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In IgG molecules, the glycan appended to N297 residue of upper CH2 region is indispensable for binding to FcγRs (Fc gamma receptors) expressed on the surface of various immune effector cells and.eliciting therapeutic effector functions. Therefore, aglycosylated antibodies expressed in bacteria have no ADCC (Antibody dependent cell mediated cytotoxicity) activity for the clearance of tumor cells. In this lecture, we will discuss a set of Fc (fragment crystallizable) engineered aglycosylated antibodies displaying unique FcγRs selectivities and therapeutic effector functions.

While subcutaneous tissue has been proposed as an optimal site for pancreatic islet transplantation, concern remains that the microvasculature of subcutaneous tissue is too poor to support transplanted islets. In an effort to overcome this limitation, we evaluated whether fibrin gel with human adipose-derived stem cells (hADSCs) and pancreatic islet cells could cure diabetes mellitus if transplanted into the subcutaneous space of diabetic mice. The fibrin gel with islet cells and hADSCs was subcutaneously implanted into diabetic mice resulting in normalization of the recipient’s blood glucose levels. These results were enhanced by co-treatment of fibroblast growth factor-2 (FGF2) in the fibrin gel. The hADSCs induced formation of new microvasculature via overexpression of vascular endothelial growth factor (VEGF), reducing apoptosis of the co-transplanted islet cells (enhancement of Bcl-2 expression in islets). The new microvasculature was likely secondary to induction by hypoxia-induced factor-1a (HIF-1a) in hADSCs, but may also be derived from recipient endothelial cells. Secondary to insulin contained within the co-transplanted islets, the hADSCs did not directly differentiate into endothelial cells (no detection of biomarkers of human endothelial cells), but instead into insulin-secreting cells (detection of human insulin). Mice receiving islet cell transplantation alone did not become normoglycemic. Collectively, cotransplantation of fibrin gel with islet cells and hADSCs will expand the indications for islet cell transplant therapy and the potential clinical application of cell-based therapy.

Nanoparticles and hydrogels with designed physical and chemical properties can exhibit unique characteristics which have high potentials to overcome various problems in current clinical approaches to treat diseases. This presentation will introduce our recent study in the application of designed nanobiomaterials for therapeutic growth factor and cell delivery. Targeted delivery of gold nanoparticles conjugated with angiogenic growth factor to ischemic tissues resulted in recovery of blood perfusion through enhanced angiogenesis. Transplantation of designed alginate capsule-in-capsules containing pancreatic islets and nanoparticle contrast agents allowed immunoprotected insulin-producing cell delivery and multimodal imaging. Active ferrogels that can be remotely controlled by a magnetic field was fabricated to deliver various biological agents including anticancer drug, DNA, growth factor, and cells on demand.

Electrically conducting biomaterials have garnered new attentions from bioengineers since they can electrically modulate cellular functions and communicate with cells via electrical signals at the material interface, which can utilized for various biomedical applications such as tissue engineering scaffolds and prosthetic electrodes. My research has been focused on engineering of biomaterials with electrically conducting organic materials (i.e., polypyrrole and graphene) and investigation of cell and tissue responses. First, biocompatible three dimensional conducting nanofibrous scaffolds were produced from poly(lactide-co-glycolide) nanofibers and polypyrrole as neural tissue scaffolds. Electrical stimulation and scaffold structures could enhance neuronal cell growth and differentiation. Further modification of the conducting nanoscaffolds with nerve growth factor allowed for inherent neurotrophic activity. Neurite formation and elongation was observed with multiple stimuli including ‘electrical’, ‘physical’, and ‘biochemical’ cues. In addition, novel electrically polymerizable pyrrole-hyaluronic acid conjugates (PyHA) were synthesized and utilized for the development of biocompatible neural prosthetic probes. The modified electrode interfaces prevented adhesion and migration of inflammatory cells (i.e., reactive astrocytes). In vivo implantation revealed that the PyHA modified probes reduced inflammatory astrocyte activity in brain compared to unmodified probe controls. Lastly, an electrical elastomer, graphene-layered PDMS (polydimethylsiloxane), was fabricated as a flexible platform capable of modulating cellular responses with external electrical stimulation and tunable material mechanical properties. Cellular interactions with these materials were demonstrated with skeletal muscle cell growth and myotube formation. I envision that the ultimate progression of these studies will facilitate the use of electrically conducting materials as bio-interfaces to modulate tissue function.

