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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 16, Issue 12 - Dec 2006
Volume 16, Issue 11 - Nov 2006
Volume 16, Issue 10 - Oct 2006
Volume 16, Issue 9 - Sep 2006
Volume 16, Issue 8 - Aug 2006
Volume 16, Issue 7 - Jul 2006
Volume 16, Issue 6 - Jun 2006
Volume 16, Issue 5 - May 2006
Volume 16, Issue 4 - Apr 2006
Volume 16, Issue 3 - Mar 2006
Volume 16, Issue 2 - Feb 2006
Volume 16, Issue 1 - Jan 2006
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Electrochemically Active Bacteria (EAB) and Mediator-Less Microbial Fuel Cells
Chang In-Seop ; Moon Hyun-Soo ; Bretschger Orianna ; Jang Jae-Kyung ; Park Ho-Il ; Nealson Kenneth H. ; Kim Byung-Hong ;
Journal of Microbiology and Biotechnology, volume 16, issue 2, 2006, Pages 163~177
Development of a Meta-Information System for Microbial Resources
Yu Jae-Woo ; Chung Won-Hyong ; Sohn Tae-Kwon ; Park Yong-Ha ; Kim Hong-Ik ;
Journal of Microbiology and Biotechnology, volume 16, issue 2, 2006, Pages 178~183
Microbes are one of the most important bioresources in bioindustry and provide high economic values. Although there are currently about 6,000 bacterial species with validly published names, microbiologists generally assume that the number may account for less than 1% of the bacterial species present on Earth. To discover the remaining species, studies of metagenomes, metabolomes, and proteomes related to microbes have recently been carried out in various fields. We have constructed an information system that integrates various data on microbial resources and manages bioinformation to support efficient research of microorganisms. We have designated this system 'Bio-Meta Information System (Bio-MIS).' Bio-MIS consists of an integrated microbial resource database, a microbial resource input system, an integrated microbial resource search engine, a microbial resource online distribution system, a portal service, and management via the Internet. In the future, this system is expected to be connected with various public databases. We plan to implement useful bioinformatics software for analyzing microbial genome resources. The Web site is accessible at http://biomis.probionic.com.
Biological Control and Plant-Growth Promotion by Bacillus Strains from Milk
Nautiyal Chandra Shekhar ; Mehta Sangeeta ; Singh Harikesh Bahadur ;
Journal of Microbiology and Biotechnology, volume 16, issue 2, 2006, Pages 184~192
Six-hundred bacterial strains from human milk and milk from Sahiwal cows, Holstein Friesian cows, and buffaloes were screened for their ability to suppress phytopathogenic fungi under in vitro conditions. A consortium of 3 strains, viz., Bacillus lentimorbus B-30486 (B-30486), B. subtilis B-30487 (B-30487), and B. lentimorbus B-30488 (B-30488), isolated from Sahiwal cow milk resulted in better biological control and plant-growth promotion than single-strain treatments. For commercial-scale production of a bioinoculant, the solid-state fermentation of sugarcane agro-industrial residues, i.e., molasses, press mud, and spent wash, using the consortium of B-30486, B-30487, and B-30488, resulted in a value-added product, useful for enhancing plant growth. The application of the consortium to sugarcane fields infested with Fusarium moniliforme and Colletotrichum falcatum resulted in a reduction of mortality and significantly higher (P=0.05) plant height, number of tillers, and cane girth when compared with the control. Furthermore, under field conditions, the treatment of sugarcane with the consortium resulted in significantly (P=0.05) greater plant growth compared with nonbacterized plants. Accordingly, this is the first report on the effective use of bacteria isolated from milk for biological control and enhancing plant growth under field conditions. Furthormore, a solid-state fermentation technology was developed that facilitates the economic utilization of agro-industrial residues for environmental conservation and improving plant and soil health.
