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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 15, Issue 6 - Dec 2005
Volume 15, Issue 5 - Oct 2005
Volume 15, Issue 4 - Aug 2005
Volume 15, Issue 3 - Jun 2005
Volume 15, Issue 2 - Apr 2005
Volume 15, Issue 1 - Feb 2005
Selecting the target year
Assessment of Characteristics of Biofilm Formed on Autotrophic Denitrification
JANG AM ; BUM MINSU ; KIM SUNGYOUN ; AHN YEONGHEE ; KIM IN S ; BISHOP PAUL L ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 455~460
A pilot-scale sulfur particle autotrophic denitrification (SPAD) process for the treatment of municipal wastewater was operated for 10 months at Shihwa, Korea, and higher than
removal efficiency was observed. Plate counting showed that the lower part of the denitrifying column reactor had the most autotrophic denitrifiers. The biofilm thickness formed on sulfur particles from the SPAD reactor was approximately
, measured by DAPI (4,6-diamidino-2-phenylindole) staining. The presence of bacteria inside the highly porous sulfur particle was also monitored by SEM observation of the internal surfaces of broken sulfur particles. Biofilm extracellular polymeric substances (EPS) analysis showed that the ratio of carbohydrate to protein decreased with the reactor heights at which biofilm-formed sulfur particles were obtained.
Biological Fixation of
by Chlorella sp. HA-1 in a Semi-Continuous and Series Reactor System
LEE JAE-YOUNG ; KWON TAE-SOON ; BAEK KITAE ; YANG JI-WON ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 461~465
Characteristics of biological
fixation by Chlorella sp. HA-1 were investigated in a semi-continuous and series reactor system using an internally illuminated photobioreactor to overcome shortcomings of physicochemical technologies such as adsorption and membrane separation. High
fixation rate was achieved in the semi-continuous reactor system, in which the dilution ratios of the culture medium were controlled. The average
fixation rate was maintained almost constantly when the dilution ratio increased by 0.1 increment from the initial value of 0.5. The total removal efficiency of
was enhanced by employing a series reactor system. The average
fixation rate increased until 4.013 g
in a series operation of four reactors, compared to 0.986 g
in a batch operation mode. The total
fixation rate was proportional to the number of reactors used in the series reactor system. In the series reactor system of semi-continuous operation, a large amount of
was removed continuously for 30 days. These results showed that the present reactor systems are efficient and economically feasible for a biological
Overexpression and Characterization of appA Phytase Expressed by Recombinant Baculovirus-Infected Silkworm
CHEN YIN ; ZHU ZHONGZE ; LIN XU'AI ; YI YONGZHU ; ZHANG ZHIFANG ; SHEN GUIFANG ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 466~471
An Escherichia coli strain with high phytase activity was screened from pig excreta. The phytase gene, appA, was amplified by PCR technique. To obtain large amounts of appA phytase, the appA gene was subcloned into the baculovirus transfer vector pVL1393 under the control of the Polyhedrin promoter. The recombinant baculovirus harboring the appA gene was obtained after co-transfection and screening. The early
instar larvae of silkworm were infected with the recombinant virus. Using this system, the appA phytase was overproduced up to 7,710 U per ml hemolymph. SDS-PAGE analysis revealed the baculovirus-derived appA phytase to be approximately 47 kDa in size. The optimal temperature and pH of the expressed phytase were
and pH 4.5, respectively. The enzymatic activity was increased by the presence of 1 mM
, 1 mM
Preparation of Feather Digests as Fertilizer with Bacillus pumilis KHS-1
Kim, Jin-Man ; Choi, Yang-Mun ; Suh, Hyung-Joo ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 472~476
The present study was untaken to assess the capacity of Bacillus pumilis KHS-1 to grow on chicken flour and to prepare feather digest as fertilizer. To increase keratinolytic activity, the addition of cysteine (5.0 mM) showed the highest keratinolytic activity (245 unit) among the reducing agents tested. The production of soluble protein (feather digests) paralleled the tendency to the production of keratinolytic protease. In the growth curve of B. pumilis KHS-1 at
in the feather medium with 5 mM cysteine, the maximum keratinolytic activity of B. pumilis was about 161 units/ml after 84 h of incubation. The maximum enzyme activities were observed at the late logarithmic growth phase, and remained thereafter with little changes. Using 27-day plant growth assays on carrot and Chinese cabbage, feather digests and reference fertilizer were compared. In terms of the length and the weight of the above-ground vegetations, feather digests showed the same effect as that of the fertilizer. Therefore, our investigation shows that the feather digests can be used in agriculture.
Thymidine Production by Corynebacterium ammoniagenes Mutants
Song, Kyung-Hwa ; Kwon, Do-Young ; Kim, Sang-Yong ; Lee, Jung-Kul ; Hyun, Hyung-Hwan ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 477~483
Corynebacterium ammoniagenes ATCC 6872, which does not accumulate pyrimidine nucleoside or nucleotide, was metabolically engineered to secrete a large amount of thymidine. Characteristics of 5-fluorouracil resistance (
), hydroxyurea resistance (
), trimethoprim resistance (
), thymidylate phosphorylase deficiency (
), inosine auxotrophy (
), 5-fluorocytosine resistance (
), thymidine kinase deficiency, and thymidine resistance (
) were successively introduced into mutant strains KR3 and DY5T9-5, and shake-flask cultures were able to accumulate 408.1 mg/l and 428.2 mg/l of thymidine, respectively, as a major product. The mutant strains did not accumulate thymine at all and accumulated less than 10 mg/l of other pyrimidine nucleosides, such as cytosine, cytidine, and deoxycytidine, as byproducts.
