Earticle

Comparative proteome analysis has been carried out to elucidate the physiological and metabolic changes responsible for succinic acid overproduction and cell growth. The proteome of the metabolically engineered succinic acidoverproducing bacterium, Mannheimia succiniciproducens LPK7, were compared with that of the wild type strain (MBEL55E). ATP synthesis, pyruvate metabolism, glycolysis, and amino acid biosynthesis of the LPK7 strain were much changed in expression level. Based on the proteome changes, we could reduce the formation of pyruvic acid, a newly formed byproduct in the LPK7 strain, by adding pantothenate and L-cysteine, serving as precursors of CoA biosynthesis, to the medium. These results clearly suggest that further comparative proteome profiling under various genetic and/or environmental perturbations will reveal more new insights into the physiology and metabolic characteristics and, therefore, will be used to develop metabolic engineering strategies. [This work was supported by the Genome-Based Integrated Bioprocess Development Project of the Ministry of Education, Science and Technology (MEST). Further supports by the LG Chem Chair Professorship, Microsoft, IBM SUR, WCU (World Class University) program through the National Research Foundation of Korea funded by MEST (R32-2008-000-10142-0) are ppreciated.]

Clopidogrel is a platelet aggregation inhibitor widely administered to atherosclerotic patients with the risk of a heart attack or stroke that are caused by the formation of a clot in the blood. Plavix (clopidogrel bisulfate) is marketed worldwide in nearly 110 countries, with sales of $6.4 billion per year. It had been the second top selling drug in the world for a few years and was still growing by over 20%. Methyl (R)-2-chloromandelate is key intermediate of clopidogrel and should be enantiopure. Considering the enantioselective nature of enzyme, biological transformation of ketones into optically active alcohols could be an efficient strategy. In the present study, we investigate the factors that influence the reduction of methyl-2-chlorobenzoylformate to find optimum conditions enabling the efficient production of methyl (R)-2-chloromandelate.[This work was supported by the Small & Medium Business Administration]

β-agarase hydrolyzes β-1,4 linkages of agarose, yielding neoagarooligosaccharides. The neoagaro-oligosaccharides inhibit the growth of bacteria and slow down the degradation of starch and they can reduce the caloric value as additives. Therefore, economically feasible production process of β-agarase enzyme is required. The β-agarase gene (agaB, 1 kb ORF) from Zobellia galactanivorans was subcloned into Pichia expression vector, pPIC9. The constructed plasmid pPICAgaB (9 kb) was integrated into HIS4 and AOX1 locus of P. pastoris genome, respectively, resulting in Mut+(AOX1) and Muts (aox1Δ, methanol utilization slow) strain. The transformed cells showed red halos around its colonies in methanol agar plate by adding iodine solution, indicating the secretory expression of agaB in P. pastoris. Also, the protein and activity of secreted β-agarase were confirmed by using SDS-PAGE and zymographic analysis. When transformed cells (Mut+ and Muts) were cultured on medium containing each 0.1%, 0.5% and 1% methanol, the extracellular activity of β-agarase were increased from about 1.3 unit/ml to 1.6 unit/ml according to elevate methanol concentration. However, the expression level of β-agarase in Mut+ strain was indistinguishable from that of Muts strain.

Due to limiting fuel reservoirs, high oil prices and politico-strategic problems it is urging to produce biofuels as transportation energy. Fossil fuel is a complex mixture of hydrocarbons including linear, branched, and cyclic alkanes, and aromatics. Terpenoid based biofuel is promising alternative to replace the present hydrocarbon fuels. The terpenoid biosynthesis pathways could produce branched-chain and cyclic alkanes, alkenes, and alcohols with desired chain lengths. But their mass production is hampered due to their toxicity to host strain, E. coli. So it is very pressing to develop tolerant host strain to noxious terpenoid biofuels.In our study we have over expressed different stress responsive genes (marA, cls, imp ,and cti) which are suppose to provide tolerance against hostile solvents. Plate overlay, broth overlay, and viable cell count and gas chromatography analysis were conducted to observe any improved tolerance by the tested genes. In present work hexane is used as dilution solvent of terpenoids. Overexpression of marA increased tolerance of E. coli to geraniol, farnesol, and myrcene but showed no effect on isopentenol. Mutant strain with marA deletion was much more sensitive to the terpenoids, compared to its wild type strain. This work was supported by the 21C Frontier Microbial Genomics and Applications Center Program, EB-NCRC (Grant No. R15-2003-012-02001-0), and BK21 program of Korea.

