Earticle

Bacillus sp. strain BCNU 5001 and BCNU 5008 were isolated from industrial wastewater in Korea. The strain BCNU 5001 and BCNU 5008 were able to utilize anthracene, naphthalene, 4-nitrophenol, phenanthrene, pentachlorophenol and toluene as a sole source of carbon and energy under aerobic condition. Based on morphological, physiological and biochemical characteristics, and 16S rDNA sequence, strain BCNU 5001 and BCNU 5008 were identified as B. amyloliquefaciens, and B. subtilis, respectively. Strains BCNU 5001 and BCNU 5008 completely degraded naphthalene with initial concentration of 250, 500 and 1000 ppm within 70, 96, 120 and 196 h, respectively. Bacillus sp. strain BCNU 5001 and BCNU 5008 were also tolerant to various solvents. Finally, Biodegradation of naphthalene by strain BCNU 5001 and BCNU 5008 was also elucidated by LC-MS.

Due to the global warming, fuel security and economics, reusable fuels for transportation have been urgently needed. Marine biomass such as macroalgae has been attracted offering a huge amont of energy substrate for biofuel production. There has been remarkable interest in conversion of macroalgae into fuels in Korea lately. At the same time, interest in butanol fermentation using solventogenic clostridium also has been renewed due to high demands of alternative fuels and disadvantages of ethanol.We examined butanol fermentation by Clostridium beijerinckii NCIMB 8052 using Laminaria japonica (kelps) as substrates. In order to increase sugars content of the hydrolysates, pretreatment with acid or base was applied before enzymatic hydrolysis using laminarinase, alginate lyase and cellulase. The maximum amount of sugars (9 g/L) was produced in 0.5 M H2SO4 in acid pretreatment step, followed by enzyme treatment. Fermentation test was prepared in the hydrolysate of 25 g/L and 50 g/L kelp, using different hydrolysis process. Both growth and butanol production have seen in the most of hydrolysate cultures but it was noticeable that water soluble material without any pretreatment was found to be a good culture medium for clostridium. Fermentation of major sugar componets (alginate, lamininarin, D-mannitol, fucoidan, etc) in macroalgea hydrolysate were also tested at the concentration of 10 g/L and 30 g/L in 96 well cell culture test system.

Pseudomonas aeruginosa, an opportunistic pathogen controls the production of many exoproteins and secondary metabolites via a hierarchical quorum sensing (QS) mechanism. This highly regulated cascade involves LuxR-like proteins LasR, RhlR and their cognate signal molecules. A third LasR-RhlR homologue, QscR, is a regulator of quorum sensing in P. aeruginosa and plays a role in controlling the virulence. Unlike previously reported LasR and TraR, QS receptor proteins of P. aeruginosa, QscR can be obtained as an apo-protein and can reversibly form an active complex in vitro with its cognate signal molecule, 3-oxododecanoyl-homoserine lactone (3OC12-HSL), and subsequently binds to the target promoter DNA equences. In order to search for potential QS inhibitors, an in vitro gel retardation assay was developed using the purified QscR. The in vitro assay and the in vivo cell-based assay were carried out using QscRoverproducing recombinant strains in screening process. Among >100 furanones tested, three compounds showed strong and dose-dependent inhibitory effects on QscR in both assay systems. One compound in particular, designated as F2, completely inhibited the 3OC12-HSL-dependent QscR activity in vitro at a concentration of 50-fold molar in excess to 3OC12-HSL. However, a significant decrease in activity was noticed with furanones F3 and F4, which were structurally similar to F2 with a nitro group in place of the amine moiety. These results suggest that (i) the in vitro assay is a sensitive and reliable tool for screening QS inhibitors, and (ii) furanones are potentially important QS inhibitors for many LuxR-type receptor proteins.

