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REFERENCE LINKING PLATFORM OF KOREA S&T JOURNALS
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Journal of Microbiology and Biotechnology
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Journal DOI :
The Korean Society for Applied Microbiology and Biotechnology
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Volume & Issues
Volume 11, Issue 6 - Dec 2001
Volume 11, Issue 5 - Oct 2001
Volume 11, Issue 4 - Aug 2001
Volume 11, Issue 3 - Jun 2001
Volume 11, Issue 2 - Apr 2001
Volume 11, Issue 1 - Feb 2001
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Proteomics and Microarrays in Cancer Research
Kondabagil, Kiran-Rojanna ; Kwon, Byoung-Se ;
Journal of Microbiology and Biotechnology, volume 11, issue 6, 2001, Pages 907~914
A whole genome analysis for monitoring specific changes in gene expression, using microarrays or proteome profiling of the same, are the two tools that have already revolutionized current approaches for studying disease. These methods are particularly important in cancer research as there are many overexpressed genes, and their products remain uncharacterized. This article presents a general overview of these technologies and their applications for studying cancer.
Enhanced and Targeted Expression of Fungal Phytase in Saccharomyces cerevisiae
LIM, YOUNG-YI ; EUN-HA PARK ; JI-HYE KIM ; SEUNG-MOON PARK ; HYO-SANG JANG ; YOUN-JE PARK ; SEWANG YOON ; MOON-SIK YANG ; DAE-HYUK KIM ;
Journal of Microbiology and Biotechnology, volume 11, issue 6, 2001, Pages 915~921
Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals, and reduces the phosphorus pollution of animal waste. In order to express a high level of fungal phytase in Saccharomyces cerevisiae, various expression vectors were constructed with different combinations of promoters, translation enhancers, signal peptides, and terminator. Three different promoters fused to the phytase gene (phyA) from Aspergillus niger were tested: a galactokinase (GAL1) promoter, glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter, and yeast hybrid ADH2-GPD promoter consisting of alcohol dehydrogenase II (ADH2) and a GPD promoter. The signal peptides of phytase, glucose oxidase (GO), and rice amylase 1A(RAmy1A) were included. Plus, the translation enhancers of the
sequence and UTR70 from the tobacco mosaic virus (TMV) and spinach, respectively, were also tested. Among the recombinant vectors, pGphyA06 containing the GPD promoter, the
sequence, RAmy1A, and GAL7 terminator expressed the highest phytase activity in a culture filtrate, which was estimated at 20 IU/ml. An intracellular localization of the expressed phytase activity in a culture filtrate, which was estimated at 20 IU/ml. An intracellular localization of the expressed phytase was also performed by inserting an endoplasmic reticulum (ER) retention signal, KDEL sequence, into the C-terminus of the phytase within the vector pHphyA-6. It appeared that the KDEL sequence directed most of the early expression of phytase into the intracellular compartment yet more than
of the total phytase activity was still retained within the cell even after the prolonged (>3 days) incubation of the transformant. However, the intracellular enzyme activity of the transformant without a KDEL sequence was as high as that of the extracellular one, thereby strongly suggesting that the secretion of phytase in S. cerevisiae appeared to be the rate-limiting step for the expression of a large amount of extracellular recombinant phytase, when compared with other yeasts.
Certification of Gibroblase Cell Adhesion and Spreading Mediated by Arg-Gly-Asp (RGD) Sequence on Thermo-Reversible Hydrogel
NA, KUN ; DONG-WOON KIM ; KEUN-HONG PARK ;
Journal of Microbiology and Biotechnology, volume 11, issue 6, 2001, Pages 922~927
In an effort to regulate the mammalian cell behavior in entrapment with a gel, we have functionalized hydrogels with the putative cell-binding (-Arg-Gly-Asp-)(RGD) domain. An adhesion molecule of Gly-Arg-Gly-Asp-Ser (GRGDS) peptides, a cell recognition ligand, was induced into thermo-reversible hydrogels, composed of N-isopropylacrylamide with small amounts of acrylic acid (typically 2-5
in feed), as a biomimetic extracellular matrix (ECM). The GRGDS containing a p(NiPAAm-co-AAc) copolymer gel was studied in vitro for its ability to promote the spreading and viability of cells by introducing a GRGDS sequence. Hydrogel with no adhesion molecule was a poor ECM for adhesion, permiting spreading of only
of the seeded cells for 36h. By immobilizing the peptide linkage into the hydrogel, the conjugation of RGD promoted
of proliferation for 36h. However, the GREDS sequence, nonadhesive peptide linkage, conjugated hydrogel showed only
of the seeded cell for the same time period. In addition, with the serum-free medium, only GRGDS peptides conjugated to hydrogel was able to promotecell spreading, while there was no cell proliferation in the hydrogel without GRGDS. Thus, the GRGDS peptide-conjugated thermo-reversible hydrogel specifically mediated the cell spreading. This result suggests that utilization of peptide sequences conjugating with the cell-adhesive motifs can enhance the degree of cell surface interaction and influence the long-term formation of ECM in vitro.
Degradation of Polyvinyl Alcohol by Brevibacillus laterosporus: metabolic Pathway of Polyvinyl Alcohol to Acetate
Lim, Joong-Gyu ; Park, Doo-Hyun ;
Journal of Microbiology and Biotechnology, volume 11, issue 6, 2001, Pages 928~933
Approximately 0.1 mg/ml of polyvinyl alcohol (PVA) was degraded by the growing cell, Brevibacillus laterospours, for 30 h, and 0.2 mg/ml of PVA was degraded by the cell-free extract that was isolated from Brevibacillus laterosporus. Approximately
/ml of acetic acid was produced from PVA by using the cell-free extract as a catalyst for 40 min.
value of purified PAV-degradation enzyme was 3.75g/l and 2.75 g/l/min in reaction with EDTA and 3.99 g/l and 2.98 g/l/min in reaction without EDTA, respectively. Molecular weight of the purified enzyme determined by SDS-PAGE was 63,000 Da. Alcohol dehydrogenase and aldehyde dehydrogenase activities were qualitatively detected on a native acrylamide gel by an active staining method, indicating the existence of the metabolic pathway to use PVA as a substrate.