DNA Repair Activity of Human rpS3 is Operative to Genotoxic Damage in Bacteria
JANG CHANG-YOUNG ; LEE JAE YUNG ; KIM JOON ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 484~490
Human ribosomal protein S3 (rpS3), which has a DNA repair endonuclease activity, is a multifunctional protein. This protein is involved in DNA repair, translation, and apoptosis. In particular, rpS3 has a lyase activity, which cleaves the phosphodiester bond of damaged sites such as cyclobutane pyrimidine dimers and AP sites. Here, using deletion analysis, we identified that the repair endonuclease domain resides in the C-terminal region (165-243 aa) of rpS3. We also found that ectopic expression of GST-rpS3 in bacterial strain BL21 promoted the resistance of these cells to ultraviolet (UV) radiation and hydrogen peroxide (
) treatment. The repair domain of rpS3 was sufficient to exhibit the resistance to UV irradiation and recover cell growth and viability, showing that the repair activity of rpS3 is responsible for the resistance to UV irradiation. Our study suggests that rpS3 is able to process DNA damage in bacteria via its repair domain, showing the resistance to genotoxic stress. This implies that rpS3-like activity could be operative in bacteria.
Isolation and Characterization of Kasugamycin Biosynthetic Genes from Streptomyces kasugaensis KACC 20262
JO YOU-YOUNG ; LIU JING ; JIN YING-YU ; YANG YOUNG-YELL ; SUH JOO-WON ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 491~496
The biosynthetic gene cluster for the aminoglycoside antibiotic kasugamycin was isolated and characterized from the kasugamycin producing strain, Streptomyces kasugaensis KACC 20262. By screening a fosmid library using kasA, the gene encoding aminotransferase, we isolated a 22 kb DNA fragment. The fragment contained seventeen complete open reading frames (ORFs); one of these ORFs, kasD, was identified as the gene for dNDP-glucose 4,6-dehydratase, which catalyzes the conversion of dNDP-glucose to 4-keto-6-deoxy-dNDP-glucose. The enzyme showed a broad spectrum of substrate specificity. In addition, ksR was overexpressed in E. coli BL21 and proved to be a self-resistance gene against kasugamycin. These findings suggest that the isolated gene cluster is highly likely responsible for the biosynthesis of kasugamycin.
Mercury Ion Removal Using a Packed-Bed Column with Granular Aminated Chitosan
JEON, CHOONC ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 497~501
This study deals with the removal of mercury species using a packed-bed column with spherical aminated chitosan material. These adsorbents revealed a high adsorption capacity for mercury species. Experiments with feed solutions of 10 ppm Hg dissolved in distilled water showed an excellent removal with a sharp increase of the filter effluent concentration after a total throughput of 900 bed volumes of feed water. Up to
desorption was reached by using 3 bed volumes of 0.01 N EDTA solution. EDTA could be recovered by means of sulfuric acid with about
efficiency. Almost the same results were obtained in repeated sorption and desorption experiments at identical conditions. The experiments demonstrated that the sorbents possessed practically no sorption capacity for alkaline earth ions (
). Their influence on the sorption of mercury was negligible. In experiments with spiked tap water of the Karlsruhe Research Centre and a feed mercury concentration of 0.01 mg/l, the breakthrough of Hg was observed only after a total throughput of about 6,000 bed volumes of feed water.
A Genetic Marker Associated with the A1 Mating Type Locus in Phytophthora infestans
KIM KWON-JONG ; EOM SEUNG-HEE ; LEE SANG-PYO ; JUNG HEE-SUN ; KAMOUN SOPHIEN ; LEE YOUN SU ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 502~509
Sexual reproduction plays an important role in the biology and epidemiology of oomycete plant pathogens such as the heterothallic species Phytophthora infestans. Recent worldwide dispersal of A2 mating type strains of P. infestans resulted in increased virulence, gene transfer, and genetic variation, creating new challenges for disease management. To develop a genetic assay for mating type identification in P. infestans, we used the Amplified Fragment Length Polymorphism (AFLP) technique. The primer combination E+AT/M+CTA detected a fragment specific to A1 mating type (Mat-A1) of P. infestans. This fragment was cloned and sequenced, and a pair of primers (INF-1, INF-2) were designed and used to differentiate P. infestans Mat-A1 from Mat-A2 strains. The Mat A1-specific fragment was detected using Southern blot analysis of PCR products amplified with primers INF-1 and INF-2 from genomic DNA of 14 P. infestans Mat-A1 strains, but not 13 P. infestans Mat-A2 strains or 8 other isolates representing several Phytophthora spp. Southern blot analysis of genomic DNAs of P. infestans isolates revealed a 1.6 kb restriction enzyme (EcoRI, BamHI, AvaI)-fragment only in Mat-A1 strains. The A1 mating type-specific primers amplified a unique band under stringent annealing temperatures of
, suggesting that this PCR assay could be developed into a useful method for mating type determination of P. infestans in field material.
PLGA Microspheres in Hyaluronic Acid Gel as a Potential Bulking Agent for Urologic and Dermatologic Injection Therapies
KANG SUN-WOONG ; CHO EUI RI ; KIM BYUNG-SOO ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 510~518
In this study, we investigated whether PLGA microspheres in combination with hyaluronic acid (HA) gel have appropriate properties as a bulking agent for urologic injection therapies and whether the implantation of PLGA microspheres and HA gel induces angiogenesis in the newly formed tissues. In order to investigate whether this bulking agent is injectable, this material was injected through 24-gauge needles into the subcutaneous dorsum of the mouse. The bulking agent was easily injected without needle obstruction. Histological analyses of the hybrid tissues at 2 weeks showed that host cells at the surrounding tissues migrated into the spaces between the implanted PLGA microspheres and formed tissue-like structures. An inflammatory response to the implants was mild at 2 weeks and diminished at 8 weeks. Importantly, extensive ingrowth of blood vessels was observed in the hybrid tissues formed by the injection of PLGA microspheres and HA, whereas blood vessels rarely formed in the hybrid tissues formed by the injection of PLGA microspheres only. The implant volume was conserved for almost the entire implantation period. Histological analyses of the distant organs of the bulking agent-implanted animals, such as the lungs, liver, heart, brain, kidney, and spleen, showed no evidence of the injected microsphere migration. These results show that PLGA microspheres in combination with HA possess the appropriate characteristics for a bulking agent for urologic injection therapies and induce extensive blood vessel formation in the hybrid tissues.
