Earticle

생체고분자나 여러 단백질체 (프로테옴) 분석을 위해 그 하위단계인 펩타이드의 구조적 정보를 확인하고 이를 토대로 어미 단백질을 동정하거나 단백질의 반응을 연구하는 액체크로마토그래피 다중텐덤 전자분무이온화 질량분석(LC-MS/MS) 기술은 매우 빠르게 발전하고 있다. 그 중 가장 최상위 기술의 Orbitrap 및 Thermo Ion Trap MS 를 소개하고 이를 이용한 Proteomics와 metabilomics 및 Biosimilar 분야와 같은 다양한 최신 응용 사례 또한 소개하고자 한다.

With the LightCycler® family of PCR systems, Roche Applied Science has set a standard in real-time PCR. LightCycler® Instruments are well known for their speed, accuracy, and flexibility, which are due to the symmetrical "one-well–like"design, air-based heating and cooling, and the use of capillaries as reaction vessels. Following along the same lines, the new LightCycler® 480 System efficiently facilitates the delivery of higher sample throughputs when performing gene expression or mutation analysis in life-science research. For the first time, a real-time PCR platform now offers the LightCycler® Systems’ unique combination of accuracy and speed for multiwell-plate based assays with comparable performance with 96- or 384 sample throughputs, and for all relevant qPCR applications. In addition, the highly modular concept of the LightCycler® 480 System hardware and software allows scientists to customize the system to best suit their laboratory’s specific research needs.

Streptomyces which is Gram‐positive, filamentous and soil dwelling bacteria produce various secondary metabolites including antibiotics, peptides and various pigments 1. In the research of Streptomyces, study of regulators related to producing antibiotics has potential to supplement the existing method like random mutagenesis and change more global metabolic flux than single enzyme in metabolic pathway. In addition, many examples of regulators such as ActII‐ORF4 of S. coelicolor 2, StrR of S. griseus 3 and DnrI of S. peucetius 4, TylS and TylR from S. fradiae 5, WblA from S. peucetius 6 also show possibility that the results of characterizing regulators can be applied to develop more efficient industrial strain by directly up‐ and down‐regulating antibiotic biosynthetic gene clusters. To find out unknown regulators affecting primary and secondary metabolisms in Streptomyces coelicolor, we applied different omics approaches for screening of regulators and characterization of new regulators, and characterized several regulators such as NdgR and AbsC.

It is well known that sulfur compounds in beer make significant contributions to flavor and aroma. Sulfite plays an important role in flavor stability. Although breeding of bottom‐fermenting yeast strains that produce high levels of SO2 is desirable, it is complicated by the fact that undesirable H2S is produced as an intermediate in the same pathway. We developed a high SO2‐producing bottom‐fermenting yeast strain by integrated metabolome and transcriptome analysis. This analysis revealed that Oacetylhomoserine (OAH) is the rate‐limiting factor for production of SO2 and H2S. Appropriate genetic modifications were then introduced into a prototype strain to increase metabolic fluxes from aspartate to OAH and from sulfate to SO2, resulting in high SO2 and low H2S production. Spontaneous mutants of an industrial strain resistant to both methionine and threonine analogs were then analyzed for similar metabolic fluxes. One promising mutant produced much higher levels of SO2 than the parent, but parental levels of H2S. Furthermore, on the other application of CE‐MS to a bottom‐fermenting yeast, we investigated the function of bottom‐fermenting yeast‐specific (BFY) genes that have no significant homology with sequences in the budding yeast. One of the BFY genes, AMI1 encodes a protein with homology to an amidase conserved among plants and fungi. By metabolome analysis of intracellular compounds followed by differential analysis, we found that the amount of histidine and arginine is increased, and the amount of threonine, lysine, and nicotinic acid is decreased in the Ami1p‐overproducing strain as compared with the control. This suggests that Ami1p may hydrolyze some amides related to amino acid and niacin metabolism in the cell. These results indicate that metabolome analysis is powerful approach to understand the mechanism of cellular metabolisms and breed strains with preferable characteristics for industrial use.

