Earticle

In this study, we evaluated the efficiency of different adsorbents for the removal of plant-derived impurities during the pre-purification of paclitaxel from plant cell cultures. Using the synthetic adsorbents sylopute and active clay and their major components SiO2 and MgO, we performed adsorbent treatment and analyzed the paclitaxel precipitates recovered from hexane precipitation. When SiO2 was used, the highest purity (~58.1%) and yield (~91.5%) of paclitaxel were obtained. We also determined differences in the effectiveness of the adsorbent treatment according to changes in the surface area, pore volume and pore diameter of SiO2. Adsorbent treatment was more effective when pore diameter was larger (silica I [2.19 nm] <silica II [4.92 nm] <silica III [9.07 nm]). The highest purity (~74.3%) and yield (~92.9%) of paclitaxel were obtained when silica III was used in the adsorbent treatment. Pore diameter had a greater effect on the removal of plant-derived impurities during the pre-purification of paclitaxel compared with surface area and pore volume. This result could be confirmed by HPLC analysis of the absorbent after treatment and TGA of the organic substances that were bonded to the adsorbent.

In recent times, foot and mouth disease have occurred in all parts of the country. Disease animals are buried in the ground. In addition, the concentration of buried animals could affect ground or groundwater. We are presumably supposed that treatment of Corynebacterium glutamicum is a feasible method that may provide to reduce the risk of carcass decomposition affecting human or the environment. Therefore, this study shows to investigate that influence of C. glutamicum on the soil environment with carcass by HPLC and plant test, respectively. In the results, composition of amino acids in soil treated C. glutamicum is generally higher than that of amino acids in untreated soils. Also, Root length in untreated samples was elongated relative value 28.1 %, however oat root in samples treated C. glutamicum was similar to control; 84.0 % relative value. Therefore, this work suggests the possibility that could be reduce toxicity of pollutants contaminated soils from grave land with disease animals.

This study was performed to elucidate an antibacterial mechanism of Eisenia bicyclis extracts against methicillin-resistant Staphylococcus aureus (MRSA). Among of the extracts, the ethyl acetate soluble extract of E. bicyclis exhibited the highest anti-MRSA activity ranging in 128 μg/mL of minimum inhibitory concentration (MIC) against MRSA. Indeed, ethyl acetate extract was assessed its synergistic effects in combination with ß-lactam, ampicillin, penicillin and oxacillin against MRSA. Based on these results, we hypothesized that the ethyl acetate extraxct might inhibit a gene(s) or protein(s) related the drug resistance. It was observed that the ethyl acetate extract inhibited the mRNA expression of the resistant genes (mecI, mecR1 and mecA) in a dosedependent manner. Moreover, the results of Western blotting assays indicated that the ethyl acetate extract inhibited the synthesis of PBP2a protein. These results will provide a possible anti-MRSA mechanism by the ethyl acetate soluble extract of E. bicyclis.

Fermented foods contain various beneficial microorganisms. Lactic acid bacteria (LAB) including Lactobacillus, Bifidobacterium, and Pediococcus are most popular microorganisms in fermented foods. Theses bacteria exist in gastrointestinal (GI) tract and affect digestion system maintaining microorganism balance. In this study, we investigated the effect of LAB culture extracts on the nematode Caenorhabditis elegans focusing on host animals’ digestion system. In order to verify whether the small molecules or metabolites produced by LAB are beneficial for host animals’ digestion system, the extracellular extract was administered into C. elegans together with their food bacteria, E. coli. As a result, extracellular non-protein part of extracts significantly reduced the indigestion of foods in intestine and improved the defecation. Enhanced digestion and defecation increased the body growth of adult animals and improved the survival ratio of middle-aged adults. We have also found that LAB extracts inhibited the growth of E. coli when the extract was added to the culture of E. coli. These results demonstrate that LAB extracts have a great potential as health beneficial supplements by improving digestion and killing harmful bacteria in GI tract.

