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Human morphogenesis is a complex process involving distinct microenvironmental and physical signals that are manipulated in space and time to give rise to complex tissues and organs. Advances in pluripotent stem cell (PSC) technology have promoted the in vitro recreation of processes involved in human morphogenesis. The development of organoids from human PSCs represents one reliable source for modeling a large spectrum of human disorders, as well as a promising approach for drug screening and toxicological tests. Based on the “self-organization” capacity of stem cells, different PSC-derived organoids have been created; however, considerable differences between in vitro-generated PSC-derived organoids and their in vivo counterparts have been reported. Advances in the bioengineering field have allowed the manipulation of different components, including cellular and noncellular factors, to better mimic the in vivo microenvironment. In this review, we focus on different examples of bioengineering approaches used to promote the self-organization of stem cells, including assembly, patterning, and morphogenesis in vitro, contributing to tissue-like structure formation.

We developed a three-dimensional (3D) cellular microarray platform for the high-throughput (HT) analysis of human neural stem cell (hNSC) growth and differentiation. The growth of an immortalized hNSC line, ReNcell VM, was evaluated on a miniaturized cell culture chip consisting of 60nl spots of cells encapsulated in alginate, and compared to standard 2D well plate culture conditions. Using a live/dead cell viability assay, we demonstrated that the hNSCs are able to expand on-chip, albeit with lower proliferation rates and viabilities than in conventional 2D culture platforms. Using an in-cell, on-chip immunofluorescence assay, which provides quantitative information on cellular levels of proteins involved in neural fate, we demonstrated that ReNcell VM can preserve its multipotent state during on-chip expansion. Moreover, differentiation of the hNSCs into glial progeny was achieved both off- and on-chip six days after growth factor removal, accompanied by a decrease in the neural progenitor markers. The versatility of the platform was further demonstrated by complementing the cell culture chip with a chamber system that allowed us to screen for differential toxicity of small molecules to hNSCs. Using this approach, we showed differential toxicity when evaluating three neurotoxic compounds and one antiproliferative compound, and the null effect of a non-toxic compound at relevant concentrations. Thus, our 3D high-throughput microarray platform may help predict, in vitro, which compounds pose an increased threat to neural development and should therefore be prioritized for further screening and evaluation.

Controlled pore glass was activated by transition metal salts, FeCl 2 , FeCl 3 , VCl 3 and ZrCl 4 , and derivatized by the method of Cabral et al. 1 Glucoamylase (exo-1,4-α- d -glucosidase, EC 3.2.3.1) was coupled to the aldehyde derivative, the resulting initial activities being 61.7, 71.8, 57.6 and 63.2 U g −1 of matrix, respectively. The immobilized glucoamylase preparations in which the carrier was activated with zirconium (IV) chloride displayed a very stable behaviour, with a half-life of 1709 h, in contrast with the preparations activated with iron(II) chloride ( t 1 2 = 43 7 h , iron(III) chloride ( t 1 2 = 518 h ) and vanadium(III) chloride ( t 1 2 = 893 h ). Several biocatalysts, fungal α-amylase (EC 3.2.1.1), bacterial α-amylase (EC 3.2.1.1), β- d -fructofuranosidase (invertase, EC 3.2.1.26), papain (EC 3.4.22.2) and yeast cells of Saccharomyces cerevisiae with β- d -fructofuranosidase activity were immobilized on aldehyde derivatives of titanium(IV)-activated porous silica (Spherosil), half-lives of 140, 157, 980, 236 and 413 h being obtained, respectively.

Publisher Summary A number of methods of the immobilization of biological molecules exist but no one method is perfect for all molecules or purposes. A method where matrix derivatization, after preparation, can be avoided and instantaneous coupling can be achieved under simple condition is the metal-link chelation process. This method is based on the chelation properties of transition metals—namely, of titanium and zirconium that seem particularly attractive because of the nontoxicity of their oxides. This chapter discusses the way in which transition metal compounds chelate biopolymers from the viewpoint of a simple system of titanium (IV) chloride and cellulose. A proportion of the titanium in the titanium chloride solution is octahedrally coordinated with molecules or ionic species that are essentially the ligands of the complex ion. Chelation may be achieved for many molecule-containing groups appropriate to replace the ligands of the titanium bound to cellulose.

