Earticle

Cytochrome P450, the heme-thiolate proteins are the ubiquitous mono-oxygenase enzymes which plays a key role in almost all living forms; prokaryotes to eukaryotes. The P450 enzymes catalyze versatile oxidation reactions and have wide substrate specificity. Despite, the P450 enzyme plays a crucial role in fungi by its hydrophobic conversion of the primary and secondary metabolites and in the adaption of diverse ecological niches by degrading the ecological pollutants. Indeed, genome sequencing projects has revealed the presence of several thousands of putative P450 genes from various families in the fungal kingdom. Bioinformatic annotations showcase the presence of 169 putative P450 genes and 2 putative Cytochrome P450 reducatase (CPR) genes from the whole genome sequence of Fusarium oxysporum f. sp. lycopersici. We initiated the functional genomic research of this fungus by screening and selecting the target putative novel P450s based on the multiple alignment and the phylogenetic analysis with the reported P450 genes of other organisms with various novel functionalities. So far, the target P450s selected includes the Sterol 14α-Lanosterol demethylase, Hydroxylation of Flavonoids (flavine and naringenin), Hydroxylation of Benzoates, Oxidation of polycyclic aromatic hydrocarbons (PAH) and Toluene, etc. To characterize the functional FoCYPs, cDNAs are synthesized by optimizing the culture conditions and the transcriptional parameters. To date, about 20 P450 genes and CPR genes are been isolated and cloned, which will be expressed to analyze biochemically. Consequently, the FoCYP P450 gene library will be constructed, which drives us towards an intensive approach to determine the functionalities of novel P450s.

Stem cells have become very important resources for basic research on cell replacement therapy and other medical applications. Especially, human embryonic stem (hES) cell is derived from the inner cell mass cells of early mammalian blastocyst. This cell is pluripotent and generally retain their long-term proliferative potential in an undifferentiated state. Also, hES cell can differentiate into derivatives of all three embryonic germ layers upon transfer to in vitro differentiation culture conditions or an in vivo environment. Recently, Our team established induced pluripotent stem (iPS) cell that minimizes the ethical problem associated with the use of human embryos. These cells become a powerful tool for in vitro investigation of developmental processes at both cellular and organism levels, and offer tremendous potential for clinical application as an unlimited source of cells for transplantation and tissue generation therapies. This lecture will focused on I) Establishment of hES and iPS cells II) Tansplantation of hES cell into disease animal model III) Things to do in the near future.

2010년 세계 바이오의약품 규모는 1490억불 규모로 전체 의약품 시장의 약 17%를 점하고 있으 며 2015년에는 그 규모가2390억불에 이르러 연평균 10%의 높은 성장율을 보이고 전체 의약품 시장에서의 비중도 크게 확대될 것으로 예측되고 있다. 2009년 국내 바이오의약품 시장 규모는 총 2조 5363억원으로 세계시장의 약 1.6%에 해당하는 미미한 규모이다. 우리나라 제약기업들은 시장과 규모의 영세성에도 불구하고 신약 개발에 많은 자원과 노력을 투 자하여 현재까지 15개의 신약을 상업화하는데 성공하였으나, 정작 큰 시장을 만들어 내는 데에는 많은 아쉬움이 있는 형편이다. 이와 같이 신약의 개발이 시장의 창출로 이어지지 못하고 있는 이유로는 우리나라 제약기업들이 강점을 가질 수 있는 분야에 대한 선택과 집중이 되지 못하였고, 시장 창출에 대한 구체적이고 실 현 가능한 전략 없이 물질의 개발에만 매달렸으며, 영세한 국내시장을 벗어나 글로벌 시장으로 나 아가려는 전략 부재 등이 그 원인으로 꼽히고 있다. 이번 발표에서는 실질적인 가치 창출이 가능한 제품 개발을 위한 한국 바이오의약 기업의 전략 사 례에 대하여 논의하고자 한다.

Currently, chemicals come as a side product of the refining of petroleum to fuels. As biomass replaces petroleum as the fuel feedstock, chemicals will also come from biomass. Some chemicals will come from fermentation of the sugars in feedstocks; others will come from the lignin in biomass. Enzyme-catalyzed reactions could be a key to convert lignin to chemicals. Enzymes could fragment lignin and can convert the resulting the mixture of aromatic compounds into chemical intermediates. However, nature has not evolved enzymes for this purpose. To find suitable enzymes, we have adapted natural enzymes and engineered new ones for these reactions. Perhydrolases make peracetic acid for lignin fragmentation, while converting the aromatic compounds to chemicals can involve decarboxylations, additions to carbonyl compounds and hydroxylations. This lecture will review current progress in this area.

