Scale-up of fermentation and purification of recombinant allophycocyanin over-expressed in Escherichia coli

Phycobiliprotein is a photosynthetic antenna pigment found in cyanobacteria, rhodophytes, cryptophytes and certain dinoflagellates, which has been found to have anti-oxidative and anti-tumour activities. In this paper, a recombinant allophycocyanin (rAPC) had been expressed in Escherichia coli for anti-tumour effect. E. coli cells were cultured using glucose fed-batch method to achieve high cell densities. The biomass of rAPC was up to 3.52 g/L broth. The rAPC was purified from soluble E. coli cell lysate employing hydrophobic interaction chromatographic (HIC) method developed at the bench scale using 20 mL column. The process was performed at the pilot scale using 500 mL column for evaluation of scale-up. An amylose affinity column was used to improve the purity of final product in pilot scale purification. The purification process resulted in greater than 98% pure product and yielded up to 2.0 g/kg wet cells at the bench scale and 1.2 g/kg wet cells at the pilot scale. Peptide mapping was used to prove the identity of rAPC purified from bench scale and pilot scale process. Purified rAPC at the pilot scale was found to have remarkable inhibition on S-180 carcinoma in mice. (c) 2005 Elsevier Ltd. All rights reserved.

One step isolation and purification of liquiritigenin and isoliquiritigenin from Glycyrrhiza uralensis Risch using high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) technique in semi-preparative scale has been successfully applied to the separation of bioactive flavonoid compounds, liquiritigenin and isoliquiritigenin in one step from the crude extract of Glycyrrhiza uralensis Risch. The HSCCC was performed using a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-acetonitrile-water (2:2:1:0.6:2, v/v). Yields of liquiritigenin (98.9% purity) and isoliquiritigenin (98.3% purity) obtained were 0.52% and 0.32%. Chemical structures of the purified liquiritigenin and isoliquiritigenin were identified by electrospray ionization-MS (ESI-MS) and NMR analysis. (c) 2005 Published by Elsevier B.V.

Algae-Based Purification of Landfill Biogas Using a CO2 Removal System and Helical Photobioreactor in Series

Biogas is a mixture of methane and other gases. In its crude state, it contains carbon dioxide (CO2) that reduces its energy efficiency and hydrogen sulfide (H2S) that is toxic and highly corrosive. Because chemical methods of removal are expensive and environmentally hazardous, this project investigated an algal-based system to remove CO2 from biogas. An anaerobic digester was used to mimic landfill biogas. Iron oxide and an alkaline spray were used to remove H2S and CO2 respectively. The CO2-laden alkali solution was added to a helical photobioreactor where the algae metabolized the dissolved CO2 to generate algal biomass. Although technical issues prevented testing of the complete system for functionality, cost analysis was completed and showed that the system, in its current state, is not economically feasible. However, modifications may reduce operation costs.

Rational design of a dual-mode optical and chemical prodrug

Purpose. The purpose of this study is to demonstrate the rational design and behaviour of the first dual mode optical and chemical prodrug, exemplified by an acetyl salicylic acid-based system. Methods. A cyclic 1,4-benzodioxinone prodrug was synthesised by reaction of 3,5-dimethoxybenzoin and acetyl salicoyl chloride with pyridine. After purification by column chromatography and recrystallization, characterization was achieved using infrared and NMR spectroscopies, mass spectrometry, elemental analysis and single crystal X-ray diffraction. Light-triggered drug liberation was characterised via UV-visible spectroscopy following low-power 365 nm irradiation for controlled times. Chemical drug liberation was characterised via UV-visible spectroscopy in pH 5.5 solution. Results. The synthetic method yielded pure prodrug, with full supporting characterisation. Light-triggered drug liberation proceeded at a rate of 8.30 10j2 sj1, while chemical, hydrolytic liberation proceeded independently at 1.89 10j3 sj1. The photochemical and hydrolytic reactions were both quantitative. Conclusions. This study demonstrates the first rational dual-mode optical and chemical prodrug, using acetyl salicylic acid as a model, acting as a paradigm for future dual-mode systems. Photochemical drug liberation proceeds 44 times faster than chemical liberation, suggesting potential use in drug-eluting medical devices where an additional burst of drug is required at the onset of infection.

Purification of imidazolium ionic liquids for spectroscopic applications

Ionic liquids are often contaminated by colored impurities. These impurities can be problematic for spectroscopic studies or for monitoring organic reactions by UV/Vis spectroscopy. The effect of different purification methods on the optical quality of colored ionic liquids was studied and compared. Yellowish ionic liquids can partially be decolorized by treatment with active charcoal or by recrystallization. Our experiments show column liquid chromatography is not always a good technique to prepare spectrograde imidazolium halide ionic liquids. Colorless and UV-transparent ionic liquids were synthesized by a method that can exclude the need for further purification steps. (c) 2005 Elsevier B.V. All rights reserved.

