29 results on '"Lopez-Gallego F"'
Search Results
2. Optimizing the biological activity of Fab fragments by controlling their molecular orientation and spatial distribution across porous hydrogels
- Author
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Ferrari, M., Barreto, R., Jackson, E., Guisan, J.M., Lopez-Gallego, F., and Betancor, L.
- Published
- 2015
- Full Text
- View/download PDF
3. Intraparticle Macromolecular Migration Alters the Structure and Function of Proteins Reversibly Immobilized on Porous Microbeads
- Author
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Diamanti, E, Arana-Pena, S, Ramos-Cabrer, P, Comino, N, Carballares, D, Fernandez-Lafuente, R, and Lopez-Gallego, F
- Abstract
While migration of reversibly immobilized proteins across the volume of supports is investigated in conditions where an external force is applied or under fluid flow conditions, their passive migration upon sample storage and its effect on the protein functionality remain unexplored. Understanding such intraparticle macromolecular migration is essential to develop protein functionalized biomaterials with a longer life span. This work investigates the spatiotemporal migration of His-tagged immobilized fluorescent proteins inside porous agarose microbeads under different storage conditions. A tool that assesses the intraparticle protein migration across the surface of the porous supports is developed. Differences in migration patterns between different proteins suggest that binding dynamics between proteins and their supports play a key role in their migration. The effect of macromolecular migration on the functional and structural properties of bound proteins and enzymes is also explored. Therefore, single-particle measurements to understand how the migration process affects the functionality of immobilized enzymes are performed. Evaluating protein migration and understanding the reason behind such phenomena allows gaining control over immobilization processes and design immobilization chemistries that either prevent or promote intraparticle macromolecular diffusion upon storage, depending on the desired final application.
- Published
- 2022
4. Cell-enzyme tandem systems for sustainable chemistry
- Author
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Betancor, L and Lopez-Gallego, F
- Abstract
The combination of isolated enzymes and whole cells for chemical biomanufacturing has recently arose as an alternative with multiple industrial advantages. Both isolated enzymes and whole-cell biocatalysis have benefits of their own that can be synergistically used in more efficient and sustainable bioprocesses. Those benefits range from decreasing the production times to generating products that are otherwise unobtainable. In this review we have studied the reports of cell-enzyme tandem systems applied as biocatalysts focusing on the different architectures used for their coupling. Combination of extracellular enzymes and microorganisms, enzyme display on whole cell walls and integration of enzymes and microorganisms into different materials are presented as the available alternatives for tandem enzyme-cell systems' biotransformations.
- Published
- 2022
5. Interfacial activity of modified dextran polysaccharide to produce enzyme-responsive oil-in-water nanoemulsions dagger
- Author
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Navascuez, M, Gracia, R, Marradi, M, Diaz, N, Rodriguez, J, Loinaz, I, Lopez-Gallego, F, Llop, J, and Dupin, D
- Abstract
Herein, we report the evaluation of dextran (DXT) derivatives bearing hydrophobic or hydrophilic functional groups as stabilisers of oil-in-water (O/W) emulsions. All investigated modifications conferred interfacial activity to produce stable O/W emulsions, methacrylate(MA)-functionalised DXT being the most promising stabiliser. A minimum amount of MA was required to obtain stable O/W nanoemulsions, which could be degraded in the presence of lipases.
- Published
- 2021
6. O-185 Parameterisation of winter lamb production in the extensive sheep production system with rotational and holistic grazing
- Author
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López Gallego, F. and Aceituno, O.
- Published
- 2023
- Full Text
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7. Transaminase from Pseudomonas fluorescens
- Author
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Smith, P., primary, Roura Padrosa, D., additional, Lopez-Gallego, F., additional, Paradisi, F., additional, and Dreveny, I., additional
- Published
- 2019
- Full Text
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8. Structure of FPT bound to DATFP-DH-GPP
- Author
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Hovlid, M.L., primary, Edelstein, R.L., additional, Henry, O., additional, Ochocki, J., additional, DeGraw, A., additional, Lenevich, S., additional, Talbot, T., additional, Young, V., additional, Hruza, A.W., additional, Lopez-Gallego, F., additional, Labello, N.P., additional, Strickland, C.L., additional, Schmidt-Dannert, C., additional, and Distefano, M.D., additional
- Published
- 2009
- Full Text
- View/download PDF
9. Purification of a Catalase from Thermus thermophilus via IMAC Chromatography: Effect of the Support
- Author
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Hidalgo, A., primary, Betancor, L., additional, Mateo, C., additional, Lopez-Gallego, F., additional, Moreno, R., additional, Berenguer, J., additional, Guisan, J.M., additional, and Fernandez-Lafuente, R., additional
- Published
- 2004
- Full Text
- View/download PDF
10. Use of Physicochemical Tools to Determine the Choice of Optimal Enzyme: Stabilization of -Amino Acid Oxidase
- Author
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Betancor, L., primary, Hidalgo, A., additional, Fernandez-Lorente, G., additional, Mateo, C., additional, Rodriguez, V., additional, Fuentes, M., additional, Lopez-Gallego, F., additional, Fernandez-Lafuente, R., additional, and Guisan, J.M., additional
- Published
- 2003
- Full Text
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11. Co-aggregation of Enzymes and Polyethyleneimine: A Simple Method To Prepare Stable and Immobilized Derivatives of Glutaryl Acylase
- Author
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Lopez-Gallego, F., Betancor, L., Hidalgo, A., Alonso, N., Fernandez-Lafuente, R., and Guisan, J. M.