Inhibitory Effects of Acetic Acid and Temperature on Growth of Campylobacter jejuni ATCC 33291
Kim, Wang-june ; Shin, Soon-Young ; Hwang, Han-Joon ;
Journal of Microbiology and Biotechnology, volume 11, issue 6, 2001, Pages 934~939
The growth inhibition of Campylobacter jejuni ATCC 33291 in the presence of
acetic acid at 4, 25, and
, followed by
, at pH 5.5 and pH 6.5, and by the addition of
acetic acid aat 4, 25, and
were determined to be 22, 8.5, and 1.4 min, respectively, in an FBP-SBB medium. The D values of C. jejuni were increased by the addition of chicken and did not follow the linear relationship observed in the FBP-SBB media without chicken. When using distilled water instead of FBP-SBB in the model system, the death rate of C. jejuni was dramatically accelerated. The injured or low cell numbers that were impossible to enumerate using the plate count method, were detected by a polymerase chain reaction and enrichment culture procedure. These results suggested that acetic acid is reliable and effective as a disinfectant, however, it is necessary to take additional care at refrigeration temperatures due to the potential of injred cells during poultry processing.
Estimation of Distribution of a Commensal Thermophile in Soil by Competitive Quantitative PCR and Terminal Restriction Fragment Length Polymorphism Analysis
Rhee, Sung-Keun ; Hong, Seung-Pyo ; Bae, Jin-Woo ; Jeon, Che-Ok ; Lee, Seung-Goo ; Song, Jae-Jun ; Poo, Ha-Ryoung ; Sung, Moon-Hee ;
Journal of Microbiology and Biotechnology, volume 11, issue 6, 2001, Pages 940~945
Symbiobacterium toebii has been previously reported as a novel commensal thermophile exhibiting a commensal interaction with thermophilic Geobacillus sp. SK-1. We investigated the distribution of this commensal thermophile in various soils using molecular methods, such as quantitative PCR and terminal restriction fragment polymorphism analysis. Based on a nested competitive quantitative PCR the 16S rDNA of the commensal thermophile was only detected in compost soils at about
cpoies per gram of soil, corresponding to
cells per gram of soil. However, in an enrichment experiment at
copies of 16S rDNA molecules were detected per ml of enriched culture broth for all the soils, and more than 0.1 mM indole accumulated as the product of commensal bacterial growth. When incubated at
, neither the 16S rDNA of the commensal bacterium nor any indole accumulation was detected. Accordingly, even though the 16S rDNA of the bacterium was only detected in the compost soils by a nested PCR, the presence of the 16S rDNA molecules of commensal thermophile and accumulation of indole in all the enriched cultures appeared to indicate that the commensal thermophile is widely distributed in various soils.
Purification and Characterization of Extracellular and Intracellular Glutamine Synthetases from Mycobacterium bovis BCG
SUH, CHANG-IL ; JUN-MAN LIM ; HA-CHIN SUNG ;
Journal of Microbiology and Biotechnology, volume 11, issue 6, 2001, Pages 946~950
Slow-growing pathogenic mycobacterium species, including Mycobacterium bovis BCG, secrete a large amount of glutamine synthetase into culture media. Extracellular and intracellular glutamine synthetases were purified from M. bovis BCG. While the native molecular weights of both glutamine synthetases were estimated to be 370.2 kDa, those of the subunits were 61.7 kDa, indicating that the native forms were composed of 6 subunits. The enzymes showed a hhigh thermal stability and high degree of sequence similarity with the glutamine synthetase from M. tuberculosis in the N-terminal amino acid sequence. Western blotting analysis indicated that the antibodies prepared against both the extracellular and intracellular enzymes exhibited common antigen determinants.
Purification and Characterization of Lipase from Trichosporon sp. Y-11and Its Use in Ester Synthesis of Unsaturated Fatty Acids and Alcohols
Song, Xin ; Qu, Yinbo ; Shin, Dong-Hoon ; Kim, Eun-Ki ;
Journal of Microbiology and Biotechnology, volume 11, issue 6, 2001, Pages 951~956
A 28-kDa extracellular lipase (pI 8.7) was purified to homogeneity from the culture supernatant of Trichosporon sp. Y- 11 by mmonium sulfate precipitation, DEAE-Sephadex A-50, Bio-Gel P-30, CM- Sephadex C-50, and Bio-Gel P- 10 chromatographies. The purified enzyme exhibited a specific activity of
based on the hydrolysis of triolein, and the optimal hydrolysis activity was dentified at pH 8.0 and
. The enzyme activity was inhibited by
and enhanced by
. The enzyme activity exhibited for the hydrolysis of both tributyrin and trilinolein. The ester synthesis of unsaturated fatty acids with various alcohols catalyzed by the purified lipase in a nonaqueous medium or microaqueous system was also investigated. The esterification activity of the lipase increased with an increase of the carbon chain length in the alcohol. The synthesis rate of linoleic acid and oleyl alcohol was the highest with an optimal temperature and pH of
and 8.0, respectively. The water content and agitation also affected the esterification activity of the lipase.
Purification and Characterization of Streptococcus mutans Cell Wall Hydrolase from Bacillus subtilis YL-1004
OHK, SEUNG-HO ; YUN-JUNG YOO ; DONG-HOON BAI ;
Journal of Microbiology and Biotechnology, volume 11, issue 6, 2001, Pages 957~963
Bacillus subtilis YL-1004 was isolated from soil for the development of agents to control dental caries. This strain produced an extracellular lytic enzyme that hydrolyzed the Streptococcus mutans cell wall. The lytic enzyme was purified to homogeneity by affinity chromatography and gel permeation chromatography to give a single band on SDS-PAGE and non-denaturing polyacrylamide gel electrophoresis. The molecular weight of the enzyme was deduced from SDS-PAGE and gel chromatography to be 38 kDa and the PI to be 4.3 from isoelectric focusing. Sirty
of its lytic activity remained after incubation at
for 30 min, and its optimal temperature was
. The enzyme showed its highest activity at pH 8.0 and was stable at pHs ranging from 4.0 to 9.0. Treatment with several modifiers showed that a cysteine residue was involved in the active site of the enzyme. This lytic enzyme from Bacillus subtilis YL-1004 exhibited specificity towards Streptococci and also showed autolytic activity on Bacillus subtilis YL-1004.
