Earticle

Bioethanol is one of the most common biofuels that can be obtained from various types of biomass. Recently, seaweed biomass has received great attention as a new potent feedstock for bioethanol production because of its abundance in coastal area and high CO2 capturing ability during the growth. To produce ethanol from seaweed, polysaccharides contained in the seaweed biomass should be first converted into easily-fermentable monosaccharides. This saccharification step is usually considered as the bottleneck of the ethanol production so that it is important to develop an efficient method, whatever it would be biochemical or physicochemical one. In this study, we isolated several new bacteria capable of performing saccharification from the gut of Batillus cornutus, and evaluated their enzymatic abilities to hydrolyze the polysaccharides of a brown alga, Laminaria Japonica. Using DNSA (dinitrosalicylic acid) method, we determined the enzyme activities of the isolated bacteria, which encompassed cellulase, alginate lyase and laminarinase.

A micelle-fractional precipitation hybrid process was developed for the effective pre-purification of the anticancer agent paclitaxel extracted from plant cell cultures. By removing waxy substances originating from plant cells using the adsorbent sylopute, the efficiency of the subsequent pre-purification process could be remarkably enhanced. Paclitaxel yield was improved and the fractional precipitation time was shortened compared to the control by increasing the surface area per working volume (S/V) of the reacting solution through the addition of a cation exchange resin (Amberlite IR120 or Amberlite 200), an anion exchange resin (Amberlite IRA400 or Amberlite IRA96), or glass beads. Most of the paclitaxel (>98%) could be obtained after about 12 h of fractional precipitation using Amberlite 200. Purity increased with increasing fractional precipitation time up to 9 h to about 85%, after which it showed little change. On the other hand, no paclitaxel precipitate was formed using either of the nonionic exchange resins because paclitaxel, which is hydrophobic, was strongly adsorbed on the hydrophobic resin surface. Since high purity paclitaxel can be obtained in high yield and the precipitation time can be reduced by combining micelle formation with fractional precipitation, this hybrid method is expected to significantly enhance the final purification process.

Expression level of protein is affected by various factors including mRNA stability, codon usage and productive binding between RBS and ribosome. It is well-known that direct interactions between the Shine-Dalgarno (SD) sequence and ribosome directly influence translation initiation, that is, protein expression level. Here, we proved that secondary structures of the 5’ proximal coding regions are also crucial for translational efficiency by exploring how expression levels of RFP (DsRed-monomer) were dependent on mRNA secondary structures in 5’ proximal coding region. Random silent mutation of 5’ proximal coding region (+4~+48), which makes 13 possible silent mutation, has shown various levels of gene expression. Also we found that the gene expression increased when its mRNA secondary structure was destabilized. Among them, we chose four RFP variants which showed the highest gene expression and found that the RFP fluorescence increases more than 10-fold. From these results, we propose that manipulating 5’ proximal coding region is a powerful methodology to optimization of protein expression.This work was supported by BK21 program from the Korean Ministry of Education and Seoul R&BD Program (NT080612, KU080657).

Chlorella sp. was grown to produce biodiesel under mixotrophic condition in a recycling culture system (3 ℓ working volume) at 0.03 (ℓ/day) of recycling rate for 90days. Optimal culture conditions of mixotrophic growth were decided to be 25-30℃, 180 rpm and 3%(v/v) of CO2 supply. The effect of acetate as an organic carbon source was observed by adding various acetate concentrations: 5, 10, 15, 20 (g/L), respectively, and methanol, 0%, 0.05%, 0.1%, 0.5%, 1.0% and 3.0% (v/v), respectively. 10 (g/L) of acetate showed the maximum cell concentration as 4.8 (g-dry wt./L) with 19.8 (%, w/w) of total lipid production. While, at 5 (g/L) of acetate concentration, after 50 days the cell concentration showed relatively constant values as 2.9 (g-dry wt./L) of cell concentration. The optimal concentration of methanol was also determined as 1.0% (v/v), maintaining 4.2 (g-dry wt./L) of maximum cell concentration and 17.5% (w/w) of the total lipid production. Particularly, there was almost no cell growth and total lipid production in adding 3.0% (v/v) of methanol because of highly toxicity. In observing residual concentrations of 10 (g/L) of acetate or 1.0(% (v/v)) of methanol, it was shown that the organic carbon source consumption rate was fast during the first period of cultivation and CO2 consumption rate was increased more during the last period of cultivation. This study confirmed that optimal concentrations of acetate or methanol as an organic substrate would be a important factor to maintain high the cell concentration and total lipid production.

