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In this study, the enhanced solubilization performance of a poorly soluble drug, beclomethasone dipropionate (BDP), was investigated using hydroxypropyl-β-cyclodextrin (HP-β-CD) and ethanol. The enhanced solubility of the drug was determined using the phase solubility method and correlated as a function of both HP-β-CD and ethanol concentrations. The effective progress of drug solubility originated from the formation of cyclodextrin and BDP inclusion complexes and increase in the lipophilicity of the medium, by aqueous ethanol, for hydrophobic BDP. BDP and HP-β-CD composite particles were produced using supercritical assisted atomization (SAA) with carbon dioxide as the spraying medium, 54.2% (w/w) aqueous ethanol as the solvent, and an optimal amount of the dispersion enhancer leucine. The effect of the mass ratio of HP-β-CD to BDP (Z) on the in vitro aerosolization and in vitro dissolution performance of BDP–HP-β-CD composite particles was evaluated. The aerosolization performance showed that the fine particles fraction (FPF) of the composite particles increased with increasing mass ratio. The water-soluble excipient (HP-β-CD) effectively enhance the dissolution rate of BDP from composite particles. This study suggests that BDP–HP-β-CD composite particles produced using SAA can be employed in immediate-release drug formulations for pulmonary delivery.

In this study, hydroxypropyl-beta-cyclodextrin (HP-β-CD) particles were produced using supercritical assisted atomization (SAA) with carbon dioxide as the spraying medium or co-solute and aqueous ethanol solution as the solvent. The effects of several key factors on the morphology and size of the HP-β-CD particles were investigated. These factors included the solvent effect, temperatures of the precipitator and saturator, concentration of the HP-β-CD solution, and flow rate ratio of carbon dioxide to the HP-β-CD solution. The conducive conditions for producing fine spherical particles were 54.2% (w/w) aqueous ethanol as the solvent, precipitator and saturator temperatures of 373.2 K and 353.2 K, respectively, a flow rate ratio of carbon dioxide to HP-β-CD solution of 1.8, and low concentrations of HP-β-CD solution. The addition of leucine (LEU) enhanced the aerosol performance of the HP-β-CD particles, and the fine particle fraction (FPF) of the HP-β-CD particles with the addition of 13.0 mass% LEU was 1.8 times higher than that of the HP-β-CD particles without LEU. This study shows that LEU can act as a dispersion enhancer and that HP-β-CD particles produced using SAA can be used as pulmonary drug carriers.

Mannitol carrier and drug-carrier composite particles were produced via low-temperature supercritical assisted atomization (LTSAA). The enhanced aerosolization of the mannitol carrier particles with added L-leucine could be attributed to the formation of fine corrugated particles, which reduced the interparticle cohesion and led to better flowability of the mannitol carrier particles. A poorly water-soluble drug, beclomethasone dipropionate (BDP), and mannitol co-precipitated during LTSAA with the optimal addition of 9.1 mass % L-leucine. The in vitro aerosolization and dissolution experiments indicated that the fine particles fraction (FPF) of the drug-carrier composite particles was 3.4 times (FPF = 49.9 %) higher than that of the as-received BDP (FPF = 14.7 %) and the 63.2 % drug release time of the drug-carrier composite particles was 15.6 times faster than that of as-received BDP. This study suggests that the drug-carrier composites produced using LTSAA can be employed in the instant formulation of inhalable dry powder.

In this study, the formation of sub-micrometric chitosan (CS) particles via supercritical assisted atomization (SAA) was investigated using supercritical CO2 spraying medium and aqueous ethanol solution (50%, v/v) as solvent. Results indicated that smaller CS particle were obtained by using low concentrations of the CS solution, high saturator temperatures, and an optimized CO2-to-CS solution flow ratio. Using the mixed-suspension, mixed-product-removal (MSMPR) population balance model, the precipitation kinetics parameters were determined from CS particle size distributions and the mass-weighted mean sizes of CS particles, which are a function of the precipitation parameters. An efficient tight junction opening effect was induced by the interaction of CS microparticles and Caco-2 cell monolayers as indicated by the marked decrease in the transepithelial electrical resistance (TEER) of Caco-2 cell monolayers incubated with CS particle suspensions. In addition, the reversibility of the TEER changed depending on the viability of the Caco-2 cells, and cell viability was high at low concentrations of CS suspensions.

Microalgae have been recognized as one of the potential resources for biodiesel production based on its fast growth or its high total lipid content depending on species. Expression of Kennedy pathway genes, which encodes GPAT, LPAAT, PAP, and DGAT for increasing the metabolic flux towards the TAG storage in Chlorella sp. from 20 to 46 wt% and total lipid accumulation from 35 to 60 wt.% corresponding to each specific gene combination under autotrophy, compare to the wild type (vector only). The highest TAG content was found in cells expressing a quadruple-gene construct (GPAT-LPAAT-PAP-DGAT) in the Kennedy pathway, corresponding to 46 wt.% of TAG and 60 wt.% of total lipid content. This work provides the optimization of TAG production in Chlorella sp. can be achieved by manipulating the selected genes, in turns making commercially producing biodiesel practical.

