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Aurelio Hidalgo

Publications and source records attributed to Aurelio Hidalgo.

At least 19 recordsLinked to original sources
PubMed2025

Ultrahigh-throughput screening assay for PET-degrading enzymes.

In recent years, several PET-degrading enzymes have been identified from both known microorganisms and metagenomic sources in response to the growing environmental issue of polyethylene terephthalate (PET) accumulation. Despite this progress, there is a limited number of (ultra)high-throughput screening methods for assessing PET-hydrolyzing activity without relying on surrogate substrates. This method utilizes the coupled activity of ketoreductases (KREDs) and diaphorase to produce a fluorescent compound (resorufin) in the presence of PET degradation products, offering a more direct and efficient screening approach. A metagenomic KRED was coupled with the diaphorase from Clostridium kluyveri to enable the detection of the hydrolysis of PET degradation products catalyzed by the Bacillus subtilis BS2 esterase. The coupled reaction was established in water-in-oil microdroplets, encapsulating a single E. coli cell per droplet, demonstrating its potential for use in the ultrahigh-throughput screening of metagenomic libraries or randomized libraries for directed evolution campaigns.

High-Throughput Screening Assays↗
PubMed2007

A versatile esterase from Bacillus subtilis: cloning, expression, characterization, and its application in biocatalysis.

An esterase from Bacillus subtilis DSM402 (BS2) was cloned and functionally expressed in E. coli. The enzyme is active up to 50 degrees C, and the V(max) (1449 mM/min) and K(M) values (119 mM) were determined using p-nitrophenyl acetate as substrate. BS2 belongs to the few hydrolases that can act on tertiary alcohols and was therefore used to resolve racemic acetates of selected tertiary alcohols, but also to selectively remove the tert-butyl ester protecting group from peptides. In addition, the enzyme shows promiscuous amidase activity.

Alcohols↗
PubMed2006

Immobilization of enzymes in microtiter plate scale.

Different immobilization methods were adapted to the 96-well microtiter plate scale using esterases as model enzymes. The methods tested were based on adsorption, coprecipitation, aggregation and covalent bonding. The protein covered microcrystals proved to be the best method in terms of yield and expressed activity for the test reaction, which was the alcoholysis of p-nitrophenyl acetate with 1-propanol under anhydrous conditions.

Adsorption↗
PubMed2005

Enzyme stabilization by glutaraldehyde crosslinking of adsorbed proteins on aminated supports.

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.

Adsorption↗
PubMed2005

Preparation of a very stable immobilized biocatalyst of glucose oxidase from Aspergillus niger.

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.

Aspergillus niger↗
PubMed2004

Optimization of an industrial biocatalyst of glutaryl acylase: stabilization of the enzyme by multipoint covalent attachment onto new amino-epoxy Sepabeads.

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.

Adsorption↗
PubMed2004

Prevention of interfacial inactivation of enzymes by coating the enzyme surface with dextran-aldehyde.

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.

Aldehydes↗
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↗
PubMed2004

Thermus thermophilus as a cell factory for the production of a thermophilic Mn-dependent catalase which fails to be synthesized in an active form in Escherichia coli.

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.

Catalase↗
PubMed2004

Use of an antisense RNA strategy to investigate the functional significance of Mn-catalase in the extreme thermophile Thermus thermophilus.

The expression of an antisense RNA revealed that an Mn-catalase was required in Thermus thermophilus for aerobic but not for anaerobic growth. The antisense system is based on the constitutive expression of a "bicistronic" transcript consisting of the kanamycin resistance gene mRNA followed by the antisense RNA against the selected target.

Aerobiosis↗
PubMed2002

Degradation of phenol by Rhodococcus erythropolis UPV-1 immobilized on Biolite in a packed-bed reactor.

A strain of Rhodococcus erythropolis has been isolated and identified by 16S rRNA sequencing. Cells acclimated to phenol can be adsorbed on the external surface of beads of the ceramic support Biolite where they grow forming a network of large filaments. Exponentially-growing cells were adsorbed faster than their stationary-phase counterparts. Immobilization resulted in a remarkable enhancement of the respiratory activity of cells and a shorter lag phase preceding the active phenol degradation. Under optimum operation conditions, the immobilized cells in a laboratory-scale column reactor packed with support beads were able to degrade completely phenol in defined mineral medium at a maximum rate of 18 kg phenol m(-3) per day. The performance of the bioreactor in long-term continuous operation was characterized by pumping defined mineral medium which contained different concentrations of phenol at different flow-rates. Once phenol biodegradation in defined mineral medium was well established, an industrial wastewater from a resin manufacturing company, which contained both phenol and formaldehyde, was tested. In this case, after wastewater conditioning (i.e. pH, nitrogen source and micronutrient amendments) the immobilized cells were able to remove completely formaldehyde and to partly biodegrade phenols at a rate of 1 kg phenol m(-3) per day.

Biodegradation, Environmental↗
PubMedDate not supplied

Epoxy-amino groups: a new tool for improved immobilization of proteins by the epoxy method.

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).

Adsorption↗
PubMedDate not supplied

Cross-linked aggregates of multimeric enzymes: a simple and efficient methodology to stabilize their quaternary structure.

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

Electrophoresis, Polyacrylamide Gel↗
PubMedDate not supplied

Advantages of the pre-immobilization of enzymes on porous supports for their entrapment in sol-gels.

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.

Aspergillus niger↗
PubMedDate not supplied

Co-aggregation of enzymes and polyethyleneimine: a simple method to prepare stable and immobilized derivatives of glutaryl acylase.

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.

Electrophoresis, Polyacrylamide Gel↗
PubMedDate not supplied

Use of physicochemical tools to determine the choice of optimal enzyme: stabilization of D-amino acid oxidase.

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.

D-Amino-Acid Oxidase↗
PubMedDate not supplied

Preparation of a stable biocatalyst of bovine liver catalase using immobilization and postimmobilization techniques.

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.

Animals↗