Earticle

Most of the biological mechanisms such as DNA replication, transcription, and gene regulation are based on molecular interactions between DNA and protein (or enzyme). It has been demanded to develop more functional probe molecules, which are useful and proper for analyzing molecule-level study between DNA and protein. In this study, we presented new approaches using DNA oligomer with unnatural nucleobases as novel DNA probes. In particular, nitric oxide (NO)- damaged nucleobases such as oxanine (Oxa) and hypoxanthine (Hyp) were explored in terms of their biotechnological applications. We prepared Oxa- or Hyp-containing DNA oligomers and employed as novel substrates for DNA-recognizing enzymes or DNA-binding proteins. These methods and the obtained results will be useful for elucidation of molecular studies based on DNA-protein interactions.

A nanoparticle is a small particle with at least one dimension less than 100 nm. Recently, nanoparticle research attracts public attention due to its potential to be applied to biomedical, optical, and electronic fields. Many scientist and engineer make efforts to develop new nanomaterials to be effective on illness and disease like cancer beyond current imagination. Polymer-based nanoparticles are designed to improve the property of drugs in terms of pharmacokinetics which determines the strength of drug delivery system. We have recently found that orally administered nano-size polystyrene beads pass through the pharynx and are taken up into the intestine. When we fed animals with 50, 100, 200, and 500 nm polystyrene beads, only 50 and 100 nm particles were mobilized beyond the gut and were found to accumulate in the gonad and epidermis. To explore the requirements for nanoparticle transport, we tested uptake of 50 nm particles in worms defective for genes involved in apical transport or endocytosis in intestinal lumen and found which proteins enable nanoparticles to move from the intestine to other sites in the animals. These findings suggest that the upper limit in this size of nanoparticles that can undergo endocytic transport from the lumen to gut and from gut to other organs may be less than 200 nm.

Most lipases cannot hydrolyze amides although their active sites are similar to those of serine-proteases that catalyze hydrolysis of amides. Recently, we proposed molecular basis for the enhanced lipasecatalyzed N-acylation of 1-phenylethanamine with methoxyacetate. We suggested that the hydrogen atom connected to the nitrogen atom of the amine substrate may interrupt formation of the key hydrogen bond between the catalytic histidine and the nitrogen atom of the substrate. The disruption could be avoided by using methoxyacetate as an acyl donor. The methoxy group of the acyl donor can hydrogen bond with the nitrogen atom and thus the proton does not interrupt the key hydrogen bond. We introduced a residue to serve a similar role of the methoxy group into a lipase. We prepared a mutant of Candida antarctica lipase B (CAL-B) to construct a hydrogen bond between the residue introduced and the nitrogen atom. The mutant enzyme clearly showed improved hydrolysis activity toward p-nitroacetanilide.

Drug resistance of cancer cells has emerged as one of the cardinal issues in cancer therapy for effective customizable treatment for cancer patients. Epidermal growth factor receptor (EGFR) mutations are potential markers and determinants for predicting responses of cancer patients to tyrosine kinase inhibitors (TKIs, e.g.; gefitinib and erlotinib) therapy. Here, we have evaluated the clinical applicability of detecting lung cancer cells using molecular beacon probes targeting mutant EGFR with the real-time PCR based assay and in situ fluorescence image approach. These methods provide a faster and easy method to monitoring for clinically relevant mutations and response to EGFR-specific tyrosine kinase inhibitor treatment in lung cancer

