Discovery of cSNPs in Pig Using Full-length Enriched cDNA Libraries of the Korean Native Pig as a Source of Genetic Diversity

Dirisala, Vijaya R.,Kim, Ju-Hyun,Park, Kwang-Ha,Lee, Hoon-Taek,Park, Chan-Kyu Korean Society for Biotechnology and Bioengineerin 2007 Biotechnology and Bioprocess Engineering Vol.12 No.4

Clones from full-length enriched cDNA libraries serve as valuable resources for functional genomic studies. We analyzed 3,210 chromatograms obtained from sequencing the 5'-ends of brainstem, liver, neocortex, and spleen clones derived from full-length enriched cDNA libraries of Korean native pigs. In addition, 50,000 pig EST sequence trace files were obtained from Genbank and combined with our sequencing information for SNP identification in silica. For the SNP analysis, neocortex, and liver libraries were newly constructed, whereas the sequencing results from brainstem and spleen libraries were from previously constructed libraries. The putative SNPs from the in silica analysis were confirmed by genomic PCR from a group of 20 pigs of four different breeds. Using this approach, 86% of cSNPs identified in silico were confirmed and the SNP detection frequency was 1 SNP per 338 bp. Interestingly, we found a valine deletion at amino acid position 126 of the neuronal and endocrine protein gene in the Korean native pig. We confirmed that this deletion was caused by alternative splicing at the NAGNAG acceptors. Our study shows that large-scale EST sequencing of Korean native pigs can be effectively employed for natural polymorphism-based pig genome analysis.

Surface Displayed Expression of a Neutralizing Epitope of Spike Protein from a Korean Strain of Porcine Epidemic Diarrhea Virus

Park, Seung-Moon,Mo, Ae-Young,Lim, Jung-Gu,Chung, Hea-Jong,Kim, Tae-Geum,Kim, Kang-Ju,Cho, Dong-Ha,Yang, Moon-Sik,Kim, Dae-Hyuk Korean Society for Biotechnology and Bioengineerin 2007 Biotechnology and Bioprocess Engineering Vol.12 No.6

The neutralizing epitope (K-COE) of the spike protein from a Korean strain of porcine epidemic diarrhea virus (PEDV) has been shown to prevent and foster an immune response to PED, when orally adjusted. The cell surface of the budding yeast, Saccharomyces cerevisiae, was engineered to anchor the K-COE on the outer layer of the cell, and consequently, the altered yeast was applied as a dietary complement for animal feed, with immunogenic functions. In this study, the K-COE gene (K-COE) of the Korean strain of PEDV with the signal peptide of rice amylase 1A (Ramy1A), was fused with the gene encoding the carboxyterminal half (320 amino acid residues from the C terminus) of yeast ${\alpha}-agglutinin$, a mating associated protein that is anchored covalently to the cell wall. The glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter was selected in order to direct the expression of the fusion construct, and the res 내 ting recombinant plasmid was then introduced into S. cerevisiae. The surface display of K-COE was visualized via confocal microscopy using a polyclonal antibody against K-COE as the primary antibody, and FITC (fluorescein isothiocyanate)-conjugated goat anti-mouse IgG as the secondary antibody. The display of the K-COE on the cell surface was further verified via Western blot analysis using the cell wall fraction after the administration of ${\alpha}-1,3-glucanase/PNGase\;F/{\beta}-mannosidase$ treatment.

Cultural Characteristics and Extraction of the Fungal Pigment Phleichrome from the Phytopathogenic Fungus Cladosporium phlei

Lee, Joong-Keun,Kim, Beom-Tae,Kim, Jung-Ae,Chung, Hea-Jong,Park, Seung-Moon,Yang, Moon-Sik,Hwang, Ki-Jun,Kim, Dae-Hyuk Korean Society for Biotechnology and Bioengineerin 2007 Biotechnology and Bioprocess Engineering Vol.12 No.5

The cultural characteristics of the fungus Cladosporium phlei were assessed in order to develop an improved method for the production of the fungal pigment, phleichrome, which is an intermediate in the production of a photodynamic therapeutic agent. The growth of C. phlei, as measured by the hyphal growth rate and increase in biomass, varies significantly depending on the culture media utilized (V8 juice-based medium proved optimal for both growth rate and biomass increase). However, even on a V8 juice plate, the growth of C. phlei occurred slowly and in a limited fashion, in that the colony covered only 75% of the agar surface after more than 4 weeks of cultivation at $20^{\circ}C$. Supplementations of glucose, fructose, galactose, and sucrose increased both hyphal expansion and mass production, whereas supplementations of other carbon sources, including glycerol and sorbitol, exerted no detectable effects. The effect of inorganic nitrogen supplementation was negligible, whereas organic nitrogen evidenced significant effects, with enhanced growth with malt extract and growth inhibition with yeast extract and tryptone. Sporulation was enhanced under conditions of continuous light, and a minimum of $10^3$ spores per mL of liquid media was found to be necessary for the optimal mass increase. A simple extraction procedure was established in order to isolate the deep red pigment which was subsequently identified as phleichrome via NMR analysis. When C. phlei was cultured on V8 medium containing 5% glucose and 2% malt extract, the quantity of mycelial mass was estimated as 20.6g (dry weight) per liter of culture. The expected phleichrome yields from the mycelia and culture filtrates were estimated to be 43 and 2 mg/L, respectively.

