Earticle

A critical requirement for achieving a total micro-analytical system of cells and their constituent proteins is to develop the cell lysis and solid phase extraction (SPE) steps on-chip. This work was developed by miniaturized sample preparation systems, in which cell lysis was needed to obtain intracellular materials for analysis of intracellular protein. We adopt the micro-magnetic stirring method by an external magnetic stirrer for cell lysis of Escherichia coli. The intracellular sample and lysis buffer were well mixed by micro-magnetic stirring in a chamber, and the efficient-lysis with mixing time introduced into the SPE chamber, subsequently. We also analyzed the immobilization efficiency of intracellular protein after cell lysis onto the micro-patterned gold surface in microchamber. Using this microfluidic mixer system, we developed a strategy for fabricating micro-patterned protein chip. The principle of this strategy is that the gold binding polypeptide (GBP) as a fusion partner is specifically immobilized onto the gold-patterned surface. The severe acute respiratory syndrome coronavirus envelope was used as a model protein for the specific immobilization of GBP-fusion proteins onto the gold-patterned surface. This micro-mixer fluidic system was combined with Lab-on-a-Chip applications on the gold surface and used for studying antigen-antibody interactions. [This work was supported in part by MIC & IITA through IT Leading R&D Support Project, by the KOSEF through the Center for Ultramicrochemical Process Systems, and the Basic Research Program of the Korea Science and Engineering Foundation.]

Here we present a sensitive DNA detection protocol using quantum dots (QDs) and micro-magnetic beads (MBs) for large volume samples. In this study, Qdots, conjugated with streptavidin, were used to produce fluorescent signals while magnetic beads (MBs) were used to isolate and concentrate the signals.1) The presence of target DNAs lead to the hybridization between the DNA functionalized QDs, MBs and the target DNA. In fact, the QDs-MBs complex can be isolated and then concentrated. The binding of the QDs to the MBs was confirmed by confocal microscopy and Cd elemental analysis. It was found that the fluorescent intensity was proportional to concentration of the target DNA, while the presence of non-complementary DNA produced no significant fluorescent signal. In addition, the presence of low copies of target DNAs such as 0.5pM in large volume samples up to 40ml were successfully detected by using a magnet-assisted concentration protocol which consequently results in the enhancement of the sensitivity more than 100-fold.

Retinal and its derivatives represent essential compounds in many biological systems. In animals, they are synthesized through a symmetrical cleavage of β-carotene catalyzed by a monooxygenase. We have cloned β-carotene 15,15'-monooxygenase genes from 4 bacteria of Synechococcus elongatus, Salinibacter ruber, Natronomonas pharaonis and Halobacterium salinarum and 2 vertebrates of Homo sipiens and Mus musculus. We also synthesized an artificial E. coli codon optimized gene, based on the amino acid sequence of blh gene of uncultured marine bacterium 66A03. The β-carotene 15,15'-monooxygenase expression in E. coli engineered to produce β-carotene led to accumulation of retinal at the expense of β-carotene. Retinal production of 2.2mg/L was obtained in recombinant E. coli harboring blh, monooxygenase genes of H. salinarum while the other monooxygenase genes cloned here produced trace amount of retinal. The highest retinal amount of 8.9 mg/L was produced by the artificial E. coli codon optimized monooxygenase genes. This work was supported by the EB-NCRC (Grant No. R15-2003-012-02001-0), BioGreen 21 Program (Grant No. 2005041034590), Brain Pool program and BK21 program of Korea.

Aptamers are ssDNA or RNA oligonucleotides bind to a wide range of target molecules with high affinity and specificity and they are applicated useful sensing molecule. The target, RBP4 secreted from adipocytes is known to be potential biomarker for type 2 diabetes. RBP4 aptamers have been selected by FluMag-SELEX process from random DNA library, composed of approximately 3.0 x 1016 DNA molecules. To analyze aptamers' affinity and specificity for RBP4, Surface Plasmon Resonance(SPR) was used. No.38 aptamer was found to show highest specificity with low Kd value 226 nM among others. Detection of the target protein using aptamer was compared with Western blotting analysis and ELISA Assay for serum samples. The data shows that this aptamer biosensor is specific and effective for the detection of RBP4 in serum samples and could be used as a diagnosis tool.

