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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 14, Issue 6 - Dec 2004
Volume 14, Issue 5 - Oct 2004
Volume 14, Issue 4 - Aug 2004
Volume 14, Issue 3 - Jun 2004
Volume 14, Issue 2 - Apr 2004
Volume 14, Issue 1 - Feb 2004
Selecting the target year
Induction of the T7 Promoter Using Lactose for Production of Recombinant Plasminogen Kringle 1-3 in Escherichia coli
Lim, Hyung-Kwon ; Lee, Shi-Uk ; Chung, Soo-Il ; Jung, Kyung-Hwan ; Seo, Jin-Ho ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 225~230
A plasminogen kringle domain 1 to 3, rKl-3, was expressed in Escherichia coli under the control of T7 promoter. For the cost-effective production of rKl-3, the induction process was analyzed and optimized. Induction characteristics with lactose were analyzed in terms of induction time and inducer concentration in various culture conditions including batch and high-cell-density fed-batch cultures. In the fed-batch culture, the induction around 6 h after initiation of the DO-stat fed-batch culture resulted in the highest expression level of rKI-3 among the induction points examined. The highest demand of oxygen at this point was crucial for the maximum expression level of rKI-3. As the lactose concentration increased, the expression level also increased, though the expression level showed a plateau above a concentration of 14 mM of lactose. Lactose acted less specifically than IPTG since most of it was hydrolyzed to glucose and galactose. However, using lactose, the cell growth and the maximum expression level of rKl-3 increased by 20% and 24%, respectively, compared with those using IPTG in the fed-batch culture. The lactose seemed to be hydrolyzed by intracellular and extracellular
-galactosidase liberated by cell lysis at the same time. Residual concentration of glucose was maintained to a a limit of detection by high performance liquid chromatography, and galactose was not consumed by the host strain Escherichia coli BL2l(DE3).
One-step Purification of Poly-His Tagged Penicillin G Acylase Expressed in E. coli
Kim, Jin-Hee ; Kang, Hye-Jin ; Kim, Eung-Soo ; Kim, Jeong-Ho ; Koo, Yoon-Mo ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 231~236
The inexpensive large-scale production of pure PGA (Penicillin G Acylase) has been a commercial goal. PGA has been used as a model enzyme in the development of simple one-step purification methods. In this study, the purification of poly-His tagged PGA protein secreted into the periplasmic space was carried out by using immobilized metal-ion affinity chromatography (IMAC). The PGA gene was obtained from E. coli ATCC 11105. Codons encoding histidines were fused at the C-terminus of the PGA gene by PCR. E. coli JM109 harboring pPGA-HIS6 vector produced active his-tagged acylases in the presence of lac promoter during cultivation at
. The maximum specific activity of the acylase purified by using one-step chromatography after osmotic shock was 38.5 U/mg and was recovered with the yield of 70％. Both 23 kDa (
) and 62 kDa (
) subunits were recovered by using IMAC with just C-terminus tagging of the
subunit. The purification of the periplasmic fraction by osmotic shock and that of purified acylase was increased by 2.6-fold and 19-fold, respectively, compared to the crude extract.
Mass Production of Pullulan with Optimized Concentrations of Carbon and Nitrogen Sources by Aureobasidium pullulans HP-2001 in a 100-L Bioreactor with the Inner Pressure
Seo, Hyung-Pil ; Chung, Chung-Han ; Kim, Sung-Koo ; Richard A. Gross ; David L. Kaplan ; Lee, Jin-Woo ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 237~242
Cell growth and the production of pullulan by Aureobasidium pullulan HP-2001, the UV-induced mutant of A pullulans ATCC 42023, increased with increased concentration of glucose up to 15.0% (w/v). Maximal production of pullulan in the flask scale was 27.65 g/l, when concentrations of glucose and yeast extract were 15.0 and 0.25% (w/v), respectively. Maximal conversion rate of pullulan from glucose as the carbon source was 0.37, when those of glucose and yeast extract were 5.0 and 0.15% (w/v), respectively. On the basis of total amount of pullulan, the conversion rate of pullulan from glucose, and utilization rate of glucose to cell mass and pullulan by A. pullulans HP-2001, the optimal concentrations of glucose and yeast extract for the mass production of pullulan were determined to be 10.0 and 0.25% (w/v), respectively, at which concentrations the production of pullulan and its conversion rate were 27.14 g/l and 0.27, respectively. Maximal production of pullulan with optimized concentrations of carbon and nitrogen sources by A. pullulans HP-200l in a 7-1 bioreactor was 32.12 g/l for 72 h culture, and that in a 100-1 bioreactor with the inner pressure of
was 36.87 g/l. Increased inner pressure of a 100-1 bioreactor resulted in a higher concentration of dissolved oxygen in the medium, which might enhance the production of pullulan by A. pullulans HP-2001.
Direct Evidence of Intracellular Alkalinization in Saccharomyces cerevisiae KNU5377 Exposed to Inorganic Sulfuric Acid
Yun, Hae-Sun ; Paik, Sang-Kyoo ; Kim, Il-Sup ; Jin, Ing-Nyol ; Sohn, Ho-Yong ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 243~249
The toxicity of inorganic sulfuric acid as a stressor was characterized in Saccharomyces cerevisiae KNU5377. In this work, we examined physiological responses to low extracellular pH
caused by inorganic
, which could not affect cell growth after pH was adjusted to an optimum with Trizma base. The major toxicity of sulfuric and was found to be reduction of environmental pH, resulting in stimulation of plasma membrane
, which in turn contributed to intracellular alkalinization. Using a pH-dependent fluorescence probe, 5-(and-6)-carboxy SNARF-1, acetoxymethyl ester, acetate (carboxy SNARF-1 AM acetate), to determine
, we found that color was dependent on the changes of intracellular pH which coincided with calculated
of alkalinization up to approximately pH 7.3. This alkalinization did not seem to affect survival of these cells exposed to 30 mM sulfuric acid, which lowered the
of the glucose containing growth media up to approximately pH 3.0; however, the cells could grow only up to 70% of the maximum growth in the same media, when 30 mM sulfuric acid was added.