Enhanced Tolerance against Freezing Stress in Escherichia coli cells Expressing an Algal Cyclophilin Gene

Cho, Eun-Kyung Korean Society for Biotechnology and Bioengineerin 2007 Biotechnology and Bioprocess Engineering Vol.12 No.5

Members of the cyclophilin (Cyp) family are known to function as co-chaperones, interacting with chaperones such as heat shock protein 90, and perform important roles in protein folding under high temperature stress. In addition, they have been isolated from a wide range of organisms. However, there have been no reports on the functions of algal Cyps under other stress conditions. To study the functions of the cDNA GjCyp-l isolated from the red alga (Griffithsia japonica), a recombinant GjCyp-1 containing a hexahistidine tag at the amino-terminus was constructed and expressed in Escherichia coli. Most of the gene product expressed in E. coli was organized as aggregate insoluble particles known as inclusion bodies. Thus, the optimal time, temperature, and concentration of L(+)-arabinose for expressing the soluble and nonaggregated form of GjCyp-1 in E. coli were examined. The results indicate that the induction of Cyp, at 0.2% L(+)-arabinose for 2 h at $25^{\circ}C$, had a marked effect on the yield of the soluble and active form of the co-chaperone as PPlase. An expressed fusion protein, $H_6GjCyp-1$, maintained the stability of E. coli proteins up to $-75^{\circ}C$. In a functional bioassay of the recombinant $H_6GjCyp-1$, the viability of E. coli cells overexpressing $H_6GjCyp-1$ was compared to that of cells not expressing $H_6GjCyp-1$at $-75^{\circ}C$. For all the cycles of a freeze/thaw treatment, a significant increase in viability was observed in the E. coli cells overexpressing $H_6GjCyp-1$. The results of the GjCyp-1 bioassays, as well as in vitro studies, strongly suggest that the algal Cyp confers freeze tolerance to E. coli.

Application of Sodium Propionate to the Suspension Culture of Chinese Hamster Ovary Cells for Enhanced Production of Follicle-Stimulating Hormone

Yoon, Sung-Kwan,Ahn, Yong-Ho Korean Society for Biotechnology and Bioengineerin 2007 Biotechnology and Bioprocess Engineering Vol.12 No.5

In this study, we found that adding sodium propionate to batch culture medium resulted in cell growth suppression in a dose- dependent manner, while it enhanced follicle-stimulating hormone (FSH) production in Chinese hamster ovary (CHO) cells. In a pseudo-perfusion culture, with the exchange of fresh medium containing 2.5mM sodium propionate, a final FSH of $532{\mu}g$ was obtained, which was approximately 1.5-fold higher than the control. Overall, the results demonstrate that the application of sodium propionate for the commercial production of recombinant proteins in rCHO cells is feasible.

Lipase Catalyzed Production of Monoacylglycerols by the Esterification of Fish Oil Fatty Acids with Glycerol

Byun, Hee-Guk,Eom, Tae-Kil,Jung, Won-Kyo,Kim, Se-Kwon Korean Society for Biotechnology and Bioengineerin 2007 Biotechnology and Bioprocess Engineering Vol.12 No.5

In this study, we attempted the efficient production of monoacylglycerols (MAG) via the lipase-catalyzed esterification of glycerol with fatty acids obtained from sardine oil. The reaction factors that influenced MAG synthesis were the glycerol to fatty acid mole ratio, amount of enzyme, organic solvent, temperature, and the type of lipase used Porcine pancreas lipase was selected to catalyze this reaction. The optimum conditions we determined for MAG synthesis were a glycerol to fatty acid mole ratio of 1:6, 100 mg/mL of lipase, and $30^{\circ}C$ in dioxane. Under these conditions, the MAG content was 68% (w/w) after 72h of reaction. The MAGs synthesized via the lipase-catalyzed esterification of glycerol with fatty acids included monomyristin, monopamiltin, and monoolein, as identified by GC-MS.

