Earticle

최근 전 세계적으로 well-being화 경향에 따라 보다 질 좋고 건강에 좋은 새로운 식품 혹은 기능성 소재에 대한 수요가 급증하고 있는 실정이다. 기능성 소재에 관한 수많은 연구 결과들이 나오고 있으며, 이들 중 상당수는 기술적으로나 사회적으로나 의미가 크고, 활용 가능성이 높은 좋은 성과들이 도출되고 있다. 하지만 이런 기능성 소재 연구의 대부분이 농수산 자원들을 대상으로 하고 있고, 이로부터 새로운 생리 활성 탐색이나 신규 기능성 물질 분리 및 응용에 관한 연구가 대다수를 차지하고 있다. 이에 비해 임산 자원을 대상으로 하는 기능성 소재에 대한 연구는 산에 생산되는 버섯류나 기타 수종의 임산 작목에 그치고 있는 실정이다. 우리나라의 70% 정도가 산지인 점을 고려하면 임산 자원의 활용에 관한 연구나 산업화가 거의 전무하다고 할 수 있다. 이같이 초본류들에 비해 상대적으로 부가가치를 증진 시킬 수 있는 잠재성이 높음에도 불구하고 임산 자원의 특성상 대부분의 활성물질들이 가장 딱딱한 수피 부분에 주로 분포하고, 그 종류들도 초본류나 수산 자원에 비해 다양하지 못한 이유도 있다. 또한 생산되는 임산 자원 그 자체를 이용하거나 1,2 차 단순 가공을 통하여 얻어지는 자원들의 부가 가치 증진이나 창출에는 한계가 있게 마련이다. 따라서 임산 자원의 특성을 살려 유용 자원 및 소재들을 얻고 평가하고 활용하는 기술들을 도입해야 만이 획기적인 소득 창출이 가능할 것이다. 이를 위해 기존의 가공 공정으로는 얻을 수 없는 기능들의 도출을 위해 초고압이나 고온 액화 등의 최신 공정의 적용이 필요할 것으로 예상된다. 이와 함께 극한 공정을 통해 얻어진 물질의 생체 활용성 증진 기술과의 접목도 임산 자원의 소득 증진을 위한 가장 효과적인 기술로 평가되고 있다. 이를 위해 최근 그 효용성이 부가되고 있는 고로쇠나무와 매자나무 등을 대상으로 극한 공정과 생체 활용성 증진 기술에 대한 실례를 소개하고자 한다.

인삼은 식물학적으로 五加科(Araliaceae), 人蔘屬(Panax)에 속하는 식물을 말하며, 주로 뿌리를 약용으로 이용하고 있다. “Panax"라는 어원은 희랍어로 Pan(總, 汎: all)과 Axos(치료: cure)의 복합어로 만병을 치료한다는 뜻이다. 세계적으로 인삼속의 식물 종은 6-7 종이 알려지고 있다. 인삼의 성분은 유기물의 경우 사포닌(ginsenosides), 함질소 화합물, 지용성 성분, 비타민, 탄수화물(다당체 등) 등으로 구성되어 있다. 이러한 인삼의 약리효과로는 비정상적인 신체 기능을 정상화 시켜주는 효과, 항암효과, 항당뇨효과, 고혈압 치료효과, 항스트레스 및 항피로 효과, 항방사선 효과 등이 알려져 있다. 우리나라에서는 삼을 일반적으로 산삼(천종, 지종), 산양산삼, 장뇌삼, 인삼 등으로 크게 구분하고 있다. 이러한 삼들의 형태학적 특성과 성분분석 결과를 비교설명하고자 한다.

국내에 분포하는 식물종 중 약용으로 가능한 식물종은 약 1,000 여종으로 보고되고 있다. 국토면적이 협소한 우리나라는 상대적으로 부존자원도 빈약한 편으로 이를 총 망라한 대대적 관심과 개발이 필요하다. 다행스럽게도 국내 연구자들의 관심이 전통식물의 활용에 집중되고 있고 정부의 각종 R&D 투자가 상당부분 이 분야에 할애되고 있지만 괄목할만한 성과는 나타나고 있지 않다. 식물자원에 대한 정보, 추출물 정보, 활성정보에 대한 국가차원의 DB 도 필요하다. 본 고에서는 전통약용식물을 활용한 신성장동력으로서의 새로운 소재개발을 위한 기초적 정보를 논의코자 하였다.