EFFECT OF PH ON THE STABILITY OF TIO2 COATINGS ON GLASS PHOTOCATALYSIS REACTORS FOR WATER-PURIFICATION

TiO2 coated glass shows excellent stability in the range pH 2-9, however, there is rapid and complete stripping of the TiO2 coating between pH 11 and 12.

WATER-PURIFICATION BY SEMICONDUCTOR PHOTOCATALYSIS

The basic principles of the photooxidative mineralization of organic pollutants by O2, sensitized by TiO2, are described. The kinetics of this process as a function of [TiO2], [organic pollutant], [O2], light intensity, temperature, pH, and the type of anion present are discussed, and a general kinetic model is presented. Standard test and demonstration systems for water purification by TiO2 photocatalysis are described and other novel applications of semiconductor photocatalysis are outlined.

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.

The use of biodiversity as source of new chemical entities against defined molecular targets for treatment of malaria, tuberculosis, and T-cell mediated diseases: a review

The modern approach to the development of new chemical entities against complex diseases, especially the neglected endemic diseases such as tuberculosis and malaria, is based on the use of defined molecular targets. Among the advantages, this approach allows (i) the search and identification of lead compounds with defined molecular mechanisms against a defined target (e.g. enzymes from defined pathways), (ii) the analysis of a great number of compounds with a favorable cost/benefit ratio, (iii) the development even in the initial stages of compounds with selective toxicity (the fundamental principle of chemotherapy), (iv) the evaluation of plant extracts as well as of pure substances. The current use of such technology, unfortunately, is concentrated in developed countries, especially in the big pharma. This fact contributes in a significant way to hamper the development of innovative new compounds to treat neglected diseases. The large biodiversity within the territory of Brazil puts the country in a strategic position to develop the rational and sustained exploration of new metabolites of therapeutic value. The extension of the country covers a wide range of climates, soil types, and altitudes, providing a unique set of selective pressures for the adaptation of plant life in these scenarios. Chemical diversity is also driven by these forces, in an attempt to best fit the plant communities to the particular abiotic stresses, fauna, and microbes that co-exist with them. Certain areas of vegetation (Amazonian Forest, Atlantic Forest, Araucaria Forest, Cerrado-Brazilian Savanna, and Caatinga) are rich in species and types of environments to be used to search for natural compounds active against tuberculosis, malaria, and chronic-degenerative diseases. The present review describes some strategies to search for natural compounds, whose choice can be based on ethnobotanical and chemotaxonomical studies, and screen for their ability to bind to immobilized drug targets and to inhibit their activities. Molecular cloning, gene knockout, protein expression and purification, N-terminal sequencing, and mass spectrometry are the methods of choice to provide homogeneous drug targets for immobilization by optimized chemical reactions. Plant extract preparations, fractionation of promising plant extracts, propagation protocols and definition of in planta studies to maximize product yield of plant species producing active compounds have to be performed to provide a continuing supply of bioactive materials. Chemical characterization of natural compounds, determination of mode of action by kinetics and other spectroscopic methods (MS, X-ray, NMR), as well as in vitro and in vivo biological assays, chemical derivatization, and structure-activity relationships have to be carried out to provide a thorough knowledge on which to base the search for natural compounds or their derivatives with biological activity.

Functional shikimate dehydrogenase from Mycobacterium tuberculosis H37Rv: Purification and characterization

Tuberculosis (TB) poses a major worldwide public health problem. The increasing prevalence of TB, the emergence of multi-drug-resistant strains of Mycobacterium tuberculosis, the causative agent of TB, and the devastating effect of co-infection with HIV have highlighted the urgent need for the development of new antimycobacterial agents. Analysis of the complete genome sequence of M. tuberculosis shows the presence of genes involved in the aromatic amino acid biosynthetic pathway. Experimental evidence that this pathway is essential for M. tuberculosis has been reported. The genes and pathways that are essential for the growth of the microorganisms make them attractive drug targets since inhibiting their function may kill the bacilli. We have previously cloned and expressed in the soluble form the fourth shikimate pathway enzyme of the M. tuberculosis, the aroE-encoded shikimate dehydrogenase (mtSD). Here, we present the purification of active recombinant aroE-encoded M. tuberculosis shikimate dehydrogenase (mtSD) to homogeneity, N-terminal sequencing, mass spectrometry, assessment of the oligomeric state by gel filtration chromatography, determination of apparent steady-state kinetic parameters for both the forward and reverse directions, apparent equilibrium constant, thermal stability, and energy of activation for the enzyme-catalyzed chemical reaction. These results pave the way for structural and kinetic studies, which should aid in the rational design of mtSD inhibitors to be tested as antimycobacterial agents. (c) 2005 Elsevier B.V. All rights reserved.