- Abstract
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 °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.
- Published
- 2005
12. Advantages of the Pre-Immobilization of Enzymes on Porous Supports for Their Entrapment in Sol−Gels
- Author
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Betancor, L., Lopez-Gallego, F., Hidalgo, A., Fuentes, M., Podrasky, O., Kuncova, G., Guisan, J. M., and Fernandez-Lafuente, R.
- Abstract
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.
- Published
- 2005
13. Epoxy-Amino Groups: A New Tool for Improved Immobilization of Proteins by the Epoxy Method
- Author
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Mateo, C., Torres, R., Fernandez-Lorente, G., Ortiz, C., Fuentes, M., Hidalgo, A., Lopez-Gallego, F., Abian, O., Palomo, J. M., Betancor, L., Pessela, B. C. C., Guisan, J. M., and Fernandez-Lafuente, R.
- Abstract
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).
- Published
- 2003
14. Use of Physicochemical Tools to Determine the Choice of Optimal Enzyme: Stabilization of <SCP>d</SCP>-Amino Acid Oxidase
- Author
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Betancor, L., Hidalgo, A., Fernandez-Lorente, G., Mateo, C., Rodriguez, V., Fuentes, M., Lopez-Gallego, F., Fernandez-Lafuente, R., and Guisan, J. M.
- Abstract
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 μg/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 μg/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 H2 O2 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.- Published
- 2003
15. New biotechnological perspectives of a NADH oxidase variant from Thermus thermophilus HB27 as NAD+-recycling enzyme
- Author
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Rocha-Martín Javier, Vega Daniel, Bolivar Juan M, Godoy Cesar A, Hidalgo Aurelio, Berenguer José, Guisán José M, and López-Gallego Fernando
- Subjects
NAD+ ,extremophiles ,dehydrogenase ,immobilization ,Biotechnology ,TP248.13-248.65 - Abstract
Abstract Background The number of biotransformations that use nicotinamide recycling systems is exponentially growing. For this reason one of the current challenges in biocatalysis is to develop and optimize more simple and efficient cofactor recycling systems. One promising approach to regenerate NAD+ pools is the use of NADH-oxidases that reduce oxygen to hydrogen peroxide while oxidizing NADH to NAD+. This class of enzymes may be applied to asymmetric reduction of prochiral substrates in order to obtain enantiopure compounds. Results The NADH-oxidase (NOX) presented here is a flavoenzyme which needs exogenous FAD or FMN to reach its maximum velocity. Interestingly, this enzyme is 6-fold hyperactivated by incubation at high temperatures (80°C) under limiting concentrations of flavin cofactor, a change that remains stable even at low temperatures (37°C). The hyperactivated form presented a high specific activity (37.5 U/mg) at low temperatures despite isolation from a thermophile source. Immobilization of NOX onto agarose activated with glyoxyl groups yielded the most stable enzyme preparation (6-fold more stable than the hyperactivated soluble enzyme). The immobilized derivative was able to be reactivated under physiological conditions after inactivation by high solvent concentrations. The inactivation/reactivation cycle could be repeated at least three times, recovering full NOX activity in all cases after the reactivation step. This immobilized catalyst is presented as a recycling partner for a thermophile alcohol dehydrogenase in order to perform the kinetic resolution secondary alcohols. Conclusion We have designed, developed and characterized a heterogeneous and robust biocatalyst which has been used as recycling partner in the kinetic resolution of rac-1-phenylethanol. The high stability along with its capability to be reactivated makes this biocatalyst highly re-useable for cofactor recycling in redox biotransformations.
- Published
- 2011
- Full Text
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16. Enzyme-support interactions and inactivation conditions determine Thermomyces lanuginosus lipase inactivation pathways: Functional and florescence studies.
