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ABSTRACT: A new support for enzyme immobilisation, an alloy of niobium ore and graphite, was tested with: invertase from baker's yeast and inulinase from Aspergillus niger. The efficiency of immobilisation was about 30% for invertase and 72% for inulinase. The maximum activities values were observed for both enzymes at: pH 4.5, 50°C and 500 g/L of sucrose. The hydrolysis of inulin (5% w/v) by the free inulinase and invertase presented specific productivity of reducing sugars lower after immobilisation, about 15 and 1.5 times, respectively; with the hydrolysis of sucrose (50% w/v) the reductions observed were of 14 and 3.5 times, respectively
The Canadian Journal of Chemical Engineering 03/2013; 91:432-440. · 0.75 Impact Factor
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01/2012: pages 573-576; , ISBN: 9789400708839
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ABSTRACT: We have studied the effect of low oxygen levels (2% O<sub>2</sub>), or hypoxia, in the expansion and neural commitment of mouse embryonic stem (ES) cells. When ES cells were maintained in culture with leukemia inhibitory factor (LIF), cell proliferation was reduced at low oxygen levels and a simultaneous reduction in cell viability was also observed. Morphological changes and different cell cycle patterns also occurred, suggesting some early differentiation under hypoxic conditions. However, when cells were maintained in a ground state of pluripotency, by inhibition of autocrine FGF4/ERK and GSK3 signaling, hypoxia did not affect cell proliferation, and did not induce early differentiation. Nevertheless, during neural commitment, low oxygen tension exerted a positive effect on early differentiation of ES cells, resulting in a faster commitment towards neural progenitors. Overall our results demonstrate the need to specifically regulate the oxygen content, especially hypoxia, along with other culture conditions, when developing new strategies for ES cell expansion and/or controlled differentiation.
Bioengineering (ENBENG), 2011. ENBENG 2011. 1st Portuguese Meeting in; 04/2011
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ABSTRACT: Microporation is an efficient method for delivering plasmid DNA molecules into cultured cells. Herein, we present the optimization of gene delivery by microporation using a Central Composite Design methodology. It was given relevance not only to the transfection efficiency but also to the cell recovery. Different amounts of DNA (1 and 3 μg) mainly affected cell viabilities and cell recoveries, which decrease from 93 to 76% and from 47 to 25% respectively, when higher DNA quantity is used. With this work we suggest an easy methodology to improve transfection of mammalian cells underlining the feasibility to achieve 60% of gene delivery efficiencies whilst recovering 50% of cells, with 90% of viability.
Biotechnology Letters 10/2010; 32(10):1393-9. · 1.68 Impact Factor
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ABSTRACT: Microchannel reactor technologies are gaining widespread use in a large range of areas, which comprise biotechnology and chemistry. The small volumes involved and the favorable mass and heat transfer inherent to these devices make them particularly useful for the screening of biocatalysts and rapid characterization of bioconversion systems.In the present work, the enzymatic oxidation of cholesterol to 4-cholesten-3-one performed within microchannels by cholesterol oxidase, was studied in a two-phase system, comprising an organic phase as substrate and product pool and an aqueous phase with dissolved enzyme. A mathematical model based on mass balances for cholesterol, 4-cholesten-3-one and dissolved oxygen concentrations, comprising double-substrate Michaelis–Menten kinetics and the velocity profile of two immiscible fluids, was developed in order to describe and predict the process of cholesterol oxidation. The numerical procedure of solving the non-linear 3D model was based on an implicit finite-difference method improved by non-equidistant differences.In a Y-shape microreactor geometry, roughly up to 70% conversion of cholesterol was achieved at residence times below 1 min. The suitable adjustment of the ratio of the fluid flow rates was performed by taking into account the viscosity of the fluids involved. This allowed for phase separation to be reestablished at the Y-shaped exit from the microreactor and thereby enabled in situ product separation from the aqueous phase containing the enzyme.