In butyric acid fermentation, a high concentration of butyric acid is a key factor which makes the recovery of the product economically. However, toxic effects of acids prevent the cell from producing ca. 50 g/L in batch or fed-batch process. To solve this problem, several insitu recovery processes have been developed. Ion-exchanger and adsorbent are able to remove organic acids which reduce their toxic effects in a fermentor, but low adsorption capacity together with the inhibitory effects on the cell growth when using large amount of ionexchanger must be addressed. In our research, new inhibition mechanism by adsorbent was found for anaerobic fermentation. Iron and manganese are key components for anaerobic respiration. The addition of adsorbent removed these components resulting in the decrease in fermentation productivity. To prevent and alleviate the inhibition effect, small amount of these minerals were added after butyric acid adsorption. This addition was able to recover the productivity after adsorption process which resulted in the increase of 20% in overall productivity compared to that in the culture in the absence of adsorbent.

A spindle pole body-based regulatory network, septation initiation network(SIN), is required to initiate cytokinesis in late anaphase. ARP, actin-related proteins are essential components of chromatin remodeling complexes. The yeast RSC complexes contain Arp9. To confirm the existence of the complex in A. nidulans, we tagged Arp9 with FIAG tag and purified them. Also, we test how ARP9-interactors interact with components of SIN pathway in A. nidulans. To do this, we made various double mutants by genetic crosses. As a results, Δarp9 mutation suppressed the conidiation defect caused by down regulation of mobA expression. Also, we asked whether the Δarp9 mutation could suppress the sidB null mutation. sidB gene is essential function in septation and conidiation. On rich medium containing glucose, this double mutant showed a restored conidiation phenotype. Thus, Δarp9 mutation could suppress the conidiation defect caused by the loss of SIDB.

It is reported that carotenoid production can be affected by carbon sources used for growing metabolically engineered Escherichia coli expressing heterologous carotenoid pathway enzymes. In order to systematically study the effect of carbon sources on cell growth and carotenoid formation in metabolically engineered E. coli, six carbon sources (glucose, glycerol, galactose, lactose, mannose and maltose) and four carotenoid structures were chosen. The biosynthetic pathways for four carotenoids, C30 diapolycopene, C30 diapotorulene, C40 lycopene, and C40 beta-carotene, were engineered in E. coli and then the recombinant strains were grown in mineral media supplemented with the carbon sources. Among the six carbon sources, glycerol showed highest performance in the production of all four carotenoids in E. coli. Worst performance was observed in a medium containing glucose as a carbon source. Although the growth rate and final cell density were similar in media containing carbon sources, the highest carotenoid productivity was obtained in E. coli cells grown on glycerol as a carbon source.

Enterohemorrhagic (EHEC) Escherichia coli O157:H7 is a human pathogen responsible for outbreaks of hemorrhagic colitis causing bloody diarrhea that can lead to the hemolytic-uremic syndrome in many countries. Among EHEC serotype O157:H7, two strains of EDL933 (ATCC43895 Stx1+ and Stx2+) and 86-24 (Stx2+) were investigated in regards to biofilm formation on abiotic surface. Strikingly, EDL933strain forms a strong biofilm while 86-24 strain forms no biofilm. In order to investigate the mechanisms of biofilm formation of these two EHECs, DNA microarrays were performed. In the comparison of the EDL933 strain versus 86-24 strain, genes (csgBAC and csgDEFG) involved in curli biosynthesis were distinctively induced (17- to 84-fold) in the EDL933 strain while genes involved in tryptophan (a substrate of indole synthesis) synthesis were repressed. Our whole transcriptomic data suggest genetic mechanisms of biofilm formation since it has been reported that curli formation positively influences biofilm formation and indole reduces biofilm formation in Escherichia coli. Assay of curli formation clearly corroborates the microarray data as the EDL 933 produces a large amount of curli, while 86-24 produces no curli. Also, the production rate of indole in the EDL933 was 2-times lower than that of 86-24, which partially explains the difference of biofilm formation between two EHECs.

