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

• 분석과학
• /
• 제15권5호
• /
• pp.482-487
• /
• 2002

Carteolol은 국제올림픽위원회 (IOC)에서 금지약물로 규정하고 있는 ${\beta}$-차단제 중의 하나이다. 본 실험에서는 GC/MS를 사용하여 사람의 소변으로부터 carteolol의 복용여부를 확인하기 위한 검출방법 및 대사와 배설에 대해서 고찰하였다. 이를 위하여 효소 가수분해방법과 산 가수분해방법을 비교하여 보았는데 효소가수분해방법이 더 좋은 결과를 얻을 수 있었다. 효소 가수분해방법을 이용하여 실험한 결과 RSD 10%내외의 정밀도와 $0.05{\mu}g/m{\ell}$를 제외하고는 오차 5%이내의 좋은 정확도를 보여주었으며 회수율은 78.5%로 나타났다. Carteolol은 소변에서 대부분이 free carteolol과 conjugated carteolol로 존재하며 소량의 p-OH-carteolol로 대사됨을 알 수 있었으며 모 약물로 배출되는 양 중 conjugated form이 59.4%로 나타났다. 소변으로 배출된 carteolol의 양을 측정한 결과 복용양의 49%가 복용 후 72시간까지 모 약물로 배출됨을 알 수 있었다.

• 한국미생물·생명공학회지
• /
• 제17권3호
• /
• pp.213-218
• /
• 1989

Cloning을 위한 host와 vector의 이용 가능성을 타진하기 위해 호알칼리성 Bacillus속 K-17을 host로, pUB110과 pBD64를 vector로 사용하여 Bacillus속 K-17의 protoplast 형질전환조건을 검토하였다. 원형질체의 형성은 200$\mu\textrm{g}$/$m\ell$의 Iysozyme 을 처리하였으며, 원형질체 형성의 최적 온도, PH및 배양시간은 각각 4$0^{\circ}C$, 7.0 및 4시간으로 나타났다. 원형질체는 DM3 재생배지에서 재생시켰으며 0.8% agar, 0.5M sodium succinate 농도에서 가장재생이 좋았다. 형질전환시 PEG의 농도는 40%(w/v) PEG 6,000 30%(v/v)가 최적으로 나타났다. 형질전환체의 특성을 조사한 결과, plasmid 안정성은 pUB110이 pBD64보다 더 안정하였으며, 최대 효소활성은 비슷하였지만 효소 분비시간은 pUB110 은 2.5일, pBD64의 경우는 3일로 Bacillus속 K-17의 2일에 비해 약간 지연되었다.

• 원예과학기술지
• /
• 제31권6호
• /
• pp.677-686
• /
• 2013

Edible canna is a productive starch source in some tropical and semitropical regions. In these regions, water deficit stress is one of factors that limit the crop yield. In the present study, we investigated seven physiological indexes and photosynthetic responses of three edible canna (Canna edulis Ker.) cultivars ('PLRF', 'Xingyu-1', and 'Xingyu-2') under 35 days drought stress. Our results indicated that drought treatment caused visible wilting symptoms in all cultivars, especially in 'Xingyu-1'. Coupled with the increase of wilting symptoms, relative water content (RWC) and chlorophyll content decreased progressively, malondialdehyde (MDA) content gradually increased, and key antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) activities increased first and then decreased in all three cultivars. The effect of water stress was more pronounced in 'Xingyu-1' than in 'PLRF' and 'Xingyu-2', and in lower leaves than in upper leaves. In addition, 35 days drought stress also significantly reduced the photosynthetic capacity. Consistent with antioxidant parameters, photosynthetic changes of 'Xingyu-2' were less than those of the other cultivars under water deficit stress. Drought stress caused a significant increase of water use efficiency (WUE) in 'Xingyu-2', but little in 'PLRF', and obvious decrease in 'Xingyu-1'. These results indicated that 'Xingyu-2' was more tolerant to drought stress than 'PLRF' and 'Xingyu-1' by maintaining lower lipid peroxidation and higher antioxidant enzyme activities.

