Optimal criterion for the scale-up production of schizophyllan, a fungal polysaccharide secreted by Schizophyllum commune, was investigated. For the production of the polysaccharide in a 150-l bioreactor, the culture conditions optimized in a 15-l bioreactor were applied to a 150-l bioreactor with scale-up process, by changing impeller speed and airflow rate. The optimized impeller speed in the 15-l bioreactor was 50 rpm in a technical medium based on barley. For establishment of the scale-up process, 3 kinds of criteria were used while the gas throughput number was kept constant, as follows; constant volume-related power input, constant tip speed of stirrer, and constant Reynolds number. In the 150-l bioreactor, the highest values for the maximum specific growth rate (1.17/day) and productivity (0.63 g/L${\cdot}$day) were achieved in the culture condition from constant volumerelated power input criterion.

Chitosan-alginate capsules were formed by electrostatic interactions and exhibited an appropriate mechanical strength, permeability, and stability for the culture of hepatocytes. Pig hepatocytes were isolated and hepatocyte spheroids formed and immobilized in chitosan-alginate capsules. An encapsulation procedure of 3 min and spheroid formation period of 24 h were the optimum conditions for the best liver functions. Pig hepatocytes with a cell density of $6.0{\tomes}10^6$ cells/ml in the capsules were found to be most suitable for application in a bioartificial liver support system. The encapsulated pig hepatocyte spheroids exhibited stable ammonia removal and urea secretion rates in a bioreactor for 2 weeks. Accordingly, chitosan-alginate encapsulated hepatocyte spheroids in a packed-bed bioreactor would appear to have potential as a bioartificial liver.

By using PCR with nirS gene primers, three nirSharboring denitrifying bacteria (strain N6, strain N23, and strain R13) were newly isolated from activated sludge of a weak municipal wastewater treatment plant. Small-subunit rRNA gene-based analysis indicated that strain N6, strain N23, and strain R13 were closely related to Arthrobacter sp.,Staphylococcus sp., and Bacillus sp., respectively. In an attempt to identify their roles in biological nitrate and nitrite removal from sewage, we investigated their specific denitrification rates (SDNRs) for $NO_-^3$ - and $NO_-^2$ - in various cultures. All purecultures of each isolated nirS-harboring bacterial strain could remove $NO_-^3$ - and $NO_-^2$ - simultaneously in high efficiency, and the carbon requirements for $NO_-^3$ - removal of strain N6 and strain R13 were effectively low at 3.1 and 4.1 g COD/g $NO_3N$, respectively. In the case of mix-cultures of the strains (N6+N23, N6+R13, N23+R13, and N6+N23+R13), their SDNRs for $NO_-^3$ - were also effective, and their carbon requirements for $NO_-^3$ - removal were also effective at 3.0- 3.8 g COD/g NO3N. However, all tested mix-cultures accumulated $NO_-^2$ - in their culture media. On the other hand, the continuous culture of activated sludge mixed with strain N6 showed no significant increase of $NO_-^3$ - removal in comparison with strain N6's pure culture. These results suggest that nitrate and nitrite removal in biological wastewater treatment might be dependent on complicated bacterial interactions, including several effective denitrifying bacteria isolated in this study, rather than the specific bacterial types present and the number of bacterial types in activated sludge.

Gibberellins (GAs) play important roles in plant growth and development. Fifty-four fungi were isolated from the roots of 4 kinds of Sesamum indicum plants, and the production of GAs was spectrophotometrically examined. The number of GA-producing fungi was two strains from S. indicum, four strains from Gold S. indicum, and five strains from Brown S. indicum. Eleven fungi with GAs-producing activity were incubated for seven days in 40 ml of Czapek's liquid medium at $25^{\circ}C$ and 120 rpm, and the amount of each GA in the medium was measured by gas chromatographymass spectrometery (GC-MS). Penicillium commune KNU5379 produced more $GA_3$, $GA_4$, and $GA_7$ than Gibberella fujikuroi, Fusarium proliferatum, and Neurospora crassa which are known as GAs-producing fungi. GAs-producing activity of the P. commune KNU5379 was shown to produce 71.69 ng of $GA_1$, 252.42 ng of $GA_3$, 612.00 ng of $GA_4$, 259.00 ng of $GA_7$, and 202.69 ng of $GA_9$ in 25 ml of liquid medium. Bioassay of culture fluid of GA-producing fungi was also performed on rice sprout.