Halotolerant Spore-Forming Gram-Positive Bacterial Diversity Associated with Blutaparon portulacoides (St. Hill.) Mears, a Pioneer Species in Brazilian Coastal Dunes
Barbosa Deyvison Clacino ; Irene Von Der Weid ; Vaisman Natalie ; Seldin Lucy ;
Journal of Microbiology and Biotechnology, volume 16, issue 2, 2006, Pages 193~199
Halotolerant spore-forming Gram-positive bacteria were isolated from the root, rhizosphere, and non-rhizosphere soil of Blutaparon portulacoides. The different isolates were characterized genetically using an amplified ribosomal DNA restriction analysis (ARDRA), and phenotypically based on their colonial morphology, physiology, and nutritional requirements. Three different 16S rRNA gene-based genotypes were observed at a 100% similarity using the enzymes HinfI, MspI, and RsaI, and the phenotypic results also followed the ARDRA groupings. Selected strains, representing the different ARDRA groups, were analyzed by 16S rDNA sequencing, and members of the genera Halobaeillus, Virgibacillus, and Oceanobacillus were found. Two isolates showed low 16S rDNA sequence similarities with the closest related species of Halobacillus, indicating the presence of new species among the isolates. The majority of the strains isolated in this study seemed to belong to the species O. iheyensis and were compared using an AP-PCR to determine whether they had a clonal origin or not. Different patterns allowed the grouping of the strains according to Pearson's coefficient, and the resulting dendrogram revealed the formation of two main clusters, denoted as A and B. All the strains isolated from the soil were grouped into cluster A, whereas cluster B was exclusively composed of the strains associated with the B. portulacoides roots. This is the first report on the isolation and characterization of halotolerant spore-forming Gram-positive bacteria that coexist with B. portulacoides. As such, these new strains may be a potential source for the discovery of bioactive compounds with industrial value.
Enzymatic Activities in Petroleum Wastewater Purification System by an Activated Sludge Process
Li Yin ; Chrost Ryszard J. ;
Journal of Microbiology and Biotechnology, volume 16, issue 2, 2006, Pages 200~204
The enzymology of an activated sludge system for a petroleum wastewater purification process was investigated. Leucine-aminopeptidase (L-AMP),
), and lipase (LIP) were selected for the study. It was found that more than 81.7% of enzymatic activity was associated with microbial cells in the activated sludge floc. The metabolic response of a mixed microbial population to increased phenol concentration showed that L-AMP activity increased in the activated sludge, whereas activities of
and LIP decreased, due to the inhibitory effect of the phenol which varied from 100 mg/l to 500 mg/l.
Quantitative Analysis of Two Genetically Modified Maize Lines by Real-Time PCR
Lee Seong-Hun ; Kang Sang-Ho ; Park Yong-Hwan ; Min Dong-Myung ; Kim Young-Mi ;
Journal of Microbiology and Biotechnology, volume 16, issue 2, 2006, Pages 205~211
A quantitative analytical method to detect new lines of genetically modified (GM) maize, NK603 and TC1507, has been developed by using a real-time polymerase chain reaction (PCR). To detect these GM lines, two specific primer pairs and probes were designed. A plasmid as a reference molecule was constructed from an endogenous DNA sequence of maize, a universal sequence of a cauliflower mosaic virus (CaMV) 35S promoter used in most GMOs, and each DNA sequence specific to the NK603 and TC1507 lines. For the validation of this method, the test samples of 0, 0.1, 0.5, 1.0, 3.0, 5.0, and 10.0% each of the NK603 and TC1507 GM maize were quantitated. At the 3.0% level, the biases (mean vs. true value) for the NK603 and TC1507 lines were 3.3% and 15.7%, respectively, and their relative standard deviations were 7.2% and 5.5%, respectively. These results indicate that the PCR method developed in this study can be used to quantitatively detect the NK603 and TC1507 lines of GM maize.
Occurrence of Microcystin-Containing Toxic Water Blooms in Central India
Agrawal Manish K. ; Ghosh Shubhro K. ; Bagchi Divya ; Weckesser Juergen ; Erhard Marcel ; Bagchi Suvendra N. ;
Journal of Microbiology and Biotechnology, volume 16, issue 2, 2006, Pages 212~218
Three out of fourteen Microcystis-dominant cyanobacterial blooms in Central India were found to be toxic to mice (
ranging from 35-450 mg bloom dry mass/kg body weight). The liver architecture of the treated mice showed characteristic symptoms of hepatotoxicity relative to the untreated controls, with increased enzyme activities of serum lactate dehydrogenase (LDH), serum glutamate oxaloacetate transaminase (SGOT), alkaline phosphatase (ALP), and serum glutamate pyruvate transaminase (SGPT). RP-HPLC revealed the presence of microcystin-LR, microcystin-RR, and desmethyl microcystin-RR in the given region to maximum amounts of 390, 1,030, and
bloom dry weight, respectively, corresponding to a maximum of 2.8 mg/l microcystin-LR in the lake water. Further confirmation of the microcystin variants was conducted using a MALDI-TOF MS analysis.
