Cesar Mateo

Spanish National Research Council | CSIC · Institute of Catalysis and Petrochemistry

The use of biocatalysts in chemical reactions is of great interest because reactions can be carried out under very mild and green conditions [...]

Enzymes are highly active and selective catalysts that are capable of catalyzing reactions by different catalytic mechanisms (either through different amino acids located at the catalytic site or different trace elements or cofactors involved in catalysis). Among the different trace elements, iron is key both for the life of different living organi...

The efficiency of laccase-catalyzed protein cross-linking can be impacted by substrate protein structure and competing reactions. In this study, chemical grafting of ferulic acid (FA) on protein surface was applied to modulate the cross-linking of two inflexible globular proteins, lysozyme (LZM) and ovalbumin (OVA). The extent of FA-grafting was po...

The utilization of biomaterials as novel carrier materials for lipase immobilization has been investigated by many research groups over recent years. Biomaterials such as agarose, starch, chitin, chitosan, cellulose, and their derivatives have been extensively studied since they are non-toxic materials, can be obtained from a wide range of sources...

Commercial epoxy supports may be very useful tools to stabilize proteins via multipoint covalent attachment if the immobilization is properly designed. In this chapter, a protocol to take full advantage of the support’s possibilities is described. The basics of the protocol are as follows: (1) the enzymes are hydrophobically adsorbed on the support...

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 in...

Stabilization of enzymes via immobilization techniques is a valuable approach in order to convert a necessary protocol (immobilization) into a very interesting tool to improve key enzyme properties (stabilization). Multipoint covalent attachment of each immobilized enzyme molecule may promote a very interesting stabilizing effect. The relative dist...

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...

β-glucosidases are ubiquitous, well-characterized and biologically important enzymes with considerable uses in industrial sectors. Here, a tetrameric β-glucosidase from Exiguobacterium antarcticum B7 (EaBglA) was immobilized on different activated agarose supports followed by post-immobilization with poly-functional macromolecules. The best result...

The synthesis of compounds or chiral building-blocks with the desired configuration is one of the greatest challenges of chemistry and is of great interest in different fields such as analytical chemistry and especially in fine and pharmaceutical chemistry. [...]

This study presents a rapid and simple method to screen and detect new drugs to treat Alzheimer's disease. The method uses a test system based on an immobilized acetylcholinesterase from Electrophorus electricus (AChE). The optimal AChE immobilized preparation retained 65% of the original catalytic activity; immobilization improved AChE stability b...

Genes encoding lipase LipBC (lipA) and foldase LifBC (lipB) were identified in the genome of Burkholderia contaminans LTEB11. Analysis of the predicted amino acid sequence of lipA showed its high identity with lipases from Pseudomonas luteola (91%), Burkholderia cepacia (96%) and Burkholderia lata (97%), and classified LipBC lipase in the lipase su...

Immobilization is an exciting alternative to improve the stability of enzymatic processes. However, part of the applied covalent strategies for immobilization uses specific conditions, generally alkaline pH, where some enzymes are not stable. Here, a new generation of heterofunctional supports with application at neutral pH conditions was proposed....

Levansucrase (LS) represents a key enzyme in glycoside synthesis of novel prebiotics and β-2,6-levan. The study of the effects of immobilization parameters of LS, produced from Bacillus amyloliquefaciens, onto glyoxyl agarose-IDA/Cu by response surface methodology revealed the significance of their interactive effects. Retention of activity was alt...

Immobilized and stabilized lipases are important biocatalytic tools. In this paper, different tailor-made bifunctional supports were prepared for the immobilization of a new metagenomic lipase (LipC12). The new supports contained hydrophobic groups (different alkyl groups) to promote interfacial adsorption of the lipase and aldehyde groups to react...

The use of enzymes in food processing represents a historically well-established approach that gets its deepest roots in thousands of years BC. In fact, even in an unconscious way, the mankind always applied enzyme-based bioprocesses for example in beer brewing, bread baking, and cheese and wine making. Nevertheless, throughout the centuries, proce...