Recent developments in biomaterials for tissue engineering, novel cell culture techniques, and newly discovered chemicals as a replacement of the growth factors have opened new area toward the engineered technique of biomedical applications, for use in both research and clinical applications. Ideal bio-platform must be biocompatible that is to say, they should show both proper surface stability for the promotion of cell attachment and functions with drug delivery. However, plenty of challenges are still in progress, as the reason for the high compatibility of polymer film in cell culture environment and precisely controlled functional release of their drugs. To fulfill satisfactory of these requirements, attention has turned recently to not only introduce new materials but also developing nano-size controlled film structure of drug-delivery platformsassuchfilmscanenhancethespecificfunctionsincludingstimulicell-differentiation,- expansion,fightinginfectionandpromotin gwound- healing.Muchefforthasbeendevotedtocontrollingdrugreleaseviamanipulatingthedegradationordissolutionofth efilms. Layer-by-layer (LbL) self-assembly technic has been developed and used to prepared biocompatible multilayer films and polyelectrolyte capsulesfordrugdelivery.Certaindrugmolecules,suchasactiveproteins,cytokine,growthfactors,enzymes,nucl eicacids,andDNA,haveb eenimmobilizedintonano- sizedmultilayerfilms.TheadvantagesofLbLmultilayerfilmsasdrugdeliverysystemsincludethevariationofca ndidatematerials,alsodrugmoleculesitselfcanuseaseitherfunctionaldrugsorcomponentsofthefilm,andeasyto combinetoadditionalfunct ionsduetotheirevenlydistributedstructure.Furthermoresustaineddrugreleaseispossiblethroughcontrollingthefi lmphysical&chemicalp roperties,inadditionmultilayerfilmshavethepotentialtoprotectdrugmoleculesfromlosingtheirbiologicalfunctions ,andthefilmpreparati onprocessissimpleandcanbeautomated.Thedrugreleasebehaviorofpolyelectrolytemultilayerfilmsdependsont hepermeability,thedisas semblyorerosionofthemultilayerstructure,andotherexperimentalvariables. In this presentation, we prepared the cell friendly platform by take full advantages of LbL assembly with evenly distributed drug loading by nano-sized layer assembly. The platforms are prepared by various materials including not only synthetic-, natural-polymers but also functional materials such as growth factors, cytokines which are resulting different film degradation profiles. In addition, we also introduced the functional objects into multilayer film, such as block copolymer micelle as a drug container, nanotubes for enhanced mechanical strength. The structures including functional obejcts could be very useful in achieving additional functions for various cell studies. These results lay a cornerstone for future studies to achieve the multi-functional platforms including programmed loading/release of drugs for therapeutic purposes.

Two intestinal bacterial enzymes, Escherichia coli α-xylosidase (YicI) and Lactobacillus johnsonii α-1,3-glucosidase (LJAG31), are classified to glycoside hydrolase family 31 (GH31) based on their amino acid sequence similarity. The molecular mechanism and applicability of the two enzymes were studied by analyzing reaction products and kinetics in combination with site-directed mutagenesis.The specificity of the aglycone-binding site of YicI was characterized by examining transxylosylation-catalyzing property of the enzyme. Acceptor specificity and regioselectivity were investigated. YicI preferred aldopyranosyl sugars with an equatorial 4-OH as the acceptor substrate. This observation suggests that 4-OH in the acceptor sugar ring is essential for the transxylosylation. The disaccharide transfer products formed by YicI inhibited intestinal α-glucosidases. Heterologous production of LJAG31 by E. coli was poor due to inclusion body formation. However, this problem was overcome by several strategies. LJAG31 displayed higher specificity toward nigerose, maltulose, and kojibiose than other natural substrates having anα-glucosidic linkage at the non-reducing end. To our knowledge, LJAG31 is the first bacterial α-1,3-glucosidase to be characterized with a high kcat/Km value for nigerose. By mutating Asp409, the chemical rescue and glycosynthase reactions were monitored. All D409 mutants lost their activity, but D409G among the mutants showed the restoration of activity by externally added sodium azide, resulting in the formation of β-glucosyl azide. This finding demonstrates that D409 is the catalytic nucleophile in LJAG31. In addition, glycosynthase activity of those Asp409 mutants was verified.