Biosorption of Lead
from Aqueous Solution by Rhodotorula aurantiaca
Cho, Dae-Haeng ; Yoo, Man-Hyong ; Kim, Eui-Yong ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 250~255
The aim of this work was to investigate the adsorption isotherm and kinetic model for the biosorption of lead
by Rhodotorula aurantiaca and to examine the environmental factors for this metal removal. Within five minutes of contact,
sorption reached nearly 86% of the total
sorption. The optimum initial pH value for removal of
was 5.0. The percentage sorption increased steeply with the biomass concentration up to 2 g/l and thereafter remained more or less constant. The Langmuir sorption model provided a good fit throughout the concentration range. The conformity of these data to the Langmuir model indicated that biosorption of
by R. aurantiaca could be characterized as a monolayer, single-site type phenomenon with no interaction between ions adsorbed in neighboring sites. The maximum
and Langmuir constant (b) were 46.08 mg/g of biomass and 0.04 l/mg, respectively. The pseudo second-order equation was well fitted to the experimental data. The correlation coefficients for the linear plots of t/q against t for the second-order equation were 0.999 for all the initial concentrations of biosorbent for contact times of 180 min. The theoretical
value was very close to the experimental
Detection of Chitooligosaccharides in Korean Soybean Paste by Tandem Immunoaffinity-ELISA
Kim, Soon-Young ; Kwak, Bo-Yeon ; Shim, Youn-Young ; Shon, Dong-Hwa ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 256~261
In order to detect chitooligosaccharides (COS) in soybean paste, tandem immunoaffinity chromatography and enzyme-linked immunosorbent assay (ELISA) were developed. Polyclonal anti-chitooligosaccharides mixture (CaSM) antibody specific to COSM was attached to Sepharose gel for initial sample cleanup and concentration of COS in soybean paste. COS was eluted and quantified by competitive direct ELISA (cdELISA). Average ELISA recoveries from the column using binding buffer spiked with COSM at levels of 0.5, 2.0, 5.0, and
g/ml were 79.8, 72.0, 77.7, and 60.6%, respectively, with a mean recovery of 72.5%. Mean inter-well and inter-assay coefficients of variation (CV) were 7.7% and 10.3%, respectively. Average recoveries from soybean paste spiked with COSM at levels of 2, 6, 20, and
g/g were 115, 91.7, 91, and 73.3%, respectively, with a mean recovery of 92.8%. Mean inter-well and inter-assay CV were 12.9% and 16%, respectively. The COS was detected from 24 out of 25 homemade Korean soybean paste samples at an average of
g/g (n, 25; range,
g/g) and from 13 out of 14 commercially made soybean paste samples at an average of
g/g(n, 14; range,
g/g). The tandem immunoaffinity chromatography-cdELISA that was developed in this study showed that the level of COS eluted from homemade soybean paste was higher than that of the commercially made ones. In addition, the level of COS eluted from commercially available soybean paste in Korea was higher than that of the ones in Japan.
Bacterial Regrowth in Water Distribution Systems and Its Relationship to the Water Quality: Case Study of Two Distribution Systems in Korea
Yoon, Tae-Ho ; Lee, Yoon-Jin ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 262~267
This study was done to observe the occurrence of heterotrophic bacteria in terms of free chlorine residuals in two different water distribution systems, which belongs to both K and Y water treatment plant of S city in Korea. The data analyzed in the distribution systems show that the free chlorine residuals decreased from 0.10 to 0.56 mg/l for K, and 0.51 to 0.78 mg/l for Y. The decay of free chlorine is clearly higher in both March and August than in January. The HPC in the distribution systems are ranged from 0 to 40 cfu/ml for K, 0 to 270 cfu/ml for Y, on
A medium. In particular, its level is relatively high at the consumer's ground storage tanks, taps, and the point-of-end area of Y. The predominant genera that were studied in the distribution systems were Acinetobacter, Sphingomonas (branch of Pseudomonas), Micrococcus, Bacillus, Staphylococcus. The diversity of heterotrophic bacteria increases in the end-point area. Most of them are either encapsulated cells or of Gram-positve cocci. In conclusion, the point-of-end area in distribution systems shows the longer flow distance from the water treatment plants, along with a greater diversity and a higher level of heterotrophic bacteria, due to the significant decay of free chlorine residuals.
Detection of DNA Damage in Carp Using Single-Cell Gel Electrophoresis Assay for Genotoxicity Monitoring
Jin, Hai-Hong ; Lee, Jae-Hyung ; Hyun, Chang-Kee ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 268~275
To investigate the potential application of the single-cell gel electrophoresis (SCGE) assay to carp as an aquatic pollution monitoring technique, gill, liver, and blood cells were isolated from carp exposed to a direct-acting mutagen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), or indirect mutagen,
, then the DNA strand breakage was analyzed using the assay. Based on testing 5 different cell isolation methods and 6 electrophoretic conditions, the optimized assay conditions were found to be cell isolation by filter pressing and electrophoresis at a lower voltage and longer running time (at 0.4 V/cm for 40 min). In preliminary experiments, gill and liver cells isolated from carp exposed to MNNG in vitro exhibited DNA damage signals even with 0.5 ppb exposure, which is a much higher dose than previously reported. In the gill cells isolated from carp exposed to 0.01-0.5 ppm MNNG in vivo, significant dose-and time-dependent increases were observed in the tail for 4 days. As such, the linear correlation between the relative damage index (RDI) values and time for each dose based on the initial 48-h exposure appeared to provide effective criteria for the genotoxicity monitoring of direct-acting mutagenic pollution. In contrast, the in vivo exposure of carp to 0.25-1.0 ppm of
for 7 days resulted in dose-and time-dependent responses in the liver cells, in which 24-h delayed responses for metabolizing activation and gradual repair after 48 h were also observed. Thus, the negative-sloped linear correlation between the RDI and time at each dose based on the initial 48 h appeared to provide more effective criteria for the genotoxicity monitoring of indirect mutagenic pollution.