FK506 is a 23-membered polyketide macrolide with immunosuppressant activity produced by Streptomyces species. The production of FK506 in Streptomyces clavuligerus CKD1119 (KCTC 10561BP) was improved by enhancing the supply of biosynthetic precursors. FK506 production by S. clavuligerus CKD1119 was improved approximately 2.5-fold (15 mg/l) through the supplementation of 10 mM methyl oleate, which is the probable source of acyl-CoAs, in R2YE medium. When the level of FK506 production reached its maximum, the intracellular concentration of methylmalonyl- CoA in S. clavuligerus CKD1119 supplemented with methyl oleate was 12.5 times higher than that of the unsupplemented strain, suggesting that an increased methylmalonyl-CoA level caused the highlevel production of FK506. The following three pathways for the production of (2S)-methylmalonyl- CoA were evaluated to identify the effective precursor supply pathway that can support the high production of FK506 in S. clavuligerus CKD1119: propionyl-CoA carboxylase, methylmalonyl-CoA mutase (MCM), and malonyl/methylmalonyl-CoA ligase. Of the three pathways examined, the MCM pathway supported the highest levels of FK506 production. The expression of MCM in S. clavuligerus CKD1119 led to a 3-fold and 1.5-fold increase in the methylmalonyl-CoA pool and FK506 production, respectively. Supplementing the culture broth of S. clavuligerus CKD1119 expressing MCM with methyl oleate resulted in an additional 2-fold increase in the FK506 titer (17.8 mg/l). Overall, these results show that the methylmalonyl-CoA supply is a limiting factor for FK506 biosynthesis, and among the three pathways analyzed, the MCM pathway is the most effective precursor supply pathway supporting the highest titer of FK506 in S. clavuligerus CKD1119.

Aflatoxin B1 (AFB1) is the most toxic component one of mycotoxins present in food and feed. Immunological detection of low molecular weight toxins, such as AFB1 using a single-chain variable fragment (scFv), is a potentially novel and safe method of diagnosing fungal infection and food contamination. Surface plasmon resonance (SPR) studies showed that its cognate scFv (82.7 X 10-7 M) has about 118-fold lower KD than the parental mAb 2c12 (0.7 X 10-8 M). To develop an AFB1 detection protein chip using scFv, a random mutagenic scFv library displayed on the surface of yeast was constructed. Five mutated scFv clones with high binding affinity to AFB1 were sorted from this library by a fluorescence activated cell sorter (FACS). The most sensitive scFv (37M-scFv) contained 6 substitutions in amino acid, compared with the wild-scFv (WT-scFv) and was characterized to retain almost the same specificities to other mycotoxins as its parental antibody 2c12 and have KD (9.2 X 10-8 M) 9-fold lower than WT-scFv. The binding affinity of 37MscFv was more improved by replacing Ala at the position 108 in the heavy chain with Thr (VH:A108T), which is known to be a framework region-4 (FR-4) of murine antibody. Finally, the BM3-scFv, reverse-mutated to Thr at the position VH:108 has the highest binding affinity (2.7 X 10-8 M) which is approximately 31- fold lower than that of WT-scFv. Even though Thr at the position VH:108, located in FR-4 of the heavy chain, connecting the variable region with the constant region also played an important role for sensitivity of anti-AFB1 scFv in this reverse mutation study and should be conserved for binding with AFB1.

Nitro compounds are relatively rare in nature, with only ~150 known examples, but include synthetic drugs, highly energetic compounds and well known antibiotics such as chloramphenicol and pyrrolnitrin. Especially, biosynthesis of aromatic nitro compounds is poorly understood, and key enzymes in catalyzing the formation of aromatic nitro group are not characterized well. Here, we present an arylamine N-oxygenase (AurF) through mechanistic and structural studies, which is involved in the biosynthesis of nitro-containing antibiotics including aureothin, SNF4435C/D and spectinabilin. Biosynthetic gene cluster of spectinabilin as an iterative type I polyketide synthase is described and compared with other gene clusters. This study gives some insights of biosynthesis of aromatic nitro compounds for medical and industrial application.

Green fluorescent protein (GFP) Nobel Prize protein from jellyfish is a versatile reporter protein for monitoring gene expression and protein localization in variety of systems. Applications using GFP reporters have expanded greatly due to the availability of mutants with altered spectral prosperities, including several blue emission variants, all of which contains the single point mutation in tyrosine analogue in green fluorescent protein chromophore. Generally, GFP variants will be generated through replacement of naturally occurring amino acids by site directed mutation or directed evolution methodology and disadvantages of this methodology is limited with 20 naturally occurring side chains. In other hand unnatural amino acid mutagenesis has been used to selectively substitute target amino acids with our interesting side chains in our objective protein. Current studies have explored incorporation of unnatural amino acid analogues into green fluorescent protein and alter the spectral emission and excitation properties. We have selected different tyrosine analogues and successfully incorporated into GFP to alter the specific characters. We have selected the tyrosine analogues due to the important role in the chromophore and structure formation. Current study not only demonstrated the importance of unnatural amino acid mutagenesis and also demonstrated new approach to generate tailormade proteins with interesting and useful spectral properties

