Biomedical subjects
Publications and source records attributed to Lorena Betancor.
The coimmobilization of nitrobenzene nitroreductase and glucose-6-phosphate dehydrogenase in silica particles enables the continuous conversion of nitrobenzene to hydroxylaminobenzene with NADPH recycling.
The nitroreductase-catalyzed conversion of a strong electron-withdrawing nitro group to the corresponding electron-donating hydroxylamine is useful in a variety of biotechnological applications. Activation of prodrugs for cancer treatments or antibiotic therapy are the most common applications. Here, we show that a bacterial nitrobenzene nitroreductase (NbzA) from Pseudomonas pseudoalcaligenes JS45 activates the dinitrobenzamide cancer prodrug CB1954 and the proantibiotic nitrofurazone. NbzA was purified by affinity chromatography and screened for substrate specificity with respect to prodrug activation. To facilitate screening of alternate potential prodrugs, polyethyleneimine-mediated silica formation was used to immobilize NbzA with high immobilization yields and high loading capacities. Greater than 80% of the NbzA was immobilized, and enzyme activity was significantly more stable than NbzA in solution. The resulting silica-encapsulated NbzA was packed into a microfluidic microreactor that proved suitable for continuous operation using nitrobenzene, CB1954, and the proantibiotic nitrofurazone. The flow-through system provides a rapid and reproducible screening method for determining the NbzA-catalyzed activation of prodrugs and proantibiotics.
The stabilization achieved by different immobilization protocols have been compared using three different enzymes (glutaryl acylase (GAC), D-aminoacid oxidase (DAAO), and glucose oxidase (GOX)): adsorption on aminated supports, treatment of this adsorbed enzymes with glutaraldehyde, and immobilization on glutaraldehyde pre-activated supports. In all cases, the treatment of adsorbed enzymes on amino-supports with glutaraldehyde yielded the higher stabilizations: in the case of GOX, a stabilization over 400-fold was achieved. After this treatment, the enzymes could no longer be desorbed from the supports using high ionic strength (suggesting the support-protein reaction). Modification of the enzymes immobilized on supports that did not offer the possibility of react with glutaraldehyde showed the same stability that the non modified preparations demonstrating that the mere chemical modification did not have effect on the enzyme stability. This simple strategy seems to permit very good results in terms of immobilization rate and stability, offering some advantages when compared to the immobilization on glutaraldehyde pre-activated supports.
Glucose oxidase (GOX) has been immobilized on different activated supports, including glyoxyl agarose, epoxy sepabeads and glutaraldehyde-activated supports. Immobilization onto supports pre-activated with glutaraldehyde rendered the most thermo-stable preparation of GOX. Therefore, as the glutaraldehyde chemistry gave a high stabilization of the enzyme, we proposed another technique for improving the multipoint attachment through glutaraldehyde: the enzyme was ionically adsorbed on cationic supports with primary amino groups and then the immobilized preparation was treated with a glutaraldehyde solution. The decrease on enzyme activity was <20%. Following this methodology, we achieved the highest stability of all the immobilization systems analyzed, showing a half-life 100 times higher than the soluble enzyme. Moreover, this derivative showed a higher stability in the presence of organic solvents (for instance methanol) or hydrogen epoxide than the ionically adsorbed enzyme or the soluble one. Therefore, the adsorption of GOX on aminated cationic support and subsequent treatment with glutaraldehyde was presented as a very successful methodology for achieving a very stable biocatalyst.
The surface carboxylic groups of penicillin G acylase and glutaryl acylase were chemically aminated in a controlled way by reaction with ethylenediamine via the 1-ethyl-3-(dimethylamino-propyl) carbodiimide coupling method. Then, both proteins were immobilized on glyoxyl agarose. In both cases, the immobilization of the chemically modified enzymes improved the enzyme stability compared to the stability of the immobilized but non-modified enzyme (by a four-fold factor in the case of PGA and a 20-fold factor in the case of GA). The chemical modification presented a deleterious effect on soluble enzyme stability. Therefore, the improved stability should be related to a higher multipoint covalent attachment, involving both the lysine amino groups and also the new amino groups chemically introduced on the enzyme. Moreover, the lower pK(a) of the new amino groups permitted to immobilize the enzyme under milder conditions. In fact, the aminated proteins could be immobilized even at pH 9, while the non-modified enzymes could only be immobilized at pH over 10.