To immobilize the effective and specific enzymes on nanomaterials such as nanoparticles, nanofiber and nanotube for stabilizing the activity of free enzymes has developed the various fields. In this results, Lysosomal enzymes isolated from hen`s egg white and sacharomyces cerevisiae were directly immobilized on titanium (IV) oxide (TiO2) particles using shaking methods (150 rpm, room temperature, 10 min), and the immobilization efficiency, antimicrobial activity using viable cell counts against Escherichia coli, and stability of lysosomal enzymes immobilized on TiO2 were evaluated. In addition, enhanced immobilization efficiency was shown in TiO2 pretreated with a divalent, positively charged ion, Ca2+, and the antimicrobial activity for E. coli increased as a function of increasing ratio of immobilized enzymes. Furthermore, the degradation of melanin for a week and by measuring contents of peroxidase in immobilized lysosomal enzyme, we could determine the decrease of melanin in lysosomal enzymes immobilized on TiO2 after examining the effect of different pH of solvent on the immobilization of lysosomal enzymes extracted from S. cerevisiae and egg white. We found that not only the immobilization efficiency but also other results of immobilized lysosomal enzymes extracted from S. cerevisiae were higher than those extracted from hen’s egg white with pH 4. Therefore, Our results showed that the various activities of lysosomal enzymes immobilized on TiO2 are facilitated by sources and the pH differences.

The goal of this research is to develop recombinant E. coli for improved fatty acid synthesis (FAS). Genes for acetyl-CoA carboxylase (accA, accB, accC), malonyl-CoA-[acyl-carrier-protein] transacylase (fabD), and acyl-acyl carrier protein thioesterase (E3.1.2.14), which are the enzymes that catalyze the key steps in the synthesis of fatty acids, were cloned and over-expressed in E. coli MG1655. The enzymes of acc family genes catalyze the addition of CO2 to acetyl-CoA to generate malonyl-CoA, enzyme of fabD gene converts malonyl-CoA to malonyl-[acp], and gene for E3.1.2.14 converts fatty acyl-ACP chain to long chain fatty acids. All the genes were identified as homologous gene of E. coli and used to improve enzyme activities for overflowing of FAS pathway through a metabolic engineering. Eleven different E. coli MG1655 strains containing various combination of genes were developed by using pTrc99A expression vector. To observe changes in metabolism, in vivo and in vitro metabolites and fatty acids were analyzed from recombinants. The recombinant strains produced more malonic acid and fatty acids than its wild type did.

Despite many powerful advantages of iron oxide nanoparticles such as imaging and targeting tools for therapeutic purposes, its biological effects have not been extensively elucidated yet. We found that iron oxide nanoparticles can enhance the neurite outgrowth in PC12 cells. PC12 cells exposed to iron oxide nanoparticles and nerve growth factor (NGF) synergistically increased the efficiency of neurite outgrowth in a dose-dependent manner. This phenomenon might result from the activation of cell adhesion molecule associated with cell-matrix interaction. Immunoblotting assay revealed that both neural specific marker protein and cell adhesion protein expression levels were upregulated by iron oxide nanoparticles compared with non-treated cells via activation of mitogen-activated protein kinase (MAPK) signaling pathway. Our findings point out the possibility that iron oxide nanoparticles can affect cell-substrate interactions and regulate cell behaviors, which provide potential clinical insights into neurologic and therapeutic applications of iron oxide nanoparticles.

The changes in bacterial communities associated with volatile fatty acids (VFAs) profiles were investigated in anaerobic batch reactors treating swine wastewater. Denaturing gradient gel electrophoresis (DGGE) and real-time QPCR were used to analyze the bacterial community structure and the methanogenic population, respectively. The three subspecies of Lactobacillus delbrueckii (bulgaricus, lactis, and indicus)along with the Clostridium-like organismswere identified.The L. delbrueckii-related organisms were assumed to be responsible for the fermentation of the organic compounds throughout the overall reaction period. The presence of C. ultunense and C. kluyveri, together with hydrogenotrophic methanogens, was essential for methane production from propionic acid in the later stage of the experiment. The order Methanomicrobiales, hydrogenotrophic methangoens, increased rapidly as the process proceeded, resulting in its dominance above 64% after day 28, among the methanogenic groups monitored. Our results showed that the variations in the VFA concentrations and methane production were complicated and dynamic. It was also demonstrated that analyzing acidogenic community along with methanogenic population was essential for better insights into understanding anaerobic digestion of swine wastewater.