Polyethylene glycol (PEG) conjugated biomolecules, such as proteins, antibody fragments, aptamers, peptides, and other small molecules, are widely used, because PEG can increase the solubility and stability of conjugated biomolecules in general. The chemical attachment of PEG to these biomolecules is referred to as “PEGylation". Various PEGylation techniques are well established so far, especially the reaction conditions, desired site and degree of PEGylation, etc. In this study, phase-separation properties of PEG or PEGylated IgG with dextran solution was compared. PEG (M.W. 10000) or PEGylated (M.W. 10000) mouse anti-human IgG was used with dextran T500 (10%, w/w) solution. The volume ratio of two phases in microchannel was compared based upon the infused volumetric flow rate of each polymer solution. Developed technique will be used for the simultaneous phase-separation and affinity separation of biomolecues.

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 there by 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 EB-NCRC (Grant No. R15-2003-012-02001-0), Technology Development Program for Agriculture and Forestry (Ministry of Agriculture and Forestry), Brain Pool program and BK21 program of Korea.

We describe here a novel method for the surface immobilization of DNA oligonucleotides using a gold nanoparticle as a mediator. The outer surface of gold nanoparticle is electrostatically attached to amine-modified DNA and, subsequently the gold-DNA complex is easily immobilized on the amine or aldehyde activated glasses through covalent bond or electrostatic force. Based on optimization of gold-DNA concentration and linker effect experiments, sensitivity and specificity of the gold-DNA complex system were tested through hybridization reaction with two kinds of capture probes consisting of different sequences. As a result of the gold-DNA complex system, the surfaces reveal high immobilization efficiency and hybridization intensity for amine-modified DNAs compared with conventional immobilization method. Furthermore, as we use gold nanoparticle as a mediator, it is possible to do silver enhancement enabling us to monitor the capture probe status just after immobilization washing step.

The identification of the substrate specificity of a protein kinase is critical in understanding its role and function in a cellular signal transduction network. [1,2] Here, a new high-throughput system was developed to identify the substrate specificity of PTKs, p60c-src and ZAP-70, using a fully randomized "one-bead one-compound"(OBOC) combinatorial peptide library constructed by ladder synthesis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). As expected from previous studies, the substrate specificity of p60c-src showed that acidic amino acids (Glu and Asp) at the +1 and +2 positions and Ile at the -1 position are necessary for efficient tyrosine residue phosphorylation. To generalize our method, the substrate sequence identification of ZAP-70 tyrosine kinase was carried out. Interestingly, glutamic acid (Glu) at the +2 and +1 position appeared most frequently (49% and 21%) according to the sequence analysis. Aspartic acid (Asp) at the +2 position was the second most frequently appearing residue (29%). Moreover, Glu and Asp were shown to be the most likely amino acids present at the -1 position, but there was no selectivity at the -2 position. In addition, the annotation of potential target proteins for the p60c-src and ZAP-70 kinase signal transduction pathway was undertaken using the SwissProt database.

본 연구에서는 미생물 발효공정의 모니터링을 위해서 졸-겔을 이용한 pH와 용존산소 검출막을 개발하였다. 졸-겔은 물리, 화학적 안정성이 우수하여 다양한 분야의 서에 적용이 가능하다. 특히, GPTMS와 MTMS 그리고 TEOS의 실란계 화합물로 제조한 졸-겔은 폴리에스테르 필름에 스핀 코팅시 열경화 건조를 하기 때문에 건조시간이 매우 짧은 이점을 가진다. 제조한 pH와 용존산소 검출막은 pH와 용존 산소량에 따라 형광세기를 측정한 결과, 센서막의 감도가 우수하며 재현성이 우수하였다. 그리고 절광층을 코팅한 검출막은 코팅하지 않은 검출막에 비해 검출 감도가 향상되었다.