Glutaryl-7-aminocephalosporanic acid acylase (GA), an industrially relevant enzyme, has been immobilized onto very different supports, including glyoxyl agarose, heterofunctional epoxy Sepabeads, glutaraldehyde and cyanogen bromide (CNBr) activated supports. Immobilization onto amino-epoxy Sepabeads rendered the most thermo stable preparation of GA, with a half-life time eight times higher than the soluble enzyme, keeping 80% of the enzyme activity. Several parameters that affect the enzyme-support interaction (pH and incubation time) were studied. It was found that after immobilization onto amino-epoxy Sepabeads, incubation at alkaline pH and low temperature exerted dramatic stabilizing effects, increasing the half-life time of the derivative 130 times with respect to the soluble enzyme, while keeping unaltered its intrinsic activity. The loading capacity of the amino-epoxy Sepabeads proved to be very good with a maximum load of 62 mg of protein per g of support with 85 IU/g at 25 degrees C and 200 IU/g at 37 degrees C which makes it a biocatalyst of possible industrial application.
Interactions between soluble enzymes and interfaces of organic solvent drops or gas bubbles have a very negative effect on the operational stability of the soluble enzymes. In this study, the formation of a hydrophilic shell around the enzyme has been attempted using dextran-aldehyde which would prevent the interaction between enzyme and hydrophobic interfaces with minimal modification of the enzyme surface. After optimizing the size of the dextran (that was found to play a critical role), three different enzymes (glucose oxidase, d-amino acid oxidase, and trypsin) have been conjugated with dextran-aldehyde and their stability towards organic-aqueous and air-liquid interfaces has been evaluated. The treatment itself proved to be very low-cost in terms of activity and was highly stabilizing for the three enzymes assayed. The conjugated preparation of the three assayed enzymes remained fully active in the presence of air-liquid interfaces for at least 10h. However, the unmodified enzymes lost more than 50% of activity within the first hour of the experiments except for trypsin which kept 38% activity after 12h while the trypsin dextran-aldehyde conjugate maintained 100% enzyme activity. Similar results were achieved in the presence of stirred organic solvent-aqueous buffer biphasic system, although in this case some activity was lost by the action of the soluble portion of the organic solvent. In fact, this treatment seems to be also effective to improve the resistance to the action of organic solvent.
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.
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.
Thermostable Mn-dependent catalases are promising enzymes in biotechnological applications as H(2)O(2)-detoxifying systems. We cloned the genes encoding Mn-dependent catalases from Thermus thermophilus HB27 and HB8 and a less thermostable mutant carrying two amino acid replacements (M129V and E293G). When the wild-type and mutant genes were overexpressed in Escherichia coli, unmodified or six-His-tagged proteins of the expected size were overproduced as inactive proteins. Several attempts to obtain active forms or to activate the overproduced proteins were unsuccessful, even when soluble and thermostable proteins were used. Therefore, a requirement for a Thermus-specific activation factor was suggested. To overcome this problem, the Mn-dependent catalase genes were overexpressed directly in T. thermophilus under the control of the Pnar promoter. This promoter belongs to a respiratory nitrate reductase from of T. thermophilus HB8, whose transcription is activated by the combined action of nitrate and anoxia. Upon induction in T. thermophilus HB8, a 20- to 30-fold increase in catalase specific activity was observed, whereas a 90- to 110-fold increase was detected when the laboratory strain T. thermophilus HB27::nar was used as the host. The thermostability of the overproduced wild-type catalase was identical to that previously reported for the native enzyme, whereas decreased stability was detected for the mutant derivative. Therefore, our results validate the use of T. thermophilus as an alternative cell factory for the overproduction of thermophilic proteins that fail to be expressed in well-known mesophilic hosts.
