A novel vortex flow reactor for the purification of B-phycoerythrin from Porphyridium cruentum

The purification of B-phycoerythrin from a concentrated extract of disrupted Porphyridium cruentum cells was carried out using a new vortex flow reactor design for protein purification. The reactor behaved as an expanded bed in the laminar vortices flow regime where the Streamline DEAE resin was expanded by the axial flow and stabilized by the vortex flow. After the broth culture was centrifuged and resuspended in the adsorption buffer, the concentrated extract of disrupted cells was directly loaded into the vortex flow reactor. The purification of B-phycoerythrin was carried out in two steps: adsorption in the expanded bed and elution from the settled bed. 142.0 mg of B-phycoerythrin was eluted representing a total recovery yield of 86.6%. Prior to B-phycoerythrin purification, the protein adsorption of the vortex flow reactor was characterized through hydrodynamic studies and a dynamic capacity measurement using a standard protein.

Exploring protein-RNA interactions with site-directed mutagenesis and phage display

The importance of RNA as a mediator of genetic information is widely appreciated. RNA molecules also participate in the regulation of various post-transcriptional activities, such as mRNA splicing, editing, RNA stability and transport. Their regulatory roles for these activities are highly dependent on finely tuned associations with cognate proteins. The RNA recognition motif (RRM) is an ancient RNA binding module that participates in hundreds of essential activities where specific RNA recognition is required. We have applied phage display and site-directed mutagenesis to dissect principles of RRM-controlled RNA recognition. The model systems we are investigating are U1A and CUG-BP1. In this dissertation, the molecular basis of the binding affinity of U1A-RNA beyond individual contacts was investigated. We have identified and evaluated the contributions of the local cooperativity formed by three neighboring residues (Asn15, Asn16 and Glu19) to the stability of the U1A-RNA complex. The localized cooperative network was mapped by double-mutant cycles and explored using phage display. We also showed that a cluster of these residues forms a “hot spot” on the surface of U1A; a single substitution at position 19 with Gln or His can alter the binding properties of U1A to recognize a non-cognate G4U RNA. Finally, we applied a deletion analysis of CUG-BP1 to define the contributions of individual RRMs and RRM combinations to the stability of the complex formed between CUG-BP1 and the GRE sequence. The preliminary results showed RRM3 of CUG-BP1 is a key domain for RNA binding. It possibly binds to the GRE sequence cooperatively with RRM2 of CUG-BP1. RRM1 of CUG-BP1 is not required for GRE recognition, but may be important for maintaining the stability of the full-length CUG-BP1.

Design of advanced reverse osmosis and nanofiltration membranes for water purification

Most commercially available reverse osmosis (RO) and nanofiltration (NF) membranes are based on the thin film composite (TFC) aromatic polyamide membranes. However, they have several disadvantages including low resistance to fouling, low chemical and thermal stabilities and limited chlorine tolerance. To address these problems, advanced RO/NF membranes are being developed from polyimides for water and wastewater treatments. The following three projects have resulted from my research. (1) Positively charged and solvent resistant NF membranes. The use of solvent resistant membranes to facilitate small molecule separations has been a long standing industry goal of the chemical and pharmaceutical industries. We developed a solvent resistant membrane by chemically cross-linking of polyimide membrane using polyethylenimine. This membrane showed excellent stability in almost all organic solvents. In addition, this membrane was positively charged due to the amine groups remaining on the surface. As a result, high efficiency (> 95%) and selectivity for multivalent heavy metal removal was achieved. (2) Fouling resistant NF membranes. Antifouling membranes are highly desired for “all” applications because fouling will lead to higher energy demand, increase of cleaning and corresponding down time and reduced life-time of the membrane elements. For fouling prevention, we designed a new membrane system using a coating technique to modify membrane surface properties to avoid adsorption of foulants like humic acid. A layer of water-soluble polymer such as polyvinyl alcohol (PVA), polyacrylic acid (PAA), polyvinyl sulfate (PVS) or sulfonated poly(ether ether ketone) (SPEEK), was adsorbed onto the surface of a positively charged membrane. The resultant membranes have a smooth and almost neutrally charged surface which showed better fouling resistance than both the positively charged NF membranes and commercially available negatively charged NTR-7450 membrane. In addition, these membranes showed high efficiency for removal of multivalent ions (> 95% for both cations and anions). Therefore, these antifouling surfaces can be potentially used for water softening, water desalination and wastewater treatment in a membrane bioreactor (MBR) process. (3) Thermally stable RO membranes. Commercial RO membranes cannot be used at temperature higher than 45°C due to the use of polysulfone substrate, which often limits their applications in industries. We successfully developed polyimides as the membrane substrate for thermally stable RO membranes due to their high thermal resistance. The polyimide-based composite polyamide membranes showed desalination performance comparable to the commercial TFC membrane. However, the key advantage of the polyimide-based membrane is its high thermal stability. As the feed temperature increased from 25oC to 95oC, the water flux increased 5 - 6 times while the salt rejection almost kept constant. This membrane appears to provide a unique solution for hot water desalination and also a feasible way to improve the water productivity by increasing the operating temperature without any drop in salt rejection.