This study investigated the production of catalase of Bacillus flexus BKBChE-3. Bacillus flexus BKBChE-3 was screened and identified from soil for the production of catalase. This strain was cultivated in shaking flasks with tryptic soy broth(TSB) complex medium at 30℃ and speed of 200 rpm. Effects of the temperature on the activity of the native catalase and whole cell viability were studied in the temperature range of 25-60℃ and pH range of 2.0-13.0 for stability. Korean natural zeolite was added to shaking flasks and mixed with microorganisms for immobilization during 24 hours. The native catalase has kept its activity over 50℃. And bacillus cell have survived at extreaming conditions of over 50℃ and pH 11. The hydrogen peroxide was removed at concentration of 10.5 mM within 1 hour.

PTA (purified terephthalic acid) wastewater contains various aromatic compounds such as phthalate isomers, p-toluate and benzoate, which can be converted into valuable biogas under methanogenic condition. Although many full-scale PTA wastewater treatment processes are currently being operated in the world, the involved microbial reactions have not yet fully elucidated especially for the p-toluate degradation. In this study, a lab-scale PTA wastewater treatment process consisting of two sequentially-connected UASB (upflow anaerobic sludge blanket) reactors was operated and the microbial community at each stage was investigated by comparing the T-RFLP (terminal restriction fragment length polymorphism) patterns together with the 16S rDNA gene clone libraries complementarily. In the first stage reactor, where most contaminants except p-toluate were completely degraded, both Syntrophus and Syntrophobacter species known to have a higher substrate affinity for benzoate were identified to be most predominant. In the second stage reactor, on the other hand, a species belonging to the family of Bacteroidales was most predominant with Syntrophus and Syntrophobacter species diminished. In the second stage reactor mainly treating p-toluate, it was further revealed that an uncultured species belonging to α-proteobacteria was newly present and the relative amounts of Syntrophorhabdaceae and Syntrophomonas species were also remarkably increased compared to the first stage reactor. Considering the substrate availability at each stage, this result strongly suggests that the last three microbial groups are closely related to the p-toluate degradation.

Microalgae are one of potential carbon resources from ocean. The microalgae have been used for live feeds, cosmetics, biomedicine and the production of biodiesel. In this study, the determination of lipid contents was carried out from 3 marine microalgae. Phaeodactylum tricornutum, Nannochloropsis oculata, Isocrysis galbana were selected due to high oil contents in cell body comparing to the other strains. Inoculation densities of the microalgae were optimized for the scale-up process. Nitrate as limiting nutrient source was monitored by UVspectrophotometer for the accumulation of lipid. Lipid contents of the strains harvested 3, 6 and 9 days after stationary phase were determined using Soxhlet extractor. Nitrate source was exhausted before stationary phase. The specific growth rates of P.tricornutum and N.oculata were 0.25 cells/ml/day, 0.27 cells/ml/day and 0.20 cells/ml/day, respectively. N.oculata could maintain high specific growth rate and cell density (1.5~10folds) in stationary phase. Further studies are focused on the increase of lipid contents in the microalgae.

Chrysin (5,7-dihydroxyflavone) is a naturally occurring flavone chemically extracted from the blue passion flower. Dietary flavonoids have many biological properties that could make them useful as chemopreventive agents. However, very poor oral bioavailability makes them largely ineffective in vivo. In previous studies, hydroxylated flavonoid compounds are certificated more effctive absorption in vivo than flavonoid compounds. We selected chrysin as substrate and more than 10 species of microorganism including Streptomyces Ceolicolor, Streptomyces Avermitilis, Streptomyces Griceus, Nocardia farcinica . Streptomyces Ceolicolor was cultivated for 6 days at 28℃ and under 220rpm with shaking incubator using R2YE media. 50ml of culture was harvested every 24 hours, and used for biotransformation of chrysin. Reaction was performed using 50ml of PBS (Phosphate buffered saline) buffer with 0.2mM of chrysin as substrate at 28℃ and under 220rpm with shaking incubator for 6 hours. Reaction product was sampled every 30 minute or 1 hour, extracted with ethyl acetate and derivatized with BSTFA (N,O-Bis(trimethylsilyl)trifluoro-acetamide). Derivatized reaction product was analyzed with LC, GC-Mass. Chrysin was ortho-hydroxylated 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 ompletely 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.