Efficient conversion of cellulosic biomass to ethanol requires utilization of both hexoses (C-6 sugar) and pentoses (C-5 sugar), primarily glucose and xylose. Saccharomyces cerevisiae which has been used for ethanol production from starch and sugarcane cannot convert xylose to ethanol because the yeast does not have a xylose metabolizing activity. S. cerevisiae was metabolically engineered for converting xylose to ethanol by introducing xylose reductase (XR) and xylitol dehydrogenase (XDH) from Pichia stipitis, a natural xylose metabolizing yeast. Even with metabolic engineering of S. cerevisiae, the recombinant S. cerevisiae cannot utilize xylose as efficiently as glucose due to competitive transport of xylose and glucose for the hexose transport system. S. cerevisiae has transporters (proteins responsible for sugar transport) with high affinity for glucose relative to xylose and hence xylose transport is strongly inhibited by the presence of glucose in the fermentation broth. A fermentation strategy has to be designed by taking into account competitive transport between xylose and glucose for the hexose transporters in S. cerevisiae. This presentation will cover metabolic engineering of the xylose assimilation pathway and optimization of a fermentation process in an attempt to improve ethanol productivity from cellulosic biomass.

Bioethanol from cellulosic biomass has the potential to become an competitive fuel resource, but currently requires many technological improvements to be economically viable. One-pot fermentation of cellulosic biomass into bioethanol through consolidated bioprocessing (CBP) using multi-functional microbes can make a major breakthrough for low cost biomass processing. Unfortunately, however, there are no natural microbes which can do this process effectively. Brewer's yeast Saccharomyces cerevisiae is often chosen as a candidate workhorse for CBP owing to its high productivity of ethanol and resistance to ethanol, even though it has no cellulolytic activity. To develop S. cerevisiae as a CBP yeast, it is essential to make yeast produce sufficient amounts of cellulases required for the complete hydrolysis of cellulosic biomass. Hyper-secretion technology using yeast translational fusion partner (TFP) have been applied to produce several fungal cellulases and hemicellulases. Grams per liter of different cellulases could be secreted into culture supernatant during fed-batch fermentation. Reconstituted cellulase cocktails using cellulases produced separately, showed cellulolytic activities on a filter paper and pretreated biomass. Recombinant yeasts of this study will be useful for the development of a cost-effective CBP system to convert cellulosic biomasses into bioethanol.

Escherichia coli has been explored as a host for an alternative biofuel butanol production because of its many advantages such as a fast growth and easy genetic manipulation. However, the low butanol-tolerance of E. coli (<1% [vol/vol] butanol) is a major obstacle for their exploitation in the butanol production. Here we developed a new method to increase the butanol-tolerance of E. coli by artificial transcription factor (ATF) libraries which consist of zinc finger (ZF) DNA-binding proteins and an E. coli cyclic AMP receptor protein (CRP). Using these ATFs, we could select a butanol-tolerant E. coli which can tolerate up to 2.5% (v/v) butanol and then introduced a butanol synthesis pathway into the selected butanol-tolerant E. coli. The resulting butanol-producing strain E. coli showed increase in butanol production by ~20% compared to a wild-type E. coli strain engineered with the same ATFs described above. As an alternative, the butanol tolerance of E. coli is also improved by regulating the fatty acid composition of the plasma membrane. In addition, DNA scaffold approach is being explored to further increase the butanol production. The strategies described herein will open up an unexplored frontier for high-yield butanol production in E. coli.

Abstract: Microalgae, the light-driven cell factory, represents a biomass and renewable resource for liquid bio-fuel production. In this study, a novel photosynthesis-fermentation model was created to merge the positive aspects of autotrophs and heterotrophs. Microalga Chlorella protothecoides was grown autotrophically for CO2 fixation and then metabolized heterotrophically for oil accumulation. Compared to typical heterotrophic metabolism, 69% higher lipid yield on glucose was achieved at the fermentation stage in the photosynthesis-fermentation model. An elementary flux mode study suggested that the enzyme Rubisco catalyzed CO2 re-fixation, enhancing carbon efficiency from sugar to oil. This result may explain the higher lipid yield. In this new model, 61.5% less CO2 was released compared with typical heterotrophic metabolism. Immunoblotting and activity assay further showed that Rubisco functioned in sugar bleaching cells at the fermentation stage. Overall, the photosynthesis-fermentation model with double CO2 fixation in both photosynthesis and fermentation stages, enhances carbon conversion ratio of sugar to oil and thus provides an efficient approach for the production of algal lipid and liquid bio-fuels.