Xylose reductase (XR) is a key enzyme in D-xylose metabolism, catalyzing the reduction of D-xylose to xylitol. However, the broad substrate spectrum of XR can also reduce L-arabinose to L-arabitol, byproduct, which requires expensive downstream process in industrial xylitol production. Some filamentous fungi have L-arabitol dehydrogenase(LAD) oxidizing L-arabitol to L-xylulose and Lxylulose reductase(LXR) reducing L-xylulose to xylitol. Genes incoding LAD and LXR were isolated from the genomic DNA of Neurospora crassa ATCC10333. Each gene was expressed in Escherichia coli BL21(DE3) using T7 promoter. Expression of LAD and LXR was confirmed by determining enzyme activity in cells grown on the LB media. The activity of LAD and LXR was 600 mU/mg of proteins and 4430 mU/mg of proteins, respectively. In vitro assay was performed for conversion of arabitol to xylitol using crude enzymes(LAD and LXR) and cofactors(NAD and NADPH). Substrates and products were analyzed by HPLC, and consequently, 49% of arabitol was converted to xylitol for two hours of enzyme reaction.

Victivallis vadense ATCC BAA-548 is an anaerobic, cellobiose-degrading bacterium isolated from a human fecal sample by combining enrichments in liquid and soft-agar basal media. From the whole genomic sequence of V. vadense, one candidate for agarase gene was identified. The gene product was composed of 1427-amino acids and its molecular weight was calculated as 159,733 Da from the deduced amino acid sequence. Because it has been annotated as a putative alpha-agarase, expression and characterization of the gene product was performed. First, anaerobic cultivation of V. vadense was performed and chromosomal DNA was isolated from the cultivated cells. And then, the gene encoding the putative alpha-agarase was cloned based on PCR. The cloned gene was expressed in Streptomyces lividans host, but no agarase activity was detected even though strong ermE promoter was used. Thus, we investigated production of agarase by using the overexpression vector pET28a and the OmpA signal peptide in E.coli. Therefore, comparison of expression systems and characterization of the expressed protein with those of original signal sequence will be presented. [This work was supported by the 21C frontier Microbial Genomics and Application Center Program]

The pentose phosphate pathway (PPP) and glycolysis comprise the most central metabolic pathways in the primary metabolism of Streptomyces. Most of the antibiotic biosynthetic pathways include reductive steps where NADPH is required as a reducing factor. NADPH is produced in PPP, by glucose-6-phosphate dehydrogenase (Zwf1 and Zwf2) and 6- phosphogluconate dehydrogenase (Zwf3). According to the report, the two genes (zwf1 and zwf2) were translationally coupled with the downstream opc1 and opc2 genes, respectively. The opc1 and opc2 genes were reported to be essential for Zwf activity in Cyanobacteria and corynebacterium. On the basis of the rationale, we postulated that the increase of NADPH concentration in the microbial cells would result in overproduction of secondary metabolites. Based on the genomic sequence of S. coelicolor, firstly, four different combinations of zwf and opc genes, were introduced into the pUWL201PW vector, and the resulting vectors were transformed into Streptomyces lividans TK24. Secondly, the carbon metabolism of S. lividans transformanrs was investigated. Especially, biomass, antibiotics, NADPH, and sedoheptulose- 7-phosphate production in the engineered cells were nalyzed. Taken as a whole, our results indicate engineering of PPP by the glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase genes is a useful way to elevate intracellular concentration of NADPH and sedoheptulose-7-phosphate as well as biomass, which may be applied to improve productivity of secondary metabolites biosynthesized from the intermediates of PPP. [supported by grant no. 2009-0073015 from the Basic Research Program of the National Research Foundation]