Sub-micrometric chitosan hydrochloride (CH) particles were produced using a supercritical assisted atomization (SAA) process with supercritical carbon dioxide as the spraying medium and 50% (v/v) ethanol aqueous solution as the solvent. Adding ethanol to the aqueous solution increased the solubility of CO2, enhanced the second atomization in SAA, and reduced CH particles size. The results revealed that low concentrations of the CH solution, high saturator temperatures, and an optimized CO2-to-CH solution flow ratio effectively minimized the mean particle size. A marked decrease in the transepithelial electrical resistance of Caco-2 cell monolayers incubated with CH suspensions demonstrated that the interaction between the fine CH particles and Caco-2 cells produced an efficient tight junction opening effect. Cytotoxicity experiments showed that a low concentration of the CH suspension and large CH particle size caused high Caco-2 cell viability. The biological properties of the CH particles were investigated and could be used in drug delivery system.

The authors report a small molecule host of 2,7-bis(diphenylphosphoryl)-9-[4-(N,N-diphenylamino)phenyl]-9-phenylfluorene (POAPF) doped with 8 wt% iridium(III)-bis[(4,6-difluorophenyl)pyridinato- N , C 2′ ]picolinate (FIrpic) for use in efficient and single-layer blue phosphorescent organic light-emitting diodes (PHOLEDs) exhibiting a maximum external quantum efficiency of ∼20.3% at brightness of 100 cd/m 2 . The high performance of such single layer PHOLEDs is attributed to the POAPF host’s high morphological stability, suitable triplet energy level, and equal charge carrier mobilities of hole and electron to form the broad carrier recombination zone in the emitting layer, thus reducing the triplet-triplet annihilation and resulting in a slight efficiency roll off of 0.5% from the brightness of 1 and 1000 cd/m 2 . This work also systematically investigated the arrangement of the POAPF:FIrpic recombination zone for optimizing the performance of the single layer PHOLED.

Using recombinant DNA technology, we constructed a dual fusion gene expression plasmid, pRCAH-30, encoding carbonic anhydrase (CA) from the cyanobacterium Synechocystis sp. PCC6803, an R5 peptide sequence, and an affinity (His)6 tag, to allow the simultaneous purification and immobilization of the encoded fusion enzyme, termed RCAH. The expressed fusion protein was approximately 30 kDa, and could be rapidly purified using affinity resins. To enhance enzyme activity, the R5 peptide facilitated immobilization by means of silicification with tetramethoxysilane; the aggregated particles were approximately 300 nm in diameter. Activity tests revealed that the enzyme functioned optimally between pH 7.0 and 7.5; maximum stability was achieved between 25 and 45°C, at pH 6.0 ∼ 8.0. Activity of the fusion enzyme persisted, even after encapsulation by biomimetic silicification. In fact, silicone embedding stabilized the enzyme structure, thereby increasing its stability and reusability rate under different environmental conditions. In addition, the silicified enzyme reduced waste CO2 gas from 800 to 42 ppm, resulting in a gas capture rate of 94.7% after conversion. Thus, the construct developed in this study can be effectively utilized for the sequestration of industrial waste CO2 gas.

Highly efficient butanol-producing bacterial microflora were isolated from hydrogen-producing sludge of a sewage treatment plant. Based on denaturing gradient gel electrophoresis (DGGE) analysis and 16s rDNA comparison, four strains from the butanol-producing microflora were identified as Clostridium saccharoperbutylacetonicum, Clostridium butylicum, Clostridium beijernckii, and Clostridium acetobutylicum. The effects of glucose, FeSO(4) · 7H(2)O and yeast extract concentrations on the butanol production by the mixture culture were investigated on batch mode. The medium composition for bio-butanol production was optimized using the Box-Behnken design and response surface methodology (RSM). The maximum butanol production rate (0.25 ± 0.02 g/L-h) and concentration (12.4 g/L) were obtained under the condition of glucose concentration, 60 g/L; FeSO(4) · 7H(2)O, 0.516 g/L; yeast extract concentration, 5.13 g/L. Addition of 6.0 g/L butyric acid significantly increased the butanol titer to 17.51 ± 0.49 g/L. Pressurized fermentation strategy (employed with a 5L fermentor) further enhanced the butanol concentration to 21.35 g/L, along with a maximum butanol rate of 1.25 g/L-h.