The production of cheese involves two stages:1) the enzymatic hydrolysis of K casein in which its micelle is desestabilized and 2) its subsequent aggregation. In most cases, this operation is carried out by microbial proteases, being used also, animal and plant enzymes.

Plant rennets are used in Portugal for the small-scale manufacture of certain varieties of cheese, namely “Serra” and “Serpa”. In the traditional preparation of these types of cheese the clotting stage is induced by immersing the flowers of the native Compositae Cynara cardunculus or Silybum marianum in the milk vats. However, the flowers are, by themselves, a potential source of microbial contamination of the product. This problem could be overcome by plant cell culture. Previous work demonstrated that in vitro cultivated cells of both species present clotting activity (Fevereiro et al. 1986, Esquivel et al. 1986), no clotting activity being found in the supernatant (Fonseca et al. 1987).

The species Cynara cardunculus is traditionally used in Portugal to produce tasteful and valuable cheese.

Pseudomonas AM1 utilizes glycine and methanol to produce L-serine aerobically. The consumption of methanol in this bioconversion is stoichiometrically in excess of L-serine production. Consequently, the oxygen requirement associated with L-serine production is higher than expected for the conversion from glycine. One method of L-serine production investigated was a technique utilizing a hollow-fiber ultrafiltration cartridge as a bioreactor. Oxygen diffusion limitations appear to impede the consumption of methanol and, consequently, the production of L-serine in such a reactor. Methanol consumption data agree with predictions based on a hollow-fiber diffusion model.

Whole cells of Saccharomyces bayanus, Saccharomyces cerevisiae and Zymomonas mobilis were immobilized by chelation/metal-link processes onto porous inorganic carriers. The immobilized yeast cells displayed much higher sucrose hydrolyzing activities (90–517 U/g) than the bacterial, Z. mobilis, cells (0.76–1.65 U/g). The yeast cells chelated on hydrous metal oxide derivative of pumice stone presented higher initial β-d-fructofuranosidase (invertase, EC 3.2.1.26) activity (161–517 U/g) than on other derivatives (90–201 U/g). The introduction of an organic bridge between the cells and the metal activator led to a decrease of the initial activity of the immobilized cells, however S. cerevisiae cells immobilized on the carbonyl derivative of titanium (IV) activated pumice stone, by covalent linkage, displayed a very stable behaviour, which in continuous operation at 30° C show only a slightly decrease on invertase activity for a two month period (half-life=470 days). The continuous hydrolysis of a 2% w/v sucrose solution at 30° C in an immobilized S. cerevisiae packed bed reactor was described by a simple kinetic model developed by the authors (Cabral et al., 1984a), which can also be used to predict the enzyme activity of the immobilized cells from conversion degree data.

Publisher Summary During recent years, a number of methods to immobilize whole cells have been developed. This chapter presents a methodology of the immobilization of whole cells by metal-link/chelation processes. This method is based on the chelation properties of transition metals—namely, titanium (IV) and zirconium (IV)––that seem particularly attractive because of the nontoxicity of their oxides. From the practical viewpoint, hydrous titanium (IV) and zirconium (IV) oxides proved most satisfactory, as they are insoluble over the normal physiological pH range, and when acting as enzyme immobilization matrices, they give good retention of enzyme activity, they thus seem to have little or no effect on the function of biologically active molecules. The effect of the chelate was not produced by a mere mixture of cellulose and hydrous titanium (IV) oxide. The added success with the chelate was therefore attributable to the altered mode of the presentation of the immobilizing titanium species. Thus, it may be concluded that the addition of hydrous titanium (IV) oxide to a bacterial fermentation will cause aggregation of the bacteria and thus give a higher bacterial concentration in the fermenter.

Publisher Summary This chapter discusses immobilized living cells, their advantages, and their applications in industry. Over the past two decades, there have been rapid developments in the use of enzymes as catalysts for industrial, analytical, and medical purposes and a new field of research called enzyme technology. Current and industrial applications of continuous single-enzyme reactions are carried out using immobilized microbial cells. Immobilized living cells should be preferred to immobilized enzymes for degradative and synthetic reactions in industry; another major advantage of immobilized cells is that the operational stability of the immobilized living cells may often be greatly enhanced by the regeneration of the enzyme activities of the immobilized cells. The use of immobilized cells enables greater control throughout the reaction. The applications of immobilized living cells are not limited to the production of chemical fermentation products but have been extended to the production of viral particles or synchronous cells, the chromatographic separation of special cells, the culturing of animal tissue, and, more recently, the immobilization and use of plant cells in the production of alkaloids.