Gluconacetobacter persimmonis sp. nov., Isolated from Korean Traditional Persimmon Vinegar
Yeo, Soo-Hwan ; Lee, Oh-Seuk ; Lee, In-Seon ; Kim, Hyun-Soo ; Yu, Tae-Shick ; Jeong, Yong-Jin ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 276~283
Screening was performed to isolate cellulose-producing microorganisms from the Korean traditional fermented persimmon vinegar. The resulting strain, KJ
, was then taxonomically investigated by phenotypic characterization, particularly chemotaxonomic, and by phylogenetic inference based on a 16S rDNA sequence analysis including other related taxa. Strain KJ
was found to grow rapidly and form pale white colonies with smooth to rough surfaces on a GYC agar. Strain KJ
also produced acetate from ethanol, and was tolerable to 10% ethanol in SM medium. In a static culture, a thick cellulose pellicle was produced, and in GYC broth, the strain grew at temperatures ranging from 28 to
with an optimum pH of 4.0. The genomic DNA G+C content of strain KJ
was 61.9 mol%, and the predominant ubiquinone was Q 10 as the major quinone and Q9 as the minor quinone. The major cellular fatty acids were
and the sum in feature 7 (
w9c, w12t and/or w7c). A 16S rRNA-targeted oligonucleotide probe specific for strain KJ
was constructed, and the phylogenetic position of the new species was derived from a 16S rDNA-based tree. When comparing the 16S rDNA nucleotide sequences, strain KJ
was found to be most closely related to G. hansenii LMG
(99.2%), although KJ
was still distinct from G. hansenii LMG
and G. xylinus LMG
in certain phenotypic characteristics. Therefore, on the basis of 16S rDNA sequences and taxonomic characteristics, it is proposed that strain KJ
should be placed in the genus Gluconacetobacter as a new species, Gluconacetobacter persimmonis sp. nov., under the type-strain KJ
Direct Identification of Vibrio vulnificus by PCR Targeting Elastase Gene
Lee, Jae-Won ; Jun, In-Joon ; Kwun, Hyun-Jin ; Jang, Kyung-Lib ; Cha, Jae-Ho ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 284~289
A PCR assay for the rapid detection of Vibrio vulnificus strains was developed using a virulence gene for elastase found in various Vibrio species. The DNA sequences in the elastase gene facilitated the identification of a species-specific probe for pathogenic V. vulnificus strains from both clinical and environmental sources. Using an elastase gene-based PCR reaction, a species-specific 507-bp PCR product was visualized by agarose gel electrophoresis. Three different DNA extraction methods were then compared to improve the simplicity and rapidity of detection. A PCR assay using the conventional DNA extraction or boiling method was able to detect as few as 25 V. vulnificus cells, making the detection limits at least 1-log-scale lower than that for the EDT A-treated DNA extraction method. In particular, the boiling method, which does not require purification of the chromosomal DNA, was very effective in terms of simple and rapid detection. Meanwhile, the detection limit in a mixed bacterial culture that included other bacteria, such as Escherichia coli or Bacillus subtilis, was two V. vulnificus cells, which was 1-log-scale lower than that for the control. Accordingly, when coupled with a new DNA extraction method, the elastase gene-based PCR can provide a rapid, specific, and sensitive method for identifying V. vulnificus in clinical and environmental samples.
K:Fe Ratio as an Indicator of Cyanobacterial Bloom in a Eutrophic Lake
Ahn, Chi-Yong ; Park, Dae-Kyun ; Kim, Hee-Sik ; Chung, An-Sik ; Oh, Hee-Mock ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 290~296
The effects of potassium, sodium, calcium, magnesium, and iron on cyanobacterial bloom potentials were investigated in Daechung Reservoir, Korea. Potassium showed the highest correlation with the cyanobacterial cell number (r=0.487, P<0.05) and phycocyanin concentration (r=0.499, P<0.05). However, it was not likely that the potassium had directly affected the bloom formation, because the variations of its concentration were not significantly large. In contrast, the Fe concentration fluctuated drastically and exhibited a negative correlation with the cyanobacterial cell number (r=- 0.388, P<0.1) and phycocyanin concentration (r=-0.446, P<0.05). Accordingly, the K:Fe atomic ratio would appear to reflect the extent of cyanobacterial bloom more precisely than K or Fe alone. The K:Fe ratio specifically correlated with cyanobacterial percentage, the cyanobacterial cell number and phycocyanin concentration (r=0.840, P<0.001; r=0.416, P<0.05; r=0.522, P<0.01, respectively). With the K:Fe atomic ratio of over 200, the chlorophyll-a concentration, cyanobacterial cell number, and phycocyanin concentration exceeded
, and 20 pM, respectively, the general criteria of eutrophic water.
Production of dTDP-4-keto-6-deoxy-D-glucose by Immobilization of dTDP-D-glucose 4,6-dehydratase
Kharel, Mandan-Kumar ; Liou, Kwang-Kyoung ; Sohng, Jae-Kyung ; Lee, Hei-Chan ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 297~301
The dTDP-D-glucose 4,6-dehydratase from Salmonella enterica was immobilized using covalent binding to cyanogen bromide activated sepharose. The immobilized enzyme was used to produce dTDP-4-keto-6-deoxy-D-glucose, a key sugar intermediate that can be used economically to produce diverse classes of unusual sugars appended in various antibiotics. The enzyme was immobilized on the sepharose after activation with cyanogen bromide. The maximum immobilization (80.03％) was achieved after 14 h of coupling. The covalently immobilized enzyme was stable, and an average of 78.4 ％ conversion was achieved until 120 h of immobilization when it was repeatedly used. Similar conversion was noticed for the first batch using the enzyme entrapped-hydrogel but activity was gradually decreased in the following batches. The production of dTDP-4-keto-6-deoxy-D-glucose by using an immobilized enzyme has high potential for commercial application.