bioadhesives. While the role of L-3,4-dihydroxyphenyl alanine (DOPA) for water-resistant strong adhesion has been extensively studied, aggregate formation and physical adsorption behavior of MAPs have not been well examined yet. Here, we investigated the degree of aggregation and attachment property using recombinant MAP having no DOPA residues. Chemical cross-linking of oxidized DOPA residues was not required for formation of aggregates, and increased kosmotropic property for anions and chaotropic property for cations allowed formation of aggregates for strong adsorption on hydrophilic surfaces. This study suggests that the initial step of MAP adhesion might be aggregation based on the physical property itself. Physical adsorption can be adjusted by salt ions in water solution for efficient attachment, followed by DOPA-mediated water-resistant strong adhesion.

Among many biodegradable polymers, poly(lactic acid) (PLA) has been used most widely for a wide variety of pharmaceutical and biomedical applications such as scaffolds for tissue engineering and carriers for controlled drug release because of its proven biocompatibility and favorable regulatory status. However, the high hydrophobicity of PLA results in some problems in particulate formulation of drugs such as too slow drug release and unfriendly environment to hydrophilic drugs. Poly(ethylene glycol) (PEG) is widely used to cloak particles and obtain stealthy properties. PEG is water-soluble, nonionic, nonantigenic and non-immunogenic. In this study, methoxy poly(ethylene glycol)-b-poly(L-lactic acid) (MPEG-PLLA) diblock copolymer was synthesized via a ring-opening polymerization, and then MPEG-PLLA microparticles were prepared by aerosol solvent extraction system (ASES) using supercritical carbon dioxide as antisolvent. The effects of temperature, pressure, initial concentration and flow rate of polymer solution on the particle size, size distribution, and morphology were investigated.

Proteomics related toxicology has provided innovative approaches to novel biomarkers screening, toxic materials screening, risk assessments, and predictive toxicology. The application of proteomic analysis in the field of aquatic toxicology has recently started to increase, but until now, little attention has been given to the integrated analysis of molecular responses and higher-level effects within the same study. In this study, protein expression profiles provided significant insights into the toxicity mechanisms for the Daphnia magna, post exposure of heavy metals. We performed proteomic analysis of Daphnia magna in response to Sodium arsenite [As(III)], Sodium arsenate [As(V)], Cadmium nitrate [Cd], As(III)+As(V), As(III)+Cd and As(V)+Cd as a heavy metal, by using two-dimensional gel electrophoresis (2-DE) and found that protein profiles from Daphnia magna were shown different expression patterns for the each heavy metals. In addition, Daphnia magna exposed to the mixture of heavy metals showed different expression patterns. Daphnia magna protein expression profile patterns of binary metal mixtures are not the simple sum of their individual compounds fingerprint.

Although pork is a important resource of protein, it occasionally caused allergy reaction to patients who have pork allergy. The ultimate purposes of this study are to search for processing conditions which will apply to hypoallergenic product. Heating, microwave and sonication have been used to process food and known to change the allergenicity of food. Each physical treated pork extract underwent ci-ELISA with the IgG in order to evaluate its allergenicity. As a result of ci-ELISA, SDS-PAGE and immunoblotting, autoclave was the most effective method in physical treatments. From these results, pork ham treated with autoclave (121℃, 10 and 30 min) and gamma-irradiation (1, 3, 10 and 20 kGy) to examine implement possibility in hypoallergenic pork. Compared with untreated ham, binding ability of pork ham treated with autoclave (10 and 30 min) and gamma-irradiation (10 and 20 kGy) was desreased.

In this work a microplate-based bioreactor with an optical online monitoring system (MABOOMS) has been studied for high-throughput screening of microorganisms and medium optimization in biotecnological processes. The MABOOMS has been constructed with a fluorescence sensing system, a 24-well microplate-based bioreactor with sensing membranes for detection of dissolved oxygen (dO2), pH, dissolved carbon dioxide (dCO2) and reflectance (RF), and an automated control system with a microprocessor. The MABOOMS has been used to optimize fermentation media in fermentation processes. A 24-well microtiter plate with sensing membranes for dO2, pH and dCO2, has been employed to online monitor the concentrations of dO2, pH and dCO2 in fermentation processes with E.coli DH5α and B.cereus 318. The trends of the online data agreed with those of the offline data. The MABOOMS was validated to be very useful to online monitor the cell growth characteristics.