- Author
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Souza PMP, Carballares D, Lopez-Carrobles N, Gonçalves LRB, Lopez-Gallego F, Rodrigues S, and Fernandez-Lafuente R
- Subjects
- Aspartic Acid chemistry, Enzymes, Immobilized metabolism, Ethylenediamines chemistry, Fungal Proteins metabolism, Glycine chemistry, Lipase metabolism, Substrate Specificity, Sulfones chemistry, Triacetin chemistry, Enzymes, Immobilized chemistry, Eurotiales enzymology, Fungal Proteins chemistry, Lipase chemistry, Microspheres, Sepharose analogs & derivatives
- Abstract
Lipase from Thermomyces lanuginosus (TLL) has been covalently immobilized on heterofunctional octyl-vinyl agarose. That way, the covalently immobilized enzymes will have identical orientation. Then, it has blocked using hexyl amine (HEX), ethylenediamine (EDA), Gly and Asp. The initial activity/stability of the different biocatalysts was very different, being the most stable the biocatalyst blocked with Gly. These biocatalysts had been utilized to analyze if the enzyme activity could decrease differently along thermal inactivation courses depending on the utilized substrate (that is, if the enzyme specificity was altered during its inactivation using 4 different substrates to determine the activity), and if this can be altered by the nature of the blocking agent and the inactivation conditions (we use pH 5, 7 and 9). Results show great changes in the enzyme specificity during inactivation (e.g., activity versus triacetin was much more quickly lost than versus the other substrates), and how this was modulated by the immobilization protocol and inactivation conditions. The difference in the changes induced by immobilization and inactivation were confirmed by fluorescence studies. That is, the functional and structural analysis of partially inactivated immobilized enzyme showed that their inactivation pathway is strongly depended on the support features and inactivation conditions., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2021
- Full Text
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17. Modulating the properties of the lipase from Thermomyces lanuginosus immobilized on octyl agarose beads by altering the immobilization conditions.
- Author
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Lokha Y, Arana-Peña S, Rios NS, Mendez-Sanchez C, Gonçalves LRB, Lopez-Gallego F, and Fernandez-Lafuente R
- Subjects
- Biocatalysis, Enzyme Stability, Kinetics, Ascomycota enzymology, Cells, Immobilized enzymology, Glyoxylates chemistry, Lipase metabolism, Sepharose chemistry
- Abstract
The lipase from Thermomyces lanuginosus (TLL) has been immobilized on octyl-agarose beads via interfacial activation under 16 different conditions (changing the immobilization pH, the ionic strength, the presence of additives like calcium, phosphate or glycerol) and using a low loading (1 mg/g support). Then, the properties of the different biocatalysts have been evaluated: stability at pH 7.0 and 70 °C and activity versus p-nitro phenyl propionate, triacetin and R- and S- methyl mandelate. Results clearly indicate that the immobilization conditions determine the final enzyme properties, altering enzyme stability (by 10 folds), activity (by 8 folds using R- methyl mandelate) and specificity (V
R /VS changed from 0.7 to 2.3 using mandelate esters). For instance, the enzymes immobilized at pH 7.0 using 5 mM buffer were the most stable preparations, while the presence of 250 mM sodium phosphate greatly decreased the final enzyme stability. The biocatalyst stability of TLL increased with increasing NaCl in the immobilization buffer at pH 5. Fluorescence studies confirmed that the conformation of the different immobilized enzymes were different, despite being a physical and reversible immobilization method. Thus, the immobilization of TLL on octyl agarose beads under different conditions produced biocatalysts with different properties, the optimal condition depends on the studied reaction and condition., (Copyright © 2019 Elsevier Inc. All rights reserved.)- Published
- 2020
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18. A roadmap for biocatalysis - functional and spatial orchestration of enzyme cascades.
- Author
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Schmidt-Dannert C and Lopez-Gallego F
- Subjects
- Metabolic Networks and Pathways, Organic Chemicals chemical synthesis, Biocatalysis, Biotechnology methods, Enzymes metabolism, Organic Chemicals metabolism
- Abstract
Advances in biological engineering and systems biology have provided new approaches and tools for the industrialization of biology. In the next decade, advanced biocatalytic systems will increasingly be used for the production of chemicals that cannot be made by current processes and/or where the use of enzyme catalysts is more resource efficient with a much reduced environmental impact. We expect that in the future, manufacture of chemicals and materials will utilize both biocatalytic and chemical synthesis synergistically. The realization of such advanced biomanufacturing processes currently faces a number of major challenges. Ready-to-deploy portfolios of biocatalysts for design to production must be created from biological diverse sources and through protein engineering. Robust and efficient multi-step enzymatic reaction cascades must be developed that can operate simultaneously in one-pot. For this to happen, bio-orthogonal strategies for spatial and temporal control of biocatalyst activities must be developed. Promising approaches and technologies are emerging that will eventually lead to the design of in vitro biocatalytic systems that mimic the metabolic pathways and networks of cellular systems which will be discussed in this roadmap., (© 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)
- Published
- 2016
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19. Selective oxidation of glycerol to 1,3-dihydroxyacetone by covalently immobilized glycerol dehydrogenases with higher stability and lower product inhibition.