Hypomycetous fungus Cladosporium phlei (C.T.Gregory) de Vries, known as a causal agent of leaf spot disease of timothy(Phleumpratense), is characterized by the production of deep red pigmented mycelia due to the presence of a fungal pigment phleichrome. Pheichrome, the secondary metabolite from the fungus Cladosporium phlei, has been known as a key precursor of photosensitizer which produce a reactive oxygen species due to the activation of photoenergy, photosensitizer is a key component of photodynamic theraphy(PDT). In our previous studies, the culture condition of C. phlei was previously achieved at 20℃ of temperature and using V8 juice-based media. Simple solvent extracting procedures using fungal mycelia as well as culture filtrate were developed to isolate the pigment from C. phlei and the resulting deep red pigment was identified to be phleichrome by NMR analysis. In the study, the fermentational features for the mass production of phleichrome have been determined and the genes related to the biosynthesis of phleichrome is defined. The improved strain to produce phleichrome using UV mutagenesis and molecular technique as a genetic manipulation is found. Mycellial mass and culture filtrates were extracted with ethyl acetate(EtOAc). The residues were analyzed via thin-layer chromatography(TLC) using a resolving solution(CH2Cl2-MeOH=95:5, v/v). TLC screening method was progressed in the rapid and easy ways of determining the phleichrome content using analytical method. Phleichrome was produced from mycellial which were harvested at 12 to 15 days. About 50 isolated strains using UV-mutagenesis have analyzed by a rapid TLC screening method to obtain the improved strain. Phelichrome is among the perylenequinone of secondary metabolites. Therefore, polyketide synthase(PKS) gene have isolated by a comparative genomic approach on the genes related to the genes involved in biopsynthesis of fungal polyketide.

Ecklonia cava is commonly used as foodstuff and pharmaceutical and nutraceutical materials. The residue of E. cava, which is a by-product of the seaweed products, has been dispose as a fertilizer or an industrial waste. However, we expect that the seaweed residue still contains many of bioactive compounds in the tissue. In order to extend the usage of E. cava residue, microbial fermentation was performed. The microbial fermentation of the residue may result in the breakdown of bioactive compounds or the production of bioactive compounds. As expected, the methanolic extract of E. cava residue fermented by Candida utillis showed strong antibacterial activity against several pathogenic bacteria. The antibacterial activity was higher than that of the extract of raw E. cava residue. Our finding may contribute to the development of a natural antibiotic agent using the seaweed waste.

High ammonia concentration involved in swine wastewater, which may cause lower performance efficiency during process due to the inhibition of microbial activity related with anaerobic process. This study investigated experimentally the significant aspects on both acidogenic and methanogenic population dynamics in anaerobic batch process treating swine wastewater. For observation of microorganisms adapted in high ammonia concentration, we conducted anaerobic batch processes with duplication for 3 months. During the reactions, the chemical oxygen demand concentration of two reactors decreased by about 68% and 75% of the input swine wastewater and 67 L and 64 L of methane was produced, respectively. During the reactions, there were maintained with high ammonia concentration from 3.4 g/L to 4.1 g/L and total kjeldahl nitrogen from 5.8 g/L to 5.3 g/L. The bacteria denaturing gradient gel electrophoresis (DGGE) profiles showed that Lactobacillus. delbrueckii were identified as major group. Therefore, L.delbrueckii was supposed that it would play an important role in acidogenesis. Also, from methanogenic DGGE profiles, we can infer that the methane gas observed in the overall process was produced by the activity of Methanoculleus marisnigri JR1 and Methanocorpusculum labreanum Z, belonging to the order Methanomicrobiales.