Hybrid lime addition/capacitive deionization process is a simple, efficient method for isolating sugars from the mixture of glucose, xylose, acetic acid, and sulfuric acid, which are the typical products from the biomass hydrolysis. In this study, we optimized the important process parameters of hybrid process to isolate sugars. In the lime addition process, the optimal lime type, lime/sulfuric acid molar ratio, agitation speed, and reaction time for removal of sulfuric acid were CaCO3, 1:1, 500 rpm, and 8 min, respectively. For the CDI process, the optimal voltage was 1.2 V, the flow rate was 20 mL/min and the efficiency of the acid removal rate increased as the initial acetic acid concentration decreased. The developed hybrid process was able to remove 98.08% of sulfuric acid and 76.97% of acetic acid from the mixture of glucose, xylose, acetic acid, and sulfuric acid. It was able to recover most of the sugar (>99%) at high purity (97.53%).

Bioenergy production from microalgae that is able to convert solar energy into biochemicals by photosynthesis is a realistic and possible way to replace diminished fossil fuels reservation such as coal, petroleum, others derived from petroleum. However, relatively high cost of production is a major obstacle prohibiting its commercialization. To cultivate mciroalgae using the nutrients dissolved in the seawater without any additional nutrients, semi-permeable membrane photobioreactor was developed for economical and eco-friendly production of microalgal bioenergy. Two major nutrients (N, P) for microalgal growth transfer rate across semi-permeable membranes were measured as function of different N, P concentration in artificial seawater. Then, microalgae were cultured using the semi-permeable membranes which have different molecular weight cut off(MWCO). Time profiles of algal biomass were increased with in proportion to nutrients transfer rate measured. Fatty acid methyl ester were analyzed and compared in all cases.

Solid freeform fabrication (SFF) is recognized as a promising tool for creating tissue engineering scaffolds due to advantages such as superior interconnectivity and highly porous structure. Despite structural support for SFF-based three-dimensional (3D) scaffolds that can lead to tissue regeneration, lack of cell recognition motifs and/or biochemical factors has been considered a limitation. Previously, recombinant mussel adhesive proteins (MAPs) were successfully demonstrated to be functional cell adhesion materials on various surfaces due to their peculiar adhesive properties. Herein, MAPs were applied as surface functionalization materials to SFF-based 3D polycaprolactone/poly(lactic-co-glycolic acid) scaffolds. We successfully coated MAPs onto scaffold surfaces by simply dipping the scaffolds into the MAP solution, which was confirmed through X-ray photoelectron spectroscopy and scanning electron microscopy analyses. Through in vitro study using human adipose tissue-derived stem cells (hADSCs), significant enhancement of cellular activities such as attachment, proliferation, and osteogenic differentiation was observed on MAP-coated 3D scaffolds, especially on which fused arginine–glycine–aspartic acid peptides were efficiently exposed. In addition, we found that in vivo hADSC implantation with MAP-coated scaffolds enhanced bone regeneration in a rat calvarial defect model. These results collectively demonstrate that facile surface functionalization of 3D scaffolds using MAP would be a promising strategy for successful tissue engineering applications.

Advances in custom de novo DNA synthesis technology have propelled the progress of modern biotechnology, embracing the recent emergence of synthetic biology. However, present cell-based protocols for the translation of synthesized genes are time and labor intensive, and thus limiting the throughput of creating protein molecules. Herein, we demonstrate a seamless consolidation of PCR-based synthetic gene assembly with fully automated cell-free protein synthesis for direct translation of sequence information into purified proteins. Chemically synthesized gene fragments were PCR-assembled, and incubated in the ExiProgenTM system (BIONEER, Korea) for automated, sequential cell-free expression and purification of the target proteins. Starting from assembled PCR products, 16 different proteins were successfully obtained in purified forms within 7 hours without manual intervention during the protein preparation process. Proposed strategy involving in silico gene design and automated cell-free expression and purification greatly reduces time and laboratorial efforts for protein preparation and thus would provide a versatile platform for the rapid and parallel materialization of sequence information.

Finding desirable phenotypes by the evolutionary method in the biological system often gives the solution to challenging obstacles in biotechnology areas, such as biofuel production, screening desirable phenotypes, etc. Conventional mutation methods, however, can be biased to certain types of mutations which restrict the accessibility to the desirable phenotypes. In this work, we tried to evaluate the potential of the artificial transcription factor library for overcoming such limits.