1-Deoxynojirimycin (DNJ) is known as a strong α-glucosidase inhibitor (1). In order to reveal the biosynthetic pathway (2), we compared biochemical properties between Bacillus subtilis MORI-91 producing DNJ and its mutant, B. subtilis MORI 3K-85 highly producing DNJ. After cultured, MORI 3K-85 strain has 2.5 fold higher α-glucosidase inhibitory activity in comparison with MORI-91 strain. The relative preferences of two strains for carbon and nitrogen sources were investigated. The results showed that both strains prefer galactose and polypeptone to the other carbon and nitrogen sources for producing DNJ. Total protein profiles in two strains were compared by SDSPAGE, Native-PAGE, and two-dimensional gel electrophoresis (2- DE). These results showed that 7 protein bands on SDS-PAGE and 5 protein bands on NATIVE-PAGE were different between two strains. In addition, six protein spots were found to be different on 2-DE and identified by MALDI-TOF/MS. As a result, three out of these spots were identified to thiol peroxidase, molybdenum cofactor biosynthesis protein A, and malate dehydrogenase. The other spots were identified to the nonfunctional proteins.

Squalene is a triterpenoic hydrocarbon (C30H50). Dietary squalene is able to stimulate nonspecific immune functions and reduce the levels of lowdensity lipoprotein as well as triglyceride. The shark liver oil, the current major natural resource, is limiting and its future supply is uncertain. In this work, Saccharomyces cerevisiae, some strains of which are known to accumulate a large amount of ergosterol, was metabolically engineered to overproduce squalene. In eukaryotes, isoprenoids are synthesized from acetyl CoA through mevalonate. The catalytic domain of the gene encoding hydroxymethylglutaryl CoA reductase (HMGR), a key regulatory step in mevalonate pathway, was over-expressed under the GAL10 promoter in S. cerevisiae 2805 strain. Over-expression of HMGR resulted in the intracellular accumulation of squalene (0.49 g/L) in a shake flask experiment. Other genes involved in isoprenoid synthesis such as ispA (a bifunctional synthase for geranyl diphosphate and farnesyl diphosphate), GGPP (geranylgeranyl diphosphate) synthase or MVAE (acetyl-CoA acetyltransferase) were also overexpressed in S. cerevisiae 2805 strain. Over-expression of these genes alone did not show any improvements in squalene accumulation. Coexpression of ispA with HMGR, however, dramatically increased the level of squalene (up to 1.8 g/L). The level of squalene accumulation varied among different strains of S. cerevisiae.

Xylose reductase (XR) is a key enzyme in xylitol production, catalyzing the reduction of D-xylose to xylitol. However, most XRs from yeasts and fungi have higher activity for L-arabinose than D-xylose and this broad substrate acceptance causes formation of byproduct, arabitol. Neurospora crassa XR(NcXR) is known to have high overall catalytic efficiency and higher activity toward D-xylose than L-arabinose significantly. C. tropicalis and N. crassa have different patterns in codon usage and three codons, CCC(P), CGC(R) and CTC(L) are particularly expected to be problems in functional expression of NcXR. Consequently, all codons of NcXR were changed into preferred codons in C. tropicalis. A promoter region of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was isolated from the genomic DNA of C. tropicalis ATCC 20913 to construct a constitutive expression cassette for this strain. The codon-optimized NcXR(NXRG) gene was placed under the control of GAPDH promoter and integrated into the genome of C. tropicalis N43 which is xyl2-disrupted and xyl1-partially disrupted mutant. Expression of NXRG was confirmed by determining enzyme activity in cells grown on a medium containing glucose as a carbon source. The resulting recombinant yeast, C. tropicalis NG4, showed higher XR activity (1287mU/mg of proteins) than that of the parental strain (26mU/mg of proteins) and 3.6 times higher substrate specificity for D-xylose than L-arabinose.