To make wider the practical use of enzymes for chemical industry, it is important to diversify available reaction types by developing multi-component enzymatic processes, which are functional combination of enzymatic reactions and complex biological systems providing energy or reducing power for the reaction. Recombinant microorganisms expressing the genes coding the desired enzymes were constructed and their potentials were evaluated in combination with their accessory components such as cofactor generation system, energy generation system, and inhibitor decomposing system. In this talk, the examples of multi- component enzymatic systems were presented in the application of stereospecific and regiospecific alcohol synthesis together with enzyme screening in microbial novel metabolisms. (1) Dioxygenase process: We obtained novel 2-oxoglutarate-dependent amino acid dioxygenases and applied them to hydroxy amino acid production. As an 2-oxoglutarate generation system, TCA-cycle of E. coli was metabolically engineered and the modified E. coli cells were used as a host cell for dioxygenase expression. As a result, a practical process for optically pure 4- hydroxyisoleucine production was established. (2) Aldolase process: Deoxyriboaldolase (DERA) from Klebsiella pneumoniae was overexpressed in E. coli. The E. coli cells produced 2-deoxyribose 5-phosphate (DR5P), a key intermediate for 2’-deoxyribonucleoside production, from acetaldehyde and fructose 1,6-diphosphate (FDP). Coupling with an efficient ATP-generating system for FDP production from glucose through partial reactions of alcoholic fermentation by baker’s yeast enabled direct production of DR5P from glucose and acetaldehyde. (3) Cytochrome P450 monooxygenase process: A mutant cytochrome P450 BM-3, F87V, catalyzed regioselective hydroxylation of various phenolic compounds. To enhance this activity, P450-stabilizing/activating factors were screened. Addition of superoxide dismutase to the P450 BM-3 reaction mixture greatly increased NADPH-consumption rate, slightly increased coupling efficiency, and resulted in acceleration of the reaction rate. (4) Enzyme screening in novel microbial metabolisms: Enzymes useful for multi- component enzyme systems were found through the analysis of novel microbial metabolisms of anaerobic fatty acid transformation, hydroxy amino acid metabolisms and oxidative pyrimidine metabolisms and applied for the production of useful chemicals.

Oligosachharides in human milk have various useful biological functions for infant and applications in industry. Among them, sialyllactose(3’/6‘-SL) and fucosyllactose(2‘/3-FL) draw our atttention. To produce these oligosaccharides in large quantity, economic supply of CMP-Neu5Ac, highly active sialyltransferase(ST) and fucosyltransferase(FT) are keys to the success of the process development. Most sialylated oligosaccharides consist of Neu5Ac attached to galactose by an α2,3- or α2,6- linkage. In this research, α2,3-ST and α2,6-ST were engineered by hybrid approach to improve production of siayllactose. Hybrid approach is combined with rational design and directed evolution. This method can reduce library size by selecting target region such as substrate binding pocket and functional residues based on alanine scanning and computational mutation analysis. Saturation mutagenesis was done for selected residues to find best hits. First, multifunctional α2,3 ST from Pasteurella multocida was engineered by this approach. We selected non-conserved residues located in substrates binding site by alignment with STs in GT80 family. And we applied alanine scanning for the selected residues. Mutants which show neutral activity were selected for saturation mutagenesis, and a single mutant interacting with lactose showed 168% of increased specific activity. Also, specific activity of a combination mutant was increased 200% compared to wild-type. In addition, α2,6 side reaction was reduced significantly for R313 mutants. Second, α2,6 ST from Photobacterium damselae showed low activity and protein expression level. Therfore, it was engineered to increase catalytic activity. Substrate binding sites were predicted through homology modelling and functional residues were selected by the same method. Several mutants which show higher activity than wild type were screened by color assay method. Among them, single mutant interacting with CMP showed 4-5 times higher activity than wild type. Thus, α2,3- and α2,6-ST mutants obtained by hybrid approach will be an efficient tool for the improvement of production of sialyllactose. In addition, Fucosyltransferase is used for the synthesis of fucosyllactose(2‘/3-FL) and sialyl-LewisX derivatives, and the same hybrid approach was undertaken. The major problem of FTs was their inclusion body formation and low level of soluble expression. How we have overcome this problem will be discussed.

Synthetic organophosphates (OPs) have been used as nerve agents and pesticides due to their extreme toxicity and have caused serious environmental and human health problems. Therefore, effective methods for detoxification and decontamination of OPs are of great significance. We have working on the development of organophosphate-degrading enzymes through directed evolution and structure-based rational design. For directed evolution, we constructed and used a high throughput screening system that was based on phenolics-responsive transcription activator for directed evolution of organophosphate-degrading enzymes. In the screening system, phenolic compounds produced from substrates by OP-degrading enzymes bind a constitutively expressed transcription factor DmpR, initiating the expression of enhanced green fluorescent protein (EGFP) located at the downstream of the DmpR promoter. Fluorescence intensities of host cells are proportional to the levels of phenolic compounds, enabling the screening of OP-degrading enzymes with high catalytic activities by fluorescence-activated cell sorting (FACS). Methyl parathion hydrolase (MPH) from Pseudomonas sp. WBC-3 and p-nitrophenyl diphenylphosphate (pNDP) were used as a model enzyme and an analogue of G-type nerve agents, respectively. The utility of the screening system was demonstrated by generating a triple mutant with a 100-fold higher kcat/Km than the wild-type enzyme after three rounds of directed evolution. We also conducted structure-based rational design, and obtained enzymes with high catalytic activity. The contributions of individual mutations to the catalytic efficiency were elucidated by mutational and structural analyses. Our approach is expected to be widely used for developing organophosphate-degrading enzymes with greater potential.