Chemical Engineering Journal. 06/2010;
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ABSTRACT: Recent developments in gene therapy have increased the need for the large-scale production of pharmaceutical-grade plasmid DNA. Regulatory agencies require final preparations to be free of host RNA, genomic DNA, proteins and lipopolysaccharides. This paper reports the application of anion-exchange high-performance liquid chromatography (HPLC) to the analysis of process streams during the downstream processing of plasmid DNA for gene therapy.An HPLC purity degree and a purification factor were defined and used to demostrate that process ion-exchange largely reduces the amount of impurities (purification factor of 70.1). However, since plasmid purity after ion-exchange was 59.3% a further gel-filtration step was included in order to reach 100% purity.
Pharmacy and Pharmacology Communications. 02/2010; 5(1):57 - 59.
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ABSTRACT: Mesenchymal stem cells (MSCs) hold a great promise for application in several therapies due to their unique biological characteristics. In order to harness their full potential in cell-or gene-based therapies it might be advantageous to enhance some of their features through gene delivery strategies. Accordingly, we are interested in developing an efficient and safe methodology to genetically engineer human bone marrow MSC (BM MSC), enhancing their therapeutic efficacy in Regenerative Medicine. The plasmid DNA delivery was optimized using a cationic liposome-based reagent. Transfection efficiencies ranged from approximately 2% to approximately 35%, resulting from using a Lipid/DNA ratio of 1.25 with a transgene expression of 7 days. Importantly, the number of plasmid copies in different cell passages was quantified for the first time and approximately 20,000 plasmid copies/cell were obtained independently of cell passage. As transfected MSC have shown high viabilities (>90%) and recoveries (>52%) while maintaining their multipotency, this might be an advantageous transfection strategy when the goal is to express a therapeutic gene in a safe and transient way.
Journal of Biomedicine and Biotechnology 01/2010; 2010:735349. · 2.44 Impact Factor
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ABSTRACT: A commercial inulinase preparation from Aspergillus niger was immobilized into
polyvinyl alcohol hydrogel lenticular particles (Lentikats®
) and into hemispheric-shaped
capsules, both based on the use of LentiKat®
liquid. The characterization of the resulting
biocatalysts, aiming at inulin hydrolysis to fructose, was performed, and the two methods
of immobilization were compared. Temperature and pH profiles, as well as kinetic constants were determined, for both free and immobilized enzyme preparations. A
broader-shaped curve was observed for the pH-activity profile when immobilized forms
were compared to the free form. The apparent KM of inulinase increased roughly 2-fold
upon immobilization in either form of the support particles, suggesting diffusion limitations of inulin inside the gel. Long-term operation with immobilized enzymes proved unfeasible above 55 °C, due to the lack of mechanical stability of the supports tested. When
the temperature of incubation was lowered to 50 °C, the hemispheric form of the immobilized enzyme displayed considerable long-term operational stability, since it allowed
20 repeated, consecutive batch-mode runs, with a final decay in product yield of 20 %.
When inulinase immobilized in Lentikats®
particles was used, the final decay in product
yield was roughly 70 %.
Chemical and Biochemical Engineering Quarterly 12/2009; 23(4):429-434. · 0.69 Impact Factor
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ABSTRACT: A series of quinone-based compounds were tested for their ability to act as external electron acceptors in the 1-dehydrogenation of-α-methyl-hydrocortisone-21-acetate, with polyurethane-entrapped Arthrobacter simplex cells in buffer-saturated n-decan-1-ol. This organic solvent was needed to solubilize the steroid substrate. In aqueous medium, the conversion with free cells virtually stopped after one hour, probably due to substrate limitation. All the tested quinones acted as external electron acceptors, increasing the bioconversion rate. The process kinetics were complex. However, when keeping the concentration of one of the substrates (steroid or quinone) constant and varying that of the other, Michaelis-Menten kinetics provided a reasonably good model for the initial reaction rates, and apparent kinetic constants were estimated. The most effective of the tested external electron acceptors were 2,6-dimethyl-p-benzoquinone and menadione. Mass transfer limitations seemed to appear after some hours of reaction, with low concentrations of the more efficient quinones, when the biocatalyst microenvironment was quinone- and possibly oxygen-depleted. Monosodium glutamate was included with the cells in the immobilisation foam, as an activity-stabilizing agent. It was observed that some of the quinones apparently formed complexes with this glutamate, thereby influencing the kinetics of the process. The catalytic half-life of the system depended on the quinone concentration and optimal values (60-80 h) were observed at 1 mM levels of 2,6-dimethyl-p-benzoquinone or menadione. Quinone toxicity, direct or through the formation of peroxides in the aerobic reoxidation process, may be at the origin of enzyme deactivation.