Industrial microorganisms such as Saccharomyces cerevisiae and Corynebacterium glutamicum were engineered for assembly of designer minicellulosomes by heterologous expression of a recombinant scaffolding protein from Clostridium cellulovorans and chimeric enzymes such as cellulase and agarase from different organisms. The chimeric enzymes fused with the dockerin domain of endoglucanase from C. cellulovorans was assembled with the recombinant scaffolding protein. The resulting strain was able to ferment different recalcitrant biomass including cellulose and agarose into commodity biofuels such as ethanol and biodiesel. The in vivo assembled minicellulosomes retained the synergistic effect for biomass hydrolysis. The minicellulosomes containing the cellulose binding domain was purified by crystalline cellulose affinity in a single step. This result indicates that a one-step processing of biomass in a consolidated bioprocessing configuration is carried out by recombinant microbial cells expressing functional minicellulosomes. The development of a more effective and high-yield cellulosic and/or agarosic biofuel fermentation process is required to bring about a necessary dramatic reduction of production costs. So this novel consolidated bioprocess will lead to more efficient commodity biofuel production from various sources of plant and marine biomass.

Biodiesel from microalgae is expected to be a desirable, renewable biofuel that can replace petroleum-derived transport fuels. However, the development of a cost-effective harvesting method is required to reduce the production cost and realize the economic feasibility. A partial factorial design was used to screen the main factors among several inorganic coagulants and bioflocculants. These factors were different, depending on the algal genera. Response surface methodology was used to further investigate the optimal conditions for these factors on flocculation of Scenedesmus sp. and Botryococcus braunii. Analysis of variance and other relevant tests, such as lack of fit and residual probability plots, confirmed the validity of the suggested empirical model. The optimal flocculation conditions could be easily derived using the obtained equation. The experimentally determined flocculating activities corresponded with the estimated values. The equation suggested using response surface methodology could be applied to optimize harvesting conditions and minimize the related costs of biodiesel production.

One of the major barriers for production of eukaryotic transgenic organism is very low or no expression of transgene. Transgene silencing is also a common phenomenon in Chlamydomonas reinhardtii as well as in higher plants. To develop the stable expression system of transgene in C. reinhardtii, we transformed a suppressor of RNA silencing, Cucumber mosaic virus 2b (CMV2b) and evaluated the RNA silencing activity in C. reinhardtii. Inhibition of Maa7 silencing was detected in CMV2bexpressing Maa7-IR44 strains, indicating that CMV2b suppressed siRNA pathways in C. reinhardtii as in higher plants. In addition, mRNA expression targeted for cleavage by miRNA was significantly higher in CMV2b-expressing strains, but increased accumulation of miRNA was not detected. Furthermore, reduction of AGO1, AGO2, AGO3, and DCL1 expression were detected in CMV2b-expressing strains. Taken together, our results suggest that CMV2b suppress both siRNA and miRNA pathways in C. reinhardtii, and CMV2b-expressing C. reinhardtii can be used as a host line for stable expression of transgene.

Botryococcus braunii, green microalgae, is characterized by its considerable hydrocarbon production. However, established extraction methods for hydrocarbons are limited due to low efficiency and laborious culture. This study presents solvent recycling as an extraction method to improve hydrocarbon recovery yield as well as biomass production of B. braunii. A photobioreactor was designed to operate the solvent recycling extraction process. Hydrocarbon recovery yield using this method was 2.5-fold higher than the existing two-phase extraction with high amount biomass 3.5g/L. Three-times of recycling process revealed high efficiency for hydrocarbon from B. braunii without cell loss. The method herein also successfully reduced clump formation and moreover, could be used to regenerate algal cells after three cycles of the extraction process. Accordingly, solvent recycling could recover the hydrocarbons without serious cell damage by an upstream solvent.