Streptomyces avermitilis, a well‐studied member of this genus, is used for the industrial production of the important anthelminthic macrolide avermectin. Sequencing of the S. avermitilis genome has revealed four presumptive terpene synthase genes. One of these, ptlA (sav2998), encodes a pentalenene synthase, a second, geoA (sav2163), is a germacradienol/geosmin synthase, and a third, sav3032, encodes an epiisozizaene synthase. The function of the remaining putative terpene synthase gene, sav76, has not previously been established. Two highly conserved Mg2+‐binding motifs, characteristic of essentially all terpene cyclases, are evident in the predicted SAV_76 protein as an aspartate‐rich 80DDQFD motif and the “NSE” triad motif, 239NDVYSLEKE. Incubation of purified recombinant SAV_76 with FPP in the presence of MgCl2 gave a sesquiterpene alcohol (1) (m/z 222) as the major enzymatic reaction product (85%,), accompanied by germacrene A (2) (1 %), germacrene B (3) (5 %) and the known tricyclic sesquiterpene alcohol, viridiflorol (4) (3 %), as determined by GC‐MS analysis. The in vivo activity of the sav76 gene was also demonstrated using a genome‐minimized mutant, S. avermitilis SUKA17, from which >1.5‐Mb of DNA had been deleted, including the genes for the major endogenous secondary metabolites produced by the parent strain. GC‐MS analysis of hexane extracts of cultures of S. avermitilis SUKA17 harboring sav76 under control of the native S. avermitilis promoter rpsJp (sav4925) showed the presence of avermitilol (1, 15%,), accompanied by small quantities of germacrene A (2, 10%), germacrene B (3, 5%), and viridiflorol (4, 2%). The major component of the mixture was ketone avermitilone (5, 67 %, m/z 220). The formation of avermitilone (5) may result from adventitious oxidation of 1 by an endogeneous dehydrogenase, since no dehydrogenase gene is evident in the genome of S. avermitilis immediately upstream or downstream of the native sav76 cyclase gene. We have now assigned the biochemical functions of all four terpene synthases originally revealed by the sequencing of the S. avermitilis genome. Avermitilol (1) is a new sequiterpene whose isolation has not previously been reported.

The typical chemical structure of lipids is relatively simple, but lipid diversity is very high owing to complexity arising from combinations of hydrophobic acyl chain molecular species. Generally, lipids are considered to be hydrophobic compounds. However, their hydrophilicity occasionally increases when combined with highly hydrophilic molecules such as phosphoric acid and sugar. Thus, lipids possess a wide range of polarity. Sophisticated techniques are required for separation and component analysis to identify each lipid in a lipid mixture. A supercritical fluid is a substance whose temperature and pressure are above the critical values, and which possesses features such as low viscosity and high diffusivity. Therefore, it exhibits properties suitable for its application as a mobile phase for chromatography. SFC is a suitable technique for the separation of hydrophobic compounds, and is superior to liquid chromatography/mass spectrometry (LC/MS) because of its high throughput and high resolution. Therefore, we attempted to apply SFC/MS to lipid profiling in order to establish an analysis system applicable to lipid metabolomics. Separation conditions for SFC (column, modifier, back pressure, etc.) and detection conditions in the mass spectrometer (ionization method, parameters, etc.) were investigated in order to develop a simultaneous analytical method for lipid mixtures comprising phospholipids, glycolipids, neutral lipids, and sphingolipids. When a cyanopropyl silica gel packed column was used for the separation, all the lipids were successfully detected, and the analysis was completed in less than 15 min. The use of an octadecylsilyl column resulted in separation based on the differences in the unsaturation of the fatty acid side chains and isomer distribution. Additionally, SFC/MS was applied for profiling lipids in soybeans. For this purpose, lipids extracted from 12 soybean cultivars were used. Different soybean cultivars used in the manufacture of various processed foods, such as natto, tofu, edamame, and nimame, could be distinguished from each other. In principal component analysis (PCA), triacylglycerol (TAG) was found to be the main variable for the discrimination of soybean cultivars. For a more effective discrimination of soybean cultivars, a detailed TAG profiling method was developed by using SFC/MS with 3 Chromolith Performance RP‐18e columns connected in series. TAG molecules were separated effectively within 8 min without purification. Additionally, each TAG was successfully identified by using programmed cone voltage fragmentation, even without MS/MS analysis and any standard sample. This study shows that SFC/MS is a powerful tool for studies on lipid metabolomics because it is useful not only as a fingerprinting method for screening diverse lipids, but also for the detailed profiling of individual components.

Xylose reductase (XR) and Xylitol dehydrogenase (XDH) are important enzymes for transforming hemicellulosic material, xylose, to fermentative sugar. Although several xylose-utilizing recombinant microbes of Saccharomyces cerevisiae (S. cerevisiae) have been constructed by genetically expressing XDH and XR, the results were not successful. In particular, the utilizing rate of xylose for ethanol production in recombinant S. cerevisiae was not sufficient, so that enzymatic functionalities of XR and XDH should be improved by protein engineering approach. In this study, we designed XR and XDH from Pichia stipitis (PsXDH) in terms of their cofactor preferences and thermostability. In particular, we developed appropriate design strategies via bioinformatical comparison with other polyol dehydrogenase family members. More functional PsXDH and PsXR varients were obtained by site-directed mutagenesis. Recombinant S. cerevisiae expressing the designed PsXR or PsXDH showed high perfoemance in xylose-toethnaol production.