Immobilization of antibodies on carriers in an oriented manner was shown to be critical for achieving high sensitivity in immunoassays. Here we demonstrate the increase in the detection sensitivity of a lateral flow assay for a cardiac marker troponin I (cTnI) by magnetic labels with oriented immobilization of antibodies. For the construction of sensitive magnetic labels, orientated-immobilization of anti-troponin I antibodies on magnetic beads was attempted using protein G. Magnetic beads were first conjugated with protein G followed by immobilization of anti-cTnI monoclonal antibodies. The factors affecting the detection sensitivity in the lateral flow assay system were investigated. The lateral flow assay of cTnI was carried out using the magnetic beads and a Giant magnetoresistive (GMR) sensor at the optimized conditions, and compared with that using conventional random immobilization method. The detection sensitivity in the lateral flow assays using present approach was determined to be about 0.01 ng/mL, showing significantly increased sensitivity compared with conventional immobilization method. This result clearly shows that the magnetic labels with oriented immobilization of antibodies can find wide applications in the lateral flow assays of target analytes.

Variable lymphocyte receptors (VLRs) which are leucine-rich repeat (LRR) proteins play important roles in adaptive immunity of jawless vertebrates such as lamprey and hagfish that lack Ig-based immune system. The VLRs are also the oldest immune receptors and the only non-Ig scaffold used for antigen recognition in nature. Based on their modularity and antigen binding property, we have successfully designed a engineered VLR scaffold protein, Repebody, with high physicochemical stability and mass productivity in prokaryote expression system. In order to verify whether Repebody acts as a universal molecular binder, we carried out the directed evolution of Repebody by saturation mutagenesis of concave surface and phage display technology for selection of ligand binders. The resulting binder D3 exhibits the nanomolar binding affinity for Interleukin-6 with target specificity. For general and practical applications, we further designed repeat module- based approach for affinity maturation of Repebody. Through the method, high affinity binders were successfully isolated from additional module library and the best clone D3E8C4 exhibits about 50-fold binding affinity improvement with a dissociation constant (KD) of 397pM, and which shows inhibition of IL-6 activity in human monocyte at low doses (0.1ug/ml). These results suggest that Repebody acts as not only an alternative scaffold but also a potential therapeutic agent as the artificial antibody and that module-based engineering method is effective for library design and affinity maturation of repeat proteins.

A system for expression and in situ display of recombinant proteins on microbead surface is described. Biotinylated PCR products were immobilized on microbead surfaces, which were then embedded in a gel matrix and supplied with translation machinery and substrates. Upon the incubation of the gel matrix, target proteins encoded on the bead-immobilized DNA were expressed and captured on the same bead, thus allowing bead-mediated linkage of DNA and encoded proteins. The new method combines the simplicity and convenience of solid-phase separation of genetic information with the benefits of cell-free protein synthesis, such as instant translation of genetic information, unrestricted substrate accessibility and flexible assay configuration design.

Sacchromyces cerevisiae contains cell organelles such as vacuoles correspond to lysosomes in higher eukaryotes. Lysosomes have antimicrobial activity and anticancerous effect and those are not silent organelles, but dynamic organelles in which lysosomal enzymes are easily integrated when exposed to stressful conditions, especially oxidative. In this study, we selected a protein that aminopeptidase Y (APY) conspicuously was increased than other proteins in oxidative stress, and we cloned, over-expressed and purified the APY gene production form S. cerevisiae. As a result, cloned S. cerevisiae had antimicrobial effects more than normal cells, besides those were increased more over by galactose induction. We found that anticancerous effects of lysosomes isolated from recombinant S. cerevisiae were increased on cancer cells such as HeLa cells and melanocyte in vitro assay. In addition, bacterial growth in blood was inhibited by lysosomes when we injected lysosomes for therapy agents of bacterimia in rat model. Therefore, increase in endogenous levels of lysosomes and their activity following genetic modification may have various applications as antimicrobial agent and apoptosis-inducing materials for cancer cells and consequently in may be possible to use the organelles for improving in vitro and in vivo functions.