Photosynthetic biofuel production is one of alternative forms of energy supply to fossil fuels since its use is controversially considered as a main reason for high oil prices and global warming. In addition to traditional uses in providing food and feed application, photosynthesis will continue to be used for transport energy and chemical feedstock. Algal photosynthesis has been considered as one of the system to be maximized for energy application by means of synthetic biology. Over the years, cyanobacteria have been regarded as ideal model systems for studying fundamental biochemical processes like oxygenic photosynthesis and carbon and nitrogen assimilation. They have been also used as human foods, sources for vitamins, proteins, fine chemicals, bioactive compounds, and even as alternative energy crops. Additionally, aiming to increase plant productivity as well as nutritional values, cyanobacterial genes involved in carbon metabolism, fatty acid biosynthesis, and pigment biosynthesis have been intensively exploited as alternatives to homologous gene sources. In this talk, I’l present overview on algal photosynthesis as live fuels replacing fossil fuels.

Removal of carbon dioxide becomes one of important research area in recent green era. But, instead of just removal, carbon dioxide can be utilized as a good resource to obtain high valuable chemicals. Many marine organisms use carbon dioxide as useful source for their lifes. Some organisms fix carbon dioxide to synthesize glucose and some produce calcium carbonate shells to protect their bodies. In any cases, initial step is hydration of carbon dioxide (CO2 + H2O → HCO3-). In this step, key material is carbonic anhydrase (CA) because uncatalyzed interconversion between carbon dioxide and bicarbonate is very slow. CA is a zinc-containing metalloenzyme, catalyzing the reversible hydration of carbon dioxide. In the present work, we will present the use of recombinant CA enzymes to convert carbon dioxide biologically to valuable chemical, calcium carbonate.

One of the key factors to influence the specific productivity of whole-cell biocatalysts for the production of fine chemicals (e.g., alcohols, carboxylic acids) is efficiency of biosynthetic pathway to the target molecules from the reaction substrates as well as of background metabolic pathways to provide cofactors (e.g., NAD(P)H, ATP) required for the production of target compounds1-3. This presentation will focus on impact of the background metabolisms on oxygenation of cyclohexanone and styrene by recombinant Escherichia coli to provide the critical factors in metabolic engineering and optimization of whole-cell biocatalysts in biorefinery.

In recent years, a great deal of concern has been expressed with regard to global climate change and its link to growing atmospheric concentrations of carbon dioxide. Separation of carbon dioxide from a large amount of flue gas by chemical separation is known for many years and considered to be one of the most reliable to capture the CO2. Various studies for improving the capture process have been performed, however, the current applied technology for this separation is still expensive, both in terms of capital cost and operating cost. If an alternative separation system having less energy for CO2 capture and regeneration could be developed, it is very attractive option to install CO2 capture facility. Since recently, emerging ex-vivo applications of carbonic anhydrase for its potential use in CO2 capture technologies are attracting attentions. The objective of the present study was to investigate the feasibility of using enzyme as a biocatalyst for hydration of CO2, as well as its precipitation in the form of calcium carbonate. The bio-catalytic capture of CO2, and its precipitation as CaCO3, over bovine carbonic anhydrase (BCA) and hemocyte for diseased shell (HDS) immobilized on a pore-expanded SBA-15 support was investigated. SBA-15 support was synthesized using TMB as a pore expander, and the resulting porous silica was characterized by XRD, BET, IR and FE-SEM analysis. BCA and HDS were immobilized on support through cross-linked enzyme aggregation. The bio-catalytic activity for hydration of CO2 was calculated for BCA and HDS. The kcat/Km value was 123.8 M-1s-1 for BCA and 119.1 M-1s-1 for HDS. Considering that HDS is a water-soluble, conjugated protein, the kcat/Km value of HDS may be regarded as near to that of BCA. This means that BCA, which is expensive and difficult to extract, can be replaced by the more economical HDS biocatalyst extracted from oysters. CaCO3 was produced by adding Ca2+ ion to the CO2 hydrated solution containing CO32-. The amount of CaCO3 was also measured at different times during the reaction. The initial formation of CaCO3 required about 15 seconds in the CaCO3 precipitation experiment without the biocatalysts. In contrast, when BCA and HDS were added, CaCO3 formation occurred within 5 seconds, on average ,for a 3 times faster reaction rate. The CaCO3 structure was the calcite structure, as indicated by a calcite peak with 2θ=29o. Calcium carbonate is a common and thermodynamically stable mineral found in rocks worldwide. If the widespread transformation of CO2 to CaCO3 is possible, it will represent a stable process for long-term CO2 capture and storage. In addition, the process yields a final product, CaCO3, which can be utilized as road pavement or paper coating materials. The study presented here is only the beginning for the capture and storage of CO2 using enzyme, especially HDS. Additional studies on various conditions which include cloning of enzyme, operating conditions, and scale-up factor are underway.