The thin films of oxygen and pH sensing membranes were fabricated by entr-apping Ru( ) complex and 6-aminofluorescein in Ⅱ the sol-gel matrix. The sol-gel matrix was prepared by the silane compounds of precursors, tetraethyl-orthosilicate(TEOS), methyl-trimethoxy silane(MTMS) and 3-glycidoxypropyl-trimethoxysilane(GPTMS), in ethnol solution. To prevent light scattering, the sensing membranes were recoated light-insulating layer. The layer was prepared by tetramethyl orthosilicate(TMOS) and dimethoxy-dimethyl silane(DiMe-DMOS). These sensing membranes are suitable for a variety of applications, including environmental monitoring and cell cultivations.

The toxic modes of actions by silver nanoparticles were investigated with a panel of recombinant bioluminescent bacteria and compared with the results obtained from gold nanoparticles and silver nitrate. Based upon the changes in the bioluminescence, the silver nanoparticles resulted in a higher cellular toxicity than the same amount of gold nanoparticles. Furthermore, using the stress-specific nature of the promoters, it was determined that the silver nanoparticles cause heat shock and oxidative damage to the bacterial cells, but not DNA damage, while gold nanoparticles did not cause any bioluminescence induction at the same test concentrations. Silver nitrate, used to evaluate differences between the silver ion and the nano-sized silver particles, was also found to causes heat shock damage and oxidative stress within the bacterial cells.

D-Psicose and D-allose, a rare keto and aldo-hexose, are not abundant in nature and are difficult to prepare by chemical methods. For the production of D-allose in E. coli, we used galactose 6-phosphate isomerase coding lacAB of Staphylococcus aureus, Lactococcus lactis, and ribose 5-phosphate isomerase coding rpiB of Clostridium thermocellum, Thermotoga maritima, Corynebacterium glutamicum. Through the whole cell reaction using recombinant E. coli harboring lacAB or rpiB, psicose was not converted to allose, but conversion of allose to psicose was observed. The enzymes used here were shown to have the biased isomerization activity from allose to psicose. In the enzyme reaction using ribose 5-phosphate isomerase of C. thermocellum, which had the highest isomerization activity among the tested enzymes, isomerization of allose to psicose was only occurred.

SMB(simulated moving bed) is a chromatographic process that, unlike traditional HPLC system, operates continuously without losing the enantiomeric purity of the outlet streams. We performed separation experiments for separating D- and L-form from racemate mandelic acid mixture by using lab-scale 4(1-1-1-1)zone SMB unit with 9 operation conditions which were change of feed concentration and switching time. The operating conditions were chosen from results simulated by Aspen chromatography as well as batch chromatographic HPLC experiments. We separated approximately 94% of D-mandelic acid in extract stream. Operating costs at 9 operating conditions were also compared.

To search an effective extraction method of isoflavones from kudzu (Pueraria lobata Ohwi), superior to a conventional solvent extraction such as maceration and soxhlet extraction, an ultrasonication-assisted extraction (UAE) method was explored. The effects of different extraction conditions were determined, i.e., temperature, ultrasonic power, solvent types, and compositions of ethanol in ethanol-water mixtures. The results show that the yield increased with extraction times and extraction temperatures. The percent extraction efficiency of isoflavones using ultrasound was found to be highly dependent on the type of solvents. Furthermore, the use of ethanol-water solution as extraction solvent increased the yield of isoflavones due to the relative polarity, the swelling effect of plant tissue matrix by water, and increased sound absorption. To achieve the same recovery as that achieved by UAE, soxhlet extraction and maceration required much longer time.

The rotifer, Brachionus rotundiformis is most commonly used as live feed for fish larvae cultures at early stages. Larval fish generally have a high demand for dietary protein due to their rapid growth rates and extensive catabolism of amino acids for the production of metabolic energy1). The rotifer has been studied as a food source for marine fish because of its size and rich nutrients, which make it a good source for large quantity cultivation2). In order to identify the antioxidative peptide in the hydrolysate of the rotifer, which is hydrolyzed by Alcalase, Neutrase, -chymotrypsin, α papain, pepsin, and trypsin in a batch reactor, the antioxidative activities of hydrolysates were evaluated using direct free radical scavenging activity. Among the six hydrolysates, peptic hydrolysate, had the highest antioxidative activity compared to the other hydrolysates. The peptide showing strong antioxidative activity was isolated from the hydrolysate using consecutive chromatographic methods including Sephadex G-25 Gel-chromatography and high-performance liquid chromatography on an ODS column. The antioxidative peptide was purified and the molecular weight measured. Further studies are planned to measure the cytotoxic effect on MRC-5 and ECV304 cell lines.