The purpose of this research is to develop new strains for producing more fatty acids by improving fatty acid biosynthesis mechanism in Escherichia coli. β-Ketoacyl acyl carrier protein synthase III (fabH) is the enzyme that catalyze the first step in the synthesis of fatty acids with acetyl-CoA carboxylase (accABC) and malonyl-CoA: acyl carrier protein (fabD) acyltransferase in Escherichia coli MG1655. fabH, accABC and fabD genes were cloned and characterized. The enzyme of fabH gene leads to increase short-chain-length fatty acids and shows strong preference for acetyl-CoA, and produces only straight chain fatty acids (SCFAs). β-Ketoacyl acyl carrier protein synthase III (fabH) is also thought to play a major role in determining fatty acids type and compositions. The fabH, accABC and fabD genes were identified as homologous gene of E. coli MG1655 through a metabolic pathway. The recombinant E. coli MG1655 containing the fabH or accABC::fabD or accABC::fabD::fabH gene-inserted expression vector (pTrc99A) was constructed. To analyze the effects of genes overexpression, analysis of intermediate products and fatty acids was performed. As a result, recombinant strains produced more acetic acid, malonic acid and fatty acids than its wild type E. coli did.

The PDA vesicles-based sensors have been interest due to their unique chromatic properties, such as blue-to-red color transition by external perturbations like pH, heat, physical stresses.1-2 Moreover, the PDA vesicles simultaneously emit the red fluorescence while changing blue to red phase. Polydiacetylene (PDA)vesicles have been demonstrated as many different types of label-free biosensors by the bichromic property. The PDA-based sensors have successfully detected various biological samples, including choleratoxin, Escherichia coli, oligonucleotides, and lipopolysaccharides since influenza virus were first detected.3-4 The liposome-like PDA vesicles are possible to have different functional groups, such as amine, carboxylic acid, and biotin, etc, by using various PDA derivatives. Therefore, the label-free PDA biosensor on the solid substrate is easily possible to be constructed. Here, we report the microarrayed PDA-based biosensors with antibody or antigen-specific aptamer to detect bacteria cells using the stress-induced fluorescence emission of PDA vesicles. Through the results, we expect that the system of micro-patterned PDA-based biosensor immobilized on the solid substrate can be applied to make a novel label-free diagnostic sensor.

Nanofiber technology has been used for the fabrication of tissue engineering scaffolds that provide the three-dimensional extracellular matrix-like structure. Biodegradable synthetic polymers, which have a good mechanical property and are able to be easily manipulated, have been conventionally used as materials of nanofiber scaffold. However, these materials have limitations such as poor cell adhesion ability, and no bioactive motifs. Mussel adhesive protein (MAP), fp-151 was previously produced by recombinant DNA technology and also showed a great cell adhesion, spreading and proliferation ability when they were coated on tissue culture plate surfaces. In this work, we used the typical electrospinning procedure to fabricate the novel MAP-coated polycaprolactone and MAP / polycaprolactone composite nanofibrous membrane by varing the ratio of polycaprolactone and MAP concentrations. Characterization of nanofibrous membrane was performed by scanning electron microscopy images, FT-IR, contact angle, and tensile strength. And also, the cell behavior effect of MAP on these nanofibrous membrane was tested using MC3T3-E1, mouse pre-osteoblast cell line.

Saccharomyces cerevisiae has widely been used in the ethanol production. However, it cannot utilize the pentose sugar in feed-stocks as a carbon source such as arabinose, fructose and xylose. Many attempts to express a prokaryotic xylose isomerase with high activity in S. cerevisiae have not been successful so far. In this study, we try to clone and express an active XI from the anaerobic bacterium Clostridium phytofermentans ATCC700394 into Saccharomyces cerevisiae 2805. Recombinant plasmid pYES2-XI was constructed to produce ethanol from xylose. In this result, the recombinant S. cerevisiae strain exhibited a low xylose consumption and activity. However, when galactose was added into the medium, the gal promoter was induced and xylose isomerase was highy expressed. As a result, the xylose consumption and ethanol production were significantly increased by inducing the expression of the Gal promoter.