Effect of Aluminum on
Secretion from Murine RAW264.7 Cells for Endotoxin Detection in Hepatitis B Vaccines
Park Chul-Yong ; Lee Sun-Suk ; Rhee Dong-Kwon ;
Journal of Microbiology and Biotechnology, volume 16, issue 2, 2006, Pages 219~225
The rabbit pyrogen test and Limulus amoebocyte lysate (LAL) assay have been used to detect endotoxins present in vaccines. Currently, the rabbit pyrogen test is used to detect endotoxins in hepatitis B (HB) vaccines, even though the HB surface protein, which is the active ingredient, is overexpressed in and purified from eukaryotic cells that lack these endotoxins. Although the LAL clot assay is sensitive and reliable and can be used to replace the rabbit pyrogen test, its reaction is limited by the lack of responsiveness to the Gram-positive bacterial components. Furthermore, aluminum hydroxide in the HB vaccine can interfere with the LAL assay. In contrast, macrophages can detect the endotoxin as well as other pyrogens, and secrete
. Therefore, this study was undertaken to examine the possibility of replacing the animal tests with a more efficient
secretion assay. With this in mind, we determined if aluminum hydroxide in the HB vaccines affects the
secretion assay. HB vaccines and the HB protein solutions spiked with lipopolysaccharide (LPS) produced the same level of dose-dependent
secretion and temperature increase in rabbits, indicating that aluminum hydroxide in the HB vaccine does not interfere with the pyrogenic response in rabbits, nor does it interfere with
secretion. In addition, the
assay was found to be more sensitive than the LAL assay, and correlated well with the pyrogen test and the LAL assay. These results suggest that the
assay in RAW264.7 cells is a good substitute for the current pyrogen assays that are used for detecting LPS in HB vaccines as well as in other vaccines containing aluminum.
Effect of Nutrients on the Production of Extracellular Enzymes for Decolorization of Reactive Blue 19 and Reactive Black 5
Lee Yu-Ri ; Park Chul-Hwan ; Lee Byung-Hwan ; Han Eun-Jung ; Kim Tak-Hyun ; Lee Jin-Won ; Kim Sang-Yong ;
Journal of Microbiology and Biotechnology, volume 16, issue 2, 2006, Pages 226~231
Several white-rot fungi are able to produce extracellular lignin-degrading enzymes such as manganese peroxidase (MnP), lignin peroxidase (LiP), and laccase. In order to enhance the production of laccase and MnP using Trametes versicolor KCTC 16781 in suspension culture, the effects of major medium ingredients, such as carbon and nitrogen sources, on the production of the enzymes were investigated. The decolorization mechanism in terms of biodegradation and biosorption was also investigated. Among the carbon sources used, glucose showed the highest potential for the production of laccase and MnP. Ammonium tartrate was a good nitrogen source for the enzyme production. No significant difference in the laccase production was observed, when glucose concentration was varied between 5 g/l and 30 g/l. As the concentration of nitrogen source increased, a lower MnP activity was observed. The optimal C/N ratio was 25 for the production of laccase and MnP. When the concentrations of glucose and ammonium tartrate were simultaneously increased, the laccase and MnP activities increased dramatically. The maximum laccase and MnP activities were 33.7 U/ml at 72 h and 475 U/ml at 96 h, respectively, in the optimal condition. In this condition, over 90% decolorization efficiency was observed.
Bacterial Community Structure in Activated Sludge Reactors Treating Free or Metal-Complexed Cyanides
Quan Zhe-Xue ; Rhee Sung-Keun ; Bae Jin-Woo ; Baek Jong-Hwan ; Park Yong-Ha ; Lee Sung-Taik ;
Journal of Microbiology and Biotechnology, volume 16, issue 2, 2006, Pages 232~239
The microbial activity and bacterial community structure of activated sludge reactors, which treated free cyanide (FC), zinc-complexed cyanide (ZC), or nickel-complexed cyanide (NC), were studied. The three reactors (designated as re-FC, re-ZC, and re-NC) were operated for 50 days with a stepwise decrease of hydraulic retention time. In the re-FC and re-ZC reactors, FC or ZC was almost completely removed, whereas approximately 80-87% of NC was removed in re-NC. This result might be attributed to the high toxicity of nickel released after degradation of NC. In the batch test, the sludges taken from re-FC and re-ZC completely degraded FC, ZC, and NC, whereas the sludge from re-NC degraded only NC. Although re-FC and re-ZC showed similar properties in regard to cyanide degradation, denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rRNA gene of the bacterial communities in the three reactors showed that bacterial community was specifically acclimated to each reactor. We found several bacterial sequences in DGGE bands that showed high similarity to known cyanide-degrading bacteria such as Klebsiella spp., Acidovorax spp., and Achromobacter xylosoxidans. Flocforming microorganism might also be one of the major microorganisms, since many sequences related to Zoogloea, Microbacterium, and phylum TM7 were detected in all the reactors.
