• 한국식물병리학회:학술대회논문집
• /
• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
• /
• pp.94.2-95
• /
• 2003

E. intermedium 60-2G possessing a strong ability to solubilize insoluble phosphate, has plant growth-promoting activity, induced systemic resistance activity against scab pathogen in cucumber, and antifungal activity against various phytopathogenic fungi. The phosphate solubilizing activity of 60-2G may be mainly accomplished by production of gluconic acid through a direct extracellular oxidation of glucose by glucose dehydrogenase that required a PQQ cofactor for its activation. A pqq gene cluster conferred Phosphate-solubilizing activity in E. coli DH5${\alpha}$ was cloned and sequenced. The 6,783 bP pqq sequence had six open reading frames (from A to F) and showed 50-95％ homology to pqq genes from other bacteria. The E. coli strain expressing the pqq genes solubilized phosphate from hydroxyapatite after a pH drop to 4.0, which paralleled in time the secretion of gluconic acid. To study the role of PQQ in biocontrol traits of E. intermedium, PQQ mutants of 60-2G were constructed by marker exchangee mutagenesis. The PQQ mutants of E. intermedium were lost activities of solubilizing phosphate, growth inhibition of phytopathogenic fungi, and plant growth promotion. These findings suggest that PQQ plays an important role, possibly activation of certain enzymes, in several beneficial bacterial traits of E. intermedium by as yet an unknown mechanism.

• Journal of Microbiology and Biotechnology
• /
• 제10권1호
• /
• pp.95-98
• /
• 2000

An immunochemical method has been develooped as the most sensitive tool for studying the expression of quinoproteins containing pyrroloquinoline qinone(PQQ) in E. coli. The PQQ was conjugated to bovine serum albumin (BSA), and the conjugant was purified by using a $KwikSep^{TM}$ dextran desalting column chromatography. The PQQ-BSA conjugant was immunized to rabbits, and the IgG fractions of the antisera were purified. The most sensitive antibody against PQQ-BSA conjugant recognized some nanogram quantity of the antigen on the blot, but had little cross reactivity with BSA. Using this batch of the antibody, all the immunochemical assays of quinoproteins in E. coli were preformed. Some six different PQQ-specfic spots were detected by Western blot analysis of the soluble proteins in E. coli were performed. Some six different PQQ-specific spots were detected by Western blot analysis of the soluble proteins in E. coli after two-dimensional gel electrophoresis. Their molecular weights on the blot were estimated to be about 100-, 90-, 72-, 58-, 52-, and 50kDa. Their pI values fell in the range from 4.8 to 5.5. These results stronly suggest that quinoproteins are present in E. coli, and that the protein moieties were covalently bound to PQQ.

• Journal of Oral Medicine and Pain
• /
• 제30권3호
• /
• pp.329-336
• /
• 2005

본 연구는 superoxide의 제거물질로 알려진 pyrroloquinoline quinone (PQQ)이 파골세포의 분화 및 성숙한 파골세포의 활성에 미치는 영향을 알아보고자 시행하였다. Superoxide를 인지하는 방법인 nitroblue tetrazolium (NBT) 염색방법을 이용하여 PQQ가 HD-11 세포가 생성한 superoxide를 제거하는지 확인하였다. 본 연구에서 이용된 HD-11세포는 닭 myelomonocytic 세포주로써 1,25-dihydroxyvitamin $D_3\;[1,25(OH)_2D_3]$ 처리시 tartrate-저항성 산성인산분해효소 (tartrate-resistant acid phosphatase, TRAP)의 활성을 나타내는 등 파골세포의 특성을 지니는 세포로 알려져 있다. HD-11세포에서 TRAP 활성을 확인하기 위하여 조직화학적 염색을 시행하였다. PQQ는 NBT의 환원을 감소시켰으며 1,25(OH)2D3에 의해 유도된 TRAP 활성을 억제하였다. 또한 PQQ가 닭 골수세포에서 TRAP 양성 다핵세포의 형성에 미치는 영향도 관찰한 결과 20 ${\mu}M$의 PQQ는 TRAP 양성 다핵세포의 형성을 현저히 억제하였다. 닭 파골세포를 상아질 절편에서 배양하면서 20 ${\mu}M$의 PQQ를 처치한 경우 파골세포에 의한 상아질 흡수가 현저히 억제되었다. 따라서 본 연구결과 PQQ가 superoxide의 제거물질로 작용하여 파골세포의 분화 및 활성도에 영향을 미칠 것으로 사료되며, 이는 생리적 혹은 병적 골흡수에 억제적인 작용을 할 물질로의 가능성을 시사한다.

• Journal of Microbiology and Biotechnology
• /
• 제13권6호
• /
• pp.955-959
• /
• 2003

Rhizobium tropici CIAT 899 is capable of synthesizing inactive apo-glucose dehydrogenase (GDH). To become an active holo enzyme, the GDH requires a cofactor, PQQ. When R. tropici CIAT 899 was grown in a broth culture medium containing hydroxyapatite and pyrrolo quinoline quinone (PQQ), pH decreased while the concentration of soluble P increased. The solubilization of hydroxyapatite was associated with the production of gluconic acid and 2-ketogluconic acids. The organic acid production and P solubilization were greatly enhanced when the bacterium was grown with air supply. Effect of R. tropici CIAT 899 with (CI＋PQQ) and without PQQ (CI) on the common bean growth was examined. Shoot and root weight, and N and P contents in CI＋PQQ treatment, were significantly higher than those in control and CI treatment. Nodule weight and acetylene reducing activities were also significantly higher in CI＋PQQ treatment than in other treatments.