FACS-based purification of Arabidopsis microspores, sperm cells and vegetative nuclei

Background: The male germline in flowering plants differentiates by asymmetric division of haploid uninucleated microspores, giving rise to a vegetative cell enclosing a smaller generative cell, which eventually undergoes a second mitosis to originate two sperm cells. The vegetative cell and the sperm cells activate distinct genetic and epigenetic mechanisms to control pollen tube growth and germ cell specification, respectively. Therefore, a comprehensive characterization of these processes relies on efficient methods to isolate each of the different cell types throughout male gametogenesis. Results: We developed stable transgenic Arabidopsis lines and reliable purification tools based on Fluorescence-Activated Cell Sorting (FACS) in order to isolate highly pure and viable fractions of each cell/nuclei type before and after pollen mitosis. In the case of mature pollen, this was accomplished by expressing GFP and RFP in the sperm and vegetative nuclei, respectively, resulting in 99% pure sorted populations. Microspores were also purified by FACS taking advantage of their characteristic small size and autofluorescent properties, and were confirmed to be 98% pure. Conclusions: We provide simple and efficient FACS-based purification protocols for Arabidopsis microspores, vegetative nuclei and sperm cells. This paves the way for subsequent molecular analysis such as transcriptomics, DNA methylation analysis and chromatin immunoprecipitation, in the developmental context of microgametogenesis in Arabidopsis.

Extraction and purification of theobromine using ionic liquids

Theobromine is an alkaloid present in cocoa and it is used in the treatment of atherosclerosis, hypertension, angina, among others. Due to its importance, the aim of this work consists on the development of an efficient and sustainable technology for the extraction of theobromine from cocoa beans. For the development of a purification technique for theobromine extracted from cocoa, aqueous biphasic systems (ABS) composed of ionic liquids (ILs) were initially studied to infer on the most promising systems. Cholinium-based ILs, based on a non-toxic and biocompatible cation, were used combined with two polymers (PPG 400 and PEG 400) and an inorganic salt (K3PO4). The respective phase diagrams at 298 K and atmospheric pressure were determined, as well as their extraction efficiencies for theobromine. The results obtained indicate that K3PO4 has a greater ability to induce the formation of ABS compared to PEG 400 and PPG 400. ABS consisting of K3PO4 also have a high potential for the extraction of theobromine, with extraction efficiencies ranging between 96.4 and 99.9 %. Based on the most promising ILs for the purification step, they were further used in aqueous solution to extract theobromine from cocoa beans, with extraction yields ranging between 4.5% and 6.5 wt%. Finally, ABS were applied to the aqueous solutions containing theobromine from the cocoa extract, with extraction efficiencies ranging between 96.7 and 99.0%.

Extraction and purification of immunoglobulin G with aqueous biphasic systems

The immune system is able to produce antibodies, which have the capacity to recognize and to bind to foreign molecules or pathogenic organisms. Currently, there are a diversity of diseases that can be treated with antibodies, like immunoglobulins G (IgG). Thereby, the development of cost-efficient processes for their extraction and purification is an area of main interest in biotechnology. Aqueous biphasic systems (ABS) have been investigated for this purpose, once they allow the reduction of costs and the number of steps involved in the process, when compared with conventional methods. Nevertheless, typical ABS have not showed to be selective, resulting in low purification factors and yields. In this context, the addition of ionic liquids (ILs) as adjuvants can be a viable and potential alternative to tailor the selectivity of these systems. In this work, ABS composed of polyethylene glycol (PEG) of different molecular weight, and a biodegradable salt (potassium citrate) using ILs as adjuvants (5 wt%), were studied for the extraction and purification of IgG from a rabbit source. Initially, it was tested the extraction time, the effect on the molecular weight of PEG in a buffer solution of K3C6H5O7/C6H8O7 at pH≈7, and the effect of pH (59) on the yield (YIgG) and extraction efficiency (EEIgG%) of IgG. The best results regarding EEIgG% were achieved with a centrifugation step at 1000 rpm, during 10 min, in order to promote the separation of phases followed by 120 min of equilibrium. This procedure was then applied to the remaining experiments. The results obtained in the study of PEGs with different molecular weights, revealed a high affinity of IgG for the PEG-rich phase, and particularly for PEGs of lower molecular weight (EEIgG% of 96 % with PEG 400). On the other hand, the variation of pH in the buffer solution did not show a significant effect on the EEIgG%. Finally, it was evaluated the influence of the addition of different ILs (5% wt) on the IgG extraction in ABS composed of PEG 400 at pH≈7. In these studies, it was possible to obtain EEIgG% of 100% with the ILs composed of the anions [TOS]-, [CH3CO2]-and Cl-, although the obtained YIgG% were lower than 40%. On the other hand, the ILs composed of the anions Br-, as well as of the cation [C10mim]+, although not leading to EEIgG% of 100%, provide an increase in the YIgG%. ABS composed of PEG, a biodegradable organic salt and ILs as adjuvants, revealed to be an alternative and promising method to purify IgG. However, additional studies are still required in order to reduce the loss of IgG.