Diversity of Heterocystous Filamentous Cyanobacteria (Blue-Green Algae) from Rice Paddy Fields and Their Differential Susceptibility to Ten Fungicides Used in Korea
Kim Jeong-Dong ; Lee Choul-Gyun ;
Journal of Microbiology and Biotechnology, volume 16, issue 2, 2006, Pages 240~246
Cyanobacteria are present abundantly in rice fields and are important in helping to maintain rice fields fertility through nitrogen fixation. Many rice fields soil contain a high density of cyanobactera, and over 50% of cyanobacterial genera that are in existence in rice paddy fields are heterocystous filamentous forms. A total of 142 isolates of heterocystous filamentous cyanobacteria were screened from 100 soil samples taken from rice paddy fields in 10 different locations across Korea, classified according to their morphological characteristics under light microscopy, and their susceptibly to fungicides examined. The collected blue-green alga were classified into a total of 14 genera, including seven genera of filamentous cyanobacteria and seven genera of nonfilamentous cyanobacteria. In particular, 142 heterocystous filamentous cyanobacteria were isolated and classified into six genera, including Anabaena, Nostoc, Calothrix, Cylindrospermum, Nodularia, Scytomena, and Tolypotrix. Yet, over 90% of the heterocystous filamentous cyanobacteria isolated from the rice paddy fields belonged to two genera: Anabaena and Nostoc. The response of 129
cyanobacterial isolates, 53 Anabaena and 76 Nostoc, to 10 fungicides was then investigated. The results showed that the Nostoc spp. were more tolerant of the ten tested fungicides than the Anabaena spp., and among the ten tested fungicides, benomyl showed the highest acute toxicity to Anabaena spp. and Nostoc spp. In conclusion, although benomyl is a very useful agent to control phytopathogenic fungi, the application of this fungicide to rice fields should be considered because of its toxicity to the heterocystous filamentous cyanobacteria.
Preparation and Analysis of Yeast Cell Wall Mannoproteins, Immune Enhancing Materials, from Cell Wall Mutant Saccharomyces cerevisiae
Ha Chang-Hoon ; Yun Cheol-Won ; Paik Hyun-Dong ; Kim Seung-Wook ; Kang Chang-Won ; Hwang Han-Joon ; Chang Hyo-Ihl ;
Journal of Microbiology and Biotechnology, volume 16, issue 2, 2006, Pages 247~255
Yeast cell wall matrix particles are composed entirely of mannoprotein and
. The mannoproteins of yeast cell wall can systemically enhance the immune system. We previously purified and analyzed alkali-soluble
-(1,6)-glucans] . In the present study, a wild-type strain was first mutagenized with ultraviolet light, and the cell wall mutants were then selected by treatment with 1.0 mg/ml laminarinase (endo-
-(1,3)-D-glucanase). Mannoproteins of Saccharomyces cerevisiae were released by laminarinase, purified by concanavalin-A affinity and ion-exchange chromatography. The results indicated that the mutants yielded 3-fold more mannoprotein than the wild-type. The mannoprotein mass of mutant K48L3 was 2.25 mg/100 mg of yeast cell dry mass. Carbohydrate analysis revealed that they contained mannose, glucose, and N-acetylglucosamine. Saccharomyces cerevisiae cell wall components, mannoproteins, are known to interact with macrophages through receptors, thereby inducing release of tumor necrosis factor alpha (
) and nitric oxide. Mannoprotein tractions in the present study had a higher macrophage activity of secretion of
and nitric oxide and direct phagocytosis than positive control (
of lipopolysaccharide). In particular, F1 and F3 fractions in mannoproteins of K48L3 enhanced and upregulated the activity of nitric oxide secretion and macrophage phagocytosis by approximately two- and four-fold, respectively.