Purification and Characterization of Quercitrin-Hydrolyzing
-L-Rhamnosidase from Fusobacterium K-60, a Human Intestinal Bacterium
PARK SUN-YOUNG ; KIM JOO-HYUN ; KIM DONG-HYUN ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 519~524
-L-rhamnosidase (EC 18.104.22.168.), which transforms quercitrin to quercetin, was purified from Fusobacterium K-60, a human intestinal anaerobic bacterium. The specific activity of the purified
-L-rhamnosidase was 2.89 mol/min/mg protein.
-L-Rhamnosidase, whose molecular size was 170 kDa by gel filtration, was composed of four subunits (
41,000 Da) with pI and optimal pH values of 5.2 and 5.5-7.0, respectively. The apparent
values for p-nitrophenyl-
-L-rhamnopyranoside and quercitrin were determined to be 0.057 mM and 3.4 mol/min/mg, and 0.077 mM and 5.0 mol/min/mg, respectively. This enzyme was strongly inhibited by
, L-rhamnose, and p-chlormercuriphenylsulfonic acid. These findings suggest that the biochemical properties and substrate specificity of the purified enzyme are different from those of the previously purified
-L-rhamnosidase. This is the first reported purification of quercitrin-hydrolyzing
-L-rhamnosidase from intestinal bacteria.
Cloning and Characterization of the pyrH Gene Encoding UMP-Kinase from Lactobacillus reuteri ATCC 55739
PARK JAE-YONG ; NAM SU JIN ; KIM JONG-HWAN ; JEONG SEON-JU ; KIM JUNG KON ; HA YEONG LAE ; KIM JEONG HWAN ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 525~531
From a genomic library of Lactobacillus reuteri ATCC 55739, one clone, NE347, carrying a pyrH gene encoding UMP kinase, was identified. pNE347 carried a 1.88 kb EcoRI fragment and the pyrH was located in the middle of the insert. pyrH ORF was 723 bp in size and capable of encoding UMP kinase composed of 240 amino acid residues. tsf encoding an elongation factor-Ts and frr encoding a ribosomal recycling factor were present upstream and downstream of pyrH, respectively. When introduced into E. coli KUR1244, a pyrH-negative strain, pNE347 restored the ability to grow at
, indicating that pyrH from L. reuteri synthesized functional UMP kinase in E. coli. Northern blot experiment showed that pyrH and frr were cotranscribed as a 1.4 kb single transcript. pyrH was overexpressed in E. coli by using a pET26b(+) vector, and a major 25 kDa protein band appeared on SDS-polyacrylamide gel.
Gentisyl Alcohol Inhibits Apoptosis by Suppressing Caspase Activity Induced by Etoposide
KIM JINHEE ; KIM DONGHYUN ; KIM MEEREE ; KWON HOJEONG ; OH TAEKWANG ; LEE CHOONGHWAN ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 532~536
In the course of our screening for small molecules to inhibit apoptosis of U937 human leukemia cells induced by etoposide (
), Penicillium sp. F020150 with potent inhibitory activity was selected. The active compound was purified from ethyl acetate extract of the microorganism by Sephadex LH-20 column chromatography and HPLC, and was identified as gentisyl alcohol (2,5-dihydroxybenzyl alcohol) by spectroscopic methods. The compound inhibited caspase-3 induction with
after 8 h of etoposide treatment. The expression levels of caspase-3 and PARP were dose-dependently inhibited by the compound, suggesting that gentisyl alcohol inhibits etoposide-induced apoptosis via downregulation of caspases.
Miniscale Identification and Characterization of Subtilisins from Bacillus sp. Strains
CHOI NACK-SHICK ; JU SUNG-KYU ; LEE TAE YOUNG ; YOON KAB-SEOG ; CHANG KYU-TAE ; MAENG PIL JAE ; KIM SEUNG-HO ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 537~543
Subtilisin (EC 22.214.171.124) is the major extracellular alkaline serine protease of Bacillus species. Previously, we found that subtilisins did not migrate in the electrophoretic field in the Laemmili buffer system due to their high pI values (over 8.8); however, it formed a 'binding mode' at the top of the separating gel . Utilizing this characteristic, four subtilisins from Bacillus sp. strains (e.g., B. subtilis 168, B. subtilis KCTC 1021, B. amyloliquefaciens KCTC 3002, and Bacillus sp. DJ-1 and DJ-4) were easily and quickly identified by an over-running electrophoretic technique with a miniscale culture supernatant (less than 20 ml) without any column chromatographic steps. Two subtilisins (DJ-l and a recombinant version) from Bacillus sp. DJ-l were characterized, and the enzymatic properties were determined by SDS-fibrin zymography and densitometric analysis. Based on this observation, the recombinant pro-subtilisin DJ-l showed the same 'binding mode,' similar to native subtilisin DJ-l. On the other hand, mature subtilisin DJ -1 without pro-peptide showed no enzymatic activity.
