Lorena Betancor

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.

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 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 H2O2 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.

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

ABSTRACT Thermostable Mn-dependent catalases are promising enzymes in biotechnological applications as H2O2-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.

Reversible immobilization of glutaryl acylase on sepabeads coated with polyethyleneimine.

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 °C, and the support could be reused 10 times after the full desorption of the enzyme without decreasing loading capacity.

Solid-phase reducing agents as alternative for reducing disulfide bonds in proteins.

Disulfide reduction of Kluyveromyces lactis and Aspergillus oryzae β-galactosidases and β-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 μmol of SH groups/g of dried gel). Both reductants allowed an increase of three- (for K. lactis β-galactosidase) and fourfold (for A. oryzae β-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 β-galactosidase with thiopropyl-agarose than for the same reduction with DTT. However, for A. oryzae β-galactosidase, nearly twice as many micromoles of SH groups per milligram of protein were needed with thiopropylagarose than with DTT. Disulfide bonds in β-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.

Very Strong But Reversible Immobilization of Enzymes on Supports Coated With Ionic Polymers

In this chapter, the properties of tailor-made anionic exchanger resins based on films of large polyethylenimine polymers (e.g., molecular weight 25,000) as supports for strong but reversible immobilization of proteins are shown. The polymer is completely coated, via covalent immobilization, the surface of different porous supports. Proteins can interact with this polymeric bed, involving a large percentage of the protein surface in the adsorption. Different enzymes have been very strongly adsorbed on these supports, retaining enzyme activities. On the other hand, adsorption is very strong and the derivatives may be used under a wide range of pH and ionic strengths. These supports may be useful even to stabilize multimeric enzymes, by involving several enzyme subunits in the immobilization.

Purification of a Catalase from Thermus thermophilus via IMAC Chromatography: Effect of the Support

A hexameric Mn-catalase was purified from crude extracts of Thermus thermophilus using ammonium sulfate precipitation and ion metal-chelate affinity chromatography (IMAC). Eupergit 250 and Sepabeads FP-EP3 epoxy supports derivatized with iminodiacetic acid (IDA) and copper were used, at similar micromole/packed milliliter of support. Although Eupergit 250-IDA-Cu support adsorbed 80% of the total proteins in the extract, it exhibited a minimum affinity for the catalase. On the other hand, Sepabeads FP-EP3-IDA-Cu allowed the full adsorption of the catalase activity, which could be desorbed in fractions of different purity. This was attributed to a different geometrical congruence of the support surfaces with the enzyme surface, resulting in a different ability to form multipoint interactions with the proteins. Thus, by a cleanup step, followed by a negative chromatographic step using Eupergit 250-IDA-Cu2+ and by the adsorption of the catalase on Sepabeads-IDA-Cu2+ support, a pure enzyme fraction was obtained and its N-terminal end was sequenced.

Glutaraldehyde-mediated protein immobilization.

In this chapter, we describe different approaches for the utilization of glutaraldehyde in protein immobilization. First, we focus on the covalent attachment of proteins to glutaraldehyde-activated matrixes. We describe conditions for the synthesis of such supports and provide an example of the immobilization and stabilization of fructosyltransferase. We also describe how glutaraldehyde may be used for the cross-linking of protein-protein aggregates and protein adsorbed onto amino-activated matrixes. In these cases, glutaraldehyde bridges either two lysine groups from different proteic molecules or a lysine from the protein structure and an amine group from the support. Examples of cross-linking are given for the immobilization of DAAO on different amino-activated supports.

Stabilization of Multimeric Enzymes Via Immobilization and Further Cross-Linking With Aldehyde-Dextran

Subunit dissociation of multimeric proteins is one of the most important causes of inactivation of proteins having quaternary structure, making these proteins very unstable under diluted conditions. A sequential two-step protocol for the stabilization of this protein is proposed. A multisubunit covalent immobilization may be achieved by performing very long immobilization processes between multimeric enzymes and porous supports composed of large internal surfaces and covered by a very dense layer of reactive groups. Additional cross-linking with polyfunctional macromolecules promotes the complete cross-linking of the subunits to fully prevent enzyme dissociation. Full stabilization of multimeric structures has been physically shown because no subunits were desorbed from derivatives after boiling them in SDS. As a functional improvement, these immobilized preparations no longer depend on the enzyme.

Cellulose-Based Nanosupports for Enzyme Immobilization

Integration of biocatalysts and nanoscale materials offer multiple advantages over micro-scaled heterogeneous biocatalysts. Apart from providing reusability and sustainability of the enzyme, the use of nanosupports is aimed at increasing E. Jackson · S. Correa · L. Betancor (*) Laboratorio de Biotecnología, Facultad de Ingeniería, Universidad ORT Uruguay, Montevideo, Uruguay e-mail: betancor@ort.edu.uy # Springer International Publishing AG, part of Springer Nature 2018 Md. I. H. Mondal (ed.), Cellulose-Based Superabsorbent Hydrogels, Polymers and Polymeric Composites: A Reference Series, https://doi.org/10.1007/978-3-319-76573-0_42-1 1 the surface area available for biocatalyst immobilization and improving the yields in bioconversions through better biocatalyst mobility and less diffusional problems. Among many nanomaterials for enzyme immobilization, cellulose stands out as biocompatible, biodegradable, and environmentally-friendly regarding its biological source. In this chapter, we discuss the steady advancement in utilizing different nanostructured cellulosic materials for enzyme immobilization. We address the use of hybrid materials that include cellulose and improve the properties of the heterogeneous biocatalyst. The methodologies for functionalization and integration of enzymes on nanocellulose hydrogels are discussed including covalent linkage through chemical modification, entrapment, and crosslinking. We consider its applications to biomedicine, food industry, and environmental science with a special emphasis on the impact of the enzymatic properties caused after immobilization on cellulosic supports.