Improved reactor performance and operability in the biotransformation of carveol to carvone using a solid-liquid two-phase partitioning bioreactor

In an effort to improve reactor performance and process operability, the microbial biotransformation of (-)-trans-carveol to (R)-(-)-carvone by hydrophobic Rhodococcus erythropolis DCL14 was carried out in a two phase partitioning bioreactor (TPPB) with solid polymer beads acting as the partitioning phase. Previous work had demonstrated that the substrate and product become inhibitory to the organism at elevated aqueous concentrations and the use of an immiscible second phase in the bioreactor was intended to provide a reservoir for substrates to be delivered to the aqueous phase based on the metabolic rate of the cells, while also acting as a sink to uptake the product as it is produced. The biotransformation was previously undertaken in a two liquid phase TPPB with 1-dodecene and with silicone oil as the immiscible second phase and, although improvement in the reactor performance was obtained relative to a single phase system, the hydrophobic nature of the organism caused the formation of severe emulsions leading to significant operational challenges. In the present work, eight types of polymer beads were screened for their suitability for use in a solid-liquid TPPB for this biotransformation. The use of selected solid polymer beads as the second phase completely prevented emulsion formation and therefore improved overall operability of the reactor. Three modes of solid-liquid TPPB operation were considered: the use of a single polymer bead type (styrene/butadiene copolymer) in the reactor, the use of a mixture of polymer beads in the reactor (styrene/butadiene copolymer plus Hytrel(R) 8206), and the use of one type of polymer beads in the reactor (styrene/butadiene copolymer), and another bead type (Hytrel(R) 8206) in an external column through which fermentation medium was recirculated. This last configuration achieved the best reactor performance with 7 times more substrate being added throughout the biotransformation relative to a single aqueous phase benchmark reactor and 2.7 times more substrate being added relative to the best two liquid TPPB case. Carvone was quantitatively recovered from the polymer beads via single stage extraction into methanol, allowing for bead re-use.     

Paper-PEG-based membranes for hydrophobic interaction chromatography: purification of monoclonal antibody

This article discusses the preparation of novel Paper-PEG interpenetrating polymer network-based membranes as inexpensive alternative to currently available adsorptive membranes. The Paper-PEG membranes were developed for carrying out hydrophobic interaction membrane chromatography (HIMC). PEG is normally very hydrophilic but can undergo phase separation and become hydrophobic in the presence of high antichaotropic salt concentrations. Two variants of the Paper-PEG membranes, Paper-PEG 1 and Paper-PEG 2 were prepared by grafting different amounts of the polymer on filter paper and these were tested for their hydraulic properties and antibody binding capacity. The better of the two membranes (Paper-PEG 1) was then used for purifying the monoclonal antibody hIgG1-CD4 from simulated mammalian cell culture supernatant. The processing conditions required for purification were systematically optimized. The dynamic antibody binding capacity of the Paper-PEG 1 membrane was about 9 mg/mL of bed volume. A single step membrane chromatographic process using Paper-PEG 1 membrane gave high monoclonal antibody purity and recovery. The hydraulic permeability of the paper-based membrane was high and was maintained even after many runs, indicating that membrane fouling was negligible and the membrane was largely incompressible.     

An N-terminal 78 amino acid truncation of REIC/Dkk-3 effectively induces apoptosis

Overexpression of REIC/Dkk-3 (a tumor suppressor gene) induces cancer cell apoptosis through endoplasmic reticulum (ER) stress. Therefore, the identification of the portion of REIC/Dkk-3 that causes ER stress may be essential for the development of cancer treatment based on REIC/Dkk-3. Here, we made several truncated forms of REIC/Dkk-3 and investigated their therapeutic potentials against prostate cancer. Among three truncated forms, a variant comprising the N-terminal 78 amino acid region of REIC/Dkk-3 (^1-78REIC/Dkk-3) most strongly induced ER stress and apoptosis in human prostate cancer cells (PC3). For in vivo gene expression, we coupled a biodegradable polymer with naked DNA, which attained robust trans-gene expression in PC3-derived subcutaneous tumor. In therapeutic experiments, we demonstrated that multiple direct injections of polymer-conjugated ^1-78REIC/Dkk-3 plasmid provoke ER stress and significantly reduced the subcutaneous tumor volume compared with the control group. We suggest this non-viral strategy may be an effective alternative to viral gene therapy.     

Bioinspired polymers that control intracellular drug delivery

One of the important characteristics of biological systems is their ability to change important properties in response to small environmental signals. The molecular mechanisms that biological molecules utilize to sense and respond provide interesting models for the development of “smart” polymeric biomaterials with biomimetic properties. An important example of this is the protein coat of viruses, which contains peptide units that facilitate the trafficking of the virus into the cell via endocytosis, then out of the endosome into the cytoplasm, and from there into the nucleus. We have designed a family of synthetic polymers whose compositions have been designed to mimic specific peptides on viral coats that facilitate endosomal escape. Our biomimetic polymers are responsive to the lowered pH within endosomes, leading to disruption of the endosomal membrane and release of important biomolecular drugs such as DNA, RNA, peptides and proteins to the cytoplasm before they are trafficked to lysosomes and degraded by lysosomal enzymes. In this article, we review our work on the design, synthesis and action of such smart, pH-sensitive polymers.     