Biosorption of Copper by Immobilized Biomass of Pseudomonas stutzeri
Cho, Ju-Sik ; Hur, Jae-Seoun ; Kang, Byung-Hwa ; Kim, Pil-Joo ; Sohn, Bo-Kyoon ; Lee, Hong-Jae ; Jung, Yeun-Kyu ; Heo, Jong-Soo ;
Journal of Microbiology and Biotechnology, volume 11, issue 6, 2001, Pages 964~972
The kinetics of copper ion biosorption by Pseudomonas stutzeri cells immobilized in alginate was investigated. During the first few minutes of the metal uptake, the copper biosorption was rapid and then became progressively slower until an equilibium was rapid, and then became progressively slower until an equilibrium was reached. At a biomass concentration of 100g/l, the copper biosorption reaction reached approximately 90% of the equilibrium position within 30 min. A Freundich-type adsorption isotherm model was constructed based on kinetics with different amounts of biomass. When using this model, the experimental values only agreed well with the predicted values in a solution containing less than 200 mg/l Cu(II). Desorption of the bound copper ions was achieved using electrolytic solutions of HCl,
, EDTA, and NTA (0.1 or 0.5 M). Metal desorption with 0.1 M NTA allowed the reuse of the biosorbent for at least ten consecutive biosorption/desorption cycles, without an apparent decrease in its metal biosorption capability. A packed-bed column reactor of the immobilized biomass removed approximately 95% of the metal in the first 30 liter of wastewater [containing 100 mg/l Cu(II)] delivered at a rate of 20 L/day, and, thereafter, the rate gradually decreased.
Molecular Cloning and Expression of a Sodium-Driven Flagellar Motor Component Gene(motX) from Vibrio fluvialis
Park, Je-Hyeon ; Lee, Jong-Hee ; Kim, Young-Sook ; Hong, Yong-Ki ; Kong, In-Soo ;
Journal of Microbiology and Biotechnology, volume 11, issue 6, 2001, Pages 973~978
The bacterial flagellar motor is a molecular machine that couples proton or sodium influx to force generation, mostly for driving rotation of the helical flagellar filament. In this study, we cloned a gene (motX) encoding a component of the sodium-driven flagellar motor from Vibrio fluvialis. The nucleotide sequence of the motX gene, composed of 633 bp and 211 amino acid residues, was determined. Overexpression of the motX gene in Escherichia coli using a strong promoter induced growth inhibition and cell lysis. The lethal effect of E. coli was suppressed by adding amiloride, as a potent inhibitor for the sodium channel. Electron microscopic observation of the expressed protein indicated that MotX protein induced by isopropyl
-D-thiogalactopyranoside caused the lysis of host cell.
Fluorescence Immunoassy of HDL and LDL Using Protein A LB Film
Choi, Jeong-Woo ; Park, Jun-Hyo ; Lee, Woo-Chang ; Oh, Byung-Keun ; Min, Jun-Hong ; Lee, Won-Hong ;
Journal of Microbiology and Biotechnology, volume 11, issue 6, 2001, Pages 979~985
A fluorometric detection technique for HDL (High Density Lipoprotein) and LDL (Low Density Lipoprotein) was developed for application in a fiber-optic immunosensor using a protein A Langmuir-Blodgget (LB) film. For the fluorescence immunoassay, antibodies specific to HDL or LDL were imobilied on the protein A LB film, and a fluorescence amplification method was developed to overcome their weak fluorescence. The deposition of protein A using the LB technique was monitored using a surface pressure-are
curve, and the antibody immobilization of the protein A LB film was experimentally verified. The immobilized antibody was used to separate only HDL and LDL from a sample, then the fluorescence of he separated HDL or LDL was amplified. The amount of LDL or HDL was measured using the developed fiber optic fluorescence detection system. The optical properties resulting from the reaction of HDL or LDL with o-phtaldialdehyde, detection range, response time, and stability of the immunoassay were all investigated. The respective detection ranges for HDL and LDL were sufficient to diagnose the risk of coronary heart disease. The amplification step increased the sensitivity, while selective separation using the immobilized antibody led to linearity in the sensor signal. The regeneration of the antibody-immobilized substrate could produce a stable and reproducible immunosensor.
Translation Initiation Factor IF1-Dependent Stimulation of 30 S Preinitiation Complex Formation: Rapid Isolation and fMEt-tRNA Binging Activity of IF1
CHOIK, SANG-YUN ; HYUN-JUNG KIM ; JUNG-IK YANG ; HYO-IL CHANG ;
Journal of Microbiology and Biotechnology, volume 11, issue 6, 2001, Pages 986~993
Translation Initiation in prokaryotes involves the formation of a 30 S preinitiation complex, in which translation initiation factors play a role in the stimulation of fMet-tRNA (fMet) binding. However, the specific function and precise mechanism of initiation factor IF1 are still unclear. One a functionally active factor with a high purity. In the present study a large quantity of active IF was rapidly purified, obtained by the overexpression of the infA gene, and then used for a functional study. The induction of infA did not appreciably affect the growth rate of the protease-deficient strain E. coli AR68 harboring the IF1 overproducing plasmid. The level of IF1 obtained was approximately
of the total cell protein, which enabled the yield of highly purified IF1 (>
pure) to be increased to 0.15 mg of IF1/g of cells. The IF1 was isolated within one day by the centrifugatioin of the ribosomal washed fraction, by ammonium sulfate fractionation, chromatography on batch of phosphocellulose, and FPLC Mono S. The overexpressed IF1 was found to be comparable to the factor isolated from normal cells, as determined by migration in NEPHGE/SDS 2-D gels. For binding of fMet-tRNA(fMet) to the 30 S ribosomal subunitis, relatively high levels of binding were obtained when IF2 was present. The addition of IF1 up to 110 pmol proportionally stimulated the binding to a variable extent. This IF1-dependent stimulation of the 30 S preinitiation complex formation demonstrated that IF1 would appear to be exclusively essential for promoting the initiation phase of protein synthesis.