07/2009; 7(2):83-96.
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ABSTRACT: The influence of buffer molarity and pH, substrate and surfactant concentration on α-chymo-trypsin stability inside reverse micelles was studied. The native and modified enzymes were encapsulated in a reversed micellar system formed by the cationic surfactant tetradecyltri-methylammonium bromide (TTAB) and the organic solvent heptane/octanol (80/20). Enzyme modification was performed at the Lys residues by introducing a dianhydride compound. Enzyme stability was measured during a dipeptide (AcPheLeuNH2) synthesis reaction as the ester substrate consumption (AcPheOEt) and the ratio of peptide synthesis to substrate hydrolysis. An increase in the nucleophile substrate (LeuNH2) concentration as well as in the surfactant, together with a decrease in the buffer parameters led to the higher stability of both forms of encapsulated α-chymotrypsin. By appropriate selection of the 4 parameters, an increase by a factor of 2.4 for the native protein and a factor of 1.8 for the modified form can be obtained when the studies were performed at 30°C.
07/2009; 17(1):3-19.
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ABSTRACT: 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 compared to ester which showed no inhibitory effects at much higher concentrations. Reducing the enzyme concentration resulted in an increase of specific activity. Cutinase showed good stability in CTAB reverse micelles with hexanol as cosurfactant, (half life > 100 days), whereas in the presence of chloroform a rapid loss of much of the activity was observed in the first few hours.
07/2009; 14(2):125-146.
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ABSTRACT: The selective side-chain cleavage of β-sitosterol by free cells of Mycobacterium sp. NRRL B-3805 is a well-established multi-enzymatic process for the production of the pharmaceutical steroid precursors androstenedione (AD) and androstadienedione (ADD). In this study, bis(2-ethylhexyl) phthalate (BEHP) was used as a reaction medium for carrying out the process with freely suspended cells. The work aimed to show that microbial sitosterol side-chain cleavage is possible in this essentially mono-phasic organic medium, provided that some important parameters are adequately controlled. The effects of the biocatalyst/substrate mass ratio, system aeration rate and minimum buffer addition to the organic medium on the product yield and the reaction rate were thus evaluated.
07/2009; 22(3):189-194.
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ABSTRACT: The enzymatic production of ethyl butyrate was studied: the lipase of Candida rugosa (E.C. 3.1.1.3.) was immobilized in a polyurethane matrix and subsequently introduced in an organic medium containing the substrates in appropriate concentrations. The large majority of experiments was carried out in n-hexane. Two further solvents were tested, namely n-heptane and n-dodecane. The partition coefficients matrix/solvent were estimated for the various solvent systems. The initial esterification rate, the molar yield ester/acid and the degree of conversion were found to be solvent independent when the reaction media were designed so that similar concentrations were created in the microenvironment. Initial rate experiments indicated that in n-hexane the threshold of inhibitory substrate concentrations lies (i) between 0.40 M and 0.50 M for butyric acid, according to the purity of the enzyme preparation and (ii) at 0.30 M for ethanol. Batch operational stability tests indicate that no enzyme deactivation occurs after 20 consecutive batches.
07/2009; 5(1):21-34.
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Biotechnology Progress 09/2008; 12(3):290 - 301. · 2.34 Impact Factor
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ABSTRACT: 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.
Applied Spectroscopy 09/2008; 62(8):932-5. · 1.66 Impact Factor
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ABSTRACT: During cationic bed adsorption (EBA), with cutinase with varying length tryptophan tags (WP)(2)and (WP)(4), 33% and 10% of adsorption capacity and 80% and 32% eluted specific activity were observed in relation to wild type (wt)-cutinase in the conventional process. Therefore, as the hydrophobicity of the protein increases, it is important to integrate the EBA step with a hydrophobic interaction chromatography (HIC) process. As the length of the hydrophobic tag-(WP) increases from n = 2 to n = 4, the purification factor obtained by HIC was 1.8 and 2.2-fold higher than wt-cutinase. However, the recovery yield obtained in HIC decreases substantially as the length of hydrophobic tag increases (97%, 84% and 70% for wt-cutinase, cutinase-(WP)(2) and cutinase-(WP)(4)). The integration of two purification steps, EBA followed by HIC, resulted in the highest overall purity level for cutinase-(WP)(2), and the highest overall recovery yield for wt-cutinase. When optimizing the design of a hydrophobic tag fused to a protein secreted by Saccharomyces cerevisiae it must be considered that the cultivation parameters could impair the downstream process, and consequently the optimum tag is not necessarily the one that presents the highest purification factor in HIC.