Effects of Fomitopsis pinicola Extracts on Antioxidant and Antitumor activities

Choi, Du-Bok,Park, Sang-Shin,Ding, Ji-Lu,Cha, Wol-Suk Korean Society for Biotechnology and Bioengineerin 2007 Biotechnology and Bioprocess Engineering Vol.12 No.5

We investigated the effects of Fomitopsis pinicola extract on biological activity by examining the antioxidant and antitumor activity in vitro and in vivo. When the F. pinicola extract concentration was raised from 60 to $120{\mu}g/mL$, the DPPH scavenging rate increased from 50.3 to 88.2% and the superoxide anion radical scavenging rate increased from 45.2 to 85.3% when the F. pinicola extract concentration was raised from 500 to $700{\mu}g/mL$. After incubating F. pinicola extract for 12 h, the linoleic acid scavenging rate increased from 35.5 to 90.5%. A similar finding was observed for butylated hydroxytoluene. The total phenolic content of the F. pinicola extracts were approximately 10- to 16-fold higher than what was observed in the P. nebrodensis and A. camphorate extracts. The glutathione production, using decoctions prepared from F. pinicola, was $20.0{\mu}M/g$ of liver, which corresponded to approximately 4.0-fold higher than the control. The glutathione peroxidase activity was 8.3 U/mg of protein, which was approximately 2.8-fold higher than the activity level observed in the control rat livers. The cell viability rates of all the human cancer cells, when $100{\mu}g/mL$ of ethanol extract was used for the different types of cancer cells, decreased with increasing extract concentrations in comparison to the hot water extract. In particular, when HeLa and Hep3B cells were incubated with $1000{\mu}g/mL$ of methanol extract, the cell viability rates were 20 and 25%, respectively, which was approximately 3.0-fold higher than what was observed for the hot water extract.

Optimization of Dilute Sulfuric Acid Pretreatment of Corn Stover for Enhanced Xylose Recovery and Xylitol Production

Hong, Eunsoo,Kim, Jinyeong,Rhie, Seunggyo,Ha, Suk-Jin,Kim, Junghoe,Ryu, Yeonwoo Korean Society for Biotechnology and Bioengineerin 2016 Biotechnology and Bioprocess Engineering Vol.21 No.5

Pretreatment steps are necessary for the bioconversion of corn stover (CS) to xylitol. In order to optimize the pretreatment parameters, the sulfuric acid concentration, sulfuric acid residence time, and solid slurry concentration were evaluated, based on the glucose and xylose recovered from CS at the relatively low temperature of <TEX>$120^{\circ}C$</TEX>. The optimum conditions were found to be pretreatment with 2.5% (w/v) sulfuric acid for 1.5 h, with a solid slurry concentration of 90 g/L. Under these conditions, the hydrolysis rates of glucan and xylan were approximately 26.0 and 82.8%, respectively. High xylitol production (10.9 g/L) and conversion yield (0.97 g/g) were attained from corn stover hydrolysate (CSH) without detoxification and any nutrient addition. Our results were similar for xylitol production in synthetic medium under the same conditions. The non-necessity of both the hydrolysate detoxification step and nutrient addition to the CSH is undoubtedly promising for scale-up application on an industrial scale, because this medium-based manufacturing process is expected to reduce the production cost of xylitol. The present study demonstrates that value-added xylitol could be effectively produced from CS under optimized pretreatment conditions, especially with CSH as the substrate material.

Effect of Mobile Phase Additives on Resolution of Some Nucleic Compounds in High Performance Liquid Chromatography

Jin, Chun-Hua,Koo, Yoon-Mo,Choi, Dae-Ki,Row, Kyung-Ho Korean Society for Biotechnology and Bioengineerin 2007 Biotechnology and Bioprocess Engineering Vol.12 No.5

Here we investigate the chromatographic behavior, with reversed-phase high performance liquid chromatography (RP-HPLC), of nucleic compounds (nucleobases, nucleosides, and nucleotides) on a $C_{18}$ column in several different mobile phase additives, including 1-butyl-3-methylimidazolium tetrafuloroborate $([BMlm][BF_4])$, 1-ethyl-3-methylimidazolium methylsulfate ([EMlm][MS]) ionic liquids, ammonium formate, and potassium phosphate. The effect of the alkyl group length, the imidazolium ring, and the ionic liquid's counterions on retention and resolution of the samples were tested. The results show the potential application of a used buffer system, ion pairing system, and ionic liquid as mobile phase additives in liquid chromatography resolution of nucleic compounds.

Optimization of Medium for the Production of a Novel Aquaculture Probiotic, Micrococcus MCCB 104 Using Central Composite Design

Preetha, R.,Jayaprakash, N.S.,Philip, Rosamma,Singh, I.S. Bright Korean Society for Biotechnology and Bioengineerin 2007 Biotechnology and Bioprocess Engineering Vol.12 No.5

A marine isolate of Micrococcus MCCB 104 has been identified as an aquaculture probiotic antagonistic to Vibrio. In the present study different carbon and nitrogen sources and growth factors in a mineral base medium were optimized for enhanced biomass production and antagonistic activity against the target pathogen, Vibrio harveyi, following response surface methodology (RSM). Accordingly the minimum and maximum limits of the selected variables were determined and a set of fifty experiments programmed employing central composite design (CCD) of RSM for the final optimization. The response surface plots of biomass showed similar pattern with that of antagonistic activity, which indicated a strong correlation between the biomass and antagonism. The optimum concentration of the carbon sources, nitrogen sources, and growth factors for both biomass and antagonistic activity were glucose (17.4 g/L), lactose (17 g/L), sodium chloride (16.9 g/L), ammonium chloride (3.3 g/L), and mineral salts solution (18.3 mL/L).