Streptomyces clavuligerus NRRL3585 produces a clinically important ß-lactamase inhibitor, clavulanic acid and 5S clavam. In this study, the orf21 encodes putative sigma factor was disrupted from the clavulanic acid and 5S clavam producer Streptomyces clavuligerus NRRL3585 and characterized. The fully sequenced open reading frame encodes 201 amino acids, with a predicted molecular mass of 22.75 kDa. A orf21 mutant of Streptomyces clavuligerus was obtained by insertional inactivation via double crossover, and the effect of orf21 disruption on clavulanic acid yield was investigated by comparing antibiotic levels in culture broths of this mutant and in the wild type. Although growth and morphological analyses showed no differences between Streptomyces clavuligerus/Δorf21::neor and wild-type, the production of clavulanic acid in Streptomyces clavuligerus/Δorf21::neor was found to be slightly decreased. But the co-expression of orf21 in wildtype cells resulted in an enhanced 1.43-fold production of clavulanic acid higher than normal levels. This result demonstrates that the replication of orf21 enhances CA production.

Calcium phosphate has been used for over 30 years to deliver genetic material to mammalian cells. The traditional method using calcium phosphate strongly depends on the experimental parameters such as concentrations (DNA, salt), temperature, pH, reaction time for precipitation and also on person-to-person variation. In this study, to evaluate the effects of particle size on transfection efficiency, factors that affect transfection efficiency were carefully evaluated. Particularly, when plasmid DNA with filtered nano-particle was transfected to HEK293 cells, it was shown that the Ca-phosphate/DNA complexes were effectively transfected to the cells. We observed transfection efficiency by filtered Ca-phosphate particle with DNA varied according to the pore size filters (0.2 μm, 0.45 μm, 0.5 μm and 1 μm).When Caphosphate particles were filtered after preparation, particles could be stored for longer than one month without significant loss in transfection efficiency. This long-term stability is one of the important features that should be satisfied to be used as gene delivery tools for gene therapy applications

An aldehyde dehydrogenase (AldH), from Klebsiella pneumoniae was cloned, expressed, and characterized for its properties in E. coli BL21(DE3). The enzyme effectively converts 3-hydroxypropionaldehyde (3-HPA), an intermediary compound of glycerol metabolism in bacteria, to 3-hydroxypropionic acid (3-HP), a commercially valuable platform chemical. It also exhibited broad substrate specificity for various aliphatic and aromatic aldehydes. The enzyme showed preference towards NAD+ over NADP+ as a cofactor for the oxidation of most aliphatic aldehydes. When several aldehydes tested, the specific activity of 27.7 U mg-1 protein was recorded for 3-HPA at pH 8.0 and 45°C. The halfsaturation constant (Km) and the specificity constant (kcat) for 3-HPA in the presence of NAD+ were 0.48 mM and 41.44ⅹ103 M-1 S-1, respectively. The AldH activity was enhanced in the presence of disulfide reductants such as dithiothreitol, 2-mercaptoethanol, or semicarbazide hydrochloride. Reduction in the AldH activity was noted in the presence of certain metal ions like Hg2+, Ag+, Cu2+, Fe2+ and Zn2+. This study demonstrated that AldH of K. pneumoniae was very specific towards NAD+ and highly active in converting 3-HPA to 3-HP.

To understand microbial metabolic functionality, it is important to focus on intracellular reaction kinetics. This requires the development of methodologies to measure intracellular reactant concentrations under in vivo conditions. The analysis of intracellular metabolites, nucleotides, and cofactors has been attempted with a wide range of techniques, e.g., enzymatic assays, HPLC, GC-MS, LC-MS, and NMR. Although there are some limitations in using ESI LC-MS detection, it has a number of advantages compared to the other methods. Since glycolytic intermediates are especially difficult to analyze due to their polarity, structural similarity, and noncharacteristic UV absorption. MS detection has not only the advantage of specific detection, but also that has m/z-specific separation. Here we propose a convenient and highly selective and sensitive LCMS/MS method using tributylamine as volatile ion pair reagent. The method allows the combined separation and quantification of 9 metabolites from the most interesting biological classes in central carbon metabolism. This method can be successfully applied to identification and quantification of these intracellular metabolites in various cell extract samples. In this study determination of intracellular metabolites in E. coli was given as an example.