In this study, we developed a novel protein-free medium for CHO DG44 host cells in suspension culture. Chinese hamster ovary (CHO) DG44 host cell line is mainly used in the production of monoclonal antibody (mAB) for therapeutic use on the market. Serum has been used in culture of recombinant CHO cells for mAB production, but since serum possesses potential contaminants, e.g. viruses, mycoplasma or prions, commercial protein-free media with serum eliminated are mostly used in mAB production. The composition of commercial media is unknown and the suspension culture of rCHO DG44 cells with commercial media requires long term adaptation periods. Development of an in-house protein-free medium with stable cell growth at adaptation-free or short term adaptation for CHO DG44 host cells will enable to overcome these limitations. We have established the formulation of protein-free components such as amino acids, iron supplement, hydrolysate, energy source, inorganic salts, etc. The maximum cell density of the medium was 7.5*106 cells/ml in batch culture with 52% higher cell growth than the commercial media.

The therapeutic efficacy and in vivo biological function of a glycoprotein is significantly affected by its glycosylation profile.For the development of glycoproteins with therapeutic applications, selection of cell lines producing a glycoprotein with adequate glycoform is crucial. Here we demonstrate an array-based analysis of secreted glycoproteins for rapid and efficient selection of a single cell producing a glycoprotein with desirable glycosylation. Our approach relies on microengraving and interrogation of glycoproteins produced by individual cells in a microwell array in terms of glycosylation profile as well as the produced amount. Based on statistical analysis of the interrogation, corresponding single cell is selected, retrieved, and expanded. We applied the approach to human recombinant erythropoietin (rhEPO)-producing CHO cells, and verified the selection of a single CHO cell that produces rhEPO with a high sialylation degree. Human erythropoietin (hEPO) bearing highly sialylated oligosaccharide was shown to display a much longer plasma half-life, resulting in high therapeutic efficacy.3) This method can find widespread use in the clonal selection for the production of other glycoproteins with specific glycosylation as well as analysis of the heterogeneity in cell populations in a high-throughput manner.

We demonstrate here a highly-sensitive electrochemical immunosensor based on the combined use of substrate recycling and carbon nanotubes (CNTs) coated with tyrosinase (TYR) and magnetic nanoparticles (MNP). Both TYR and MNP were immobilized on the surface of CNTs by covalent attachment, followed by additional cross-linking via glutaraldehyde treatment to construct multi-layered crosslinked TYR-MNP aggregates (M-EC-CNT). M-EC-CNT was highly loaded with TYR and MNP, allowing for high activity and quick magnetic capture, and the good TYR stability could be achieved due to the multiple covalent linkages on the surface of TYR1,2). Magnetically-capturable, highly active and stable M-EC-CNT were further conjugated with primary antibody against a target analyte of hIgG, and used for a sandwich-type immunoassay with a secondary antibody conjugated with alkaline phosphatase (ALP). In the presence of a target analyte, a sensing assembly of M-EC-CNT and ALP-conjugated antibody was attracted onto a gold electrode using a magnet for an electrochemical assay. On an electrode, ALP-catalyzed hydrolysis of phenyl phosphate generated phenol, and successive TYR-catalyzed oxidation of phenol produced electrochemically measurable o-quinone that was recycled to catechol for substrate recycling. The combination of highly active M-EC-CNT and substrate recycling for the detection of hIgG resulted in a sensitivity of 27.6 nA ng-1 mL-1 and a detection limit of 0.19 ng mL-1 (1.2 pM), which represent better performance than any other electrochemical immunosensors based on TYR-ALP for substrate recycling. The present approach also displayed a long-term stability by showing negligible loss of electrochemical signal after the reagents were stored in an aqueous buffer at 4ºC for more than 6 months. High sensitivity and stability of the present immunosensor seem to result from stabilization of multi-layered enzyme assembly on conductive CNTs. We anticipate that the developed immunosensing system meets the current demanding in high performance electrochemical immunoassay, in terms of sensitivity, stability and reproducibility, and might be applied to detection of a variety of analytes.

PEGylation of biopharmaceutical proteins is important to increase in serum circulation stabilityand to minimize antigenicity. The most preferable method of PEGylation is a site-orterminal-specific, mono-EGylation. We propose the terminal-specific mono-PEGylationmethod using intein-mediated fusion protein technology. By exploiting the affinity taggingdomain (chitin binding domain) of a fusion protein, we were able to immobilize the C-terminusof the fusion protein to chitin matrix, exposing the N-terminus for solid-phase PEGylation.The distinct advantage of inteins is self-splicing ability by simple changes in pH and/ortemperature without the need for cleavage proteins or reagents. By this way, we couldpresent an integrated process for expression-refolding-PEGylation-purification. Rh-EGF(recombinant human epidermal growth factor) was used as a model protein. The PEGylationsite was determined by mass spectrometry, and the bioactivity of the modified rhEGF wascompared with the native and randomly PEGylated EGF using NRK cell culture assay.