Versatile Catabolic Properties of Tn4371-encoded bph Pathway in Comamonas testosteroni (Formerly Pseudomonas sp.) NCIMB 10643
Kim, Jong-Soo ; Kim, Ji-Hyun ; Ryu, Eun-Kyeong ; Kim, Jin-Kyoo ; Kim, Chi-Kyung ; Hwang, In-Gyu ; Lee, Kyoung ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 302~311
Comamonas testosteroni (formerly Pseudomonas sp.) NCIMB 10643 can grow on biphenyl and alkylbenzenes
via 3-substituted catechols. Thus, to identify the genes encoding the degradation, transposon-mutagenesis was carried out using pAG408, a promoter-probe mini-transposon with a green fluorescent protein (GFP), as a reporter. A mutant, NT-1, which was unable to grow on alkylbenzenes and biphenyl, accumulated catechols and exhibited an enhanced expression of GFP upon exposure to these substrates, indicating that the gfp had been inserted in a gene encoding a broad substrate range catechol 2,3-dioxygenase. The genes (2,826 bp) flanking the gfp cloned from an SphI-digested fragment contained three complete open reading frames that were designated bphCDorfl. The deduced amino acid sequences of bphCDorfl were identical to 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC), 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (BphD), and OrfI, respectively, that are all involved in the degradation of biphenyl/4-chlorobiphenyl (bph) by Ralstonia oxalatica A5. The deduced amino acid sequence of the orfl revealed a similarity to those of outer membrane proteins belonging to the OmpW family. The introduction of the bphCDorfl genes enabled the NT-l mutant to grow on aromatic hydrocarbons. In addition, PCR analysis indicated that the DNA sequence and gene organization of the bph operon were closely related to those in the bph operon from Tn4371 identified in strain A5. Furthermore, strain A5 was also able to grow on a similar set of alkylbenzenes as strain NCIMB 10643, demonstrating that, among the identified aromatic hydrocarbon degradation pathways, the bph degradation pathway related to Tn4371 was the most versatile in catabolizing a variety of aromatic hydrocarbons of mono- and bicyclic benzenes.
A New Thermophile Strain of Geobacillus thermodenitrificans Having L- Arabinose Isomerase Activity for Tagatose Production
Baek, Dae-Heoun ; Lee, Yu-Jin ; Sin, Hong-Sig ; Oh, Deok-Kun ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 312~316
Five strains, producing bacterial thermostable L-arabinose isomerase, were isolated from Korean soil samples obtained from compost under high temperature circumstances. Among these strains, the CBG-Al showed the highest L-arabinose isomerase activity at
and was selected as a D-tagatose producing strain from D-galactose. This strain was identified as Geobacillus thermodenitrificans based on the 16S rRNA analysis, and biological and biochemical characteristics. The isolated strain was aerobic, rod-shaped, Gram-positive, nonmotile, and an endospore-forming bacterium. No growth was detected in culture temperature below
. The maximum growth temperature and maximum temperature of enzyme activity were
, respectively. In metal ion effects,
was the most effective enzyme activator with the reaction rate by 150%. In a 5-1 jar fermentor with 3-1 MY medium, L-arabinose isomerase activity was growth-associated and pH decreased rapidly after the initial logarithmic phase.
Antimicrobial Activity of Hetero-Chitosans and Their Oligosaccharides with Different Molecular Weights
Park, Pyo-Jam ; Je, Jae-Young ; Byun, Hee-Guk ; Moon, Sung-Hoon ; Kim, Se-Kwon ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 317~323
This study was performed to investigate the antimicrobial effects of hetero-chitosans and their oligosaccharides against three Gram-negative bacteria and five Gram-positive bacteria. Nine classes of hetero-chitosan oligosaccharides consisted of partially deacetylated chitosans; 90％, 75％, and 50％ deacetylated chitosans. Based on molecular weight, they were prepared using an ultrafiltration membrane reactor system. Seventy-five percent deacetylated chitosan showed the highest antimicrobial acitivity as compared with the 90％ and 50％ deacetylated chitosan, and the activity was dependent on their molecular weights. It was apparent that the growth of Gram-negative bacteria is less inhibited in the presence of the heterochitosans and their oligosaccharides than Gram-positive bacteria. These results revealed that the antimicrobial effects of hetero-chitosans and their oligosaccharides depend on the degree of deacetylation, and their molecular weights.
Improvement of Cathode Reaction of a Mediatorless Microbial Fuel Cell
Pham, The-Hai ; Jang, Jae-Kyung ; Chang, In-Seop ; Kim, Byung-Hong ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 324~329
Oxygen diffuses through the cation-specific membrane, reducing the coulomb yield of the fuel cell. In the present study, attempts were made to enhance current generation from the fuel cell by lowering the oxygen diffusion, including the uses of ferricyanide as a cathode mediator and of a platinum-coated graphite electrode. Ferricyanide did not act as a mediator as expected, but as an oxidant in the cathode compartment of the microbial fuel cell. The microbial fuel cell with platinum-coated graphite cathode generated a maximum current 3-4 times higher than the control fuel cell with graphite cathode, and the critical oxygen concentration of the former was 2.0 mg
, whilst that of the latter was 6.6 mg
. Based on these results, it was concluded that inexpensive electrodes are adequate for the construction of an economically feasible microbial fuel cell with better performance as a novel wastewater treatment process.