Removal Behavior of Biological Nitrogen and Phosphorus and Prediction of Microbial Community Composition with Its Function, in an Anaerobic-Anoxic System form Weak Sewage
LEE, JIN WOO ; EUI SO CHOI ; KYUNG IK GIL ; HAN WOONG LEE ; SANG HYON LEE ; SOO YOOUN LEE ; YONG KEUN PARK ;
Journal of Microbiology and Biotechnology, volume 11, issue 6, 2001, Pages 994~1001
An easier way of understanding the BNR system was proposed from the study on substrate, nutrient removal tendency, microbial community and its metabolic function by applying the municipal settled sewage. During the anaerobic period, the phosphorus release rate per VFACOD we varied depending on the phosphorus content in the sludge. When the phosphorus content in the sludge was
VSS, according to influent VFACOD, the phosphorus release rate and PHA production were
and 1.0 gPHA/gVFACOD, respectively. The
requirement for the phosphorus uptake as an electron acceptor was about
based on the proposed equation with PHA, biomass, production, and the concentration of phosphorus release/uptake. Bacterial-community analysis of the sludge, as determined by FISH and 16SrDNA characterization FISH, revealed that the beta-subclass proteobacteria were the most abundant group (
of the proteobacteria-specific probe EUB338), and it was likely that representative of the beta-subclass played key roles in activated sludge. The next dominant group found was the gamma-protebacteria (
of probe EUB338). 16S rDNA clone library analysis showed that the members of
-proteobacteria were also the most abundant groups, and
(PN2 and PN4) and
(PN1 and PN5) of total clones were the genera of denitrifying bacteria and PAO, respectively. Prediction of the microbial community composition was made with phosphorus content (Pv,
P/VSS) in wasted sludge and profiles of COD, PHA,
in an anaerobic-anoxic SBR unit. Generally, the predicted microbial composition based upon metabolic function, i.e., as measured by stoichiometry, is fairly similar to that measure by the unculturable dependent method. In this study, a proposal was made on he microbial community composition that was more easily approached to analyze the reactor behavior.
Purification and Characterization of a Regulatory Protein XyIR in the D-Xylose Operon from Escherichia coli
Shin, Jae-Ho ; Roh, Dong-Hyun ; Heo, Gun-Young ; Joo, Gil-Jae ; Rhee, In-Koo ;
Journal of Microbiology and Biotechnology, volume 11, issue 6, 2001, Pages 1002~1010
The D-xylose operon in Escherichia coli is known to be regulated by a transcriptional activator protein, XyIR, which is responsible for the expression of both xylAB and xylFGH gene clusters. The XyIR was purified to homogeneity by using the maltose binding protein fusion expression and purification systems involving two chromatography steps. The purified XyIR protein was composed of two subunits of 45 kDa, which was determined by both sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration. The purified XyIR was specifically bounded to the xylA promoter, regardless of adding xylose to the reaction mixture, but binding of XylR was specifically bounded to the xylA promoter, regardless of adding xylose to the reaction mixture, but binding of XylR to the xylA promoter was enhanced by adding xylose. The enhanced binding ability of XyIR in the presence of xylose was not diminished by adding glucose. The presumed XyIR binding site is located between 120 bp to 100 bp upstream the xylA initiation codon.
Purification and Characterization of a Cytochrome P-450 from Pravastatin-Producing Streptomyces sp. Y-110.
Park, Joo-Woong ; Lee, Joo-Kyung ; Kwon, Tae-Jong ; Yi, Dong-Hee ; Park, Yong-Il ; Kang, Sang-Mo ;
Journal of Microbiology and Biotechnology, volume 11, issue 6, 2001, Pages 1011~1017
Streptomyces sp. Y-110 cytochrome P-450, induced by the addition of compactin -Na into the culture medium, was purified from the cell extract to apparent homogeniety, mainly by DEAE-Sepharose, hydroxyapatite, and Mono Q column chromatyography. The sepcific activity of purified enzyme on its substrate, compactin-Na, was determined to be 15 nmol of pravastatin per mg protein. The molecular mass of this enzyme on SDS-PAGE was
kDa, pI was 4.5, and its CO difference spectrum showed maximum absorption peaks at 452 and 550nm, respectively. The N-terminal amino acid sequence was determined to be Met>Thr>Cys>Thr>Pro>Val>Thr>Val>The>Gly>Ala>Ala>Gly>Gln>Ile>Gly>Tyr>Ala>Leu. Its apparent
on compactin-Na was
. The maximum substrate concentration (
) for reaction was
. These physicochemical characteristics and kinetic behavior of this enzyme were compared and shown to be different from those of Streptomyces cytochrome P-450 enzymes reported, suggesting that this enzyme may be an additional member of the Streptomyces cytochrome P-450 family.
Production of Oleamide, a Functional Lipid, by Streptomyces sp. KK90378
Kwon, Jeong-Ho ; Hwang, Sung-Eun ; Han, Jae-Taek ; Kim, Chang-Jin ; Rho, Jung-Rae ; Shin, Jong-Eon ;
Journal of Microbiology and Biotechnology, volume 11, issue 6, 2001, Pages 1018~1023
Oleamide (cis-9-octadecenamide) is endogenous primary amide of fatty acid that is produced in small amounts in animal brains. It is known to induce sleep and to lower temperature by destroying the lipid plasma membrane structure of cells, thereby disclosing gap junction channels. To develop a new biological production method for oleamide, a screening program was conducted to isolate a microorganism producing oleamide. Among 1,500 soil microorganisms tested, KK90378 exhibited a potent positive reaction with Dragendoff`s reagent, used to detect the primary amide of oleamide. KK90378 was identified as a Streptomyces species based on cultural and morpohological characteristics, the presence of diaminopimelic acid in the cell wall, and the sugar patterns for the whole-cell extrat. Streptomyces sp. KK90378 produced oleamide 3 days after culture at
, pH 7.2 A series of purification steps, including hexane extraction, silica gel column, and preparative thin layer chromatographies, were performed for the purification of oleamide. A spectrophotometric analysis using
-NMR, and GC-MS confirmed that the chemical structure of the purified oleamide was identical to that of authentic oleamide.