W/O/W (water-in-oil-in-water) type multiple emulsion was applied to improve the storage stability of an antagonistic microorganism, Burkholderia gladioli. Encapsulation of microorganism into a W/O/W emulsion was conducted by using a two-step emulsification method. W/O/W emulsion was prepared by the incorporation of B. gladioli into rapeseed oil and the addition of polyglycerin polyriconolate (PGPR) and castor oil polyoxyethylene (COG 25) as the primary and secondary emulsifier, respectively. Microcrystalline cellulose was used as an emulsion stabilizer. To evaluate the usefulness of W/O/W emulsion formulation as a microbial pesticide for controlling the bacterial wilt pathogen (Ralstonia solanacearum), the storage stability and antagonistic activity of emulsion formulation were tested in vitro. The storage stability test revealed that the viability of formulated cells in emulsion was higher than that of unformulated cells in culture broth. At $4^{\circ}C$, the viabilities of formulated cells and unformulated cells at the end of 20 weeks decreased to about 2 and 5 log cycles, respectively. At $37^{\circ}C$, the viability of formulated cells decreased to only 2 log cycles at the end of storage. On the other hand, the viable cells in culture broth were not detected after 13 weeks. In activity test, formulated cells in emulsion were more effective in inhibiting the growth of pathogen than unformulated cells in culture broth. Unformulated cells completely lost their antagonistic activity during storage under similar conditions. The W/O/W multiple emulsion formulation was shown to be useful as the novel liquid formulation for biological control.

To identify the activity of recombinant subtilisins (subtilisin BPN' and subtilisin Carlsberg), three different zymography methods, SDS-fibrin zymography (SDS-FZ), reverse fibrin zymography (RFZ), and isoelectric focusingfibrin zymography (IEF-FZ), were used. The recombinant subtilisins BPN' and Carlsberg did not migrate into the electrophoretic field based on a Laemmli buffer system, instead forming a "binding mode" at the top part of the separating gels with the SDS-FZ and RFZ techniques. Yet, this problem was resolved when using IEF-FZ with a pH range from 3 to 10. In addition, all these methods enabled the activity of a recombinant pro-subtilisin DJ-4 to be detected without a refolding pathway.

Modeling and simulation for simultaneous saccharification and fermentation (SSF) process in bioconversion of paper mill sludge to lactic acid was carried out. The SSF process combined the enzymatic hydrolysis of paper mill sludge into glucose and the fermentation of glucose into lactic acid in one reactor. A mathematical modeling for cellulose hydrolysis was developed, based on the proposed mechanism of cellulase adsorption deactivation. Another model for simple lactic acid fermentation was also made. A whole mathematical model for SSF was developed by combining the above two models for cellulose hydrolysis and lactic acid fermentation. The characteristics of the SSF process were investigated using the mathematical model.

A comparison of promoter fusions with the luxCDABE genes from Vibrio fischeri and Photorhabdus luminescens was made using promoters from several genes (katG, sodA, and pqi-5) of E. coli that are responsive to oxidative damage. The respective characteristics, such as the basal and maximum bioluminescence and the relative bioluminescence, were compared. E. coli strains carrying fusions of the promoters to P. luminescens lux showed higher basal and maximally induced bioluminescent levels than strains carrying the same promoter fused to the luxCDABE genes from V. fischeri. The sensitivities between the strains were similar, regardless of the luciferase used, but lower response ratios were seen from strains harboring the P. luminescens lux fusions. Furthermore, using the two katG::lux fusion strains, the bioluminescence from the P. luminescens lux fusion strain, DK1, was stable after reaching a maximum, while that of strain DPD2511 decreased very rapidly due to substrate limitation.

Sophorolipid was produced by Candida bombicola ATCC 22214 from soybean dark oil, a byproduct of soybean oil processing. With a fed-batch culture of C. bombicola for 7 days, 90 g/l of sophorolipid was obtained. The CMC (critical micelle concentration) and minimum surface tension of the sophorolipid in aqueous solution were found to be 150 mg/l and 48 mN/m, respectively. The dispersion capability of sophorolipid was higher than that of the chemical surfactants such as SDS and Brij30. The molar solubility ratio (MSR) of 4-methylnaphthalene was 0.2. Linoleic and oleic acids were the main constituents of the fatty acid composition of the sophorolipid. The sophorolipid showed antimicrobial activity against Propionibacterium acne and Bacillus subtilis.