Virulence of Environmental Urease-Positive and Kanagawa Phenomenon-Negative Vibrio parahaemolyticus
Park, Mi-Yeon ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 330~336
Fifty-two pathogenic Vibrio parahaemolyticus strains were isolated from the environments of Busan and Yeosu, Korea. Forty-three of these strains showed protease activities, whereas 4 strains showed
hemolysin activities and 6 strains had urease activities. Their pathogenic factors were not overlapping except one strain, which had both protease and hemolysin activities. The 6 urease-positive strains (V. parahaemolyticus YKB4, YKB14, S25, YFB20, YFO21, and YFO22) showed the same biochemical characteristics as a reference strain [V. parahaemolyticus KCTC 2471 (urease-negative)], except for urease production. The 6 urease-positive strains showed different urease activities in their culture supernatant during the growth. The urease activity of S25 increased sharply at the late exponential phase, and was the highest at the initial stationary phase and was kept until the late stationary phase. The other 5 isolates, except C25, showed urease activities at the mid-stationary phase and increased steadily until the late stationary phase, when the urease activity was maximal. To compare the degree of virulence of V. parahaemolyticus with different pathogenic factors, hemolysin, protease, or urease-positive strains were injected into groups of 10 each of ICR mice (7- to l0-week-old males). The lethal rates of urease-positive V. parahaemolyticus, YKB14, YKB4, and S25, were significantly high, being 50, 70, and 80%, respectively. Protease-positive V. parahaemolyticus strains FM39 and FM50 showed 40% and 60% of lethal rate, respectively. Hemolysin-positive V. parahaemolyticus strains S34 and S72 had no mortality, similar to nonpathogenic V. parahaemolyticus FM12.
Purification and Properties of Chitosanase from Chitinolytic
Yi, Jae-Hyoung ; Jang, Hong-Ki ; Lee, Sang-Jae ; Lee, Keun-Eok ; Choi, Shin-Geon ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 337~343
A bacterial strain concurrently producing extracellular chitosanase and chitinase was isolated from soil and identified as a member of the
-subgroup of Proteobacteria through its 16S rRNA analysis and some biochemical analyses. The newly discovered strain, named as KNU3, had 99% homology of its 16S rRNA sequence with chitinolytic
-Proteobacterium CTE108. Strain KNU3 produced 34 kDa of chitosanase in addition to two chitinases of 68 kDa and 30 kDa, respectively. The purified chitosanase protein (ChoK) showed activity toward soluble, colloidal, and glycol chitosan, but did not exhibit any activity toward colloidal chitin. The optimum pH and temperature of ChoK were 6.0 and
, respectively. The chitosanase was stable in the pH 4.0 to 8.0 range at
, while enzyme activity was relatively stable at below
. MALDI-TOF MS and N-terminal amino acid sequence analyses indicated that ChoK protein is related to chitosanases from Matsuebacter sp. and Sphingobacterium multivorum. HPLC analysis of chitosan lysates revealed that glucosamine tetramers and hexamers were the major products of hydrolysis.
Cloning, Expression, and Purification of Exoinulinase from Bacillus sp. snu-7
Kim, Kyoung-Yun ; Koo, Bong-Seong ; Jo, Do-Hyun ; Kim, Su-Il ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 344~349
A gene encoding inulin-degrading enzyme of Bacillus sp. snu-7 with ORF of 1536 nucleotides was cloned. And it was overexpressed as His-tagged protein in E. coli BL21(DE3) pLysS using pRSET B vector containing mature enzyme sequence. Maximum enzyme production was achieved by IPTG (0.1 mM) induction at
followed by 6 h incubation. The expressed protein purified through immobilized metal affinity chromatography showed molecular mass of 60 kDa on SDS-PAGE. Results of thin-layer chromatography using inulin as a substrate showed the enzyme to be an exotype inulinase capable of producing only monomeric fructose as a product.
, for the hydrolyses of inulin and sucrose were
mM and 358.05
, and 22.02
0.41 mM and 4619.11
, respectively. Optimal activity of the exoinulinase occurred at pH 7.0 and
Sulforhodamine B Assay to Determine Cytotoxicity of Vibrio vulnificus Against Human Intestinal Cells
Lee, Byung-Cheol ; Choi, Sang-Ho ; Kim, Tae-Sung ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 350~355
Sulforhodamine B (SRB) assay is a rapid, sensitive, and inexpensive method for measuring cell proliferation and chemosensitivity. However, the lactate dehydrogenase (LDH) release assay is generally used to measure cytototoxicity of infectious microorganisms against host cells. In this study, we investigated the possibility of applying the SRB assay to determine cytotoxicity for infectious microorganisms, and compared the results with those obtained by the LDH release assay. We used Vibrio vulnificus as a model of infectious microorganisms. The SRB assay showed that V vulnificus strongly induced cytotoxic activity against human intestinal cells, Caco-2 and INT-407 cells. The degree of cytotoxicity closely correlated with infection time and number ratios of V. vulnificus to intestinal cells (MOI, multiplicity of infection). Furthermore, cytotoxicity values obtained by SRB assay correlated well with results obtained by the LDH release assay, and both assays gave a linear response with respect to MOI Heat-inactivation of V. vulnificus for 35 min at
did not induce cytotoxic activity, indicating that viability of V. vulnificus is crucial for cytotoxic activity against intestinal cells. Although both assays are suitable as cytotoxicity endpoints, the SRB assay is recommended for measuring cytotoxicity of infectious microorganisms against host cells because of its significantly lower cost and more stable endpoint than the LDH release assay.