Bioconversion of agro-industrial wastes for the production of fibrinolytic enzyme from Bacillus halodurans IND18: Purification and biochemical characterization

Background: Agro-wastes were used for the production of fibrinolytic enzyme in solid-state fermentation. The process parameters were optimized to enhance the production of fibrinolytic enzyme from Bacillus halodurans IND18 by statistical approach. The fibrinolytic enzyme was purified, and the properties were studied. Results: A two-level full factorial design was used to screen the significant factors. The factors such as moisture, pH, and peptone were significantly affected enzyme production and these three factors were selected for further optimization using central composite design. The optimum medium for fibrinolytic enzyme production was wheat bran medium containing 1% peptone and 80% moisture with pH 8.32. Under these optimized conditions, the production of fibrinolytic enzyme was found to be 6851 U/g. The fibrinolytic enzyme was purified by 3.6-fold with 1275 U/mg specific activity. The molecular mass of fibrinolytic enzyme was determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis, and it was observed as 29 kDa. The fibrinolytic enzyme depicted an optimal pH of 9.0 and was stable at a range of pH from 8.0 to 10.0. The optimal temperature was 60°C and was stable up to 50°C. This enzyme activated plasminogen and also degraded the fibrin net of blood clot, which suggested its potential as an effective thrombolytic agent. Conclusions: Wheat bran was found to be an effective substrate for the production of fibrinolytic enzyme. The purified fibrinolytic enzyme degraded fibrin clot. The fibrinolytic enzyme could be useful to make as an effective thrombolytic agent.

Purification and renal effects of phospholipase A(2) isolated from Bothrops insularis venom

Bothrops insularis venom contains a variety of substances presumably responsible for several pharmacological effects. We investigated the biochemical and biological effects of phospholipase A(2) protein isolated from B. insularis venom and the chromatographic profile showed 7 main fractions and the main phospholipase A(2) (PLA(2)) enzymatic activity was detected in fractions IV and V. Fraction IV was submitted to a new chromatographic procedure on ion exchange chromatography, which allowed the elution of 5 main fractions designated as lV-1 to IV-5, from which lV-4 constituted the main fraction. The molecular homogeneity of this fraction was characterized by high-performance liquid chromatography (HPLC) and demonstrated by mass spectrometry (MS), which showed a molecular mass of 13984.20 Da; its N-terminal sequence presented a high amino acid identity (up to 95%) with the PLA(2) of Bothrops jararaca and Bothrops asper. Phospholipase A(2) isolated from B. insularis (Bi PLA(2)) venom (10 mu g/mL) was also studied as to its effect on the renal function of isolated perfused kidneys of Wistar rats (n = 6). Bi PLA(2) increased perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF) and glomerular filtration rate (GFR). Sodium (%TNa+) and chloride tubular reabsorption (%TCl-) decreased at 120 min, without alteration in potassium transport. In conclusion, PLA(2) isolated from B. insularis venom promoted renal alterations in the isolated perfused rat kidney. (c) 2007 Elsevier Ltd. All rights reserved.

Purification and characterization of the biological effects of phospholipase A(2) from sea anemone Bunodosoma caissarum

Sea anemones contain a variety of biologically active substances. Bunodosoma caissarum is a sea anemone from the Cnidaria phylum, found only in Brazilian coastal waters. The aim of the present work was to study the biological effects of PLA(2) isolated from the sea anemone B. caissarum on the isolated perfused kidney, the arteriolar mesenteric bed and on insulin secretion. Specimens of B. caissarum were collected from the Sao Vicente Channel on the southern coast of the State of São Paulo, Brazil. Reverse phase HPLC analysis of the crude extract of B. caissarum detected three PLA(2) proteins (named BcPLA(2)1, BCPLA(2)2 and BcPLA(2)3) found to be active in B. caissarum extracts. MALDI-TOF mass spectrometry of BcPLA(2)1 showed one main peak at 14.7 kDa. The N-terminal amino acid sequence of BcPLA(2)1 showed high amino acid sequence identity with PLA(2) group III protein isolated from the Mexican lizard (PA23 HELSU, HELSU, PA22 HELSU) and with the honey bee Apis mellifera (PLA(2) and 1POC_A). In addition, BcPLA(2)1 also showed significant overall homology to bee PLA(2). The enzymatic activity induced by native BCPLA(2)1 (20 mu g/well) was reduced by chemical treatment with p-bromophenacyl bromide (p-BPB) and with morin. BcPLA(2)1 strongly induced insulin secretion in presence of high glucose concentration. In isolated kidney, the PLA(2) from B. caissarum increased the perfusion pressure, renal vascular resistance, urinary flow, glomerular filtration rate, and sodium, potassium and chloride levels of excretion. BcPLA(2)1, however, did not increase the perfusion pressure on the mesenteric vascular bed. In conclusion, PLA(2), a group III phospholipase isolated from the sea anemone B. caissarum, exerted effects on renal function and induced insulin secretion in conditions of high glucose concentration. (C) 2009 Elsevier Ltd. All rights reserved.