- Author
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Rocha-Martin J, Acosta A, Berenguer J, Guisan JM, and Lopez-Gallego F
- Subjects
- Cellulomonas enzymology, Citrobacter enzymology, Cloning, Molecular, Cross-Linking Reagents, DNA Primers genetics, Dihydroxyacetone metabolism, Electrophoresis, Polyacrylamide Gel, Enzymes, Immobilized metabolism, Escherichia coli, Geobacillus stearothermophilus enzymology, Inhibitory Concentration 50, Oxidation-Reduction, Polyethylene Glycols, Dihydroxyacetone biosynthesis, Glycerol metabolism, Industrial Microbiology methods, Oxidoreductases metabolism
- Abstract
Glycerol dehydrogenase (GlyDH) catalyzes the regioselective oxidation of glycerol to yield 1,3-dihydroxyacetone (DHA); an important building block in chemical industry. Three recombinant GlyDHs from Geobacillus stearothermophilus, from Citrobacter braakii and from Cellulomonas sp. were stabilized by covalent immobilization. The highest activity recoveries (40-50%) of the insoluble preparations were obtained by immobilizing these enzymes in presence of polyethylene glycol (PEG). Noteworthy, these immobilized preparations were more stable and less inhibited by DHA than their soluble counterparts. In particular, GlyDH from G.stearothermophilus immobilized on agarose activated with both amine and glyoxyl groups and crosslinked with dextran aldehyde was 3.7-fold less inhibited by DHA than its soluble form and retained 100% of its initial activity after 18h of incubation at 65°C and pH 7. This is one of the few examples where the same immobilization protocol has minimized enzyme product inhibition and maximized thermal stability., (Copyright © 2014 Elsevier Ltd. All rights reserved.)
- Published
- 2014
- Full Text
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20. Oriented covalent immobilization of antibodies onto heterofunctional agarose supports: a highly efficient immuno-affinity chromatography platform.
- Author
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Batalla P, Bolívar JM, Lopez-Gallego F, and Guisan JM
- Subjects
- Biosensing Techniques instrumentation, Chelating Agents chemistry, Electrophoresis, Polyacrylamide Gel, Enzyme Assays instrumentation, Horseradish Peroxidase analysis, Horseradish Peroxidase antagonists & inhibitors, Horseradish Peroxidase metabolism, Immunoglobulin G chemistry, Immunoglobulin G metabolism, Kinetics, Models, Molecular, Porosity, Protein Binding, Silver chemistry, Antibodies, Immobilized chemistry, Antibodies, Immobilized metabolism, Biotechnology methods, Chromatography, Affinity instrumentation, Chromatography, Affinity methods, Sepharose chemistry
- Abstract
The development of new bioconjugates formed by one antibody optimally bound (through its Fc region) to fairly inert solid surfaces is of primary relevance in immuno-affinity chromatography. Immunoglobulins G (IgG) have a Fc region very rich in histidine (His) residues. In this way, immobilization of IgGs on heterofunctional metal chelate-glyoxyl supports (Ag-Me(2+)/G) takes place in two steps: firstly the antibodies are conjugated to the support via His-metal coordination bonds. Secondly, their incubation under alkaline condition promotes an intramolecular covalent attachment between lysine residues at the Fc region and glyoxyl groups on the support surface. The IgG that recognizes as antigen the HRP (antiHRP-IgG) has been conjugated to Ag-Me(2+)/G supports. The resulting bioconjugate is highly inert and able to specifically bind the antigen (HRP) without significant unspecific binding of any other proteins, resulting in an excellent HRP purification platform. The binding activity of this bioconjugate has been optimized by controlling the antibody distribution throughout the bead's surface in order to avoid high antibody densities that led to a low binding activity of the antibodies. The optimal antibody distribution has been achieved when these proteins were slowly immobilized on Ag-Cu(2+)/G in presence of imidazole. This bioconjugate was able to bind up to 1.5 moles of antigen per mole of antibody, only 1.3-fold less than the antibody in solution. Hence, we have been able to develop an optimal protocol to prepare bioconjugated composites in an oriented and irreversible fashion which results in highly efficient and specific surfaces for the exclusive biological recognition., (Copyright © 2012 Elsevier B.V. All rights reserved.)
- Published
- 2012
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21. Optimized compatible set of BioBrick™ vectors for metabolic pathway engineering.