Saccharification of Laminaria japonica (sea tangle) is an essential process for the use as a substrate for bioethanol fermentation. The seaweeds obtained from local market in Busan (South Korea) were dried in sunlight or hot-air and then ground by hammer mill to the powder with 150~750 (wet) sizes of particles. The various methods such as acid hydrolysis, industrial enzymes and isolated strains from sea were used for the saccharification of Laminaria japonica. The saccharification process by the H2SO4-thermal hydrolysis and the isolated marine bacteria treatment showed the highest yield of saccharification from Laminaria japonica. The optimal saccharification condition with the highest reducing sugar and lowest viscosity was determined as follows: 10% (w/v) seaweed slurry, 0.2% (v/v) H2SO4, heating at 121℃ for 60 min, neutralization by 5N NaOH, and then incubation at 30℃, 200 rpm for 5 days after 10% (v/v) inoculation of the isolated marine bacteria to the seaweed slurry. The reducing sugar and viscosity under the optimal condition were 40.5 g / liter and 24.9 cp, respectively. The saccharification yield of Laminaria japonica was 82.1% with the optimal saccharification condition.

Carbohydrate-protein interactions are involved in various biological events, including viral and bacterial infection, immune response, differentiation and development, the progression of tumor cell metastasis. Carbohydrate microarrays are new technology that has potentiated the high-throughput analysis of carbohydrate-protein interaction and contributes to significant advances of glycomics. Here, we proposed a facile, efficient, and cost-effective method whereby diverse carbohydrate types are modified in a single step and directly immobilized onto a glass surface, with retention of functional orientation. We modified various types of carbohydrates by reductive amination, in which reducing sugar groups were coupled with the linker. The modified carbohydrates were covalently attached to an aminereactive NHS-activated glass surface by formation of stable amide bonds. This method was applied for construction of functional carbohydrate microarray platform to analyze carbohydrate-protein interactions. This array by using our method can be powerful tool for screening the specificity of GBPs (glycan-binding proteins) such as lectins, growth factors, antibody, and microbial toxins, and identification of carbohydrateprotein interactions

Mussels inhabit seashore by attachment themselves using their foot proteins (fps) which has underwater adherent properties. L-3,4-dihydroxyphenyl alanine (Dopa) is regarded as key factor for strong and submerged adhesion. However, other essential adhesion processes including condensation of fps with as high as 30% (w/v) in vacuoles and secretion as a watery liquid with no dispersion into water are poorly understood yet. In this circumstance, we suggest potential complex coacervation process of fps due to their notable properties, such as low interfacial tension, liquid/liquid phase separation (via concentration) and viscoelasticity. Previously, we successfully produced hybrid fp proteins, fp-151 which is composed of six fp-1 decapeptide repeats at both termini of fp-5 and fp-131, containing six fp-1 decapeptide repeats at both termini of fp-3 variant A in Escherichia coli to overcome low production and purification yields from single fps. We observed successful formation of complex coacervation between fp-151 or fp-131 and hyaluronic acid (HA), an anionic partner for cationic hybrid fps. Formation of coacervation was evidenced through analyses with electrophoretic mobility, turbidity, and morphology. Interestingly, it was revealed that coacervation process improves adhesion ability of hybrid fps in bulk adhesion test and the fps coacervates can encapsulates the oil droplet to form microencapsulated particle.

The bacterial ghost system is a novel vaccine delivery system. It provides versatile carrier functions for foreign antigens with excellent natural intrinsic adjuvant properties. The optimized medium condition for the ghost bacteria, vaccines E. coli K-12/pHCE-InaN-GAPDHGhost27SDM were determined as 10 g/L glucose, 5 g/L yeast extract and 50 mg/L thiamine with Riesenberg's semi-defined medium according to one-at-a-time method. The optimal batch fermentation parameters were determined as 10% inoculum size, 300 rpm agitation and 2.0 vvm aeration for the highest production of 3.7 g dcw/L. The nutrient feeding strategy with Riesenberg's defined medium for ghost bacteria vaccine fed-batch process was evaluated. Fed-batch fermentation for the optimized ghost bacteria vaccine production was carried out in 4 phases; batch, fed-batch, induction and heat-shock phases. The heat-shock phase was high temperature holding phase at 47℃ for 2 h to increase ghost cell efficiency. Under the optimal condition of fed-batch fermentation, the cell density of ghost bacteria reached 35 g dcw/L with 99.9% of ghost formation efficiency.