Saccharomyces cerevisiae is one of the industrially important microorganisms. In our laboratory, S. cerevisiae sake K6 was developed to overproduce S‐adenosyl‐L‐methionine (SAM). S. cerevisiae K6 has not been engineered with DNA recombinant technology due to the lack of a proper genetic marker. An UV mutagenesis was conducted with S. cerevisiae sake K6 and a mutant with leucine auxotroph, K6‐1, was selected. The mutant showed similar growth rate and SAM productivity to its wild type. Using the auxotroph as a genetic marker, a SAM synthase (SAM2) and a ethionine resistant (ERC1) genes were overexpressed. The recombinant DNA successfully enhanced SAM productivity in sake yeast. We also metabolically engineered S. cerevisae to produce 1,2‐propanediol using glycerol as a main carbon source. The introduction of two genes, mgs (methylglyoxal synthase) and gldA (glycerol dehydrogenase) from Escherichia coli, not only made S. cerevisiae produce 1,2‐propanediol, but also increased the growth rate in glycerol and glycerol utilization rate of S. cerevisiae. Further increase of 1,2‐propnaediol production and glycerol utilization rate were achieved by overexpression of endogenous GUT1(glycerol kinase) and GUT2 (glycerol 3‐phosphate dehydrogenase) as well as GUP1 gene which involved in glycerol transport in S. cerevisiae. Lastly, additional glycerol dissimilation pathway was introduced by expressing a glycerol dehydrogenase gene from Pichia angusta in S. cereviaise. By engineering, 0.98 g/l of 1,2‐propanediol concentration was achieved in flask culture with 1% (v/v) glycerol as a main carbon source.

PAT is defined as a “system for the continuous analysis and control of manufacturing processes based on real‐time or rapid measurements during processing of quality and performance attributes of raw and in‐process materials and processes, to assure end product quality at the completion of the process”. Optimal manufacturing process control involves testing raw materials, in‐process monitoring and endproduct testing with stringent documentation. Critical sources of variability must be identified and managed. PAT implementation, therefore, requires innovative PAT tools or Process Analytical Instrumentation (PAI) which gives real‐time or near real‐time results by monitoring continuous and episodic operations. In order to specifically implement PAT in testing for Bacterial Endotoxins, the Portable Testing System (Endosafe®‐PTS™) offered by Charles River is an effective Process Analytical Instrument. The PTS™ uses disposable LAL cartridges approved by the USFDA (July 2006) and are pre‐loaded with existing FDA‐approved Endosafe® Chromogenic LAL reagents. The cartridges are calibrated directly to the primary standard (Reference Standard Endotoxin EC‐6). The PTS cartridges are compliant to the current harmonized compendia test for Bacterial Endotoxins. The suitability of the PTS™ Reader comes from its highly flexible platform and portability. It gives a rapid test result time of less than 15 minutes. It eliminates the need for endotoxin standards, preparation of Positive Product Controls (PPC) and validation of accessories‐ all of which need a laboratory clean room setting and extensive analyst training. The PTS™ Reader allows the test to be done near the point‐of‐use and the cartridges can be stored at ambient temperatures for 18 months. The PTS™ allows for single sample analysis, near real time results, and decentralization from QC labs – all of which ultimately contribute to manufacturing process control and a quick response to contamination issues. By implementing PAT and the PTS™ rapid endotoxin testing system, this turnaround time for sample release would be reduced to just 30 minutes allowing manufacturing to move forward without delay. Using the PTS™/MCS™ systems as Process Analytical instruments can reduce risks in the manufacturing process and improve results in the quality of end‐products.

One of the biggest trends in biopharmaceutical manufacturing over the past few years has been the increasing use of single-use system for biomanufacturing. There are many reasons companies are seeking disposable solution, including easier cleaning, faster turnaround times, easier validation, and better quality management. Contract manufacturing organization (CMO) are leading the charge, partly because of the need for flexibility in their manufacturing. By enabling flexibility and quick turnaround times between process runs, disposables enable CMOs to perform different processes more efficiently within a given facility. These efficiencies can reduce operating costs and capital investments. As the industry shift its attention to purification capacity and cost, new technologies(include Membrane Chromatography) are being developed to address downstream processing bottlenecks. In most new downstream technologies, Sartorius is incorporating single-use systems. Although no fully commercialized downstream alternative to current chromatography processes exist, a number of products are in development. once such products are available, more companies will likely increase their overall use of single-use systems. This will make it possible to begin to realize the advantages expected from fully disposable production processes.

While the development of single‐use process equipment and drug storage containers made from plastic materials has increased in line with demand, extractables and leachables from these materials have become a topic of concern for the pharmaceutical and biopharmaceutical industry. Consequently, appropriate testing strategies and practices are required to ensure the quality and safety of drug products. This Extractables / Leachables section is a new and unique platform to discuss and develop scientific based and practical approaches to extractables and leachables testing. A major focus will be on the process specific leachables testing using the actual product as test solution this is designed for individuals from development, manufacturing, validation, quality assurance and compliance who are confronted with the challenges of extractables and leachables testing in pharmaceutical and biopharmaceutical industry.