- Author
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Vick JE, Johnson ET, Choudhary S, Bloch SE, Lopez-Gallego F, Srivastava P, Tikh IB, Wawrzyn GT, and Schmidt-Dannert C
- Subjects
- Bioengineering instrumentation, Escherichia coli metabolism, Genetic Vectors metabolism, Plasmids genetics, Plasmids metabolism, Promoter Regions, Genetic, Bioengineering methods, Escherichia coli genetics, Genetic Vectors genetics, Metabolic Networks and Pathways
- Abstract
The BioBrick™ paradigm for the assembly of enzymatic pathways is being adopted and becoming a standard practice in microbial engineering. We present a strategy to adapt the BioBrick™ paradigm to allow the quick assembly of multi-gene pathways into a number of vectors as well as for the quick mobilization of any cloned gene into vectors with different features for gene expression and protein purification. A primary BioBrick™ (BB-eGFP) was developed where the promoter/RBS, multiple cloning sites, optional protein purification affinity tags and reporter gene were all separated into discrete regions by additional restriction enzymes. This primary BB-eGFP then served as the template for additional BioBrick™ vectors with different origins of replication, antibiotic resistances, inducible promoters (arabinose, IPTG or anhydrotetracycline), N- or C-terminal Histidine tags with thrombin cleavage, a LacZα reporter gene and an additional origin of mobility (oriT). All developed BioBricks™ and BioBrick™ compatible vectors were shown to be functional by measuring reporter gene expression. Lastly, a C(30) carotenoid pathway was assembled as a model enzymatic pathway to demonstrate in vivo functionality and compatibility of this engineered vector system.
- Published
- 2011
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22. Sesquiterpene synthases Cop4 and Cop6 from Coprinus cinereus: catalytic promiscuity and cyclization of farnesyl pyrophosphate geometric isomers.
- Author
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Lopez-Gallego F, Agger SA, Abate-Pella D, Distefano MD, and Schmidt-Dannert C
- Subjects
- Cyclization, Fungal Proteins chemistry, Isomerism, Models, Molecular, Polyisoprenyl Phosphates chemistry, Sesquiterpenes chemistry, Substrate Specificity, Coprinus enzymology, Fungal Proteins metabolism, Polyisoprenyl Phosphates metabolism, Sesquiterpenes metabolism
- Abstract
Sesquiterpene synthases catalyze with different catalytic fidelity the cyclization of farnesyl pyrophosphate (FPP) into hundreds of known compounds with diverse structures and stereochemistries. Two sesquiterpene synthases, Cop4 and Cop6, were previously isolated from Coprinus cinereus as part of a fungal genome survey. This study investigates the reaction mechanism and catalytic fidelity of the two enzymes. Cyclization of all-trans-FPP ((E,E)-FPP) was compared to the cyclization of the cis-trans isomer of FPP ((Z,E)-FPP) as a surrogate for the secondary cisoid neryl cation intermediate generated by sesquiterpene synthases, which are capable of isomerizing the C2--C3 pi bond of all-trans-FPP. Cop6 is a "high-fidelity" alpha-cuprenene synthase that retains its fidelity under various conditions tested. Cop4 is a catalytically promiscuous enzyme that cyclizes (E,E)-FPP into multiple products, including (-)-germacrene D and cubebol. Changing the pH of the reaction drastically alters the fidelity of Cop4 and makes it a highly selective enzyme. Cyclization of (Z,E)-FPP by Cop4 and Cop6 yields products that are very different from those obtained with (E,E)-FPP. Conversion of (E,E)-FPP proceeds via a (6R)-beta-bisabolyl carbocation in the case of Cop6 and an (E,E)-germacradienyl carbocation in the case of Cop4. However, (Z,E)-FPP is cyclized via a (6S)-beta-bisabolene carbocation by both enzymes. Structural modeling suggests that differences in the active site and the loop that covers the active site of the two enzymes might explain their different catalytic fidelities.
- Published
- 2010
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23. Multi-enzymatic synthesis.
- Author
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Lopez-Gallego F and Schmidt-Dannert C
- Subjects
- Animals, Bacterial Proteins metabolism, Biotechnology methods, Humans, Industrial Microbiology methods, Biocatalysis, Multienzyme Complexes metabolism
- Abstract
Biocatalytic conversions can involve one enzyme that carries out one specific reaction at a time, or multiple enzymes that carry out a series of conversions to yield a desired product. The use of several enzymes allows the realization of much more complex synthetic schemes. Multi-step synthesis can be carried out in biological systems by utilizing or engineering their metabolic networks for catalysis. Alternatively, multi-enzymatic catalysis can be carried out in vitro using isolated biocatalysts. Both approaches, in vivo or in vitro, have their specific advantages, problems, and challenges that will be illustrated using recent examples., (Copyright 2009 Elsevier Ltd. All rights reserved.)