Photosynthesis in aquatic environments may be limited due to the low solubility and slow diffusion rate of CO2 in water. Most unicellular algae have evolved a CO2-concentrating mechanism (CCM) to compensate for this CO2 limitation. Carbonic anhydrase (CA) is a zinc-containing enzyme catalyzing reversible hydration of carbon dioxide: CO2+H2O ↔ HCO−� 3+H+, has been suggested to play a crucial role in the CCM as well as in the CA-mediated calcification found in eukaryotic algae and protists. The green algae Chlamydomonas reinhardtii adapts to low Ci conditions by inducing various forms of CAs, which help concentrate Ci internally, resulting in much higher levels than can be obtained by diffusion alone. In C. reinhardtii, two different intracellular CAs have been identified, a low CO2-induced mitochondrial CA and a constitutively expressed CA located in the chloroplast thylakoid membranes. In the present talk, I’l briefly introduce a thylakoid-associated CA, which is localized in photosystem (PS) II membrane fractions and plays a pivotal role in determining the viability of low CO2 intolerant mutants in atmospheric CO2 through involvement in CCM.

Carbonic anhydrase (CA, EC 4.2.1) accelerates to uptake of CO2 from air into intracellular system and phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) catalyzes the conversion of HCO3 - concentrated by carbonic anhydrase (CA) into four-carbon organic acid oxaloacetate (OAA). PEPcase is also the main anaplerotic enzyme providing OAA and/or malate, replenishing the tricarboxylic acid (TCA) cycle intermediates in all photosynthetic organisms [1]. Our research objective is to develop the platform technology for high-reactivity and high-production of marine CA and PEPCase. To achieve this goal, the experts on marine biology and genetic and protein engineering are collaborated to perform high-production of marine CA and PEPCase via search, screening and fabrication of high-expression recombinant hosts. In our laboratory, three types of CA in two strains of marine microalga (Dunaliella sp., Phaeodactylum tricornutum CCMP632) were identified. In Dunaliella sp., two types of CA, namely alpha and gamma-type CA, were isolated and cloned. Two types of PEPCase were also identified from marine green algae Dunaliella sp. and Dunaliella salina. The molecular cloning of the two PEPCase genes in Dunaliella salina (DsPEPC1, DsPEPC2) and one in Dunaliella sp. (DspPEPC1) were isolated, respectively. The recombinant micro-algae and microbe containing the developed CA and PEPCase variants will be used as important CO2-utilizing bioconversion system for production of useful C4 chemicals. This platform biotechnology for high-reactive design and high-production of marine CA and PEPCase will be essentially employed for development of efficient enzyme-based CO2 fixation/sequestration process.

Cosmetics have been widely used since ancient times notably by the Egyptians nearly 4000 years ago. Most of the cosmetics used by Egyptians in those days were biotech based, as they contained beeswax, fatty substances, animal wastes and plants. After many changes, Nowadays Natural and biotechnological products lead the cosmetic industry again. Biotechnology and cosmetics have had a cyclical relationships. Skin is the final target that biotechnology is applied in cosmetics. It is an organ that forms a protective barrier against germs and other materials and keeps a boundary between the inside of the body and outside. Modern molecular biology methods have greatly advanced our understanding in the molecular and cellular mechanisms regulating skin aging. It makes the task of biotechnology clear in cosmetics field. The development of skin lightener is a typical example. Skin pigmentation involves production and dispersion of melanin by epidermal melanocytes to neighboring keratinocytes. Melanin synthesis is catalyzed by pigment-producing enzymes in the specialized organelle termed melanosome. The process is regulated by a number of factors, including UV light, hormones, growth factors, cytokines. Biotechnological achievements found the controlling agent which keep a role at the point of melanogenesis such as Kojic acid, Ascorbic acid and niacin amide. Apart from the above case of cosmeceuticals, other several biotechnologies is being applied in cosmetic industry such as transdermal delivery systems, Polymer science, Phytochemicals, Oil & fats production and actives development by microbial fermentation. Our company, COTDE,Inc., is specialized in developing cosmeceuticals with the use of enzyme & fermentation. The first innovative cosmeceuticals is the de novo vitamin C synthesis system which converts the stable vitamin C derivative, Ascorbyl -2- glucoside, to active form of vitamin C instantly after applying to the skin. The second case is making the new skin lightener from Gastrodia elata with the process of ‘uruk Fermentation’ Korean traditional method, We applied the above two patented technologies to the new brand whitening skin care in Asian emerging market.

