PubMed Health⌕ Search

Biomedical subjects

Roberto Fernandez-Lafuente

Publications and source records attributed to Roberto Fernandez-Lafuente.

At least 19 recordsLinked to original sources
PubMed2007

Improved catalytic properties of immobilized lipases by the presence of very low concentrations of detergents in the reaction medium.

The addition of a very small concentration of a detergent (in many instances under the critical micellar concentration (cmc)) has been found to greatly increase the activity of immobilized lipases, using those from Pseudomonas fluorescens (PFL) and Candida antarctica (isoform B) as model enzymes. However, the detergents may also have a negative effect on enzyme activity; in fact, for all enzyme preparations and substrates the activity/detergent concentration curve reached a maximum value and started to decrease, in many instances even under the initial value. The concentration and nature of the detergent (SDS, CTAB, Triton X-100, or X-45) that permitted the maximum hyperactivation was different depending on the substrate. The best hyperactivation values promoted by the presence of detergent were over a 20-fold factor. The presence of detergents permitted the inhibition of lipases by irreversible covalent inhibitors (e.g., 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride) (AEBSF) while the enzyme, in the absence of detergent, is not inhibited by these irreversible inhibitors. This suggested that the main effect of the detergents is to shift the conformational equilibrium of lipases toward the open form. Moreover, the presence of detergents also permitted to improve the enantioselectivity exhibited by the immobilized lipases in some cases. For example, the enantioselectivity of PFL-glyoxyl agarose increased from 40 to more than 100 in the hydrolysis of (+/-)-2-hydroxy-4-phenylbutyric acid ethyl ester by using 0.1% CTAB.

Biocompatible Materials↗
PubMed2006

Solid phase proteomics: dramatic reinforcement of very weak protein-protein interactions.

Very weak protein-protein interactions may play a critical role in cell physiology but they are not easily detectable in "in vitro" experiments. To detect these weak interactions, we have developed a strategy that included: (a) design of a rapid and very effective crosslinking of protein-protein complexes with poly-functional reagents; (b) selective adsorption of very large proteins on lowly activated ionic exchangers, based on the need of a multipoint physical adsorption to incorporate the proteins into the matrix; (c) purification by selective adsorption of protein-protein complexes formed by strong protein-protein interactions, via selective adsorption of the complexes on lowly activated ionic exchangers via multi-protein physical adsorption and leaving the non-associated proteins in the solution; (d) reinforcement of very weak protein-protein interactions by selective adsorption of the complex on lowly activated ionic exchange supports via a synergetic cooperation of the weak protein-protein interaction plus the interactions of both proteins with the support enabling the almost full shifting of the equilibrium towards the association position; (e) control of the aggregation state of proteins like BSA, formed by weak protein-protein interactions. In this last case, it seems that the interaction of the protein molecules placed on the borders of the aggregate with the groups on the support partially stabilizes the whole aggregate, although, some molecules of the aggregate cannot interact with the support. The size of the aggregates may be defined by controlling the concentration of ionised groups on the support: the less activated the supports are, the bigger the complexes. In this way, solid-phase proteomics could be a very interesting tool to detect weak protein-protein interactions.

Adsorption↗
PubMed2006

Stabilization of a formate dehydrogenase by covalent immobilization on highly activated glyoxyl-agarose supports.