Phthalocyanine derivatives have perhaps received more attention than any other class of dyes as sensitizers for photodynamic therapy and most favoured are the aluminium and zinc derivatives. Photoinduced electron transfer in donor-acceptor molecules has been extensively studied, in particular for covalently linked dyads in solution as model systems of natural photosynthesis to harvest efficiently solar energy. In this study, the characterizations for immobilization o f R. sphaeroides on two phthalocyanines were evaluated. There are two solvent for phthalocyanines, water and SDS. In order to determine electron reaction of cell bound phthalocyanines, we are confirmed cyclic voltammetry measurement. R. sphaeroides bound ZnPc in water solvent and CuPc in SDS solvent were shown active electron reactions. It was correlated with the amount of cell bound phthalocyanine by photo-luminescence at 683nm for emission and at 600nm for excitation. In addition, electron reactions of R. sphaeroides on phthalocyanines were increased for 10 days storage period. Phthalocyanines as electron donoron on R. sphaeroids can apply for improving photosynthetic efficiency.

Glycans expression behavior of mammalian cell surfaces provides valuable information for biological functions.[1] Especially, cancerous cells have considerably different from structural formation and over-expression of glycans compared with noncancerous cells. Such changes participate in metastasis by altering cancer cell proliferation, adhesion, differentiation, invasion, angiogenesis and immunity. Therefore, the alterations in cancer cell surface glycans have potential for early diagnosis and treatment. There have been tremendous efforts to develop comprehensive profiling methods for the cellular glycomes via mass spectrometry (MS), high-performance liquid chromatography (HPLC) and frontal affinity chromatography (FAC). Although the analytical technologies provide detailed quantitative and functional information about the glycans, critical challenges in the separation of cell surface glycans remain, such as time-consuming and cumbersome procedures as well as the need for a large number of cells.This article describes assay of expressed cell surface glycans using biotin-lectin (biotinylated lectin) and SA-QD (streptavidin-QD) on combined cell patterning for reliable and readily quantifiable assay that to enable high-throughput and automated assay. The SA-QD can bind to biotin-lectin combined with cell surface glycans which lectins specifically bind to glycans. The intensity of QDs shows clear difference between cancerous and normal cells depends on amount of expressed glycans on cell surface where evaluated with various lectins for various cell lines. This assay method performed with patterned cells obtained reliable and consistent quantification data. Thus, the cell surface glycan assay using biotin-lectin and SA-QD based on patterned cells provided useful assay tools for high throughput cancer diagnostics and cellular physiological studies

In this study, we demonstrated a novel method of reverse transcriptasepolymerase chain reaction (RT-PCR) using a rotary system, called Rotary Genetic Analyzer, for detecting influenza A virus with high speed. The Rotary Genetic Analyzer consists of three parts including a disposable plastic PCR microchip, thermal blocks for temperature control, and a stepper motor for precise rotating of the chip. A disposable RT-PCR microchip was fabricated by glass-PDMS hybrid with 1 ㎕ reaction volume. Three thermal blocks fitted on the rotary stage, and the temperature of each block was correspondent to the PCR thermal cycling, namely 95 ℃(denaturing), 58 ℃(annealing), 72 ℃ (extension). A stepper motor were positioned at the center and could rotate the PCR chip at each heater block as designed angle and time by LabVIEWTM. The generated amplicons by the rotary system were analyzed on a capillary electrophoretic microdevice. Target amplification was performed with only 25 min time and limit of detection was 12 ag of influenza A H3N2 virus. In addition, multiplex detection was performed confirming three influenza A virus (H1N1, H3N2, and H5N1) simultaneously.

Single-walled carbon nanotube (SWNT)-based sensors exhibit a high sensitivity to specific analytes. In order to modify the analyte-recognizing property of SWNTs, we developed a single-step immobilization method of peptide receptors which can specifically recognize trimethylamine. An immobilization occurred by π-π stacking interactions between SWNTs and aromatic rings of phenylalanines which was additionally synthesized at the C-terminus of peptide receptors. Through the immobilization of peptide receptors, SWNTs obtained new function to selectively discriminate trimethylamine with high sensitivity. This simple immobilization method can be effectively used to modify the function of SWNTs for various purposes.