제품 전환 시 Cross Contamination에 대한 우려가 증가되고, Lead Time의 감소, 초기 설비 투자비 및 기간의 단축 등 여러 가지 장점으로 인하여 전통적인 Reusable Bioreactor 대신 Disposable Bioreactor의 사용이 증가하고 있는 추세이다. 또한 Disposable Bioreactor의 최대 단점이었던 2000L 이상 Scale up이 어렵다는 문제는 현재 Upstream 공정 기술의 발전에 따라 Titer가 증가되고 있고, 5000L급 Disposable Bioreactor가 개발되고 있는 등, 이러한 단점은 극복되고 있는 상황이어서 향 후 Disposable Bioreactor의 이용 비율은 더욱 크게 증가할 것으로 보인다. 본 발표에서는 선진국에서 조사된 자료를 바탕으로 Disposable Equipment의 이용 추세를 알아보고, 다년간 Reusable Bioreactor와 Disposable Bioreactor를 이용하여 항체 의약품 생산했던 경험을 바탕으로 실제 Reusable Bioreactor와 Disposable Bioreactor의 Qualification 단계부터 준비, SIP, 배양공정, CIP등 단 단계별 두 가지 Bioreactor를 비교하여 그에 대한 장/단점을 알아보고, Disposable Bioreactor를 이용하며 공정 진행 시 주의해야 할 사항을 Case Study를 통해 알아보고자 한다.

Redox biocatalysis comprises a fast growing arsenal of synthetically and industrially relevant reactions with high selectivity and interesting value added products. For preparative purposes, the biocatalysts are mostly (living) microbial cells including complex, often multicomponent oxygenases for meeting basic requirements for cofactor regeneration and enzyme stability. In addition, living cells synthesize and regenerate the catalytically active oxygenases, e.g. after reactive inactivation during catalysis. Engineering living microbial cells with the objectives turnover number, total turnover number (specific activity and volumetric productivity) poses significant challenges, often integrating different levels like the enzyme, the microbial cell, the reaction and the overall process. Systems biotechnology offers a previously not available experimental and theoretical frame of methodologies and concepts to address these challenges. The focus on a certain product is the main difference of systems biotechnology in comparison to systems biology. Ultimately, understanding regulatory functions of physiology and metabolism on a genome scale for biocatalytic product formation will transform applications of cellular m icrobial biocatalysts from individual and case specific R&D endeavors to rational, modular, and concept based design approaches. This will be exemplified by studying the link of metabolic capacity and asymmetric epoxidation of styrene to (S)‐styrene oxide in recombinant E. coli and Pseudomonas sp. using flux balance analysis and metabolic flux analysis. Process data will also be evaluated with respect to environmental impact and economic significance.

Due to the global warming and concerns over the shortage of crude oil, bioenergy recently emerges as a promising option to overcome the problems. Among various options for the bioenergy use, the application of bioenergy in the transport sector has the greatest potential. Currently about fifty million kilo liters of biofuels are consumed in the world and the demand for the biofuels will be increased by 20% annually until 2030. However, the first generation biofuels produced from the edible materials may not meet the demand for the increase of the biofuels. Active works have been carried out to develop 2nd generation biofuel technologies in major developed countries. Recent R&D works on the 2nd generation biofuels in Korea will be introduced.

The feasibility of ethanol and butanol production from construction and demolition (C&D) wood wastes acid hydrolysate was investigated. The chemical compositions of classified C&D wood waste were analyzed. The concentrated sulfuric acid hydrolysis was performed to obtain the saccharide hydrolysates and the inhibitors in hydrolysates were also analyzed. The C&D wood waste composed of lumber, plywood, particleboard, and medium density fiberboard (MDF) have the polysaccharides (cellulose, xylan, and glucomannan) fraction of 60.7 –67.9%. The C&D wood waste showed a different sugar composition (glucose, xylose, and mannose) according to a type of wood. The additives used in the processing of wood seem not to be released into mixed sugars solution under acid hydrolysis. The C&D wood wastes carried into the landfill site could be a feedstock as the raw material for the production of bioethanol and biobutanol.