- Published
- 2010
- Full Text
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24. Synthesis, properties, and applications of diazotrifluropropanoyl-containing photoactive analogs of farnesyl diphosphate containing modified linkages for enhanced stability.
- Author
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Hovlid ML, Edelstein RL, Henry O, Ochocki J, DeGraw A, Lenevich S, Talbot T, Young VG, Hruza AW, Lopez-Gallego F, Labello NP, Strickland CL, Schmidt-Dannert C, and Distefano MD
- Subjects
- Binding Sites, Farnesyltranstransferase metabolism, Kinetics, Photoaffinity Labels, Polyisoprenyl Phosphates chemistry, Polyisoprenyl Phosphates pharmacology, Saccharomyces cerevisiae Proteins chemistry, Sesquiterpenes chemistry, Sesquiterpenes pharmacology, Structure-Activity Relationship, Substrate Specificity, Polyisoprenyl Phosphates chemical synthesis, Sesquiterpenes chemical synthesis
- Abstract
Photoactive analogs of farnesyl diphosphate (FPP) are useful probes in studies of enzymes that employ this molecule as a substrate. Here, we describe the preparation and properties of two new FPP analogs that contain diazotrifluoropropanoyl photophores linked to geranyl diphosphate via amide or ester linkages. The amide-linked analog (3) was synthesized in 32P-labeled form from geraniol in seven steps. Experiments with Saccharomyces cerevisiae protein farnesyltransferase (ScPFTase) showed that 3 is an alternative substrate for the enzyme. Photolysis experiments with [(32)P]3 demonstrate that this compound labels the beta-subunits of both farnesyltransferase and geranylgeranyltransferase (types 1 and 2). However, the amide-linked probe 3 undergoes a rearrangement to a photochemically unreactive isomeric triazolone upon long term storage making it inconvenient to use. To address this stability issue, the ester-linked analog 4 was prepared in six steps from geraniol. Computational analysis and X-ray crystallographic studies suggest that 4 binds to protein farnesyl transferase (PFTase) in a similar fashion as FPP. Compound 4 is also an alternative substrate for PFTase, and a 32P-labeled form selectively photocrosslinks the beta-subunit of ScPFTase as well as E. coli farnesyldiphosphate synthase and a germacrene-producing sesquiterpene synthase from Nostoc sp. strain PCC7120 (a cyanobacterial source). Finally, nearly exclusive labeling of ScPFTase in crude E. coli extract was observed, suggesting that [32P]4 manifests significant selectivity and should hence be useful for identifying novel FPP-utilizing enzymes in crude protein preparations.
- Published
- 2010
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25. A versatile photoactivatable probe designed to label the diphosphate binding site of farnesyl diphosphate utilizing enzymes.
- Author
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Henry O, Lopez-Gallego F, Agger SA, Schmidt-Dannert C, Sen S, Shintani D, Cornish K, and Distefano MD
- Subjects
- Animals, Carbon-Carbon Lyases chemistry, Carbon-Carbon Lyases metabolism, Drosophila melanogaster enzymology, Escherichia coli enzymology, Farnesyltranstransferase metabolism, Geranyltranstransferase metabolism, Models, Molecular, Nostoc enzymology, Photoaffinity Labels analysis, Polyisoprenyl Phosphates metabolism, Protein Conformation, Sesquiterpenes metabolism, Binding Sites, Farnesyltranstransferase chemistry, Geranyltranstransferase chemistry, Photoaffinity Labels chemistry, Polyisoprenyl Phosphates chemistry, Saccharomyces cerevisiae enzymology, Sesquiterpenes chemistry
- Abstract
Farnesyl diphosphate (FPP) is a substrate for a diverse number of enzymes found in nature. Photoactive analogues of isoprenoid diphosphates containing either benzophenone, diazotrifluoropropionate or azide groups have been useful for studying both the enzymes that synthesize FPP as well as those that employ FPP as a substrate. Here we describe the synthesis and properties of a new class of FPP analogues that links an unmodified farnesyl group to a diphosphate mimic containing a photoactive benzophenone moiety; thus, importantly, these compounds are photoactive FPP analogues that contain no modifications of the isoprenoid portion of the molecule that may interfere with substrate binding in the active site of an FPP utilizing enzyme. Two isomeric compounds containing meta- and para-substituted benzophenones were prepared. These two analogues inhibit Saccharomyces cerevisiae protein farnesyltransferase (ScPFTase) with IC(50) values of 5.8 (meta isomer) and 3.0 microM (para isomer); the more potent analogue, the para isomer, was shown to be a competitive inhibitor of ScPFTase with respect to FPP with a K(I) of 0.46 microM. Radiolabeled forms of both analogues selectively labeled the beta-subunit of ScPFTase. The para isomer was also shown to label Escherichia coli farnesyl diphosphate synthase and Drosophila melanogaster farnesyl diphosphate synthase. Finally, the para isomer was shown to be an alternative substrate for a sesquiterpene synthase from Nostoc sp. strain PCC7120, a cyanobacterial source; the compound also labeled the purified enzyme upon photolysis. Taken together, these results using a number of enzymes demonstrate that this new class of probes should be useful for a plethora of studies of FPP-utilizing enzymes.