Background. Surface camouflage using poly(ethylene glycol) (PEG) onto islet is promising approach for immunoprotection of transplanted islets. Furthermore go one step, here, we analyzed that 6-arm-PEG-catechol grafting on the islets plus low dose of FK506 are effectively improve the islets survival time under the strong rat to mouse xengenic islet transplantation model. Methods. After end group of 6-armed PEG was modified with catechol, it was grafted onto the surface of rat pancreatic islet. Then surface coverage, viability and functionality of islets were evaluated in vitro. The function of surface modified islets was also evaluated after xenotransplantation into diabetic mice. In addition, its synergistic effect with low dose of FK506 immunosuppressant was evaluated in vivo. Results. 6-arm-PEG-catechol did not affect the viability of islets for 1hr at 1% condition. The median survival time (MST) of 6-arm-PEG-catechol grafted islets was 12 days. However, the MST of unmodified islets was 10.5 days. Furthermore, the MST of 6-arm-PEG-catechol grafted islet plus 0.2 mg/kg of FK506 treatment was significantly increased compared to that of unmodified islets plus 0.2 mg/kg of FK506 treatment.(6 arm-PEG-catechol grafted islet receiving group: 21 days, unmodified islets receiving group: 10 days) Conclusion. Newly developed protocol using polymer and clinically used immunosuppressive drug would certainly be an effective combination therapy for the treatment of type 1 diabetes.

About 80% of chiral amines used as pharmaceutical intermediates are in (R) form. Despite the needs of (R) form intermediates, only one (R)-specific ω-transaminase (R-ωAT) is known. In this study, we used bioinformatical approach to search putative R-ωAT. Because R-ωAT is believed to belong to aminotransferase group III which contains D-alanine aminotransferase, branched-chain aminotransferase and 4-aminodeoxychorismate lyase (ADCL), we generated profiles of DAATs, BCATs and ADCLs and scored bacterial sequences in RefSeq database using three profiles. We plotted sequences in 3-D space whose coordinates are composed of profile analysis score against DAATs, BCATs and ADCLs. Slightly homologous to DAAT, BCAT and ADCL, but not authentic DAAT, BCAT nor ADCL, were selected for activity investigation. Reaction results using enzyme purified from recombinant E. coli showed that transaminases from Hypomonas neptunium and Saccharopolyspora erythrea showed activity to (R)-a-MBA. These two enzymes did not showed activity to (S)-a-MBA. Therefore, R-ωAT were successfully screened using evolutionary relationship by comparison of profile analysis scores against neighborhood subfamily profiles.

Human intestinal maltase (HMA) is one of these key enzymes that responsible for catalyzing the last glucose-releasing step in starch digestion and it is an important inhibition target in the treatment of type II diabetes. With three dimension structural information of HMA (PDB code: 2QMJ and 2QLY)1 with acarbose as know inhibitor was available and compare the Cα, the HMA was carried out redocking with three parameters and parameter 2 with change of maximum of energy evaluations (ga_num_evals) 2500000 satisfied with -12.62 kcal/mol and RMSD 2.02 A0 and check time to finish one docking lower than 60 minutes. This parameter was chosen for large scale docking on WISDOM production environment2. The result of data challenge showed that 7,977 compounds had lower energy than that of acarbose with energy varied up to -17.09 kcal /mol. All of this data challenge was finished at 3.2 days on 4,700 CPUs with distribution efficiency 54.4%. From 2,974 compounds that had lower energy from -13 kcal/mol were checked hydrogen interaction with key residues of HMA and 87% of compounds showed the interaction with key residue of HMA and 74 select compounds were divided into 17 groups.