PURIFICATION AND PROPERTIES OF SHIKIMATE DEHYDROGENASE FROM CUCUMBER (CUCUMIS-SATIVUS L)

Shikimate dehydrogenase (SDH, EC 1.1.1.25) extracted from cucumber pulp (Cucumis sativus L.) was purified 7-fold by precipitation with ammonium sulfate and elution from columns of Sephadex G-25, DEAE-cellulose, and hydroxyapatite. Two activity bands were detected on polyacrylamide gel electrophoresis at the last purification step. pH optimum was 8.7, and molecular weight of 45 000 was estimated on a Sephadex G-100 column. SDH was inhibited competitively by protocatechuic acid with a K(i) value of 2 x 10-4 M. K(m) values of 6 x 10-5 and 1 x 10-5 M were determined for shikimic acid and NADP+, respectively. The enzyme was completely inhibited by HgCl2 and p-(chloromercuri)benzoate (PCMB). NaCl and KCl showed partial protection against inhibition by PCMB. Heat inactivation between 50 and 55-degrees-C was biphasic, and the enzyme was completely inactivated after 10 min at 60-degrees-C. Incubation of SDH with either NADP+ or shikimic acid protected the enzyme against heat inactivation.

Partial purification and characterization of pectin methylesterase from acerola (Malpighia glabra L.)

The enzyme pectin methylesterase (PME) is present in acerola fruit and was partially purified by gel filtration on Sephadex G-100. The results of gel filtration showed different PME isoforms. The total PME (precipitated by 70% salt saturation) and one of these isoforms (fraction from Sephadex G-100 elution) that showed a molecular mass of 15.5 +/- 1.0 kDa were studied. The optimum pH values of both forms were 9.0. The total and the partially purified PME showed that PME specific activity increases with temperature, the total acerola PME retained 13.5% of its specific activity after 90 min of incubation at 98 degreesC. The partially purified acerola (PME isoform) showed 125.5% of its specific activity after 90 min of incubation at 98 degreesC. The K-m values of the total PME and the partially purified PME isoform were 0.081 and 0.12 mg/mL, respectively. The V-max values of the total PME and the partially purified PME were 2.92 and 6.21 mumol/min/mL/mg of protein, respectively.

Purification and characterization of pectin methylesterase from acerola (Malpighia glabra L.)

The enzyme pectinmethylesterase (PME) from acerola was extracted and purified by gel anion-exchange chromatography (Q Sepharose) and filtration on Sephadex G-100. The results showed two different PME isoforms (PME1 and PME2), with molecular masses of 25.10 and 5.20 kDa, respectively. PMEI specific activity increased by 9.63% after 60 min incubation at 98 degrees C, while PME2 retained 66% of its specific activity under the same conditions. The K-m values of PMEI, PME2 and concentrated PME were 0.94, 0.08 and 0.08mg mL(-1), respectively. The V-max value of PMEI, PME2 and concentrated were 204.08, 2, 158.73 and 2.92 mu mol min(-1) mg(-1) protein, respectively. (c) 2007 Society of Chemical Industry.

Magnetic Purification of Curcumin from Curcuma longa Rhizome by Novel Naked Maghemite Nanoparticles

Naked maghemite nanoparticles, namely, surface active maghemite nanoparticles (SAMNs), characterized by a diameter of about 10 nm, possessing peculiar colloidal stability, surface chemistry, and superparamagnetism, present fundamental requisites for the development of effective magnetic purification processes for biomolecules in complex matrices. Polyphenolic molecules presenting functionalities with different proclivities toward iron chelation were studied as probes for testing SAMN suitability for magnetic purification. Thus, the binding efficiency and reversibility on SAMNs of phenolic compounds of interest in the pharmaceutical and food industries, namely, catechin, tyrosine, hydroxytyrosine, ferulic acid, coumaric acid, rosmarinic acid, naringenin, curcumin, and cyanidin-3-glucoside, were evaluated. Curcumin emerged as an elective compound, suitable for magnetic purification by SAMNs from complex matrices. A combination of curcumin, demethoxycurcumin, and bis-demethoxycurcumin was recovered by a single magnetic purification step from extracts of Curcuma longa rhizomes, with a purity >98% and a purification yield of 45%, curcumin being >80% of the total purified curcuminoids.

Polyphenol oxidase from sweet potato: Purification and properties

Polyphenol oxidase (PPO, EC 1.14.18.1) extracted from sweet potato root [Ipomoea batatas (L.) Lam.] was purified 189-fold by precipitation with ammonium sulfate and elution from columns of Sephadex G-25, DEAE-cellulose, and Sephadex G-100. Polyacrylamide gel electrophoresis of the purified preparation revealed that PPO was highly purified by the procedure adopted. The purified enzyme had an estimated molecular weight of 96 000 and Km values of 26, 8, 5, and 96 mM for 4-methylcatechol, chlorogenic acid, caffeic acid, and catechol, respectively. The optimum pH varies from about 4.0 to 6.5, depending on the substrate. PPO activity was inhibited by p-coumaric and cinnamic acids, sodium metabisulfite, dithioerythritol, ascorbic acid, L-lysine, D-phenylalanine, L-methionine, glycine, L-isoleucine, and L-glutamine. Heat inactivation between 60 and 80 °C was biphasic. Sucrose, (NH4)2SO4, NaCl, and KCl appeared to be protective agents of sweet potato PPO against thermal denaturation. © 1992 American Chemical Society.