• Journal of Microbiology and Biotechnology
• /
• 제30권11호
• /
• pp.1750-1759
• /
• 2020

The characterization of cytochrome P450 CYP125A13 from Streptomyces peucetius was conducted using cholesterol as the sole substrate. The in vitro enzymatic assay utilizing putidaredoxin and putidaredoxin reductase from Pseudomonas putida revealed that CYP125A13 bound cholesterol and hydroxylated it. The calculated K_D value, catalytic conversion rates, and Km value were 56.92 ± 11.28 μM, 1.95 nmol min^-1 nmol^-1, and 11.3 ± 2.8 μM, respectively. Gas chromatography-mass spectrometry (GC-MS) analysis showed that carbon 27 of the cholesterol side-chain was hydroxylated, characterizing CYP125A13 as steroid C27-hydroxylase. The homology modeling and docking results also revealed the binding of cholesterol to the active site, facilitated by the hydrophobic amino acids and position of the C27-methyl group near heme. This orientation was favorable for the hydroxylation of the C27-methyl group, supporting the in vitro analysis. This was the first reported case of the hydroxylation of cholesterol at the C-27 position by Streptomyces P450. This study also established the catalytic function of CYP125A13 and provides a solid basis for further studies related to the catabolic potential of Streptomyces species.

• Journal of Microbiology and Biotechnology
• /
• 제25권8호
• /
• pp.1349-1360
• /
• 2015

The rhizospheric zone abutting plant roots usually clutches a wealth of microbes. In the recent past, enormous genetic resources have been excavated with potential applications in host plant interaction and ancillary aspects. Two Pseudomonas strains were isolated and identified through 16S rRNA and rpoD sequence analyses as P. fluorescens QAU67 and P. putida QAU90. Initial biochemical characterization and their root-colonizing traits indicated their potential role in plant growth promotion. Such aerobic systems, involved in gluconic acid production and phosphate solubilization, essentially require the pyrroloquinoline quinine (PQQ)-dependent glucose dehydrogenase (GDH) in the genome. The PCR screening and amplification of GDH and PQQ and subsequent induction of mutagenesis characterized their possible role as antioxidants as well as in growth promotion, as probed in vitro in lettuce and in vivo in rice, bean, and tomato plants. The results showed significant differences (p ≤ 0.05) in parameters of plant height, fresh weight, and dry weight, etc., deciphering a clear and in fact complementary role of GDH and PQQ in plant growth promotion. Our study not only provides direct evidence of the in vivo role of GDH and PQQ in host plants but also reveals their functional inadequacy in the event of mutation at either of these loci.

• Journal of Microbiology
• /
• 제35권4호
• /
• pp.295-299
• /
• 1997

Pseudomonas sp. S-47 is capable of transforming 4-chlorobenzoate to 4-chlorocatechol which is subsequently oxidized bty meta-cleavage dioxygenase to prodyce 5-chloro-2-hydroxymuconic semialdehyde. Catechol 2,3-dioxygenase (C23O) produced by Pseudomonas sp. S-47 was purified and characterized in this study. The C23O enzyme was maximally produced in the late logarithmic growth phase, and the temperature and pH for maximunm enzyme activity were $30{\sim}35^{\circ}C$ and 7.0, respectively. The enzyme was purified and concentrated 5 fold from the crude cell extracts through Q Sepharose chromatography and Sephadex G-100 gel filtration after acetone precipitation. The enzyme was identified as consisting of 35 kDa subunits when analyzed by SDS-PAGE. The C23O produced by Pseudomonas sp. S-47 was similar to Xy1E of Pseudomonas putida with respect to substrate specificity for several catecholic compounds.

• Journal of Microbiology and Biotechnology
• /
• 제16권11호
• /
• pp.1819-1825
• /
• 2006

Four naphthalene-degrading bacteria (Pseudomonas sp. strains O1, W1, As1, and G1) were isolated feom pollutant-contaminated sites. Examination of their substrate utilization and analyses of key naphthalene-catabolic regulatory genes revealed that the pathway and regulation of naphthalene-degradation in all four strains resemble those of NAH7 from P. putida G7. Superoxide anion production, superoxide dismutase activity, and catalase activity during their growth on naphthalene-amended medium increased significantly, compared with those with glucose-amended medium. Addition of ascorbate, an antioxidant, or ferrous iron ($Fe^{2+}$) increased the growth rates of all tested microorganisms on naphthalene. Northern blot and HPLC analyses showed that both nahA gene expression and naphthalene degradation increased under those conditions. Our data suggest that naphthalene degradation can impose severe oxidative stress, and defenses against oxidative stress would play an important role in the metabolism of naphthalene.

