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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 12, Issue 6 - Dec 2002
Volume 12, Issue 5 - Oct 2002
Volume 12, Issue 4 - Aug 2002
Volume 12, Issue 3 - Jun 2002
Volume 12, Issue 2 - Apr 2002
Volume 12, Issue 1 - Feb 2002
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Leakage of Cellular Materials from Saccharomyces cerevisiae by Ohmic Heating
Yoon, Sung-Won ; Lee, Chung-Young-J. ; Kim, Ki-Myung ; Lee, Cherl-Ho ;
Journal of Microbiology and Biotechnology, volume 12, issue 2, 2002, Pages 183~188
The ohmic heating of foods for sterilization provides a shorter come-up time compared to conventional thermal processes. The electric fields as well as the heat generated by ohmic heating facilitate germicidal effects. In the present study, the effect of ohmic heating on the structure and permeability of the cell membrane of yeast cells, Saccharomyces cerevisae, isolated from Takju (a traditional Korean rice-beer), was investigated. The ohmic heating was found to translocate intracellular protein materials out of the cell wall, and the amount of exuded protein increased significantly as the electric field increased from 10 to 20 V/cm. As higher frequencies were applied, more materials were exuded. Compared to conventional heating, more amounts of proteins and nucleic acids were exuded when these cells were treated with ohmic heating. The molecular weights of the major exuded proteins ranged from 14 kDa to 18 kDa, as analyzed by Tricine-SDS PAGE. A TEM study also confirmed the leakage of cellular materials, thus indicating irreversible damage to the cell wall by ohmic heating. It was, therefore, concluded that the electric fields generated by ohmic heating induced electroporation, causing irreversible damage to the yeast cell wall and promoting the translocation of intracellular materials.
Production and Characterization of Extracellular Phospholipase D from Streptomyces sp. YU100
Lim, Si-Kyu ; Choi, Jae-Woong ; Chung, Min-Ho ; Lee, Eun-Tae ; Khang, Yong-Ho ; Kim, Sang-Dal ; Nam, Doo-Hyun ;
Journal of Microbiology and Biotechnology, volume 12, issue 2, 2002, Pages 189~195
Using Streptomyces sp. YU100 isolated from Korean soil, the fermentative production of phospholipase D was attempted along with its purification and characterization studies. When different carbon and nitrogen sources were supplemented in the culture medium, glucose and yeast extract were found to be the best. By varying the concentration of nutrients and calcium carbonate, the optimal culture medium was determined as 2.0% glucose, 1.5% yeast extract, 0.5% tryptone 0.3% calcium carbonate. During cultivation, the strain secreted most of the phospholipase D in the early stage of growth within 24 h. The phospholipase D produced in the culture broth exhibited hydrolytic activity as well as transphosphatidylation activity on lecithin (phosphatidylcholine). In particular, the culture broth showed 8.7 units/ml of hydrolytic activity when cultivated at
for 1.5 days. The phospholipase D was purified using 80% ammonium sulfate precipitation and DEAE-Sepharose CL-6B column chromatography, which produced a major band of 57 kDa on a 10% SDS-polyacrylamide gel with purity higher than 80%. The enzyme showed an optimal pH of 7 in hydrolytic reaction, and at pH 4 in a transphosphatidylation reaction. The enzyme activity increased until the reaction temperature was elevated to
. The enzyme was relatively stable at high temperatures and neutral pH, but significantly unstable in the alkaline range. Among the detergents tested as emulsifiers of phospholipids, the highest enzyme activity was observed when 1.5% Triton X-100 was employed. However, no inhibitory effect by metal ions was detected. Under optimized reaction conditions, the purified enzyme not only completely decomposed PC to phosphatidic acid within 1 h, but also exhibited higher than 80% conversion rate of PC to PS by transphosphatidylation within 4 h.
Preparation of High-Purity Urokinase Using Single-Step Hydrophobic Interaction Chromatography with p-Aminobenzamidine Ligand
Cao, Xue-Jun ; Zhou, Jian-Hua ; Huang, Zhen-Hui ; Wu, Xing-Yan ; Hur, Byung-Ki ;
Journal of Microbiology and Biotechnology, volume 12, issue 2, 2002, Pages 196~203
A novel process for urokinase purification was studied using p-aminobenzamidine as the ligand and sepharose 4B as the matrix. The adsorption, washing, and elution conditions were optimized by an unusual method. An adsorption buffer containing 2.5 M NaCl and
Tween 80 facilitated the adsorption of urokinase on the affinity media and prevented contaminants from binding to the p-aminobenzamidine affinity gel. It was found that
Tween 80 removed most of the contaminants from the affinity column. A 0.2 M glycine elution buffer containing 0.5 M NaCl (pH 3.0) was found to have a strong elution ability with a high recovery and purity of urokinase. A crude urokinase material of231 IU/mg protein from human urine was purified to 124,300 IU/mg protein with a purification factor of 538 and yield of
. As a result, a high purity urokinase was obtained with only a single affinity chromatography step. The purification process was successfully scaled-up to a 2-1 chromatography column. The resulting urokinase eluate could be directly lyophilized, thereby complying with Chinese pharmacopoeia (1995 version) standards.