• Molecules and Cells
• /
• 제39권10호
• /
• pp.768-775
• /
• 2016

The Arabidopsis female gametophyte contains seven cells with eight haploid nuclei buried within layers of sporophytic tissue. Following double fertilization, the egg and central cells of the gametophyte develop into the embryo and endosperm of the seed, respectively. The epigenetic status of the central cell has long presented an enigma due both to its inaccessibility, and the fascinating epigenome of the endosperm, thought to have been inherited from the central cell following activity of the DEMETER demethylase enzyme, prior to fertilization. Here, we present for the first time, a method to isolate pure populations of Arabidopsis central cell nuclei. Utilizing a protocol designed to isolate leaf mesophyll protoplasts, we systematically optimized each step in order to efficiently separate central cells from the female gametophyte. We use initial manual pistil dissection followed by the derivation of central cell protoplasts, during which process the central cell emerges from the micropylar pole of the embryo sac. Then, we use a modified version of the Isolation of Nuclei TAgged in specific Cell Types (INTACT) protocol to purify central cell nuclei, resulting in a purity of 75-90% and a yield sufficient to undertake downstream molecular analyses. We find that the process is highly dependent on the health of the original plant tissue used, and the efficiency of protoplasting solution infiltration into the gametophyte. By isolating pure central cell populations, we have enabled elucidation of the physiology of this rare cell type, which in the future will provide novel insights into Arabidopsis reproduction.

• Journal of Applied Biological Chemistry
• /
• 제54권3호
• /
• pp.197-205
• /
• 2011

This study was carried out to investigate the dietary effects of Monascus pilosus mycelial extract on obesity in high-fat with cholesterol-induced obese rat models. It was observed that M. pilosus mycelial extract contains $25.85{\pm}1.98mg%$ of total monacolin K without citrinin by highperformance liquid chromatography (HPLC). The rats were randomly divided into 2 groups; normal control and a high-fat with cholesterol diet group. The high-fat with cholesterol diet group was fed a 5L79 diet with an added 15% lard and 1% cholesterol supplemented diet for 3 weeks for induction of obesity. After induction, obesity was confirmed by checking obesity indexes, the animals were divided into 4 groups (n=5); first, the normal control (NC), and then taken from the obese model of rats, a high-fat with cholesterol diet obesity control group (HF), 0.5% M. pilosus mycelial extract supplemented high-fat with cholesterol diet group (MPMs), 2% conjugated linoleic acid supplemented high-fat with cholesterol diet group (CLA) for 7 weeks. Body weight gains, obesity indexes, and body fat contents in the experimental groups (MPMs and CLA) were decreased compared with HF group. Feed Efficiency Ratio (FER) in MPMs was significantly lower than that of HF without change of feed intake. These results suggested that the anti-obesity effects of the M. pilosus mycelial extracts (MPMs) could prevent obesity induced by high-fat with cholesterol diet possibly via inhibition of lipid absorption.

• 미생물학회지
• /
• 제48권3호
• /
• pp.225-227
• /
• 2012

백색부후균류가 가지는 리그닌 분해효소들은 기질특이성이 넓기 때문에 다양한 난분해성 화합물들을 분해할 수 있다. 본 실험에서는 3가지 다른 방법을 사용하여 laccase와 manganese peroxidase가 각각 도입된 아교버섯 형질전환체의 배양 상등액 효소를 고정화 효소로 만들어 대표적 염료의 하나인 Remazol Brilliant Blue R (RBBR)의 탈색을 실험하였다. 그 결과 알긴산을 효소 용액과 직접 반응하여 만든 고정화 효소에서 48시간 반응 후 약 75% 탈색을 보였다. 비록 한번 사용했던 고정화 효소를 재사용하였을 경우 탈색능이 10-15% 정도 감소되었으나 본 실험에서 제시한 방법이 리그닌 분해효소의 고정화 효소 활용에 기여할 것으로 기대한다.