Vanillin (3-methoxy-4-hydroxybenzaldehyde) is quantitatively one of the most widely used aromatic molecules, particularly in food but also in the pharmaceutical, beverage and fragrance industries. Moreover, vanillin has antimicrobial and antioxidant properties. Conversion of ferulic acid to vanillin is a two step process, catalyzed by feruloyl-CoA synthase encoded by fcs and enoyl-CoA hydratase/aldolase encoded by ech, with the formation of feruloyl-CoA as an intermediate. Substrate channeling approach was performed by dimer formation between leucine- zippers of Fcs and Ech, in order to channelize feruloyl-CoA from Fcs to Ech and thereby increase vanillin production from recombinant Escherichia coli. E. coli harboring a plasmid pTBE-FP forming an efficient dimer of Bait-Ech and Fcs-Prey, produced 2.1 g/L of vanillin at an initial ferulic acid concentration of 3 g/L for 30 hours of culture, which was improved by 2.3-fold from vanillin production of 0.9 g/L of control strain harboring pTAHEF with no leucine-zipper. This result suggests that the E. coli strain harboring pTBE-FP is a potential strain for enhancing vanillin production by substrate channeling. This work was supported by the 21C Frontier Microbial Genomics and Applications Center Program, EBNCRC (Grant No. R15-2003-012-02001-0), and BK21 program of Korea.

Translation initiation, a process which forms an initiation complex by assembly of the ribosomal subunit and initiator tRNA at the start codon on the mRNA, is regarded as a rate-limiting step in protein synthesis. Three initiation factors (IF1, IF2, and IF3) are participated in translation initiation process. IF3 assisted by IF1 promotes the dissociation of vacant 70S subunit to prevent forming premature initiation complex (PIC). IF2 accelerates association of initiator tRNA, 30S subunit, and mRNA. IF2 might play a key role in determining the rate of translation initiation because it affects to the formation of the initiation complex and subsequent passage from the initiation to the elongation. However, the relationship between the structure of the IF2 and the rate of translation initiation was not revealed. In this study, we engineered chromosomal IF2 encoded by infB using error-prone PCR and Red/ET recombination to modulate the translation rate. Also, the change of the translation rate caused by engineered IF2 variants was investigated.

Mannheimia succiniciproducens, a gram-negative rumen bacterium producing succinic acid, was adapted to medium with high-concentration of succinic acid for acid tolerance. Responses of the adapted M. succiniciproducens strain to succinic acid shock were investigated based on transcriptome analysis. During the investigations, gene expression levels of the adapted M. succiniciproducens strain were compared with a parent strain and the expression patterns after acid shock are classified refer to function of genes. Standing on the transcriptome data, specific genes of M. succiniciproducens are selected as targets of genetic manipulations. To improve tolerance for succinic acid, It was tried that overexpression or knock-out experiments of target genes and finding global regulators for acid resistance. [This work was supported by the Genome-Based Integrated Bioprocess Development Project from the Korean Ministry of Education, Science and Technology (No. 2005-01304). Further supports by LG Chem Chair Professorship, and WCU (World Class University) program through the National Research Foundation of Korea are greatly appreciated.]

Mannheimia succiniciproducens MBEL55E, a gram-negative facultative capnophilic rumen bacterium, produces mainly succinic acid by metabolizing a wide range of carbon sources including pentose sugar (xylose), hexose sugars (fructose and glucose), and disaccharides (lactose, maltose, and sucrose). Fermentation studies have shown that M. succiniciproducens prefers sucrose to other carbon sources and produces equal or higher titers of succinic acid on this sugar. Hence, we identified genes involved in the sucrose transport and utilization. The sucrose-utilizing phenotype was examined both for the parent strain and the deletion mutant of each gene. The corresponding enzymes were further characterized by measuring enzyme activities using radio-labeled substrate. As a consequence, several unique features on sucrose uptake and utilization system were found in Mannheimia. Strategies evolved from this characterization of the sugar metabolism will also be suggested in detail. [This work was supported by the Genome-Based Integrated Bioprocess Development Project of the Ministry of Education, Science and Technology (MEST). Further supports by the LG Chem Chair Professorship, Microsoft, IBM SUR, WCU (World Class University) program through the National Research Foundation of Korea funded by MEST (R32-2008-000-10142-0) are appreciated.]