Identification of Streptomyces sp. Producing New Polyene Antibiotics and In Vivo Antimicrobial Activity of Tetrin C Against Phytopathogenic Fungi
CHOI, WON-CHANG ; SEOK-YEON HWANG ; TAE-KYU PARK ; SI-KWAN KIM ;
Journal of Microbiology and Biotechnology, volume 12, issue 2, 2002, Pages 204~208
A Streptomyces sp. isolated from a soil sample collected in Taejeon, Korea has previously been found to produce two new polyene antibiotics. The two new antibiotics were named "16-methyloxazolomycin (antibacterial)" and "tetrin C (antifungal)", and their chemical structures are presented elsewhere [10, 11]. In the current study, chemotaxonomy, numerical taxonomy, and ISP methods were all employed for the taxonomic study. The spore chains were spirales and the spore surface was smooth. The spore mass was a gray series and no melanin pigment was produced. On the basis of the morphological and physiological properties, the microorganism was identified to be Streptomyces erumpens, belonging to the gray series of category IV, as defined by Bergey′s Manual. Tetrin C at the concentration of 20
/ml demonstrated a potent in vivo (pot test) preventive effect against rice blast, rice sheath blight, cucumber gray mold, wheat powdery mildew, and barley leaf rust.
Physico-Chemical and Rheological Properties of a Bioflocculant BF-56 from Bacillus sp. A56
Suh, Hyun-Hyo ; Moon, Seong-Hoon ; Seo, Weon-Taek ; Kim, Kyung-Kab ; Jeon, Gee-Ill ; Park, Hyun-Geoun ; Park, Yong-Il ;
Journal of Microbiology and Biotechnology, volume 12, issue 2, 2002, Pages 209~216
Bacillus sp. A56 was studied, because of its high flocculating activity. The flocculating substance produced by this strain was purified by ethanol precipitation, cetylpyridinium chloride (CPC) precipitation, and gel permeation chromatography (GPC). The FT-IR spectrum of the purified bioflocculant, designated as BF-56, showed typical characteristics of polysaccharides. The non-sugar substituents, and sugar components of BF-56 containing glucose, fucose, glucuronic acid, and galactose in an approximate molar ratio of 2.76:1.10:1:0.12, suggested that it was a novel bioflocculant with an estimated molecular mass of over
kDa. Rheological analysis of BF-56 revealed that it was a pseudoplastic that had higher apparent viscosity rate at dilute concentrations than those of zooglan. The solution of bioflocculant BF-56 exhibited non-Newtonian characteristics and it was compatible to high concentrations of salts such as KCl, NaCl,
The present results suggested strong possibility of bioflocculant BF-56 to be fully applicable to industries such as wastewater treatment.
Function of Lysine-148 in dTDP-D-Glucose 4,6-Dehydratase from Streptomyces antibioticus Tu99
Sohng, Jae-Kyung ; Noh, Hyung-Rae ; Lee, Oh-Hyoung ; Kim, Sung-Jun ; Han, Ji-Man ; Nam, Seung-Kwan ; Yoo, Jin-Cheol ;
Journal of Microbiology and Biotechnology, volume 12, issue 2, 2002, Pages 217~221
dTDP-D-glucose 4,6-dehydratase (TDPDH) catalyzes the conversion of dTDP-D-glucose to dTDP-4-keto-6-deoxy-D-glucose, and requires
as a coenzyme for its catalytic activity. The dTDP-D-glucose 4,6-dehydratase from Streptomyces antibioticus
. In order to determine the role of lysine-148 in the
binding, the lysine of the dTDP-D-glucose 4,6-dehydratase from Streptomyces antibioticus
was mutated to various amino acids by site-directed mutagenesis. The catalytic activity of the four mutated enzymes of TDPDH did not recover after addition of
. However, the activity of K159A, the mutated enzyme of UDP-D-glucose 4-epimerase (UDPE), recovered after the addition of
. Although dTDP-glucose 4,6-dehydratase, and UDP-galactose (glucose) 4-epimerase are members of the short-chain dehydrogenase/reductase SDR family and the lysine-148 of TDPDH was highly conserved as in UDPE (Lys-159), the function of the lysine-148 of TDPDH was different from that of UDPE. The mutated enzymes showed that the lysine-148 of the dTDP-D-glucose 4,6-dehydratase played no role in the
binding. Accordingly, it is suggested that the lysine-148 of the dTDP-D-glucose 4,6-dehydratase is involved in the folding of TDPDH.