Poly-${\gamma}$-glutamic acid (PGA) is an unusual anionic polypeptide and has great potential as an environmentally and industrially significant biodegradable material. A new ${\gamma}$-PGA producer, Bacillus mesentericus MJM1, with high production capacity was isolated from Korean domestic Chungkuckjang bean paste. It produced ${\gamma}$-PGA at the level of 10 g/l in suitable media. The viscosities of 5% initially extracted mucin and purified ${\gamma}$-PGA solutions were 660 cps and 600 cps, respectively. The produced ${\gamma}$-PGA polymer consisted of 2,000 glutamic acid residues with even proportion of L and D types with molecular mass of about 200- 300 kDa. Bacillus mesentericus MJM1 displayed ${\gamma}$-glutamyltranspeptidase (${\gamma}$-GTP) activity that is known to play a key role in ${\gamma}$-PGA biosynthesis. The ${\gamma}$-GTP coding region was located on the plasmid of 5.8 kb. The plasmid, named pMMH1, is a rolling-circle replication (RCR) plasmid and additionally contained a replication origin and type I signal peptidase (sipP) coding region.

Doxorubicin is a clinically important anticancer polyketide compound that is typically produced by Streptomyces peucetius var. caesius. To improve doxorubicin productivity by S. peucetius, a doxorubicin pathway-specific regulatory gene, dnrI, was cloned into a high-copy-number plasmid containing a catechol promoter system. The S. peucetius containing the recombinant plasmid exhibited approximately 9.5-fold higher doxorubicin productivity compared with the wild-type S. peucetius. The doxorubicin productivity by this recombinant S. peucetius strain was further improved through the optimization of culture media composition. Based on the Fractional Factorial Design (FFD), cornstarch, $K_2HPO_4$, and $MgSO_4$ were identified to be the key factors influencing doxorubicin productivity. The Response Surface Method (RSM) results based on 20 independent culture conditions with varying amounts of key factors predicted the highest theoretical doxorubicin productivity of 11.1 mg/l with corn starch of 46.33 g/l, $K_2HPO_4$ of 4.63 g/l, and $MgSO_4$ of 9.26 g/l. The doxorubicin productivity of the recombinant S. peucetius strain with the RSM-based optimized culture condition was experimentally verified to be 11.46 mg/l, which was approximately 30.8-fold higher productivity compared with the wild-type S. peucetius without culture media optimization.

A new Bacillus peptidase, bacillopeptidase DJ-2 (bpDJ-2), with molecular mass of 42 kDa and isoelectric point (pI) of 3.5- 3.7, was purified to homogeneity from Bacillus sp. DJ-2 isolated from Doen-Jang, a traditional Korean soybean fermented food. The enzyme was identified as an extracellular serine fibrinolytic protease. The optimal conditions for the reaction were pH 9.0 and $60^{\circ}C$. The first 18 amino acid residues of the N-terminal amino acid sequence of bpDJ-2 were TDGVEWNVDQIDAPKAW, which is identical to that of bacillopeptidase F (bpf). However, based on their Nterminal amino acid sequence, molecular size, and pI, it is different from that of bpf and extracellular 90 kDa. The whole (2,541 bp, full-bpDJ-2) and mature (1,956 bp, mature-bpDJ-2) genes were cloned, and its nucleotide sequence and deduced amino acid sequence were determined. The expressed proteins, full-bpDJ-2 and mature-bpDJ-2, were detected on SDSPAGE at expected sizes of 92 and 68 kDa, respectively.

Electrochemical control of the metabolic flux of W. kimchii sk10 on glucose and pyruvate was studied. The growing cell of W. kimchii sk10 produced 87.4 mM lactate, 69.3 mM ethanol, and 4.9mM lactate from 83.1mM glucose under oxidation condition of the anode compartment, but 98.9 mM lactate, 84.3mM ethanol, and 0.2 mM acetate were produced from 90.8 mM glucose under reduction condition of the cathode compartment for 24 h, respectively. The resting cell of W. kimchii sk10 produced 15.9 mM lactate and 15.2 mM acetate from 32.1 mM pyruvate under oxidation condition of the anode compartment, and 71.3 mM lactate and 3.8 mM acetate from 79.8mM pyruvate under reduction condition of the cathode compartment. The redox balance (NADH/$NAD^+$) of metabolites electrochemically produced from pyruvate was 1.05 and 18.76 under oxidation and reduction conditions, respectively. On the basis of these results, we suggest that the neutral red (NR) immobilized in bacterial membrane can function as an electron channel for the electron transfer between electrode and cytoplasm without dissipation of membrane potential, and that the bacterial fermentation of W. kimchii sk10 can be shifted to oxidized or reduced pathways by the electrochemical oxidation or reduction, respectively.