Astaxanthin Production by Haematococcus pluvialis under Various Light Intensities and Wavelengths
Park, Eun-Kyung ; Lee, Choul-Gyun ;
Journal of Microbiology and Biotechnology, volume 11, issue 6, 2001, Pages 1024~1030
The key factors for high-density Haematococcus pluvialis cultures and conditions for astaxanthin induction were examined to maximize astaxanthin production. Light intensity was found to be the most important factor, and thus experiments were found to be the most important factor, and thus experiments were carried out using different light sources and intensities. A high cell density of over 2.7 g/l was obtained at
, whereas a much lower cell concentration (<1.0 g/ 1) was obtained with lower light intensities
. A high light intensity and the supplement of 470 nm photons had a more dramatic effect on the final astaxanthin concentration and per cell astaxanthin content. A maximum astaxanthin concentration of 6.5 mg/l was obtained at a light intensity of
, whereas only 1.3 and 0.7 mg/l were obtained at 30 and
, respectively. A supplement of 470 nm photons enhanced the carotenoid and chlorophyll formation.
Expression Analysis of
-Ketothiolase and Acetoacetyl-CoA Reductase of Rhodobacter sphaeroides
KHO, DHONG HYO ; CHEOL YUN JEONG ; JEONG JUG LEE ;
Journal of Microbiology and Biotechnology, volume 11, issue 6, 2001, Pages 1031~1037
By a sequential action of
-ketothiolase and acetoacetyl-CoA reductase, two molecules of acetyl-CoA re converted into D-3-hydroxybutyryl-CoA, a substrate for PHB synthase to form poly-3-hydroxybutyryl-CoA, a substrate for PHB synthase to form poly-3-hydroxybutyrate (PHB) of rhodobacter sphaeroides. The
-ketothiolase gene, phbA, and acetoacetyl-CoA reductase gene, phbB, were cloned and analyzed for their expression. Enzyme activities of
-ketothiolase and acetoacetyl-CoA reductase showed constitutive levels during aerobic and photoheterotrophic growth of R. sphaeroides. In addition, no difference of each enzyme activity was observed between cells grown aerobically and photoheterotrophically. The constitutive level of the enzyme activities are regulated according to the growth phases along with growth conditions. Thus, phbAB expression is not determinative in regulating the PB content. On the other hand, phbA-deleted cell AZI accumulated only
PHB of the wild-type, and an elevated dosage of phbAB in trans in R. sphaeroides resulted in a higher content of PHB, indicating that phbAB codes for the enzymes responsible for providing the main supply of subsyrate for PHB synthase. PHB formation by an alternative pathway that does not does not depend on the phbA-and phbB-coding enzymes is also proposed.
Bioremediation of Diesel-Contaminated Soil by Bacterial Cells Transported by Electrokinetics
LEE, HYO-SANG ; KISAY LEE ;
Journal of Microbiology and Biotechnology, volume 11, issue 6, 2001, Pages 1038~1045
The electrokinetic technology was applied in bioremediation for the purpose of supplying a Pseudomonas strain capable of degrading diesel to contaminated soil bed, and their biodegradation of diesel was carried out after a desired cell distribution was obtained. Electrokinetic injection of the strain was made possible because the cells acted as negatively charged particles at neutral pH, and thus the cells were transported with a precise directionality through the soil mostly by the mechanism of electrophoresis and in part by electroosmosis. A severe pH change in the soil bed was formed due to the penetration of electrolysis products, which was harmful to the cell viability and cell transport. To achieve a desirable cell transport and distribution, the control of pH in soil bed by a recirculating buffer solution in electrode chambers was essential during the appliation of an electric field. The judicious selections of electrolyte concentration and conductivity were also important for achieving an efficient electrokinetic cell transport since a higher electrolyte concentration favored the maintenance of pH stability in soil bed, but lowered electrophoretic mobility on the other hand. With electrolyte solution of pH 7 phosphate buffer, a 0.05 M concentration showed a better cell transport buffer, a 0.05 M concentration showed a better cell transport than 0.02 M and 0.08 M. The cell under pH 8 were obtained, compared to the cells under pH 7 or pH 9 in a given time period Up to
of diesel was degraded in 8 days by the Pseudomonas cell, which were distributed electrokinetically under the conditions of pH 8 (
, a mixture of phosphate and ammonia buffers) and 40 mA in a soil bed of 15 cm length.
16S/23S Intergenic Spacer Region as a Genetic Marker for Thiobacillus thiooxidans and T.ferrooxidans
Lee, Hye-Won ; Choi, Won-Young ; Cho, Kyung-Suk ; Choi, Won-Ja ;
Journal of Microbiology and Biotechnology, volume 11, issue 6, 2001, Pages 1046~1054
Bioleaching is the process in which insoluble metal sulfide is oxidized by specialized iron- and/or sulfur-oxidizing lithotrophic bacteria in acidic, metal-rich environments. Most of these processes are carried out by the genus Thiobacillus. Three novel Thiobacillus strains (Thiobacillus thiooxidans AZ11, Thiobacillus thiooxidans MET, and thiobacillus thiooxidans TAS) associated with bioleaching have been isolated from soil and sludge (Korean patent No. 1999-0073060 for T. thiooxidans AZ11, Korean patent No. 1999-0005798 for T. thiooxidans MET, and Korean patent No. 1999-0073059 for T. thiooxidans TAS). A partial sequence of 16S ribosomal RNA gene (16S rDNA) and the entire sequence of 16S/23S intergenic spacer region (ISR) were determined in the three above novel strains and in Thiobacillus ferrooxidans ATCC19859 as a reference strain. When phylogenetic analysis was performed based on G+C contents and sequence alignments, T. ferroxidans ATCC19859 was found to be closely related to previously registered T. ferrooxidans strains in a monophyletic manner, while the three novel T. thiooxidans strains were classified in a paraphyletic manner. Close examination on the base composition of 16S/23S ISR revealed that the 5\` part (nucleotide residues 21-200) was specific for the genus Thiobacillus. On the other end, the 3\` part (nucleotide residues 201-520) showed specificity in T. ferrooxidans strains, but not in T. thiooxidans strains. These results suggest that the proximal and distal halves of 16S/23S could be used as a genetic marker for the identification of the genus Thiobacillus and the species T. ferrooxidans, respectively.