The bio-conversion of lignocellulosic biomass to ethanol has been extensively studied in bioenergy field. When lignocellulosic biomass converts to ethanol, enzyme takes lots of part of whole cost. Therefore, cellulase production is one of the important processes for the successful enzymatic conversion of cellulosic biomass to ethanol. Cellulase is multi-complex enzyme containing endo-glucanase, exoglucanase and ß-glucosidase (cellobiase). The production of cellulase has been mainly carried out by using Trichodema reesei. Also, suitable cellulase composition was important for the effective saccharification of lignocellulosic biomass and strain having high level production of cellulase should be developed for hydrolysis. In this study, T. reesei KCTC 6950, which was mutant derived from wild type QM 6a, was used as a starting strain for mutation by proton beam irradiation. The selection of cellulase over-producing mutants was performed on agar plate of Mandels mineral salts solution including carbon source such as carboxymethylcellulose (CMC) and phosphoric acid-swollen cellulose. Positive mutants were selected on the basis of larger clear zone or the formation of very clear halo. Selected mutants were tested for the production of cellulase in liquid culture.

Quinones are key constituents of the membrane-bound respiratory electron transfer chains in both prokaryotes and eukaryotes. These lipid components, containing isoprenoid side chains of various lengths, can be divided into two major structural groups, benzoquinones and naphthoquinones. The anaerobic bacterium E. coli is capable of synthesizing octaprenyl quinones of both types, i.e. a benzoquinone, ubiquinone-8 (Q-8), and two types of naphthoquinone, menaquinone-8 (MQ-8) and demethylmenaquinone(DMQ). The first committed step in the biosynthesis of ubiquinone is the formation of 4-hydroxybenzoate from chorismate mediated by the enzyme chorismate pyruvate lyase encoded by the ubiC gene. The 4-hydroxybenzoate is subsequently prenylated by the enzyme 4-hydroxybenzoate ctaprenyltransferase encoded by the ubiA gene. In this study, Escherichia coli quinone biosynthetic pathway was metabolically engineered to improve the yield of quinone compounds. Proteomic analysis was then carried out to understand the effect of metabolic/genetic changes on the proteome of the engineered E. coli. The ubiCA deletion mutant produced higher amount of quinone compounds than wild-type E. coli, indicating than metabolic flux was redirected towards quinone formation. Enhanced quinone formation was observed when pathway enzymes ispA, idi, dxr or menD was overexpressed in the deletion mutant. Proteome analysis disclosed different proteins expression profiling between mutant and wild-type E. coli.

Glucan formed by linking of many glucose molecules has several interesting in properties, and it is structurally divided into two types of α-glucan and ß-glucan depending on the mode of bonding. Among them, ß-glucan is a powerful immune stimulator known to activate macrophages in the immune system. Especially, to enhance production of ß-glucan (exopolysaccharides, EPS), we have optimized media for EPS by Aureobasidium pullulans. The optimization of culture medium was investigated for the production of ß-glucan by statistical experimental design. In the previous study, among organic nitrogen sources, it was found that Ashbya gossypii extract was pivotal factor for the production of ß-glucan. After adaptive nefactor- at-a-time (OFAT) method experimentation was carried out, the relative importance of medium components for maximum glucan production was evaluated by using Plackett-Burman design (PBD). As the result, it was showed that sucrose, ammonium sulfate and A. gossypii extract were significant components for EPS production. Also, on the basis of PBD analysis, Box-Behnken design and Response Surface Method (RSM) were carried out to search for optimum medium of EPSs productionby using statistical analysis system (SAS) program. Using the optimized parameters from RSM, theoretical maximum production of ß-glucan resulted in about 60 g/L. Under optimized medium, EPS production of the optimum medium was ten times higher than one (5.34 g/L) of the basal medium.

Lactic acid production was investigated using Lactobacillus paracasei KCTC 13169, L-lactic acid producer, for the feedstock of biopolymer. In batch cultures, glucose and yeast extract (YE) concentrations were varied in the range of 50-250 g/L and 5-15 g/L, respectively, in order to determine maximum concentration and productivity of lactic acid as well as the optical purity of lactic acid during fermentation. Higher YE concentrations gave better cell growth affecting on the growthassociated lactic acid production so that the productivity of lactic acid increased up to more than 4 g/L.hr under 15 g/L of yeast extract. The maximum lactic acid concentration of more than 180 g/L was obtained when 250 g/L of glucose was fed while a high concentration of glucose inhibited cell growth rate. Several kinds of fed-batch strategy including constant feeding, intermittent feeding, and pH-stat feeding were applied in order to increase the productivity and lactic acid concentration.