Strain M-18 was isolated from the rhizosphere soil of sweet melon, using 1-aminocyclopropane-1-carboxylate (ACC) as a sole nitrogen source. Its phenotypic characteristics, metabolic tests, and 16S rDNA sequence were analyzed. The antibiotics secreted by strain M-18 were determined to be phenazine 1-carboxylic acid and pyoluteorin. These data showed that strain M-18 was a new fluorescent Pseudomonas strain that produced both phenazine 1-carboxylic acid and pyoluteorin, some features being similar to Pseudomonas aeruginosa and Pseudomonas fluorescens. Therefore, the strain M-18 appears to be the first pseudomonad described to date that is capable of producing both phenazine 1-carboxylic acid and pyoluteorin.

The hyoscyamine 6${\beta}$-hydroxylase (h6h) gene was introduced into the genome of Scopolia parviflora through the Agrobacterium rhizogenes binary vector system. The enzyme was expressed ally and tissue specific selectively in roots, resulting in five transgenic hairy root lines. The presence of the h6h gene in kanamycin-resistant hairy roots and its overexpression were confirmed by polymerase chain reaction (PCR), Northern blotting, and Western blotting, respectively. In the transgenic hairy root lines which constitutively expressed the H6H enzyme, hyoscyamine and scopolamine accumulated in high concentration. Among the transgenic hairy root lines that expressed the H6H enzyme, only two were more productive. The levels of tropane alkaloids in transgenic hairy root varied greatly: The best transgenic line (#5) contained 8.12 mg of scopolamine per g dry weight, which produced the compound three times more than wild-type root. These results suggest a possibility of improving the yield of tropane alkaloids in hairy root lines by genetic and metabolic engineering.

Microbacterium laevaniformans levansucrase synthesized various hetero-oligosaccharides by transferring fructosyl residue from sucrose to various saccharides as acceptors. The acceptor specificity test showed that reducing saccharides were more favorable acceptors than nonreducing saccharides. The transfructosylated product, fructosyl galactose, was produced in the presence of D-galactose as an acceptor. The chemical structure of the resulting fructosyl galactose was analyzed by yeast invertase and NMR, and identified as O-$\alpha$-D-galactosyl-(1${\to}$2)-$\beta$-D-fructofuranoside. These results indicate that the main transfructosylation activity of the enzyme is to make nonreducing transferred products via a transfer of fructosyl residue to acceptor molecules having reducing group. When nonreducing sugars, such as methyl $\alpha$-D-glucoside and methyl $\alpha$-D-galactoside, were used as an acceptor, the transfer product was also formed in spite of the reducing group blocked with methyl group. The fact that no transfer product was formed with sugar alcohols as acceptors was suggested to be due to marked conformational difference of acceptors.

End-stage renal disease is a fatal and devastating disease that is caused by progressive and irreversible loss of functioning nephrons in the kidney. Dialysis and renal transplantation are the common treatments at present, but these treatments have severe limitations. The present study investigated the possibility of reconstructing renal tissues by transplantation of renal precursor cells to replace the current treatments for end-stage renal disease. Embryonic renal precursor cells, freshly isolated from metanephroi of rat fetus at day 15 post-gestation, were seeded on biodegradable polymer scaffolds and transplanted into peritoneal cavities of athymic mice for three weeks. Histologic sections stained with hematoxylin & eosin and periodic acid-Schiff revealed the formation of primitive glomeruli, tubules, and blood vessels, suggesting the potential of embryonic renal precursor cells to reconstitute renal tissues. Immunohistochemical staining for proliferating cell nuclear antigen, a marker of proliferating cells, showed intensive nuclear expression in the regenerated renal structures, suggesting renal tissue reconstitution by transplanted embryonic renal precursor cells. This study demonstrates the reconstitution of renal tissue in vivo by transplanting renal precursor cells with biodegradable polymer scaffolds, which could be utilized as a new method for partial or full restoration of renal structure and function in the treatment of end-stage renal disease.