Formate dehydrogenase (FDH) is a stable enzyme that may be readily inactivated by the interaction with hydrophobic interfaces (e.g., due to strong stirring). This may be avoided by immobilizing the enzyme on a porous support by any technique. Thus, even if the enzyme is going to be used in an ultra-membrane reactor, the immobilization presents some advantages. Immobilization on supports activated with bromocianogen, polyethylenimine, glutaraldehyde, etc., did not promote any stabilization of the enzyme under thermal inactivation. However, the immobilization of FDH on highly activated glyoxyl agarose has permitted increasing the enzyme stability against any distorting agent: pH, T, organic solvent, etc. The time of support-enzyme reaction, the temperature of immobilization, and the activation of the support need to be optimized to get the optimal stability-activity properties. Optimized biocatalyst retained 50% of the offered activity and became 50 times more stable at high temperature and neutral pH. Moreover, the quaternary structure of this dimeric enzyme becomes stabilized by immobilization under optimized conditions. Thus, at acidic pH (conditions where the subunit dissociation is the first step in the enzyme inactivation), the immobilization of both subunits of the enzyme on glyoxyl-agarose has allowed the enzyme to be stabilized by hundreds of times. Moreover, the optimal temperature of the enzyme has been increased (even by 10 degrees C at pH 4.5). Very interestingly, the activity with NAD(+)-dextran was around 60% of that observed with free cofactor.

Biocompatible Materials↗
PubMed2006

Adsorption behavior of bovine serum albumin on lowly activated anionic exchangers suggests a new strategy for solid-phase proteomics.

Diluted solutions of bovine serum albumin (BSA) (e.g., 0.1 mg /mL) do not form detectable protein large aggregates. Using gel-filtration experiments, we determined that a diluted solution of BSA is 97% monomeric BSA and 3% dimeric. The adsorption of this diluted BSA on highly activated anionic exchangers (e,g., having 40 micromol/wet g) keeps this mainly monomeric form. When supports activated with 2 micromol/wet g are used, only dimers become adsorbed to the support, accounting for 100% of the offered BSA. When the diluted BSA solution is offered to very mildly activated anionic exchangers (even only 0.125 micromol/wet g), an unexpected adsorption of most of the BSA on the support was also observed. These very slightly activated supports are only able to adsorb very large proteins or very large protein-protein complexes, larger than BSA dimers. In fact, a rapid cross-linking of the adsorbed BSA with dextran-aldehyde reveals the formation of very large BSA-BSA complexes with molecular mass higher than 500 000 Da, complexes that may be observed for soluble BSA with very high concentrations but are not detectable at 0.1 mg/mL. Moreover, the size of the aggregates strongly depends on the concentration of the ionized groups on the support: the less activated the supports are, the higher the sizes of the complexes. It seems that the interaction of the BSA molecules on the margins of the BSA aggregate with the groups on the support may stabilize the whole protein aggregate, although some components are not interacting with the support. Aggregates could account for more than 40% of the BSA in the solution after 50 h of incubation. However, only these large BSA aggregates were adsorbed in the support.

Adsorption↗
PubMed2006

Glutaraldehyde cross-linking of lipases adsorbed on aminated supports in the presence of detergents leads to improved performance.

Lipases from Candida rugosa (CRL) and lipase isoforms A and B from Candida antarctica (CAL-A and CAL-B) were adsorbed on aminated supports in the presence of detergents to have individual lipase molecules. Then, one fraction was washed to eliminate the detergent, and both preparations were treated with glutaraldehyde. The presence of detergent during the cross-linking of the lipases to the support permitted an increase in the recovered activity (in some instances, even by a 10-fold factor). This activity was higher even than that exhibited by the just adsorbed lipases, suggesting that it was not a result of some protective effect of the detergent in the enzyme activity during glutaraldehyde chemical modification. Moreover, the enantioselectivity of the different enzyme preparations was very different if the glutaraldehyde was offered in the presence or in the absence of detergent, in some cases increasing the E value (even by a 7-fold factor in the case of CAL-A in the hydrolysis of (+/-)-2-hydroxy-4-phenylbutyric acid ethyl ester), in other cases even inverting the enantio preference (e.g., in the case of CRL). The irreversible chemical inhibition of the enzyme that was immobilized and cross-linked with glutaraldehyde in the presence of detergents was more rapid than that in the other preparations (by more than a 10-fold factor). This experiment reveals an exposition degree of the active serine in the preparation cross-linked with the support in the presence of detergent that is higher than that in the other preparations. The results suggested that different enzyme structures were "stabilized" by the glutaraldehyde treatment if performed in the presence or in the absence of detergent, and that, in the presence of detergent, a form of the lipase with the serine residue more exposed to the medium and much more active could be obtained. This strategy seems to be of general use to improve the lipase activity to be used in macroaqueous media.