-Globin Second Intron Highly Enhances Expression of Foreign Genes from Murine Cytomegalovirus Immediate-Early Promoter
KANG MOONKYUNG ; KIM SEON-YOUNG ; LEE SUKYUNG ; LEE YOUNG-KWAN ; LEE JAEHO ; SHIN HYUN-SEOCK ; KIM YEON-SOO ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 544~550
To develop a highly efficient mammalian expression vector, a series of vectors were constructed based on the murine cytomegalovirus (MCMV) immediate-early (IE) promoter and human
-globin second intron. The resulting MCMV promoter was several-fold stronger than the HCMV promoter in various mammalian cell lines, such as the NIH3T3, Neuro-2a, 293T, and HT1080 cell lines, and was only slightly weaker than the HCMV promoter in HeLa and CHO cells. The inclusion of the human
-globin second intron behind the MCMV promoter or HCMV promoter markedly enhanced the promoter activity in various mammalian cell lines, and the resultant MCMV/Glo-I expression system was stronger than the HCMV promoter from 4.7- to 11.2-fold in every cell line tested. Also, the MCMV/Glo-I promoter induced a higher level of the VSV-G protein in a transiently transfected 293T cell line, which is useful for the production of recombinant retrovirus and lentivirus vectors.
Dynamic Behavior of Regulatory Elements in the Hierarchical Regulatory Network of Various Carbon Sources-Grown Escherichia coli
Lee, Sung-Gun ; Hwang, Kyu-Suk ; Kim, Cheol-Min ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 551~559
The recent rapid increase in genomic data related to many microorganisms and the development of computational tools to accurately analyze large amounts of data have enabled us to design several kinds of simulation approaches for the complex behaviors of cells. Among these approaches, dFBA (dynamic flux balance analysis), which utilizes FBA, differential equations, and regulatory events, has correctly predicted cellular behaviors under given environmental conditions. However, until now, dFBA has centered on substrate concentration, cell growth, and gene on/off, but a detailed hierarchical structure of a regulatory network has not been taken into account. The use of Boolean rules for regulatory events in dFBA has limited the representation of interactions between specific regulatory proteins and genes and the whole transcriptional regulation mechanism with environmental change. In this paper, we adopted the operon as the basic structure, constructed a hierarchical structure for a regulatory network with defined fundamental symbols, and introduced a weight between symbols in order to solve the above problems. Finally, the total control mechanism of regulatory elements (operons, genes, effectors, etc.) with time was simulated through the linkage of dFBA with regulatory network modeling. The lac operon, trp operon, and tna operon in the central metabolic network of E. coli were chosen as the basic models for control patterns. The suggested modeling method in this study can be adopted as a basic framework to describe other transcriptional regulations, and provide biologists and engineers with useful information on transcriptional regulation mechanisms under extracellular environmental change.
Characterization of Exopolysaccharides Produced by Submerged Culture of an Entomopathogenic Fungus Paecilomyces sinclairii by Using a Multi-Angle Laser Light Scattering System
KIM SANG-WOO ; HWANG HYE-JIN ; CHOI JANG-WON ; YUN JONG-WON ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 560~567
Three groups of exopolysaccharides (EPSs) (designated as Fr-I, Fr-II, and Fr-III) were isolated from the culture filtrates of Paecilomyces sinclairii by gel filtration chromatography on Sepharose CL-4B. Their molecular characteristics were examined by multi-angle laser light scattering (MALLS) connected online to a size exclusion chromatography (SEC) and refractive index (RI) detector system. The weight-average molar mass of Fr-I, Fr-II, and Fr-III of EPSs were determined to be
, respectively. All three EPSs showed a fairly low polydispersity indice, ranging from 1.008 to 1.059 (nearly mono disperse behavior), and showed different carbohydrates and amino acids compositions; all fractions of EPSs consisted of mainly cystine, valine, and arginine in the protein moiety, and mainly ribose, galactose, and glucose in the carbohydrate moiety. The determination of gyration radii of the EPSs in SEC/MALLS analysis revealed the molecular shape of the Fr-I to be a rod-like structure, whereas the Fr-II and Fr-III had a random-coil structure in an aqueous solution.
Mass Production of Yeast Spores from Compressed Yeast
Lim, Yong-Sung ; Bae, Sang-Myun ; Kim, Keun ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 568~572
Saccharomyces yeast spores are more resistant to drying and storage than vegetative cells. For the mass production of yeast spores, compressed yeast was directly inoculated into a sporulation medium (SM). The effects of inoculum size and the addition of rice wine cake (RWC) into SM on the sporulation were examined using flasks. With
inoculum of compressed yeast,
of asci was obtained. The addition of
RWC into SM improved the cell growth and spore yield, and the number of asci formed was
. The effects of culture temperature, temperature-shift, and concentrations of inoculum, potassium acetate, and RWC on the sporulation were also evaluated using a jar fermentor. The optimum temperature for spore formation was
where the number of asci formed was
. The shift of culture temperature from initial
for 1 day to
for 3 days increased the number of asci formed to
. The use of
(w/v) inoculum of compressed yeast,
potassium acetate, and
(w/v) RWC in SM with the shift of culture temperature of initial
sporulation ratio and formation of
Purification and Characterization of Pyrimidine Nucleotide N-Ribosidase from Pseudomonas oleovorans
YU, Tae-Shick ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 573~578
Pyrimidine nucleotide N-ribosidase (pyrimidine 5'-nucleotide phosphoribo(deoxyribo)hydrolase/pyrimidine 5'-nucleotide nucleosidase, EC 126.96.36.199) catalyzes the breakdown of pyrimidine 5'-nucleotide into pyrimidine base and ribose(deoxyribo)-5-phosphate. However, detailed characteristics of the enzyme have not yet been reported. The enzyme was purified to homogeneity 327.9-fold with an overall yield of
from Pseudomonas oleovorans ATCC 8062. The enzyme catalyzed cytidine monophosphate (CMP) and uridine monophosphate (UMP), but not adenosine monophosphate (AMP) and guanosine monophosphate (GMP). The enzyme optimally metabolized CMP at pH 6.0 and UMP at around 8.5, and the optimum temperature for the overall enzyme reaction was found to be
values of the enzyme for CMP (at pH 6.0) and UMP (at pH 8.5) were 1.6 mM and 1.1 mM, respectively. AMP, deoxyCMP, and deoxyUMP were very effective inhibitors of the reaction. Double-reciprocal plots obtained in the absence and in the presence of AMP revealed that this inhibitory effect was of the mixed competitive type with respect to the breakdown of CMP and of the noncompetitive type with respect to the breakdown of UMP. In the presence of AMP, the enzyme followed sigmoid kinetics with respect to each substrate.