In our previous proteomic study, the recombinant yeast YGH2 expressing active human H-ferritin induced cellular iron deficiency and significantly decreased expression of the mitochondria heat shock protein 60(HSP60) whereas other HSPs increased. HSPs are chaperon proteins that promote the folding of proteins and are overexpressed under stress. In the study, we verified the relationship between iron depletion and HSP60 via Northern blot using the iron chelator BIP or iron supplements. The expression level of the high affinity iron transporter gene FET3 was also examined. Several recombinant strains were compared. The HSP60 expression reduced in the cells of YGH2 cultured for 12 hr. Upon treatment of the control cells with BIP, the expression of HSP60 reduced and FET3 increased. For YGH2 added with Fe2+ or Fe3+ at 0-300μM, changes in the amounts of HSP60 were little while FET3 decreased gradually. Possible explanations for such discrepancy are that (i) H-ferritins are still functional at the iron concentrations we examined. (ii) H-ferritin may function in a distinctive way in the HSP60 expression. Future study of YGH2 with higher iron levels will assist us to understand the questions.

Microbial pathogens are constantly developing resistance to antibiotics, giving impetus for the development of efficient methods for drug targeting and discovery. Genome-scale biological network is expected to play a significant role, as it has succeeded in capturing the physiological characteristics of various organisms. We herein employed two systematic approaches. One of them is constraints-based flux analysis, an optimizationbased simulation technique that calculates metabolic fluxes (1). Another important work is chokepoint analysis, which is network-topology-based method that selects enzymes or metabolites as a target that has a single ingoing and/or outgoing reaction (2). We combined these methods to generate novel drug targets in several emerging drug-resistant pathogens, including Escherichia coli, Helicobacter pylori, Mycobacterium tuberculosis and Staphylococcus aureus. This study demonstrates that drug targeting using in silico approaches enables a rational design of experiments applicable to biomedical science. [This work was supported by the Korean Systems Biology Research Project (M10309020000–03B5002–00000) of the Ministry of Education, Science and Technology. Further supports by the LG Chem Chair Professorship, Microsoft, and IBM SUR program are appreciated.]

Kinetic models are proposed for the production of succinic acid from glucose by Mannheimia succiniciproducens MBEL55E. Experimental data collected from a series of batch fermentations with different initial glucose concentrations were used to estimate parameters and also to validate the models proposed. The optimal values of the parameters were measured by minimizing the discrepancy between the model predictions and corresponding experimental data. The growth of M. succiniciproducens could be expressed by a modified Monod model incorporating inhibitions of glucose and organic acids accumulated in the culture broth. In all cases, the model simulation matched well with the experimental observations, which made it possible to elucidate the fermentation characteristics of M. succiniciproducens during succinic acid production from glucose. These models can be employed for the development and optimization of bio-based succinic acid production processes. [This work was supported by the Genome-based Integrated Bioprocess Project of the Ministry of Science and Technology (No. 2005-01294). Further supports by the LG Chem Chair Professorship, Microsoft, 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) are appreciated.]

Mannheimia succiniciproducens MBEL55E is a capnophilic gramnegative bacterium which efficiently produces succinic acid. In order to analyze metabolic pathways of M. succiniciproducens, we applied pathway analysis to the biochemical network of M. succiniciproducens, previously developed by our group. We then also analyzed the biochemical network of Escherichia coli, in the same way as above, in order to grasp the notable differences between these two organisms. We clustered the solutions of two microorganisms, and compared each other. Each of clusters showed characteristic yield of succinic acid and the number of solutions and clusters of M. succiniciproducens is greater than that of E.coli. [This work was supported by the Genome-based Integrated Bioprocess Project of the Ministry of Education, Science and Technology (No. 2005-01294). Further supports by the LG Chem Chair Professorship, Microsoft, 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) are appreciated.]