Construction of a Bacterial Artificial Chromosome Library Containing Large BamHI Genomic Fragments from Medicago truncatula and Identification of Clones Linked to Hypernodulating Genes
Park So-Yeon ; Nam Young-Woo ;
Journal of Microbiology and Biotechnology, volume 16, issue 2, 2006, Pages 256~263
In the model legume Medicago truncatula, two mutants, sickle and sunn, exhibit morphologically and genetically distinct hypernodulation phenotypes. However, efforts to isolate the single recessive and single semidominant genes for sickle and sunn, respectively, by map-based cloning have so far been unsuccessful, partly due to the absence of clones that enable walks from linked marker positions. To help resolve these difficulties, a new bacterial artificial chromosome (BAC) library was constructed using BamHI-digested genomic fragments. A total of 23,808 clones were collected from ligation mixtures prepared with double-size-selected high-molecular-weight DNA. The average insert size was 116 kb based on an analysis of 88 randomly selected clones using NotI digestion and pulsed-field gel electrophoresis. About 18.5% of the library clones lacked inserts. The frequency of the BAC clones carrying chloroplast or mitochondrial DNA was 0.98% and 0.03%, respectively. The library represented approximately 4.9 haploid M. truncatula genomes. Hybridization of the BAC clone filters with a
DNA probe revealed that approximately 37% of the clones likely carried repetitive sequence-enriched DNA. An ordered array of pooled BAC DNA was screened by polymerase chain reactions using 13 sequence-characterized molecular markers that belonged to the eight linkage groups. Except for two markers, one to five positive BAC clones were obtained per marker. Accordingly, the sickle- and sunn-linked BAC clones identified herein will be useful for the isolation of these biotechnologically important genes. The new library will also provide clones that fill the gaps between preexisting BAC contigs, facilitating the physical mapping and genome sequencing of M. truncatula.
Purification and Characterization of Two Novel Fibrinolytic Proteases from Mushroom, Fomitella fraxinea
Lee Jong-Suk ; Baik Hyung-Suk ; Park Sang-Shin ;
Journal of Microbiology and Biotechnology, volume 16, issue 2, 2006, Pages 264~271
Two fibrinolytic enzymes were purified from the culture supernatant of Fomitella fraxinea mycelia by ion-exchange and gel filtration chromatographies, and were designated as F. fraxenia proteases 1 and 2 (FFP1 and FFP2). The apparent molecular masses of the enzymes were estimated to be 32 kDa and 42 kDa, respectively, by SDS-PAGE and gel filtration chromatography. Both enzymes had the same optimal temperature (
), but different pH optima (10.0 and 5.0 for FFP1 and FFP2, respectively). FFP1 was relatively stable at pH 7.0-9.0 and temperature below
, whereas FFP2 was very stable in the pH range of 4-11 and temperature below
. FFPI activity was completely inhibited by phenylmethylsulfonyl fluoride (PMSF) and aprotinin, indicating that this enzyme is a serine protease. The activity of FFP2 was enhanced by the addition of
and inhibited by
. Furthermore, FFP2 activity was strongly inhibited by EDTA and 1,10-phenanthroline, implying that the enzyme is a metalloprotease. Both enzymes readily hydrolyzed fibrinogen, preferentially digesting the
-chains of fibrinogen over
-chain. FFP1 showed broad substrate specificity for synthetic substrates, but FFP2 did not.
values of FFP1 for a synthetic substrate, N-succinyl-Ala-Ala-Pro-Phe-pNA, were 0.213 mM and 39.68 units/ml, respectively. The first 15 amino acids of the N-terminal sequences of both enzymes were APXXPXGPWGPQRIS and ARPP(G)VDGQ(R,I)SK(L)ETLPE, respectively.