Enzymes utilizing maltodextrin and glycogen in bacteria were studied to investigate their roles in maltodextrin and glycogen metabolism. Bacillus subtilis mutants deficient maltogenic amylase (ΔyvdF) and/or pullulanase (ΔamyX) were constructed. A series of Escherichia coli mutants lacking MalP (maltodextrin phosphorylase), MalZ (maltodextrin glucosidase), MalQ (4-α-glucanotransferase), and/or GlgA (glycogen synthase) were constructed, and the glycogen produced from the mutants of E. coli and B. subtilis was analyzed. The data on the amount, the structure, and hydrolysis rate of the glycogen from Bacillus mutants revealed that the maltogenic amylase and the pullulanase have a significant role in the synthesis as well as the degradation of the glycogen. In the E. coli mutant lacking the glycogen synthase, glycogen was synthesized from the maltose by the action of MalQ. E. coli MalPmutant accumulated 20 times greater amount of glycogen than the wild type, and the result implies that MalP controlled the glycogen accumulation of the MalQ-dependant pathway. In conclusion, the maltodextrin and the glycogen metabolism in bacteria are tightly interconnected by the actions of the glycoside hydrolases.

Recombinant proteins containing an N-terminal fragment of splitinteins were selectively labeled with a fluorescent dye in live cells through an amide bond. This approach allows introduction of various ligands with diverse physical properties to a target protein. Such in situ addition of small probes perturbs the properties of target proteins much less than does green fluorescent proteins and enables monitoring the functions and locations of target proteins in live cells. Additionally, the use of photo-chemical switch allows the necessary temporal control for studying the function of proteins with increased sensitivity. This system provides a novel and effective approach to control the fluorescent tagging of proteins in live cells using light as an external stimulus.

In this presentation, I will discuss several recent achievements developed in my laboratory about electrochemical detection methods for genetic diagnosis. First, we developed novel electrochemical strategies to detect nucleic acids utilizing neutral charge property of peptide nucleic acid (PNA) orunique binding property of DNA intercalating agent.1,2New electrochemical methods to identify DNA mutation and SNP genotype were also developed based on mismatched DNA-specific cleavage activity of CEL I or S1 endonuclease.3,4 I will also discuss novel strategies for the realization of the electrochemicalreal- time PCR based on the transport control of signaling materials which include methylene blue or metal nanoparticles.5,6 Finally, I will discuss our unique and innovative biomolecular detection platformutilizing capacitive touchscreen.7 By systematically utilizing both surface capacitive and projected capacitive touchscreens, we have demonstrated the detection capability of a new technology for detection and quantitation of DNA and also verified the potential of this technology in the biosensing area. These results developed by our group will serve as the important basis for advanced electrochemical strategies for genetic diagnosis.

The photonic-based biosensing technologies have been utilized as one of the most useful tools for clinical diagnosis and bio-analysis owing to their analyzing performances with high sensitivity, wide dynamic range and simple mechanism of signal transducing. Among the diverse optical biosensors, the biosensors using evanescent field of optical wave are highly sensitive to the changes of the refractive index in the vicinity of sensor surfaces. Therefore, one can sensitively detect the biochemical substances bound to the surface without any labels in real time. Surface plasmon resonance (SPR), localized surface plasmon resonance (LSPR) [1,2] interferometric biosensors and optical waveguide sensors such as metal clad waveguide (MCW) [3-5] are categorized into this type of biosensor.The current presentation introduced highly sensitive detection of biomolecules using SPR and LSPR for the applications of bioassay and clinical diagnosis, including nanofabrication technologies realizing LSPR biosensor. In addition, we show that the MCW can be utilized for monitoring large biosubstances such as cells as well as for analyzing the biomolecular interaction.

Now a day much attention pay on planar Hall resistance (PHR) sensors due to its high signal-to-noise ratio, small offset voltage and very linear response at low field range compare to other magnetoresistive sensors [1]. In the past few years, multilayer sensor structures (bilayer, spinvalve or trilayer) have been investigated to progress the sensitivity of PHR sensor using cross-shaped sensor junction and the sensitivities of the PHR approximately 3, 7 and 12 uV/Oe respectively. Among these structures, the higher sensitivity has been obtained for trilayer due to small exchange bias field and small shunt current. For further improvement of the sensitivity, we have developed a new geometry (ring-shaped) of the sensor with Wheatstone bridge configuration over conventional cross-shaped. Both the output voltage and the sensitivity increases with the ring radius for a constant ring width. In addition, we have integrated multiple rings in a one ring junction of the sensor called multi-ring sensor, where the sensitivity of the sensor with 7 rings was found to be more than 600 uV/Oe. The interaction of biotin and streptavidin has been exploited using PHR sensor where this interaction is used in many protein and nucleic acid detection due to the streptavidin-biotin complex is the strongest known non-covalent interaction. We have performed the detection of streptavidin conjugated with magnetic label and biotin immobilization on the sensor surface modifying by APTES. We have succeeded to detect a 2.9 femto-molar (fM) concentration using PHR sensor. Figure: 3D-microscopic images of (a) cross-shaped, single-ring, multi-ring with 5 & 7 rings of PHR sensors, (b) schematic of biotin-streptavidin conjugation with magnetic label for detection, (c) detection of femtomolar streptavidin.