Under the induced fit hypothesis, enzyme structure is changed to fit substrate and is recovered to its original form after catalysis occurred, which resembles elastic body motion. We propose novel model to analyze mechanical change of enzyme. This modeling method uses 2 forms of pdb files, apo and holo form of enzyme structure, to analyze the forces by substrate binding. Validation was performed from the data of bacteriophage T4 lysozyme, HIV-1 protease and Candida antarctica lipase B. Elastic body model was applied to select target mutation sites with the aim of activity enhancement. It was discovered that some mutants having change at the forced region showed enhancement of activity in bacteriophage T4 lysozyme. The mutants of Candida antarctica lipase B showed about 4 times enhanced activity when flexible amino acid were introduced in the forced region. And the activity enhancement was due to the increase of kcat value. It inferred that activity enhancement was achieved by flexibility and dynamics increase.

Haematococcus pluvialis synthesizes a high yield of astaxanthin using CO2 in a photoautotrophic culture without contaminant heterotrophs; however, it takes too long to induce astaxanthin production. In this study, a highly-photosensitive mutant strain was attained by conventional random mutagenesis to shorten induction time. Sensitivity to photoinhibition in this mutant was raised by a partial lesion in the photosystem II (PSII) of photosynthesis, thereby prompting a change in cellular morphology as well stimulating carotenogenesis (astaxanthin production). As a result, the concentrations of cell biomass and astaxanthin were dramatically increased by 27% and 53% under strong light and 40% and 137% under moderate light, respectively. This Haematococcus mutant would be useful for the economical production of astaxanthin to reduce the light energy expenses in a photoautotrophic culture system, particularly in light-insufficient area.

Recent cancer therapies have developed multi-functional modulated medications with enhanced therapeutic efficacy and safety.1,2 Inspired by these trends, we demonstrate a novel anti-cancer therapeutics composed of diversified medicinal subdivisions containing both DNA/RNA/doxorubicin cassettes and gold nanorods. It enables to exhibit a chemotherapy and a photothermal therapy at the same time, Owing to the synergic effects, as compared with traditional anti-cancer methods based on a singlet anti-cancer strategy, the improved therapeutic efficacy against a PSMA expressing prostate cancer is observed in in vitro and even in vivo. The therapeutic efficacy is regulated depending on the concentration of gold nanorods and doxorubicin incorporated DNA/RNA nanostructures. These system mediate dramatical regression of prostate tumors in athymic mice, relative to control groups including free AuNRs and doxorubicin alone. It may be due to several therapeutic factors representing a PSMA-specific A10 aptamer-based active targeting, a doxorubicin-induced gene transcription inhibition, and a gold nanorods-derived localizing thermal ablation. These studies demonstrate that the selective replacement of other nucleic acids specific and active drugs on the therapeutics may have it easily applied to various infectious diseases.

Marine microalgae produce large array of metabolic products. Therefore, marine microalgae is used in different fields of application including nutraceutical, pharmaceuticals and cosmetic ingredient. However, there are less reports in use of microalgae on isolating active metabolites. So, this study has focused on investigating the apoptosis inductive effects of active compound, stigmasterol isolate from marine microalgae Navicula incerta extracts. There are several reports on anti-cancer effects of plant sterols on breast cancer and prostate cancer, but no reports on liver cancer. Thus, we investigated the apoptosis inductive effects of stigmasterol on hepatocarcinoma HepG2 cells. According to the results obtained stigmasterol induce the apoptosis on HepG2 cell by down regulating Bcl-2 and by upregulating caspase-8, 9 and FASL. In conclusion, we can suggest that the stigmasterol isolated from marine microalgae shows potent apoptosis inductive effects and could be developed as a anti-cancer therapeutic against liver cancer.

We developed a simple, ultrasensitive, and quantitative detection method for the reduced form of nicotinamide adenine dinucleotide (NADH), based on carbon nanotube field effect transistors (CNT-FETs). Following the injection of NADH at different concentrations, we obtained different electrical signals from semiconductor characterization system mimicking biological catalysis of NADH dehydrogenase (CoI). Here, FET was fabricated via photolithography, attached silicon well as the detection chamber on the channel area of the single wall carbon nanotube (SWNT). SWNTs were functionalized with phenazine derivant, a counterpart of the key functional prosthetic group of CoI enzyme. In the presence of NADH, electrons transferred to phenazine derivant through SWNT, by analogous means of the electron transport chain formed by a series of iron-sulfur (FeS) clusters in CoI. Using this method, the limit of detection was as low as 1 pM, and the range of linear response was 10 pM to 500 nM. Significantly, this approach possesses a great potential for applications in real-time detection of NADH at extremely low concentrations, and rigorous analysis for NADH in electrochemical fields.