Identification of a Cytochrome P450 Hydroxylase Gene Involved in Rifamycin Biosynthesis by Amycolatopsis mediterranei S699
Lee, Sang-Kil ; Choi, Cha-Yong ; Ahn, Jong-Seog ; Cho, Jae-Yong ; Park, Cheon-Seok ; Yoon, Yeo-Joon ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 356~365
In analyzing the region of the Amycolatopsis mediterranei S699 chromosome responsible for the biosynthesis of the ansamycin antibiotic rifamycin, we identified a gene, designated orj0, which is located immediately upstream of the rifamycin polyketide synthase (PKS). Orj0 encodes a protein, on the basis of sequence-comparative analysis, that is similar to several cytochrome P450 monooxygenases from different sources. The rifamycin producer, A. mediterranei, predominantly produces rifamycin B from its macrocyclic intermediate, proansamycin X, through dehydrogenation and hydroxylation steps. However, an A. mediterranei strain, deleted in orj0 by gene replacement, no longer produced rifamycin B. Furthermore, a versatile replicative vector in A. mediterranei was constructed and rifamycin B production was restored in a complementation experiment of orj0 using this novel vector. These consecutive results verified that the arf0 protein, which is a P450 hydroxylase, is required for the production of rifamycin B in A. mediterranei.
Breeding of L(+)-Lactic Acid Producing Strain by Low-Energy Ion Implantation
Ge, Chun-Mei ; Gu, Shao-Bin ; Zhou, Xiu-Hong ; Yao, Jian-Ming ; Pan, Ren-Rui ; Yu, Zeng-Liang ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 363~366
In order to obtain an industrial strain with higher L(+)-lactic acid yield, the wild type strain Rhizopus oryzae PW352 was mutated by means of Nitrogen ions implantation (l5 Kev,
and two mutants RE3303 and RF9052 were isolated. After 36 h shake-flask cultivation, the concentration of L(+)-lactic acid reached 131-136 g/l, the conversion rate of glucose was as high as 86%-90% and the productivity was 3.61 g/l.h. It was almost a 75% increase in lactic acid production compared with the wild type strain. Maximum fermentation temperature of RF9052 was increased to
. At the same time, the preferred range of fermentation temperature of RF9052 was broadened compared with PW352.
Dithiothreitol Attenuates the Pathogenic Interaction Between Pseudomonas aeruginosa and Drosophila melanogaster
Lee, Ji-Sun ; Kim, Seol-Hee ; Cho, You-Hee ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 367~372
Infection of Drosophila melanogaster adults with Pseudomonas aeruginosa (PA14) can kill the flies within 48h. We found that the virulence of PA14 was significantly attenuated when treated with a reducing agent, dithiothreitol (DTT), prior to infection. Infection with DTT-treated PA14 elevated Metchnikowin expression at 22 h post-infection and the virulence of DTT-treated PA14 was not attenuated in Dif and Relish mutants. These results suggest that DTT pre-treatment of PA14 can aggravate certain virulence factors that may be required to paralyze fly immune responses, triggering Metchnikowin expression via Dif and Relish activations.
Regulation of Enzymes Involved in Methionine Biosynthesis in Corynebacterium glutamicum
Yeom, Hye-Jin ; Hwang, Byung-Joon ; Lee, Myong-Sok ; Kim, Youn-Hee ; Lee, Heung-Shick ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 373~378
The regulatory mechanism of methionine biosynthesis in Corynebacterium glutamicum was analyzed at the protein arid gene expression level. O-Acetylhomoserine sulfhydraylase (encoded by metY) was inhibited by 10 mM methionine to a residual activity of 10% level, whereas no such inhibition was found with cystathionine
-synthase (encoded by metB) and cystathionine
-lyase (encoded by metC). The enzymatic activity of homoserine acetyltransferase (encoded by metX) was repressed to a residual activity of 25% level by 10 mM methionine which was added to the growth medium. Cystathionine
-synthase and cystathionine
-lyase were also repressed by 10 mM methionine, but only to a residual activity of 50-70% level. O-Acetylhomoserine sulfhydrylase was very sensitive to repression by 10 mM methionine, showing residual activity of 13%. In addition, homoserine acetyltransferase was also repressed by 10 mM cysteine to 50% of its original activity. No repression of the enzymes by S-adenosyl methionine was observed. The pattern of repression by methionine indicated that the metB and aecD genes might be regulated by a common mechanism, while the metA and metY genes are differently regulated.
Effect of Fibroblast Growth Factor-2 on Migration and Proteinases Secretion of Human Umbilical Vein Endothelial Cells
Oh, In-Suk ; Kim, Hwan-Gyu ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 379~384
Fibroblast growth factor-2 (FGF-2) is known to modulate numerous cellular functions in various cell types, including cell proliferation, differentiation, survival, adhesion, migration, and motility, and also in processes such as wound healing, angiogenesis, and vasculogenesis. FGF-2 regulates the expression of several molecules thought to mediate critical steps during angiogenesis. This study examines the mechanisms underlying FGF-2-induced cell migration, using human umbilical vein endothelial cells (HUVECs). FGF-2 induced the nondirectional and directional migration of endothelial cells, which are inhibited by MMPs and plasmin inhibitors, and induced the secretion of matrix metalloproteinase-3 (MMP3) and MMP-9, but not MMP-l and MMP-2. FGF-2 also induced the secretion of the tissue inhibitor of metalloproteinase-l (TIMP-I), but not of TIMP- 2. Also, the pan-PKC inhibitor inhibited FGF-2-induced MMP-9 secretion. It is, therefore, suggested that FGF-2 induces the migration of cultured endothelial cells by means of increased MMPs and plasmin secretion. Furthermore, FGF-2 may increase MMP-9 secretion by activating the PKC pathway.