The autodisplay technology is a protein expression method which produces a target protein as a fusion protein on the outer membrane of E. coli. This work presents the application of autodisplayed proteins such as Z-domain and streptavidin for the hyper sensitive immunaffinity biosensors. Usually, immunoaffinity (IA) biosensors have been made by immobilizing antibodies on metal surface of transducers as a molecular recognition layer. As the antigen-binding sites of antibodies are located at Fab region, only 20% of the immobilized antibodies have active orientation for the binding of analytes. In this work, (1) Z-domain was autodisplayed on the outer membrane of E. coli for the orientation control of immobilized antibodies [1-2], and (2) streptavidin was also autodisplayed for the immobilization of antigens with controlled orientation and high density [3]. For the application of such autodisplayed proteins to biosensors, two kinds of assay configurations were used: (1) E. coli cells with autodisplayed proteins were directly used for immunoassay [4-5], and (2) the outer membrane with autodisplayed proteins was separated and then layered on the transducers based on surface plasmon resonance (SPR), fluorescence microarray, flow cytometry (FACS) and amperometric analysis [6-8]. The biosensors with autodisplayed proteins showed far improved sensitivity as well as limit of detection, and such biosensors were demonstrated to be feasible to medical diagnosis of acute and chronic inflammatory diseases [8-10].

A manufacturing process use raw materials through appropriate processing, and end up with final products of our needs. Mechanical and electronics industries use many parts that can be assembled into final products such as automobiles and televisions, etc. Chemical and biotech industries use chemicals and biochemical to make their own products from raw materials such as coals, oils, natural gases; starch bearing plants, sugar canes, and their derivative products. Biotech industries needs raw materials (mainly biological but some chemical) to produce products of their needs from small molecules (MW less than 100) to very large molecules such as proteins, macromolecules (PHBs, PHAs) and even stem cells for tissue engineering for regenerative medicine. Biotech products are very diverse these days. Here I like to focus on two products of bioethanol and biodiesels by chemical catalysts, and fermentation using unconventional raw materials (low cost waste organics); unconventional fermentation technologies of mixed culture; unconventional bioreactor technologies (multi-stage continuous high cell density culture: MSC-HCDC) and downstream processing of water removing technologies. In this way perhaps we can make microbial diesel, currently known as uneconomical, economical.

Nepal is full with rich and diverse resources of animals, both domesticated and wild. Most population live in rural areas where their livelihood is dependent heavily on livestock wealth that comprise some 17 domestic livestock species (Wilson, 1997), including five Bovidae, seven Aves, two Equidae, pig (Sus scrofa), rabbit (Oryctolagus cuniculus) and elephant (Loxodonta africana). Among these species, cattle (Bos taurus and Bos indicus) are the largest in number of quadrupeds, while buffaloes (Bubalus bubalis) are economically the most important ones. Local cattle and buffalo are quite large in count (Cattle 8.1Million, Buffalo 4.1M) but very poor production with an average of less than 1400 liters per lactation cattle and 1500 liters per lactation of buffalo respectively. To meet the increasing demand of milk and dairy product has been a great challenge to the department of Livestock Services of Nepal. Cross breeding of local cattle by using Artificial Insemination (AI) in the farmers shed even in remote villages is the beauty of this program in Nepal. By the end of 2010, Nepal only covered artificial insemination to 6-7% of the total breeding population which is urgent to increase. Nepal is launching Dairy Cattle improvement intensively by using semen from jersey, Holstein-Friesian (HF) bulls and Murrha buffalo bull which is the choice of the farmers. To intensify the AI coverage private veterinary officers and paravet practinecers are heavily involved and the number of annual AI has significantly increased with hope that cross bred heifers will increase and milk production will go up with improved herd production record.

Jatropha curcas is non-edible oil plant which has great potential to be used as the raw material for biofuels production. Currently, the seed yield of Jatropha curcas is not high comparing to the oil palm. Therefore, many researchers work intensively to increase the yield of Jatropha curcas. Some researchers have focused on the hybridization or using the chemical growth promoter or inter-crossing and genetic engineering to increase the yield or to have the new high yield varieties. However, this paper presents the sustainable yield development of Jatropha curcas through combination scheme. This scheme combines the variety selection, yield trial, cultural practice and plantation management. It takes about 10 years to have good sustainable varieties which farmers can plant with the seed without physiological and productivity changes.

