Cultivation of E. coli carrying a plasmid-based Measles vaccine construct (4.2 kbp pcDNA3F) employing medium optimisation and pH-temperature induction techniques

<p>Abstract</p> <p>Background</p> <p>Plasmid-based measles vaccines offer great promises over the conventional fertilised egg method such as ease of manufacture and mimic wild-type intracellular antigen expression. The increasing number of clinical trials on plasmid-based measles vaccines has triggered the need to make more in less time.</p> <p>Results</p> <p>In this work, we investigated the process variables necessary to improve the volumetric and specific yields of a model plasmid-based measles vaccine (pcDNA3F) harboured in <it>E. coli </it>DH5<it>α</it>. Results from growth medium optimisation in 500 mL shake flasks by response surface methodology (RSM) generated a maximum volumetric yield of 13.65 mg/L which was 1.75 folds higher than that of the base medium. A controlled fed-batch fermentation employing strategic glycerol feeding and optimised growth conditions resulted in a remarkable pcDNA3F volumetric yield of 110 mg/L and a specific yield of 14 mg/g. In addition, growth pH modification and temperature fluctuation between 35 and 45°C were successfully employed to improve plasmid production.</p> <p>Conclusion</p> <p>Production of a high copy number plasmid DNA containing a foreign gene of interest is often hampered by the low plasmid volumetric yield which results from the over expression of foreign proteins and metabolic repressors. In this work, a simple bioprocess framework was employed and successfully improved the production of pcDNA3F.</p

Production polystyrene micro-emulsion as template for monolith synthesis

Monolith have received much attention as high-performance chromatographic matrices due to its convective mass transfer and interconnected porous structure. Biodegradable polymers, free radicals and cross-linkers are among the templates used to form pore structure. However, poor heat dissipation and uneven pore size distribution across monolith remain as a key challenge in monolith fabrication. Therefore, this study aims to synthesize and characterize polystyrene micro-emulsion as template for monolith. The operating conditions for the synthesis of the polymeric micro-emulsion, that includes polymer concentration (14 - 35 wt %), surfactant concentration (1 - 9 wt %), temperature (30 - 70oC) and stirring rate (500 - 1000 rpm), were designed using Response Surface Methodology (RSM). The characterizations of resulting particles were observed using Scanning Electron Microscopy (SEM) and Inverted Microscope. The sizes of the particles were determined within range of 5.9 - 11.7 µm. Out of the 30 tested samples with different operating conditions, observation under the inverted microscope indicated homogenous particles of polystyrene microemulsion while some forming aggregations. Sample that was synthesized using 21 wt% polymer, 3 wt% surfactant, stirring rate at 875 rpm and heated at 40 oC resulted homogenous particles with particle diameter ranging from 7.92 µm to 8.80 µm. Good particle homogeneity was also obtained at a higher polymer concentration (35 wt %) using similar surfactant concentration and operating temperature at slower stirring rate (625 rpm). Samples aggregation were observed when using 35 wt % polymer, 7 wt % surfactant heated at 50oC at 750 rpm as well as sample under parameter of 25 wt% polymer wt % surfactant for 60oC stirring at 875 rpm. The findings of the study provide useful insights on the feasibility of polymeric micro-emulsion particles as a directing template for monolith fabrication with structured pores

Green biodiesel production: a review on feedstock, catalyst, monolithic reactor, and supercritical fluid technology

The advancement of alternative energy is primarily catalyzed by the negative environmental impacts and energy depletion caused by the excessive usage of fossil fuels. Biodiesel has emerged as a promising substitute to petrodiesel because it is biodegradable, less toxic, and reduces greenhouse gas emission. Apart from that, biodiesel can be used as blending component or direct replacements for diesel fuel in automotive engines. A diverse range of methods have been reported for the conversion of renewable feedstocks (vegetable oil or animal fat) into biodiesel with transesterification being the most preferred method. Nevertheless, the cost of producing biodiesel is higher compared to fossil fuel, thus impeding its commercialization potentials. The limited source of reliable feedstock and the underdeveloped biodiesel production route have prevented the full-scale commercialization of biodiesel in many parts of the world. In a recent development, a new technology that incorporates monoliths as support matrices for enzyme immobilization in supercritical carbon dioxide (SC-CO2) for continuous biodiesel production has been proposed to solve the problem. The potential of SC-CO2 system to be applied in enzymatic reactors is not well documented and hence the purpose of this review is to highlight the previous studies conducted as well as the future direction of this technology