Reserve lipids of microalgae are promising for biodiesel production. However, optimization of cultivation conditions for both biomass yield and lipid production of microalgae is a contradictory problem because required conditions for both targets are different. In this study, a two-stage cultivation strategy is proposed to enhance lipid production of the microalga Nannochloropsis oculata. Biomass growth and lipid production were carried out in two separate and non-interacting stages. In first-stage cultivation, microalgae were cultivated in optimal conditions for cell growth. Then, microalgae were harvested and transferred into a growth-limited environment, thus enhancing lipid production of microalgae. Here, optimization of the lipid production stage (second stage) with respect to different levels of inoculum concentration, salinity of culture broth, and intensity of irradiance was performed. The results show that irradiance exhibits a significant influence on lipid production. The highest lipid productivity of 0.324 g L−1 day−1 was obtained with an inoculum concentration of 2.3 g L−1, a salinity of 35 g L−1, and an irradiance of 500 μmol photons m−2 s−1. The final yield of lipid obtained from the two-stage process was 2.82-times higher than that from traditional single-stage batch cultivation systems.

Biofilms have been successfully applied for biotransformation for decades, especially in the area of bioremediation due to the feature of harsh reaction condition resistance. Acetobacter xylinum is known for its cellulose pellicle forming capability. Like biofilm, A. xylinum cells are immobilized by simultaneously produced cellulose nanofibers in the pellicle. A recombinant A. xylinum was constructed with the expectation that the cells could be self-immobilized and achieve a desired and stable biotransformation. d -Amino acid oxidase (DAAO) of Rhodosporidium toruloides was used as the model enzyme to be expressed in the recombinant A. xylinum . The constructed recombinant A. xylinum not only successfully produced DAAO activity but also self-immobilized by cellulose nanofibers in both the static and shaken culture. Although self-immobilized cells demonstrated a DAAO activity approximately 10% of the cell crude extract activity, it provided the benefits of improved thermal stability, operational stability, and easy retrieval for repeated use.

Rapid recovery, immobilization, and silica encapsulation of a dual-fusion enzyme was achieved by using iminodiacetic acid (IDA) modified magnetic nanoparticle as a carrier. D-amino acid oxidase (DAAO) of Rhodosporidium toruloides was used as a model enzyme in which a silica-precipitating peptide R5 and a metal ion complexing peptide (His)(6) were fused to its N- and C-terminal, respectively. After charging the magnetic particle with Cu(2+), the dual-fusion DAAO of 0.43 g could be directly recovered from the recombinant E. coli crude extract and immobilized on 1 g of the magnetic particle. Once in contact with hydrolyzed tetramethoxysilane (TMOS), the homogeneously dispersed immobilized dual-fusion DAAO was biosilicificated to form aggregates with size about 50 microm. The silica-encapsulated immobilized DAAO demonstrated a pyruvic acid production rate comparable with that of the naked immobilized DAAO in five repeated batch reactions when D-alanine was used as substrate. Furthermore, 85% of its activity remained after incubation at 60 degrees C for 1 h while the naked immobilized DAAO lost all its activity. This process provides the advantages that recombinant fusion enzyme can be directly recovered from crude extract, silica encapsulation protects the enzyme from leakage and denaturation, and the enzyme activity can be easily retrieved by applying a magnetic field.

Vitreoscilla hemoglobin (VHb) gene driven by the constitutive bla promoter was expressed in the cellulose-producing Acetobacter xylinum. The expressed VHb was biochemically active and could enhance cell growth in a shaken culture containing cellulase. VHb-expressing A. xylinum (VHb + ) exhibited a specific growth rate 50% higher than that of the host strain (VHb - ). Probably because of its faster growth rate, the size of tentacled cellulose beads produced by VHb + was about 20% of that produced by VHb - after 2 days cultivation in a shake-flask. When cultured statically, the amount of cellulose pellicle produced by VHb + could be 2-fold that produced by VHb - . Cellulose pellicle concentration of 11 g/L was obtained for VHb + , whereas 6 g/L was obtained for VHb - after 6 days of microaerobic incubation.

Recombinant Escherichia coli with self-disruption characteristic was developed. The cell harbored a d -amino acid oxidase (DAAO) expressing plasmid resulted in an obvious self-disruption. When T7 lysozyme instead of DAAO was used as a lysis gene, no appreciable self-disruption was observed. In the presence of two compatible plasmids, which expressed DAAO and T7 lysozyme, respectively, E. coli BL21(DE3) showed a significant self-disruption. A lysis plasmid containing both inducible DAAO and constitutive T7 lysozyme genes were, therefore, constructed to make the transformed cells self-disrupt more effectively. Green fluorescent protein (GFP), a model target protein, was co-expressed in this self-disruptive E. coli cell. About 60% of the cells were self-disrupted when the lysis genes were induced by isopropyl thiogalactoside (IPTG). The cell disruption efficiency reached to 90% after freeze/thawing treatment.