- Published
- 2009
- Full Text
- View/download PDF
26. Diversity of sesquiterpene synthases in the basidiomycete Coprinus cinereus.
- Author
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Agger S, Lopez-Gallego F, and Schmidt-Dannert C
- Subjects
- Alkyl and Aryl Transferases genetics, Cloning, Molecular, Coprinus genetics, Cytochrome P-450 Enzyme System genetics, Cytochrome P-450 Enzyme System metabolism, Escherichia coli genetics, Escherichia coli metabolism, Fungal Proteins genetics, Genes, Fungal, Phylogeny, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Alkyl and Aryl Transferases metabolism, Coprinus enzymology, Fungal Proteins metabolism, Sesquiterpenes metabolism
- Abstract
Fungi are a rich source of bioactive secondary metabolites, and mushroom-forming fungi (Agaricomycetes) are especially known for the synthesis of numerous bioactive and often cytotoxic sesquiterpenoid secondary metabolites. Compared with the large number of sesquiterpene synthases identified in plants, less than a handful of unique sesquiterpene synthases have been described from fungi. Here we describe the functional characterization of six sesquiterpene synthases (Cop1 to Cop6) and two terpene-oxidizing cytochrome P450 monooxygenases (Cox1 and Cox2) from Coprinus cinereus. The genes were cloned and, except for cop5, functionally expressed in Escherichia coli and/or Saccharomyces cerevisiae. Cop1 and Cop2 each synthesize germacrene A as the major product. Cop3 was identified as an alpha-muurolene synthase, an enzyme that has not been described previously, while Cop4 synthesizes delta-cadinene as its major product. Cop6 was originally annotated as a trichodiene synthase homologue but instead was found to catalyse the highly specific synthesis of alpha-cuprenene. Coexpression of cop6 and the two monooxygenase genes next to it yields oxygenated alpha-cuprenene derivatives, including cuparophenol, suggesting that these genes encode the enzymes for the biosynthesis of antimicrobial quinone sesquiterpenoids (known as lagopodins) that were previously isolated from C. cinereus and other Coprinus species.
- Published
- 2009
- Full Text
- View/download PDF
27. Solid-phase chemical amination of a lipase from Bacillus thermocatenulatus to improve its stabilization via covalent immobilization on highly activated glyoxyl-agarose.
- Author
-
Fernandez-Lorente G, Godoy CA, Mendes AA, Lopez-Gallego F, Grazu V, de Las Rivas B, Palomo JM, Hermoso J, Fernandez-Lafuente R, and Guisan JM
- Subjects
- Adsorption, Amination, Bacillus classification, Butyrates chemistry, Catalysis, Enzyme Stability, Ethylenediamines chemistry, Glutarates chemistry, Hydrogen-Ion Concentration, Hydrolysis, Hydrophobic and Hydrophilic Interactions, Models, Molecular, Molecular Structure, Phenylacetates chemistry, Stereoisomerism, Surface Properties, Time Factors, Bacillus enzymology, Enzymes, Immobilized chemistry, Enzymes, Immobilized metabolism, Glyoxylates chemistry, Lipase chemistry, Lipase metabolism, Sepharose chemistry
- Abstract
In this paper, the stabilization of a lipase from Bacillus thermocatenulatus (BTL2) by a new strategy is described. First, the lipase is selectively adsorbed on hydrophobic supports. Second, the carboxylic residues of the enzyme are modified with ethylenediamine, generating a new enzyme having 4-fold more amino groups than the native enzyme. The chemical amination did not present a significant effect on the enzyme activity and only reduced the enzyme half-life by a 3-4-fold factor in inactivations promoted by heat or organic solvents. Next, the aminated and purified enzyme is desorbed from the support using 0.2% Triton X-100. Then, the aminated enzyme was immobilized on glyoxyl-agarose by multipoint covalent attachment. The immobilized enzyme retained 65% of the starting activity. Because of the lower p K of the new amino groups in the enzyme surface, the immobilization could be performed at pH 9 (while the native enzyme was only immobilized at pH over 10). In fact, the immobilization rate was higher at this pH value for the aminated enzyme than that of the native enzyme at pH 10. The optimal stabilization protocol was the immobilization of aminated BTL2 at pH 9 and the further incubation for 24 h at 25 degrees C and pH 10. This preparation was 5-fold more stable than the optimal BTL2 immobilized on glyoxyl agarose and around 1200-fold more stable than the enzyme immobilized on CNBr and further aminated. The catalytic properties of BTL2 could be greatly modulated by the immobilization protocol. For example, from (R/S)-2- O-butyryl-2-phenylacetic acid, one preparation of BTL2 could be used to produce the S-isomer, while other preparation produced the R-isomer.