Background Levansucrase (LS)-catalyzed-transfructosylation reaction is a potential approach for the synthesis of fructooligosaccharides (FOSs) and levan as health promoting compounds. This biocatalytic approach is hindered by low thermal stability of LS and its high rate of hydrolysis. In the present study, LS from Bacillus amyloliquefaciens was im...

The aldol reactions catalyzed by aldolases are an important tool in the synthesis of different products or building blocks. However, despite its advantages, aldolases, as enzymes, have some troubles as their low activity against synthetic substrates or the low stability, doing difficult their application. There are different tools to improve the pr...

The preparation and performance of a suitable chimeric biosensor based on antibodies (Abs) immobilized on lipase-coated magnetic particles by means of a standing orienting strategy are presented. This novel system is based on hydrophobic magnetic particles coated with modified lipase molecules able to orient and further immobilize different Abs in...

The production of xylooligosaccharides (XOS) using a packed-bed enzymatic reactor was studied at lab-scale. For this, a xylanase from Aspergillus versicolor was immobilized on different supports. The optimal derivative was xylanase immobilized on glyoxyl-agarose supports. This derivative preserved 85% of its catalytic activity; it was around 700-fo...

The immobilization of proteins on epoxy activated supports is discussed in this chapter. Immobilization on epoxy supports is carried out as a two-step mechanism: in the first step the adsorption of the protein is promoted and in the second step the intramolecular covalent linkage among epoxy groups and nucleophiles of the protein is produced. Based...

Stabilization of enzymes via immobilization techniques is a valuable approach in order to convert a necessary protocol (immobilization) into a very interesting tool to improve key enzyme properties (stabilization). Multipoint covalent attachment of each immobilized enzyme molecule may promote a very interesting stabilizing effect. The relative dist...

Novel heterofunctional glyoxyl-agarose supports were prepared. These supports contained the maximal concentration of glyoxyl groups to promote maximization of covalent immobilization and groups' capability to adsorb proteins by various mechanisms (e.g., ionic exchange, metal-chelate formation). Immobilization on various supports makes it possible t...

A highly active and stable derivate of immobilized Bacillus circulans β-galactosidase was prepared for the synthesis of galacto-oligosaccharides (GOS) under repeated-batch operation. B. circulans β-galactosidase was immobilized on monofunctional glyoxyl agarose and three heterofunctional supports: amino-, carboxy-, and chelate-glyoxyl agarose. Glyo...

The hydrolysis of phenolic compounds using an immobilized and highly active and stable derivative of laccase from Trametes versicolor is presented. The enzyme was immobilized on aldehyde supports. For this, the enzyme was enriched in amino groups by chemical modification of its carboxyl groups. The aminated enzyme was immobilized with a high recove...

β-Xylosidases have important applications in many biotechnological processes. In this context, the aim of this work was the purification, immobilization and characterization of a β-xylosidase produced by a new isolate of Aspergillus niger USP-67. β-Xylosidase was produced on static conditions in liquid Benassi medium supplemented with xylan birchwo...

Epoxide hydrolase (EH) is an interesting tool in different processes of organic chemistry. In this review the different available techniques to improve the properties (activity, stability and selectivity) and to enhance the applicability of this enzyme at a large scale will be studied. In particular, the review focuses on immobilization techniques...

The enantioselectivity of a lipase from Geobacillus thermocatenulatus (BTL2) has been altered by a site-directed immobilization on tailor-made disulfide supports. The enzyme was genetically modified introducing a unique cysteine into different positions of the protein surface. These new enzyme variants (phi-BTL2) maintained the catalytic properties...

An endoxylanase from Streptomyces halstedii was stabilized by multipoint covalent immobilization on glyoxyl-agarose supports. The immobilized enzyme derivatives preserved 65% of the catalytic activity corresponding to the one of soluble enzyme that had been immobilized. These immobilized derivatives were 200 times more stable 200 times more stable...