To build an energy and material secure future, we must pioneer the next generation of renewable fuels and chemicals using environmentally benign production process. Biomass represents an abundant carbon‐neutral renewable resource for the production of biofuels. Numerous environmental and social benefits could result from the replacement of petroleum‐based transport fuels with bio‐ethanol converted from lignocellulosic materials such as agricultural residues and industrial waste. One of our key technologies is cell surface engineering, which is a powerful tool to engineer and functionalize many organisms. Using the technology, various kinds of functional proteins such as enzymes can be expressed on the cell surface without loss of their activities. The display of cellulolytic enzymes on the surface of Saccharomyces cerevisiae has accomplished direct ethanol production from cellulosic biomass. Also, the display of hemicellulase on the surface of S. cerevisiae that has xylose‐assimilating metabolic pathway has enabled production of ethanol from hemicellulosic biomass. The yeast is reusable for the hydrolysis and fermentation by collecting the cells. Thus, the cell surface technology has advantages on the process integration of enzyme hydrolysis and fermentation. For the efficient production of ethanol from cellulosic materials with S. cerevisiae, the improvement of both its fermentation ability and tolerance to inhibitors resulted from the thermochemical pretreatment has been required. The demands on the yeast that perform this reaction are more complicated than those for conventional ethanol production from hexoses or their disaccharides, because lignocellulosic hydrolysates contain high concentrations of inhibitors that negatively affect metabolism and ethanol yields compared with current corn starch‐ or sugarcane‐derived feed stocks. To circumvent the difficulties, we are trying to breed robust S. cerevisiae strains that efficiently ferment mixtures of hexose and pentose sugars in various chemical contexts for industrial ethanol production from lignocellulosic feed stocks through synthetic biology approaches. A combination of a cell surface displayed enzyme system and an intracellular metabolic engineering system is a very effective approach to develop cells with novel fermentation ability for industrial applications. The technology will open up the various new applications of cell factories to the industrially important processes.

The carbon dioxide concentration of the atmosphere is projected to increase by almost 50% over the first 50 years of this century. The major cause of this increase is continued combustion of fossil fuels. As a result, the significant changes in climate that have already occurred will be amplified, in particular a global temperature increase. Renewable energy production has a central role to play in abating net CO2 emissions to a level that will arrest the development of global warming. Especially, biomass crops are becoming increasingly important as concerns grow about climate change and the need to replace carbon dioxide producing fossil fuels with carbon‐neutral renewable sources of energy. To succeed in this role, biomass crop has to grow rapidly and yield a reliable, regular harvest. A prime candidate is Miscanthus and Phragmites, a perennial species that produces over 3 metres of bamboolike stems in a year. Here I will detail the Miscanthus and Phragmites characteristics desired of a biomass fuel crop.

Cattail (Typha L.), a phytoremediation grass in the wetland, was studied for the development of biorefinery processing to produce value-added products. After the pretretment of dilute sulfuric acid, the lignosulfonate and the non-bleached pulp were firstly produced and the hemicelllulose was remained in the wastewater. The linosulfonate is used as plasticizers in making concrete and for the production of plasterboard. Non-bleached pulp is also a commercial product and it is comprised of cellulose and lingin. After bleaching process, the bleached pulp was produced and ready for making a variety of papers. Non-bleached pulp and the residual hemicellulose can be used for the bioethanol production. Under optimized condition, acid-pretreated cattail showed 0.72 g/g of saccharification yield. The concentrated hydrolysates were tested for the bioethanol production using Saccharomyces cerevisiae and the maximum ethanol yield and fermentation efficiency in 30 L batch fermentation was 0.47 g/g and 92%, respectively. It might be expected to produce up to 162 L/ton of cattail by mass analysis during overall pretreatment-saccharification-fermentation processes. For further studies on the bleached pulp, cattail bleached pulp can be used for textile industry. Taken together, cattail is a good non-wood biomass with various industrial applications.

Due to recent huge consumptions of fossil fuels, followed by natural resource depletion and environmental problems, our attention has been on the concept of "sustainable development". For reducing global carbon emissions, many countries are seeking to find renewable and environmentally friendly energy resources, especially in the production of biofuels. As a result of bioenergy policies of developed countries, economic advancement of developing countries, and limitations on agricultural productivity, there is an increasing demand for grains. Increasing biofuel production and supply is a driving factor accounting for increasing grains prices. Therefore, it is difficult to secure the food supply worldwide. To reduce the negative impact of biofuels, major developed countries are focusing on the use of environmentally friendly, non‐food energy resources in their biofuel policies, which include total biofuel production and use (from feedstock transport to final product distribution). Due to the limited cultivation area of biomass, the world supply of biofuels will be lower than needed worldwide. To overcome these limitations, it is essential to develop new technologies in the field of biomass feedstock production and supply, as well as bioenergy production processes. Development and use of low cost cellulose‐derived biofuels will eventually improve the world's use of them. "Low carbon green growth" is one of the goals of the Korean government's policies for seeking to increase the rate of domestic production of biofuels. Korea has established regulations regarding the production and use of biofuels. The Korean researches are focused upon the development of non‐food biomass feedstocks. As the technological advancement of the efficiency of biomass and biofuel production improves, the cost of biofuel production and their effect on the environment will decrease. If we consider the huge consumption of fossil fuels, the ripple effect on biofuels, and potential biofuels markets, supportive biofuel policies would be required. The short term goal of the biodiesel supply in Korea is 540,000kl. To meet the demand of feedstock, we are planning to grow rapeseed in a 45,000ha area during the winter season of 2011.