Hypocholestrolemic Effect of CJ90002 in Hamsters: A Potent Inhibitor for Squalene Synthase from Paeonia moutan
Park, Jong-Koo ; Cho, Hi-Jae ; Lim, Yoon-Gho ; Cho, Youl-Hee ; Lee, Chul-Hoon ;
Journal of Microbiology and Biotechnology, volume 12, issue 2, 2002, Pages 222~227
Squalene synthase catalyzes the reductive dimerization of two molecules of farnesyl diphosphate to form squalene at the final branch point of the cholesterol biosynthetic pathway. Due to the unique position of this enzyme in the pathway, its inhibitors may have advantages as antihypercholesterolemic agents. Therefore, selective inhibitors of squalene synthase do not prevent the formation of the essential branch products of the isoprene pathway, such as dolichol, coenzyme-Q, and prenylated proteins, as might be expected for inhibitors of enzymes earlier in the pathway; for example, lovastatin and mevalotin. The current study reports that CJ90002, a pentagalloylglucose isolated from Paeonia moutan SIM (Paeoniaceae), which is an important Chinese crude drug used in many traditional prescriptions, was a potent inhibitor of rat microsomal squalene synthase, and also a potent inhibitor of cholesterol biosynthesis in vitro. In addition, the intraperitoneal and oral administration of CJ90002 had a significant lowering effect on plasma cholesterol levels in hamsters.
Characterization of Subtilein, a Bacteriocin from Bacillus subtilis CAU131 (KCCM 10257)
Park, Sung-Yong ; Yang, Yong-Jae ; Kim, Young-Bae ; Hong, Jae-Hoon ; Lee, Chan ;
Journal of Microbiology and Biotechnology, volume 12, issue 2, 2002, Pages 228~234
Bacillus subtilis CAU131 (KCCM 10257) isolated from a fermented shrimp product produces subtilein, tentatively named as a bacteriocin, which exhibited a bactericidal effect against closely related species such as Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 11778, and several other strains of Bacillus sp. The purification of the subtilein was achieved by applying a mono-Q anion exchange chromatography on FPLC and
reverse-phase chromatography on HPLC. After purification, specific activity of subtilein was increased about 3,000-fold compared with culture broth and its molecular mass was about 5,000 Da on SDS-PAGE. The antimicrobial activity of subtilein was well maintained at acidic and neutral pHs between 3 and 8. Subtilein was relatively heat stable, and its antimicrobial activity remained for 2 h at
. However, the activity was reduced after heating at
, and about
of the activity was found after 1 h incubation at
. The treatment of Bacillus subtilis ATCC 6633 with subtilein led to morphological changes in stationary-phase cells and most cells appeared to be lysed.
Characteristics of Sophorolipid as an Antimicrobial Agent
KIM, KAPJUNG ; DALSOO YOO ; YOUNGBUM KIM ; BAEKSEOK LEE ; DOONHOON SHIN ; EUN-KI KIM ;
Journal of Microbiology and Biotechnology, volume 12, issue 2, 2002, Pages 235~241
Sophorolipid, a biosurfactant produced from Candida bombicola ATCC 22214, showed antimicrobial activity against Bacillus subtilis, Staphylococcus xylosus, Streptococcus mutans, and Propionibacterium acne at 4, 1, 1, 0.5 ppm, respectively. Also, 100 ppm of sophorolipid inhibited
of cell growth of plant pathogenic fungus, Botrytis cineria. However, sophorolipid showed no effect on Escherichia coli, indicating that its selective antimicrobial activity depended on the cell wall structure. Treatment of B. subtilis with sophorolipid increased leakage of intracellular enzyme, malate dehydrogenase, indicating a possible interaction of sophorolipid with a cellular membrane. Comparing lactone-type and acid-type sophorolipids, the former showed a higher antimicrobial activity. Supplementing other surfactants showed no significant effects on the antimicrobial activity. Animal study showed that 5 g of sophorolipid per kg body weight by oral administration caused no toxicity, and sophorolipid induced no irritation on the skin. These results show potential use of sophorolipid as an active ingredient in healthcare products.