Cloning, Expression, and Characterization of a Hyperalkaline Phosphatase from the Thermophilic Bacterium Thermus sp. T351
Choi Jeong-Jin ; Park Jong-Woo ; Shim Hye-Kyung ; Lee Suk-Chan ; Kwon Moo-Sik ; Yang Joo-Sung ; Hwang Heon ; Kwon Suk-Tae ;
Journal of Microbiology and Biotechnology, volume 16, issue 2, 2006, Pages 272~279
The gene encoding Thermus sp. T351 alkaline phosphatase (T351 APase) was cloned and sequenced. The gene consisted of 1,503 bp coding for a protein with 500 amino acid residues including a signal peptide. The deduced amino acid sequence of T351 APase showed relatively low similarity to other Thermus APases. The T351 APase gene was expressed under the control of the T7lac promoter on the expression vector pET-22b(+) in Escherichia coli BL21 (DE3). The expressed enzyme was purified by heat treatment, and
Heparin HP column chromatographies. The purified enzyme exhibited high activity at extremely alkaline pHs, reaching a maximum at pH 12.0. The optimum temperature of the enzyme was
, and the half-life at
was approximately 103 min. The enzyme activity was found to be dependent on metal ions: the addition of
increased the activity, whereas EDTA inhibited it. With p-nitrophenyl phosphate as the substrate, T351 APase had a Michaelis constant (
. The enzyme catalyzed the hydrolysis of a wide variety of phosphorylated compounds.
Antifungal Activity Against Colletotrichum spp. of Curcuminoids Isolated from Curcuma longa L. Rhizomes
Cho Jun-Young ; Choi Gyung-Ja ; Lee Seon-Woo ; Jang Kyoung-Soo ; Lim He-Kyoung ; Lim Chi-Hwan ; Lee Sun-Og ; Cho Kwang-Yun ; Kim Jin-Cheol ;
Journal of Microbiology and Biotechnology, volume 16, issue 2, 2006, Pages 280~285
Methanol extract of the rhizomes of turmeric, Curcuma longa L., effectively controlled the development of red pepper anthracnose caused by Colletotrichum coccodes. In addition three antifungal substances were identified from the methanol extract of C. longa rhizomes as curcumin, demethoxycurcumin, and bisdemethoxycurcumin using mass and
spectral analyses. The curcuminoids in a range
effectively inhibited the mycelial growth of three red pepper anthracnose pathogens, C. coccodes, C. gloeosporioides, and C. acutatum. The three curcuminoids inhibited mycelial growth of C. coccodes and C. gloeosporioides to an extent similar to the synthetic fungicide dithianon did, but the synthetic agent was a little more effective against C. acutatum. The curcuminoids also effectively inhibited spore germination of C. coccodes, and bisdemethoxycurcumin was the most active. Among the three curcuminoids, only demethoxycurcumin was effective in a greenhouse test in suppressing red pepper anthracnose caused by C. coccodes.
Cloning and Characterization of the Urease Gene Cluster of Streptococcus vestibularis ATCC49124
Kim Geun-Young ; Lee Mann-Hyung ;
Journal of Microbiology and Biotechnology, volume 16, issue 2, 2006, Pages 286~290
A genomic library of Streptococcus vestibularis ATCC49124 was constructed in an E. coli plasmid vector, and the urease-positive transformants harboring the urease gene cluster were isolated on Christensen-urea agar plates. The minimal DNA region required for urease activity was located in a 5.6 kb DNA fragment, and a DNA sequence analysis revealed the presence of a partial ureI gene and seven complete open reading frames, corresponding to ureA, B, C, E, F, G, and D, respectively. The nucleotide sequence over the entire ure gene cluster and 3'-end flanking region of S. vestibularis was up to 95% identical to that of S. salivarius, another closely related oral bacterium, and S. thermophilus, isolated from dairy products. The predicted amino acid sequences for the structural peptides were 98-100% identical to the corresponding peptides in S. salivarius and S. thermophilus, respectively, whereas those for the accessory proteins were 96-100% identical. The recombinant E. coli strain containing the S. vestibularis ure gene cluster expressed a high level of the functional urease holoenzyme when grown in a medium supplemented with 1 mM nickel chloride. The enzyme was purified over 49-fold by using DEAE-Sepharose FF, Superdex HR 200, and Mono-Q HR 5/5 column chromatography. The specific activity of the purified enzyme was 2,019 U/mg, and the Michaelis constant (
) of the enzyme was estimated to be 1.4 mM urea. A Superose 6HR gel filtration chromatography study demonstrated that the native molecular weight was about 196 kDa.