Intracellular Responses of Antibody-Producing H69K-NGD Transfectoma Subjected to Hyperosmotic Pressure
Bae, Sung-Won ; Lee, Gyun-Min ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 579~586
When subjected to hyperosmotic pressure by NaCl addition, H69K-NGD transfectoma, like KR12H-2 transfectoma, displayed decreased specific growth rate (
) and increased specific antibody productivity (
): Elevation of medium osmolality from 280 mOsm/kg to 415 mOsm/kg decreased
in batch cultures of H69K-NGD transfectoma, while it increased
. However, unlike KR12H-2 tranfectoma, enhanced
of H69K-NGD transfectoma at hyperosmolalities was not due to elevated levels of Ig mRNAs. In hyperosmotic cultures of H69K-NGD transfectoma, heavy-chain mRNA per cell was not enhanced with increasing osmolality. Hyperosmotic pressure was found to preferentially enhance immunoglobulin (Ig) translation rates of H69K-NGD transfectoma. However, under hyperosmotic pressure, the translation rate of Ig polypeptides was not enhanced as much as
. This result suggests that hyperosmotic pressure also influences the post-translational process. Taken together, the results obtained show that intracellular response of transfectomas to hyperosmotic pressure, in regard to the main intracellular steps of the antibody secretory pathway, is cell-line dependent.
Relationship Between Enhancement of Electrostriction and Decrease of Activation Energy in Porcine Pancreatic Lipase Catalysis
PARK HYUN ; LEE KI-SEOG ; PARK SEON-MI ; LEE KWANG-WON ; KIM AUGUSTINE YONGHWI ; CHI YOUNG-MIN ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 587~594
The contribution of electrostriction of water molecules to the stabilization of the negatively charged tetrahedral transition state of a lipase-catalyzed reaction was examined by means of kinetic studies involving high-pressure and solvent dielectric constant. A good correlation was observed between the increased catalytic efficiency of lipase and the decreased solvent dielectric constant. When the dielectric constant of solvents was lowered by 5.00 units, the losses of activation energy and free energy of activation were 7.92 kJ/mol and 11.24 kJ/mol, respectively. The activation volume for
decreased significantly as the dielectric constant of solvent decreased, indicating that the degree of electrostriction of water molecules around the charged tetrahedral transition state has been enhanced. These observations demonstrate that the increase in the catalytic efficiency of the lipase reaction with decreasing dielectric constant resulted from the stabilization of electrostatic energy for the formation of an oxyanion hole, and that this stabilization was caused by the increase of electrostricted water around the charged tetrahedral transition state. Therefore, we conclude that the control of solvent dielectric constant can stabilize the tetrahedral transition state, thus lowering the activation energy.
Validation of One-Step Real-Time RT-PCR Assay in Combination with Automated RNA Extraction for Rapid Detection and Quantitation of Hepatitis C Virus RNA for Routine Testing in Clinical Specimens
KIM BYOUNG-GUK ; JEONG HYE-SUNG ; BAEK SUN-YOUNG ; SHIN JIN-HO ; KIM JAE-OK ; MIN KYUNG-IL ; RYU SEUNG-REL ; MIN BOK-SOON ; KIM DO-KEUN ; JEONG YONG-SEOK ; PARK SUE-NIE ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 595~602
A one-step real-time quantitative RT-PCR assay in combination with automated RNA extraction was evaluated for routine testing of HCV RNA in the laboratory. Specific primers and probes were developed to detect 302 bp on 5'-UTR of HCV RNA. The assay was able to quantitate a dynamic linear range of
HCV RNA copies/reaction (
). The synthetic HCV RNA standard of
copies developed in this study corresponded to 1 international unit (IU) of WHO International Standard for HCV RNA (96/790 I). The detection limit of the assay was 3 RNA copies/reaction (81 IU/ml) in plasma samples. The assay was comparable to the Amplicor HCV Monitor (Monitor) assay with correlation coefficient r=0.985, but was more sensitive than the Monitor assay. The assay could be completed within 3 h from RNA extraction to detection and data analysis for up to 32 samples. It allowed rapid RNA extraction, detection, and quantitation of HCV RNA in plasma samples. The method provided sufficient sensitivity and reproducibility and proved to be fast and labor-saving, so that it was suitable for high throughput HCV RNA test.