BMP-7(bone morphogenetic protein-7) mature form(amino acids 293-431) was inserted into the pET28a vector to produce the plasmid pBMP57. The BMP-7 which was expressed using IPTG inducer in E.coli could be detected in SDS-PAGE. Protein expression in E. coli often results in the formation of inclusion body. The inclusion bodies were denatured and diluted into a refolding buffer. Optimization of refolding buffer conditions for the BMP-7 was performed. A few experimental variable such as pH, DTT (dithiothreitol) concentration, salt concentration, Guanidine-HCl concentration and protein concentration are investigated. The concentration of the refolded BMP-7 was investigated to perform ELISA.

Microbial persistence is one of the mechanisms that exhibit tolerance to antibiotics. Unlike resistance, persistence has involves no genetic modification. In other words, persisters (survivors after antibiotics treatment) are not genetically different from the dead cells. Though mechanism of persistence still remains as black box, it is well accepted that microbial persistence is the cause of secondary infection of pathogen. After antibiotics degradation, persister population grows again and makes second infection.Inhibition of persister formation is believed to be useful in treating bacterial pathogenesis that is not well cured by antibiotics and will reduce the chance of resistant cell emergence. We screened persistence inhibitor using 6,800 chemical compounds library. Exponential phase cultures in 96well plate were exposed to chemical compound (5uM) and ampicillin at 50ug/ml concentration, which killed normal cells but did not kill persister (~0.1% of total cells). In first screening, we screened 52 chemical compound which killed bacteria almost completely. To exclude chemicals which do not act as persistence inhibitor but function as simple antibiotics, second screening was performed. Nine chemical compounds were selected and indentified. Currently, we are investigating persistence mechanism using these chemicals.

Fumaric acid is the chemical compound with the formula HO2CCH=CHCO2H. This white crystalline compound is one of two isomeric unsaturated dicarboxylic acids, the other being maleic acid wherein the carboxylic acid groups are cis. The commercial demand for fumaric acid has increased because of its extensive applications such as the food industry, the paper industry, the pharmaceutical industry and the feed industry.The purpose of this study is to optimize the main medium for the production of fumaric acid by R. oryzae mutant. Response surface methodology (RSM) was conducted to determine the optimal concentrations of medium components affecting the production of fumaric acid. The independent variables (Glucose, NZ-amine A, KNO3, CaCO3) were selected on the basis of preliminary experiments. The optimal concentrations of each variable yielding the maximal production of fumaric acid were 7.5% glucose, 0.11% NZ-amine A, 0.11% KNO3 , and 2.6% CaCO3. The maximum value of fumaric acid predicted from the model was 27.4 g/L. Using the optimum medium obtained by RSM, the maximum fumaric acid concentration value achieved in the flask culture was 26.2 g/L, fairly close to the 27.4 g/L predicted by the model. The highest concentration of fumaric acid (32.1 g/L) was produced by R. oryzae in the stirred tank reactor (STR).

Flavonoids are classified as Chalcone, Flavone, Flavanon and etc. Phloretin is a kind of chalcone compounds, and Apigenin and Chrysin are a flavone that are known for their antitumor, antioxidant, antiarthritic, anti-amyloid and anti-inflammatory properties. However, very poor oral bioavailability makes them largely ineffective in vivo. In previous studies, Omethylated or hydroxylated flavonoid compounds are certificated more effective absorption in vivo than flavonoid compounds. Twelve microorganisms were initially screened for their abilities to catalyze biotransformation of chrysin and phloretin. Chrysin was orthohydroxylated by Streptomyces ceolicolor to produce baicalein (5,6,7-tetrahydroxyflavone), which was confirmed using authentic baicalein with mass analysis. Maximum conversion was 12 %, which was achieved for 4 hours of reaction, and the substrate (chrysin) and reaction product (baicalein) was completely metabolized after 6 hours of reaction. Three kinds of Cytochrome P450 inhibitor, Coumarin, Erythromycin and Quinidine was added with 0.5mM final concentration. Coumarin had no effect on the bioconversion of chrysin and Quinidine inhibited conversion of chrysin into baicalein about 45 ~ 70 %, and Erythromycin completely blocked the bioconversion, which suggest bioconversion of chrysin was mediated through P450 system of Streptomyces Ceolicolor. Addition of different kinds of detergent (Triton X- 100, Tween 80, Brij 35) into reaction mixture, was not effective for the enhancement of bioconversion using Streptomyces Ceolicolor.In the biotransformation phloretin using Streptomyces avermitilis, two main products were identified in GC/MS analysis. They were interpreted as hydroxylated products of phloretin in A-ring at different position.