Recently, National Coordination Center for Global Cosmetic R&D (NCR) has established, which aims to promote cosmetic technologies in Korea. The projects of NCR will be focused on development of novel cosmetic ingredients for both in the fields of additives and base materials for cosmetic formulations, convergence based technologies for skin analyses, HTP screening system for bioactives development and alternative methods for replacing animal tests. In this regards, timely collection and analyses of information about current and emerging technologies is required at regular base. Information obtained from 2011 In-Cosmetics, Milan and ASCS, Seoul will be reviewed. And also future direction of NCR in supporting cosmetic R&D will be presented.

Jeju Island is the largest island located in the southernmost part of Korea. This island has diversity of a plant community and about 1,800 plant species exist around Halla mountain. We are searching plants to find natural products which are applicable as ingredients of cosmetic preparations.Sasa quelpaertensis Nakai (Korean name, Jejujoritdae), belonging to a family Gramineae, is an edible bamboo grass endemic to Jeju Island. S. quelpaertensis is one of the most abundant plant species among the plant varieties in the area of Halla Mountain. Its dried leaves are being used as a tea for the treatment of diabetes and gastritis in the local community. Our phytochemical studies on the leaves of this plant resulted in the isolation of 15 constituents including three new compounds. Among the isolates, anti-tyrosinase activities were identified in derivatives. In addition, a new phenylpropanoid isolate was identified to have anti-inflammation activities. It inhibits nitric oxide (NO) and prostaglandin E2 (PG E2) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. ESQ10 attenuated LPS-induced synthesis of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in parallel, and inhibited LPS-induced interleukin-6 (IL-6) production, as determined by an enzyme-linked immunosorbent assay(ELISA), in the macrophages. Distylium racemosum Sieb. Et Zucc (Korean name: Joroknamu) belongs to a Hamamelidaceae family. This evergreen tree is distributed over the mountain area in Jeju. Twenty compounds were isolated from the ethanol extract of Distylium racemosum branches. The isolated compounds were identified as dibenzofurans, abscisic acid, 6’O-galloylsalidroside, catechin derivatives, gallic acid derivatives, tyrosol, flavonoids, lupeol, and 1,2,3,6-tetragalloylglucose. Among these isolates, four dibenzofurans were new compounds. Inhibitory activities on tyrosinase, elastase, and free radicals evaluated for the isolated compounds. M. fauriei (Leguminosae) is a large deciduous tree, endemic to Halla Mountain, Jeju Island. The EtOH extract of this plant was selected for bioactivity-guided fractionation due to its strong inhibition activities on mushroom tyrosinase. Bioassay-guided investigation of the stems of Maackia fauriei led to the isolation of seven flavonoid constituents, such as formononetin, genistein, daidzein, texasin, tectorigenin, odoratin, and mirkoin. Tyrosinase inhibition activities were carried out for the isolated compounds. Among these, mirkoin was identified as a potent tyrosinase inhibitor. It inhibited mushroom tyrosinase with an IC50 value of 0.005 mM, which is ten times more active than kojic acid (IC50 = 0.045 mM). The inhibition kinetics, analyzed by Lineweaver-Burk plots, indicated mirkoin to be a competitive inhibitor of tyrosinase when L-tyrosine was used as a substrate.

Stem Cell Cosmetics are advanced cosmetics that are claimed to develop based on the stem cell technology. In a narrow meaning, they are specific products that used main ingredients originated from stem cell research. Even Stem cells are known beneficial to human, stem cell cosmetics are very slow growing. Almost main cosmetics manufacturers are afraid to involve in biological, legal and ethical problems. It is valuable to review history of stem cell cosmetics and developing direction of them.