The engineering of the desired phenotypes in organisms of interest is a major goal in biotechnology and basic research towards engineering complex biological systems and understanding their natural counterparts. For the phenotypic engineering of microorganisms, we have developed novel artificial transcription factors (ATFs) capable of reprogramming innate gene expression circuits in Escherichia coli. These ATFs are composed of zinc finger (ZF) DNA-binding proteins, with distinct specificities, fused to an E. coli cyclic AMP receptor protein (CRP). By randomly assembling 40 different types of ZFs, we have constructed more than 6.4 x 104 ATFs that consist of 3 ZF DNA-binding domains and a CRP effector domain. Using these ATFs, we induced various phenotypic changes in E. coli and selected for industrially important traits, such as resistance to heat shock, osmotic pressure, and cold shock. Genes associated with the heat-shock resistance phenotype were then characterized. These results and the general applicability of this platform clearly indicate that novel ATFs are powerful tools for the engineering of complex regulatory transcription networks in microorganisms and can elicit new phenotypes in E. coli.

The growth of Au nanoparticles (NPs) catalyzed by H2O2 was performed in Ca-alginate beads immobilizing Au NPs and glucose oxidase (GOx) in the presence of AuCl4 - and cetyltrimethylammonium chloride. As a result of the Au growth reaction, the color of Au NP-immobilized beads changed redder and UV absorbance at 535 nm was augmented along with increasing concentration of hydrogen peroxide. Au NPs in alginate hydrogel beads were analyzed with the transmission electron microscopy and indicated that Au NPs in alginate were successfully enlarged with growth solution and hydrogen peroxide. The growth of Au NPs was then conducted with alginate beads co-immobilizing GOx and Au NPs in which H2O2 was produced through biocatalytic oxidation of glucose by GOx. The absorbance of resulting alginate beads at 535 nm was enlarged with increasing glucose content after the growth procedure and it proved that the glucose can be detected by the enlargement of Au NPs in alginate beads.

Capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) analysis is known as a simple and rapid molecular marker detection technique due to its high sensitivity and great reproducibility with conformation dependent separation of single strand DNA.However, conventional linear polyacrylamide (LPA) blending polymer matrix generally used for CE-based analysis has poor resolution in CESSCP analysis since it is optimized for molecular weight dependent separation. Therefore, new polymer matrix suitable for CE-SSCP analysis is required. In this study, we checked a number of polymers for CESSCP analysis based on its resolving potential using 255bp DNA fragments in variable region of 16S rRNA gene of Yersinia enterocolitica, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae. Among three polymers tested in this study, poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer (PEO-PPO-PEO) showed the high resolution. After carrying out further optimization including molecular weight, PEO composition, and concentrations, high resolving CE-SSCP analysis system could be obtained. The results also displayed a strong potentials in many applications in CE-SSCP.

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. In this work, response surface methodology (RSM) was employed to optimize the cultivation of Bacillus subtilis pBPT62 for the enhancement of rhBMP-7 production. A fractional factorial design was applied to study the main factors that affect the rhBMP-7 production. A few such as incubation time, starch concentration and lactose concentration were identified as most significant for high production of rhBMP-7 and the central composite design were adopted to derive a statistical model with these variables. In addition, the time course of rhBMP-7 production was also described under the optimized medium composition.

Arg-Gly-Asp sequence from fibronectin and laminin has been widely used for cell adhesion and identified to interact with integrin receptor and enhance the focal adhesion of the target cell to modulate the cellular behaviors. Previously, mussel adhesive protein, fp-151-RGD was constructed and produced successfully and also, fp-151-RGD showed a potential as a biocompatible tissue engineering material due to its great cell adhesion, spreading and proliferation ability. In this study, fp-151-RGD was prepared and used as a substrate for the in vitro culture of mouse fibroblastic NIH3T3 cells in order to investigate the effect of GRGDSP modification. The fp-151- RGD played a significant role in NIH3T3 cell behaviors. Cell spreading and proliferation rate were remarkably greater than other commercially used cell-adhesion materials. Phosphorylated FAK protein expression and CycD1 gene expression level was also increased by fp-151-RGD. These results suggest that fp-151-RGD has a positive effect on NIH3T3 and can be used for tissue culture material.