Biotechnology Letters 09/2008; 30(8):1353-8. · 1.68 Impact Factor
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ABSTRACT: Cutinase is an enzyme suitable for detergent applications as well as for organic synthesis in non-aqueous solvents. However, its inactivation in the presence of anionic surfactants is a problem which we have addressed by creating a complete saturation library. For this, the cutinase gene from Fusarium solani pisi was mutated to incorporate all 19 possible amino acid exchanges at each of the 214 amino acid positions. The resulting library was screened for active variants with improved stability in the presence of the anionic surfactant dioctyl sulfosuccinate sodium salt (AOT). Twenty-four sites in cutinase were discovered where amino acid replacements resulted in a 2-11-fold stability increase as compared to the wild-type enzyme.
Protein Engineering Design and Selection 07/2008; 21(6):387-93. · 2.94 Impact Factor
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ABSTRACT: The activity of various lipases was compared, in both free and immobilized forms, using the kinetics of the hydrolysis reaction of p-nitrophenyl butyrate, which was followed with in situ UV/Vis diode array spectrophotometry. Several enzymes were used to catalyze the reaction, namely Candida antarctica lipase B and Fusarium solani pisi cutinase wildtype and three single-mutation variants. The enzymes were tested in three different forms: free, immobilized as cross-linked aggregates and supported on zeolite NaY. A simple kinetic model was used to allow a quantitative comparison of the behavior of the different catalysts. It was concluded that although immobilization reduces the activity of the enzyme, the zeolite offers a much higher specific activity when compared to the cross-linked aggregates, thus supplying a heterogeneous catalyst with promising catalytic properties.
Bioprocess and Biosystems Engineering 07/2008; 31(4):323-7. · 1.81 Impact Factor
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ABSTRACT: The reactivity, stability and unfolding of wild-type (WT) Fusarium solani pisi cutinase and L153Q, S54D and T179C variants were studied in the absence and presence of the dioctyl sulfosuccinate sodium salt (AOT) surfactant. In the absence of surfactant the S54D variant catalytic activity is similar to that of the WT cutinase, whereas L153Q and T179C variants show a lower activity. AOT addition induces an activity reduction for WT cutinase and its variants, although for low AOT concentrations a small increase of activity was observed for S54D and T179C. The enzyme deactivation in the presence of 0.5 mM AOT is relatively slow for the S54D and T179C variants when compared to wild-type cutinase and L153Q variant. These results were correlated with secondary and tertiary structure changes assessed by the CD spectrum and fluorescence of the single tryptophan and the six tyrosine residues. The WT cutinase and S54D variant have similar secondary and tertiary structures that differ from those of T179C and L153Q variants. L153Q, S54D and T179C mutations prevent the formation of hydrophobic crevices responsible for the unfolding by anionic surfactants, with the consequent decrease of the AOT-cutinase interactions.
Biochimica et Biophysica Acta 06/2008; 1784(9):1326-34. · 4.66 Impact Factor
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ABSTRACT: Silicone rubbers are hydrophobic, a feature that may prove advantageous if this material is to be used as immobilization matrix in bioconversion systems where hydrophobic species are present, such as sterols and mycobacterial cells. Mycobacterium sp. cells with sitosterol side chain cleavage activity were accordingly effectively adsorbed onto silicone and the potential application of the concept was assessed by matching the behavior of the resulting immobilized biocatalyst with free cells and Celite immobilized cells. Mass transfer, kinetics, thermal and storage stability characterization of a biotransformation system based in the use of the silicone immobilized biocatalyst was performed. The feasibility of biocatalyst reutilization was tentatively explored.
Bioresource Technology 06/2008; 99(7):2304-11. · 4.98 Impact Factor