• 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.

• The Plant Pathology Journal
• /
• 제22권4호
• /
• pp.323-328
• /
• 2006

Serratia marcescens W1, isolated from cucumber-cultivated soil in Suwon, Korea, evidenced profound antifungal activity and produced the extracellular hydrolytic enzymes, chitinase and protease. In order to isolate the antifungal genes from S. marcescens W1, a cosmid genomic library was constructed and expressed in Escherichia coli. Transformants exhibiting chitinase and protease expression were selected, as well as those transformants evidencing antifungal effects against the rice blast fungus, Magnaporthe grisea, and the cucumber leaf spot fungus, Cercospora citrullina. Cosmid clones expressing chitinase or protease exerted no inhibitory effects against the growth of fungal pathogens. However, two cosmid clones evidencing profound antifungal activities were selected for further characterization. An 8.2 kb HindIII fragment from these clones conditioned the expression of antagonistic activity, and harbored seven predicted complete open reading frames(ORFs) and two incomplete ORFs. The deduced amino acid sequences indicated that six ORFs were highly homologous with genes from S. marcescens generating pyrroloquinoline quinone(PQQ). Only subclones harboring the full set of pqq genes were shown to solubilize insoluble phosphate and inhibit fungal pathogen growth. The results of this study indicate that the functional expression of the pqq genes of S. marcescens W1 in E. coli may be involved in antifungal activity, via as-yet unknown mechanisms.

• 전기화학회지
• /
• 제2권1호
• /
• pp.27-30
• /
• 1999

Self-Assembled monolayers of carboxyl-terminated alkanethiols, which is negatively charged in pH 7.0, were usually used to facilitate the electron transfer between the positively charged protein and the electrode. In case of L-cysteine, as it has both positive and negative group, it can be a candidate for a new modifier to facilitate positively charged protein or negatively charged protein. Our investigation of L-cysteine shows that the electron transfer occurs successfully to both cytochrome c (cyt. c) and pyrroloquinoline quinone (PQQ). By using 1-ethyl-3-(3-dime-thlyaminopropyl) carbodiimide (EDC), we made a covalent bond between cyt. c and monolayer. Then PQQ was electrostatically adsorbed to the same monolayer. Cyclic voltammograms show that both molecules do not interfere each other and electron transfer is appreciable.

• Bulletin of the Korean Chemical Society
• /
• 제31권6호
• /
• pp.1711-1714
• /
• 2010

• 한국토양비료학회지
• /
• 제35권1호
• /
• pp.59-67
• /
• 2002

강한 인산 가용력을 가진 인산 용해 세균인 균주 60-2G를 잔디의 근권에서 분리하였다. GC-FAME구조와 탄소이용형태 및 16S rRNA의 부분 염기서열 분석을 통해 균주 60-2G는 Enterobacter intermedium으로 동정되었다. Hydroxyapatite를 첨가한 배지와 생장 시킨 균주 60-2G는 gluconic acid 와 2-ketogluconic acid 및 소량의 lactic acid를 생성하였다. 균주 60-2G의 생장 기간동안 배지의 pH는 3.8 까지 낮아지는 반면에 배지의 유효 인산 농도는 증가하였다. 배지의 낮은 pH와 유효인산농도의 증가는 역 상관관계이며, 이는 균주 60-2G가 생성하는 유기산에 의한 영향이다. E. intermedium 60-2G 균주는 유기산 생성에 관여하는 glucose dehydrogenase의 co-factor인 PQQ를 생성하였으며, pqq의 부분 염기서열 분석 결과 기존에 보고된 서열과 85% 이상의 상동성을 가지고 있었다.

• Journal of Microbiology and Biotechnology
• /
• 제26권11호
• /
• pp.1908-1917
• /
• 2016

Wild strain L-6 was subjected to combined mutagenesis, including UV irradiation, atmospheric and room temperature plasma, and ion beam implantation, to increase the yield of 1,3-dihydroxyacetone (DHA). With application of a high-throughput screening method, mutant Gluconobacter oxydans I-2-239 with a DHA productivity of 103.5 g/l in flask-shake fermentation was finally obtained with the starting glycerol concentration of 120 g/l, which was 115.7% higher than the wild strain. The cultivation time also decreased from 54 h to 36 h. Compared with the wild strain, a dramatic increase in enzyme activity was observed for the mutant strain, although the increase in biomass was limited. DNA and amino acid sequence alignment revealed 11 nucleotide substitutions and 10 amino acid substitutions between the sldAB of strains L-6 and I-2-239. Simulation of the 3-D structure and prediction of active site residues and PQQ binding site residues suggested that these mutations were mainly related to PQQ binding, which was speculated to be favorable for the catalyzing capacity of glycerol dehydrogenase. RT-qPCR assay indicated that the transcription levels of sldA and sldB in the mutant strain were respectively 4.8-fold and 5.4-fold higher than that in the wild strain, suggesting another possible reason for the increased DHA productivity of the mutant strain.