The production of ethanol from feedstock other than agriculture materials has been promoted in recent years. Some microalgae can accumulate a high starch content (about 44% of dry base) via photosynthesis. Algal biomass, Chlamydomonas reinhardtii UTEX 90, was converted into a suitable fermentable feedstock by two commercial hydrolytic enzymes. The results showed that almost all starch was released and converted into glucose without steps for the cell wall disruption. Various conditions in the liquefaction and saccharification processes, such as enzyme concentration, pH, temperature, and residence time, have been investigated to obtain an optimum combination using the orthogonal analysis. As a result, approximately 235 mg of ethanol was produced from 1.0 g of algal biomass by a separate hydrolysis and fermentation (SHF) method. The main advantages of this process include the low cost of chemicals, short residence time, and simple equipment system, all of which promote its large‐scale application.

In recent years, biodiesel production has increased considerably because it is an environmentally friendly and renewable alternative diesel fuel. During the production of biodiesel, a large amount of raw glycerol is generated as a by-product in the range of 10% (w/w) of biodiesel production. Biodiesel manufactures are faced with limited options for managing raw glycerol, which may become an environmental problem since it cannot be directly disposed of in the environment. The tremendous increase in the glycerol supply has led to a remarkable drop in its price about 2.5 cents/l b of raw glycerol in 2006, making it attractive to develop novel technical methods using glycerol as a raw material. One of the possible applications is its use as a carbon and energy source for microbial growth in industrial biotechnology. Glycerol can be biologically converted to various chemicals such as 1,3-propanediol, butanol, 2,3-butanediol, 3-hydroxypropionic acid, ethanol, etc. In this talk, biological production of biofuels and chemicals from glycerol will be presented and discussed.

Retaining glycosidases, which are originally glycoside hydrolases, are efficient synthetic catalysts for the assembly of glycosides. However, yields of the transglycosylation reactions are typically low due to hydrolysis of the transfer product. A class of mutant glycosidase termed glycosynthase that largely obviates this hydrolytic problem by lacking of the catalytic nucleophile, thus the ability to form a reactive glycosyl enzyme intermediate, is complemented by the use of glycosyl fluorides of opposite stereochemistry to that of the natural substrates as donor sugars. While these strategies have proved to work well for β-glycosidases, their application to α-glycosidases has been less successful. Thus, for α-glycosynthases, only two examples derived from retaining α-glycosidases have been published, along with one from an inverting α -glycosidase. Recently, it was found that an α-xylosidase mutant that lacks its acid/base residue performs efficiently and regioselective transglycosylation reactions using α-xylosyl fluoride as a donor in yields of over 95%. It will be interesting to see how generally applicable this acid/base muation strategy turns out to be to α-glycosidases

The main functions of glycosylation are stabilization, detoxification and solubilization of substrates and products. To produce glycosylated products, Escherichia coli was engineered by overexpression of UDPxylose, UDP-glucoronic acid, TDP-L-rhamnose and TDP-6-deoxy-Dallose biosynthetic gene clusters, and flavonoids were glycosylated by the overexpression of the glycosyltransferase gene from Arabidopsis thaliana. For the glycosylation, these flavonoids (ficetin, quercetin, kaempferol and other one) were exogenously fed to the host in a biotransformation system. The products were isolated, analyzed and confirmed by HPLC, LC/MS, NMR and ESI-MS/MS analyses. Several conditions, substrate concentration, incubation time) were optimized to increase the production level. We successfully isolated approximately 24 mg/L 3-O-rhamnosyl quercetin and 12.9 mg/L 3-O-rhamnosyl kaempferol upon feeding of 0.2 mM of the respective flavonoids and were also able to isolate 3-O-allosyl quercetin. Thus, this study reveals a method that might be useful for the biosynthesis of rhamnosyl and allosyl flavonoids and for the glycosylation of related compounds.