• 한국지하수토양환경학회지:지하수토양환경
• /
• 제18권4호
• /
• pp.32-39
• /
• 2013

This research focused on the effects of heavy metals on the biomonitoring activity of genetically engineered bioluminescent bacteria, Pseudomonas putida mt-2 KG1206. KG1206 was exposed to single or binary mixtures of different heavy metals as well as soils contaminated with heavy metals. In case of single exposure with different inducer pollutant, the toxicity order was as followings : As(III) > Cd, As(V) >> Cu, Cr(VI). The toxic effects of the binary mixtures was compared to the expected effect based on a simple theory of probabilities. The interactive effects were mostly additive, while in few cases antgonistic and synergistic mode of action was observed for some concentration combinations. No considerable correlation was found between the total metal contents in soils and the bioluminescence activity of each sample. However, statistically significant differences (p = 0.0102) were observed between two groups, classified based on arsenite contamination. These results demonstrate the usage of recombinant bioluminescent microorganism in biomonitoring and the complex interactive effects of metals.

• 미생물학회지
• /
• 제29권6호
• /
• pp.408-415
• /
• 1991

TOL plsmid size of Flavobacterium odoratum SUB53 was estimated as 83 Md and the optimum concentration of m-toluate degradation by TOL plasmid was 5 mM. $H_{2}$ production by Rhodopseudomonas sphaeroides KCTC1425 was largely dependent on nitrogenase activity and showed the highest at 30 mM malate with 7 mM glutamate as nitrogen source. Nitrogenase activities were inhibited by 0.3 mM $NH_{4}^{+}$ions, to be appeared the decrease of $H_{2}$ production. Conjugation of TOL plasmids from F. odoratum SUB53 and Pseudomonas putida mt-2 to R. sphaeroides showed the optimum at the exponential stage of recipient cells in presence of helper plasmid pRK2013. According to the investigation of catechol-1,2-oxygenase (C-1, 2-O) and catechol-2,3-oxygenase (C-2,3-O) activities of R. sphaeroides C1 (TOL SUB53) and C2 (TOL mt-2), the gene for C-2,3-O is located on TOL plasmid and gene for C-1, 2-O on the chromosome of R. sphaeroides. m-Toluate was biodegraded by TOL plasmid in R. sphaeroides C1 and C2, presumably to be produced $H_{2}$ gas from the secondary metabolites of m-toluate.e.

• Journal of Microbiology and Biotechnology
• /
• 제28권6호
• /
• pp.938-945
• /
• 2018

Salinity stress is an important environmental problem that adversely affects crop production by reducing plant growth. The impacts of rhizobacterial strains to alleviate salinity stress on the germination of Lactuca sativa and Raphanus sativus seeds were assessed using different concentrations of NaCl. Plant growth-promoting rhizobacteria (PGPR) strains were also examined to improve the early germination of Chinese cabbage seeds under normal conditions. Lactobacillus sp. and P. putida inoculation showed higher radicle lengths compared with non-inoculated radish (Raphanus sativus) seeds. LAP mix inoculation increased the radicle length of lettuce (Lactuca sativa) seedlings by 2.0 and 0.5 cm at salinity stress of 50 and 100 mM NaCl concentration, respectively. Inoculation by Azotobacter chroococcum significantly increased the plumule and radicle lengths of germinated seeds compared with non-inoculated control. A. chroococcum increased the radicle length relative to the uninoculated seeds by 4.0, 1.0, and 1.5 cm at 50, 100, and 150 mM NaCl concentration, respectively. LAP mix inoculation significantly improved the radicle length in germinated radish seeds by 7.5, 1.3, 1.2, and 0.6 cm under salinity stress of 50, 100, 150, and 200 mM NaCl concentration, respectively. These results of this study showed that PGPR could be helpful to mitigate the salinity stress of different plants at the time of germination.