To develop sustainable, low-input but highly productive agricultural practices for better management of soil resources, to combat climate change by reducing the input of energy-intensive chemical fertilizers in Indian soils and resultantly improve soil quality, a sustainable biofertilizer technology is required to facilitate product delivery to enhance crop productivity. Fluorescent pseudomonad strain R81 is a root colonizing rhizobacterium which has a growth- promoting effect on many plants. The PGPR produced a hydroxamate-type siderophore that chelates iron and makes it available to the plant roots and 2, 4- diacetylphloroglucinol (DAPG), an antibiotic that suppresses fungal pathogens. The presence of the siderophore and DAPG in the bioinoculants formulations was found to limit the presence of natural contaminants in non-sterile carriers to acceptable limits, thereby obviating the need of normally-used costly three-time sterilization of the carriers. The purpose of the work was, therefore, to develop suitable culture strategies which can be easily applied at an industrial scale. Accordingly, both a simple batch and a more productive fed-batch culture strategy were developed for mass production of the pseudomonad. In batch procedure, splitting of nitrogen source to NH4Cl and urea had a stabilizing effect on pH which obviated the need of pH control during cultivation. During batch cultivation of the strain in a bioreactor, a maximum of 5 g/L of dry cell mass was achieved when glycerol was 15 g/L and C/N ratio was maintained at 12.5. At higher concentrations of glycerol the biomass yield was lowered due to substrate inhibition. Therefore, the cultivation of the strain was carried out in fed-batch mode with the objective to produce increased biomass, siderophore, and DAPG. LabVIEW software- based computer controlled system was established which achieved flexible and automatic monitoring and control of process parameters. Open loop feeding strategy such as exponential feeding of nutrients and closed loop feedback strategies using dissolved oxygen and pH signals were used in a bioreactor for fed-batch cultivation of the pseudomonad for maximal production of cell mass and DAPG. Compared to batch culture which produced viable cell counts of the order of 5.0 x 109 cfu/ml in a run, the fed-batch culture using pH-based signals produced about 2.7 x 1011cfu/ml, thereby significantly enhancing the productivity of the process by about 50 times. The harvested culture broth was used for preparing a bio-inoculant formulation. The effectiveness of the application of the PGPR formulation has been demonstrated for wheat (Triticum aestivum) crop.

Carbon capture technology development is one of the main research topics in the field of climate change mitigation due to continuous increase of CO2 concentration in the atmosphere that caused “green house” effects leading to global warming. However, industries and businesses are reluctant in implementing the developed carbon capture technology because it needs capital investment and operational expense. There is a need to find carbon capture technology that can recover part of the investment and operational cost to make it more sustainable. In this paper, a system that captures CO2 and at the same time have the potential to recover capital and operation cost of the CO2 capture process was developed and characterized. This carbon capture system combined gas absorption combined with biofixation by converting the captured CO2 to algal biomass. In conventional system, CO2 is absorbed by an absorber which is usually a gas-liquid contacting system. The problem of this system is, what to do with the captured CO2?. Aside from that, the solution that absorbed CO2 may contain nitrogen in the form of ammonia or organic amines, which are fertilizers. If this solution is released to the receiving bodies of water, it may cause algal bloom and possible euthropication that may result in the eventual release of CO2 to the atmosphere. The system that we studied solves this problem by combining the absorption with biomass fixation system that converts the captured CO2 to algal biomass in a controlled environment. The harvested algal biomass was characterized for its suitability as biodiesel feedstock or as a biorefinery feedstock. The developed system may have a potential to be sustainable because part of the capital investment and the operating cost can be recovered from the potential revenue derived from the algal biomass.

The aerobic degradation enzyme system of biphenyl has been characterized well in a variety of bacteria. It is involved in the degradation of polychlorinated biphenyls (PCBs), which have caused environmental problems by their long-lasting environmental contamination. At the initial step of this system biphenyl is hydroxylated by a biphenyl dioxygenase to generate a dihydrodiol intermediate, which is converted to a diol intermediate, 2,3-dihydroxybiphenyl by a dehydrogenase. The aromatic ring of 2,3-dihydroxybiphenyl is cleaved by an extradiol ring-cleavage dioxygenase to form a phenylhexadienoate, whose side chain is trimmed by a hydrolase to produce a pentadienoate and a benzoate. Pentadienoate is metabolized by the actions of a hydratase and an aldolase via oxovalerate to pyruvate and acetoaldehyde, which is converted to acetyl-CoA by acetoaldehyde dehydrogenase. Benzoate is metabolized through the independent benzoate pathway via a dihydroxybenzoate, catechol and 3-oxoadipate to succinate and acetyl-CoA. Pyruvate, succinate and acetyl-CoA formed from biphenyl are utilized in TCA cycle. PCBs are co-metabolized by this biphenyl degradation system. In gram-negative biphenyl-degrading bacteria the upper and lower catabolic pathway enzyme genes responsible for the enzyme steps from biphenyl to benzoate and pentadienoate and those from pentadienoate to acetyl-CoA, respectively, are coded in a single gene cluster. On the other hand, the upper and lower pathway enzymes of biphenyl catabolism are coded by at least five separate gene clusters and multiple isozymes are responsible for each enzyme step in the gram-positive biphenyl-degrading bacterium, Rhodococcus jostii RHA1. In addition a couple of regulatory systems are simultaneously involved in the induction of these catabolic pathway enzyme genes in RHA1. The strain RHA1 grows on a variety of aromatics such as benzene and alkylbenzenes. The multiple enzyme and regulatory systems are appeared to be responsible for the growth on and degradation of a variety of aromatics in RHA1. The advantages and disadvantages of multiple enzyme systems will be discussed.