In this study, we intended to increase mass production of Bacillus subtilis SCD121067 through medium optimization by statistical experimental method. For the seed culture of B. subtilis SCD121067, tryptic soy broth and 15 hours were selected as the basic medium and inoculum age respectively. Final dry cell weight of B. subtilis SCD121067 by basic production medium was 1.56 g/L. First, galactose, yeast extract and potassium phosphate dibasic were selected as carbon, nitrogen and phosphate sources for mass production of B. subtilis SCD121067 by using one factor at a time method. Second, Plackett-Burman experimental design was used to determine the key factors which are critical to maximizing culture the cell mass by fermentation. According to the result of Plackett-Burman experimental design, key factors was yeast extract and K2HPO. Finally, the response surface methodology was performed to obtain the optimum concentrations of two selected variables. The optimized medium composition was consisted of 20 g/L galactose, 36 g/L yeast extract, 0.41 g/L K2HPO4, 0.25 g/L Na2CO3, 0.4g/L MgSO4 and 0.01g/L CaCl2. Dry cell weight (15.4 g/L) by optimum production medium were increased 10 times, as compared to these determined with basic production medium. Fermentation conditions were examined for the mass production of B. subtilis. The effect of temperature, agitation speed, pH and aeration rate on the mass production of B. subtilis were also studied in a batch fermenter which was carried out in a 2.5 L bioreactor with a working volume of 1.5 L containing optimized production medium.

Pseudomonas sp. BCNU 2001 was isolated from soil samples in Tae-Baek Mountain. The taxonomical position of bacterium was investigated using morphological, biochemical and genetic techniques. These results strongly indicated that it was most closely related to Pseudomonas aeruginosa. The effects of nutrients and influence of environmental parameters on growth and production of bioactive metabolites by Pseudomonas sp. BCNU 2001 were evaluated in submerge culture. Disc diffusion method was employed to study the effect of different culture media, various carbon and nitrogen sources, inoculum size, temperature, pH and incubation period on the production of bioactive metabolite in flask cultures by Pseudomonas sp. BCNU 2001. Growth was determined as dry cell weight in a fixed volume of culture broth. Dextrose, maltose, citrate, mannitol, trehalose, fructose and glycerol were found to be the suitable carbon soures. Especially, BCNU 2001 isolated from Tae-baek mountain was highly active to M. luteus, P. mirabilis and S. cereviea. (Acknowledgments; This research was financially supported by the Korean Ministry of Education, Science Technology (MEST) and Korea Industrial Technology Foundation (KOTEF) through the Human Resource Training Project for Regional Innovation)

The symbiosis of Effective Microorganisms (EM) is investigating. EM consists of eighty different species and generally, there are three main genera of EM which are phototrophic bacteria, lactic acid bacteria, and yeast. Recently, EM is applied in various fields such as in industry, agriculture and environmental cleanup. Thus, quantitative analysis of EM is necessary for more efficient use because the characteristics and efficacy of EM is depended on distribution ratio of its main genera. Three strains that used in this study are Lactobacillus plantarum, Saccharomyces cerevisiae, and Rhodopseudomonas palustris. Across the study of the cell interaction, co-culture is obviously necessary. Thus, as the beginning stage, we are examining the symbiosis of the EM. The compositions of culture medium and condition are: Glucose 5g l-1, Peptone 4gl-1, NZ-case(casein hydrolysate) 4gl-1, K2HPO4 1g l-1, MgSO4 0.5 l-1, and 32℃ in incubator for 72hr with light up the tungsten lamp. The pH adaptation test on medium is measured by arranging the pH at 3.5-6 separately. Optical density of cell by using Spectrophotometer at 600 nm and plate counting on selective pressure are used to analyze the cell growth. Furthermore, the coexistence of EM is observed by using an optical microscope.