A Microbial D-Hydantoinase is Stabilized and Overexpressed as a Catalytically Active Dimer by Truncation and Insertion of the C-Terminal Region
KIM, GEUN-JOONG ; HAK-SUNG KIM ;
Journal of Microbiology and Biotechnology, volume 12, issue 2, 2002, Pages 242~248
Previously, it was reported that the nonhomologous C-terminal regions of the D-hydantoinases are nonessential for catalysis, but affect the oligomeric structure of the enzyme . In an effort to further confirm the above observation, the C-terminal region-inserted enzyme was constructed by attaching a peptide (22 residues) at the C-terminal of the D-hydantoinase from Bacillus thermocatenulatus GH2, and its structural and biochemical properties were compared with both the wild-type and C-terminal region-truncated enzymes. As a result, native tetrameric D-hydantoinase was dimerized as the truncated enzyme, and the inserted mutant with a new sequence was expressed as a catalytically active form in E. coli. Expression level of the inserted and truncated enzymes were found to be significantly increased compared to the level of the wild-type enzyme, and this appears to be due to the reduced toxic effect of the mutant enzymes on host cells. Dimerized enzymes exhibited increased thermo- and pH stabilities considerably when compared with the corresponding wild-type enzyme. Comparison of the substrate specificity between the mutant and wild-type enzymes suggests that the substrate specificity of the D-hydantoinase is closely linked with the oligomeric structure.
A Plant Growth-Promoting Pseudomonas fluorescens GL20: Mechanism for Disease Suppression, Outer Membrane Receptors for Ferric Siderophore, and Genetic Improvement for Increased Biocontrol Efficacy
LIM, HO SEONG ; JUNG MOK LEE ; SANG DAL KIM ;
Journal of Microbiology and Biotechnology, volume 12, issue 2, 2002, Pages 249~257
Pseudomonas fluorescens GL20 is a plant growth-promoting rhizobacterium that produces a large amount of hydroxamate siderophore under iron-limited conditions. The strain GL20 considerably inhibited the spore germination and hyphal growth of a plant pathogenic fungus, Fusarium solani, when iron was limited, significantly suppressed the root-rot disease on beans caused by F. solani, and enhanced the plant growth. The mechanism for the beneficial effect of strain GL20 on the disease suppression was due to the siderophore production, evidenced by mutant strains derived from the strain. Analysis of the outer membrane protein profile revealed that the growth of strain GL20 induced the synthesis of specific iron-regulated outer membrane proteins with molecular masses of 85- and 90 kDa as the high-affinity receptors for the ferric siderophore. In addition, a cross-feeding assay revealed the presence of multiple inducible receptors for heterologous siderophores in the strain. In order to induce increased efficacy and potential in biological control of plant disease, a siderophore-overproducing mutant, GL20-S207, was prepared by NTG mutagenesis. The mutant GL20-S207 produced nearly 2.3 times more siderophore than the parent strain. In pot trials of beans with F. solani, the mutant increased plant growth up to 1.5 times compared with that of the parent strain. These results suggest that the plant growth-promoting P. fluorescens GL20 and the genetically bred P. fluorescens GL20-S207 can play an important role in the biological control of soil-borne plant diseases in the rhizosphere.
Flux Regulation Patterns and Energy Audit of E. coli B/r and K-12
Lee, Jin-Won ; Goel, Akshay ; Ataai, Mohammad-M. ; Domach, Michael-M. ;
Journal of Microbiology and Biotechnology, volume 12, issue 2, 2002, Pages 258~267
A flux determination methodology has been built which enables to develop constrained stoichiometric relationships and metabolic balances. The analysis differs from those developed for anaerobic growth conditions in that cell mass formation is a significant sink for carbon. When combined with experimental measurements, a determined system of equations results yielded tricarboxylic acid (TCA) cycle and glycolytic fluxes. The methodology was implemented to determine the fluxes of E. coli B/r and K12, and it was found that as the growth rate in a glucose minimal medium increased, the cells became increasing glycolytic and the TCA fluxes either leveled off or declined. The pattern identified for the TCA fluxes corresponded to
-ketoglutarate dehydrogenase's induction-repression pattern, thereby suggesting that the induction-repression of the enzyme could result in significant flux changes. When the minimum flux solution was contrasted to the glycolytic and TCA fluxes determined, two observations were made. First, the minimum flux could provide the cell's biosynthetic ATP requirements. Second, at a high growth rate in a glucose medium, the excess glycolytic flux exceeded that of the TCA cycle, which appeared to more closely match the biosynthetic needs.
Oxidation by Acidithiobacillus ferrooxidans Using Total Organic Carbon Measurement
Lom, In-Soo ; Jang, Hyun-Young ; Lee, Jong-Un ;
Journal of Microbiology and Biotechnology, volume 12, issue 2, 2002, Pages 268~272
Kinetic experiments on the biological oxidation of
by Acidithiobacillus ferrooxidans were conducted by measuring the total organic carbon content. The total organic carbon in the solution was determined with different initial concentrations of
(4, 9, 15, and 20 mg/ml). The growth of At. ferrooxidans and substrate utilization were described by the Monod expression. The total organic carbon was found to be an indicator of the biomass concentration and thus may be effectively utilized for estimating cell growth rates in kinetic model development.