Who will support the world population of more than 9 billion people in 2050? To cope with global crises over food and energy supplies as well as environmental problems, it is urgently required to develop new crop varieties to be grown on marginal lands including dry, high salty and contaminated lands. Sweetpotato is an important food and industrial crop to produce starch and useful components including antioxidants. The nonprofit CSPI (2007) designated sweetpotato as one of ten super foods for better health. USDA (2008) reevaluated that it is the best starch crop on marginal lands. Little research has been carried out on sweetpotato antioxidants at the molecular level, despite its importance as the well-being food source. In the presentation, our recent results on metabolic engineering of pigment antioxidants in transgenic sweetpotato by down-regulation of β-carotene hydroxylase (CHY- β) and lycopene ε-cyclase (LCY-ε), and by overexpressing IbOrange gene will be introduced. We anticipate that rational sweetpotato biotechnology will lead to development of industrial sweetpotato plants with enhanced tolerance to environmental stress on marginal lands.

Oil palm sector generates the largest amount of biomass. About 90 million dry tonnes was estimated in 2010 and expected to increase to about 100 million tonnes by 2020. Oil palm industry generated abundance of renewable biomass such as 13.97 million tons of trunks (OPT), 44.84 million tonnes of fronds (OPF), 6.93 million tonnes oil palm empty fruit bunch (OPEFB), 4.21 million tonnes shell, 7.29 million tonnes mesocarp fiber (OPMF), 3 million tonnes of decanter cake (OPDC) and 60.3 million tonnes of palm oil mill effluent (POME). The conventional practice to treat the POME is by using a series of low cost ponds require a large area of land, long retention time and release huge amount of methane gas to environment. As a sustainability of palm oil industry, we proposed for the development of a modern and complete methane fermentation system by changing lagoons into a profitable area. The sealed area will become the available area for new business to which biomass energy can be supplied from palm oil industry at a reasonable price. Thus the generation of green renewable electricity from methane will give an alternative for new biomass business towards zero emission and create new waste to wealth phenomenon. With the appropriate downstream biomass processing technologies, it contributes to the reduction of greenhouse gases emission by sealing the lagoons, prevention of undesirable smell and water pollution by modern treatment plus water recycling and finally local employment can be encouraged from a new business.

Pseudomonas aeruginosa have been known as one of biosurfactant producers. We have been utilizing P. aeruginosa to utilize glycerol waste from a palm oil industry, whey waste from dairy industry and used cooking oil to produce biosurfactant. The aim of this research is to lower the oil-water surface tension of oil trapped in rock pores of an oil well, known as Microbial Enhanced Oil Recovery. Previous application of our biosurcfactant in several wells (P-02 and P-03) observed changes in the surface tension of oil-water. Inoculation in both wells were made in anaerobic conditions. Population of microbes, nutrients and pH and its influence on the surface tension changes were observed in the period 0-72 hours. Decline in the oil-water surface tension was observed for wells P-02 at 72 that was equal to 23.7%. As for the P-03 wells at 24 it was equal to 39.5%. Observations indicate the formation of salts of organic acids and inorganic electronics afterward for both wells, rendering increase in the surface tension. Because of differences in the characteristics of the two wells, there was no obvious relationship between various parameters that were observed. The acidity of the two wells was sufficient to support microbial activity in the range pH 6.7 to 7.8 scale. Nutritional changes did not show any significant changes in surface tension. P-02 wells microbes consumed more sulfate and phosphate. On going research is utilizing ozone to increase the substrate availability of wastes.

Systems biology is an interdisciplinary field of science that emphasizes on complex interactions within biological systems. It uses a more “complete” perspective approach to understand biological and biomedical contexts such molecular profiling technologies as microarray based transcriptomics and mass spectrometry based proteomics. However, changes in gene expression level may not directly be interpreted to alterations in proteins abundances. There are many ways how proteins, and especially enzymes, can be regulated to manage metabolic fluxes and ultimately, phenotypes, from protein translation, post-translational modifications and allosteric actors (metabolites). Thus, the read-out of cellular regulation must include metabolite analysis to explain a systemic view of responsive metabolism, called metabolomics. Here, various applications of metabolomics to study metabolic responses to genetic and/or environmental perturbation are presented as stands-alone research and also integrated analysis with mass-spectrometry based semi-quantitative proteomic approaches.