The collagen peptides can be extracted from skin, sinew, cartilage, muscles, and the internal organs of cows or pigs1). According to molecular structure and physical chemistry characteristics, the collagen peptides have been used in various industries such as quality foods, medicines and cosmetics2). In the case of fish, collagen peptides are the main component of bone, fin and skin. In this study, we isolated the bioactive peptides related to antioxidative activity and ACE inhibitor activity. The major amino acids found in skate skin were glycine, glutamic acid and serine, and others contained aspartic acid, arginine and leucine. The major amino acids were contained in more than 50% of the total amino acids. The skate skin hydrolysates were obtained by enzymatic hydrolysis using Alcalase, -chymotrypsin, α Neutrase, pepsin, papain, and trypsin. Among the six hydrolysates, the papain hydrolysate had the highest DPPH radical scavenging activity compared to other hydrolysates of the skate skin. In addition, the ACE inhibitor activity of the hydrolysates, obtained from the skate skin, using α-chymotrypsin, trypsin, Alcalase, and papain were 75.72%, 65.79%, 63.99%, and 57.35%, respectively. In future studies the peptides with strong antioxidative and ACE inhibitory activity will be purified from the hydrolysate using consecutive chromatographic methods including sephadex G-25 gel-chromatography and high-performance liquid chromatography. In addition, the purified peptides will be analyzed to identify the amino acid sequences using an amino acid sequencer.

Recently, functional materials have been screened from protein hydrolysates of various fishery sources, and a number of functional peptides have been identified. The peptides isolated from protein hydrolysates were studied to determine the bioactive substances such as antioxidative activity, angiotensinⅠ converting enzyme (ACE) inhibitor activity and antibiotic activity1). The peptides regulating blood pressure are known to be potent inhibitors of ACE. Rotifers are commonly used as live feed for fish. Larval fish generally have a high demand for dietary protein due to their high growth rates, and extensive catabolism of amino acids for the production of metabolic energy2). In this study, the hydrolysates of the peptides from the rotifer, Brachionus rotundiformis were investigated for ACE inhibitors. The proximate compositions of the rotifer were measured to be 63.25% crude protein, 17.77% lipid, 7.72% carbohydrate, 6.54% moisture, and 4.72% ash. In addition, the major amino acid composition of the rotifer was glutamic acid (11.14%), aspartic acid (9.93%), leucine (9.69%), and glycine (8.47%). The rotifer protein was hydrolyzed using Alcalase, α-chymotrypsin, Neutrase, pepsin, papain, and trypsin in a batch reactor. Among the six hydrolysates, the peptic hydrolysate had the highest ACE inhibitory activity compared to the other hydrolysates. The peptides with strong ACE inhibitory activity were isolated from the hydrolysate using consecutive chromatographic methods including Sephadex G-25 Gel-chromatography and high-performance liquid chromatography. Further study is planned to analyze the amino acid sequence of peptides purified from the peptic hydrolysates of the rotifer.

Lycopene, a precursor of various carotenoids can be synthesized from FPP in E. coli by introduction of crtE, crtB, and crtI. Moreover, the lycopene-producing E. coli is able to produce β-carotene, zeaxanthin, or astaxanthin by introduction of crtY, crtZ, or crtW. The plasmids, pT-LYCm4 for lycopene, pT-HB for β-carotene, and pT-ZUM6 for zeaxanthin were constructed to produce each carotenoid in E. coli. β-Carotene can be converted to astaxanthin via zeaxanthin by crtZ, or via canthaxanthin by crtW. For production of astaxanthin in E. coli (pT-ZUM6), crtW and crtZ genes were cloned from 6 bacteria, Brevundimonas aurantiaca, Brevundimonas vesicularis, Novosphingobium aromaticivorum, Parvularcula burmudensis, Robiginitalea biformata, and Xanthobacter autotrophicus. Astaxanthin of 5.4 mg/L and other intermediate carotenoids were produced from the E. coli (pT-ZUM6) harboring crtW of B. vesicularis. There are various intermediate carotenoids on the biosynthetic pathway from β-carotene to astaxanthin. The crtZ and crtW cloned from the other bacteria did not show significant activities. This work was supported by the EB-NCRC (Grant No. R15-2003-012-02001-0), BioGreen 21 Program (Grant No. 2005041034590), Brain Pool program and BK21 program of Korea.