Two-Step Fed-Batch Culture of Recombinant Escherichia coli for Production of Bacillus licheniformis Maltogenic Amylase
Kim, Myoung-Dong ; Lee, Woo-Jong ; Park, Kwan-Hwa ; Rhee, Ki-Hyeong ; Seo, Jin-Ho ;
Journal of Microbiology and Biotechnology, volume 12, issue 2, 2002, Pages 273~278
Two-step fed-batch fermentations were carried out to overproduce Bacillus licheniformis maltogenic amylase (BLMA) in recombinant Escherichia coli. The first step was to increase the cell mass by controlling the feeding of a glucose solution, while the second step was designed to improve the amylase expression efficiency by supplementing organic nitrogen sources. The linear gradient feeding method was successfully adopted to maintain the glucose concentration below 0.2 g/l during the fed-batch mode, as effectively minimizing acetic acid formation. When the dissolved oxygen (DO) level became limiting, an accumulation of acetic acid and drastic decrease in specific BLMA productivity were observed. Glucose and organic nitrogen sources consisting of yeast extract and casein hydrolysate were simultaneously supplied in the pH-stat mode to further increase the specific BLMA expression efficiency. An organic nitrogen source consisting of 200 g/1 yeast extract and 100 g/1 casein hydrolysate was found to be the best among the various combinations tested. The feeding of an organic nitrogen source in the second-step fed-batch period was highly beneficial in enhancing the BLMA production. The optimized two-step fed-batch culture resulted in 78 g/l maximum dry cell mass and 443 U/ml maximum BLMA activity, corresponding to 1.5-fold increase in the dry cell mass and 3.7-fold enhancement in BLMA production, compared with the simple fed-batch fermentation.
Isolation of Phytase-Producing Pseudomonas sp. and Optimization of its Phytase Production
Kim, Young-Hoon ; Gwon, Moon-Nam ; Yang, Si-Yong ; Park, Tae-Kyu ; Kim, Chan-Gil ; Kim, Chang-Won ; Song, Min-Dong ;
Journal of Microbiology and Biotechnology, volume 12, issue 2, 2002, Pages 279~285
Phytase (myo-inositol hexakisphosphate phospho-hydrolase, EC 22.214.171.124) catalyzes the hydrolysis of phytate (myo-inositol hexakisphosphate) to release inorganic phosphate. A bacterial strain producing phytase was isolated from soil around a cattle shed. To identify the strain, cellular fatty acids profiles, the GC contents, a quinine-type analysis, and physiological test using an API 20NE kit were carried out. The strain was identified to be a genus of Pseudomonas sp. and named as Pseudomonas sp. YH40. The optimum culture condition for the maximum productivity of phytase by Pseudomonas sp. YH40 were attained in a culture medium composed of
(w/v) peptone, and
. Within the optimal medium condition, the production of phytase became highest after 10 h of incubation, and the maximal phytase production by Pseudomonas sp. YH40 was observed at
and pH 6.0.
New Yeast Cell-Based Assay System for Screening Histone Deacetylase 1 Complex Disruptor
Jeon, Kwon-Ho ; Kim, Min-Jung ; Kim, Seung-Young ;
Journal of Microbiology and Biotechnology, volume 12, issue 2, 2002, Pages 286~291
Histone deacetylase I (HDAC1) works as one of the components in a nucleosome remodeling (NuRD) complex that consists of several proteins, including metastasis-associated protein 1 (MTA1). Since the protein-protein interaction of HDAC1 and MTA1 would appear to be important for both the integrity and functionality of the HDAC1 complex, the interruption of the HDAC1 and MTA1 interaction may be an efficient way to regulate the biological function of the HDAC1 complex. Based on this idea, a yeast two-hybrid system was constructed with HDAC1 and MTA1 expressing vectors in the DNA binding and activation domains, respectively. To verify the efficiency of the assay system, 3,500 microbial metabolite libraries were tested using the paper disc method, and KB0699 was found to inhibit the HDAC1 and MTA1 interaction without any toxicity to the wild-type yeast. Furthermore, KB0699 blocked the interaction of HDAC1 and MTA1 in an in vitro GST pull down assay and induced morphological changes in B16/BL6 melanoma cells, indicating the interruption of the HDAC1 complex function. Accordingly, these results demonstrated that the yeast assay strain developed in this study could be a valuable tool for the isolation of a HDAC1 complex disruptor.