This Communication presents the development of a novel strategy for the easy conjugation of biomolecules to hydrophobic magnetic microparticles via reversible coating with previously functionalized lipase molecules. First, the ability of lipase to be strongly adsorbed onto hydrophobic surfaces was exploited for the stabilization of microparticles i...

β-galactosidase from Kluyveromyces lactis was covalently immobilised on a Glyoxyl Sepharose (GS) support by multi-point attachment. The enzyme immobilisation process was very efficient; the supports immobilised almost all the protein responsible for the catalytic activity in a short period of time, retaining approximately 82% of the activity in the...

Tyrosinase and Tyrosinase Inhibitors From a commercial enzyme extract juice with purpose to stabilize it in the industry, has been able to extract an extract rich in beta-galactosidase activity. It has been designed to extract and purification protocol based on polyethyleneimine SUPPORTS agarose activated with next purification yields 80%.

An endo-1,5-arabinanase (abnA) encoding gene from Aspergillus niveus was identified, cloned and successfully expressed in Aspergillus nidulans strain A773. Based on amino acid sequence comparison, the 34-kDa enzyme could be assigned to CAZy GH family 43. Characterization of purified recombinant endo-1,5-arabinanase (AbnA) revealed that it is active...

An endo-1,5-arabinanase (abnA) encoding gene from Aspergillus niveus was identified, cloned and successfully expressed in Aspergillus nidulans strain A773. Based on amino acid sequence comparison, the 34-kDa enzyme could be assigned to CAZy GH family 43. Characterization of purified recombinant endo-1,5-arabinanase (AbnA) revealed that it is active...

Staphylococcus warneri strain EX17 produces three lipases with different molecular weights of 28, 30, and 45 kDa. The 45 kDa fraction (SWL-45) has been purified from crude protein extracts by one chromatographic step based on the selective adsorption of this lipase by interfacial activation on different hydrophobic supports at low ionic strength. T...

The immobilization of Candida antarctica (fraction B) lipase expressed in Pichia pastoris, a selective glycosylated protein at Asn 74, on a new heterofunctional support consisted of phenylboronic acid and aldehyde groups (Borald) has been performed. This method occurs via a two step mechanism: first orientation by organoborane interaction at neutra...

The bioconversion of lignocellulosic biomass to fermentable sugars for production of ethanol requires a multienzyme system named cellulase. This system contains enzymes that act synergistically in the hydrolysis of cellulose: endoglucanase, cellobiohydrolase and β-glucosidase. The first two enzymes act directly on cellulose, yielding mainly glucose...

Sucrose laurate is a detergent that is useful for various biochemical applications because it is a green compound and is easily degradable after hydrolysis with a lipase or esterase. One problem observed in the process of sucrose laurate degradation is that most commercial detergent preparations are impure, necessitating the hydrolysis of all of th...

Commercial glucoamylase immobilizes very slowly on highly activated glyoxyl-agarose supports. The resulting derivatives were only 6-fold more stable than soluble enzyme. The unmodified glucoamylase, highly glycosylated, seems to have a low number of Lys able to react with glyoxyl groups on the support. Thus, the enzyme surface was highly enriched i...

For the immobilization-stabilization of multimeric enzymes, we propose a novel heterofunctional support containing a very low concentration of ionized amino groups and a very high concentration of very poorly reactive glyoxyl (aldehyde) groups. A large tetrameric enzyme, β-galactosidase from Thermus sp., was purified and dramatically stabilized wit...

Novel heterofunctional glyoxyl-agarose supports were prepared. These supports contain a high concentration of groups (such as quaternary ammonium groups, carboxyl groups, and metal chelates) that are capable of adsorbing proteins, physically or chemically, at neutral pH as well as a high concentration of glyoxyl groups that are unable to immobilize...

A novel approach is proposed to prepare a set of immobilized derivatives of a enzyme covalently rigidified through different regions of its surface. Six different variants of penicillin G acylase (PGA) from Escherichia coli (which lacks Cys) were prepared by introducing a unique Cys residue via site-directed mutagenesis in six different enzyme regi...