• 한국식물병리학회지
• /
• 제12권4호
• /
• pp.381-388
• /
• 1996

맥류세균성 줄무늬병균의 선택배양기(KM-1)를 개발하여 이병식물체 및 토양으로부터 Xanthomonas campestris pv. translucens를 선택적으로 분리할 수 있는 효율성을 검토하였다. KM-1배양기의 구성성분은 증류수 1 L당 lactose 10 g, D(+)trehalose 4.0 g, thiobarbituric acid 0.2 g, K\ulcornerHPO\ulcorner 및 KH\ulcornerPO\ulcorner 각각 0.8 g, yeast extract 30 mg, NH\ulcornerCl 1 g, cycloheximide 100 mg, tobramycin 8.0 mg, ampicillin 1.0 mg 및 Bacto agar 15 g이며 1 N NaOH로 pH 6.6으로 조절하였다. X. c. t.의 균주별 KM-1의 배양효율은 비선택성 농후배지인 Wilbrinks agar에 비하여 1.30정도였으며, 기타 토양전염성식물병원세균 Agrobacterium tumefaciens, Agrobacterium rhizogenes, Erwinia carotovora var. atroseptica, Erwinia carotovora var. carotovora, Corynebacterium insidiosum, 및 기타 토양생존 부생세균 Bacillus cereus, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Pseudomonas putida 등의 생장을 완벽하게 억제하였다. KM-1의 저장기간(shelf-life)도 5$^{\circ}C$에서 2개월 동안 선택성을 유지하였다. 따라서 본 병원균의 전염원의 생존 등 발생생태연구에 활용될 수 있는 가치가 충분히 인정되었다.

• KSBB Journal
• /
• 제16권6호
• /
• pp.603-608
• /
• 2001

낮은 농도 (0.41 g/㎥)에서 바이오필터 장치의 제거효율은 5 L/min과 10L/min에서 각각 30~70% 이상을 보여주는 반먼에 고농도 (1.23 g/㎥)에서의 효율은 10~60%이다. 그리고 Synthesized media를 담체로 사용한 바이오필터의 성능이 다른 바이오필터에 비하여 좋지 않았으며, barks를 사용한 바이오필터에 비하여 좋지 않았으며, barks를 사용한 바이오필터의 제거율이 입구농도 0.8g/㎥에서 약 70%로 가장 좋은 성능을 보였다. 이는 합성담체가 porous하여 그 보습능력이 다른 담체들에 비하여 떨어지기 때문임을 알 수 있었다. 바이오필터가 제거할 수 있는 공기중의 톨루엔양은 입구의 단위 시간당 농도를 증가시킴으로써 제거량이 증가함으로 알 수 있었으며, barks를 담체로 사용한 바이오필터의 제거량이 유입 부하량 40 g/㎥에서 최대 30 g/㎥으로 가장 우수한 것으로 나타나, 수분 및 활성의 유지가 안돼는 악의 조건하에서의 바이오필터 적응능력 면에서는 bark를 사용하는 것이 가장 바람직함을 알 수 있었다. 한편 바이오필터의 우수한 거동을 위해서는 필터층의 적당한 수분함량 조절이 가장 필수적이며, 각 바이오필터의 pH 변화는 7~8로써, 중성영역을 유지하는 것이 바람직하였다. 모든 바이오필터 시스템에서 압력강하는 0.054 cm$H_2O$/m로서 무시할 만큼 낮았고, 특히 합성담체의 경우에 다른 담체들에 비하여 압력손실이 현저히 낮았다.저히 낮았다.

• 한국환경농학회지
• /
• 제16권2호
• /
• pp.124-129
• /
• 1997

중금속오염폐수처리에 미생물 이용 가능성을 검토하기 위하여 중금속에 강한 내성을 지님과 동시에 균체내 중금속 축적능력이 우수한 중금속 내성균을 분리하여 온도, pH, 및 균체 전배양시간 등과 같은 외부 요인에 따른 균체내 중금속 축적변화를 조사한 결과는 다음과 같다. 중금속화하물 종류에 따른 균체내 중금속 축적량은 $-nitrate(NO_3)$ group을 가진 중금속화합물에서 약간 높았으나 화합물 종류에 따라서 큰 차이는 없었다. 균체 전배양시간에 따른 중금속 축적은 24시간 전배양한 균체에서 높게 나타났으나 전반적으로 전배양시간에 따라서 큰 차이는 없었다. 중금속 내성균의 균체내 중금속 축적은 온도, pH등 외부환경요인에 영향을 받았으며, 중금속 내성균의 중금속 최적축적 온도는 전반적으로 $20{\sim}37^{\circ}C$ 범위였고, 중금속 최적축적 pH는 전반적으로 $6{\sim}8$ 범위였다. 중금속을 처리한 용액중 중금속 내성균의 균체량을 증가시킬수록 균체내 축적된 총 중금속양은 증가되었으나 균체 단위 g당 중금속 축적량은 감소되었으며, 균체량의 증가에 비례해서 중금속 축적량이 증가되지는 않았다.