BKS strain No. 14, 15 and 39 were cultivated in shaking flasks with TSB(Tryptic Soy Broth) complex medium at 30℃ and speed of 200 rpm. Korean nutral zeolite was added to shaking flasks and mixed with microorganisms. They were immobilized for 24 hours. Morphology of immobilized whole cell was investigated by FE-SEM(Field Emission Scanning Electron Microscopy). Hydrogen peroxide concentration was measured by photometric method at 350 nm. The hydrogen peroxide at concentration of 10 mM was removed within 1 hour.

Understanding molecular self-assembly of peptides into ordered nanostructures is recently getting much attention from researchers in materials science as well as in chemistry and biology since it can provide an alternative route for fabricating novel bio-inspired materials. Among a number of peptide building blocks reported to date, the simplest building block peptide is diphenylalanine, which constitutes the core-recognition motif of Alzheimer's beta-amyloid peptide and readily assembles into various nanostructures such as nanotubes and organogels. In this presentation, we report the solidphase self-assembly of diphenylalanine peptides into nanostructures, starting from amorphous thin film. We could control the morphology and structures of peptide thin film by inducing the self-assembly of peptide upon exposure to solvent vapor. In particular, we could grow vertically well-aligned peptide nanowires, which are thermally and chemically very stable, by treating the amorphous peptide thin film with aniline vapor at temperatures as high as 100ºC. Lastly, some examples of applications of our peptide nanowire film in nanobiotechnology will be suggested, such as creation of smart surface and templated synthesis of functional nanomaterials.

Mussel adhesive protein (MAP) fp-151 designed and constructed by our research laboratory has the potential to be used as a cell or tissue bioadhesive at various cell lines in biocompatible aspect. To improve the cell-adhesion and proliferation properties of fp151, we designed an extracellular matrix (ECM) mimics based on mussel adhesive protein fused with biofunctional peptides such as RGD, laminin, type Ⅳ collagen, growth factor called substance P, which is fused at Cterminus of fp151. The ability of adhesion and proliferation properties is tested in several cell lines by direct cell count (DCC) method and MTT assay. Our ECM mimics showed better or similar adhesion and proliferation properties compared with other commercially produced cell-adhesion materials such as poly-L-lysine (PLL) and the naturally extracted MAP mixture Cell-Tak in MC3T3-E1, ATDC5 and 3T3-L1 cells. These data showed that these mimics can be successfully used in cell culture and tissue engineering and extended to other tissuespecific cell recognition motifs to allow attachment of target cells to artificial ECM surfaces.

In this study, production of ornithine, which is a multi-functional amino acid, was enhanced by ornithine cyclodeaminase under prolinesupplemented Corynebacterium glutamicum. In addition it was demonstrated that transcriptional factor ArgR is binds to the upstream of argC, argB, argF, and argG gene by in vivo chromatin immunoprecipitation assay. ArgR binding affinity was significantly reduced upstream of argB under proline-supplementation resulting in increased ornithine production. The ArgR-binding affinity also was altered to the upstream of argB by supplemented metabolites such as ornithine, arginine and proline and the result was consisted with the change in ornithine production. Furthermore, this study identified the important enzymes involved in the production of glutamate and proline from phenol. Based on these results, the expression of gltB and gdh were regulated by the transcriptional regulator, ArgR, under the culture condition with phenol, and its regulatory effect was enhanced by iron supplementation.

The mammalian cells contain lysosomes, and the lysosomal enzymes are localized intralysosomally. In response to certain signals they are trigger apoptotic cell death and degradation of bacteria. Therefore, in this study, HeLa cells were exposed by H2O2 and 6-hydroxydopamine (6-OHDA), we confirmed that fluorescent intensities and size of each lysosomes were changed as concentration of H2O2 and 6-OHDA. In addition, activity of cathepsinB, D as lysosomal enzymes in lysosomes have shown to be different each concentrations. In addition, the lysosomes isolated from HeLa cells showed antimicrobial activity to Escherichia coli that positively correlated with the pH of the phosphate buffer as a dissolving solvent. As a results of the lysosomes isolated from H2O2 and 6- OHDA treated HeLa cells showed higher antimicrobial activity than those isolated from normal HeLa cells. We found that the lysosomes were activated by H2O2 and 6-OHDA. Moreover, activated lysosomes are enhanced antimicrobial effect. Therefore, it suggests that lysosomes isolated from HeLa cells can be used as antimicrobial agents. Acknowledgement: 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 supports.

