Mucor miehei lipase was immobilized on magnetic polysiloxane-polyvinyl alcohol particles by covalent binding. The resulting immobilized biocatalyst was recycled by seven assays, with a retained activity around 10% of its initial activity. K m and V max were respectively 228.3 lM and 36.1 U mg of protein )1 for immobilized enzyme. Whereas the optimum temperature remained the same for both soluble and immobilized lipase (45°C), there was a shift in pH profiles after immobilization. Optimum pH for the immobilized lipase was 8.0. Immobilized enzyme showed to be more resistant than soluble lipase when assays were performed out of the optimum temperature or pH.

The influence of four variables, specifically PEG molar mass (400, 1,000, and 8,000 g/mol), concentrations of PEG and phosphate salts (15, 20, and 25% for both), and agitation intensity (110, 150, and 200 rpm), on clavulanic acid (CA) extraction by extractive fermentation with PEG/phosphate salts aqueous two-phase system was investigated in shaken flasks using a 2(4-1) -fractional factorial design. After selection of the two most significant variables (agitation intensity and PEG molar mass), an optimization study conducted according to a 2(2) -central composite design revealed that 25% PEG 8,000 g/mol and phosphate salts at 240 rpm (run 6) were the best conditions for the extractive fermentation, leading to the best results in terms of partition coefficient (k = 8.2), yield of CA in the PEG-rich phase (η(T) = 93%) and productivity (P = 5.3 mg/Lh). As a first attempt to make a scale-up of these results, the effectiveness of the extractive fermentation was then checked in a bench-scale bioreactor under conditions as close as possible to the optimum ones determined in flasks. The highest CA concentration obtained in the PEG-rich phase (691 mg/L) was 30% higher than in flasks, thus demonstrating the potential of such a new process, integrating the production and extraction steps, as a promising, low-cost tool to obtain high yields of this and similar products.

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A continuous perforated rotating disc contactor was used to extract bovine serum albumin (BSA) with aqueous two-phase systems based on polyethylene glycol (PEG) and phosphate salts. The dispersed phase holdup and mass transfer coef®cient were determined. It was found that the dispersed phase holdup increased with increasing PEG phase velocity. The overall mass transfer coef®cient for BSA was independent of the PEG phase velocity.List of symbols C concentration of BSA in phosphate-rich phase (mg/ml) C u concentration of BSA in PEG-rich phase (mg/ml) C ui concentration of BSA in PEG-rich phase at the column inlet (mg/ml) C uo concentration of BSA in PEG-rich phase at the column outlet (mg/ml) H dispersed phase holdup K da mass transfer coef®cient (min )1 ) K partition coef®cient VD dispersed phase¯ow rate (ml/min) IntroductionAqueous two-phase (biphasic) systems have been extensively used for separation and analytical studies of biomaterials since Albertsson [1] developed the technique in the 1950s. Although similar in principle to conventional liquid-liquid extraction, aqueous two-phase extraction has uncommonly wide ranges of physical properties. For instance, the density difference between the two phases varies over the range 10±100 kg/m 3 , the interfacial tension over the range 10 )3 ±10 )2 mN/m, and the viscosities over the range 10±1000 mPa. Conventional liquid-liquid extraction has no property ranges of this magnitude [2]. It has been shown that conventional extraction techniques can be adequately applied to aqueous two-phase systems such as polymer-salt or polymer-polymer systems [1].Aqueous two-phase systems have an important potential in downstream processing such as continuous operation for the large-scale separation of biomolecules. A number of different conventional contactors, e.g. spray columns and packed columns, can be used for aqueous two-phase extraction [3]. Extraction columns with rotatory motion, namely a rotating disc contactor (RDC) and a perforated rotating disc contactor (PRDC), with free areā ow [4] have been used in industry as alternative apparatus to conventional separations equipment. With this type of extractor, high mass transfer rates between the phases were obtained to recover a recombinant cutinase from an aqueous solution to a reversed micellar phase of AOT in isooctane [5].For the design of commercial scale columns, data on the following parameters are needed: protein mass transfer coef®cients, dispersed phase holdup and the extent of mixing in both the phases. Usually, these parameters depend on the velocities of the two phases, their physical properties, the column diameter and the sparger design. In this work it is analyzed bovine serum albumin (BSA) mass transfer data from a laboratory perforated rotating disc contactor (PRDC) using the PEG-phosphates system. The BSA transfer took place from the PEG phase to the salt phase. Speci®cally, the dispersed phase holdup (H) is measured and the mass transfer coef®cients determined as well as the correlations for different disperse...

Cell differentiation/dedifferentiation includes changes in oligosaccharide composition and distribution in the cell surface glycoconjugates. Lectins have been used as auxiliary tools in histopathological diagnosis of mammary, uterus and brain pathologies. Acridinium ester (AE) conjugated to biomolecules has been employed in chemiluminescent analytical applications. This work aimed to use a lectin, concanavalin A (Con A), conjugated to AE as a chemiluminescent histochemistry tool. Biopsies of normal and infiltrating duct carcinoma (IDC) of mammary tissues were treated by a Con A-AE derivative. Photon emission, observed during the breakage of the chemical bound between Con A and AE, was quantified, expressed in relative light units (RLU) and correlated to the labelling of the normal and transformed tissues. The results demonstrated that RLU presented a linear relationship with the labelled tissue area in the range 0.125-1.0 cm2 (r=0.98). Furthermore, RLU was much higher for the IDC (1283.920x103+/-220.621x103) than the normal tissue (2.565x103+/-0.247x103), namely, about 500 times higher. The Con A-AE conjugation efficiency, differential staining of normal and IDC tissues, and quantification of results contribute to a decrease in the subjectivity in routine histopathological diagnoses and indicate that acrydinum ester can join other lectin marker to be used in histochemistry.