Adsorption↗
PubMed2006

Chemical modification of protein surfaces to improve their reversible enzyme immobilization on ionic exchangers.

The enzyme penicillin G acylase (PGA) is not adsorbed at pH 7 on DEAE- or PEI-coated supports, neither is it adsorbed on carboxymethyl (CM)- or dextran sulfate (DS)-coated supports. The surface of the enzyme was chemically modified under controlled conditions: chemical amination of the protein surface of carboxylic groups (using soluble carbodiimide and ethylendiamine) and chemical succinylation (using succinic anhydride) of amino groups. The full chemical modification produced some negative effects on enzyme stability and activity, although partial modification (mainly succinylation) presented negligible effects on both enzyme features. The chemical amination of the protein surface permitted the immobilization of the enzyme on CM- and DS-coated support, while the chemical succinylation permitted the enzyme immobilization on DEAE- and PEI-coated supports. Immobilization was very strong on these supports, mainly in the polymeric ones, and dependent on the degree of modification, although the enzymes still can be desorbed after inactivation by incubation under drastic conditions. Moreover, the immobilization on ionic polymeric beds allowed a significant increase in enzyme stability against the inactivation and inhibitory effects of organic solvents, very likely by the promotion of a certain partition of the organic solvent out of the enzyme environment. These results suggest that the enrichment of the surface of proteins with ionic groups may be a good strategy to take advantage of the immobilization of industrial enzymes via ionic exchange on ionic polymeric beds.

Adsorption↗
PubMed2005

Purification, stabilization, and concentration of very weak protein-protein complexes: Shifting the association equilibrium via complex selective adsorption on lowly activated supports.

Very weak protein-protein interactions are very difficult to detect because these complexes could be under the detection limit or they tend to dissociate. Here, using as a model the antibody-antigen interaction weaken by the presence of dioxane, we have shown a strategy for the protein complexes purification by selective adsorption of the associated proteins. This strategy is based on the use of poorly activated anionic exchanger supports to selectively adsorb large complexes. This selective adsorption of the associated proteins shifted the association equilibrium of the soluble proteins toward the associated form. Thus, in the presence of 15% v/v dioxane, a concentration that is able to almost fully break the immunocomplex (less that 3% of the immunocomplex appeared associated when soluble antigen-antibody mixture was cross-linked with aldehyde-dextran), we can obtain more than 90% of the fully pure immunocomplex from the non-associated protein, adsorbed on anionic exchanger supports having a very low activation. This simple strategy may be a very useful tool to solve one of the most relevant challenges in the modern proteomics, the detection of very weak protein-protein interactions.

Adsorption↗
PubMed2004

Detection and purification of two antibody-antigen complexes via selective adsorption on lowly activated anion exchangers.

Taken advantage of the mechanism of adsorption of macro-molecules on ionic exchangers, (a multipoint interaction between the protein and the support), it is possible to selectively adsorb large proteins leaving small ones in the supernatant. Associated proteins should present a significant difference in its size as compared to the non-associated forms. Thus, the protein complexes may have much larger surfaces to interact with the support. Here, by selecting the support with the highest activation degree that was unable to adsorb the non-associated proteins, we have shown the simple and selective adsorption of immuno complexes (as a model), while antibodies and antigens remained in the supernatant. Therefore, it was possible to selectively adsorb on lowly activated supports (e.g., agarose 4BCL having only 1 micromol of amino groups per g of support) rabbit IgG/anti-rabbit immunoglobulins (immuno complex), while these supports were unable to adsorb the individual immunoglobulines. Similarly, horseradish peroxidase (HRP)/anti-HRP were selectively adsorbed on lowly activated supports, while the individual proteins were not adsorbed at all. Afterwards, the adsorbed associated proteins (purified at least from the non-associated counterparts and concentrated by the adsorption on the support) may be cross-linked with aldehyde-dextran and be desorbed from the matrix for their analysis. This strategy may permit very simple experiments to detect the presence of protein-protein complexes. Finally, we have shown the advantages of this technique compared to the use of one of the proteins previous immobilized on a support.