Safety Assessment of Lactobacillus fermentum PL9005, a Potential Probiotic Lactic Acid Bacterium, in Mice
PARK JONG-HWAN ; LEE YEONHEE ; MOON ENPYO ; SEOK SEUNG-HYEOK ; BAEK MIN-WON ; LEE HUI-YOUNG ; KIM DONG-JAE ; KIM CHANG-HWAN ; PARK JAE-HAK ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 603~608
We recently isolated a novel probiotic strain, Lactobacillus fermentum PL9005 (KCCM-10250), from infant feces and showed that it had a potential immunoenhancing effect. In the present study, a safety assessment of the bacteria was performed using a BALB/c mouse model. Mice were administered with L. fermentum PL9005 daily for 28 days. There were no detectable changes in body weight, feed intake, or clinical signs, and no significant difference in hematological parameters or blood biochemistry between the L. fermentum PL9005-fed and control groups. Bacterial translocation was detected in the mesenteric lymph nodes, liver, and spleen of some mice with and without L. fermentum PL9005 feeding, however, the organisms were not related to ingestion of L. fermentum PL9005; this was confirmed by PCR using a species-specific primer. No gross lesions were detected in the liver, spleen, or intestine of L. fermentum PL9005-fed or control mice. Mucosal thickness in the ileum, cecum, and colon of L. fermentum PL9005-fed mice was not significantly different from that of corresponding organs in control mice. No inflammation or epithelial cell degeneration in the intestines was observed in any mice. These results indicate that ingestion of L. fermentum PL9005 is safe in mice and can be applied in the functional food market.
Safety and Immunogenicity of Salmonella enterica Serovar Typhimurium llaB in Mice
CHO SUN-A ; LEE IN-SOO ; PARK JONG-HWAN ; SEOK SEUNG-HYEOK ; LEE HUI-YOUNG ; KIM DONG-JAE ; BACK MIN-WON ; LEE SEOK-HO ; HUR SOOK-JIN ; BAN SANG-JA ; LEE YOO-KYOUNG ; PARK JAE-HAK ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 609~615
The safety and immunogenicity of an attenuated recombinant Salmonella vaccine strain, Salmonella enterica serovar Typhimurium llaB, was assessed. This vaccine strain could survive in low pH condition, and its ability of intracellular survival did not differ from that of S. enterica serovar Typhimurium UK1, which is the wild-type of the vaccine strain. The mortality of the mice orally administered with the vaccine strain was
at the dose of
CFU. All mice administered with
CFU of the vaccine strain survived for 3 days postinoculation (pi). However, all mice administered with more than
CFU of the vaccine strain died within 3 days pi. To examine the protective effect of the vaccine strain, mice were orally immunized with
CFU of the bacteria. Control mice were given with 0.5 ml of phosphate buffered saline (PBS). After 8 days, the mice were challenged with
CFU of S. enterica serovar Typhimurium UK1, and mortality was examined for 5 days. The survival rates of the mice immunized with
CFU of the vaccine strain were
, respectively, whereas all control mice died within 2 days after challenging. To investigate the immunogenicity of S. enterica serovar Typhimurium llaB, mice were orally immunized with
CFU ml of the vaccine strain. Five mice of each group were sacrificed at 5 and 12 days after immunization, and results showed that immunization of the vaccine strain led to increases of IgG1, IgG2, and IgM titers against S. enterica serovar Typhimurium UK1 in mouse sera, cytokine expressions such as IL-2, IL-4, IL-6, and IL-10 in spleen, and the lymphocyte proliferation response to mitogens (concanavalin A or LPS) stimulation.
Identification and Characterization of the Vibrio vulnificus malPQ Operon
LIM MOON SUB ; LEE MYUNG HEE ; LEE JEONG HYUN ; JU HYUN-MOK ; PARK NA YOUNG ; JEONG HYE SOOK ; RHEE JEE EUN ; CHOI SANG HO ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 616~625
It is likely that maltose could provide a good substrate for the bacteria in the intestine, when the pathogenic bacteria invade and colonize in human gut. For better understanding of this organism's maltose metabolism, a mutant that was not able to grow with maltose as a sole carbon source was screened from a library of mutants constructed by a random transposon mutagenesis. By a transposon-tagging method, malPQ genes encoding a maltodextrin phosphorylase and a 4-
-glucanotransferase, were identified and cloned from Vibrio vulnificus. The deduced amino acid sequences of malPQ from V. vulnificus were 48 to
similar to those of MalP and MalQ reported from other Enterobacteriaceae. Functions of malPQ genes were assessed by the construction of mutants whose malPQ genes were inactivated by allelic exchanges. When maltose was used as the sole carbon source, neither malP nor malQ mutant was able to grow to a substantial level, revealing that the MalP and MalQ are the only enzymes for metabolic utilization of maltose. The malQ mutant exhibited decreased adherence toward intestinal epithelial cells in vitro, but there was no difference in the
of the wild-type and the malQ mutant in mice. Therefore, it appears that MalQ is less important in the pathogenesis of V. vulnificus than would have been predicted by considering maltose as a most common sugar in the intestine, but not completely dispensable for virulence in mice.
Novel Cationic Microbial Polyglucosamine Biopolymer from New Enterobacter sp. BL-2 and Its Bioflocculation Efficacy
SON MI-KYUNG ; SHIN HYUN-DONG ; HUH TAE-LIN ; JANG JIN-HO ; LEE YONG-HYUN ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 626~632
A new bacterium BL-2 excreting a novel cationic polyglucosamine biopolymer was isolated from the spoiled leaves of Chinese cabbage and identified as Enterobacter sp. BL-2. The isolated Enterobacter sp. BL-2 was cultivated in pH-stat fed-batch culture using acetic acid as the feeding stock at pH 8.0, resulting in 17.11 g/l of cells and 1.53 g/l of an extracellular biopolymer after 72 h. The excreted biopolymer was purified by a three-step procedure, involving ethanol precipitation and deproteinizations, to a nearly homogeneous state, and its molecular weight was found to be 106 kDa. It was composed of glucosamine, rhamnose, and galactose at a molar ratio of 86.4:1.6:1.0, respectively, indicating a rarely found novel high-glucosamine-containing biopolymer. The FT-IR and
spectra of the novel cationic polyglucosamine biopolymer PGB-l revealed a close identity with chitosan from crab shell. It can effectively flocculate various suspended solids, including kaolin clay,
, active carbon, microbial cells, and acidic dyes.