Genetic modification of human mesenchymal stem cells (MSC) is a powerful tool to improve the therapeutic utility of these cells and to increase the knowledge on their regulation mechanisms. In this context, strong efforts have been made recently to develop efficient nonviral gene delivery systems. Although several studies addressed this question most of them use the end product of a reporter gene instead of the DNA uptake quantification to test the transfection efficiency. In this study, we established a method based on quantitative real-time PCR (RT-PCR) to determine the intracellular plasmid DNA copy number in human MSC after lipofection. The procedure requires neither specific cell lysis nor DNA purification. The influence of cell number on the RT-PCR sensitivity was evaluated. The method showed good reproducibility, high sensitivity, and a wide linear range of 75-2.5 x 10⁶ plasmid DNA copies per cell. RT-PCR results were then compared with the percentage of transfected cells assessed by flow cytometry analysis, which showed that flow cytometry-based results are not always proportional to plasmid cellular uptake determined by RT-PCR. This work contributed for the establishment of a rapid quantitative assay to determine intracellular plasmid DNA in stem cells, which will be extremely beneficial for the optimization of gene delivery strategies.

The profile of batch ethanol production of Jerusalem artichoke tuber mash by a strain of Kluyveromyces fragilis was compared with that of the extracted juice and of the remaining pulp. Alcoholic fermentations were carried out at pH 4.5 without additives. Although K. fragilis is inhibited at added ethanol concentrations above 8% (v/v), the juice allowed the production of 12.8% (v/v) of ethanol in 70 h with the consumption of 95% of initial sugars and an ethanol yield 77% of the theoretical maximum. These high concentrations of ethanol are explained by protein supplementation mechanisms. Mash and pulp, although more difficult to handle than juice, led to the production in 70 h of 11.3 and 8.3% (v/v) of ethanol respectively, with the consumption of 87 and 80% of the initial sugars and ethanol yields of 81 and 84% of the theoretical maximum. Results indicate that the production of ethanol from the mash with and without prior extraction is feasible and may increase the total ethanol obtained by unit mass of tubers.

Description d'un modele base sur les resultats experimentaux de l'hydrolyse de l'amidon soluble et des maltodextrines. Comparaison des performances de l'enzyme dans des reacteurs a lit fixe et a lit fluidise

The use of the uspA and uspB promoters (universal stress promoters) for heterologous protein production in Escherichia coli is described. Best results were obtained with a moderate copy number vector (15-60 copies) bearing the uspA promoter, reaching 4.6 mg/g dry cell weight (DCW) of ZZ-proinsulin secreted to the periplasm and 1.9 mg/g DCW secreted to the culture medium. These values are about 1.7-fold higher than those previously reported with the same ZZ fusion tag and the SpA leader peptide showing that these stress promoters are potentially valuable for recombinant protein secretion in E. coli. It is further demonstrated that the use of M9 minimal medium is advantageous for protein secretion as compared to LB rich medium.

A study of the immobilization of yeast cells with invertase activity by the metal link method was performed. Baker's yeast cells were immobilized on titanium activated porous silica support and on its alkylamine and aldehyde derivatives, their initial activities being 19.6, 39.9 and 10.6 U/ml of reactor respectively. When crosslinking of the immobilized cells was performed, an initial activity of 48.2 U/ml was achieved on the titanium activated support. Batch long-term stability tests were car ried out for 400 hours and the crosslinked preparations showed an unsta ble behaviour compared with the very stable preparations obtained with the simple metal-link method.

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.

The recovery of a recombinant intracellular protein, cytochrome b5, from Escherichia coli TB1 cells was carried out by bead mill disintegration in a discontinuous smallscale instrument. This process was optimized by the use of experimental factorial design. Several parameters were studied: operating time, amount and size of beads, cellular suspension concentration, and presence of toluene and lysozyme. For the experimental conditions used, only the time of treatment and bead load had significant effects. The optimal values of these variables were found by applying the response surface methodology., Junta Nacional de Investigação Científica e Tecnológica (JNICT).