Disulfide reduction of Kluyveromyces lactis and Aspergillus oryzae beta-galactosidases and beta-lactoglobulin was assessed. Reduction was performed using one of two thiol-containing agents: dithiothreitol (DTT) or thiopropyl-agarose with a high degree of substitution (1000 micromol of SH groups/g of dried gel). Both reductants allowed an increase of three- (for K. lactis beta-galactosidase) and fourfold (for A. oryzae beta-galactosidase) in the initial content of SH groups in the lactases. Nearly sevenfold fewer micromoles of SH groups per milligram of protein were needed to perform the reduction of K. lactis beta-galactosidase with thiopropyl-agarose than for the same reduction with DTT. However, for A. oryzae beta-galactosidase, nearly twice as many micromoles of SH groups per milligram of protein were needed with thiopropylagarose than with DTT. Disulfide bonds in beta-lactoglobulin were not accessible to thiopropyl-agarose, since this reduction was only possible in the presence of 6 M urea. These results proved that highly substituted thiopropyl-agarose is as good a reducing agent as DTT, for the reduction of disulfide bonds in proteins. Moreover, excess reducing agent was very simply separated from the reduced protein by filtration, making it easier to control the reaction and providing reduced protein solutions free of reductant. All these advantages substantially cut down the time required and therefore the cost of the overall process.
The properties of a new commercially available amino-epoxy support (amino-epoxy-Sepabeads) for immobilizing enzymes have been compared to those of conventional epoxy supports. The new support has a layer of epoxy groups over a layer of ethylenediamine that is covalently bound to the support. Thus, this support has a great anionic exchanger power and a high number of epoxy groups. We have found a number of advantages to this new heterofunctional support. Immobilization proceeds at low ionic strength using amino epoxy Sepabeads while requiring high ionic strength using conventional monofunctional epoxy supports. Immobilization is much more rapid using amino-epoxy supports than employing conventional epoxy supports. The possibility of achieving immobilized preparations in which the enzyme orientation may be different to that obtained using the traditional hydrophobic supports (with likely effects in terms of activity or stability). Stability of the immobilized enzyme has been found to be much higher using the new support than in preparations using the conventional ones in many cases. Here we show some examples of these advantages using different enzymes (beta-galactosidases, lipase, glutaryl acylase, invertase, and glucoamylase).
In this manuscript, we show that the immobilization of proteins following the technique of cross-linked protein aggregates (CLEAS) may permit the stabilization of the most complex multimeric enzymes by preventing their dissociation. To illustrate that, we have first prepared CLEAS with two tetrameric catalases. Activity recovery was over 40%, and no protein subunit could be desorbed from the CLEAS after boiling in SDS. More interestingly, the enzyme stability, which in its soluble form strongly depends on the enzyme concentration, becomes fully independent of this parameter. This permitted the enzyme stability to greatly increase under diluted conditions. In fact, diluted CLEAs presented a higher stability than those of their glyoxyl derivatives counterparts, which were unable to fully stabilize the multimeric structure of these tetrameric enzymes
In this work, we have compared the entrapment of free or previously immobilized glucose oxidase using a sol-gel technique. The preimmobilization was carried out on Sepabeads (a porous support) derivatized with glutaraldehyde as the functional group. The prior immobilization of the enzyme permitted to maintain the enzyme activity intact after the formation of the sol-gel. In fact, only 10% of the enzyme activity was lost whereas the soluble enzyme lost 60% of its initial activity. Additionally, enzyme leakage from the sol-gel matrix was avoided, which was relatively high when entrapping the soluble enzyme (39% of the enzyme activity was released after 16 h of incubation in a buffered solution). Moreover, the immobilized enzyme, inside the porous support, cannot be in contact with the sol-gel, and, therefore, it maintained the stability achieved by means of the multipoint covalent attachment on the Sepabeads support.
We have developed a novel methodology that allowed the preparation of cross-linked enzyme aggregates (CLEAs) of glutaryl acylase (GAC) by co-aggregation of the enzyme with an aminated polymer: polyethyleneimine (PEI). The preparation of CLEAs of GAC from Pseudomonas sp. is not possible when using poly(ethylene glycol) and glutaraldehyde directly as precipitating and cross-linking agent, respectively. This problem arises probably from the low content of surface Lys groups of GAC which prevents an efficient cross-linking of the enzyme molecules in the aggregate. This fact was proven by the release of enzyme molecules from the aggregate and the solubilization of the enzyme when eliminating the precipitating agent. Our new co-aggregation system favors the cross-linking between the very reactive and abundant primary amino groups of the PEI and the primary amino groups on the enzyme surface. The use of PEI prevents the release of enzyme molecules from the aggregate. By this methodology, we prepared a very stable immobilized derivative of GAC. After optimization of the glutaraldehyde treatment conditions, the stability of the enzyme was significantly improved. It kept more than 60% of its initial activity after 72 h of incubation at 45 degrees C, whereas the soluble enzyme was fully inactivated in 2.5 h of incubation in the same conditions. Therefore, we have a new protocol for carrying out the preparation of cross-linked aggregates of enzymes with a low number of lysines on its surface.