β-Carotene synthesis plasmid pT-HB was used for cotransformation with pSdxs and pSSN12Didi which increased IPP and DMAPP synthesis. β-Carotene production significantly increased 1.5-fold with the amplification of the dxs gene through pSdxs and 4-fold with the mevalonate bottom pathway of pSSN12Didi in the presence of 3.3 mM mevalonate. The pT-DHB, constructed by integrating the dxs gene into pT-HB, was used for co-transformation of E. coli harboring pSSN12Did, resulting into β-carotene production of 141 mg/L. Recombinant E. coli harboring pT-DHB and pSSN12Didi was used to maximize β-carotene production by adjusting the available amounts of glycerol, a carbon source, and mevalonate, the precursor of the mevalonate bottom pathway. When recombinant E. coli was given 16.5 mM mevalonate and 2.5% (w/v) glycerol, β-carotene production of 503 mg/L in concentration was obtained at 144 hours. This work was supported by the EB-NCRC (Grant No. R15-2003-012-02001-0), BioGreen 21 Program (Grant No. 2005041034590), Brain Pool program and BK21 program of Korea.

Covalent conjugation of poly(ethylene glycol) (PEG) to therapeutic proteins increases their in vivo stability by protecting the protein from degradation, masking its immunogenic sites and reducing clearance1). Frequently, PEGylation results in non-site specificity and multiple attachments, which might mask or interfere with the receptor binding sites, causing a dramatic decrease in in vitro bioactivity. To solve this problem, site-specific PEGylation has been employed2). Epidermal growth factor (EGF) has been used as a medical treatment, since it controls and accelerates the epidermal cell's proliferation3). In this study, we demonstrated the PEGylation of the thiolated EGF using 2․iminothiolane․HCl, which can improve the binding interaction between a PEG molecule and a EGF protein and yield of the PEGylation. The site of PEGylation was identified through tryptic peptide mapping and Matrix Assisted Laser Desorption-Time of Flight (MALDI-TOF) techniques, which shows mass analysis and identified through Reversed Phase-HPLC, which can confirm the existence of tri-, di-, mono-EGF PEGylates and their purity levels.

Simulated moving bed(SMB) chromatography is a chromatographic process to solve several difficult separation problems in batch chromatography. We carried out pulsed input method(PIM) to find adsorption isotherms of L-ribose and L-arabinose in NH2 column. Adsorption isotherm is calculated by Aspen chromatography, and compared with the simulation for separation of L-ribose and L-arabinose. SMB operation is a formidable task since it is a periodic cyclic process. To find the most suitable separation condition in SMB, we carried out simulations in m2-m3 plane base on the triangle theory and calculated operating parameters (flow rates of four zones, switching time and feed concentration and so on) using Aspen chromatography.

Tetragonia tetragonioides has long been used as a traditional remedy for stomach cancer and furuncle. We investigated antioxidant, acetylcholinesterase (AChE) and lipoxygenase (Lox) activity for the solvent extracts from Tetragonia tetragonioides. The CH2Cl2 and EtOAc extracts of Tetragonia tetragonioides were found to exhibit a distinctive antioxidant activity. The fractions of methanol, n-hexane, CH2Cl2, EtOAc and n-buOH were caused concentration-dependent inhibition of soybean Lox and AChE activity. The results indicate that the fractions of Tetragonia tetragonioides containing these compounds inhibit Lox and AChE activity. The MeOH extracts of Tetragonia tetragonioides exhibited moderate inhibition of the enzyme, defined as more than 40% at 1 mg/ml. Thus, these compounds or fractions containing them may be beneficial for the treatment of inflammatory conditions and Alzheimer's desease.