Ultraviolet (UV) irradiation is a major environmental factor responsible for a high incidence of skin aging, which is referred to as photoaging, as well as skin cancer and melanoma. UVA (320-380 nm) irradiation represents 90% of the solar UV light that reaches the earth’ surface and is able to deeply penetrate into subcutis; therefore, its contribution to human life may be significant. In addition, Adipocyte dysfunction is strongly associated with the development of obesity, which is a major risk factor for many disorders including diabetes, hypertension and heart disease. This study shows that ultraviolet A (UVA) inhibits adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells (hAMSCs) and its action mechanisms. The mRNA levels of peroxidase proliferator-activated receptor (PPAR) g and CCAAT/enhancer binding protein (C/EBP) a, but not C/EBP b and d, were reduced by UVA. Moreover, the mRNA levels of PPAR g target genes (lipoprotein lipase (LPL), CD36, Protein(aP2)andliverXreceptors(LXR)a) were down-regulated by UVA. Additionally, attempts to elucidate a possible mechanism underlying the UVA-mediated effects revealed that UVA induced migration inhibitory factor (MIF) gene expression, and that this was mediated through activation of activator protein (AP)-1 (especially, Jun N-terminal Kinase (JNK) and p42/44 (mapk))and (NF-kB). In addition, reduced adipogenesis by UVA was recovered upon the treatment with anti-MIF antibodies. AMP-activated protein kinase (AMPK) phosphorylation and upregulation of Kruppel-like factor 2 (KLF2) were induced by UVA. Taken together, these findings suggest that the inhibition of adipogenic differentiation of hAMSCs by UVA occurs primarily through the reduced expression of PPAR g, which is mediated by upregulation of KLF2 via the activation of MIF-AMPK signaling.

Among matrix proteins, fibronectin (Fn) has been known to adhesion protein containing binding site to extracellular matrix proteins (collagen, fibulin) and cell surface receptor (integrin). Although the expression of MMP by fibronectin fragments (Fn-frs) have been investigated in chondrocyte, little is known about the roles of Fn-frs in aged human skin and normal human fibroblasts (NHFs), and the roles of cathepsin G that digested Fn. Therefore, the purpose of this study was to investigate the relationship between cathepsin G-mediated fragmentation of Fn and induction of MMPs that leads to increased extracellular matrix degradation during intrinsic aging and photoaging in skin.Fn-frs were measured in young and aged skin, and then compared with cathepsin G. We also investigated whether Fn-frs increase MMP-1 expression and cathepsin G stimulate proMMP-1 to convert active MMP-1. We found that cathepsin G expression is higher in aged skin than in young skin, and that this is correlated with increased fragmentation of Fn. Studies with cultured normal human fibroblasts (NHFs) showed that specific Fn fragments (Fn-f45:N-terminal gelatin binding fragment, Fn-f70:N-terminal heparin and gelatin binding fragment) induced MMP-1 expression and MMP-2 activity.Thus, the generation of Fn fragments by cathepsin G contributes to damage of matrix in aged skin.

Cosmetics were created for woman's beauty. Development of cosmetic technology was closely related with the alteration of social culture. New flow of organic and oriental herb was made in company with well-being trend. Recently the fermentation technology of the food industry was applied for cosmetic technique. LG Household &Health Care Co. Ltd. attempted to combine natural cosmetics with fermented materials. It is based on the fermented ingredients which have many efficacies such as anti-inflammation, anti-oxidation, cell-activation, whitening effect and collagen synthesis. Also, we made some specific formula which is best to deliver unique effectiveness to the skin. Our fermented cosmetics were Sooryehan hyo and Su:m37. In this presentation, we will show our development of the fermentation cosmetics and some examples of fermented cosmetic materials. *This study was supported by a grant of the Korea Healthcare technology R&D Project, Ministry of Health &Welfare, Republic of Korea.(Grant No.: A103017)

Cosmetic industry which is based on chemical industry and chemical engineering recently breaks the boundary of technical areas by introducing bio-nano technology which is fused between bio-technology and nano-technology. In recent study of cosmetic industry, development of specific targeting material based on skin metabolism was introduced by bio-technology while nano-technology introduced formulation and evaluation technology by applying selective targeting technology such as stabilization of active ingredient and stimulation of skin penetration. For example, cosmeceutical and nutricosmetics are newly emerged by adding its functionality from beauty oriented cosmetics to the health care such as disease mitigation. Kolmar korea which is specified its R&D area to cosmetics, medical supplies and functional foods is hardly working on fusion technology to strengthen the advantage of each field by interchanging our information and technologies. For examples, studies regarding anti-aging and isoflavone saponin from soy bean oil by using bio-fusion technology were conducted as well as studies regarding anti allergenic and NONI adventitious root treatment materials were conducted by using biomass technology. In addition, study on non aqueous liquid crystal was proceeded to improve stability of active ingredients and skin penetration. Recently, series of work regarding astaxanthin for optimizing degree of purity, extraction concentration and extraction method were conducted. In this recent study, New vesicle which is consist of skin compatible component is used rather than conventionally used hydrogenated lecithin for better cosmetic use. Particle size, color, zeta potential, TEM and FF-SEM analysis were operated to check its stability and CLSM was used for checking skin penetration. Therefore, It is now leading the new functional cosmetic field by establishing formulatory stabilization technology using nano capsulation and developing DDS technology for effective skin penetration.