Detection of Aspergillus, Penicillium, and Fusarium Species by Sandwich Enzyme-Linked Immunosorbent Assay Using Mixed Monoclonal Antibodies
Kwak, Bo-Yeon ; Kwon, Byung-Joon ; Kweon, Chang-Hee ; Shon, Dong-Hwa ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 385~389
The antibody-mix sandwich enzyme-linked immunosorbent assay (Ab-mix sELlS A) system was developed in order to simultaneously detect the extracellular polysaccharide (FPS) of Aspergillus, Penicillium, or Fusarium species using one detection system. The detection limit and detection range of the Ab-mix sELISA towards EPS of Penicilliun citrinum were not changed, and those towards Fusarium moniliforme EPS were changed a little compared to that of individual sandwich ELISA [9, 10]. The fungal culture filtrates of Aspergillus and Penicillium species showed nearly similar reactivity towards Ab-mix sELISA as that of sELISA using the MAb lB8 alone . Also, the fungal culture filtrates of Fusarium species showed nearly the same reactivity towards Ab-mix sELISA as that of sELISA using the MAb lB8 alone . Thus, this ELISA system showed that the three genera of molds, Aspergillus, Penicillium, or Fusarium, which are three major important molds producing mycotoxins in food or agricultural commodities, could be detected at the same time, using one detection system.
Detection of Recombinant Marker DNA in Genetically Modified Glyphosate- Tolerant Soybean and Use in Environmental Risk Assessment
Kim, Young-Tae ; Park, Byoung-Keun ; Hwang, Eui-Il ; Yim, Nam-Hui ; Lee, Sang-Han ; Kim, Sung-Uk ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 390~394
The genetically modified glyphosate-tolerant soybean contains the following introduced DNA sequences: the EPSPS (5-enol-pyruvylshikimate-3-phosphate synthase) gene from Agrobacterium sp. strain CP4, the 35S promoter from the cauliflower mosaic virus, and the NOS terminator from Agrobacterium tumefaciens. In the present study, detection of these introduced DNAs was performed by amplification using the polymerase chain reaction (PCR). A multiplex PCR method was also applied to prevent false positive results. When primers for 35S promoter, nos3', CTP(chloroplast transit peptide), and CP4 EPSPS (EPSPS from Agrobacterium sp. CP4) were used, positive results were obtained in PCR reactions using DNA from genetically modified glyphosate-tolerant soybeans. There were no false positive results when using DNA from non-genetically modified soybeans. The CP4 EPSPS gene was detected when less than 125 pg glyphosate-tolerant soybean DNA was amplified. Lectin Lel and psb A were amplified from both non-genetically modified and genetically modified glyphosate-tolerant soybean DNA. Multiplex PCR was performed using different primer sets for actin Sacl, 35S promoter and CP4 EPSPS. The actin gene was detectable in both non-genetically modified and glyphosate-tolerant soybeans as a constant endogenous gene. Target DNAs for the 35S promoter, and CP4 EPSPS were detected in samples containing 0.01-0.1% glyphosate-tolerant soybean, although there were variations depending on primers by multiplex PCR. Soybean seeds from five plants of non-genetically modified soybean were co-cultivated for six months with those of genetically modified soybean, and they were analyzed by PCR. As a result, they were not positive for 35S promoter, nos3' or CP4 EPSPS. Therefore, these results suggest there was no natural crossing of genes between glyphosate-tolerant and non-genetically modified soybean during co-cultivation, which indicates that gene transfer between these plants is unlikely to occur in nature.
An Immobilized Culture of Choristoneura fumiferana Cells for High Production of CfMNPV
Son, Jeong-Hwa ; Buchholz, Rainer ; Kim, Sung-Koo ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 395~400
Choristoneura fumiferana (Cf-2Cl) insect cells were cultured and immobilized by using cellulosesulfate (NaCS) and polydiallyldimethylammoniumchloride (PDADMAC). A concentration of CfMNPV (Choristoneura fumiferana multiple-mucleopolyhedrovirus) and a Cf-2Cl cell density in the microspheres have been achieved at the densities of
cells/ml, respectively. Additionally, MTT-test was used to measure the viable cell density in the microspheres, and the confidence of MIT-test was investigated before and after baculovirus infection in the immobilized cell culture.
Distribution and Antimicrobial Susceptibility of Clostridium Species in Soil Contaminated with Domestic Livestock Feces of Korea
Kim, Jeong-Dong ; Lee, Dae-Weon ; Lee, Kyou-Seung ; Choi, Chang-Hyun ; Kang, Kook-Hee ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 401~410
Soil samples from five different areas in Korea were collected during 2001/02 and examined for presence of the genus Clostridium. Direct immuno-fluorescent assay (IFA) examination showed that Clostridium septicum, Cl. novyi and Cl. chauvoei were detected in the soil of specific areas in Korea. Sixteen species of Clostridium were isolated and cultivated from the soil samples. Cl. peifringens was detected in all sampling locations, while the other species were not. The in vitro activity of 14 antibiotic agents was determined against 421 clostridia isolated from the soil contaminated with animal feces in Korea. Trovafloxacin was effective against all isolates of the genus Clostridium except one isolate of Cl. subterminale, two of Cl. tetani, and three of Cl novyi with
. Thirteen species of Clostridium were resistant to vancomycin except for Cl. perfringens, Cl. sporogenes, and Cl. subterminale. Imipenem and trovafloxacin showed high antimicrobial activities (>95%) against all strains in the clostridia investigated. Therefore, antibiotic agents such as imipenem and trovafloxacin are the most suitable agents for polymicrobial infection as broad-spectrum monotherapy.
In Vivo Antitumor Activity of Hydrophilic Arginine-Conjugated Linoleic Acid Complex
Kim, Young-Jun ; Lee, Ki-Won ; Kim, Dae-Ok ; Kim, Tae-Wan ; Lee, Seong-Kweon ; Lee, Hyong-Joo ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 411~414
Although conjugated linoleic acid (CLA) exerted potent antitumor activities in several animal models, application of CLA as a bioactive ingredient has been limited due to its hydrophobicity. This study was designed to determine the antitumor activity of arginine-CLA complex (Arg-CLA), a hydrophilic form of CLA. Mouse forestomach cancer was induced by gavage with benzo(a)pyrene (B(a)p) for 4-weeks prior to Arg-CLA (0.2 and 0.5％) feeding. Complete necropsies were performed to determine the number, size and locations of all the forestomach tumors at 20 weeks post-B(a)P administration. All mice in the B(a)P group developed tumors, and tumor incidences were decreased by 31 ％ and 44％ in 0.2％ and 0.5％ Arg-CLA-fed groups, respectively, whereas no decrease was observed when Arg or com oil was given alone. Our results suggest that Arg-CLA suppresses mouse forestomach cancer.