모유(Human Milk)에는 분유(Cow Milk)에 미량으로 존재하거나 존재하지 않는 다양한 종류의 기능성 올리고당 (Human Milk Oligosaccharides, HMO)들이 존재한다. 이들은 갓 태어난 유아를 감염성 유해 미생물 및 바이러스 등으로부터 보호 할 뿐 아니라 두뇌개발, 면역시스템 구축 및 세포 간 신호전달에 관여하여 아이가 건강하게 자라는 데에 매우 중요한 역할을 하는 것으로 알려져 있다. 최근 들어 소위 현대병이라고 불리는 아토피, 천식등 allergies 뿐만 아니라, 소아당료, 관절염, 알츠하이머, 등 각종의 자가면역질환 환자들이 급격히 증가 하고 있는데, 이들 질병 발생의 원인 중 일부는 모유수유 되신 지나친 분유의 사용으로 성장하는 유아에게 이들 다양한 올리고당들이 충분히 공급되지 않아서 생기는 것으로 알려져 있다. 이들 HMO들은 그 중요성에도 불구하고 정제에 의한 공급은 source가 한정되어 충분한 양의 공급이 어렵고 합성 또한 용의치 않아 몇몇 HMO 만이 연구용으로 공급될 뿐 기능성 nutrition source로서 산업적 응용은 이루어지지 않고 있다. 심지어 대부분의 HMO들은 연구용으로서 공급조차 어려워 생체 내에서의 역할이 제대로 알려지지 않고 있다. (주)진켐에서는 지난 십여 년 동안의 당 관련 연구를 통해 축적된 기술을 이용하여 이들 HMO들 중 가장 연구가 많이 된 시알릴올리고당 들을 저렴한 가격으로 대량생산할 수 있는 공법 개발을 완료하였으며, 시알릴올리고당들을 이용하여 인간의 건강을 지켜주는 식품 소재 및 치료제로 개발하고자 한다. 이를 위해 (주)진켐은 세계최초로 2009년 1월에 시알릴락토오스를 미국 FDA에 식품첨가제로서 등록하였으며, 현재 국내 식약청에 기능성 식품 등록을 위한 임상을 진행 중에 있다. 이와 관련된 내용을 발표하고자 한다.

In this seminar I will briefly introduce about current research progress in our Lab related with glycoside synthesis using glycansucrases and carbohydrase inhibitor screening using Grid. We are synthesizing various glycosides of the hydroquinone for the improvement of antioxidant activity. Hydroquinone (HQ) functions as a skin whitening agent, but it has the potential to cause dermatitis. We synthesized a novel HQ fructoside (HF), HQ galactoside and HQ glucoside as potential skin whitening agents by reacting glycansucrases from Leuconostoc mesenteroides with HQ as an acceptor and sucrose as a donor. HF synthesis was determined using a response surface methodology and HF showed anti-oxidation activities and inhibition against tyrosinase. The IC50 of DPPH scavenging activity was 5.83-mM, showing higher anti-oxidant activity compared to β-arbutin (IC50 = 6.04-mM). The Ki value of HF (0.67-mM) against tyrosinase was smaller than that of β-arbutin (Ki = 2.8-mM). Virtual screening (VS) was applied for discovery of new inhibitors for the human intestinal maltase (HMA) enzyme. VS of 308 307 compounds was performed with HMA using 4700 CPUs and AutoDock 3.0.5 in a WISDOM (Wide In Silico Docking On Malaria) production environment. The 42 best ranked compounds containing hydrogen bond interaction with key residues from VS were tested in vitro for their inhibitory activities against the recombinant HMA from Pichia pastoris. Compounds 17 and 18 were identified as competitive inhibitors for enzyme inhibition. In contrast to acarbose, the two compounds showed no inhibition on human pancreatic α -amylase, suggesting potential inhibitors with fewer side effects, including abdominal discomforts.

Monosaccharides have recently attracted much attention due to their many uses, such as low-calorie sweeteners, bulking agents, immunosuppressants in allogenic orthotopic liver transplantation, and potential inhibitors of various glycosidases, ischemia/reperfusion injury of the rat liver, and segmented neutrophil production without other detrimental clinical effects. Monosaccharides can be made by microbial and enzymatic reactions with ketose epimerase, aldose isomerase, aldose-phosphate isomerase, aldose reductase, and oxidoreductase. Among sugar converting enzymes, sugar isomerases including L-arabinose isomerase, L-rhamnose isomerase, L-fucose (D-arabinose) isomerase, and D-lyxose isomerase; and sugar-phosphate isomerases including D-ribose-5-phophate isomerase, D-mannose-6-phophate isomerase, D-glucose-6-phophate isomerase, and D-galactose-6-phophate isomerase are of interest for the production of monosaccharides because they have various types of substrate specificities. In this study, the biochemical properties and substrate specificities of sugar isomerases and sugar-phosphate isomerases are investigated and the production of monosaccharides is attempted using these enzymes. Increases in the production of monosaccharides are realized by genetic improvements via directed evolution and by structural modification of the determinant residues on or near the active site, based on homology models or the determined structure of the enzymes.