Green fluorescent protein (GFP) is the fluorescent protein that is extensively used as reporter protein for the protein expression, as a biosensor, and fusion partner in the field of cell biology. It is reported that the some variants of GFP quenches it fluorescence with respect the concentration of Metal ions such as Zinc and Calcium. Here, we report that our GFP exhibit a Mercury and bismuth sensing property. The fluorescence of the protein diminishes with respect to metal ion concentration. In order to identify the key residues for the metal binding, we carried out structural analysis using bioinformatics tools and we developed point mutants to identify the key residues responsible for the metal binding. Based on this study, we successfully demonstrate that the Cysteine64 of our GFP exhibit mercury sensing property and the residue Thr203 is one of the residues important for the Bismuth sensing.

Recent medical technologies have primarily paid attention to the development of new diagnostic material with intrinsic biocompatibility and specific selectivity and strong intensity. Several bio-inspired hybrid materials containing natural components fused with either fluorescent organics or inorganics have been intensively considered for the design of new diagnostic tool kits. A number of candidates have been dealing with such technical issues.[1, 2]. Here, we presented a synthetic lipid supported fluorescence encoding nucleic acids particle, resembling a natural and biofriendly light-emitting bacterial cells. It composed of three subdivisions including outer membrane, interfacial polymeric wall, inner fluorescence labelled DNA template. With the evaluation of compartmental color emissions and external morphologic studies, it concluded that they produced size-tunable spherical shapes with distinct color-labelings. Besides its applications to bioimaging modules, owing to the versatile design strategies, it may have much potential at the field of system biology or biomimetic engineering.

The co-production of 3-hydroxypropionic acid (3HP) and 1,3-propanediol (PDO) from glycerol was studied using the resting cells of a recombinant Klebsiella pneumoniae J2B strain [1] that overexpresses an aldehyde dehydrogenase. Active biomass was produced in M9 under a range of aeration conditions, and shifted to potassium phosphate buffer containing glycerol for bioconversion. The microaerobic or anaerobic conditions were favorable for both the production of active biomass and subsequent bioconversion. At flask level, the recombinant strain grown under microaerobic conditions produced 43.2 mM 3HP and 59.0 mM PDO from glycerol (117 mM) in 30 min with a cumulative yield of 0.87 (mol/mol). The fed-batch bioconversion performed in a 1.5 L bioreactor at a constant pH 7.0 under anaerobic conditions, resulted in 125.6 mM 3HP and 209.5 mM PDO in 12 h with a cumulative overall productivity, yield and maximum specific production rate of 27.9 mmol/L/h, 0.71 (mol/mol) and 128.5 mmol/g CDW/h, respectively.

Depending on the species, microalgae produce many different kinds of lipids, hydrocarbons and other complex oils. The biomass and overall lipid productivities are some of the key parameters affecting the economic feasibility of utilizing algal oil for biofuel production. Therefore, an ideal process should be able to produce lipid at the highest productivity with the high biomass concentration, which can be modulated by varying growth conditions. Botryococcus braunii, a green microalga that produces lipid and hydrocarbons by itself, has already been proposed as a future renewable source of fuel. However, Botryococcus braunii is very slow growing strain that compared with the others. In this present study, the biotechnological exploitation for growth and lipid production of this microalga has been carried out. Statistical optimization of culture conditions was performed for faster growth and to obtain a higher concentration of lipid from Botryococcus braunii LB572. The effect of culture conditions such as light intensity, flow rate and CO2 concentration on cell growth and lipid production was examined with bubble column photobioreactors using a central composite design. The optimum points obtained for light intensity were 50 μE/m2/s, with 2% (v/v) CO2 at a flow rate of 0.2 vvm. Using the above optimal culture conditions, lipid productivity of B. braunii LB572 reached 0.181 g/L/day and 60% w/w with an average biomass productivity of 0.302 g/L/day.