Laccase, one of the ligninolytic enzymes, received attention from researchers for the past decades due to its broad substrate specificity. Unlike peroxidases, it use molecular oxygen as the final electron acceptor. The potential application of laccase includes pulping, detergents, textile dyes, biosensors, detoxification of pollution and enzymatic conversion of chemical intermediates. Laccase production from repeated batch cultures of immobilized Pleurotus ostreatus was investigated. Immobilization of P. ostreatus was conducted by polyurethane foam (PUF). The effect of pore size of PUF was evaluated by laccase activity. Among 25, 40 and 90 pores per inch (PPI), PUF with 40 PPI showed the highest activity in submerged cultures. Replacement of the medium was conducted at different phases of its growth in submerged fermentation; the exponential, early stationary and death phases. It showed that laccase production of repeated batch culture was significantly affected by the time of replacement of medium.

A tannic acid-inducible and mycoviral-regulated laccase3 (lac3) from the chestnut blight fungus Cryphonectria parasitica has recently been identified, but further characterization was hampered because of the precipitation of protein products by tannic acid supplementation. The present study investigated the heterologous expression of the functional laccase3 using a yeast Saccharomyces cerevisiae. Laccase activity in the culture broth of transformants measured using a laccase-specific substrate suggested that the lac3 gene was successfully expressed and the corresponding protein product secreted into the culture media. In addition, activity staining and Western blot analysis of a native gel revealed that the enzyme activity coexisted with the protein product specific to anti-laccase3 antibody, confirming that the cloned lac3 gene is responsible for the laccase activity. When transformants were grown on plates containing tannic acidsupplemented media, brown coloration was observed around transformed cells, indicating the oxidation of tannic acid. However, the enzymatic activity was measurable only in the selective ura- media and was negligible in nonselective YEPD culture conditions. This was in part because of the increased plasmid instability in the nonselective media. Moreover, the protein product of lac3 appears to be sensitive to the cultured YEPD broth, because a rapid decline in enzymatic activity was observed when the cultured broth of ura- media was mixed with that of YEPD. In addition, constitutive expression of the lac3 gene resulted in a reduced cell number of the lac3 transformants compared to that of mock-transformants. However, the presence of recombinant vector without lac3 induction did not affect the growth of transformants. These results suggest that expression of the lac3 gene has an inhibitory effect on the growth of transformed S. cerevisiae and that the controlled expression of lac3 is appropriate for the possible application of recombinant yeast to the treatment of phenolic compounds.

Streptomyces natalensis produces natamycin that is predominantly a strong antifungal agent, inhibiting the growth of both yeasts and molds and preventing the formation of aflatoxin in filamentous fungi. Because of the low toxicity of natamycin to mammalian cells compared to other antifungal compounds, it found many applications in the treatment of many fungal diseases. S-Adenosyl-L-methionine synthtase (SAM-s) catalyzes the biosynthesis of SAM from ATP and L-methionine. SAM plays important roles in the primary and secondary metabolism of the cell. A metK encoding a SAM-s was searched from S. natalensis with PCR. PCR using the primers designed from the two highly conserved regions for metK genes of Streptomyces strains gave 854-bp bands. The sequence of these bands had a high similarity to the expected region of a metK gene. An intact 1.2-kb metK gene of S. natalensis was obtained by genomic Southern hybridization with PCR product as a probe, and then studied. [This work was supported by the Korea Science and Engineering Foundation(KOSEF) grant funded by the Korean government(MEST) (No. 2009-0058974)]