A novel aqueous two-phase system, based on polyethyleneglycol (PEG) and monosodium glutamate, was tested for the 1-dehydrogenation of hydrocortisone-based substrates. This system led to higher substrate solubilities and biocatalyst/steroid separation levels when compared with alternative systems. The addition of short-chain monohydric alcohols resulted in higher solubilities and more favourable partition coefficients for the tested substrates. Bioconversion activities in PEG/glutamate systems with 2,5% (v/v) methanol were comparable to those measured in monophasic buffer-methanol medium.

Penicillin acylase (EC 3.5.1.11) was immobilized by covalent binding on a silica gel carrier activated by silanization. High immobilization yields, operational stability, enzyme loading and support reutilization capacity were obtained. The effects of several variables on the activation of the support and on the immobilization method were studied. Other supports and immobilization methods were assessed for the immobilization of penicillin acylase and compared with the basic process used. Activity versus temperature, pH, buffer molarity and penicillin concentration profiles were determined and compared for the free, crosslinked and covalently-bound silica carrier penicillin acylase preparations. The hydrolysis performance of the latter preparation was assessed in a batch basket reactor and the time course of the hydrolysis reaction modelled by a two parameter model equation. The operational stabilities of penicillin acylase coupled covalently to the silica gel support and immobilized by a crosslinking procedure using glutaraldehyde were also compared.

A simple procedure for the purification of an IgG-type monoclonal antibody by affinity precipitation using Eudragit S-100 is presented. The ligand, a microbial lipase previously used as antigen, was coupled to the polymer at a concentration of 40 mg lipase/g Eudragit. This macroligand was reversibly precipitated by manipulating the pH at values higher and lower than 4.8. The effects of polymer concentration and dilution of hybridoma culture supernatant on the overall precipitation process were evaluated. The best purification factor was achieved with a polymer concentration of 0.1% (w/v) and a supernatant dilution of 1:3. The preliminary studies reported here enabled the purification of a monoclonal antibody in one step with an activity yield (by ELISA) of 50%– 55% and a purification factor of ca 6. Copyright © 1998 John Wiley & Sons, Ltd.

A hollow-fiber supported gas membrane process for the separation of NH{sub 3} from aqueous solutions containing both NH{sub 3} and CO{sub 2} was investigated theoretically and experimentally. A lumen laminar flow and radial diffusion model was applied to calculate the membrane wall transfer coefficient from the data stripping a single volatile component, NH{sub 3} or CO{sub 2}, from their individual aqueous solutions. Influence of the type of membranes and operating conditions on mass-transfer rate were discussed, especially the influence of the membrane transfer coefficient on the film mass-transfer coefficient in the lumen. Appropriate configurations of the hollow-fiber modules for stripping of a single component were analyzed to optimize mass transfer. To predict the stripping of NH{sub 3} from a solution containing NH{sub 3} and CO{sub 2}, a mathematical model incorporating local chemical equilibria and Nernst-Planck diffusion was developed to describe the mass transport. The models described the experimental data fairly well. The experimental results showed that the supported gas membrane process can be used to remove NH{sub 3} effectively from aqueous media containing NH{sub 3} and CO{sub 2}.

The lipolytic activity of Chromobacterium viscosum lipase B (EC 3.1.1.3.; triacylglycerol hydrolase) solubilized both in water and AOT/isooctane reversed micelles has been investigated using triolein as a substrate. The influence of relevant parameters in the catalytic activity such as temperature, pH, surfactant and substrate concentrations, and water content was tested and compared in both media. A study of stability of the lipase was carried out, with particular reference to the influence of pH. Three major effects of the encapsulation of the lipase in the micelles were observed: increased activity (up to 7 times higher than in water), greater stability, specially at pH 7, and higher resistance to thermal deactivation.

The kinetics of the hydrolysis reaction of p-nitrophenyl butyrate catalyzed by a single-site mutant of Fusarium solani pisi cutinase, supported on zeolite NaY by adsorption, was followed using ultraviolet–visible (UV/Vis) in situ diode array spectrophotometry. Spectral band-fitting techniques allowed the use of in situ diode array spectrophotometry to obtain quantitative data on the concentrations of the various species present in an aqueous reaction medium even in the presence of the solid catalyst particles, which interfere with the absorption measurements. This technique overcame the problems arising from the variable light scattering produced by these highly dispersible catalyst particles. Kinetic modeling of the reaction system was used to compute estimates of the reaction rate constants involved.