Obesity is associated with heath risks for chronic diseases such as heart disease, hypertension, diabetes, and cancer. Excess body weight is the result of imbalance between energy intake and energy expenditure, by which stored as triacylglycerol (TAG) in adipose tissue. Recently edible diacylglycerol (DAG) oil has synthesized to reduce serum TAG levels, to decrease hemoglobin A1C level in patients with type 2 diabetes and to prevent the accumulation of body fat in animals and humans with long term safety. Therefore, we synthesized DAG from tuna oil using lipase, and investigated their anti-obesity effects in C57/BL6J mice. Briefly, DAG was produced by glycerolysis of tuna oil with glycerol in a batch bioreactor using a 1,3-regioselective immobilized lipase (Lipozyme RM-IM). Consumption of DAG diet effectively reduced body weight gain, serum cholesterol and triacylglycerol levels as plasma biochemical markers of obesity in C57 BL/6J mice, compared to the high TAG fed group. Moreover, we compared changes in several gene expressions in hepatic and adipose tissue in normal, TAG and DAG diet intake mice models. DAG fed group had a potential effect on several gene expressions in hepatic and adipose tissue.

Ecklonia cava is commonly used as foodstuff, 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 reuse the residue, new approach using microbial fermentation was performed. The microbial fermentation of the residue may result in the break-down of bioactive compounds or the production of bioactive compounds. As expected, the methanolic extract of E. cava residue fermented by Aspergillus oryzae showed strong antibacterial activity against several pathogenic bacteria.The antibacterial activity was higher than that of the extract of raw E. cava. Our finding may contribute to the development of a natural antibiotic agent using the seaweed waste.

Rapamycin is a macrocylic polyketide with immunosuppressive, antifungal, and anticancer activity. The production level of rapamycin in Streptomyces hygroscopicus ATCC29253 was enhanced by classical mutagenesis and metabolic engineering approach. This study describes the development of a novel highthroughput method for rapid screening of rapamycin-producing strains using agar diffusion method and metabolic engineering strategy to enhanced rapamycin production. Rapamycin production by a mutant strain UV2-2 induced by Ultraviolet (UV) mutagenesis was improved by approximately 3.2-fold compared to that of wild-type strain. The comparative analysis of gene expression and intracellular acyl-CoA pool between wild-type and the UV2-2 strains revealed that the increased production of rapamycin in UV2-2 was due to the prolonged expression of rapamycin biosynthetic genes but the low concentration of intracellular methylmalonyl-CoA was limiting the rapamycin biosynthesis in both strains. In an attempt to promote intracellular pool of methylmalonyl-CoA, propionyl-CoA carboxylase pathway was introduced into the mutant strain UV2-2 along with supplementation of methyloleate, resulting in an additional 1.7-fold improvement of rapamycin titers. These results demonstrate that the combined approach involving traditional mutagenesis and metabolic engineering can be successfully applied for the rapamycin-producing strain.

The small coenzyme Biotin (vitamin H) functions as a cofactor that aids in the transfer of CO2 groups to various target macromolecules. A modification of biotin carboxylase (BC) or alpha subunit of acyl-CoA carboxylase (ACCase) via biotin attachment regulates the catabolic enzyme activity at the posttranslational level. The biotinylation of apo-BC to holo-BC is conducted by biotin apo-protein ligase (BPL) via utilization of a molecule ATP. The identified in Streptomyces toxytricini chromosome stbpl gene encodes a 29,870 (31,320) Da protein (StBpl) showing 20% identity with Escherichia coli BirA (EcBirA) protein. the StBpl showed very low homology with the N-terminal sequence of EcBirA, DNA binding motif involved in transcriptional regulation of bio operon. The protein is also smaller than EcBirA with 15-30 amino acid residues. In this report we cloned, overexpressed and biochemically characterized the StBpl protein from S. toxytricini. accA1 gene identified in a different DNA region of the S. toxytricini genome encodes 70 kDa protein (StAccA1), an biotin carboxylase of ACCase gene involved in primary (fatty acids) metabolism. The apo-StAccA1 or apo-BC recombinant protein was also expressed, purified and used as a substrate for biotinylation assay in vitro by StBpl and/or in vivo by the host EcBirA.