Application of Oxygen Uptake Rate Measured by a Dynamic Method for Analysis of Related Fermentation Parameters in Cyclosporin A Fermentation:Suspended and Immobilized Cell Cultures
Chun, Gie-Taek ; Agathos, S.N. ;
Journal of Microbiology and Biotechnology, volume 11, issue 6, 2001, Pages 1055~1060
Experimental data for the on-line estimation of cell concentration and growth rate are presented. For this purpose, we utilized the on-line calculation of the oxygen uptake rate (OUR), which was derived from a liquid phase dynamic mass balance for the oxygen during the active growth phase in cyclosporin A (CyA) fermentation. The cell yield coefficient, based on the oxygen
for both suspended and immobilized cells of Tolypocladium inflatum, was estimated as
from a very good linear correlation between the cell mass produced and the total oxygen consumed. The calculated yield showed a good agreement with the value of
generated from the correlation between the cell growth rate and the oxygen uptake rate. In addition, further experimental data are given, which were also applied to determine the specific oxygen uptake rate of T. inflatum cells during the exponential phase of CyA fermentation. A theoretical basis for the analysis of these fermentation parameters is also provided.
Effect of Scutellariae Radix as a Novel Antibacterial Herb on the ppk(Polyphosphate Kinase) Mutant of Salmonella typhimurium
Hahm, Dae-Hyun ; Yeom, Mi-Jung ; H.Lee, Eun-Joo ; Shim, In-Sop ; Lee, Hye-Jung ; Kim, Hong-Yeoul ;
Journal of Microbiology and Biotechnology, volume 11, issue 6, 2001, Pages 1061~1065
The antibacterial effects of water extracts of Scutellariate Radix (a dried root of Scutellaria baicalensis GEORGI) and its major flavonoid components, Baicalin and Baicalein, on Salmonella typhimurium, a representative enteric pathogen, were studied. Through a Kriby-Bauer disc analysis, the growth-inhibition activity of Scutellariae Radix against. S. typhimurium was found to be compatible with commercial antibiotics, such as ampicillin, chloramphenicol, and streptomycin. In contrast, the growth of a nonpathogenic E. coli strain was unaffercted by Scutellariae Radix. To examine the effect of polyphosphate kinase (ppk), a putative virulence factor, on the antibacterial activity of Scutellariae Radix, the growth profile of a ppk mutant of S. typhimurium was investigated in a tryptic soy broth containing different concentrations of water extracts of Scutellariae Radix. The ppk mutant was able to grow in 6 mg/ml of water extracts of Scutellariae Radix, whereas in 6 mg/ml of water extracts of Scutellariae Radix, whereas the wild-type could not, implying that the inactivation of ppk made S. typhimurium more resistant to the antibacterial activity of Scutellariae Radix. No enhanced resistance was observed in a ppk mutant of S. typhimurium complemented with a ppk expression vector. The attenuation of the virulence by ppk inactivation was also observed in a virulence assay using BLAB/c mice. Neither Baicalin nor Baicalein exhibited any growth-inhibition activity against S. typhimurium. The water extracts of Scutellariae Radix stimulated the transcription of ppk, especially in the early growth-stage of S. typhimurium.
Expression of the Aspergillus niger var. awamori Phytase Gene in Pichia pastoris, and Comparison of Biological Properties
CHOI, JAE-MUN ; DOO-SANG KIM ; MOON-SICK YANG ; HYUNG-RAK KIM ; JAE-HO KIM ;
Journal of Microbiology and Biotechnology, volume 11, issue 6, 2001, Pages 1066~1070
The PhyA gene, encoding myo-inositol hexakisphosphate phosphohydrolase in Aspergillus niger var. awamori (wild-type), was cloned and sequenced. The cDNA was overexpressed by a multicopy gene expression system in Pichia pastoris KM71. Recombinant, wild-type and commercial phytase from Aspergilus ficuum NRRL 3135 (Natuphos) were purified. The PhyA gene of Aspergillus niger var awamori showed perfect homology to the phytase of Aspergillus ficcum and
homology to A. niger var awamori (L02421). Wild-type phytase was highly glycosylated and more thermostable than the other two, while deglycosylated farms of three phytases showed identical molecular weight, 507 kDa. After heating at
, wild-type, commercial, and recombinant phytases retained
of their original activities, respectively. In conclusion, glycosylation plays a key role in the thermostability of phytase and its enzymatic characterization.
In Vitro Cancer Chemopreventive Activities of Polysaccharides from Soybeans Fermented with Phellinus igniarius or Agrocybe cylindracea
Shon, Yun-Hee ; Nam, Kyung-Soo ;
Journal of Microbiology and Biotechnology, volume 11, issue 6, 2001, Pages 1071~1076
Chemopreventive activities of polysaccharides from soybeans fermented with either Phellinus igniarius or Agrocybe cylindracea were investigated by measuring the induction of quinone reductase (QR), glutathione S-transferase (GST) activities, and glutathione (GSH) levels in the cell culture along with inhibition of polyamine biosynthesis. The polysaccharides from soybeans fermented with P. igniarius strongly (p<0.005) induced QR activity at all concentrations tested. The extract not only induced GST activity in a dose-dependent manner in the concentration range of 0.1-1.0 mg, but significantly induced GSH revels in cultured Hepa 1c1c7 cells with a maximal 1.4-fold increase at 0.1 mg. The polysaccharides from soybeans fermented with A. cylindracea were effective in inhibiting polyamine metabolism. These results suggest that polysaccharides from soybeans fermented with P. igniarius or A. cylindracea have cancer chemopreventive activities in in vitro models and, therefore, could be considered as potential agents for cancer chemoprevention.