Reverse micelles formed using the cationic surfactant, hexadecyltrimethylammoniumbromide (CTAB) were applied as a reaction medium for esterification and transesterification (alcoholysis) reactions catalysed by a recombinant lipolytic enzyme, cutinase. Reactions were initially studied with either the alcohol substrate (acyl acceptor) or chloroform as cosurfactant. Chloroform was inhibitory to the enzyme. The enzyme demonstrated specificity towards short chain substrates and these were used to study some relevant parameters affecting specific activity. Optimum values for Wo, buffer concentration, pH, CTAB concentration and temperature were identified. Optimum value for Wo was the same for the two reactions whereas the effects of buffer concentration, pH, CTAB concentration and temperature were different. An optimum level of alcohol was identified, this was the same for both reaction types but the value was dependent on the alcohol. Fatty acids showed inhibitory effects at comparatively low concentrations co...

The enzymatic hydrolysis of olive oil using Chromobacterium viscosum lipase B encapsulated in reversed micelles of dioctyl sodium sulfosuccinate (AOT) in isooctane was investigated in an ultrafiltration ceramic membrane reactor of tubular type, operating in a batch mode. Water concentration was found to be a critical parameter in the enzyme kinetics and hydrolysis yield of the reaction. The size of micelles, recirculation rate, and substrate concentration were found to be the major factors affecting the separation process. A correlation that enables the prediction of final conversion degrees in this bioreactor from the initial reaction conditions was established.

Membrane processes have recently become an accepted unit operation for a wide variety of separations in industry and in environmental applications. Hollow-fiber membrane processes with a constant external resistance having a constant or variable shell concentration resulting from an operational mode of cocurrent or countercurrent are studied. By solving numerically the continuity mass-conservation equation with the corresponding boundary conditions, the lumen laminar mass-transfer coefficients for both cases are correlated. The correlations greatly improve the calculating accuracy of the overall mass-transfer coefficient and can be used to obtain the lumen mixed-cup concentration by an algebraic equation substituting the partial differential equation. A separation factor m{prime} is introduced to characterize the effect of the operational mode. Calculation results demonstrate that the lumen mass-transfer coefficient is independent of the real lumen and shell concentrations, but it is greatly influenced by m{prime}. The countercurrent mode, compared to the cocurrent mode, provides not only a higher mean driving force, but a higher lumen mass-transfer coefficient. This conclusion is novel and valid for the tube-shell heat or mass-transfer processes and is supported by the experimental data in the literature and the authors` gas membrane separation experiments.

The influence of several biocompatible antifoam agents on the performance of ultrafiltration membranes for yeast cell concentration is described. Flux rates of water solutions and cell suspensions decreased in the presence of the antifoam agents. The anti-foam fouling effect was cumulative. Water was ineffective as a cleaning substance, while 0.1 NaOH and ethanol were used successfully for membrane washing.

A mild and reproducible method has been developed for the surface-immobilization of enzymes on glutaraldehyde crosslinked gelatin beads. In this method glutaraldehyde is used in a dual capacity, as crosslinking agent and as the enzyme coupling agent. Glucoamylase (exo-α-1,4- d -glucosidase, EC 3.2.1.3), β- d -fructofuranosidase (invertase, EC 3.2.1.26) and β- d -glucoside (cellobiase, β- d -glucoside glucohydrolase, EC 3.2.1.21) have been successfully immobilized by this method, on the surface of the crosslinked gelatin particles. The method can be combined with the existing technology for the production of gelatin-entrapped enzymes. Thus, dual immobilized enzyme conjugates of glucoamylase and invertase have been prepared using this method, by entrapment of one enzyme in, and surface-binding of the other to, the gelatin matrix. The coupling of glucoamylase onto cross-linked gelatin particles by precipitation with poly(hexamethylenebiguanide hydrochloride) was also tested.