For oligonucleotide microarray, target gene is generally amplified by polymerase chain reaction (PCR) and purified. In the present work, we suggest fast and non-labeling direct 16S rRNA detection of pathogenic bacteria without amplification and purification of target gene. Because 16S rRNA has many copies in all living bacteria, we successfully used 16S rRNA as a target for microarray. For simple and fast direct detection of pathogens, we used our previously constructed 16S rDNA-based oligonucleotide microarray detection system. The bacterial total RNAs from cell lysis were hybridized to a specific 16S rRNA probes and detected using fluorescent-labeled detector probe without labeling reactions of target RNAs. We found that hybridization specificity and sensitivity were enhanced using the fragmented RNAs. We also investigated the limit of detection (LOD) using Vibrio vulnificus.

Tautomycetin (TMC), which is produced by Streptomyces sp. CK4412, is a novel activated T cell-specific immunosuppressive compound with an ester bond linkage between a terminal cyclic anhydride moiety and a linear polyketide chain bearing an unusual terminal alkene. Previously, we isolated and characterized the entire TMC biosynthetic gene cluster from Streptomyces sp. CK44121). And also, TMC pathway-specific gene, tmcN was successfully utilized to increase TMC productivity by introducing its extra copies, implying the importance of the regulatory network optimization for strain improvement2). It was previously reported that a 64- amino-acid encoding wblA inhibited the biosynthesis of doxorubicin in S. peucetius as well as the antibiotics production in S. coelicolor, suggesting that wblA and its homologs act globally among streptomycetes as putative down-regulators of antibiotic biosynthesis3). Here, we identified a wblA ortholog gene (named wblA-tmc) via genomic DNA library screening using the degenerated primers based on wblA sequence from S. coelicolor A3(2). A 0.39kb wblA-tmc showed 96% amino acid identity compared to a previously-known S. coelicolor wblA. A targeted gene disruption of wblAtmc from the TMC-producing strain, which was confirmed by PCR and Southern-hybridization, exhibited significantly different morphological pattern as well as TMC productivity.. The more detailed biological significance of wblA-tmc will be discussed.

In recent years the inflorescences of Humulus lupulus have received attention for their biological effects such as phytoestrogens and cancer prevention. Supercritical fluid extraction of essential oil is one of conventional separation techniques. SFE is useful of separating specific compounds from natural resources effectively by its unique properties and process design. In this study, extractions were performed to recover essential oil from Humulus lupulus at temperature of 30, 40 and 50℃ and pressure of 100bar and 250bar for 2hrs and used semi-batch type of 200mL stainless steel vessel. The essential oils obtained each conditions were identified by GC/MS and the data were compared with that of the organic solvent extraction. The main chemical compounds identified in the extracts showed terpenes. The best extraction condition was 40℃ and 250bar. The composition in the oil recovered from the separator with and without a cold-trap was different. The contents of main terpenes in the oil recovered the separator with a cold-trap was myrcene 29.6%, humulene 29.1% and beta-caryophyllene 7.4% which show higher content than the oil recovered from the separator without a cold-trap process.

In previous studies, recombinant 30K protein from Bombyx mori (silkworm) had shown many favorable effects on animal cells such as anti-apoptotic effect, cell growth and glycosylation pattern of recombinant proteins produced by cells. In this study, it was assumed that the beneficial effects of recombinant 30K protein on animal cells could come from the enhancement of cell’s mitochondrial activity. In the MTT assay, normalized with cell counting, HeLa cells treated with recombinant 30Kc19 protein showed dramatically the enhancement in the MTT reduction. To see whether this activity increase actually came from the enhancement of mitochondrial activity, in vitro mitochondrial complex I / III activity assay was performed. The analysis showed a noticeable increase in the mitochondrial activity when r30Kc19 was added to isolated mitochondria. These results highly imply that mitochondrial activity enhancement is closely related with beneficial effects of 30K protein on animal cells.