Immunological Relationship of Crystal Proteins of Six Bacillus thuringiensis Serovarieties; B. thuringiensis serovar. coreanensis (H25), konkukian (H34), leesis (H33), seoulensis (H35), sooncheon (H41) andyosoo (H18a18c)
Lee, Kwang-Yong ; Kang, Eun-Young ; Kwon, Hyuk-Han ; Lee, Hyung-Hoan ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 415~416
Crystals of six new Bacillus thuringiensis serovarieties [coreanensis (H25), konkukian (H34), leesis (H33), seoulensis (H35), sooncheon (H41), and yosoo (HI8a18c)] with different H-antigens, which are toxic to Bombyx mori and/or mosquito larvae, were serologically quite distinct from each other.
Cloning, Sequencing, and Expression of cDNA Encoding Bovine Prion Protein
Kang, Sang-Gyun ; Kang, Sung-Keun ; Lee, Deog-Yong ; Park, Yong-Ho ; Hwang, Woo-Suk ; Yoo, Han-Sang ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 417~421
A normal prion protein (PrPc) is converted to a protease resistant isoform (PrPsc) by an apparent self-propagating activity in bovine spongiform encephalopathies (BSE), which is a neurodegenerative disease. The cDNA encoding bovine PrP open reading frame (ORP) in Korean cattle was cloned by polymerase chain reaction (PCR). The cloned cDNA had a length of 795 base pairs which coded for a protein of 264 amino acid residues with a calculated molecular mass of 28.6 kDa. Identities of 90, 90, 79 and 78% on nucleotide and 94, 94, 84, and 84% on amino acid sequence were shown to PrP genes from sheep, goat, human, and mouse, respectively. The cloned DNA was ligated into the pQE30 expression vector and transformed into E. coli M15. The PrP was expressed by induction with isopropyl-
-D-thiogalactoside (IPTG) and purified on the Ni-NTA affinity column. High specific activities of the recombinant PrP were observed in the fraction of pH 5.8 eluate and showed a molecular mass of-29 kDa on SDS-PAGE and Western blot analysis.
Antifungal Activity of Streptomyces sp. Against Puccinia recondita Causing Wheat Leaf Rust
Yi, Yong-Sub ; Kim, Seung-Hyun ; Kim, Min-Woo ; Choi, Gyung-Ja ; Cho, Kwang-Yun ; Song, Jae-Kyeong ; Lim, Yoong-Ho ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 422~425
To discover a potent strain against wheat leaf rust, soil samples collected from Ilgamho, Seoul, Korea were tested in vivo and a strain belonging to Streptomyces sp. was found to show good antifungal activity when fermented in a broth. The identification of the strain was carried out based on 16S rDNA analysis, and the active compound was separated from the fermented broth. Even though its structure was not determined completely, the authors report the results obtained so far indicate that the fermented broth of the strain showed activity against wheat leaf rust. Therefore, we propose that this may be a potential novel strain showing antifangal activity against Puccinia recondita.
Catechin Degradation by Several Fungal Strains Isolated from Mexican Desert
Aguilar Cristobal Noe ; Mario Cruz ; Raul Rodriguez ; Gerardo Gutierrez-Sanchez ; Ascencion Ramirez-Coronel ; Christopher Augur ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 426~429
Eleven fungal strains previously isolated from the Mexican desert were evaluated for their capacity to use catechin as carbon source in submerged cultures. At 2 g/l of catechin, all strains grew better than the control strains, Aspergillus niger Aa-20. Aspergillus niger PSH and Penicillium commune EH2 degraded 79.33％ and 76.35％ with degradation rates of 0.0065 and 0.0074 g/l/h, respectively, when an initial catechin concentration of 3 g/l was used. Obtained results demonstrated the potential biotechnological capacity of these fungal strains to use condensed tannins as carbon source.
Expression of a Bacillus subtilis Endoglucanase in Protease-Deficient Bacillus subtilis Strains
Yang, Mi-Jeong ; Jung, Sun-Hwa ; Shin, Eun-Sun ; Kim, Jung-Ho ; Yun, Han-Dae ; Wong, Sui-Lam ; Kim, Ho-On ;
Journal of Microbiology and Biotechnology, volume 14, issue 2, 2004, Pages 430~434
Three extracellular protease-deficient Bacillus subtilis strains were transformed with the plasmid pCK98 containing the endo-
-1,4-glucanase (Eng) gene of B. subtilis BSE616. The three transformants, B. subtilis DB104 (pCK98), WB600 (pCK98) and WB700 (pCK98), produced the same high level of enzyme activity and showed similar patterns of cell growth and enzyme production. When B. subtilis DB 104 (pCK98), a two-extracellular protease deficient strain, was cultured for 22 h, almost all the secreted enzyme was found to be in the completely cleaved form by both activity staining and Western blotting studies. B. subtilis WB600 (pCK98), a six-extracellular protease-deficient strain, produced a partially cleaved form in addition to the intact form of the enzyme, although the degree of internal cleavage of the enzyme was greatly reduced. With B. subtilis WB700 (pCK98), a seven-extracellular protease-deficient strain, almost all the enzyme was produced as the intact uncleaved form. This study illustrates that a role of the V pr protease is to degrade foreign proteins produced in B. subtilis and WB700 is a suitable expression system for producing the intact form of the Eng and other foreign proteins that may lose at least part of their efficacy due to internal proteolytic cleavage.