An adequate knowledge on molecular mechanism of melanogenesis provides an opportunity to find the novel molecular targets for the new cosmetics discovery and development. These known targets are involved in other essential metabolic processes thus inhibition of their function affects the skin health. A nonpathogenic albinism caused by selective mutation or deletion of the OCA-2 gene have motivated us to target this gene and respective protein (P-protein) for new kind of cosmetics. The aim of the present work was to screen the molecule which specifically binds to this protein, resulting as inhibition of the melanin synthesis. Unfortunately, the structure of protein is not available, thus Dock based screening is pity hard. To resolve this we used a pattern search for amino acid sequence, based on homology analysis. The role of this protein have been cited more versatile than the regulating the tyrosine transport to melanosome. Thus, we have tried to generate a molecular library of a homologue to tyrosine and tyrosine-transporter inhibitor. Based on the dock-score five molecules have been subjected to ADME/TOX analysis. The molecules were predicted as non-carcinogenic by START program. Based on dock score a 20 molecule have been selected and 8 were found to be quite effective as anti melanogenic without having any toxicity. Among these compound, compound #1, #2, #3, #4, #8, #13, #16, #17 showed the significant antimelanogenic properties at a concentration of 20 mM. Further investigation in relation to mechanism of action is under process.

Several recent studies have highlighted the inhibitory effects different plant hydrolysate compounds have on the conversion of plant cellulose to biofuels. These compounds include phenolic acids and aldehydes as well as furans. In this talk, I will discuss research out group has performed to (1) Select biomarker genes from E. coli that are responsive to these compounds; (2) Clone the promoters regulating expression of these genes to generate transcriptional fusions with the bioluminescence genes; (3) Characterize the responses from bioluminescent strains of E. coli to a wide number if chemical effectors and actual rice straw hydrolysates. The results show that we can detect these compounds, including ferulic acid, vanillin and furfural, at concentrations below their published inhibitory concentrations, making these strains a quick and effective tool to assess for these and other chemical effectors within biomass hydrolysates samples.

In this present study, a Pipes Inserted Microalgae Reactor(PIMR) containing pretreatment system including Ca &Mg was developed and employed for microalgae remediation for heavy metals. The pretreatment system could reduce the initial high concentrations of Fe and Mn released from AMD and served to supply into the PIMR. The hybrid system combined with pretreatment system and PIMR could enhance the reduction of heavy metal in AMD and provide an economic means of improving bioremediation for enhancing microalgae production.The study concludes that PIMR was developed and operated for microalgae cultivation. The pipes in PIMR served to delivery light deeper into the photobioreactor and distribute light to microalgae. And PIMR could be used as an efficient reactor for the removal of iron from AMD and cultivation of microalgae. Batch studies showed that this reactor and microalgae can adsorb iron with an uptake of 63.21 mg/L iron. Continuous studies also prove that PIMR can maintain to remediation of metal and cultivation of microalgae.

High functional cosmetics originated from plants have been developed and become a major trend all over the world in these days. Various papersshowed that the extractions and stabilizations of active ingredients from plants, converting them into cosmetic products, have been investigated extensively. Active ingredients from 100 domestic wild plants were separated and done in vivo test to find out whitening and anti-aging efficacies. First, five highly effective ingredients were selected and converted to low molecular ingredient to improve skin penetration by using bioconversion technology. Second, the stabilization and the enhanced solubility of active ingredients were obtained by using nano liposome technology. Those active ingredients were further stabilized by inclusion complex with silica. In addition to previous technologies, multilamellar liquid crystal and W/O/Wtechnologies have been adopted to formulas stabilization for whitening and anti-wrinkle products. Finally, the establishment of large scale-up production condition through process standardization was achieved for active ingredients.

To reduce skin irritation and/or inflammation induced by irritants and/or allergens which may be from environmental factors or cosmetics is one of main issues that most cosmetic companies concern. It has become increasingly important to develop safer cosmetics because most people think that cosmetics are one of daily necessaries regardless of age and gender. We performed this project to develop new cosmetic ingredients from natural origins, which can reduce skin irritation and/or inflammation, and which are useful for sensitive skin, atopic-like skin, and troubled skin. Several in vitro screening methods to evaluate the anti-inflammatory effects of natural ingredients were set up, and the safety and anti-irritancy of screened ingredients were confirmed clinically through skin patch test. We also evaluated skin barrier function of screened ingredients that are expected to prevent from weakening the skin barrier. Finally, screened ingredients were formulated in cosmetic products as active materials. Therefore, it is expected both that we can provide the market with new cosmetics which are useful for sensitive skin, atopic-like skin, and troubled skin, and that the screened ingredients can be formulated to reduce the irritation of paraben-free products or sun-care products which may have a possibility to include irritative ingredients.