Bacillus sp. stains 2002 and 2003 were isolated from a forest soil samples collected at Taebaek mountain of Gangwon province, Korea. On the basis of the comprehensive taxonomic data from morphological, physiological and biochemical characteristics, and 16S rDNA sequence analysis, BCNU 2002 and 2003 were identified as B. amyloliquefaciens. The in vitro antimicrobial activities against bacteria such as B. subtilis and M. luteus and pathogenic fungi such as Aspergillus niger, Candida albicans, Epidermophyton floccosum, Saccharomyces cerevisiae, Trichophyton mentagrophytes and Trichophyton rubrum were elucidated. The effect of medium components (i.e. carbon and nitrogen sources) and other culture requirements (i.e. initial pH and temperature) on production of antimicrobial antibiotics by Bacillus sp. 2002 and Bacillus sp. 2003 were also investigated in this study. Maximum production of bioactive metabolites in a synthetic medium was found on the seven day of incubation. Furthermore, maintaning the incubation temperature at 28℃ under shaking condition was found to be optimum for maximum antimicrobial activity. In addition, BCNU 2002 and BCNU 2003 were determined to have the ability to produce enzymes such as amylase, protease, gelatinase and catalase. (Acknowledgments; This research was financially supported by the Korean Ministry of Education, Science Technology (MEST) and Korea Industrial Technology Foundation (KOTEF) through the Human Resource Training Project for Regional Innovation)

Diagnostics and disease-management strategies require technologies to enable the multiplex detection and quantification of a wide range of pathogenic microorganisms. However, most multiplex· quantitative detection methods available suffer from compromises between the level of multiplexing, throughput and accuracy of quantification. Here, we demonstrate the novel multiplex pathogen detection method using capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) coupled with multiplex ligation-dependent probe amplification (MLPA). MLPA-CE-SSCP is composed of four major steps which are probe hybridization, probe ligation, multiplex amplification and detection. Using five clinically important pathogens as a model set, all the steps were carefully optimized to precisely quantify by MLPA-CE-SSCP. We could obtain the results which illustrate a strong potential in clinical diagnosis, food safety, and biosafety.

In this study, we isolated Shewanella putrefaciens from shellfish and determined the optimum growth conditions of the isolates. S. putrefaciens is Gram-negative marine bacterium which is able to produce hydrogen sulfide from thiosulfate. Initially, the isolation of S. putrefaciens was carried out by obtaining black colonies on spreadplated LB agar supplemented with ferrous sulfate and sodium thiosulfate after incubation for 24hr. All black colonies were tested for their catalase activity and examined Gram-staining. Subsequently catalase positive and Gram-negative colonies were confirmed by PCR assays. The 16S rRNA gene and gyrB gene were used as S. putrefaciens-specific PCR primers. From three species of shellfish – oyster, short-neck clam and mussel samples tested, a total of 27 strains of S. putrefaciens were isolated and ten of them were tested to optimum growth conditions. To determine the optimum growth conditions, we selected the range of temperature (4, 25, 30, 37 and 42℃), pH (3, 4, 5, 6, 7, 8, and 9) and NaCl concentration (0, 0.085, 0.25, 0.5, 0.7, and 1M). According to our results, all isolates grew optimally at 25℃, pH 7.0 and 0.085 M NaCl concentration.

A bacterial flavin-containing monooxygenase (FMO) gene was cloned from Methylophaga aminisulfidivorans MPT, and a plasmid pBlue 1.7 was constructed to express the bacterial fmo gene in E. coli. The recombinant E. coli harboring the pBlue 1.7 plasmid produced 95 mg/L of indirubin and 5 mg/L of indigo in tryptophan medium (2g tryptophan, 10g NaCl, 5g yeast extract per liter) containing several amino acids (cysteine 0.05%, aspratic acid 0.005% and arginine 0.005%, w/v). The aim of this study was the investigation of the optimum indirubin production conditions through batch culture. The optimum conditions were as follows: tryptophan concentration 1g/L, pH 7.0, air supply 1.0 vvm and culture temperature 35℃. Under these conditions, 100 mg/L indirubin and 4mg/L indigo were produced.