A new anion exchanger support has been designed for the selective adsorption of small proteins. This has been achieved activating an aminated support with glutaraldehyde and further coating the support surface with bovine serum albumin (BSA). In this support, "wells" are generated by two neighborhoods BSA molecules, on the bottom of those "wells" g...

Highly activated glyoxyl-supports rapidly immobilize proteins at pH 10 (where the ɛ-amino groups of the Lys groups of the protein surface are very reactive), and stabilize them by multipoint covalent attachment. However, they do not immobilize proteins at pH 8. This paper shows that the enzyme immobilization at this mild pH value is possible by inc...

This research describes the immobilization on glyoxyl, cyanogen bromide or octyl agarose beads of a purified lipase from Staphylococcus warneri strain EX17 (SWL), and the effect on its properties. The immobilization on glyoxyl-agarose at pH 10 and 25°C, conditions in which the enzyme is readily inactivated, required the stabilization of the soluble...

It has been found that the enzymes penicillin G acylase from Escherichia coli (PGA) and lipase from Bacillus thermocatenulatus (BTL) did not significantly adsorb on highly activated amino-agarose beads at pH 7 (a support where 85–90% of a crude extract of proteins become adsorbed). Moreover, it has been found that these enzymes do not covalently im...

The immobilization of a glutamate dehydrogenase from Thermus thermophilus (GDH) on glyoxyl agarose beads at pH 7 has permitted to perform the immobilization, purification and stabilization of this interesting enzyme. It was cloned in Escherichiacoli and a first thermal shock of the crude preparation destroyed most mesophilic multimeric proteins. Gl...

This paper shows a simple and effective way to avoid the dissociation of multimeric enzymes by coating their surface with a large cationic polymer (e.g., polyethylenimine (PEI)) by ionic exchange. As model enzymes, glutamate dehydrogenase (GDH) from Thermus thermophilus and formate dehydrogenase (FDH) from Pseudomonas sp. were used. Both enzymes ar...

Glutamate dehydrogenase (GDH) from Thermus thermophilus is a homotrimeric enzyme that tends to dissociate at acidic pH values. GDH is readily adsorbed on highly activated anionic exchangers (HAAE), but hardly adsorbed on lowly activated supports (LAAE) or on highly activated epoxy supports. When using amino-epoxy supports, GDH immobilized on HAAE-e...

Immobilization of anti-horseradish peroxidase on glyoxyl-agarose proceeds rapidly, and after the immobilization, it was found that the antibody captured almost the same amount of peroxidase than the free antibody. After boiling the antibodies in the presence of SDS and mercaptoethanol, more than 95% of the immobilized antibodies presented the four...

Different carrier-free and carrier-bound penicillin acylases were evaluated in the thermodynamically controlled enzymatic synthesis in organic medium of deacetoxycephalosporin G (using phenylacetic acid and 7-amino-deacetoxycefalosporanic acid) used as a model reaction system. Stability of all biocatalysts was determined in a strong (dioxane) and a...

Agarose coated with polyethyleneimine (PEI) or sulfate-dextran, monoaminoethyl-N-aminoethyl-agarose, DEAE-agarose and carboxymethyl-agarose was used to immobilize by ionic exchange and α-galactosidase from Thermus sp. T2 under different conditions. Supports coated with polymers (PEI or sulfate-dextran) allowed higher immobilization yields (by 10–20...

The genome of Thermus thermophilus contains two genes encoding putative glutamate dehydrogenases. One of these genes (TTC1211) was cloned and overexpressed in Escherichia coli. The purified enzyme was a trimer that catalyzed the oxidation of glutamate to alpha-ketoglutarate and ammonia with either NAD+ or NADP+ as cofactors. The enzyme was also abl...

The correct immobilization of antibodies is one of the most critical steps in the preparation of immunosensors and immunochromatography matrices. In addition, the final support has to be chemical and physically inert to avoid the unspecific adsorption of proteins that can reduce the sensitivity of the biosensor or the purification achieved by the c...