Therapeutic Effect of Astaxanthin Isolated from Xanthophyllomyces dendrorhous Mutant Against Naproxen-Induced Gastric Antral Ulceration in Rats
KIM JEONG-HWAN ; KIM SEUNG-WOOK ; YUN CHEOL-WON ; CHANG HYO-IHL ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 633~639
Frequently used for humans as a nonsteroidal anti-inflammatory drug, naproxen has been known to induce ulcerative gastric lesions. The present study was undertaken to investigate the in vivo therapeutic effect of astaxanthin, isolated from a Xanthophyllomyces dendrorhous mutant, against naproxen-induced gastric antral ulceration in rats. The rats were treated with three doses of astaxanthin [1, 5, and 25 mg/kg body weight (B.W.), respectively] once daily for 2 weeks after pretreatment of 80 mg of naproxen/kg B.W. twice daily for 3 days, while the control rats received only 80 mg of naproxen/kg B.W. twice daily for 3 days. The oral administration of astaxanthin (1,5, and 25 mg/kg B.W.) showed a curative effect against naproxen (80 mg/kg B.W.)-induced gastric antral ulcer and reduced the elevated lipid peroxide level in gastric mucosa. In addition, astaxanthin treatment resulted in significant increase in the activities of radical scavenging enzymes such as superoxide dismutase, catalase, and glutathione peroxidase. A histologic examination clearly proved that acute gastric mucosal lesion induced by naproxen nearly disappeared after the astaxanthin treatment. These results suggest that astaxanthin eliminated the lipid peroxides and free radicals induced by naproxen and may be a potential candidate for remedy of gastric ulceration.
The Role of a Second Protein (Des VIII) in Glycosylation for the Biosynthesis of Hybrid Macrolide Antibiotics in Streptomyces venezuelae
HONG JAY SUNG JOONG ; KIM WON SEOK ; LEE SANG KIL ; KOH HWA SOO ; PARK HEE SUB ; PARK SU JIN ; KIM YOUN SANG ; YOON YEO JOON ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 640~645
The function of the desVIII gene in the pikromycin producer Streptomyces venezuelae was characterized by gene deletion and complementation analysis. In addition to the DesVII glycosyltransferase, the desVIII gene that has previously been suggested to be required for the incorporation of endogenous deoxysugar, TDP-D-desosamine, into the aglycone of pikromycin is also required for the transfer of exogenous deoxysugars, TDP-D-quinovose and TDP-D-olivose.
Antimicrobial Activity of Quinoline Derivatives Isolated from Ruta chalepensis Toward Human Intestinal Bacteria
CHO JANG-HEE ; LEE CHI-HOON ; LEE HOI-SEON ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 646~651
The growth responses of Ruta chalepensis leaf-derived materials toward human intestinal bacteria were examined. The biologically active constituent of the R. chalepensis extract was characterized as quinoline-4-carboxaldehyde(
). The growth responses varied depending on the bacterial strain, chemicals, and dose tested. At 0.25 and 0.1 mg/disk, quinoline-4-carboxaldehyde strongly inhibited the growth of Clostridium perfringens and weakly inhibited the growth of Escherichia coli without any adverse effects on the growth of three lactic acid bacteria. Furthermore, at 0.05 and 0.025 mg/disk, this isolate showed moderate activity against C. perfringens. In comparison, chloramphenicol at as low as 0.01 mg/disk significantly inhibited the growth of all bacteria tested, and cinnamaldehyde at 0.25 mg/disk did not inhibit Bifidobacterium bifidum, B. longum, E. coli, and Lactobacillus acidophilus, with the exception of C. perfringens. The structure-activity relationship revealed that quinoline-3-carboxaldehyde had strong growth inhibition against C. perfringens, but quinoline, quinoline-3-carboxylic acid, and quinoline-4-carboxylic acid did not inhibit the growth of B. bifidum, B. longum, C. perfringens, E. coli, and L. acidophilus. These results indicate that the carboxyl aldehyde functional group of quinolines seems to be required for growth-inhibiting activity against C. perfringens, thus indicating at least one of the pharmacological actions of R. chalepensis leaf.
Cloning and Expression of Isocitrate Lyase, a Key Enzyme of the Glyoxylate Cycle, of Candida albicans for Development of Antifungal Drugs
SHIN DONG-SUN ; KIM SANGHEE ; YANG HYEONG-CHEOL ; OH KI-BONG ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 652~655
This paper describes the development of an enzymatic assay system for the identification of inhibitors of isocitrate lyase (ICL), one of the key enzymes of the glyoxylate cycle that is considered as a new target for antifungal drugs. A 1.6 kb DNA fragment encoding the isocitrate lyase from Candida albicans ATCC10231 was amplified by PCR, cloned into a vector providing His-Patch-thioredoxin-tag at the N-terminus, expressed in Escherichia coli, and purified by metal chelate affinity chromatography. The molecular mass of the purified ICL was approximately 62 kDa, as determined by SDS-PAGE, and the enzyme activity was directly proportional to incubation time and enzyme concentration. The effects of itaconate-related compounds on ICL activity were also investigated. Among them, itaconic acid, 3-nitropropionate, and oxalate had strong inhibitory activities with
values of 5.8, 5.4 and
, respectively. These inhibitors also exhibited antifungal activity on YPD agar media containing acetate as a sole carbon source, albeit at high concentration. The results indicate that the C. albicans ICL may be a regulatory enzyme playing a crucial role in fungal growth and is a prime target for antifungal agents.