Expression of the gene (daao) encoding D-amino acid oxidase (DAAO) in Escherichia coli typically results in a marked decrease of cell viability, and it has generally been assumed that the consumption of intracellular D-alanine by DAAO is responsible for this effect. Vitreoscilla hemoglobin (VHb) gene (vgb) was coexpressed with Rhodosporidium toruloides D-amino acid oxidase in E. coli BL21(DE3) and BL21(DE3)pLysS, expression hosts differing in the stringency of suppressing basal transcription. Not only was the toxic effect of DAAO on cell growth relieved but also the pronounced cell lysis of BL21(DE3)pLysS caused by the expression of DAAO was prevented by coexpressing VHb with DAAO. As a result of the higher cell density achieved, DAAO activity about 1.5-fold higher than that of DAAO-expressing control strains could be obtained by DAAO/VHb-coexpressing strains. The relieving effect of VHb on DAAO toxicity resulted from its oxygen-binding ability. The low availability of free intracellular oxygen reduced DAAO activity and consequently its toxicity.

This study aimed to clone and characterize a thermal resistant glucose dehydrogenase (GDH) and investigate its clinical potential. A Sulfolobus -like thermophilic microbe was first isolated from a hot spring in Taitung, Chihpen County, Taiwan. The gene encoding GDH was cloned from the bacterium and expressed in Escherichia coli . The molecular weight of the enzyme was found to be approximately 39,000 kDa. The enzyme is stable over pH 4.0 to 11.0 and has an optimum pH of 8.0. The thermostability range of the enzyme correlated well with that of the natural environment for Sulfolobus . The GDH showed high substrate specificity for glucose. GDH could be useful in biotechnological applications because of its higher thermostability and substrate specificity when compared with that of other glucose-degrading enzymes. Keywords: Glucose dehydrogenase, sequencing, glucose test strip, blood glucose meter, diabetes mellitus

Discovery of an alternative fuel is now an urgent matter because of the impending issue of oil depletion. Lipids synthesized in algal cells called triacylglycerols (TAGs) are thought to be of the most value as a potential biofuel source because they can use transesterification to manufacture biodiesel. Biodiesel is deemed as a good solution to overcoming the problem of oil depletion since it is capable of providing good performance similar to that of petroleum. Expression of several genomic sequences, including glycerol-3-phosphate dehydrogenase, glycerol-3-phosphate acyltransferase, lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, diacylglycerol acyltransferase, and phospholipid:diacylglycerol acyltransferase, can be useful for manipulating metabolic pathways for biofuel production. In this study, we found this approach indeed increased the storage lipid content of C. minutissima UTEX 2219 up to 2-fold over that of wild type. Thus, we conclude this approach can be used with the biodiesel production platform of C. minutissima UTEX 2219 for high lipid production that will, in turn, enhance productivity.

Bacillus subtilis strains that can produce hyaluronic acid (HA) were constructed by integrating the HA synthase gene (hasA) and the UDP-glucose dehydrogenase gene of group C Streptococcus (hasB) or of B. subtilis itself (tauD) into the amyE locus of the B. subtilis chromosome. All of the inserted genes were under the control of a strong constitutive vegII promoter of B. subtilis. Although HA production could be achieved by expressing hasA alone, coexpressing hasB or tauD with hasA could enhance HA production at least 2-fold. To replenish the energy consumed for HA biosynthesis, Vitreoscilla hemoglobin (VHb) was coexpressed with the HA-expressing genes. With the expression of VHb, not only the cell concentration was enhanced 25%, but also HA production was further increased by 100%. About 1.8 g/L of HA was obtained by the recombinant strain B. subtilis carrying VHb, hasA, and tauD genes in the expression cassette after 30 h cultivation.

Vitreoscilla hemoglobin (VHb) was constitutively expressed in Acetobacter xylinum to enhance bacterial cellulose (BC) production. A pronounced enhancement of BC production in static culture was observed. Reducing O(2) tension in gaseous phase of the culture by tightly sealing the culture tube could also enhance BC production by 70%. O(2) tension in gaseous phase reduced from 21 to 15% in the sealed and static culture of VHb-expressing A. xylinum after 7 days cultivation, while 7.36g/l of BC with yield of 0.44 were obtained. BC pellicle production by VHb-expressing A. xylinum was successfully scaled-up in a sealed 4l disposable zip lock plastic bag with BC yield of 0.38 and concentration of 6.73g/l.

The PsADH2-promoter of Pichia stipitis alcohol dehydrogenase II (ADH II) gene was employed to control the expression of Vitreoscilla hemoglobin (VHb) gene in Pichia pastoris. As in P. stipitis, the promoter was also induced microaerobically in P. pastoris. The expression level of VHb in P. pastoris at low O2 tension (