Production of lectin-affinity matrices for process-scale glycoprotein purification

A selection of prokaryotic lectins with a variety of glycan specificities and affinities have been identified, cloned, expressed in Eschericia coli and characterised. The aims of this project are to: - express the lectins at 1L scale to produce sufficient quantities for immobilisation studies (~100 mg) - immobilisethelectinsonSepharose - evaluate lectin performance on column by monitoring their ability toreproducibly capture and elute glycoprotein glycoforms

Polymerase-endonuclease amplification reaction for large-scale enzymatic production of antisense oligonucleotide

Synthetic oligonucleotides are contaminated with highly homologous failure sequences. Oligonucleotide synthesis is difficult to scale up because it requires expensive equipments, hazardous chemicals, and tedious purification process. Here we report a novel thermocyclic reaction, polymerase-endonuclease amplification reaction (PEAR), for the amplification of oligonucleotides. A target oligonucleotide and a tandem repeated antisense probe are subjected to repeated cycles of denaturing, annealing, elongation and cleaving, in which thermostable DNA polymerase elongation and strand slipping generate duplex tandem repeats, and thermostable endonuclease (PspGI) cleavage releases monomeric duplex oligonucleotides. Each round of PEAR achieves >100-fold amplification. The product can be used in one more round of PEAR directly, and the process can be further repeated. In addition to avoiding dangerous materials and improved product purity, this reaction is easy to scale up and amenable to full automation, so it has the potential to be a useful tool for large-scale production of antisense oligonucleotide drugs

A metallurgical route to solar-grade silicon

The aim of the process is to produce silicon for crystallization into ingots that can be sliced to wafers for processing into photovoltaic cells. If the potential purity can be realized, the silicon will also be applicable for ribbon pulling techniques where the purification during crystallization is negligible. The process consists of several steps: selection and purification of raw materials, carbothermic reduction of silica, ladle treatment, casting, crushing, leaching, and melting. The leaching step is crucial for high purity, and the obtainable purity is determined by the solidification before leaching. The most difficult specifications to fulfill are the low contents of boron, phosphorus, and carbon. Boron and phosphorus can be excluded from the raw materials, but the carbothermic reduction will unavoidably saturate the silicon with carbon at high temperature. During cooling carbon will precipitate as silicon carbide crystals, which will be harmful in solar cells. The cost of this solar silicon will depend strongly on the scale of production. It is as yet premature to give exact figures, but with a scale of some thousand tons per year, the cost will only be a few times the cost of ordinary metallurgical silicon

Large-scale purification of factor VIII by affinity chromatography: optimization of process parameters

The optimization of a new process for the extraction of human coagulation factor VIII (FVIII) from plasma with the tailor-made affinity matrix dimethylaminopropylcarbamylpentyl-Sepharose CL-4B (C3---C5 matrix) is described. First, plasma is applied to DEAE-Sephadex A-50 anion exchanger in order to separate a number of proteins, including coagulation factors II, IX and X (prothrombin complex), from FVIII. Subsequently, the unbound fraction of the ion exchanger, containing FVIII, is contacted with the C3---C5 affinity matrix. Optimization of the FVIII affinity chromatographic procedure is accomplished in terms of the ligand density of the matrix, adsorption mode (batch-wise versus column-wise adsorption and matrix to plasma ratio), and conditions of pH and conductivity to be applied on washing and desorption. In scale-up experiments, by processing 20 1 of plasma, the recovery (340 U VIII:C/kg plasma) and the specific activity (s.a.) (1.2 U VIII:C/mg protein) are better than those obtained by cryoprecipitation (recovery 300 U VIII:C/kg plasma, s.a. O.3 U VIII:C/mg protein). The newly developed process using the specially designed C3---C5 affinity matrix has potential application in the process-scale purification of FVIII

Water Purification by Shock Electrodialysis: Deionization, Filtration, Separation, and Disinfection

The development of energy and infrastructure efficient water purification systems are among the most critical engineering challenges facing our society. Water purification is often a multi-step process involving filtration, desalination, and disinfection of a feedstream. Shock electrodialysis (shock ED) is a newly developed technique for water desalination, leveraging the formation of ion concentration polarization (ICP) zones and deionization shock waves in microscale pores near to an ion selective element. While shock ED has been demonstrated as an effective water desalination tool, we here present evidence of other simultaneous functionalities. We show that, unlike electrodialysis, shock ED can thoroughly filter micron-scale particles and aggregates of nanoparticles present in the feedwater. We also demonstrate that shock ED can enable disinfection of feedwaters, as approximately $99\%$ of viable bacteria (here \textit{E. coli}) in the inflow were killed or removed by our prototype. Shock ED also separates positive from negative particles, contrary to claims that ICP acts as a virtual barrier for all charged particles. By combining these functionalities (filtration, separation and disinfection) with deionization, shock ED has the potential to enable more compact and efficient water purification systems

