• Title/Summary/Keyword: Insoluble phosphate

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Risk Evaluation of Monopotassium Phosphate (MKP) and Bentonite Application via the Mobility Reduction of Soil TNT and Heavy Metals (제일인산칼륨과 벤토나이트 처리를 통한 토양 내 TNT와 중금속 이동성 및 인체위해도 저감 기술)

  • Jung, Jae-Woong;Yu, Gihyeon;Nam, Kyoungphile
    • Journal of Soil and Groundwater Environment
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    • v.20 no.6
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    • pp.28-36
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    • 2015
  • Simultaneous mobility reduction of explosives and heavy metals in an operational range by monopotassium phosphate (MKP) and bentonite spreading technology was investigated. Potassium ion and phosphate ion in MKP act as explosives sorption enhancer and insoluble heavy metal phosphate formation, respectively, while bentonite acts as the explosives adsorbent. Then, the decrease in surface water concentration of the pollutants and resulting risk reduction for local residents of the operational range, by MKP/bentonite application was estimated. Under untreated scenario, the noncancer hazard index (HI) exceeded unity on February, July and August, mainly due to 2,4,6-trinitrotoluene (TNT); however, MKP/bentonite treatment was expected to lower the noncancer hazard index by decreasing the surface water concentration of explosives and heavy metals (especially TNT). For example, on July, estimated surface water concentration and HI of TNT were 0.01 mg/L and 1.1, respectively, meanwhile the sorption coefficient of TNT was 3.9 mg1−nkg−1Ln. However, by MKP/bentonite treatment, the TNT sorption coefficient increased to 113.8 mg1−nkg−1Ln and the surface water concentration and HI decreased to about 0.002 mg/L and 0.2, respectively. Based on the result, it can be concluded that MKP/bentonite spreading is a benign technology that can mitigate the risk posed by the pollutants migration from operational ranges.

Mechanism of Free Phosphate Production by Penicillium sp. GL-101, Phosphate Solubilizing Fungus, in the Submerged Culture (인산가용화균 Penicillium sp. GL-101의 유리인산 생성기작에 관한 연구)

  • Kang, Sun-Chul;Yang, Mi-Ok;Tae, Un-Hee
    • Korean Journal of Environmental Agriculture
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    • v.20 no.1
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    • pp.1-7
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    • 2001
  • We investigated the capability of the phosphate-solubilizing fungus, Penicillium sp. GL-101, to solubilize in vitro some insoluble rock phosphate via possible mechanisms: acidification of the medium, production of chelating metabolites, redox activity, and so on. GL-101 was able to solubilize rock phosphate (mostly calcium phosphate) in a liquid potato dextrose broth(PDB) medium, as determined by spectrophotometric analyses. Acidification was the major mechanism of solubilization since the pH of cultures fell below 4.0 and in cultures containing 1.0%(w/v) loess the pH dropped from 7.0 to 3.2. More than 10 mg/mL concentrations of citric acids were detected by high-performance liquid chromatography(HPLC) in the culture supernatants. Also this fungus showed the phosphatase activity (over 1.3 unit) to contribute partially releasing phosphate from rock phosphate, when supplemented with 1.0% loess in culture broth. The chelating activity of GL-101 in culture supernatants was not present because 2-ketogluconic acid, a chelating agent for the phosphate, was produced only a basal level. Therefore, the solubilization mechanism of rock phosphate by Penicillium sp. GL-101 involves both acidification due to citric acid production and phosphatase activity.

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Molecular and Biochemical Characteristics of ${\beta}$-Propeller Phytase from Marine Pseudomonas sp. BS10-3 and Its Potential Application for Animal Feed Additives