Structure Prediction of the Peptide Synthesized with the Nonribosomal Peptide Synthetase Gene from Bradyrhizobium japonicum
JUNG BO-RA ; LEE YUKYUNG ; LIM YOONGHO ; AHN JOONG-HOON ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 656~659
Small peptides synthesized by nonribosomal peptide synthetases (NRPSs) genes are found in bacteria and fungi. While some microbial taxa have few, others make a large number and variety. However, biochemical characterization of the products synthesized by NPRS demands a great deal of efforts. Since the completion of genome projects of numerous microorganisms, the numbers of available NRPSs genes are being expanded. Prediction of the peptides encoded by NRPS could save time and efforts. We chose the NRPS gene from Bradyrhizobium japonicum as a model to predict the peptide structure encoded by NRPS genes. Using computational analyses, the domain structure of this gene was defined, and the structure of a peptide synthesized by this NRPS was deduced. It was found that it encoded a tripeptide consisting of proline-serine-phenylalanine. This method would be helpful to predict the structure of small peptides with various NPRS genes from the genome sequence.
Analysis of In Vivo Interaction of HCV NS3 Protein and Specific RNA Aptamer with Yeast Three-Hybrid System
HWANG BYOUNGHOON ; LEE SEONG-WOOK ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 660~664
We have previously isolated specific RNA aptamers with high affinity against the helicase domain of hepatitis C virus (HCV) nonstructural protein 3 (NS3). The RNA aptamers competitively and efficiently inhibited the helicase activity, partially impeding HCV replicon replication in human hepatocarcinoma cells. In this study, the RNA aptamers were tested for binding to the HCV NS3 proteins in eukaryotic cells, using a yeast three-hybrid system. The aptamers were then recognized by the HCV NS3 proteins when expressed in the cells, while the antisense sequences of the aptamers were not. These results suggest that the in vitro selected RNA aptamers can also specifically bind to the target proteins in vivo. Consequently, they could be potentially utilized as anti-HCV lead compounds.
Cell Signaling Mechanisms of Sperm Motility in Aquatic Species
Kho, Kang-Hee ; Morisawa, Masaaki ; Cho, Kap-Seong ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 665~671
Initiation and activation of sperm motility are prerequisite processes for the contact and fusion of male and female gametes at fertilization. The phenomena are under the regulation of cAMP and
in vertebrates and invertebrates. Mammalian sperm requires
and cAMP for the activation of sperm motility. Cell signaling for the initiation and activation of sperm motility in the ascidians and salmonid fishes has drawn much attention. In the ascidians, the sperm-activating and attracting factors from unfertilized egg require extracellular
for activating sperm motility and eliciting chemotactic behavior toward the egg. On the other hand, the cAMP-dependent phosphorylation of protein is essential for the initiation of sperm motility in salmonid fishes. A decrease of the environmental
concentration surrounding the spawned sperm causes
influx through the specific
channel and dihydropyridine-sensitive L-/T-type
channel, respectively, thereby leading to the membrane hyperpolarization. The membrane hyperpolarization induces synthesis of cAMP, which triggers further cell signaling processes, such as cAMP-dependent protein phosphorylation, to initiate sperm motility in salmonid fishes. This article reviews the studies on the physiological mechanisms of sperm motility and its cell signaling in aquatic species.
Diversity and Genotypic Structure of ECOR Collection Determined by Repetitive Extragenic Palindromic PCR Genome Fingerprinting
HWANG KEUM-OK ; JANG HYO-MI ; CHO JAE-CHANG ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 672~677
The standard reference collection of strains for E. coli, the ECOR collection, was analyzed by a genome-based typing method. Seventy-one ECOR strains were subjected to repetitive extragenic palindromic PCR genome fingerprinting with BOX primers (BOX-PCR). Using a similarity value of 0.8 or more after cluster analysis of BOX-PCR fingerprinting patterns to define the same genotypes, we identified 28 genotypes in the ECOR collection. Shannon's entropy-based diversity index was 3.07, and the incident-based coverage estimator indicated potentially 420 genotypes among E. coli populations. Chi-square test of goodness-of-fit showed statistically significant association between the genotypes defined by BOX-PCR fingerprinting and the groups previously defined by multi-locus enzyme electrophoresis. This study suggests that the diversification of E. coli strains in natural populations is actively ongoing, and rep-PCR fingerprinting is a convenient and reliable method to type E. coli strains for the purposes ranging from ecology to quarantine.ine.
Efficient Expression of a Carbon Starvation Promoter Activity Under Nutrient-Limited Chemostat Culture
KIM DAE-SUN ; PARK YONG-IL ; LEE HYANG BURM ; KIM YOUNGJUN ;
Journal of Microbiology and Biotechnology, volume 15, issue 3, 2005, Pages 678~682
The promoter region of a carbon starvation gene isolated from Pseudomonas putida was cloned and analyzed for its potential use for in situ bioremediation and bioprocessing. We constructed a recombinant plasmid pMKD101 by cloning the 0.65 kb promoter region of the gene into the promoter proving vector, pMK301, which contains the lacZ for
-galactosidase activity as a reporter gene. pMKD101 was transformed into the wild-type P. putida MK1, resulting in P. putida RPD101, and analyzed for
-galactosidase activity under different culture conditions. When RPD101 was grown on the minimal medium plus
glucose as a sole carbon source in batch cultures,
-galactosidase activity was found to be 3.2-fold higher during the stationary phase than during the exponential phase. In chemostat cultures,
-galactosidase activity was found to be 3.1-fold higher at the minimal growth rate (dilution rate=
) than at the maximal growth rate (dilution rate=
). The results suggest that a carbon starvation promoter can be utilized to maximize the expression of a desired gene under nutrient limitation.