Abstract Griseofulvin has been used as an antifungal antibiotic for the treatment of mycotic diseases of humans and veterinary animals. The purpose of this work was to identify a griseofulvin-producing endophytic fungus from Abies holophylla and evaluate its in vivo antifungal activity against plant pathogenic fungi. Based on nuclear ribosomal ITS1-5.8SITS2 sequence analysis, the fungus was identified and labeled as Xylaria sp. F0010. Two antifungal substances were purified from liquid cultures of Xylaria sp. F0010, and their chemical identities were determined to be griseofulvin and dechlorogriseofulvin through mass and NMR spectral analyses. Compared to dechlorogriseofulvin, griseofulvin showed high in vivo and in vitro antifungal activity, and effectively controlled the development of rice blast (Magnaporthe grisea), rice sheath blight (Corticium sasaki), wheat leaf rust (Puccinia recondita), and barley powdery mildew (Blumeria graminis f. sp. hordei), at doses of 50 to 150 ${\mu}$g/ml, depending on the disease. This is the first report on the production of griseofulvin and dechlorogriseofulvin by Xylaria species.

In this research, we examined the effect of NaCl on the growth, energy metabolism, and proton motive force of Halomonas salina, and the effect of compatible solutes on the bacterium growing in the high salinity environment. H. salina was isolated from seawater and identified by 16srDNA sequencing. The growth of H. salina was not enhanced by the addition of external compatible solutes (choline and betaine) in the high salinity environment. The resting cells of H. salina absorbed more glucose in the presence of 2.0 M NaCl than in its absence. H. salina did not grow in the medium with either KCl, RbCl, CsCl, $Na_2SO_4$, or $NaNO_3$, in place of NaCl. The optimal concentration of NaCl for the growth of H. salina ranged from 1.4 M to 2.5 M, and the growth yield was decreased in the presence of NaCl below 1.4M and above 2.5M. The activity of isocitrate dehydrogenase, pyruvate dehydrogenase, and malate dehydrogenase of H. salina was not inhibited by NaCl in in vitro test. The proton translocation of H. salina was detected in the presence of NaCl only. These results indicate that NaCl is absolutely required for the normal growth and energy metabolism of H. salina, but the bacterial growth is not enhanced by the compatible solutes added to the growth medium.

The growth and phosphatase (phosphomonoesterase) activity of Chroococcidiopsis culture isolated from the cryptoendoliths of the Antarctic were compared with a similar isolate from the Arizona hot desert. Such cyanobacteria living inside rocks share several features with the immobilized cells produced in the laboratory. This study has relevance because the availability of phosphorus is a key factor influencing the growth of these cyanobacteria in nature, in such unique ecological niches as the hot and cold deserts. Phosphatase activity therefore is of particular importance for these organisms if they are to survive without any other source of phosphorus availability. Also, there is paucity of knowledge regarding this aspect of study in cyanobacterial cultures from these extreme environments. The salient feature of this study shows the importance of specific pH and temperatures for growth and phosphatase activity of both cultures, although there were marked differences between the two isolates. The pH and temperature optima for growth and phosphatase activity (PMEase) of Chroococcidiopsis 1 and 2 were 9.5, $240^{\circ}C$ and 8.5, $40^{\circ}C$ respectively. The $K_m and V_max$ values of cultured Chroococcidiopsis 1 showed lower affinity of PMEase for the substrate compared to the enzyme affinity of the same organism when found within the rocks; Chroococcidiopsis 2 and Arizona rocks containing the same alga however showed similar affinity of PMEase for the substrate. An interesting observation was the similarity in response of immobilized Chroococcidiopsis 1 culture and the same organism in the Antarctic rocks to low light and low temperature stimulation of PMEase. This thermal response seems to be related to the ability of the immobilized Antarctic isolate and the rocks to either cryoprotect the PMEase or undergo a change to save the enzyme from becoming nonfunctional under low temperatures. The free cells of Chroococcidiopsis 1 culture however did not show such responses.

Under sulfur deprived conditions, PS II and photosynthetic $O_2$ evolution by Chlamydomonas reinhardtii UTEX 90 are inactivated, resulting in shift from aerobic to anaerobic condition. This is followed by hydrogen production catalyzed by hydrogenase. We hypothesized that the photosynthetic capacity and the accumulation of endogenous substrates such as starch for hydrogen production might be different according to cell age. Accordingly, we investigated (a) the relationships between hydrogen production, induction time of sulfur deprivation, increase of chlorophyll after sulfur deprivation, and residual PS II activity, and (b) the effect of initial cell density upon sulfur deprivation. The maximum production volume of hydrogen was 151 ml $H_2$/l with 0.91 g/l of cell density in the late-exponential phase. We suggest that the effects of induction time and initial cell density at sulfur deprivation on hydrogen production, up to an optimal concentration, are due to an increase of chlorophyll under sulfur deprivation.