In the present work, we report the results of a study at evaluating the acetylcholinesterase (AChE) and β-secretase (BACE) inhibitory activity of the solvent extracts from Alnus sp. The fractions of methanol, n-hexane, CH2Cl2, EtOAc and n-buOH extracts were tested for AChE inhibitory activity using the Ellman colorimetric method. The results showed that the MeOH and EtOAc fraction of Alnus sp. at concentration of 1 mg/ml inhibited more than 50% of AChE activity. The EtOAc extract of Alnus sp. also exerted an inhibitory effect against BACE activity. These finding may contribute to biological significance in that AChE inhibitor has been used as a drug for the symptomatic treatment of Alzheimer's desease.

The aim of this study was to identify antifungal active bacteria with purify the strong biological active compounds. The identification of isolated bacteria was carried out by 16s ribosomal DNA sequence analysis. Strain 320 showed most active for Rhizoctonia solani, Botrytis cinerea and Fusarium oxysporium. Ethyl Acetate fraction of culture broth was confirmed the inhibitory zone by plate assay the compound of Rf = 0.4 by silicagel thin layer chromato graphy (TLC, n-exane : Ethyl Acetate (5 : 1, v/v)) represented high antifungal activity against at broad range of plant pathogenic fungi.The aim of this study was to identify antifungal active bacteria with purify the strong biological active compounds. The identification of isolated bacteria was carried out by 16s ribosomal DNA sequence analysis. Strain 320 showed most active for Rhizoctonia solani, Botrytis cinerea and Fusarium oxysporium. Ethyl Acetate fraction of culture broth was confirmed the inhibitory zone by plate assay the compound of Rf = 0.4 by silicagel thin layer chromato graphy (TLC, n-Hexane : Ethyl Acetate (5 : 1, v/v)) represented high antifungal activity against at broad range of plant pathogenic fungi.

Natural indigo dye is the earliest dye obtained from leaves of plants. Various chemical synthesis methods for indigo dye had been reported. Using the chemical synthesis, indigo is produced with higher productivity and simpler process than obtainment from leaves of plants. The chemical synthesis of indigo, however, makes toxic materials which make pollution and bad effect on the human health. The indigo fermentation using microorganism has good characteristics to overcome the aforementioned problems of chemical synthesis and obtainment from plants. While indigo dye is produced by cell cultivation, indirubin is produced as one of byproducts. Indirubin and its derivatives are the inhibitor of cyclin dependent kinase (CDK) which participates in cell differentiation. Therefore, many studies to investigate the virtue of indirubin as the anticancer drug are performed. Before the purification of indirubin by using chromatography, a pretreatment step for crude indigo dye is necessary. Due to the low solubility of indigo and indirubin in water, several organic solvents were tested for selective batch extraction of indirubin from crude indigo dye. For efficient extraction, extraction process was developed by continuous operation.

A simple and rapid microwave-assisted extraction procedure was developed and optimized for the extraction of paclitaxel from the plant cell culture of Taxus chinensis. The samples, immersed in a methanol-water mixture, were irradiated with microwaves in a closed-vessel system. The microwave-assisted extraction was compared with the existing conventional solvent extraction in terms of yield, purity, throughput, and solvent consumption.

Rotifers are a valuable and indispensable food source for industrial larviculture and crustaceans worldwide. Rotifers possess several characteristics that make them suitable live prey for newly hatched fish larvae, e.g. relative small size, slow swimming behavior, rapid reproduction rate, ability to be cultured at high densities1). However, the rotifers are an incomplete food source, because they are low in n-3 highly unsaturated fatty acids, DHA and EPA that are required for survival and successful development of marine fish larvae2). In this study, lipid was extracted from the rotifer by the method of Bligh and Dyer3). The proximate composition of the rotifer were measured to be 63.25% crude protein, 17.77% lipid, 7.72% carbohydrate, 6.54% moisture, and 4.72% ash. Among the fatty acids in the rotifer lipids, palmitic, linoleic, vaccenic, and stearic acids were the pre-dominant fatty acids, accounting for almost 35.8%, 21.5%, 15.9%, and 7.7%, respectively. The different lipids extracted from the rotifer were isolated including fatty acids, monoacylglycerol, diacylglycerol, and triacylglycerol acids by thin-layer chromatography. The neutral lipids, glycolipids and phospholipids, were fractionated using organic solvents by silica gel column chromatography. To improve the lipid content, we modified the rotifer lipids by hydrolysis and glycerolysis using lipases such as porcine pancrease and Candida rugosa.

