The recovery of proteins using reversed micelles is a liquid-liquid extraction process that has received increasing attention since proteins were shown to be solubilized in organic solvents with surfactants, maintaining their functional properties, and to be transferred between an aqueous solution and a reversed micellar organic phase. This article reviews the application of reversed micellar systems as a bioseparation technique for isolation and purification of proteins. The parameters that affect protein solubilization into the reversed micelles and the equilibrium and kinetics aspects that are involved in the extraction and back-extraction of proteins are discussed. Several examples are also described including the application of this technique for purification of recombinant proteins: cytochrome b 5 and a cutinase from Fusarium solani pisi.

This review analyses the role of cutinases in nature and their potential biotechnological applications. The cloning and expression of a fungal cutinase from Fusarium solani f. pisi, in Escherichia coli and Saccharomyces cerevisiae hosts are described. The three dimensional structure of this cutinase is also analysed and its function as a lipase discussed and compared with other lipases. The biocatalytic applications of cutinase are described taking into account the preparation of different cutinase forms and the media where the different types of enzymatic reactions have been performed, namely hydrolysis, esterification, transesterification and resolution of racemic mixtures. The stability of cutinase preparations is discussed, particularly in anionic reversed micelles considering the role of hexanol as substrate, co-surfactant and stabilizer. Process development based on the operation of cutinase reactors is also reviewed.Cutinases are hydrolytic enzymes that degrade cutin, the cuticular polymer of higher plants, which is a polyester composed of hydroxy and epoxy fatty acids (Purdy and Kolattukudy, 1975). The fatty acids of cutin are usually n-C 16 and n-C 18 and contain one to three hydroxyl groups. Ester bonds predominate in the cutins, although peroxide bridges and ether linkages have also been presented.Cutin plays a key role in protection against the entry of pathogens into plants, and its enzymatic degradation has proved to be one of the first steps in the infection process.

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Fluorescence resonance energy transfer (FRET) is a potential method for the characterization of DNA-cationic lipid complexes (lipoplexes). In this work, we used FRET models assuming a multilamellar lipoplex arrangement. The application of these models allows the determination of the distance between the fluorescent intercalator on the DNA and a membrane dye on the lipid, and/or the evaluation of encapsulation efficiencies of this liposomal vehicle. The experiments were carried out in 1,2-dioleoyl-3-trimethylammonium-propane/pUC19 complexes with different charge ratios. We used 2-(3-(diphenylhexatrienyl)propanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (DPH-PC) and 2-(4,4-difluoro-5-octyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (BODIPY-PC) as membrane dyes, and ethidium bromide (EtBr) and BOBO-1 as DNA intercalators. In cationic complexes (charge ratios (+/-) >or= 2), we verified that BOBO-1 remains bound to DNA, and FRET occurs to the membrane dye. This was also confirmed by anisotropy and lifetime measurements. In complexes with all DNA bound to the lipid (charge ratio (+/-) = 4), we determined 27 A as the distance between the donor and acceptor planes (half the repeat distance for a multilamellar arrangement). In complexes with DNA unbound to the lipids (charge ratio (+/-) = 0.5 and 2), we calculated the encapsulation efficiencies. The presented FRET methodology is, to our knowledge, the first procedure allowing quantification of lipid-DNA contact.

Fusarium solani pisi recombinant cutinase, solubilized in AOT/isooctane-reversed micelles, was used to catalyze the esterification of fatty acids with aliphatic alcohols. Some relevant parameters for the enzyme activity such as pH, W(o) (water/surfactant molar ratio), temperature, and substrate concentration were optimized. Maximal specific activity was obtained for hexanol. The cutinase showed selectivity for short-chain fatty acids. The stability of the microencapsulated cutinase was investigated at various concentrations of water and different values of pH. Oleic acid had a negative effect on the cutinase stability, while hexanol proved to be a strong stabilizer increasing the half-life of the enzyme about 45 times.

graphic steps within established downstream processes would result in a significant decrease of overall production cost [4,5]. The concept of process intensification refers to any process adaptation or optimization that results in a less resource-intensive biomanufacturing scheme, including, but not limited to, lower water consumption, energy demand and environmental burden. This review will primarily focus on multifaceted approaches addressing process integration and intensification based on the utilization of non-traditional chromatographic or extractive methods, which include advances in bioprocess material development, hardware design and advantageous modes of operation. While adsorptive methods, such as chromatography, are widely used in industry as a key purification technology, liquid-liquid extraction technologies, such as aqueous twophase separation (ATPS) systems, are elegant emerging examples of process integration. In this technique, direct extraction of bioproducts from crude feedstocks can be accomplished by the use of two incompatible polymers or of a polymer and a salt in an aqueous environment. It is

Fusarium solani pisi recombinant cutinase, immobilized by entrapment in calcium alginate and by covalent binding on porous silica, was used to catalyze the hydrolysis of tricaprylin. The influence of relevant parameters on the catalytic activity such as pH, temperature, and the substrate concentration were studied. Cutinase immobilized by entrapment presented a Michaelis-Menten kinetics for tricaprylin concentrations up to 200 mM. At higher concentrations of substrate, inhibition was observed. For covalent binding immobilization, diffusional limitations were observed at low substrate concentrations and substrate inhibition occurred for concentrations higher than 150 mM. The stability of immobilized cutinase was also evaluated. The enzyme immobilized by entrapment showed a high stability, in contrast to the immobilization on porous silica.