An evaluation of the stability of several forms (including soluble and two immobilized preparations) of d-amino acid oxidases from Trigonopsis variabilis (TvDAAO) and Rhodotorula gracilis (RgDAAO) is presented here. Initially, both soluble enzymes become inactivated via subunit dissociation, and the most thermostable enzyme seemed to be TvDAAO, which was 3-4 times more stable than RgDAAO at a protein concentration of 30 microg/mL. Immobilization on poorly activated supports was unable to stabilize the enzyme, while highly activated supports improved the enzyme stability. Better results were obtained when using highly activated glyoxyl agarose supports than when glutaraldehyde was used. Thus, multisubunit immobilization on highly activated glyoxyl agarose dramatically improved the stability of RgDAAO (by ca. 15,000-fold) while only marginally improving the stability of TvDAAO (by 15-20-fold), at a protein concentration of 6.7 microg/mL. Therefore, the optimal immobilized RgDAAO was much more stable than the optimal immobilized TvDAAO at this enzyme concentration. The lower stabilization effect on TvDAAO was associated with the inactivation of this enzyme by FAD dissociation that was not prevented by immobilization. Finally, nonstabilized RgDAAO was marginally more stable in the presence of H(2)O(2) than TvDAAO, but after stabilization by multisubunit immobilization, its stability became 10 times higher than that of TvDAAO. Therefore, the most stable DAAO preparation and the optimal choice for an industrial application seems to be RgDAAO immobilized on glyoxyl agarose.
Bovine liver catalase was immobilized on different supports. The tetrameric nature of this enzyme was found to cause its rapid inactivation in diluted conditions due to subunit dissociation, a fact that may rule out its industrial use. Multi-subunit immobilization using highly activated glyoxyl agarose was not enough to involve all enzyme subunits. In fact, washing the derivative produced a strong decrease in the enzyme activity. Further cross-linking of previously immobilized enzyme with tailor-made dextran-aldehyde permitted the multimeric structure to be fully stabilized using either multisubunit preparations immobilized onto highly activated glyoxyl-agarose support or one subunit enzymes immobilized onto poorly activated glyoxyl-agarose. The highest stability of the final biocatalyst was observed using the multisubunit immobilized derivative cross-linked with dextran-aldehyde. The optimal derivative retained around 60% of the immobilized activity, did not release any enzyme subunits after boiling in the presence of SDS, and did not lose activity during washing, and its stability did not depend on the dilution. This derivative was used for 10 cycles in the destruction of 10 mM hydrogen peroxide without any decrease in the enzyme activity.
The immobilizaton of the enzyme glutaryl-7-aminocephalosporanic acid acylase (GA) was performed via ionic adsorption onto several supports: a new anionic exchange resin, based on the coating of Sepabeads internal surfaces with polyethyleneimine (PEI) of different molecular weights, and conventional EC-Q1A-Sepabeads and DEAE-agarose. Immobilization occurred very rapidly in all cases, but the adsorption strength was much higher in the case of PEI-Sepabeads than in the other supports at pH 7 (e.g., at 150 mM NaCl, 90% of the enzyme was eluted from the DEAE agarose and 15% was eluted from the EC-Q1A-Sepabeads, whereas no desorption was detected with the best PEI-Sepabeads). Interestingly, the adsorption strength of the GA was increased when it was immobilized on PEI-Sepabeads with higher molecular weights. For instance, enzyme desorption was detected from 75 mM NaCl for the derivative prepared onto Sepabeads coated with PEI 700 Da, whereas in the derivative prepared with the highest molecular weight PEI (600 000 Da) no enzyme desorption was detected below 150 mM NaCl. Optimal PEI-Sepabeads (prepared with PEI of 600 000 Da) was even much more thermostable than the covalent derivative prepared onto cyanogen bromide agarose. Moreover, this derivative presented a half-life 26-fold higher than that of the soluble enzyme at 45 degrees C, and the support could be reused 10 times after the full desorption of the enzyme without decreasing loading capacity.