PCR-Based Detection and Molecular Genotyping of Enterotoxigenic Clostridium perfringens Isolates from Swine Diarrhea in Korea
Kim Sang-Bum ; Lim Hyeong-Jun ; Lee Wan-Kyu ; Hwang In-Gyun ; Woo Gun-Jo ; Ryu Sang-Ryeol ;
Journal of Microbiology and Biotechnology, volume 16, issue 2, 2006, Pages 291~294
Clostridium perfringens strains were isolated from swine diarrhea in Korea. Three out of nineteen (15.8%) isolates of C. perfringens were found to be enterotoxigenic by PCR analysis. PCR-based genotyping of the three enterotoxigenic isolates of C. perfringens revealed that they were types A, C and D, respectively. These results suggest that various types of enterotoxigenic C. perfringens can cause swine diarrhea, and that the presence of enterotoxigenic type A strain, known to be strongly associated with food poisoning, may cause public health problem in Korea.
Heterologous Expression of Novel Cytochrome P450 Hydroxylase Genes from Sebekia benihana
Park Nam-Sil ; Park Hyun-Joo ; Han Kyu-Boem ; Kim Eung-Soo ;
Journal of Microbiology and Biotechnology, volume 16, issue 2, 2006, Pages 295~298
Actinomycetes are ubiquitous Gram-positive soil bacteria and a group of the most important industrial microorganisms for the biosynthesis of many valuable secondary metabolites as well as the source of various bioconversion enzymes. Cytochrome P450 hydroxylase (CYP), a hemebinding protein, is known to be involved in the modification of various natural compounds, including polyketides, fatty acids, steroids, and some aromatic compounds. Previously, six different novel CYP genes were isolated from a rare actinomycetes called Sebekia benihana, and they were completely sequenced, revealing significant amino acid similarities to previously known CYP genes involved in Streptomyces secondary metabolism. In the present study, these six CYP genes were functionally expressed in Streptomyces lividans, using an
promoter-containing Streptomyces expression vector. Among six CYP genes, two S. benihana CYP genes (CYP503 and CYP504) showed strong hydroxylation activities toward 7-ethoxycoumarin. Furthermore, the recombinant S. lividans containing both the S. benihana CYP506-ferredoxin genes as well as the S. coelicolor feredoxin reductase gene also demonstrated cyclosporin A hydroxylation activity, suggesting potential application of actinomycetes CYPs for the biocatalysts of natural product bioconversion.
Enhancing the Solubility of Recombinant Akt1 in Escherichia coli with an Artificial Transcription Factor Library
Park Kyung-Soon ; Lee Ho-Rim ; Kim Jin-Soo ;
Journal of Microbiology and Biotechnology, volume 16, issue 2, 2006, Pages 299~302
A combinatorial library of artificial transcription factors (ATFs) was introduced into the bacterial cells that expressed the Akt1-GFP fusion protein. By measuring the level of fluorescence generated by the transformed E. coli cells, we were able to obtain clones in which ATFs increased the solubility of the Akt1. Our results show that ATF library is a useful tool for increasing the solubility of selected recombinant proteins in E. coli.
Selection and Characterization of Peptides Specifically Binding to
Seo Min-Hee ; Lee Jong-Ho ; Kim Min-Soo ; Chae Hee-K. ; Myung Hee-Joon ;
Journal of Microbiology and Biotechnology, volume 16, issue 2, 2006, Pages 303~307
We have screened phage display peptide libraries to select for peptides binding to various sized
nanoparticles. Phage libraries displaying random 7mer, 12mer, and C-7-Cmer peptides were used for screening. The size of target
particles used were 7 nm, 15 nm, and 25 nm in diameter. We could select peptides binding each nanoparticles from all 3 libraries. Their binding was confirmed by transmission electron microscopy (TEM). Each peptide investigated was also shown to bind the other sized particles, meaning that the binding was specific for the nature of the particle rather than for the size of it. One of the 7mer peptides (PEP9, SVSPISH) was chosen for further analysis. The binding was shown to be in a dose-dependent manner, suggesting a specific interaction.