Removal of Chromate by White Rot Fungus, Inonotus cuticularis
LEE, DONG-HEUB ; YONG-WON MIN ; HAE-IK RHEE ; JAE E. YANG ; GIE-TAEK CHUN ; YEON-HO JEONG ;
Journal of Microbiology and Biotechnology, volume 12, issue 2, 2002, Pages 292~295
A chromate-resistant white rot fungus, Inonotus cuticularis, abundant in oak trees, was isolated for chromate removal and detoxification of chromate. Inonotus cuticularis was also investigated for an optimal waste treatment system. The screened cells were able to reduce an initial chromate concentration of as high as 1,300 ppm. Cell growth kinetics showed that the optimum culture conditions in flasks were at
and pH 4.2. Furthermore, the cells were able to remove
of the initial chromate by a two-stage operation based on the combination of a fermentor and airlift reactor.
Involvement of Lipopolysaccharide of Bradyrhizobium japonicum in Metal Binding
Oh, Eun-Taex ; Yun, Hyun-Shik ; Heo, Tae-Ryeon ; Koh, Sung-Cheol ; Oh, Kye-Heon ; So, Jae-Seong ;
Journal of Microbiology and Biotechnology, volume 12, issue 2, 2002, Pages 296~300
Bacterial cell surface components are the major factors responsible for pathogenesis and bioremediation. In particular, the surface of a Gram-negative bacterium cell has a variety of components compared to that of a Gram-positive cell. In our previous study, we isolated an isogenic mutant of Bradyrhizobium japonicum, which exhibited altered cell surface characteristics, including an increased hydrophobicity. Polyacrylamide gel electrophoretic analysis of the lipopolysaccharide (LPS) in the mutant demonstrated that the O-polysaccharide part was completely absent. Meanwhile, a gel permeation chromatographic analysis of the exopolysaccharide (EPS) in the mutant demonstrated that it was unaltered. Since LPSs are known to have several anion groups that interact with various cation groups and metal ions, the mutant provided an opportunity to examine the direct role of LPS in metal binding by B. japonicum. Using atomic absorption spectrophotometry, it was clearly demonstrated that LPS was involved in metal binding. The binding capacity of the LPS mutant to various metal ions
was 50-70% lower than that of the wild-type strain. Also, through an EPS analysis and desorption experiment, it was found that EPS and centrifugal force had no effect on the metal binding. Accordingly, it would appear that LPS molecules on B. japonicum effect the properties, which precipitate more distinctly metal-rich mineral phase.
Cloning and Expression of the Gene for Inorganic Pyrophosphatase of Thermus caldophilus GK24 and Properties of the Enzyme
Hoe, Hyang-Sook ; Jo, In-Geun ; Shin, Hea-Jin ; Jeon, Hyo-Jeong ; Kim, Hyun-Kyu ; Lee, Jin-Sung ; Kim, Yong-Sung ; Lee, Dae-Sil ; Kwon, Suk-Tae ;
Journal of Microbiology and Biotechnology, volume 12, issue 2, 2002, Pages 301~305
The gene (ppaT) encoding Thermus caldophilus GK24 pyrophosphatase (Tca pyrophosphatase) was cloned and sequenced. The gene was found to contain an open reading frame encoding 175 amino acids with a calculated mass of 19,155 Da. The ppaT gene was expressed under the control of the tac promoter in Escherichia coli. The recombinant Tca pyrophosphatase was purified 21.4-fold with
yield and specific activity of 25.7 U
, following a combination of heating (to denature the E. coli proteins) and one step of DEAE-Sephacel column chromatography. The native enzyme was found to have an approximate molecular mass of 110,000 Da and consisted of six subunits. The enzyme exhibited maximal activity at pH of 8.0-8.5 and was stable at
. A divalent cation was absolutely required for the enzyme activity, with
. being the most effective.
Production of O-GlcNAc Modified Recombinant Proteins in Escherichia coli
LIM, KI HONG ; CHANG HOON HA ; HYO IHL CHANG ;
Journal of Microbiology and Biotechnology, volume 12, issue 2, 2002, Pages 306~311
O-linked N-acetylglucosamine (O-GlcNAc) is an abundant posttranslationally modified compound in eukaryotic cells. Human O-GlcNAc transferase (OGT) was produced as a maltose binding protein (MBP) fusion protein, which showed significant catalytic activity to modify recombinant Sp1, transcription factor. To facilitate the production of O-GlcNAc modified proteins, instead of using the tedious in vitro glycosylation reaction or expression in eukaryotic cells, a MBP-fusion OGT expression vector (pACYC184-MBPOGT) was constructed using pACYC184 plasmid, which could coexist with general prokaryotic expression vectors containing ColE1 origin. By cotransforming pACYC184-MBPOGT and pGEX-2T vectors into Escherichia coli BL21, intracellular O- GlcNAcylated proteins could be obtained by a simple purification procedure. It is expected that this may be a useful tool for production of O-GlcNAc modified proteins.