Adsorption↗
PubMed2004

Selective and mild adsorption of large proteins on lowly activated immobilized metal ion affinity chromatography matrices. Purification of multimeric thermophilic enzymes overexpressed in Escherichia coli.

A strategy to selectively adsorb large proteins on immobilized metal ion affinity chromatography supports is presented. It is based on the fact that large proteins have a large surface that permits the long distance interaction with groups placed quite far apart (very dispersed onto the support surface) in the support, therefore, even using lowly activated supports, these proteins may be able to yield multiple interactions with the support, which is not possible for smaller proteins. This has been shown using a crude extract from Escherichia coli, where only large proteins were adsorbed on supports having 0.25 micromol of metallic groups/g of support. Then, these lowly activated supports have been used for purifying multimeric enzymes from thermophilic organisms (alpha- and beta-galactosidases from Thermus sp. strain T2) cloned and over-expressed in mesophilic ones. A previous heating step of the crude extract destroyed the quaternary structure of all multimeric enzymes from the host (E. coli). Thus, the only large protein remaining in the supernatant of this heated extract are the cloned multimeric thermophilic enzymes, permitting their very simple purification by using only one chromatographic step.

Adsorption↗
PubMed2004

Use of immobilized lipases for lipase purification via specific lipase-lipase interactions.

Lipase from Pseudomonas fluorescens (PFL), an enzyme with a great tendency to yield bimolecular aggregates, was immobilized via multipoint covalent attachment on glyoxyl-agarose in the presence of Triton X-100. This strategy permitted to obtain the enzyme with the active center oriented towards the reaction medium. This immobilized enzyme presents the capacity of specifically adsorbing PFL molecules, that can be easily desorbed by the use of detergents. More interesting, the enzyme was also able to adsorb other lipases. That is, the lipase from Bacillus thermocatenulatus (BTL2) cloned in Escherichia coli was selectively adsorbed on this immobilized enzyme, enabling a very simple purification strategy. Similar results were achieved with some other lipases (those from Rhizomucor miehei (RML), Rhizopus oryzae (ROL), and Humicola Lanuginosa (HLL)). In all cases, the enzyme could be easily desorbed by incubation with Triton X-100. The matrix could be used several cycles without any detrimental effect on the adsorption capacity.

Electrophoresis, Polyacrylamide Gel↗
PubMed2004

Ion exchange using poorly activated supports, an easy way for purification of large proteins.

Ion-exchange chromatography using commercial ionic supports is a commonly used technique for protein purification. However, selective adsorption of a target protein from a given extract onto commercial ion exchangers seems to be quite complex since they are designed to adsorb the maximum percentage of proteins with the opposite charge. In this paper, ion-exchanger supports with different activation degrees (from 1 to 40 micromol of amino groups per g of agarose) have been prepared and used for the purification of large proteins. These kinds of proteins have large surfaces to interact by many points with the support. Therefore, it was possible to purify large proteins as beta-galactosidase from Thermus sp. strain T2 from a crude extract from Escherichia coli or bovine liver catalase from a commercial preparation, with tailor-made ion-exchanger supports. A simple step of adsorption/desorption on lowly activated supports rendered both enzymes rather pure as confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Moreover, this strategy makes also easy the desorption step that requires rather low NaCl concentrations, which may become a serious problem for desorption of large proteins when using conventional supports, due to their ability of generating a very strong adsorption.