Glycation of collagen causes skin yellowing via a spontaneous reaction among protein and sugar in the presence of reactive oxygen. Advenced glycosylation end products(AGEs) are produced by attaching an aldehyde group of glucose to amino group of a collagen molecule. These AGEs are yellowish in color and contribute to the yellow or sallow appearance of skin.(1-4) We know that fruit extracts contain a lot of antioxidant compounds and functional cosmetic raw materials. So we studied the effect of gold kiwifruit as anti aging agent and glycation inhibitor in collagen. We screened for in vitro antioxidant and anti-glycation activity of whole kiwifruit extract, kiwi pulp and rind extract. Rind portion exhibited more effective than pulp and whole kiwi extract in result. Thus it was selected to be further fractionated hexane, chloroform, ethyl acetate and butanol. Among these solvent fractions, kiwi rind extract of ethyl acetate (KRE-E) was observed to be the greatest. Immunofluorescence staining for the presence of AGEs in glycated collagen matrix showed that the glycation inhibitory effect of KRE-E and 5 mM of amnoguanidine were similar. Finally a phenolic compound of KRE-E was identified as quercetin-3-rhamnoside by employing HPLC. Recently using agricultural wastes as low cost source of potential raw material in cosmetics is an active research domain. Because these wastes give adventages of low cost and the possibility to reduce environmental problem caused by waste disposal.

Circular permutation of enzymes offers an alternative protein engineering strategy forchanging the substrate specificity, as well as improving enantioselectivity and catalyticperformance of biocatalysts. In previous work on lipase B from Candida antarctica (CALB),we demonstrated that the reorganization of the polypeptide sequence results in significant rateenhancements. These functional changes were linked to structural rearrangements alteringactive site accessibility and backbone flexibility. To more broadly explore the potential functional benefits of circular permutatedenzymes, we have now expanded our engineering studies to include two new biocatalystswith distinct folds: the xylanase from Bacillus circulans (BcX) and old yellow enzyme fromSaccharomyces cerevisiae (OYE). BcX is an endo-acting glycosyl hydrolase with a betajellyrollfold while OYE is a member of the flavin-dependent oxidoreductase family with analpha/beta barrel topology. The results from our random circular permutation experimentsand the structural and functional characterization of selected enzyme variants will bepresented. In the case of OYE, the effects of active site reorganization are not limited tosubstrate specificity and enantioselectivity but can potentially extend to changes in chemistry, due to the remodeling of the flavin cofactor environment. Besides highlighting the method’ ability for creating novel biocatalysts, our new studies also demonstrates that not all enzymesare created equal when it comes to engineering by circular permutation.

While the expression and screening of genetic libraries for protein engineering is generally carried out in microbial cells, the use of living cells sets intrinsic limitations in numerous dimensions including the throughput of process and choice of assay substrates. These limitations associated with cell-based expression screening can be effectively overcome by expressing enzyme libraries in the cell-free protein synthesis system. Not being subject to the constraints of maintaining cell viability and intact cell membranes, cell-free protein synthesis offers greater flexibility in designing expression and screening procedures. In this study, we will discuss different approaches to express and screen genetic libraries in a cell-free synthesis system derived from Escherichia coli. In particular, the discussion will be focussed on the development of effective methods to assay the expression of individual genes constituting the targeted library.

The light-oxygen-voltage (LOV) domain is a member of the PAS (Per-ARNT-Sim) domain superfamily mediating light sensing signals in the cell. Allosteric control of dihydrofolate reductase (DHFR) was explored by creating PAS-DHFR light switch, a chimeric protein that connects a light-sensing signaling domain from A. sativa phototropin (LOV2) with E. coli DHFR. Here, we demonstrate that a light dependent allosteric effect is introduced through linkage of the domain to the coupled catalytic network in DHFR. Light activation of the chimera triggered a characteristic spectral shift to 390 nm due to formation of a covalent thiol-FMN adduct that initiates a conformational change. Among the chimeras, LOV domain insertion into a specific position in the bF-bG loop of the DHFR exhibits light-dependent modulation of the hydride transfer rate. This work clearly proved the presence of unique network(s) in proteins through which a signal is transmitted. The engineering of a light-dependent control represents an initial step for the general development of proteins subject to allosteric regulation.