xylene (X) on the cytochrome-450(CYP)-mediated metabolizing capacity, induction of CYP isozymes and the excretion of their metabolites in urine. Animal were adults male Sprague-Dawley (SD) rats and divided into 4 groups such as control, T (treated with 63.7 mg/body kg), X (treated with 65.9 mg/body kg) and TX(T=X). Organic solvents was administrated by intraperitoneal injection for 3 days. The contents of protein and CYP in liver microsomes of control group were $16.48{\pm}0.56 mg/m{\ell}$ and $0.744{\pm}0.025$ nmol/mg protein, respectively, and they contents were significantly lower than in derived from treated groups (p<0.01). The activities of PROD and ${\rho}NPH$ were significantly higher in single treated groups than in control and combined group (TX). When Western immunoblotting were carried out with two monoclonal antibodies (MAb 1-98-1 and MAb 2-66-3) which were specific against CYP2B1/2 and CYP2E1, respectively, a strong signal corresponding to CYP2B1/2 was observed in microsomes obtained from rats treated with X and TX. The color density against CYP2E1 was slightly increased in T and TX groups compared with C and X groups. The amounts of urinary hippuric acid in T single treated group was $3.29{\pm}1.97$ g/g creatinine and TX combined group was $2.91{\pm}1.76$ g/g creatinine, but was not significant. However, amount of urinary methy hippuric acid in X single treated group ($1.62{\pm}0.72$ g/g creatinine) was significantly higher than TX combined group ($0.93{\pm} 0.63$ g/g creatinine)(p<0.01). These results suggested that CYP2E1 isozyme might be responsible for the metabolism of T, and CYP2B1/2 isozyme is for X. And also, difference of metabolites level between single and combined group may be speculated that the intermediates of T and X interacted each other in the process of their metabolite formation reaction.

• Preventive Nutrition and Food Science
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• 제2권4호
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• pp.333-337
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• 1997

Trans-cinnamic acid-4-hydroxylase(tC4H) is the first cytochrome P450-dependent monooxygenase of the phenylpropanoid pathway. The roots of avocado seedlings were wounded and examined to determine whether the tC4H would be activated in response to wounding and/or whether tC4H activity be modulated by the application of exogenous p-coumarate. At the specified length of times, the wounded and treated roots were either frozen in liquid nitrogen or used immediately to extract microsomal proteins. The microsomal proteins were subjected to immunoblot analysis using polyclonal antibodies against CYP73 of tC4H gene. In this study, tC4H was induced in wounded roots sealed in bags within 6 hours, and in low level({TEX}$10^{-8}${/TEX}M) of p-coumarate solution within 24 hours, whereas the olution without p-coumarate and high levels of p-coumarate solution repressed tC4H induction in wounded roots. These results indicate that tC4H is induced by wounding in the root of avocado, and is inhibited by the application of exogenous p-coumarate.

• BMB Reports
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• 제32권3호
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• pp.232-238
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• 1999

Sex differences in the induction of microsomal cytochrome P-450 (CYP) and the activities of several related enzymes of Sprague-Dawley rats treated with 1-bromopropane (1-BrP) were investigated. Male and female rats were exposed to 50, 300, and 1800 ppm of 1-BrP per kg body weight (6 h a day,S days a week, 8 weeks) by inhalation. The mean body weight of 1-BrP treated groups increased according to the day elapsed, but four and five weeks respectively after the start of the exposure, the mean body weight of male and female rats had significantly reduced in the group treated with 1800 ppm 1-BrP compared with the control group (p<0.01). While the relative weights of liver increased in both sexes, statistical significance in both sexes was found only in the group receiving 1800 ppm/kg of 1-BrP (p<0.01). The total contents of CYP, $b_5$, NADPH-P-450 reductase, NADH $b_5$ reductase, ethoxyresorufin-O-deethylase (EROD), pentoxyresorufin-O-dealkylase (PROD), and p-nitrophenol hydroxylase (pNPH) activities were examined for the possible effects of 1-BrP. No significant changes in the CYP and $b_5$ contents, NADPH-P-450 reuctase, NADH $b_5$ reductase, ethoxyresorufin-O-deethylase (EROD), and pentoxyresorufin- O-dealkylase (PROD) were observed between the control and treated groups. The activity of pNPH increased steadily with the increase in the concentration of 1-BrP in both sexes, but was significantly increased only in the 1800 ppm-treated group of male rats (p<0.05). When Western blottings were carried out with three monoclonal antibodies (MAb 1-7-1, MAb 2-66-3, and MAb 1-98-1) which were specific against CYP1A1/2, CYP2B1/2, and CYP2E1, respectively, a strong signal corresponding to CYP2E1 was observed in microsomes obtained from rats treated with 1-BrP. Glutathione S-transferase (GST) activity and the content of lipid peroxide significantly increased in the treated groups compared with the control group (p<0.05). These results suggest that 1-BrP can primarily induce CYP2E1 as the major form and that GST phase II enzymes play important roles in 1-BrP metabolism, showing sex-dependence in the metabolic mechanism of 1-BrP in the rat liver.