Chromatography, Gel↗
PubMed2004

Determination of protein-protein interactions through aldehyde-dextran intermolecular cross-linking.

A very simple strategy, based on the intermolecular cross-linking of associated proteins by using aldehyde-dextrans, has been proposed to detect protein-protein interactions. Aldehyde-dextran was able to cross-link different enzymes composed of several polypeptide chains (e.g., trypsin and penicillin G acylase), proteolyzated proteins (e.g., extracts from porcine pancreas) and finally, an immunocomplex (horseradish peroxidase/anti-horseradish peroxidase). This cross-linked immunocomplex could be selectively adsorbed on immobilized anti-rabbit IgG. The presence of unspecific covalent attachment between unrelated protein molecules was not detected. Thus, this strategy permits the cross-linking of different protein components and avoids the formation of nonspecific protein-protein associations.

Aldehydes↗
PubMed2003

Enzymatic transformations. Immobilized A. niger epoxide hydrolase as a novel biocatalytic tool for repeated-batch hydrolytic kinetic resolution of epoxides.

Studies aimed at immobilization of the Aspergillus niger epoxide hydrolase were performed. The use of conventional approaches, i.e. of commercially available supports and classical methodologies, only led to low stabilisation and unsatisfactory enzymatic activity recovery. Therefore, a new strategy based on the use of a "second generation" type of epoxy-activated supports allowing multi-point covalent immobilization, i.e. Eupergit C, partially modified with ethylene diamine (Eupergit C/EDA), and of an adequate experimental procedure was set up. This allowed us to prepare an immobilized biocatalyst with 70%, retention of the initial enzymatic activity and a stabilisation factor of about 30. Interestingly, this biocatalyst also led to a noticeable increase of the E value for the resolution of two test substrates, styrene oxide 1 and p-chlorostyrene oxide 2. This was improved from about 25 to 56 and from 40 to 100, respectively. A typical repeated batch experiment indicated that the thus immobilized enzyme could be re-used for over 12 cycles without any noticeable loss of enzymatic activity or change in enantioselectivity. This therefore opens the way for the use of an 'heterogeneous catalysis' methodology for achieving the preparation of various enantiopure epoxides via biocatalysed hydrolytic kinetic resolution.

Aspergillus niger↗
PubMed2003

Inhibitory effects in the side reactions occurring during the enzymic synthesis of amoxicillin: p-hydroxyphenylglycine methyl ester and amoxicillin hydrolysis.

Penicillin G acylase immobilized on glyoxyl-agarose is used to catalyse the reaction between p -hydroxyphenylglycine methyl ester (POHPGME) and 6-aminopenicillanic acid (6-APA). Inhibitory effects affecting the side reactions that occur during the synthesis of amoxicillin have been reported and need to be considered when proposing a kinetic model for the enzymic synthesis. In this work, we present a semi-empirical kinetic model that successively includes different inhibitory effects in the rate equations. The model performance was always compared with experimental data on amoxicillin synthesis. Enzyme load and stirring rate were chosen to prevent diffusional effects. Our results indicate that POHPGME and amoxicillin were competitive inhibitors of the hydrolysis of amoxicillin and POHPGME, respectively. 6-APA was a competitive inhibitor of the hydrolysis of amoxicillin. POHPG was a competitive inhibitor and methanol a non-competitive inhibitor of the hydrolysis of both ester and antibiotic, but the action of methanol was only noticeable at very high concentrations. Adding inhibitory effects to the kinetic model led to a significant increase in the accuracy of the simulations of the overall process of synthesis.

Amoxicillin↗
PubMed2002

Enzymatic synthesis of amoxicillin: avoiding limitations of the mechanistic approach for reaction kinetics.