Guanosine tetraphosphate (ppGpp) responding to carbon and energy starvation in bacteria acts as positive regulator for antibiotic production in some Streptomyces. The relA gene of Streptomyces natalensis producing a natamycin, which is widely used in food industry to prevent mold contamination of cheese and other non-sterile foods, was searched with PCR using the primers designed from the two highly conserved regions of RelA in Streptomyces species. In a previous study, 832-bp product was gained by PCR and had a high similarity to the expected region of a relA gene. An intact 2.6-kb relA gene of S. natalensis was obtained by Southern blot as well as Colony PCR using the 832-bp PCR product as probe. The obtained relA was inserted into pSET152ET, over-expression vector, and then introduced into Streptomyces strains. Also, a relA expression was assayed in the exconjugant and its phenotype change was compared with the control strain containing only pSET152ET vector. [This work was supported by the Korea Science and Engineering Foundation(KOSEF) grant funded by the Korean government(MEST) (No. 2009-0058974)]

Prenylated flavonoids and dihydroxynapthalenes are natural products that exhibit diverse biological effects and often represent the active components of various medicinal plants. Aromatic prenyltransferases catalyze the transfer of prenyl moieties to aromatic acceptor molecules and give rise to an astounding diversity of primary and secondary metabolites in plants, fungi and bacteria. We recently amplified, cloned and expressed the aromatic prenyltransferase sp8959 from Streptomyces peucetius ATCC 27952 in E. coli to validate their functions in vivo. A novel prenyltransferase sp8959 gene is located upstream of THNS gene cluster (type III PKS).This study demonstrated the production of prenylated flavonoids and dihydroxynaphthalenes by biotransformation without feeding of prenyl donors. This method provides the possibility of generating prenylated flavonoids and dihydroxynapthalenes that occur rarely in nature.

Nattokinase is a potent fibrinolytic enzyme that is considered to be a promising agent for thrombosis therapy. The enzymes were discovered from various sources, such as Japanese natto, Korean Chungkookjang soy sauce, and Chinese douchi. Based on its food origin and relatively strong fibrinolytic activity, nattokinase has advantages over other commercially used medicine in preventative and prolonged effects, convenient oral administration, and stability in the gastrointestinal tract.In this study, Bacillus subtilis was cultivated in flask and fermentor to produce nattokinase. Glucose and NH4Cl were used as carbon and nitrogen sources, respectively, with peptone, yeast extract, or tryptone supplementation. Glucose concentration was fixed at 10 g/L. New method of assaying nattokinase activity was suggested. In flask culture, the highest cell growth (optical density monitored at 600 nm) and nattokinase activity were obtained with 50 g/L of peptone supplementation. At this condition, nattokinase activity was 630 unit/ml. In batch culture of B. subtilis in fermentor, optical density increased to 10 at 6 h and then gradually decreased. Nattokinase activity also increased with cell growth, showing growth-associated production. With 50 g/L of peptone supplementation, the highest nattokinase activity of 3400 unit/ml was obtained at 10 h.

Rapid and quantitative identification of bacterial pathogen has been considered as important criteria for its clinical and hygienical requirements. Among various methods, capillary electrophoresissingle strand conformation polymorphism (CE-SSCP) analysis which can separate target DNA in sensitive and rapid manner is applied successfully for multiplex quantitative detection of bacteria. However, conventional poly(N,N-dimethylacrylamide) polymer matrix generally used for CE-based analysis has limited resolution in CE-SSCP since it is optimized for molecular weight dependent separation.In this study, multiplex quantitative etection of bacterial pathogens using CE-SSCP was performed with noble high resolution polymer matrix. The marker sequences of targets were amplified with single set of universal PCR primers from conserved region of 16S rRNA gene. PCR products lengths were 238-265bp, and further modification of DNA was not necessary. The results showed that 9-plex quantitative detection was successfully achieved with high resolution and sensitivity, and simple procedure. The results also displayed the potential of new polymer matrix for various genetic analyses.

To investigate effect of cellular fatty acids composition on ethanol tolerance in Escherichia coli, Bacillus subtilis des encoding fatty acid desaturase, E. coli fabA encoding b-hydroxydecanoyl thio ester dehydrase or both genes were overexpressed. E. coli recombinant cells harboring fabA showed elevated tolerance against ethanol compared to wild type strain. In contrast, des encoding fatty acid desaturase resulted in a decreased resistance against ethanol and coexpression of des with fabA complemented the ethanol tolerance of E. coli. This result could give a clue to engineer bacterial strains resistant to higher concentration of ethanol.