- Published
- 2008
- Full Text
- View/download PDF
28. Identification of sesquiterpene synthases from Nostoc punctiforme PCC 73102 and Nostoc sp. strain PCC 7120.
- Author
-
Agger SA, Lopez-Gallego F, Hoye TR, and Schmidt-Dannert C
- Subjects
- Alkyl and Aryl Transferases chemistry, Alkyl and Aryl Transferases isolation & purification, Bacterial Proteins chemistry, Bacterial Proteins isolation & purification, Cloning, Molecular, Cytochrome P-450 Enzyme System genetics, Cytochrome P-450 Enzyme System metabolism, Escherichia coli genetics, Escherichia coli metabolism, Gene Expression, Kinetics, Nostoc chemistry, Nostoc genetics, Open Reading Frames, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Sesquiterpenes chemistry, Alkyl and Aryl Transferases genetics, Alkyl and Aryl Transferases metabolism, Bacterial Proteins genetics, Bacterial Proteins metabolism, Nostoc enzymology, Sesquiterpenes metabolism
- Abstract
Cyanobacteria are a rich source of natural products and are known to produce terpenoids. These bacteria are the major source of the musty-smelling terpenes geosmin and 2-methylisoborneol, which are found in many natural water supplies; however, no terpene synthases have been characterized from these organisms to date. Here, we describe the characterization of three sesquiterpene synthases identified in Nostoc sp. strain PCC 7120 (terpene synthase NS1) and Nostoc punctiforme PCC 73102 (terpene synthases NP1 and NP2). The second terpene synthase in N. punctiforme (NP2) is homologous to fusion-type sesquiterpene synthases from Streptomyces spp. shown to produce geosmin via an intermediate germacradienol. The enzymes were functionally expressed in Escherichia coli, and their terpene products were structurally identified as germacrene A (from NS1), the eudesmadiene 8a-epi-alpha-selinene (from NP1), and germacradienol (from NP2). The product of NP1, 8a-epi-alpha-selinene, so far has been isolated only from termites, in which it functions as a defense compound. Terpene synthases NP1 and NS1 are part of an apparent minicluster that includes a P450 and a putative hybrid two-component protein located downstream of the terpene synthases. Coexpression of P450 genes with their adjacent located terpene synthase genes in E. coli demonstrates that the P450 from Nostoc sp. can be functionally expressed in E. coli when coexpressed with a ferredoxin gene and a ferredoxin reductase gene from Nostoc and that the enzyme oxygenates the NS1 terpene product germacrene A. This represents to the best of our knowledge the first example of functional expression of a cyanobacterial P450 in E. coli.
- Published
- 2008
- Full Text
- View/download PDF
29. Immobilization of enzymes on heterofunctional epoxy supports.
- Author
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Mateo C, Grazu V, Palomo JM, Lopez-Gallego F, Fernandez-Lafuente R, and Guisan JM
- Subjects
- Bioreactors, Biotechnology methods, Enzymes, Immobilized chemistry, Epoxy Compounds chemistry, Proteins chemistry
- Abstract
Immobilization of enzymes and proteins on activated supports permits the simplification of the reactor design and may be used to improve some enzyme properties. In this sense, supports containing epoxy groups seem to be useful to generate very intense multipoint covalent attachment with different nucleophiles placed on the surface of enzyme molecules (e.g., amino, thiol, hydroxyl groups). However, the intermolecular reaction between epoxy groups and soluble enzymes is extremely slow. To solve this problem, we have designed "tailor-made" heterofunctional epoxy supports. Using these, immobilization of enzymes is performed via a two-step process: (i) an initial physical or chemical intermolecular interaction of the enzyme surface with the new functional groups introduced on the support surface and (ii) a subsequent intense intramolecular multipoint covalent reaction between the nucleophiles of the already immobilized enzyme and the epoxy groups of the supports. The first immobilization may involve different enzyme regions, which will be further rigidified by multipoint covalent attachment. The design of some heterofunctional epoxy supports and the performance of the immobilization protocols are described here. The whole protocol to have an immobilized and stabilized enzyme could take from 3 days to 1 week.
- Published
- 2007
- Full Text
- View/download PDF
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