Coexpression of Protein Disulfide Isomerase (PDI) Enhances Production of Kringle Fragment of Human Apolipoprotein(a) in Recombinant Saccharomyces cerevisiae
Cha Kwang-Hyun ; Kim Myoung-Dong ; Lee Tae-Hee ; Lim Hyung-Kweon ; Jung Kyung-Hwan ; Seo Jin-Ho ;
Journal of Microbiology and Biotechnology, volume 16, issue 2, 2006, Pages 308~311
In an attempt to increase production of LK8, an 86-amino-acid kringle fragment of human apolipoprotein(a) with three disulfide linkages, protein disulfide isomerase (PDI) was coexpressed in recombinant Saccharomyces cerevisiae harboring the LK8 gene in the chromosome. Whereas overexpression of the LK8 gene without coexpressing PDI was detrimental to both host cell growth and LK8 production, coexpression of PDI increased the LK8 production level by 2.5-fold in batch cultivation and 5.0-fold in fed-batch cultivation compared with the control strain carrying only the genomic PDI gene.
Functional Anaylsis of sprD Gene Encoding Streptomyces griseus Protease D(SGPD) in Streptomyces griseus
Choi Si-Sun ; Kim Joung-Hoon ; Kim Jong-Hee ; Kang Dae-Kyung ; Kang Sang-Soon ; Hong Soon-Kwang ;
Journal of Microbiology and Biotechnology, volume 16, issue 2, 2006, Pages 312~317
The chromosomal sprD gene encoding Streptomyces griseus protease D (SGPD), a chymotrypsin-like protease, was disrupted in Streptomyces griseus by insertion of the neomysin-resistance gene. The production of chymotrypsin activity of sprD disruptant was not completely abolished, but delayed by 24 h, compared with that of wild-type strain. The aerial mycelial formation of sprD disruptant was retarded, and specifically the formation of spores was not observed in the central region of colonies. However, normal morphological development into spores was observed in the marginal region of colonies. In addition, the production of yellow pigment that might be dependent on A-factor was also decreased in the sprD disruptant, compared with that of the wild-type strain. Introduction of the sprD gene, which was placed on a high copy-numbered plasmid into S. griseus
, partially restored the ability of morphological development, and a significant level of sporulation was observed. When the overexpression vector for sprD, pWHM3-D, was introduced in S. griseus, there was no significant change in the chymotrypsin activity or colonial morphology, in contrast to Streptomyces lividans, indicating the presence of a tight regulation system for the overexpression of the sprD gene in S. griseus.
Cloning and Expression of hpaA Gene of Korean Strain Helicobacter pylori K51 in Oral Vaccine Delivery Vehicle Lactococcus lactis subsp. lactis MG1363
Kim Su-Jung ; Jun Do-Youn ; Yang Chae-Ha ; Kim Young-Ho ;
Journal of Microbiology and Biotechnology, volume 16, issue 2, 2006, Pages 318~324
In order to develop an oral vaccine to prevent H. pylori infection, we have expressed the hpaA gene of H. pylori K51 isolated from Korean patients, encoding 29-kDa HpaA that is known to be localized on the cell surface and flagella sheath, in a live delivery vector system, Lactococcus lactis. The hpaA gene, amplified by PCR using the genomic DNA of H. pylori K51, was cloned in the pGEX-2T vector, and the DNA sequence analysis revealed that the hpaA gene of H. pylori K51 had 99.7% and 94.8% identity with individual hpaA genes of the H. pylori 26695 strain (U.K) and the J99 strain (U.S.A). A polyclonal anti-HpaA antibody was raised in rats using GST-HpaA fusion protein as the antigen. The hpaA gene was inserted in an E. coli-L. lactis-shuttle vector (pMG36e) to express in L. lactis. Western blot analysis showed that the expression level of HpaA in the L. lactis transformant remained constant from the exponential phase to the stationary phase, without extracelluar secretion. These results indicate that the HpaA of H. pylori K51 was successfully expressed in L. lactis, and suggest that the recombinant L. lactis expressing HpaA may be applicable as an oral vaccine to induce a protective immune response against H. pylori.
Production of Glucooligosaccharides and Mannitol from Leuconostoc mesenteroides B-742 Fermentation and its Separation from Byproducts
Chung Chang-Ho ;
Journal of Microbiology and Biotechnology, volume 16, issue 2, 2006, Pages 325~329
Leuconostoc mesenteroides B-742 fermentations with maltose as an acceptor were tested for glucooligosaccharides and mannitol co-production. Leuconostoc oligosaccharides were produced that were oligomers with a size range of DP 2 to 7 and were primarily DP 3, 4, 5, and 6, containing mainly
linkages. Maltose was linked to the reducing end of the isomaltosyl residues. The
form of cation-exchange column could separate glucooligosaccharides from byproducts.