Butyric acid is an important raw material in various industries including chemistry, food, cosmetics and pharmaceutics. It has also become an increasingly attractive in the field of renewable energy in case of production from bio-based natural ingredients due to the depletion of non-renewable resources. Brown algae is a good substrate in Korea that can be grown and obtained easily from the seas surrounding Korea. In this study, butyrate was produced from a model compound, glucose, in clostrial growth medium (CGM) using Clostridium tyrobutyricum ATCC 25755 in fed-batch fermentation at pH 6.0 and 37℃. In this process the total butyrate obtained was higher than 50 g/L. Then, brown algae were used as substrates for butyrate fermentation. So far 7 g/L of butyrate was produced from Laminaria japonica, one of the well-known brown algae, further experiment is going on to explore the optimal condition for butyrate fermentation. This result showed the possibility of the usage of brown algae as substrate to produce butyric acid.

Saccharomyces cerevisiae is effective for industrial scale production of bioethanol, but it cannot utilize xylose present in high amounts in lignocellulosic biomass. The effects of two different sugars on the expression levels and patterns of xylose reductase (xyl1), xylitol dehydrogenase (xyl2) and xylulokinase (xyl3) genes were analyzed using Pichia stipitis. A significant increase in mRNA levels of xyl1 was observed in culture conditions using xylose as a sole carbon source, but expressions of the three genes were not influenced by culture media with glucose. It also was found that the expression level of xyl1 increased as a function of the xylose concentration used in this study. Therefore, we constructed new recombinant S. cerevisiae harboring xyl1, xyl2 and xyl3 gene isolated from P. stipitis. Proteomic and fermentaion analyses revealed that three foreign genes were successfully expressed in S. cerevisiae grown on xylose or on glucose/xylose mixtures and ethanol production also was observed in S. cerevisiae grown on xylose. In addition, the expression level of xyl1, xyl2 and xyl3 proteins in cells grown on a xylose and glucose/xylose mixture was increased. These results suggest that regulation of xylose utilization in recombinant S. cerevisiae is successfully performed as seen in S. cerevisiae, which originally cannot utilize xylose.

Bone morphogenetic protein-7 (BMP-7) is a member of the BMP subfamily of the transforming growth factor ß (TGF-ß) superfamily. It induces bone formation and thus renders it to a protein of pharmaceutical importance. Determination of rhBMP-7 based on ELISA involves many laborious and time - consuming procedures. In this work, immuno-optical sensor has been developed to measure the concentration of rhBMP-7 by sequential injection analysis and to investigate the repeatability and stability with different condition. An optical immuno-sensor was prepared with an optical fiber immobilized with quantum dot (QD)-conjugated antibody. The antibody for the rhBMP-7 was first conjugated with CdSe/ZnS QDs via EDC/NHS and then immobilized on the tip of an optical fiber with a diameter of 2 mm and a quartz glass plate. The fluorescence intensity changed with the quantity of samples, which were attached to the QD-conjugated antibody.

Here, we studied the potential for molecular hydrogen production of [NiFe]-hydrogenase 1. The Escherichia coli hyaAB genes were homologously overexpressed in E. coli BL21, which is not capable of producing hydrogen gas. Recombinant [NiFe]- hydrogenase 1-possesing BL21 showed active hydrogen production, in contrast to the wild type strain that did not produce. Under microaerobic condition where oxygen labile [FeFe]-hydrogenase is known to be totally inactivated, the recombinant strain could make about 12.5 mL H2/(h·L) gas in 400 mL M9 minimal medium supplemented with glucose. It is inferred by the effects of formate on hydrogen production that the homologously expressed [NiFe]-hydrogenase 1 is not directly related to the formate hydrogenlyase complex pathway and is the major factor contributing the in vivo hydrogen production in recombinant E. coli BL21. Only small fraction of the protein was in active condition, majority of them being translocated to the membrane. Metal contents effects were also investigated and even when either Fe ions or Ni ions exist exclusively, the enzyme displayed the activity to some extent. These experimental results suggest that each metal on catalytic active site may be substituted by the other in the recombinant enzyme. The purified E. coli [NiFe]-hydrogenase 1 possessed relatively high oxygen tolerance showing significant activity under an ambient aeration environment. Unlike [FeFe]-hydrogenase, we expect this relatively O2 tolerant [NiFe]- hydrogenase can be successfully applied in biohydrogen production under microaerobic conditions.