This study demonstrates the ability of metals uptake by bacterial biosorbents immobilized in cross-linked chitosan fiber and the competition among metals in ternary metals system containing Pd(II), Rh(III) and Pt(IV). Batch experiments were performed with mixed metals solution in 0.1 M HCl, where metals exist as anionic chlorometal complexes. Langmuir and pseudo-first-order models were used to optimize the batch experiments. The maximum uptakes of Pd(II), Rh(III) and Pt(VI) were estimated to be 45.52, 23.96 and 37.78 mg/g, respectively. The rank of sorption uptake rate in mixed metals solutions was Pd(II) > Pt(IV) > Rh(III). The adsorption time for equilibrium was 90, 60 and 40 min, for Pd(II), Pt(IV) and Rh(III), respectively. In adsorption kinetic experiment, the equilibrium uptakes of Pd(II), Rh(III) and Pt(IV) were predicted to be 27.42, 4.76 and 15.29 mg/g, respectively. Desorption kinetic study with thiourea solution as an eluent revealed that Pd(II) could be eluted very easily within 20 min while Pt(IV) within 60 min. Only 64.79 and 49.20% desorption efficiency of Pd(II) and Pt(IV) were obtained, respectively. However Rh(III) could not be desorbed from the biosorbents, which was considered to be recovered by incineration of the biosorbents after removing Pd(II) and Pt(IV).

In the diatom Cylindrotheca fusiformis, modified peptides called silaffin polypeptides are responsible for silica deposition in vivo at ambient conditions. Recently, it is discovered that the synthetic R5 peptide, the repeat unit of silaffin polypeptide without post-translational modification, was capable of precipitating silica in vitro as well at ambient conditions. Herein, green fluorescent protein (GFP) chimeric proteins were generated by incorporating synthetic silaffin R5 peptides and related unmodified silaffin domains (R1-R7) from Cylindrotheca fusiformis onto GFP by recombinant DNA technology and their ability to cause silicification examined. GFP chimeric proteins showed silicification at very low concentrations (600-700 μg/ml), compared to adding excess amounts of R5 peptides (10 mg/ml) as previously reported. Sensitive to pH conditions, only the GFP-R1 chimera showed silicification activity at pH 8.0. The protein immobilization efficiencies of these chimeras were unexpectedly high ranging from 75 to 85%, with the R1 silaffin-protein construct showing excellent immobilization efficiency and a constant molar ratio of silica to protein ranging from 250-350 over a wide pH range. The average silica particle sizes had a tendency to decrease as pH increased to basic conditions. This study demonstrated the production of nanoscale immobilized protein, fabricated via silaffin-fused proteins.

Translational efficiency in Escherichia coli is known to be strongly influenced by the secondary structure around the ribosome-binding site and the initiation codon in the translational-initiation region of the mRNA. Several quantitative studies have reported that translational efficiency is attributable to effects on ribosome accessibility predominantly caused by the secondary structure surrounding the ribosome-binding site. However, the influence of mRNA secondary structure around regions downstream of the initiation codon on translational efficiency after ribosome-binding step has not been quantitatively studied. Here, we quantitatively analyzed the relationship between secondary structure of mRNA surrounding the region downstream of the initiation codon, referred to as the downstream region (DR), and protein expression levels. Modified hairpin structures containing the initiation codon were constructed by site-directed mutagenesis, and their effects on expression were analyzed in vivo. The minimal folding free energy (dG) of a local hairpin structure was found to be linearly correlated with the relative expression level over a range of 0.3- to 1.2-fold that of the wild-type structure. These results demonstrate that expression can be quantitatively controlled by changing the stability of the secondary structure surrounding the DR.

The three groups of methanogens in methanogenesis (automated anaerobic continuously stirred tank reactors (CSTR)), which were operated at different hydraulic retention time (HRT) for around 80 days, were investigated quantitatively using quantitative PCR (QPCR) technology. Through Redundancy Analysis (RDA), statistical evaluation of the data in relation to the operation conditions of the bioreactors showed that the four parameters containing pH, Acetic acid (HAc), Propionate acid (HPro) and free ammonia (FA) concentration together explained 48.4% of the variations in the population change of the methanogens. The results of RDA revealed that the changing HAc and HPro concentrations in the methanogenesis were the most significant environmental variables, which strongly influenced hydrogentrophic and aceticlastic methanogens at different levels. Among the four environmental variables, FA concentration was the least important factor, but showed inhibition to the three analyzed groups, while less affecting on hydrogentrophic methanogens. pH was another important factor, which together with FA concentration had highly inhibition effects on aceticlastic methanogens.