The hypoglycemic effect of Collybia confluens exobiopolymer was investigated in streptozotocin (STZ)-induced diabetic rats. In a dose-dependent study, the exobiopolymer, at 150 mg/kg body weight (BW) dose substantially lowered the plasma glucose level by 29.3%, as compared to the control group. It also lowered the plasma total cholesterol and triglyceride levels by 23.3 and 30.7%, respectively, and reduced the liver total cholesterol and triglyceride levels by 23.0 and 33.5%, respectively. The activity of alanine transaminase (ALT) and aspartate transaminase (AST) was reduced 34.6% and 23.6% respectively, by the exobiopolymer administration, compared to the control group. The exobiopolymer was found to contain 83.2% carbohydrate and 16.8% protein. The sugar and amino acid of the exobiopolymer were also analyzed in detail.

A new system for displaying proteins on the surface of Escherichia coli was developed using the Salmonella typhimurium outer membrane protein C (OmpC) as an anchoring motif. The C-terminal deletionfusion strategy was developed to fuse the polyhistidine peptides and green fluorescent protein (GFP) to the Cterminal of the truncated functional portion of OmpC. The polyhistidine peptides of up to 243 amino acids could besuccessfully displayed on the E. coli cell surface, which allowed recombinant E. coli to adsorb up to 34.2 μmol of Cd2+ per gram dry cell weight. The GFP could also be successfully displayed on the E. coli cell surface. These results suggest that the C-terminal deletion-fusion strategy employing the S. typhimurium OmpC as an anchoring motif provides a new efficient way for the display of large proteins on the surface of E. coli.

A survey for vesicular-arbuscular mycorrhizae (VAM) status was undertaken in three different industrially polluted sites at Uyyakondan channel of Senthanneerpuram area in Trichy, India. The soils and the effluents were acidic, and contained higher Zn (621 to 711 ppm) than the other heavy metals, such as Cu, Pb, and Ni. Eighteen plant species were collected from the rhizosphere soils, and 13 species were positive for VAM colonization. Fifteen VAM fungal species were isolated from the plant species. The number of VAM fungal spores from the soils ranged from 45 to 640 per 100 g of soil. There was a significant correlation observed between the number of spores and percentage root colonization, as exemplified by Acalypha indica (45 and 20%, respectively) and Paspalum vaginatum (640 and 98%, respectively). Hostspecific and site-specific associations were observed in site 2; particular VAM species, Gigaspora gigantea and Glomus fasciculatum, were specific to particular host plants, Phyllanthus maderaspatensis and A. indica, respectively, even though Eclipta prostrata and Physalis minima were maximally associated with 8 VAM species. G. fasciculatum was found in 11 plant species and predominant VAM species. These results led us to conclude that VAM fungi are associated with a majority of the plants in the industrial polluted sites and support the plants to survive in the acidic soils, polluted with heavy metals of the industrial effluents.

Abstract A direct approach was used to retrieve active lipases from Paenibacillus polymyxa genome databases. Twelve putative lipase genes were tested using a typical lipase sequence rule built on the basis of a consensus sequence of a catalytic triad and oxyanion hole. Among them, six genes satisfied the sequence rule and had similarity (about 25%) with known bacterial lipases. To obtain the six lipase proteins, lipase genes were expressed in E. coli cells and lipolytic activities were measured by using tributyrin plate and pnitrophenyl caproate. One of them, contig 160-26, was expressed as a soluble and active form in E. coli cell. After purifying on Ni-NTA column, its detailed biochemical properties were characterized. It had a maximum hydrolytic activity at $30^{\circ}C$ and pH 7- 8, and was stable up to $40^{\circ}C$ and in the range of pH 5- 8. It most rapidly hydrolyzed pNPC$_6$ among various PNPesters. The other contigs were expressed more or less as soluble forms, although no lipolytic activities were detected. As they have many conserved regions with lipase 160-26 as well as other bacterial lipases throughout their equence, they are suggested as true lipase genes.