A recurrent doubt that occurs to the enzyme-kinetics modeler is, When should I stop adding parameters to my mechanistic model in order to fit a non-conventional behavior? This problem becomes more and more involving when the complexity of the reaction network increases. This work intends to show how the use of artificial neural networks may circumvent the need of including an overwhelming number of parameters in the rate equations obtained through the classical, mechanistic approach. We focus on the synthesis of amoxicillin by the reaction of p-OH-phenylglycine methyl ester and 6-aminopenicillanic acid, catalyzed by penicillin G acylase immobilized on glyoxyl-agarose, at 25 degrees C and pH 6.5. The reaction was carried on a batch reactor. Three kinetic models of this system were compared: a mechanistic, a semi-empiric, and a hybrid-neural network (NN). A semi-empiric, simplified model with a reasonable number of parameters was initially built-up. It was able to portray many typical process conditions. However, it either underestimated or overestimated the rate of synthesis of amoxicillin when substrates' concentrations were low. A more complex, full-scale mechanistic model that could span all operational conditions was intractable for all practical purposes. Finally, a hybrid model, that coupled artificial neural networks (NN) to mass-balance equations was established, that succeeded in representing all situations of interest. Particularly, the NN could predict with accuracy reaction rates for conditions where the semi-empiric model failed, namely, at low substrate concentrations, a situation that would occur, for instance, at the end of a fed-batch industrial process.

Amoxicillin↗
PubMed2002

Regioselective enzymatic hydrolysis of acetylated pyranoses and pyranosides using immobilised lipases. An easy chemoenzymatic synthesis of alpha- and beta-D-glucopyranose acetates bearing a free secondary C-4 hydroxyl group.

Protected sugars with only one free hydroxyl group are useful building blocks for the synthesis of a large number of glycoderivatives. In order to avoid the problems of the classical chemical synthesis, we studied the regioselective enzymatic hydrolysis of different fully acetylated glycopyranoses and glycopyranosides. The main challenge was to obtain the hydrolysis of the substrates in only one position, with high regioselectivity, while avoiding any further hydrolysis towards partially acetylated sugars. Candida rugosa (CRL) and Pseudomonas fluorescens (PFL) lipases (EC 3.1.1.3) immobilised on octyl agarose afforded regioselective hydrolysis only in the 6- and 1-positions, respectively. Furthermore, a new one-pot chemoenzymatic approach has been developed in order to obtain alpha- and beta-protected glucopyranoses bearing a free secondary C-4 hydroxyl group. For instance, 1,2,3,6-tetra-O-acetyl-alpha-D-glucopyranose was easily synthesised in good overall yield (70%) starting from 1,2,3,4,6-penta-O-acetyl-alpha-D-glucopyranose by regioselective enzymatic hydrolysis in the 6-position, catalysed by CRL, followed by a temperature- and pH-controlled acyl migration.

Acetates↗
PubMedDate not supplied

Immobilization of rennet from Mucor miehei via its sugar chain. Its use in milk coagulation.

A successful strategy for the immobilization of rennet from Mucor miehei has been developed. The strategy is based on the immobilization of the enzyme, via their sugar chains at high ionic strength on aminated supports having primary amino groups with a very low pK value. The rennet was covalently immobilized via sugar chains (previously oxidized with periodate), which act as natural spacer arms and allow a very high percentage of rennet activity to be kept against small (H-Leu-Ser-p-nitro-Phe-Nle-Ala-Leu-OMe.TFA (98%)) and macromolecular substrates (k-casein) (78%). The use of tailor-made aminated support was critical to obtain good stability values, because using fully aminated supports achieved much lower thermostability values than using 50% aminated supports. The optimized derivative was utilized to hydrolyze casein in milk. To prevent the coagulation of the milk in the presence of the derivative, the reaction was performed at 4 degrees C (where hydrolyzed casein did not precipitate). Then the hydrolyzed milk was filtered and latter on heated to 30 degrees C, achieving a similar aggregate to the one achieved with soluble rennet.

Animals↗