Expressed as insoluble forms in E. coli, native CWPO_C (cell wall peroxidase from poplar tree) and mutated variants were successfully reactivated throughout refolding experiments and used for elucidating the previously presumed existence of electron transfer pathway in CWPO_C structure. Their catalytic properties were fully characterized through analysis such as steady state kinetics, direct oxidation of lignin macromolecules respectively accompanying with their stabilities during polymerization reactions, which proved strongly that the 74th residue on the CWPO_C structure plays the important role in catalyzing the macromolecules throughout electron transfer mechanism. This study also contributed deeper understanding about role of aromatic amino acids in electron transfer mechanism usually manipulated by peroxidase/H2O2 catalyst system. Furthermore, we also successfully synthesize the highly bioactive poly(catechin) by applying radical robust CWPO_C mutant in extension of poly(catechin). Anti-oxidation activity of synthesized poly(catechin) was confirmed by xanthine oxidase assay. The elucidation of uniquely catalytic mechanism in CWPO_C may improve applicability of peroxidase/H2O2 catalyst in conducting green polymer chemistry.

Protein engineers devoted lot of efforts to enhance the stability and biophysical properties of proteins through rational or irrational approaches. These approaches are limited by the restricted pool of 20 canonical amino acids. Recently, alternative approaches were developed to enrich and manipulate the functional and the biophysical properties of protein through incorporation of non-canonical amino acids (NCAA). For the purpose, genetic code engineering is the most frequently used methodology which reassigns the canonical amino acid of sense codon with NCAA. The advantage of this methodology is acquiring the synergistic effect through multiple site incorporation of NCAA can alter the biophysical properties of proteins. On the other hand, L-proline (Pro) plays a critical role in the protein structure by forming cis and trans peptidyl-proline bond conformation. The pyrrolidine ring of Pro structure adopts two alternative conformations as the Cγ-exo and Cγ- endo puckering. The replacement of proline with fluoroproline (FP) had been proven as a choice to tune or alter the biophysical properties of proteins. Fluorescent protein applications are widespread in the field of cell imaging as a model and reporter proteins. Among them DSRed have advantageous over other proteins, because of its emission and excitation in the red region of spectrum (longer wavelength) and exhibit less autofluorescence background in cellular imaging. The main disadvantage of DSRed is slow maturation and tendency to oligomerization. To circumvent this problem, protein engineers used directed evolution method for generating monomeric form DSRed variants with faster maturating property. Among them, mRFP1 is generated by introduction of 33 mutations into DSRed which showed ~10 times faster maturation speed. Here we used the genetic code engineering method to further enrich the stability and manipulate the biophysical properties of mRFP1by the incorporation of fluoroproline. A remarkable characteristic feature of this methodology is incorporation of NCAA to alter the protein properties without modifying the primary sequences. The global replacement of Pro residue with (4R)-FP into mRFP1 leads to fluorescent loss which was overcome by introducing canonical amino acid mutagenesis at Pro63 residue to Ala. Structural analysis of mRFP1 provide an insights into the key role of Pro63 for the red fluorescence emission by maintaining the planarity of the chromophore moiety. Here we showed ~2 fold enhanced thermal and chemical stability along with faster maturation of mRFP1 through a combination of canonical and non-canonical amino acid mutagenesis. Our study showed that a successful combination of canonical and non-canonical amino acid mutagenesis can enhance the protein biophysical property.

Biodegradable cationic poly(β-amino esters) (PBAE) nanoparticles are promising tool for delivering therapeutic genes into defective tissues. PBAE nanoparticles not only can deliver gene non-virally but also mediate significant gene enhancement. Here, we demonstrated the delivery of therapeutic genes using amine-end modified PBAE nanoparticles into defective tissue model with skin wound. A prototypical morphogen called Sonic hedgehog (SHH) was applied as the therapeutic gene to heal and regenerate wounded area of skin. SHH is known to regulate various organ and tissue development during embryogenesis, but is also expected to promote tissue regeneration via angiogenesis. The complexes of SHH gene and PBAE nanoparticles were administered into full-thickness skin wound mouse model by intradermal injection. Wound closure of mice receiving PBAE-SHH gene delivery was accelerated compared to control group with intradermal delivery of b-galactosidase plasmid using PBAE nanoparticles. The quantitative real-time polymerase chain reaction assay and histological analysis revealed that there were significant improvement of epidermis regeneration and blood vessel formation in the PBAE-SHH group. In conclusion, SHH gene delivery using PBAE nanoparticles may be able to provide the potential gene therapy for wound healing.