The study is comprised of multiple experiments for determining the adsorption capacity of silver using active/live and inactive/dead cells of Corynebacterium glutamicum. Langmuir and Freundlich isotherms were used to optimize the design of biosorption system. The increase in pH amplified the biosorption of silver, furthermore achieving equilibrium in less than 30 min. The maximum experimental uptake by active cells was 50.1 mg/g where as maximum uptake by inactive cells was 52.5 mg/g for the initial concentration of 1000 mg/L. As a part of our study, the bioreduction capacity of active and inactive cells of C. glutamicum to reduce Ag+ was determined. Transmission electron microscopy (TEM), Energy dispersive X-ray (EDX) and Xray diffraction (XRD) were performed for determining the formation of silver nanoparticles. The results revealed evident difference in the formation of nanoparticles by live and inactive cells. The surface of inactive cells showed large amount of reduction in contrast to the active cells. Nanoparticles acquired from these experiments showed irregularity in shape and size ranging between 5 and 50 nm in size. Our present study extends to explore the possibility of nanoparticles formation in the absence of enzymes and metabolites.

Quantification of RNA provides the crucial information in various fields of biology, such as transcriptomics, pathogen detection. The major technology for RNA quantification is real-time PCR, which is known as the most accurate method of nucleic acids quantification. However, application of real-time PCR on RNA quantification is limited by single target per an analytical run because of reduced quantification power in multiplex detection and limitation on fluorescence dye. Here, we report a novel multiplex RNA quantification method using capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) coupled with asymmetric PCR using common primers. There are three major steps which are template modification, multiplex amplification, and simultaneous detection. Firstly, RNA targets are modified for multiplex amplification step. After the first step, all targets have the same end-sequences, and the sequences are the sites on which the common primers hybridize. Second step is multiplex amplification of targets using asymmetric PCR with the common primer pair. Finally, the amplicons are separated and quantified by CE-SSCP analysis.

A cost-effective bioconversion technology was developed to produce biogas from high strength organic waste such as swine waste. Swine waste was biologically treated to produce methane both in a pilot-scale anaerobic sequencing batch reactor and in continuous upflow anaerobic sludge blanket digesters. Dynamics of methanogenic populations weremonitored in the both digesters, at order and family levels, using real-time PCR based on the 16S rRNA gene (Yu et al., 2006). A high abundance of hydrogenotrophic methanogens was observed, with Methanobacteriales as the major group in the digesters, suggesting that hydrogenotrophic methanogenesis, with the syntrophic oxidation of volatile fatty acids, was a major route of methane formation in anaerobic process treating swine waste. This phenomenon was mainly attributed to the high ammonia and VFAs concentrations which are characteristics of swine waste (Bonin and Boone, 2006; Demirel and Scherer, 2008). These substances have a severe inhibitory effect mainly on aceticlastic methanogens. Consequently, the dynamics of methanogen population deduced by QPCR analysis showed good agreement with the digester conditions and major chemical profiles. These results emphasize that more attention should be devoted to quantitative investigation of methanogenic community dynamics and structure for better understanding of their roles and behaviors in anaerobic digesters treating swine waste (McHugh et al., 2003; Zheng et al., 2006).

This experiment is concerned with optimization of culture medium by C. lytica LBH-14. The one-factor-at-a-time method was used to investigate the effect of carbon & nitrogen sources, and initial pH on production of carboxymethylcellulase (CMCase) and cellobiase by C. lytica LBH-14. Base on the result of one-factor-at-a-time experiment, L25-53 was performed to measure combined effects of carbon source & nitrogen source, and initial pH on production of CMCase and cellobiase. Optimal conditions for production of CMCase were 10.0% (w/v) rice bran, 0.75% (w/v) ammonium nitrate, and initial pH 6.00, whereas those of cellobiase were 10.0% rice bran, 0.50% ammonium nitrate, and the initial pH of 7.50. On basis of intuitive analysis and statistical calculations using data from an orthogonal experiment, ammonium nitrate was found to be the most important factor for production of the CMCase by C. lytica LBH-14, whereas the most important factor for production of cellobiase was rice bran.