Recombinant human H- and L-ferritins(FNs) were purified from Saccharomyces cerevisiae and characterized. Molecular properties were examined with respect to its electrophoretic mobility, isoelectric point (pI), iron uptake kinetics and reconstitution yield of the ferritin core. On SDS-PAGE, the yeast-derived FNs showed bands with the similar mobilities to the corresponding E. coli-derived ones. The pI pattern of yeast-derived H-FN was relatively comparable to that of E. coli-derived while pI of L-FN was more acidic than that of E. coliderived. The recombinant H- and L-FNs are non-glycosylated as proven by cells treated with tunicamycin. To confirm whether the recombinant FNs were phosphorylated, Western blot was performed using antibodies for Ser, Thr and Tyr. The H-FN was determined to be phosphorylated at Ser and Tyr sites while L-FN was not at all. Decrease in the initial rate of the iron uptake kinetics may be caused by such phosphorylation as the residues of Ser and Tyr are known to be closely related to the ferroxidase sites of this protein. In addition, the H-FN exhibited the highest reconstitution yield among the FNs examined, which means it is advantageous to the formation of the inorganic nano particles using the protein.

The scavenging activity of Magnolia denudata Desrousseaux on reactive oxygen species (ROS) were evaluated using 2’,7’-dichlorofluorescin diacetate (DCFH-DA) in HT 1080 cells. Methanol (MeOH) and dichloromethane (CH2Cl2) extracts inhibited dose-dependently generation of ROS in the cellular system. MeOH and CH2Cl2 extracts were combined and fractionated with n-hexane, 85% aqueous MeOH, and n-butanol (n-BuOH). Both n-hexane-soluble and 85% aqueous-soluble fractions showing strong radical-scavenging activity in the cellular system were further separated by diverse chromatographic methods to give five known lignans. All these compounds exhibited significant radical-scavenging effect on intracellular ROS in a dose-dependent manner. Their scavenging activity on various reactive oxygen species (ROS) was also evaluated using electron spin resonance (ESR) spintrap techniques.

Whole plants of Corydalis heterocarpa were collected at Heunri Jeollanamdo, Korea, in July, 2003. The collected samples were dried under the shade and extracted twice with dichloromethane and methanol, in turn. The combined crude extracts were concentrated in vacuo and then partitioned between dichloromethane and water. The organic layer was fractionated with n-hexane and 85% aq. methanol, and the aqueous fraction was also further fractionated with n-butanol and water, successively. The antioxidant activities of crude extracts and their solvent fractions were determined by evaluating DPPH radical, authentic ONOO-, and ONOO- generated from SIN-1 (3-morpholinsydnonimine) in vitro as well as the degree of occurrence of intracellular nitric oxide (NO). Scavenging activities of solvent fractions on authentic ONOO- increased in the order of n-butanol > 85% aq. methanol > H2O > n-hexane fractions, while those on ONOOgenerated from SIN-1 increased in the order of n-butanol > H2O > 85% aq. methanol > n-hexane fractions. In addition, all solvent fractions effectively inhibited the intracellular NO levels. These results suggested that C. heterocarpa contain compounds with strong radical scavenging and antiradical-generating effects.

Fatty acids play important roles of many biological processes. Especially, n-3 polyunsaturated fatty acids (PUFA) are effective against several diseases such as diabetes, cardiovascular disease and cancer. In this study, we analyzed the fatty acid contents of 12 species of Korean salt marsh plants and found high fatty acid contents from Salsola komarvii (76.67 μg/mg) and Limonium tetragonum (50.00 μg/mg). In the case of saturated fatty acids composition, palmitic acid is richest in most salt marsh plants. On the other hand, oleic acid and linoleic acid are major components of monounsaturated fatty acids and n-6 PUFA, respectively. In addition, n-3 PUFA acids such as LNA (linolenic acid), EPA (eicosapentaenoic acid), and DHA (docosahexaenoic acid) known as the main fatty acid components of fish oils and seaweeds, were also found in Salicornia oleracea, Salsola komarvii, Corydalis heterocarpa, Messerschmidia sibirica, Artemisia capillaris and Tetragonia tetragonoides.