• 생명과학회지
• /
• 제27권10호
• /
• pp.1145-1151
• /
• 2017

세제에 의하여 대장균의 periplasm에서 penicillin G amidase (PGA)를 방출하는 방법을 연구하였다. 결과적으로 세제와 lysozyme의 혼합 작용이 효과적인 것으로 나타났다. 세포 투과성의 최적 조건을 알아보기 위하여 세제의 종류, 농도, pH, 반응 시간, 온도 등의 영향을 살펴보았다. 그리하여 대장균에서 재조합 PGA를 periplasm에서 방출하는 모델을 만들 수 있었고 방출된 PGA를 농축할 수 있었다. 실리카 구슬을 이용한 고정화 시스템으로 PGA 용액을 농축할 수 있었으며, 더 이상의 정제 과정 없이 순수하게 추출 할 수 있었다. 고정화된 PGA는 penicillin G 생성의 원료인 6-APA를 생산하는데 사용할 수 있었다. 이 방법은 대장균으로부터 재조합 단백질을 추출하는 간단한 방법이며 고정화 PGA를 이용하여 ${\beta}-lactam$ 항생물질의 산업적 생산 이용될 수 있을 것으로 사료된다.

• Preventive Nutrition and Food Science
• /
• 제18권2호
• /
• pp.98-103
• /
• 2013

In this study, we evaluated the hypolipidemic and antioxidative effects of biotransformed soybean powder (BTS; Phellinus baumii-fermented soybean) on lipid metabolism in rats. Sprague-Dawley (SD) male rats were divided into basal diet group (BA), high fat diet group (HF), high fat diet containing 10% BTS group (10 BTS), and high fat diet containing 20% BTS group (20 BTS). Changes in the content of various isoflavones, including daidzein and genistein, within the soybean after fermentation to BTS were investigated. The levels of daidzein and genistein were $149.28{\mu}g/g$ and $364.31{\mu}g/g$, respectively. After six weeks experimental period, Food efficiency ratio in the 10 and 20 BTS group was significantly lower than the HF group (P<0.05). Total serum levels of cholesterol, triglycerides, low-density lipoprotein cholesterol, and atherogenic index ratio in the 10 or 20 BTS group were significantly lower than the HF group. The levels of alanine aminotransferase, aspartate aminotransferase and thiobarbituric acid reactive substance were significantly lower in the groups that received 10% and 20% BTS than the HF. The activities of SOD and CAT were significantly higher in the 10 and 20 BTS group than the HF group. The activity of XO in the 10 and 20 BTS group was significantly lower than in the HF group by 20% and 23%, respectively. In conclusion, these data suggest that BTS is an effective agent in improving lipid metabolism and antioxidant enzyme system.

• 동아시아식생활학회지
• /
• 제19권3호
• /
• pp.363-368
• /
• 2009

In this experiment, three different diets were produced to investigate the effects of Jeju native chicken meat, from chickens that were fed a tangerine by-product, on physiological activities in rats. The first diet did not contain any chicken (TS), the second diet contained 10% chicken that had not been fed the tangerine by-product (T0), and the third diet contained 10% that had been fed the by-products (T1). These diets were provided to 11-week-old male rats for four weeks. Weight gain, feed intake, feed efficiency, liver, kidney, and epididymis fat weights were not significantly different among the TS, T0, and T1 groups. Total lipid, triglycerides, and cholesterol levels in the liver were significantly lower in T0 and T1 than in TS (p<0.05). And total lipid, phospholipid, triglycerides, total cholesterol, HDL-cholesterol, DL+VLDL-cholesterol, HDL-cholesterol/total cholesterol, and atherogenic index levels in the blood serum were similar between T0 and T1, which did not present any significant differences. The feed containing the tangerine by-product did not cause any statistically significant differences in serum protein, glucose, or hemoglobin. Finally, T0 and T1 showed similar trends in terms of $\gamma$-GTP, ALT, AST, and ALP activities, which again did not present any statistically significant differences.