  • Nam, Seung-Jeung;Kim, Young-Ok;Ko, Tea-Kyung;Kang, Jin-Ku;Chun, Kwang-Hoon;Auh, Joong-Hyuck;Lee, Chul-Soon;Lee, In-Kyu;Park, Sunghoon;Oh, Byung-Chul
    • Journal of Microbiology and Biotechnology
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    • v.24 no.10
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    • pp.1413-1420
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    • 2014
  • Phytate is an antinutritional factor that impacts the bioavailability of essential minerals such as $Ca^{2+}$, $Mg^{2+}$, $Mn^{2+}$, $Zn^{2+}$, and $Fe^{2+}$ by forming insoluble mineral-phytate salts. These insoluble mineral-phytate salts are hydrolyzed rarely by monogastric animals, because they lack the hydrolyzing phytases and thus excrete the majority of them. The ${\beta}$-propeller phytases (BPPs) hydrolyze these insoluble mineral-phytate salts efficiently. In this study, we cloned a novel BPP gene from a marine Pseudomonas sp. This Pseudomonas BPP gene (PsBPP) had low sequence identity with other known phytases and contained an extra internal repeat domain (residues 24-279) and a typical BPP domain (residues 280-634) at the C-terminus. Structure-based sequence alignment suggested that the N-terminal repeat domain did not possess the active-site residues, whereas the C-terminal BPP domain contained multiple calcium-binding sites, which provide a favorable electrostatic environment for substrate binding and catalytic activity. Thus, we overexpressed the BPP domain from Pseudomonas sp. to potentially hydrolyze insoluble mineral-phytate salts. Purified recombinant PsBPP required $Ca^{2+}$ or $Fe^{2+}$ for phytase activity, indicating that PsBPP hydrolyzes insoluble $Fe^{2+}$-phytate or $Ca^{2+}$-phytate salts. The optimal temperature and pH for the hydrolysis of $Ca^{2+}$-phytate by PsBPP were $50^{\circ}C$ and 6.0, respectively. Biochemical and kinetic studies clearly showed that PsBPP efficiently hydrolyzed $Ca^{2+}$-phytate salts and yielded myo-inositol 2,4,6-trisphosphate and three phosphate groups as final products. Finally, we showed that PsBPP was highly effective for hydrolyzing rice bran with high phytate content. Taken together, our results suggest that PsBPP has great potential in the animal feed industry for reducing phytates.

Expressed Protein Ligation of 5-Enolpyruvylshikimate-3-phosphate (EPSP) Synthase: An Application to a Protein Expressed as an Inclusion Body

  • Kim, Hak-Jun;Shin, Hee-Jae;Kim, Hyun-Woo;Kang, Sung-Ho;Kim, Young-Tae
    • Bulletin of the Korean Chemical Society
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    • v.28 no.12
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    • pp.2303-2309
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    • 2007
  • Expressed protein ligation (EPL) technique, joining recombinantly expressed proteins to polypeptides, has been widely adopted for addressing various biological questions and for drug discovery. However, joining two recombinant proteins together is sometimes difficult when proteins are expressed insoluble and unrefoldable, because ligation-active proteins via intein-fusion are obtainable when they are folded correctly. We overcame this limitation coexpressing target protein with additional methionine aminopeptidase (MAP) which enhances removal of the initiation methionine of recombinantly expressed protein. Our approach demonstrated that two domains of 46 kDa 5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase, a target of herbicide glyphosate, were successfully joined by native chemical ligation, although its C-terminal domain was expressed as an inclusion body. The intein-fused N-terminal fragment of EPSP synthase (EPSPSN, residues 1-237) was expressed and the ligation-active thioester tagged N-terminal fragment (EPSPSN-thioester) was purified using a chitin affinity chromatography and mercapto-ethanesulphonate (MESNA) as intein thiolysis reagent. Its Cterminal fragment (EPSPSC, residues Met237-238CYS-427), expressed as an inclusion body, was prepared from an additional MAP-expressing strain. Protein ligation was initiated by mixing ~1 mM of EPSPSN-thioester with ~2 mM of EPSPSCCYS (residues 238CYS-427). Also we found that addition of 2% thiophenol increased the ligation efficiency via thiol exchange. The ligation efficiency was ~85%. The ligated full-length EPSP synthase was dissolved in 6 M GdHCl and refolded. Circular dichroism (CD) and enzyme activity assay of the purified protein showed that the ligated enzyme has distinct secondary structure and ~115% specific activity compared to those of wild-type EPSP synthase. This work demonstrates rare example of EPL between two recombinantly expressed proteins and also provides hands-on protein engineering protocol for large proteins.