Subunit dissociation of multimeric proteins is one of the most important causes of inactivation of proteins having quarternary 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...

In this chapter, the combined use of the selectivity of metal chelate affinity chromatography with the capacity of epoxy supports to immobilize poly-His-tagged proteins via multipoint covalent attachment is shown. This has been achieved by designing tailor-made chelate-epoxy supports. In order to selectively adsorb the poly-His-tagged proteins, a v...

The use of glutaraldehyde and supports containing primary amino groups is one of the most frequently used techniques for enzyme immobilization. However, glutaraldehyde is a very versatile reagent. Using low-ionic strength, the cationic nature of the surface permits the rapid ionic immobilization of the proteins. There are two different possibilitie...

Immobilization of antibodies by their oxidized sugar chain on aminated supports is a very efficient methodology to have a properly oriented antibody. However, these supports may behave as anionic exchangers, producing the unspecific adsorption of other proteins and reducing the selectivity of the system. To overcome this problem, we have proposed t...

The enzyme penicillin G acylase (PGA) is currently employed at an industrial scale in the hydrolysis of penicillin and cephalosporin G. Here, we describe the preparation of a new immobilized preparation of the enzyme that yields derivatives that are very thermostable and resistant in the presence of organic solvents. The stabilization is obtained v...

Commercial epoxy supports may be very useful tools to stabilize proteins via multipoint covalent attachment if the immobilization is properly designed. In this chapter, a protocol to take full advantage of the support’s possibilities is described. The basics of the protocol are as follows: (1) the enzymes are hydrophobically adsorbed on the support...

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 is shown. The polymer is completely coated, via covalent immobilization, the surface of different porous supports. Proteins can int...

The prospects of a new commercially available support (amino-epoxy-Sepabeads® ) for enzyme immobilization are discussed in this chapter. These supports have a layer of epoxy groups over a layer of ethylenediamine that is covalently bound to the support. Thus, the support has a high anionic exchanger power and a high number of epoxy groups. Some rel...

Chemical modification and immobilization of proteins have been usually utilized as parallel techniques to improve enzyme stability. In this chapter, we show that chemical modification of the protein surface to greatly increase its reactivity with the groups of a support activated with glyoxyl residues may be a very good alternative for greatly incr...

Immobilization of lipases on hydrophobic supports at low ionic strength permits one- step purification, immobilization, hyperactivation, and stabilization of most lipases. This selective adsorption occurs because the hydrophobic surface of the supports is able to promote the interfacial activation of the lipases, yielding enzyme preparations having...

An alpha-galactosidase from Thermus sp. T2, a hexameric protein, has been immobilized on cyanogen bromide agarose, retaining its activity almost intact, but without any significant improvement in enzyme stability. In fact, enzyme subunits could be desorbed from the immobilized preparation by boiling the solution in the presence of SDS (detected by...

A very stable α-galactosidase from Thermus sp. T2 has been immobilized on different supports activated with glyoxyl, epoxy or glutaraldehyde groups. Although all preparations retained very high activity (usually over 90%) and all immobilization protocols improved the enzyme stability, the best stability was obtained by immobilization on glutaraldeh...

Classical cordierite monoliths and acicular mullite (ACM) monoliths were used as support material for ionic adsorption of β-galactosidase from Aspergillus oryzae. Monoliths were silica-coated, then functionalized with polyethyleneimine (PEI) using different methods. In addition to direct adsorption of PEI, monoliths were modified with (3-glycidoxyp...

Multipoint covalent immobilization of enzymes (through very short spacer arms) on support surfaces promotes a very interesting 'rigidification' of protein molecules. In this case, the relative positions of each residue of the enzyme involved in the immobilization process have to be preserved unchanged during any conformational change induced on the...

Covalent immobilization of cyclodextrin glycosyltransferase on glyoxyl-agarose beads promotes a very high stabilization of the enzyme against any distorting agent (temperature, pH, organic solvents). For example, the optimized immobilized preparation preserves 90% of initial activity when incubated for 22 h in 30% ethanol at pH 7 and 40 degrees C....