Overproduction of Streptomyces griseus Protease A and B Induces Morphological Changes in Streptomyces lividans
Chi, Won-Jae ; Kim, Jung-Mee ; Choi, Si-Sun ; Kang, Dae-Kyung ; Hong, Soon-Kwang ;
Journal of Microbiology and Biotechnology, volume 11, issue 6, 2001, Pages 1077~1086
The sprA and sprB gene encoding chymotrypsin-like proteases Streptomyces griseus protease A (SGPA) and Streptomyces griseus protease B (SGPB) and the sprT gene that encodes Streptomyces griseus trypsin (SGT) were cloned from Streptomyces griseus ATCC10137 and overexpressed in Streptomyces lividans TK24 as a heterologous host. The chymotrypsin activity of tole culture broth measured with the artificial chromogenic substrate , N-succinyl-ala-ala-pro-phe-p-nitroanilide, was 10, 14 and 14 units/mg in the transformants haboring the sprA, sprB and sprD genes, respectively. The growth of S. lividans reached the maximum cell mass after 4 days of culture, yet SGPA and SGPD production started in the stationary phase of cell growth and kept increasing for up to 10 days of culture in an R2YE medium. The trypsin activity of the culture broth measured with the artificial chromogenic substrate , N-
-benzoyl-DL- arginine-p-nitroanilide , was 16 units/mg and SGT production started in the stationary phase of cell growth and kept increasing for up to 10 days of culture in an R2YE medium. The introduction of the sprA gene into S, lividans TK24 triggered the biosynthesis of pigmented antibiotics, actinorhodin and undecylprodigiosin, and induced significant morphological changes in the colonies in Benedict, R2YE, and R1R2 media. In addition, the introduction of the sprT gene also induced morphological changes in the colony shape without affecting the antibiotic production, thereby implying that certain proteases would appear to play very important and specific roles in secondary-metabolites formation and morphological differentiation in Streptomyces.
Effect of Sodium Bytyrate on Glycosylation of Recombinant Erythropoietin
Chung, Bo-Sup ; Jeong, Yeon-Tae ; Chang, Kern-hee ; Kim, Jong-Soo ;
Journal of Microbiology and Biotechnology, volume 11, issue 6, 2001, Pages 1087~1092
The effect of Sodium Butyrate (NaBu) on the N-linked oligosaccharide structure of Erythropoietin (EPO) was investigated. Recombinant human EPO was produced by CHO cells grown in an
medium with or without 5 mM NaBu, and purified from the culture supernatants using a heparin-sepharose affinity column and immunoaffinity column. The N-linked oligosaccharides were released enzymatically and isolated by paper chromatography. The isolated oligosaccharides were then labeled with a fluorescent dye, 2-aminobenzamide, and analyzed with MonoQ anion exchange chromatography and GlycosepN amide chromatography for the assignment of a GU (glucose unit) vague. A glycan analysis by HPLC showed that the most significant characteristic effect of NaBu was a reduction in the proportion of glycans with Sri-and tetrasialylated oligodaccharides from
(tetra-) in the control cultures (without NaBu) to
(tetra-) in the NaBu-treated cultures, respectively. It was also found that the proportion of asialo-glycan increased from
when treated with NaBu.
Molecular Clonging and Hyperexpression of a Bt Gene, cryIAc, in Escherichia coli
: Production and Usage of Anti-CryIAc Antibody
RYOU, CHONGSUK ; TAEYOUNG CHUNG ; MOOSIK KWON ;
Journal of Microbiology and Biotechnology, volume 11, issue 6, 2001, Pages 1093~1098
The gene coding for a Lepidoptera-specific insecticidal crystalline (or control) protein (ICP), recognized as cryIAc, from Bacillus thuringiensis subsp. kurstaki HD-73, was cloned into the vector pBluscript ll SK-, and then transformed in Escherichia coli
. The clone was named EBtIAc and the chimeric phagemid, as pEBtIAc. Hyperexpression of CryIAc protoxin was observed in the extract of the culture of E. coli harboring pEBtIAc. Crystalline protoxin was purified by differential solubility. It was dissolved in alkaline pH, and exposed to trypsin to be activated. The molecular weights of the pro- and activated toxins on SDS-PAGE were estimated to be ca. 130 kDa and 60 kDa, respectively. The toxicity was tested by force-feeding larvae of gypsi moth (Lymantria diapar) with trypsinized protoxin. Using the batch of biologically active form of the toxin as an immunogen, anti-CryIAc antiserum was raised in a New Zealand white rabbit. Immunoglobulin G was fractionated from the seam by Protein-A sepharose affinity chromatography. Immunoreactivity of the antibody was examined by dot and Westerns blottings. It has been found that the anti- CryIAc antibody recognized the purified toxin at a level below a nanogram in terms of quantity. Using the antibody some of Bt-corns were able to be differentiated from tons of corn kernels which were imported from America as forage crops.
Citrinin Hydrate Inhibit Serotonin N-Acetyltransferase Catalyzing the Conversion of Serotonin to N-Acetylserotonin
Lee, In-Kyoung ; Yun, Bong-Sik ; Kim, Kyong-Tai ; Choi, Bo-Hwa ; Park, Tae-Ju ; Kim, Young-Ho ; Yoo, Ick-Dong ;
Journal of Microbiology and Biotechnology, volume 11, issue 6, 2001, Pages 1099~1101
In an attempt to search for serotonin N-acetyltransferase (arylalkylamine N-acetyltransferasem, AA-NAT) inhibitors from microbial metabolites, we fecund the culture broth of Penicillium sp. 80722 which showed a strong inhibitory activity against AA-NNT. The active principle has been identified as citrinin hydrate through bioassay-guided fractionation of cultural broth, and structure elucidation derived by spectroscopic analyses. Citrinin hydrate inhibits AA-NAT with an
in a dose-dependent manner. Although citrinin hydrate was previously isolated as human rhinovirus 3C-protease inhibitor, this was recognized as the first AA-NAT inhibitor isolated from natural sources.