Molecular Identification of Vaginal Lactobacillus spp. Isolated from Korean Women
CHANG, CHUNG EUN ; SYLVIA I. PAVLOVA ; LIN TAO ; EUN-KI KIM ; SEUNG CHUL KIM ; HYUN SHIK YUN ; JAE-SEONG SO ;
Journal of Microbiology and Biotechnology, volume 12, issue 2, 2002, Pages 312~317
Indigenous lactobacilli were isolated from vaginas of Korean women for possible use in ecological treatment of bacterial vaginosis. Vaginal swab samples were obtained from a gynecological clinic and streaked on Rogosa SL agar plates to select the most predominant lactobacilli in each sample. The preliminary identification of the isolates as lactobacilli was based on microscopic observation of Gram-positive rod-shaped cell morphology. The initial characterization was performed on 108 isolates in terms of their cell surface hydrophobicity (CSH), antimicrobial activity, and hydrogen peroxide (H₂O₂) production capability, and 10 isolates were then selected for further molecular identification. For a rapid procedure to identify lactobacilli, polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses of the l6S rRNA genes were applied. The 10 selected lactobacilli and 9 different reference strains of Lactobacillus spp. were characterized by PCR-RFLP where the amplified l6S rDNA was digested with 7 different restriction endonucleases prior to analysis. DNA sequencing of the 16S rRNA gene of one particular isolate, KLB 46, that had been identified as L. crispatus by the PCR-RFLP analysis, further confirmed its identity as L. crispatus.
Identification and Functional Analysis of the putAP Genes Encoding Vibrio vulnificus Proline Dehydrogenase and Proline Permease
Kim, Hye-Jin ; Lee, Jeong-Hyun ; Rhee, Jee-Eun ; Jeong, Hye-Sook ; Choi, Hyun-Kyung ; Chung, Hee-Jong ; Ryu, Sang-Ryeol ; Choi, Sang-Ho ;
Journal of Microbiology and Biotechnology, volume 12, issue 2, 2002, Pages 318~326
The pathogenic marine bacterium Vibrio vulnificus is the causative agent of food-borne diseases such as life-threatening septicemia. To better understand this organism's strategies to survive osmotic stress, a mutant that was more sensitive to high osmolarity was screened from a library of mutants constructed by a random transposon mutagenesis. By a transposon-tagging method, putAP genes encoding a proline dehydrogenase and a proline permease were identified and cloned from V. vulnificus. The amino acid sequences deduced from nucleotide sequences of putAP from V. vulnificus were 38 to
similar to those of PutA and PutP reported from other Enterobacteriaceae. Functions of putAP genes were assessed by the construction of mutants, whose putAP genes were inactivated by allelic exchanges. When proline as the sole carbon or nitrogen source was used, the putA mutant was not able to grow to the substantial level, revealing the proline dehydrogenase is the only enzyme for metabolic conversion of proline into other amino acids. Although the growth rate of the putP mutant on proline as the sole carbon or nitrogen source was significantly reduced, the mutant still grew. This indicated that at least one more proline permease is produced by V. vulnificus. The putP mutant decreased approximately
CFU/ml after a hyperosmotic challenge, while the parent strain decreased approximately
CFU/ml. This result suggests that the gene product of putP contributes to the osmotic tolerance of V. vulnificus.
Identification of FM001 as Plant Growth-Promoting Substance from Acremonium strictum MJN1 Culture
JUNG, JAE-HAN ; DONG-MIN SHIN ; WOO-CHUL BAE ; SOON-KWANG HONG ; JOO-WON SUH ; SANGHO KOO ; BYEONG-CHUL JEONG ;
Journal of Microbiology and Biotechnology, volume 12, issue 2, 2002, Pages 327~330
A plant growth-promoting substance, FM001, was isolated from the culture broth of Acremonium strictum MJN1. The purification steps included solvent extraction, adsorption chromatography using Diaion HP20, TLC on silica, and HLPC using a C-18 column. The purified FM001 enhanced rice seedling growth by
of the dried weight of the shoots and roots, and also radish growth by
of the top length and dried weight. FM001 also significantly promoted the growth of red pepper by increasing
of fruit weight and
as regards the height. FM001 consisted of C, H, O, N, and S, and its molecular weight was determined to be 537.78 Da. The structure of FM001 resembled brassinosteriods, and it would appear to have great potential as an effective bio-fertilizer.
Effect of Heat Treatments on the Antimicrobial Activities of Garlic (Allium sativum)
Kim, Jeong-Youn ; Lee, Young-Chun ; Kim, Keun-Sung ;
Journal of Microbiology and Biotechnology, volume 12, issue 2, 2002, Pages 331~335
Aqueous extracts of garlic (Allium sativum) preparation were prepared after the samples were exposed to various heat treatments. A quantitative assessment of antimicrobial activities was carried out by determining the minimum inhibitory and microbicidal concentrations (MICs and MMCs) of the various extracts against some selected bacteria and fungi. The antimicrobial activity of garlic decreased as the heating temperature increased. This fact implies that alliinase may be the most critical rate-determinant to produce the activity when garlic is heated.