Abstract Glycolysis has a main function to provide ATP and precursor metabolites for biomass production. Although glycolysis is one of the most important pathways in cellular metabolism, the details of its regulation mechanism and regulating chemicals are not well known yet. The regulation of the glycolytic pathway is very robust to allow for large fluxes at almost constant metabolite levels in spite of changing environmental conditions and many reaction effectors like inhibitors, activating compounds, cofactors, and related metal ions. These changing environmental conditions and metabolic reaction effectors were focused on to understand their roles in the metabolic networks. In this study, we have investigated for construction of the regulatory map of the glycolytic metabolic network and tried to collect all the effectors as much as possible which might affect the glycolysis metabolic pathway. Using the results of this study, it is expected that a complex metabolic situation can be more precisely analyzed and simulated by using available programs and appropriate kinetic data.

Abstract Salmonella pathogenicity island 1 (SPI1) gene expression is regulated by many environmental signals such as oxygen, osmolarity, and pH. Here, we examined changes in the expression level of various regulatory proteins encoded within SPI1 in response to three different concentrations of NaCl, using primer extension analysis. Transcription of all the regulatory genes tested was activated most when Salmonella were grown in Luria Broth (LB) containing 0.17 M NaCl. The expression of hilA, invF, and hilD was decreased in the presence of 0.47 M NaCl or in the absence of NaCl, while hilC expression was almost constant regardless of the NaCl concentration when Salmonella were grown to exponential phase under low-oxygen condition. The reduced expression of hilA, invF, and hilD resulted in lower invasion of hilC mutant to the cultured animal cells when the mutant was grown in the presence of 0.47 M NaCl or in the absence of NaCl prior to infection. Among the proteins secreted via the SPI1-type III secretion system (TTSS), the level of sopE2 expression was not influenced by medium osmolarity. Various effects of osmolarity on virulence gene regulation observed in this study is one example of multiple regulatory pathways used by Salmonella to cause infection.

Abstract The cancer chemopreventive potential of chitosanoligosaccharides was investigated by measuring the induction of quinone reductase and glutathione S-transferase activities and inhibition of cytochrome P450 1A1, 2B1, and 2E1 activities. Chitosanoligosaccharide I (1-${\kappa}$Da${\kappa}$Da) significantly induced glutathione S-transferase activity with a maximal 1.5-fold increase at 500 ${\mu}$g/ml, while chitosanoligosaccharide II (3-${\kappa}$Da${\kappa}$Da) (500 ${\mu}$g/ml) strongly induced quinone reductase (p<0.01) and glutathione S-transferase (p<0.005) activities. The in vitro incubation of rat liver microsomes with chitosanoligosaccharides I and II (2.5, 5, 50, and 500 ${\mu}$g/ml) showed a dose-dependent inhibiton of cytochrome P450 1A1, 2B1, and 2E1 activities. Chitosanoligosaccharide II was a more potent inhibitor of cytochrome P450 2B1 activity than chitosanoligosaccharide I. Accordingly, these findings suggest that chitosanoligosaccharides are potential chemopreventive agents.

Abstract Cyclosporins are a family of clinically-important immunosuppressive cyclic peptides produced by Tolypocladium inflatum. The structural modification of cyclosporins via hydroxylation at various positions of N-methyl leucines in cyclosporin A leads to a dramatic change of their bioactive spectra. Among over 100 soil actinomycetes screened, two actinomycetes species, Sebekia benihana and Pseudonocardia autotrophica, were identified to contain superior cyclosporin A hydroxylation activities. A HPLC-based cyclosporin A hydroxylation assay revealed that each strain possesses distinctive hydroxylation specificity and regiospecificity; mono-hydroxylation at the 4th N-methyl leucine of cyclosporin A by S. benihana, and di-hydroxylations at both 4th and 9th N-methyl leucines of cyclosporin A by P. autotrophica. The conversion yields for cyclosporin A hydroxylation by both S. benihana and P. autotrophica were significantly improved from less than 10% and 18% up to 58% and 45%, respectively, in the optimized culture containing molybdenum with 0.05 g/l of cyclosporin A concentration. An ancymidol-specific inhibition of cyclosporin hydroxylation also suggested that the regiospecific cyclosporin hydroxylation might be catalyzed by a putative cytochrome P450 mono-oxygenase enzyme.