Therapeutic Agents against Bacteria Causing Porcine Pneumonia
Lee, Joo-Yong ; Lyoo, Young-Soo ; Park, Dong-Ki ; Jung, Ji-Hyun ; Lee, Chul-Hoon ; Kim, Min-Kyun ; Lim, Yoon-Gho ;
Journal of Microbiology and Biotechnology, volume 11, issue 6, 2001, Pages 1102~1105
In order to find therapeutic agents for porcine pneumonia, we screened far antibacterial activities of methanol extracts of 81 higher plants against four pathogenic microorganisms of Heamophilus parasuis, Pasteurella multocida, Actinobacillus pleuropneumonia, and Bordetella bronchiseptica, and found the bark of Cinnamomi cortex showed potent activities. Since this was inexpensive, we purified active compounds from it. The structures of the final active fractions were obtained through an activity-guided fractionation and their antibacterial activities are reported here.
Electron Microscopic Evidence of Paraporal Crystal Inclusion Biogenesis in Bacillus sphaericus Strain 1593
Lee, Young-Ju ; Lee, Hyung-Hoan ;
Journal of Microbiology and Biotechnology, volume 11, issue 6, 2001, Pages 1106~1110
The parasporal biogenesis of crystal inclusion during the sporulation of Bacillus sphaericus strain 1593 was observed using transmission electron microscopy. The crystal biogenesis and sporulation process involved a sequence of events talking about 10 h. The sporulation Precesses were found to be similar to previous findings. The crystal biogenesis of B. sphaericus was initiated at the start of engulfment and nearly completed by the time of exosporium formation. The crystal formation was clearly associated with the outer forespore membrane from stages III through VI, and the crystals grew from polypeptide-like chains originated from the outer forespore membrane. These observations are different from previous findings, which report no association with the forespore membrane. The crystals were located adjacent to the outer membrane of the spore until the release stage. The axes size of the bipyramidal crystal was approximately
. During crystal biogenesis, the crystal development could be classified into four stages; initiation stage Cl (sporulation stage . III), growth stage C2 (sporulation III to V), envelopment and maturation C3 (sporulation V to V), and finally release stage C4 (sporulation Vll).
Identification of Total Extracellular Fibrinase from Bacillus sp. DJ Using One-or Two-Dimensional Fibrin Zymography for Proteomic Approach
CHOI, NACK-SHICK ; JIN-YOUNG LEE ; KAB-SEOG YOON ; KYOUNG-YOEN HAN ; SEUNG-HO KIM ;
Journal of Microbiology and Biotechnology, volume 11, issue 6, 2001, Pages 1111~1114
An extracellular fibrinolytic-enzyme-producing bacterium was isolated from Doen-Jang, a Korean traditional fermented flood, and identified as Bacillus sp. DJ based on its morphology and cellular fatty acid composition. The total extracellular fibrinase (EF) from Bacillus sp. DJ was analyzed using three fibrin zymographic techniques, SDS-fibrin zymography (SDS-FZ), isoelectrofocucing-fibrin zymographs(IEF-FZ), and a two-dimensional SDS-fibrin zymographic analysis (2D SDS-FZ). As a result, the EP map of Bacillus sp. DJ was established. The results suggest that the 2D SDS-FZ method will be a useful tool for the proteomic approach for many other bacterial pretenses.
Identification of a Gene for Aerobic Growth with a SoxS Binding Sequence in Escherichia coli by Operon Fusion Techniques
Lee, Yong-Chan ; Kwon, Hyung-Bae ; Lee, Sang-Ho ; Kwon, Hye-Won ; Sung, Ha-Chin ; Kim, Joon ; Choe, Mu-Hyeon ;
Journal of Microbiology and Biotechnology, volume 11, issue 6, 2001, Pages 1115~1119
Eight Escherichia coli cells with aerobic growth deflects were isolated by the insertion of
, a hybrid bacteriophage of
and Mu, which created transcriptional fusion to lacZY. Two of these mutants, CLIO and CLl2, were irradiated with UV to obtain specialized transducing phages. The phages that took out the neighboring chromosomal DNA of the related gene responsible for deflective aerobic growth were identified. The in vivo cloned chromosomal sequence revealed that the mutated gene of CLIO was located at min 34.5 on the Escherichia coli linkage map and 1,599,515 on the physical map. The physical map indicated that there were 7 cistrons in the operon. We named this operon oxg10. The promoter sequence of oxg10 exhibited a possible binding site far SoxS, a transcriptional regulator that activates the transcription of various SoxRS regulon genes. Transferring the oxg10::
mutation into the wild-type strain, RZ4500, resulted in the inhibition of normal aerobic growth, while the salute mutation in strain MO inhibited aerobic cell growth completely. The full operon sequences of oxg10 were cloned from the Excherichia coli genomic library. The mutated gene of CLl2 was identified to be a sucA gene encoding the
-ketoglutarate dehydrogenase El component in the TCA cycle.
Streptomyces Showing Antifungal Activities against Six Plant Pathogenic Fungi
KIM, BUM-JOON ; MOONJAE CHO ; JIN-CHEOL KIM ; KWANG YUN CHO ; GYUNG JA CHO ; CHUL-HOON LEE ; YOONGHO LIM ;
Journal of Microbiology and Biotechnology, volume 11, issue 6, 2001, Pages 1120~1123
Screening tests against six plant pathogenic fungi were performed in order to develop biopesticides. Actinomycetes were used to discriminate Bacillus thuringiensis for wide use as a microbial pesticide. From more than 100 actinomycetes tested, twelve strains showed potent antifungal activities. We report in vivo screening results from fermentation broths of these twelve strains and identification of the strain taxa.