Marine mussels attach to substrates using adhesive proteins. Mussel adhesive proteins (MAPs) have received increased attention as potential biomedical and environmentally friendly adhesives. However, practical applications of MAPs have been severely limited by uneconomical extraction and unsuccessful genetic production. Developing new adhesives requires access to large quantities of material and demonstrations of bulk mechanical properties. Previously, we designed fp‐151, a fusion protein comprised of six MAP type 1 (fp‐1) decapeptide repeats at each MAP type 5 (fp‐5) terminus and successfully expressed it in Escherichia coli. This recombinant hybrid protein exhibited high level expression, a simple purification, and high biocompatibility as well as strong adhesive ability on a micro scale. In the present work, we performed investigations on the bulk adhesive properties of purified fusion fp‐151 in air. The unmodified recombinant fp‐151, as expressed, contains tyrosine residues and showed significant shear‐adhesive forces. Adhesion strength was increased after enzymatic oxidation of tyrosine residues to L‐3,4‐dihydroxyphenylalanine (DOPA) groups. Addition of cross‐linkers generally enhanced adhesion, although too much addition decreased adhesion. Taken together, we present the first bulk‐scale adhesive force measurements for an expressed recombinant hybrid mussel adhesive protein. It has also been suggested that complex coacervation (liquid‐liquid phase separation via concentration) might be involved in the highly condensed and non‐water dispersed adhesion process of MAPs. However, as purified natural MAPs are difficult to obtain, it has not been possible to experimentally validate the coacervation model. In the present work, we demonstrate complex coacervation in a system including recombinant MAPs and hyaluronic acid (HA). We observed successful complex coacervation using cationic MAPs and an anionic HA partner. Importantly, we found that highly condensed complex coacervates significantly increased the bulk adhesive strength of MAPs in both dry and wet environments. Collectively, our results indicate that a complex coacervation system based on MAPs shows superior adhesive properties, combined with additional valuable features including liquid/liquid phase separation and appropriate viscoelasticity.

Antifreeze proteins (AFP) are proteins that depress the freezing point but not the melting point of aqueous solution by inhibiting the growth of ice crystals. This phenomenon is described as thermal hysteresis (TH), a difference between the freezing point and melting points and provides organisms in subzero environments with one of cold adaptation mechanisms. Ice recrystallization (IR), a process of forming a larger grain of ice in expense of smaller ones is also hampered by AFP. IR inhibition is another characteristic of AFP. The ice-controlling characteristics make AFPs a potential biomedical, food, agricultural and industrial appications. TH activity, or antifreeze activity was investigated using nanolitre osmometer from various organisms such as bacteria, fungi, insects, and coastal fish from East Sea. Of these, arctic yeast, arctic insects and coastal fish showed moderate antifreeze activity. All had antifreeze activity as well as IR inhibition activity. To further characterize AFP, we isolated arctic yeast AFP using ice-affinity chromatography. Here we present the functional and structural characterization of arctic yeast AFP.

Algae commonly infest and grow in bodies of water, particularly water exposed to the sun or other sources of light. Algae overgrowth threatens seas, rivers, lakes, ponds, reservoirs, spas, pools, industrial water systems, sea farms and other bodies of water. Especially in seashore, the overgrowth of harmful algae leads to the harmful algal blooming (HAB) or red‐tide. The HABs caused by blooming of Cochlodinium polykrikoides, Chattonella marina, and Heterosigma akashiwo have continuously threatened the fisheries as well as public health. Chemicals have long been added to water bodies to remove the algae problem in a small scale. Such chemicals are referred to “algae control agents” if they either kill algae (i.e. are algicidal) or arrest the growth of algae (i.e. are algistatic) when an effective amount of the chemical in question is dispersed in a body of water. The traditional method to remove the HABs in the sea is the spraying of yellow soil (clay). The purpose of our research project is to develop an epoch‐making and fundamental technology for the eradication of HABs. Novel methods includes the synthesis of ecofriendly synthesized algicidal compounds, sol‐gel synthesized organoclays, production of recombinant algicidal peptides and encapsulation of these compounds into the virus capsid that could be targeted to specific HAB.

Marine organisms are equipped with a variety of biotools, which help them against with the physical challenges of survival. These include teeth, fangs, beaks, spines, claws etc, essential in feeding, defense, and hunting. In contrast to the tools of human being made of metal alloys or inorganic materials, some biotools from marine organisms such as the polychaetes jaws, the squid beaks and the cuticle of the marine hydra are mainly composed of organic materials. Strikingly these organic biotools maintain stiffness, hardness and resistance to wear that is comparable to that of highly mineralized biotools of human beings. One reason for their appeal is lighter weight than industrial and biomedical tools made of various alloys. Compare a worm jaw and a steel awl: assuming that both perform equally well for a given function, for a given volume the worm jaw would weigh only a seventh of the steel awl since protein density is about 1.4 g/mL compared to 8 g/mL for steel. As the cost of energy continues to spiral upwards, weight reduction in high performance high impact materials is in need of increasing attention. Understanding physics and chemistry of light marine biotools will provide the scientists useful insights for designing energy efficient materials. Eventually, mimicking marine biotools from the organisms will replace heavy and energy consuming alloy tools.