Polylactic acid (PLA) is a promising biomass-derived polymer, but is currently synthesized by a two-step process: fermentative production of lactic acid followed by chemical polymerization. Here we report production of PLA homopolymer and its copolymer, poly(3-hydroxybutyrate-co-lactate), P(3HB-co-LA), by direct fermentation of metabolically engineered Escherichia coli. Introduction of the heterologous metabolic pathways involving engineered propionate CoA-transferase and polyhydroxyalkanoate (PHA) synthase for the efficient generation of lactyl-CoA and incorporation of lactyl-CoA into the polymer, respectively, allowed synthesis of PLA and P(3HB-co-LA) in E. coli, but at relatively low efficiency1. In this study, the metabolic pathways of E. coli were further engineered based on in silico genome-scale metabolic flux analysis in addition to rational approach2. Using this engineered strain, PLA homopolymer and P(3HB-co-LA) copolymers containing up to 70 mol% lactate could be produced up to 11 wt% and 46 wt% from glucose, respectively. Thus, the combined approaches of systems-level metabolic engineering and enzyme engineering allowed efficient bio-based one-step production of PLA and its opolymers. This work was supported by LG Chem and by the Korean Systems Biology Research Project (20090065571) of the Ministry of Education, Science and Technology. Further supports by the LG Chem Chair Professorship, Microsoft, and IBM SUR, WCU (World Class University) program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (R32-2008-000-10142-0) program are appreciated.

Cancer is one of serious human diseases that causes angiogenesis, metastasis, and abnormal cell divisions including genetic change. Thus, lots of researchers have been investigated to inhibit cancer. Human 90K (h90K) protein has very crucial role for inhibition of cancer metastasis. h90K is a glycoprotein having potential 7 Nglycosylation sites (4 sites are clear but remaining 3 sites are not yet). In this work, we performed expression of recombinant h90K glycoprotein in insect Drosophila S2 cell system. We constructed stably-transfected S2-h90K cells by antibiotic selection and confirmed h90K expression using Western Blot and in vitro bioactivity analyses. We also identified N-glycan patterns of recombinant h90K glycoprotein using HPLC and MALDI-TOF MS. Based on the identified N-glycan structures, we next engineered N-glycosylation pathway to develop advanced N-glycan patterns using multiple expression platform strategy with recombinant baculoviruses. We found that infection with recombinant baculovirus having galactosyltransferase resulted advanced bi-antennary galactosyl form of N-glycans in h90K glycoprotein.

It is usually known that abnormal accuired hyperpigmentation, melasma is due to excessive melanin production. Till now, finding effective tyrosinase inhibitors, either from synthetic or natural sources, have been tried to reduce melanin-related hypermelanosis, because tyrosinase is a key enzyme for melanin biosynthesis in the cells. However, it sometimes reported that severe side effects after treating tyrosinase inhibitors, such as allergic sensitization or exogenous ochronosis were observed. In 2006, Massey et al. reported that disrupted lysosomal function over aging may further contribute to pigment accumulation. Therefore, in this study, we isolated lysosome-related organelles from Saccharomyces cerevisiae, HeLa cell lines and Hen’s egg white, and then used organelles extract for the reduction of melanin causing hypermelanosis. We found that as a period of 20 days, 90% of melanin was reduced by treatment of organelles extract, as measured by the reduction of melanin pigmentary intensity. Our results indicate that lysosome-related organelles extract may be useful agents for cosmeceutical skin lightening and treatment of hyperpigmentation disorders. [This study was supported by the 21C Frontier Microbial Genomics and Applications Center Program, grant No. 11-2008-10-002-00, Ministry of Education, Science & Technology, Republic of Korea. The authors are grateful for their support.]