In search for novel bioactive secondary metabolites from salt marsh plants, a number of halophytes were collected in western coastal area of Korea and screened for a series of biological activities. Among the collected halophytes, Whole plants of Vitex rotundifolia were extracted for 2 day with dichloromethane and methanol, respectively. The combined crude extracts were concentrated in vacuo and then fractionated into n-hexane, 85% aq. MeOH, n-BuOH, and water fractions. The antiproliferative effects of the crude extracts and their solvent fractions against HT1080, AGS and HT-29 human cancer cells were compared with the control at the concentrations of 10, 50, and 100 μg/mL by using MTT assay. In the comparative analysis, the 85% aq. MeOH fraction exhibited the strongest antiproliferative effects on human cancer cell lines in a dose-dependent manner (p < 0.05). The growth inhibition rate on HT1080, AGS, HT-29 human cancer cells was 74.0%, 79.9%, and 31.6%, respectively, when the 85% aq. MeOH fraction (10 μg/mL) was added to them. These results suggested that active compounds having a strong anticancer effect can be isolated from Vitex rotundifolia

Inonotus obliquus has been known to cinder conk and fungus in the family Hymenochaetaceae. Inonotus obliquus has been traditionally used for both medicinal and food purpose. Fruiting body hot-water extract of Inonotus obliquus (IFHWE), especially beta-glucan constituent, has been associated with anti-tumor and immunomodulatory activity. Macrophages are phagocytic acting in innate immunity and cellmediated immunity. Phagocytosis is involved in the acquisition of nutrients for some cells, which is the major mechanism used to remove cell debris and pathogens in the immune system. Inonotus obliquus has been known to be related with macrophage-mediated innate immune responses. Activated macrophages such as the phagocytic uptake and release of toxic molecules including nitric oxide (NO) and reactive oxygen species (ROS) were investigated. At the last, direct cytotoxicity of IFHWE was evaluated to examine the anti-tumor effect on HeLa cells and B16-F10 cells. The purpose of this study was to investigate the immunomodulatory activity of hot-water extracts from Inonotus obliquus in RAW 264.7 cell line.

The goals of this study were to produce the useful materials such as amino acid by subcritical water hydrolysis from supercritical extracted krill residues and to compare the results with raw krill. Subcritical water hydrolysis efficiency from raw and deoiled krill was examined over the temperature range of 200 to 280ºC, ratio of material to water for hydrolysis was 1:50 and for water-sample contact equilibration times of 5 min to decrease the decomposition of amino acids. The hydrolysis efficiencies of glycine, alanine, and valine were observed to increase with increasing water temperature, consistent with higher solubility at higher temperatures. The highest yield of amino acids in deoiled krill hydrolyzate was at 280ºC. While, the highest amino acid yield in raw krill hydrolyzate was at low temperature 200ºC. The presence of oil in the raw material may interfere the break down of peptide bond of protein by subcritical water hydrolysis at high temperature. Oil may form on complex with protein that may decrease the protein hydrolysis by subcritical water. Also, reducing sugars content was analyzed in both and the result showed that the reducing sugar yield in deoiled krill hydrolyzate was higher than that of raw krill hydrolyzate.

Brown alga Sargassum siliquastrum was extracted for 2 day with a mixture of acetone-dichloromethane (1:1), followed by extraction of the residue for an additional 2 days with methanol. The combined crude extracts of Sagassum siliquastrum were fractionated with nhexane, 85% aq MeOH, n-BuOH, and H2O. Further purification of 85% aq MeOH fraction led to the isolation of two isomeric chromanols, sargachromanols D and E, whose structures were determined by 2 D NMR experiments such as 1H COSY, TOCSY, gHMQC, and gHMBC. Sargachromanols D and E exhibited potent scavenging activity on the authentic ONOO- as well as ONOO- induced from SIN-1. Also, they showed good scavenging effect on DPPH radical, comparable to BHA as a positive control. Sargachromanol E showed higher scavenging acitvities on the DPPH radical and peroxynitrite than sargachromanol D.