Abstract Oligotrophic microbial fuel cells (MFCs) were tested for the continuous monitoring of low biochemical oxygen demand (BOD) by using artificial wastewater, containing glucose and glutamate, as check solution. Ten times diluted trace mineral solution was used to minimize the background current level, which is generated from the oxidation of nitrilotriacetate used as a chelating agent. The feeding rate of 0.53 ml/min could increase the sensitivity from 0.16 to 0.43 ${\mu}$A/(mg BOD/l) at 0.15 ml/min. The dynamic linear range of the calibration curve was between 2.0 and 10.0 mg BOD/l, and the response time to the change of 2 mg BOD/l was about 60 min. The current signal from an oligotroph-type MFCs increased with the increase in salts concentration, and the salt effect could be eliminated by 50 mM phosphate buffer.

A novel chitinase-producing bacterial strain of Pantoea dispersa was isolated from the sea near Bhavnagar, India for efficient disposal of chitinous waste from the seafood processing industry. The medium components were optimized by using a cubic model in the central composite design for increasing chitinase production. The optimal concentrations for higher production of chitinase were (g l-1) chitin, 10.0; urea, 0.35; MgSO4 7H2O, 0.08, and CaCl2, 0.15. Here, peptone (0.05 g l-1) was used as a constant variant in all trials. Using a statistical optimization method, the chitinase production was found to increase from 108 to 486.4 units ml-1. Chitin was prepared from the crustacean waste, and Fourier Transform Infrared (FTIR) Spectroscopy was used to characterize the isolated chitin. Chitinous waste degradation was studied in terms of chitinase production.

Our previous study showed that the overexpression of carboxypeptidase Y (CPY) of Saccharomyces cerevisiae in Escherichia coli resulted in the formation of insoluble inclusion bodies. To produce soluble CPY, we designed a novel Pichia pastoris expression system, in which the following were inserted into expression vectors: three different signal sequences derived from the mating factor a1 of S. cerevisiae, an inulinase of Kluyveromyces marxianus, and the endogenous signal sequence of CPY. The expression vector pHIL-D2-SSinul-proCPY was the most effective in the production of proCPY among the vectors examined. The purified active CPY was obtained from proCPY by treating with proteinase K, followed by QExcellose ion-exchange column chromatography.

Polyhydroxyalkanoates (PHAs) are homo or hetero polyesters of (R)-hydroxyalkanoates accumulated in various microorganisms under growth-limiting condition in the presence of excess carbon source. They have been suggested as biodegradable substitutes for chemically synthesized polymers. Recombinant Escherichia coli is one of the promising host strains for the economical production of PHAs, and has been extensively investigated for the process development. The heterologous PHA biosynthetic pathways have been established through the metabolic engineering and inherent metabolic pathways of E. coli have been redirected to supply PHA precursors. Fermentation strategies for cultivating these recombinant E. coli strains have also been developed for the efficient production of PHAs. Nowadays, short-chain-length (SCL) PHAs are being re-invited due to its improved mechanical properties and possible applications in the biomedical area. In this article, recent advances in the development of metabolically engineered E. coli strains for the enhanced production of SCL-PHAs are reviewed. Also, medical applications of SCL-PHAs are discussed.

Periconicin A and B, two new fusicoccane diterpenes originally isolated from the cultures of endophytic fungus Periconia sp. OBW-15, were tested by several biological assays. Periconicin A was consistently more active than periconicin B. In an antifungal activity assay, periconicin A showed potent inhibitory activity against the agents of human mycoses, including Candida albicans, Trichophyton mentagrophytes, and T. rubrum, with minimum inhibitory concentration (MIC) in the range of 3.12- 6.25 $\mug$ /ml. In a plant growth regulatory activity assay, periconicins inhibited hypocotyl elongation and root growth of Brassica campestris L. and Raphanus sativus L. At concentrations below 1 μg/ml, however, both compounds accelerated root growth by 110- 135%. From these results, it is apparent that a methyl group positioned in a cyclopentane ring may play an important role in plant and fungal growth inhibitory activity.

A Staphylococcus sp. EY-3 with the capability of decolorizing Congo Red was isolated from soil at an effluent treatment plant of a textile and dyeing industry. This strain was able to almost completely decolorize a high concentration of Congo Red in 48 h under aerobic conditions. Optimal color removal (more than 96%) was achieved at 30- 40oC, and no noticeable effects of different pH values (5.5- 8.0) on decolorization were observed. This strain also exhibited a remarkable decolorization capability against azo dyes under aerobic conditions, even at a high concentration (dyes 1 g/l) of dye. The metabolic product of Congo Red degradation by this strain was identified by gas chromatography with mass selective detection (GC/MSD) to be an amine derivative benzidine.