Nano-Doped Monolithic Materials for Molecular Separation

Monoliths are continuous adsorbents that can easily be synthesised to possess tuneable meso-/macropores, convective fluid transport, and a plethora of chemistries for ligand immobilisation. They are grouped into three main classes: organic, inorganic, and hybrid, based on their chemical composition. These classes may also be differentiated by their unique morphological and physicochemical properties which are significantly relevant to their specific separation applications. The potential applications of monoliths for molecular separation have created the need to enhance their characteristic properties including mechanical strength, electrical conductivity, and chemical and thermal stability. An effective approach towards monolith enhancement has been the doping and/or hybridization with miniaturized molecular species of desirable functionalities and characteristics. Nanoparticles are usually preferred as dopants due to their high solid phase dispersion features which are associated with improved intermolecular adsorptive interactions. Examples of such nanomaterials include, but are not limited to, carbon-based, silica-based, gold-based, and alumina nanoparticles. The incorporation of these nanoparticles into monoliths via in situ polymerisation and/or post-modification enhances surface adsorption for activation and ligand immobilisation. Herein, insights into the performance enhancement of monoliths as chromatographic supports by nanoparticles doping are presented. In addition, the potential and characteristics of less common nanoparticle materials such as hydroxyapatite, ceria, hafnia, and germania are discussed. The advantages and challenges of nanoparticle doping of monoliths are also discussed

Synthesis and characterization of polymeric microspheres template for a homogeneous and porous monolith

Monolith is an emerging technology applicable for separation, filtration, and chromatography due to its interconnected pore structure. However, the current templates used to form monolith pores are associated with poor heat dissipation, uneven pore size distribution, and relatively low mechanical strength during monolith scale-up. Templates made from polymeric microsphere particles were synthesized via a solvent evaporation technique using different types of polymer (polystyrene, polycaprolactone, polypropylene, polyethylene, and poly (vinyl-alcohol) at varied polymer (10–40 wt%) and surfactant (5–10%) concentrations. The resulting microsphere particles were tested as a monolith template for the formation of homogenous pores. Among the tested polymers, polystyrene at 10 wt% concentration demonstrated good particle morphology determined to around 1.94–3.45 µm. The addition of surfactant at a concentration of 7–10 wt% during microsphere synthesis resulted in the formation of well-shaped and non-aggregating microsphere particles. In addition, the template has contributed to the production of porous monoliths with enhanced thermal stability. The thermogravimetric analysis (TGA) indicated monolith degradation between 230 ◦C and 450 ◦C, implying the material excellent mechanical strength. The findings of the study provide insightful knowledge on the feasibility of polymeric microsphere particles as a pore-directing template to fabricate monoliths with desired pore structures

Effect of Crosslinkers on Immobilization of ß-Galactosidase on Polymethacrylate Monolith.

Advances in biotechnology unfold a new frontier for the development of enzyme-catalysed bioprocess which is green and sustainable in contrast with chemical processes. Immobilization technology appears as a beneficial solution to the uneconomical cost of enzyme operation. Immobilization of enzyme via crosslinking approach has become a technology interest due to the more concentrate enzyme activity in the catalyst compared to other techniques. In this study, two types of crosslinker, glutaraldehyde and hexamethylene diisocyanate at different concentration was investigated in immobilizing β-galactosidase on polymethacrylate monolith. The enzyme activity upon immobilization was measured spectrophotometrically at 405 nm. The immobilized enzyme was further characterized using Fourier Transform Infrared Spesctroscopy (FTIR) and Zeiss Axio Fluorescence Microscope. The findings showed that the optimum enzyme activity was achieved when using 0.05% and 0.01% glutaraldehyde hexamethylene diisocyanate respectively. Beyond that concentration, a significant reduction of enzyme activity was observed. It was found that glutaraldehyde was preferable as crosslinking agent as hexamethylene diisocyanate exhibited stronger effect in reducing enzyme activity. A successful binding of β-galactosidase on polymethacrylate monolith was observed using Fourier-transform infrared spectroscopy (FTIR) and Zeiss Axio Fluorescence microscope. The outcomes of this study indicate the potential of enzyme immobilization on monolith via crosslinking method

An optimal blend of single auto displayed cellulases for cellulose saccharification – a proof of concept

BACKGOUND: To date, it has been challenging to determine the optimal proportions of the individual cellulases in crude extracts and multienzyme systems to promote enzyme synergism. The application of single surface-display of enzymes may obviate such challenges. This article presents a proof of concept of the optimal mixing of single surface-displayed cellulases for cellulose hydrolysis using Simplex lattice mixture design. RESULTS: The recently discovered maximized autotransporter-mediated expression system was used to express the three cellulases. The biochemical screening analysis revealed that the enzymes have a broad range of functional pH (4–9) and temperature (30–100 ∘C) characteristics. The evaluated optima were pH 6 and 60 ∘C. Through the statistical design of experiment, a blend ratio of 1: 1.6: 1.4 of endoglucanase: exoglucanase