For the past decades, the Genetic code engineering has dramatically expanded it scope to design proteins with novel functional and biophysical property. Genetic code engineering is a recent developing method that helps to design and engineer proteins with additional chemical moiety through incorporation of non canonical amino acids. To date, several groups successfully incorporated more than 50 unnatural amino acids into recombinant proteins through invivo and invitro approach. Each report demonstrates the different characteristic functionality of unnatural amino acids and its application when incorporated into a protein. Even though, a lot of characteristic features of unnatural amino acid were reported, we are the first to demonstrate development novel fluorescent based copper biosensor through unnatural amino acid mutagenesis. In this study we show the incorporation of unnatural amino acid, L-DOPA into Green fluorescent protein render the protein metal binding property especially copper sensing property. The Copper binding property of the engineered protein was characterized by the combination of fluorescent spectroscopy, absorption spectroscopy and circular dichroism. Overall this study demonstrates that the development of selective, sensitive and robust fluorescent based metal biosensor using unnatural amino acid mutagenesis

This study will attempt to clarify the impact of two pharmaceuticals (i.e. verapamil, tramadol) and two pesticides (i.e., glyphosate, methidathion) on non-target organism in aquatic environment. We investigated the toxic effects of these chemicals on physiological responses such as viability, growth, and reproduction as well as molecular responses such as gene expression in water flea, Daphnia magna. Through RT-PCR, the expression of five genes selected as representative biomarkers such as Arnt, Vtg, CYP4, CYP314 and Dhb was analyzed in D. magna exposedto hazardous materials. Our results showed that the expression of these genes in D. magna were significantly down-regulated in response to pesticides. Among five examined genes, Cyp4 and Cyp314 genes, expressed differently in exposure of glyphosate and methidathion, respectively. This revealed that these two genes could be considered as potential biomarkers. Unlike pesticides, pharmaceuticals did not significantly affect on gene expression in D. magna in short-time exposure, but inhibited the expression of Vtg gene, a biomarker of animal reproduction, after 21d exposure. In addition, total number of D. magna neonates decreased strongly in the chronic toxicity test by both kinds of pharmaceuticals. Therefore, this study suggests that these pharmaceuticals and pesticides have distinguishable toxic modes between them. While the pesticides cause molecular damages such as significantly reduced of five genes, the pharmaceuticals lead to a chronic toxicity; not the effect of gene expression caused by acute exposure.

Lipase B from Candida antarctica (CalB) showing several excellent characteristics such as high stereo-selectivity and stability in organic solvent has attracted much attention for various enzymatic bioconversion reactions [1]. To apply lipase to commercial process, the thermal stability is one of the prerequisites. In this study, the thermal stability of CalB was improved through increasing the number of disulfide bridges based on B-factor and MODIP program. Among amino acid pairs showing highest probability to form disulfide bridges in grade A of MODIP, four candidates (S50-A273, Q156-L163, A162-K308, N169-F304) were selected based on B-factor of its residues. Four double CalB mutants were constructed and evaluated their thermal stability. As a result, CalB A162C-K308C mutant showed greatly improved thermal stability compared to its wild type. The residual activity of CalB A162C_K308C was 3.2 times greater than that of CalB wild type at 50oC after incubating 150 minutes. Increase of 8.5oC in the aspect of temperature at which 50% its activity remaining after 60 minute incubation (T50 60) was observed for CalB A162C_K308C mutant compared with that of CalB wild type. This mutant can be anticipated to apply for commercial purposes. References:1. Stefan Lutz, Engineering Lipase B from Candida Antarctica (2004), Tetrahedron:Asymmetry 15, 2743-2748.

Genetic code engineering is a recent developing method that became an indispensable quest to design and manipulate target proteins with novel functionality embedded within unnatural amino acid (UAA). Frequently, two experimental approaches were used for reassignment of sense (residue specific incorporation) and non sense codon (site specific incorporation) with UAA through in vivo expression of recombinant proteins. Even though, both of these methods successfully alter the protein functionality, the limitations of above methods is that they allow only incorporation of a single unnatural amino acid into the recombinant protein. Here we developed a promising alternative approach through coupling of both sense and suppression method in single protein. To explore the possibility for MUAA incorporation, we selected green fluorescent protein (GFP) as a model protein that contain five methionine codons and these codons will be reassigned with methionine surrogate l-homopropargylglycine (Hpg) whose functionality can be chemoselectively modified with specific alkyne bearing reagent by means of a copper mediated azide-alkyne cycloaddition (click chemistry). Simultaneously, tyrosine analogue L-3, 4-dihydroxyphenylalanine (L-DOPA) will be site specifically incorporated into chromophore tyrosine (Y66) by an evolved Methanococcus jannaschii tRNA/synthetase pairs (mutant TyrRS). This is the first study to demonstrate in vivo incorporation of two unnatural amino acids into a recombinant protein through combination of two different methodologies. This combination will offer extraordinary opportunity for protein engineers to create protein with novel functionality which expand the proteome of the cell. This approach will drive us toward a post proteomics era as well as it will also open a new door for synthetic biologist to generate multi characteristic proteins.