Utilization of lacZ to Isolate Regulatory Genes from Corynebacterium glutamicum
KIM, HYUNG-JOON ; JOON-SUNG PARK ; HEUNG-SHICK LEE ; YOUNHEE KIM ;
Journal of Microbiology and Biotechnology, volume 12, issue 2, 2002, Pages 336~339
A total of 100 Corynebacterial clones exerting a regulatory effect on the aceB promoter of Corynebacterium glutamicum were isolated by utilizing a reporter carrying the enteric lacZ gene fused to the promoter. The isolated clones were classified into 3 groups of A, B, and C, according to their color of colonies. Escherichia coli cells carrying clones in groups A and B showed a
-galactosidase activity, respectively. The introduction of group A clones into C. glutamicum also resulted in an almost complete reduction in the expression of the aceA and aceB genes, suggesting that the clones express repressor-like proteins for the genes. Although white colonies were formed on plates containing X-gal, E. coli cells carrying one of the clones in group C exhibited intact
-galactosidase activity. The result suggests that the clone may encode proteins that prevent the cells from accumulating the chromogenic compound, X-gal.
Cloning and Expression of a Paenibacillus sp. Neopullulanase Gene in Saccharomyces cerevisiae Producing Schwanniomyces occidentalis Glucoamylase
Kim, Hyo-Jeong ; Park, Jeong-Nam ; Kim, Hee-Ok ; Shin, Dong-Jun ; Chin, Jong-Eon ; Blaise Lee, Hwang-Hee ; Chun, Soon-Bai ; Bai, Suk ;
Journal of Microbiology and Biotechnology, volume 12, issue 2, 2002, Pages 340~344
A gene, npl, encoding neopullulanase from Paenibacillus sp. KCTC 8848P was cloned and expressed in Escherichia coli. It consisted of an open reading frame of 1,530 bp for a protein that consisted of 510 amino acids with a molecular weight of 58,075 Da. The deduced amino acid sequence of the neopullulanase gene had
identity with the neopullulanase of Bacillus polymyxa. The npl gene was also expressed in Saccharomyces cerevisiae secreting Schwanniomyces occidentalis glucoamylase (GAM1) under the control of the yeast actin gene (ACT1) promoter. Secretion of the neopullulanase was directed by the yeast mating pheromone
) prepro region. Enzyme assays confirmed that co-expression of npl and GAM1 enhanced starch and pullulan degradation by S. cerevisiae.
Development of a Rapid Spectrophotometric Method for Detecting Bacterial Mucinase Complex
Kim, Yoon-Hee ; Cha, Jae-Ho ;
Journal of Microbiology and Biotechnology, volume 12, issue 2, 2002, Pages 345~348
A rapid spectrophotometric method for detecting the mucinase complex was developed. Bovine submaxillary mucin is cleaved by commercial mucinase between the oligosaccharide chain and the side chain of peptide linkage, thereby liberating the N-acetyl neuraminic acid (NANA). The release of NANA resulted in an increase of absorbance at 280 nm. The susceptibility to NANA by the new method was found to be at least 10-fold more sensitive than the thiobarbituric acid method. Moreover, the quantification of NANA released from mucin by commercial neuraminidase and partially purified Vibrio parahaemolyticus mucinase showed a good linear correlation in proportion to the concentration of the enzyme used. These results demonstrate that the rapid identification of mucin degradation can be determined by a spectrophotometric assay, thereby providing a new, fast, and sensitive method for assaying the bacterial mucinase complex.
Effect of Trehalose on Bioluminescence and Viability of Freeze-Dried Bacterial Cells
PARK, JI-EUN ; KYU-HO LEE ; DEOKJIN JAHNG ;
Journal of Microbiology and Biotechnology, volume 12, issue 2, 2002, Pages 349~353
Two recombinant bacteria containing luxAB showed an increased tolerance to stresses associated with lyophilization, when the cells were freeze-dried in the presence of trehalose. In the case of a recombinant, UV2, only
of the original bioluminescence and
of the cell viability were restored after 4 h of freeze-drying without trehalose, which implies that the cells were heavily damaged during the dehydration. To improve these losses, trehalose was added before freeze-drying using different modes. Trehalose increased the bioluminescence and the viability of freeze-dried UV2 under all conditions tested, and it was also observed that the addition of trehalose to the cultures (final concentration of 0.08 M) for 15 min before the freeze-drying resulted in the restoration of
of the original bioluminescence and
of the cell viability. Trehalose also showed a similar efficacy with the other luminescent recombinant, YH9. Therefore, it was tentatively concluded that trehalose played a role as a protective agent in the freeze-drying of bacterial cells.