Effects of Loess on the Mycelial Pellet Formation of Phosphate Dissolving Fungus, Penicillium sp. GL-101 in the Submerged Culture (유리인산 생성균 Penicillium sp. GL-101의 액침배양중 Pellet 형성에 미치는 황토의 영향)

  • 강선철;이동규
    • KSBB Journal
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    • v.14 no.3
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    • pp.337-341
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    • 1999
  • In order to investigate effects of loess on the mycelial pellet formation a phosphate dissolving fungus, Penicillium sp. GL-101, was cultured in potato dextrose broth containing loess. The strain formed an amorphous pellet or loose aggregates agitated at a low speed(50rpm) while spherical and regular pellets at a high speed(150rpm). The higher concentration of loess, the smaller size of a pellet in the medium formed by the strain. Cultured in the medium supplemented with 1.5% loess the pellet size was reduced to a seventh compared to the control. In the case of addition of several insoluble salts, which are main components of loess, to the culture medium the higher concentrations of salts, the smaller sizes of pellet formed by the strain and the smallest pellet was formed by the addition of calcium sulfate.

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Biocontrol of Red Pepper Using Mixed Culture of Antagonistic Bacterium and Phosphate Solubilizing Yeast (항진균 세균과 난용성 인산염 가용화 효모의 혼합 배양액을 이용한 고추 병해의 생물학적 방제)

  • Lee, Gun Woong;Min, Byung-Dae;Park, Sujeong;Jheong, Weonhwa;Go, Eun Byeul;Lee, Kui-Jae;Chae, Jong-Chan
    • Korean Journal of Microbiology
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    • v.49 no.4
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    • pp.398-402
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    • 2013
  • This study was to investigate beneficial effects of microbial mixture on red pepper which was capable of promoting plant growth by solubilizing insoluble phosphate as well as protecting plants from pathogenic attack. Saccharomyces sp. L13 was isolated for phosphate solubilizing activity on aluminium phosphate, tricalcium phosphate, calcium hydrophosphate, and magnesium hydrophosphate. On the other hand, Bacillus sp. L32 was isolated for antagonistic activity against Phytophthora capsisi and Colletotrichum gloeosporioides, causing Phytophthora blight and Anthracnose disease in pepper, respectively. The strain L32 exhibited antagonistic activities both under dual culture assays and detached leaves assays. The each strain under the condition of mixed cultivation exhibited the same growth rates as one under pure cultivation. In greenhouse study, the mixed culture showed the both effect of plant growth promotion and reduction of disease symptom development against P. capsisi and C. gloeosporioides providing a potential as effective microbial agent for plant husbandry.

Optimization of Culture Conditions for Production of Helicobacter pylori Adhesin Protein Genetically Linked to Cholera Toxin A2B in Escherichia coli JM101

  • Kim, Byung-Oh;Pyo, Suh-Kneung
    • Biomolecules & Therapeutics
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    • v.9 no.3
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    • pp.162-166
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    • 2001
  • Helicobacter pylori is a major cause of gastric-associated diseases. In our previous study, the Adhesin/CTXA2B was expressed as insoluble recombinant chimeric protein derived from the H. pylori adhesin genetically coupled to CTXA2B subunit in Escherichia coli. Since it is very important to optimize IPTG concentration, culture temperature and composition of medium to maximize cell growth and productivity, these conditional growth factors were determined for increasing the productivity of the expressed Adhesin/CTXA2B chimeric protein in Escherichia coli JM101 carrying pTEDhpa/ctxa2b. Our data demonstrate that optimal medium for increased production of chimeric protein was a YCP/Glu medium composed of 2% yeast extract, 1% casamino acid, phosphate solution [0.3% $KH_2P0_4$, 0.4% $Na_2HP0_4$, 0.25% ($NH_4)_2HPO_4$], and 0.5% glucose. In addition, optimal concentration of IPTG was 1 mM and culture temperature, $37^{\circ}C$.