A Dynamic Folded Hairpin Conformation Is Associated with α-Globin Activation in Erythroid Cells

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Biological response of protists Haematococcus lacustris and Euglena gracilis to conductive polymer poly (3,4-ethylenedioxythiophene) polystyrene sulfonate

Improving the growth and pigment accumulation of microalgae by electrochemical approaches was considered a novel and promising method. In this research, we investigated the effect of conductive polymer poly (3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) dispersible in water on growth and pigment accumulation of Haematococcus lacustris and Euglena gracilis. The results revealed that effect of PEDOT:PSS was strongly cell-dependent and each cell type has its own peculiar response. For H. lacustris, the cell density in the 50 mg·l⁻¹ treatment group increased by 50·27%, and the astaxanthin yield in the 10 mg·l⁻¹ treatment group increased by 37·08%. However, under the high concentrations of PEDOT:PSS treatment, cell growth was significantly inhibited, and meanwhile, the smaller and more active zoospores were observed, which reflected the changes in cell life cycle and growth mode. Cell growth of E. gracilis in all the PEDOT:PSS treatment groups were notably inhibited. Chlorophyll a content in E. gracilis decreased while chlorophyll b content increased in response to the PEDOT:PSS treatment. The results laid a foundation for further development of electrochemical methods to promote microalgae growth and explore the interactions between conductive polymers and microalgae cells.     

Segmental mobility of polymers in starch granules at low moisture contents

The objective of this study was to determine the moisture content at which the segmental mobility of polymers within a starch granule is restricted. Common corn, waxy corn and high amylose corn starch samples were equilibrated to a final water activity of 0.15, 0.33, 0.75 or 0.97. The samples were then exposed to iodine vapor for 24 h and the color, absorption spectra and X-ray diffraction patterns were measured. Stained and unstained granules were also viewed under a bright field and polarized light microscope. The results demonstrate that successive local transitions occur within a granule with increasing moisture contents. Furthermore, the data shows that at moisture contents of about 13%, iodine is able to penetrate the granule and the resulting complex disrupts the crystalline arrangement within the granules. The differences in extent of mobility of polymers between different starch types can potentially illuminate differences in starch structure and architecture.     

The physical characteristics of anaerobic granular sludges in relation to their internal architecture

A range of granular sludges was taken from industrial anaerobic sludge blanket reactors treating a wide variety of wastewaters and a comparison was made between the polymers which were extractable from the granules and their internal structures. The study of the internal structure, using sequential staining of ultra-thin sections, showed the complexity of granular sludges. Much of the area was occupied by Gram-negative cells and the area which stained positive for protein was found to increase nearer the centre of the granules. This was accompanied by a decrease in the carbohydrate positive areas. Positive areas for lipid were widespread throughout the granules. Changes in the internal structure were observed when the type of wastewater treated by the granules was changed and a comparison between sludges treating the same type of wastewater showed that factors other than the nature of the substrate must be considered as parameters which will affect the structure of the granules. Although an appreciable variation in the granule strengths was noted, it was not possible to relate these differences, on an overall basis, to either the internal structure or the chemical composition of the extracted polymers. However, an examination of data for granules produced during the treatment of nominally similar wastes did suggest that there would be a relationship between polymer composition and granule strength in these cases.     

A database-based approach for predicting coupled cascade reaction kinetics in polymers: application to oxidative degradation kinetics of high-performance polymers

Coupled cascade reactions forming complex reaction networks can be commonly found in polymerisation reactions and other reactions involving radical intermediates. Predicting the mechanism and kinetics of such reactions requires proper modelling of complex reaction networks. This becomes particularly difficult when coupled cascade reactions occur in polymeric systems containing different types of residues. Here, we propose a residue-based database approach to model such reactions in polymers, with the aid of a visual interface developed here. We demonstrate this approach by predicting the oxidative degradation kinetics of high-performance polymers (HPPs). First, we show that residue-based reaction database can be linked to construct the whole reaction network. For this purpose, we developed a database for oxidation reactions of commonly occurring residues in industrially important HPPs. Then we implement a visual interface which takes inputs from a user about residues in a polymer of interest and subsequently link appropriate databases to build reaction network. Finally, this program executes numerical integration of rate equations in the back-end. Application of this approach and the developed program is demonstrated by studying the oxidative degradation kinetics of three well-known HPPs- PMR-15, HFPE-30 and PMR-II, where the computations took less than a minute in a conventional desktop computer.