The fermentation of glucose by several bacteria such as Klebsiella oxytoca, Klebsiellar pneumonia, Bacillus subtilis, Aeromonas hydrophilia and several species of Serratia yields 2,3-Butanediol as one of the products while secondary products formed acetoin, ethanol, lactic acid, and acetic acid. 2,3-Butanediol is a versatile bio-product for synthesis rubber polymers and solvents with a carbon chain length of four and two reactive sites. However the efforts of changing metabolic fluxes radically is known to be more challenging to determine which pathways should be modified in the classic genetic engineering. In this work, we constructed a model based on pyruvate distribution yielding 2,3-Butanediol and other byproducts. Our aim is to find a best mutant yielding 2,3-butanediol with the help of in silico experiments, thus the direct in silico experiments simulate the time scale concentration profiles based on the enzyme kinetics in combination with metabolic control analysis and metabolic flux analysis,. Based on Escherichia coli and Klebsiella bacteria enzyme database, several virtual mutants has been developed, simulated as well as combined with experiments confirmation. The results showed that the knockout of lactate dehydrogenase could decrease the production of lactate while increase the 2,3-Butanediol yield; NAD and NADH balance had fierce control on the reaction network; in addition other byproducts’ deletions would as well enlarge the productivity of 2,3-Butanediol.

In this study, (R)-3-fluoroalanine was asymmetrically synthesized from 3-fluoropyruvate (F-pyruvate) and (S)-a-methylbenzylamine (MBA) using recombinant w-transaminase (TA) from Vibrio fluvialis JS17. The reaction was severely inhibited by acetophenone (deaminated product of a-MBA). In the presence of 5 mM acetophenone, the reactivity of the enzyme towards F-pyruvate decreased by 22 %. To overcome product inhibition by acetophenone, a biphasic reaction was successfully used. The conversion of F-pyruvate into (R)-3-fluoroalanine (enatiomeric exess (e.e.) >99%) was about 95% in the biphasic system (75 mM F-pyruvate, 100 mM (S)-a-MBA, and 3.0 U/ml), whereas only 31% was obtained without product extraction. The use of racemic a-MBA as an amino donor instead of (S)-a-MBA can not only reduce the reaction cost but can also produce chiral amines by kinetic resolution. When the kinetic resolution of racemic a-MBA (40 mM) was carried out with F-pyruvate (30 mM) and w-TA(3.0 U/ml) in 100 mM phosphate buffer (pH 7.0), the e.e. of (R)-■a-MBA reached 98.4% with a 52.2% conversion for 10 h and 21 mM (R)-3-fluoroalanine was produced with a 70 % conversion and an e.e. >99 %. }

Copper is one of the most essential transition metal in human body which serves as an essential cofactor for oxidative scavenging enzymes. Due to its redox property it has the ability to generate reactive oxygen species and play an important role in metabolism. Imbalance of copper homeostasis leads to diseases such as Menkes, Wilson’s disease and also in Alzheimer’s disease. So, it would be highly advantageous if we develop an efficient method for measuring copper through spectroscopic /fluoro spectroscopic methods without addition of any reagents. As we know that GFP is already used as fluorescent tag in prokaryotic system as well as in mammalian systems, so developing GFP based in vivo copper biosensing tag will help in identifying the copper based diseases and localization of copper ions in cell. In the current study we have developed a metal biosensor through unnatural amino acid mutagenesis where the metal chelating amino acid, L-DOPA was metabolically incorporated into GFP. The fluorescence of DOPA incorporated GFP specifically quenches in the presence of copper and fluorescence of the protein was recovered after adding a metal chelator. This strategy helps to develop a fluorescent protein based copper biosensor and also helps in developing microarray based copper quantitation