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Organic acid production and phosphate solubilization by Enterobacter intermedium 60-2G (Enterobacter intermedium 60- 2G의 유기산 생성과 불용성인의 가용화)

  • Kim, Kil-Yong;Hwangbo, Hoon;Kim, Yong-Woong;Kim, Hyo-Jeong;Park, Keun-Hyung;Kim, Young-Cheol;Seong, Ki-Young
    • Korean Journal of Soil Science and Fertilizer
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    • v.35 no.1
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    • pp.59-67
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    • 2002
  • A phosphate solubilizing bacterium. strain 60-2G, possessing a strong ability to solubilize insoluble phosphate was isolated from the rhizosphere of grass. On the basis of GC-FAME profile, carbon utilization pattern, and the DNA sequence of a conserved partial 16S rRNA gene, the 60-2G was identified as Enterobacter intermedium. The analysis by HPLC revealed that the strain 60-2G produced mainly gluconic and 2-ketogluconic acids with small amounts of lactic acid in broth culture medium containing hydroxyapatite. During the incubation period of the strain 60-2G in broth culture, pH of the medium decreased upto 3.8 while the soluble phosphate concentration increased. The reversed correlation between pH and soluble phosphate concentration indicated that the solubility of P was due to the produced organic acids. The sequence homology of the deduced amino acids suggested that E. intermedium 60-2G synthesized PQQ which is essential for the oxidation of glucose by glucose dehydrogenase.

Phosphate Solubilization and Plant Growth Promotion by Crop Associated Bacteria (인산용해미생물에 의한 불용성 인의 용해와 식물생장에 미치는 영향)

  • Na, Jung-Heang;Choi, Jin-Ho;Jin, Rong-De;Ko, Hyun-Sun;Park, Ro-Dong;Kim, Kil-Yong
    • Korean Journal of Soil Science and Fertilizer
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    • v.42 no.1
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    • pp.29-36
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    • 2009
  • Fourteen bacterial strains were isolated from crop rhizosphere and identified as phosphate solubilizing bacteria (PSB) by 16S rRNA analysis. Only 3 strains exhibited a strong ability to solubilize insoluble phosphate in agar medium containing a hydroxyapatite. The rates of P solubilization by isolates were ranged from 200 and $2300\;mg\;L^{-1}$, which are inversely correlated with pH in culture medium. Furthermore, HPLC analyses reveal the production of organic acid from the culture filtrates of PSB. Among these, strain Acinetobacter sp. released only gluconic acid, Pseudomonas orientalis produced gluconic acid which was subsequently converted into 2-ketogluconic acid, and Enterobacter asburiae released acetic acid and succinic acid. On the other hand, P. orientalis and E. asburiae released $372\;mg\;L^{-1}$ and $191\;mg\;L^{-1}$ of IAA into broth culture, respectively, while Acinetobacter sp. did not produce IAA. Furthermore, in vivo study showed that plant growth promoting effect by bacteria generally seemed to be increased IAA production and phosphate solubilization.

Effect of Recombinant Olive Flounder Stanniocalcin on Serum Calcium Levels (혈청 칼슘 농도에 미치는 넙치 유전자 재조합 스타니오칼신의 효과)

  • Shin, Ji-Hye;Jung, Yu-Jung;Han, Yoon-Hee;Lee, Kyun-Young;Lee, Kyung-Mi;Kaneko, Toyoji;Sohn, Young-Chang
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.43 no.4
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    • pp.307-313
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    • 2010
  • Stanniocalcin 1 (STC1) is a glycoprotein hormone that is important in the maintenance of calcium and phosphate homeostasis in both fish and mammals. STC1 and its paralog STC2 are expressed in multiple tissues in fishes, although the physiological roles of piscine STCs are still unclear compared with those of mammals. In this study, we cloned olive flounder STC1 (ofSTC1) and ofSTC2 cDNAs into pET28a vector and used E. coli Rosetta (DE3) as the host strain for protein expression. Expression experiments were carried out using isopropyl-$\beta$-D-thiogalactoside (IPTG) and nickel affinity chromatography. We could identify the recombinant proteins as single 29.5 kDa (ofSTC1) and 33.2 kDa (ofSTC2) bands in the insoluble fraction on sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE). These results indicate that ofSTC1 and ofSTC2 were expressed as insoluble proteins in E. coli. Furthermore, the injection of ofSTC1 protein into juvenile tilapia resulted in a decrease of the serum calcium level. These results suggest that the purified fish STC1 and STC2 proteins may be used to elucidate the physiological role of STCs in fishes.