The production of ethanol for potable preparations is well established and the traditional methods used therein are changing. More important, however, has been the advent of fuel alcohol, first in Brazil, but now with ever-increasing possibilities of being adopted in North America and Europe. Production of alcohol by fermentation has therefore come to stay, and it is essential to discover ways of improving the productivities of such fermentations. The effect of organic (cellulose and corn stover) and inorganic matrices (silica gel, porous silica, Celite and derivatised Celite) on the performance of ethanol fermentation by Saccharomyces bayanus was tested. The hydrophilicity degree of the support seems to play an important role in the ability of reaching high ethanol concentrations in the fermentation of 300 g dm−3 of glucose. Cells immobilised on carriers with low hydrophilicity degree, namely Celite, lead to better results (residual glucose: 56 g dm−3; ethanol: 111 g dm−3) than their free counterparts (residual glucose: 136 g dm−3; ethanol: 71 g dm−3). Celite was selected for further cell immobilisation studies, including time of adsorption, whole cell concentration, initial glucose concentration and time of addition of the support to the fermentation medium. The latter has revealed to be a very important factor, improving the performance of ethanol fermentation by reducing the fermentation time. Previous immobilisation seems to have no effect when compared with the simple addition of Celite to the inoculated medium and the eficiency of Celite did not increase when the concentration of glucose was enhanced.

Separation of a commercial preparation of Chromobacterium viscosum by hydrophobic interaction chromatography yields two active fractions, one corresponding to a lipase of 33.0 ± 1.0 kDa by SDS-PAGE and the other to a high molecular weight aggregate (> 250 kDa) of the lipase with some impurities absorbing at 436 nm. Partial disaggregation of this complex occurs on gel filtration chromatography in the presence of 1% (w/v) CHAPS. On gel filtration under non reducing conditions the lipase behaves like a 17 kDa protein; in the presence of a strong denaturant and of a reducing agent a molecular size of 36 kDa is obtained, in accordance with SDS-PAGE results.

Phosphorus-31 nuclear magnetic resonance ((31)P NMR) was used to compare the anaerobic metabolism of glucose by suspended and gel-entrapped Saccharomyces bayanus cells. The fermentation of glucose was carried out in a reaction system with continuous circulation through the NMR sample tube. The intracellular pH and the levels of some phosphorylated compounds were the levels of some phosphorylated compounds were noninvasively monitored by (31)P NMR while glucose, fermentation products, and biomass were determined by analytic techniques comparisons showed that no significant differences are observed in the relative concentrations in the spectra, but distinct profiles for the variation of both intracellular and extracellular pH are found. The internal pH of immobilized cells is maintained at a constant value throughout the fermentation as opposed to freely suspended cells for which a steady decrease in the internal pH occurs. A faster and stronger acidification is also observed in the external medium of the assays with suspended cells. Furthermore, higher yields for ethanol and biomass production and lower yields of fermentation by-products are obtained with immobilized cells. It is concluded that the higher intracellular pH achieved in the presence of the gel matrix had a regulatory effect on the metabolism which favored the ethanol production pathway.

Hollow-fiber membrane separation or reaction processes, with a nonlinear boundary condition at the lumen interface, are analyzed numerically. Sixteen different nonlinear boundaly conditions in the literature were simplified to four dimensionless boundary conditions, in which only two parameters are needed to incorporate the effect on mass transport of membrane and shell resistances, lumen inlet Concentration, shell concentration, phase and/or chemical equilibria, and interface reaction kinetics. The lumen mass-transfer coefjicients were obtained by solving the continuity mass-conservation equation with the corresponding boundary conditions. The calculated results demonstrate that the lumen mass-transfer coefjicient is a function of lumen-inlet concentration and shell concentration, as opposed to the case when a linear boundary condition occurs at the lumen interface. By using the empirical correlations of the lumen local mass-transfer coefjicient, an ordina ry differential equation substituting a partial differential equation can be applied to obtain the lumen dimensionless mixed-cup concentration with an absolute error of less than 0.005 in most cases, thus making it possible to design and evaluate the hollow-fiber membrane separation or reaction processes.

The monomolecular film technique previously used to study the kinetics of lipase hydrolysis was adapted to synthesizing oleoyl glycerides (monoolein, diolein, and triolein). The water subphase was replaced by glycerol, and a film of oleic acid was initially spread on the glycerol surface. In this system a recombinant cutinase from Fusarium solani was able to catalyze oleoyl glyceride synthesis. More than 50% of the oleic acid film was acylated after 7 min of reaction. The surface pressure applied to the monomolecular film acts as a physical selectivity factor since glyceride synthesis can be steered so as to produce either diolein or triolein.

A continuous perforated rotating disc contactor was used for the extraction of a recombinant cutinase from an aqueous solution to a reversed micellar phase of AOT in isooctane. Cutinase was extracted to the organic phase with protein yield of 78% after 70 minutes of operation.