Haematococcus pluvialis is an unicellualr photosynthetic green microalgae and it produce a large amount astaxanthin under various environmental conditions such as light intensity, nitrogen deficiency and so on. In this study, we investigated that their growth and astaxanthin accumulation on red, blue and white light using LED. The growth of H. pluvialis was carried out at 25 ℃, 0.5 vvm, pH 7±0.5, light intensity from 60 μmol․m-2․s-1 to 120 μmol․m-2․s-1 on the erlenmeyer flasks. Further experiment was performed for the growth and astaxanthin accumulation in the 20 L plate photobioreactor on the red and blue light. Astaxanthin accumulation was performed at 25 ℃, 0.5 vvm, pH 7±0.5 and light intensity of 250 μmol․m-2․s-1. From the growth to the astaxanthin, light source was changed red to bule light and also light intensity was gradually increased by lumostatic method. As our results, the maximum cell numbers was 8.5×105 cells․ml-1 and the maximum astaxanthin concentration was 35 mg․L-1 on the blue LED. Astaxnathin concentration (from 2.7% to 3.3%) in our experiment was similar to the literature results (3%). Therefore, blue LED was the most efficient light source for mass cultivation and astaxanthin formulation.

Proteins composed of twenty essential canonical amino acids play important biological roles in a cell and their biological activities. Recently, great efforts have been devoted to expanding the genetic code for the in vivo biological incorporation of non-canonical amino acids into proteins. The biological approaches for the incorporation of unnatural amino acids into target proteins in vivo may give us more dimensions to engineer the physico-chemical properties of proteins which are generally limited by canonical amino acids. Frequently, two experimental approaches were used for reassignment of sense (residue specific incorporation) and non sense codon (site specific incorporation) with UAA through in vivo expression of recombinant proteins. Even though, these methods successfully alter the protein functionality, the limitations of above methods is that they allow only incorporation of a single unnatural amino acid into the recombinant protein. Taking this into consideration the importance and limitation of above methods as well as giving indispensable creation of multifunctional protein, we developed a promising alternative approach through coupling of both sense and suppression method in single protein. Herein, we report novel and facile method for incorporation of two UAA into single protein. This combination will offer extraordinary opportunity for protein engineers to create protein with novel functionality which expand the proteome of the cell. This approach will drive us toward a post proteomics era as well as it will also open a new door for synthetic biologist to generate multi characteristic proteins

Biomimetic synthesis raises a concern because it can take place under mild conditions with more facilitation (1). Biosilicification is one of biomimetic syntheses and we recently efficiently immobilized GFP (green fluorescence protein) using biosilicification by silaffin-fused proteins (GFP-Rx) (2). Glucose oxidase (GOx, EC 1.1.3.4) is one of broadly used enzymes for biosensors with high enzyme activity to detect glucose concentration such as to track blood sugar levels. We constructed R5-GOx-fused protein and it was expressed on Pichia pastoris. Secreted GOx from Pichia was immobilized by R5 (fused with GOx) in pH 5 citric buffer using 0.1 M tetramethoxysilane. To apply silaffin-GOx-silica nanoparticles for a biosensor, biosilicification occurred on a grassy carbon electrode. Electric cell consisted of Ag/AgCl reference electrode, platinum counter electrode (cathode), and working electrode (anode) modified with poly(neutral red)(PNR)/GOx/nafion. Electrochemically formed PNR was used as an electron carrier from GOx to an electrode. Scanning electron microscopy image supported a formation of PNR/GOx complex. A current was increased to 2 mA·cm-1 by 1 mM glucose at -0.4 V applied potential. A biosensor of GC/PNR/GOx/nafion showed a substrate selectivity to detect glucose concentration in mixed glucose samples with other carbohydrates.

Crystallization is a simple, energy-efficient and environmentally friendly process for purifying vancomycin from fermentation broth. However, the crystallization process has been inherently problematic due to the lengthy crystallization time that is required. An improved crystallization process could significantly reduce the crystallization time by increasing the surface area available for crystallization. Vancomycin yield was improved and the crystallization time was shortened compared to the control by increasing the surface area per working volume (S/V) of the reacting solution through the addition of a cation exchange resin (Amberlite 200), an anion exchange resin (Amberlite IRA-400), or glass beads. Most of the vancomycin could be obtained after about 12 hr of crystallization using Amberlite 200 and Amberlite IRA-400. Since high purity (>97%) vancomycin can be obtained in high yield (>96%) and the crystallization time can be reduced, this improved method is expected to significantly enhance the final purification process. In addition, the feeding and mixing method of crystallization were evaluated in terms of the yield, purity, and crystallization time of vancomycin. The highest purity (>97%) and yield (>95%) could be obtained when the sample was fed all at once and mixed only during the initial one-step feeding.