A strain producing biosurfactant, GBM 3309 was isolated from the soil sample of Kyebang Mountain by the antifungal and emulsification assessment. From 16s rDNA analysis, strain GBM 3309 was related to Bacillus subtilis. The growth, surface tension, emulsifying activity and stability of the isolate were investigated by physical and chemical tests. The emulsifying stability reached a highest degree when tributyrin was used as a substrate, suggesting that its function was similar to Tween 20 and Tween 40, compared with a variety of synthetic surfactant. The high emulsifying activity was also obtained when soybean oil and crude oil were used as the substrate. The biosurfactant substance from Bacillus subtilis. GBM 3309 were also analyzed by ESI-MS, GC-MS and MALDI-TOF mass spectra, respectively.

Homoharringthonine was purified from Cephalotaxus koreana1-2) by a combination of extraction, synthetic adsorbent treatment, low-pressure chromatography, and high-performance liquid chromatography(HPLC). A crude extract was obtained by methanol extraction of biomass, followed by liquid-liquid extraction using chloroform. The waxy compounds were efficiently removed by adsorbent(active clay P-1) treatment. The extract was purified to greater than 52% with 86.4% step yield by silica gel low-pressure chromatography. High-performance liquid chromatography steps, which were composed of an HPLC step with silica column and an HPLC step with ODS column, were applied to give 98% purity with high yield. Amorphous homoharringtonine, with a fine particle size, was simply made by dissolving homoharringtonine in methylene chloride/methanol(98/2, v/v), followed by spray drying. Residual solvents, methylene chloride and methanol were easily removed by to less than concentration limit in ICH guidance by spray drying. Amorphous homoharringtonine was markedly more soluble in water than crystalline homoharringtonine. This information is very useful for production and quality control for pharmaceutical in commercialization step.

The 1st anion exchange chromatography component of the r-hGH manufacturing process was validated in this study, using a validation protocol consistent with both policy and SOP1, 2). Three consecutive lots of crude r-hGH solutions were generated via anion exchange chromatography, which was operated within the operating parameters pre-established via in-process control. For quality control, the buffer solutions employed in 1st anion exchange chromatography were first assessed via tests of pH, endotoxin and bioburden tests. Following 1st anion exchange chromatography, tests for endotoxin, ECP, IEF, Native-PAGE, and protein purity and yield were conducted in order to validate the eluate. All three tested lots satisfied the acceptance criteria pre-established for the following purification process (the 2nd anion exchange chromatography) endotoxin: ≤350 EU/mg; residual host proteins: ≤ 42 ppm; purification yield: 32~36%; purity: ≥ 86%. Therefore, the 1st anion chromatography process was validated in this study, and consistently yields crude r-hGH solutions that fulfill the pre-established criteria for the next purification process.

Fractional precipitation is a simple, efficient method for pre-purifying paclitaxel from plant cell cultures. The pH of fractional precipitation was optimized in terms of the yield and purity of paclitaxel with a fixed methanol concentration in water (61.5%, v/v), paclitaxel content in the crude extract (0.5%, w/v), and storage time (3 days). The maximum yield and purity of paclitaxel were obtained at pH 8.0 with potassium phosphate buffer. At a constant storage temperature (4℃), both the precipitate yield and purity increased rapidly up to 18 h, and then increased more slowly. This pre-purification process serves to minimize solvent usage, and the size and complexity of the high-performance liquid chromatography (HPLC) operation required for paclitaxel purification.