The harnessing of peptide-monolith constructs for single step plasmid DNA purification

The availability of synthetic peptides has paved the way for their use in tailor-made interactions with biomolecules. In this study, a 16mer LacI-based peptide was used as an affinity ligand to examine the scale up feasibility for plasmid DNA purification. First, the peptide was designed and characterized for the affinity purification of lacO containing plasmid DNA, to be employed as a high affinity ligand for the potential capturing of plasmid DNA in a single unit operation. It was found there were no discernible interactions with a control plasmid that did not encode the lacO nucleotide sequence. The dissociation equilibrium constant of the binding between the 16mer peptide and target pUC19 was 5.0 ± 0.5 × 10-8 M as assessed by surface plasmon resonance. This selectivity and moderated affinity indicate that the 16mer is suitable for the adsorption and chromatographic purification of plasmid DNA. The suitability of this peptide was then evaluated using a chromatography system with the 16mer peptide immobilized to a customized monolith to purify plasmid DNA, obtaining preferential purification of supercoiled pUC19. The results demonstrate the applicability of peptide-monolith supports to scale up the purification process for plasmid DNA using designed ligands via a biomimetic approach

Preparative Synthesis of dTDP-L-Rhamnose Through Combined Enzymatic Pathways

dTDP-L-rhamnose, an important precursor of O-antigen, was prepared on a large scale from dTMP by executing an one-pot reaction in which six enzymes are involved. Two enzymes, dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase and dTDP-4-keto-rhamnose reductase, responsible for the conversion of dTDP-4-keto-6-deoxy- D-glucose to dTDP-L-rhamnose, were isolated from their putative sequences in the genome of Mesorhizobium loti, functionally expressed in Escherichia coli, and their enzymatic activities were identified. The two enzymes were combined with an enzymatic process for dTDP-4- keto-6-deoxy-D-glucose involving TMP kinase, acetate kinase, dTDP-glucose synthase, and dTDP-glucose 4,6- dehydratase, which allowed us to achieve a preparative scale synthesis of dTDP-L-rhamnose using dTMP and glucose-1-phosphate as starting materials. About 82% yield of dTDP-L-rhamnose was obtained based on initial dTMP concentration at 20 mM dTMP, 1 mM ATP, 10 mM NADH, 60 mM acetyl phosphate, and 80 mM glucose-1- phosphate. From the reaction with 20 ml volume, approximately 180 mg of dTDP-L-rhamnose was obtained in an overall yield of 60% after two-step purification, that is, anion exchange chromatography and gel filtration for desalting. The purified product was identifiedbyHPLC, ESI-MS,andNMR,showingabout95%purity

Looking for a psychology for the inner rational agent

Research in psychology and behavioural economics shows that individuals’ choices often depend on ‘irrelevant’ contextual factors. This presents problems for normative economics, which has traditionally used preference-satisfaction as its criterion. A common response is to claim that individuals have context-independent latent preferences which are ‘distorted’ by psychological factors, and that latent preferences should be respected. This response implicitly uses a model of human action in which each human being has an ‘inner rational agent’. I argue that this model is psychologically ungrounded. Although references to latent preferences appear in psychologically-based explanations of context-dependent choice, latent preferences serve no explanatory purpose

Optimization study of the fouling build-up on a RO membrane for pretrated olive mill wastewater purification

Even though membranes are considered in many aspects a mature technology, a range of features are still in development and under investigation. Regarding this, the main handicap of this technology is inevitably membrane fouling. Fouling issues have investigated by many research groups in the last years to convince investors to implement membranes as substitutes of a range of unit operations at industrial scale. In the wastewater treatment field, this is especially problematic, given the low economic value of the product, that is, treated water. On another hand, the management of the effluents generated by olive oil industries, olive mill wastewaters (OMW), is a task of global concern not anymore constrained to a specific region. These wastewaters represent an ever-increasing problem still unresolved. The present work was aimed for the modelling and optimization of a reverse osmosis (RO) membrane operation for the purification of pretreated olive mill wastewater, with a focus on the dynamic fouling development minimization on the selected membrane as a function of the set-up of the operating conditions. For this goal, beforehand a factorial design was implemented for the optimization of the RO treatment of the OMW stream. The results gathered were thereafter interpreted by means of the response surface methodology. A significant impact was noted to be driven by the operating pressure and the tangential velocity on the fouling rate on the RO membrane. The response surfaces withdrawn from the experimental data support the previous results, and the optimised parameters - ambient temperature range (24 - 25 °C), moderate operating pressure (25 - 30 bar) and turbulent tangential flow (3.1 - 3.5 m s -1 ) - were found to provide a stable permeate flux of 32.3 - 38.5 L h -1 m -2 . These results reveal the proposed process could be operated successfully at ambient temperature conditions and medium operating pressure, boosting the economic efficiency of the RO purification of this effluent. Finally, the parametric quality standards stablished to reuse the purified effluent for irrigation purposes were checked and found to be satisfactory