Instituto Universitario Of Nanociencia Of Aragon

Molina de Aragón, Spain

Instituto Universitario Of Nanociencia Of Aragon

Molina de Aragón, Spain

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Godoy C.A.,CSIC - Institute of Catalysis | Rivas B.D.L.,Institute Fermentaciones Industriales | Grazu V.,CSIC - Institute of Catalysis | Grazu V.,Instituto Universitario Of Nanociencia Of Aragon | And 3 more authors.
Biomacromolecules | Year: 2011

A new strategy has been developed for site-directed immobilization/ rigidification of genetically modified enzymes through multipoint covalent attachment on bifunctional disulfide-glyoxyl supports. Here the mechanism is described as a two-step immobilization/rigidification protocol where the enzyme is directly immobilized by thiol-disulfide exchange between the β-thiol of the single genetically introduced cysteine and the few disulfide groups presented on the support surface (3 μmol/g). Afterward, the enzyme is uniquely rigidified by multipoint covalent attachment (MCA) between the lysine residues in the vicinity of the introduced cysteine and the many glyoxyl groups (220 μmol/g) on the support surface. Both site-directed immobilization and rigidification have been possible only on these novel bifunctional supports. In fact, this technology has made possible to elucidate the protein regions where rigidification by MCA promoted higher protein stabilizations. Hence, rigidification of vicinity of position 333 from lipase 2 from Geobacillus thermocatenulatus (BTL2) promoted a stabilization factor of 33 regarding the unipunctual site-directed immobilized derivative. In the same context, rigidification of penicillin G acylase from E. coli (PGA) through position β201 resulted in a stabilization factor of 1069. Remarkably, when PGA was site-directed rigidified through that position, it presented a half-life time of 140 h under 60% (v/v) of dioxane and 4°C, meaning a derivative eight times more stable than the PGA randomly immobilized on glyoxyl-disulfide agarose. Herein we have opened a new scenario to optimize the stabilization of proteins via multipoint covalent immobilization, which may represent a breakthrough in tailor-made tridimensional rigidification of proteins. © 2011 American Chemical Society.


Lomba M.,University of Zaragoza | Oriol L.,University of Zaragoza | Sanchez C.,University of Zaragoza | Grazu V.,Instituto Universitario Of Nanociencia Of Aragon | And 4 more authors.
Carbohydrate Polymers | Year: 2012

Chemically unmodified sodium hyaluronate has been crosslinked by photoinduced decomposition of a trifunctional diazonium salt to generate new biomaterials. In addition, the photocrosslinking process does not require a photoinitiator. Thin films of formulations of sodium hyaluronate and the photocrosslinker at different percentages have been processed. Cytotoxicity has been explored and toxicity was not observed with the selected cell lines. 2D patterns of controlled geometry have been generated by direct laser writing to perform cell adhesion studies. Different adhesion behavior of the cell lines, as assessed by vinculin immunostaining and scanning electron microscopy, has been observed in the polymeric films depending on the degree of photocrosslinking. © 2012 Elsevier Ltd.


Grazu V.,CSIC - Institute of Catalysis | Grazu V.,Instituto Universitario Of Nanociencia Of Aragon | Lopez-Gallego F.,CSIC - Institute of Catalysis | Montes T.,CSIC - Institute of Catalysis | And 6 more authors.
Process Biochemistry | Year: 2010

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 regions which were rich in Lys residues. All variants exhibited a similar activity and stability compared to those of the native enzyme. Each variant was immobilized on supports having a low concentration of reactive disulfide moieties and a high concentration of poorly reactive epoxy groups. After immobilization at pH 7.0 by site-directed thiol-disulfide intermolecular exchange, derivatives were further incubated at pH 10.0 for 48 h to promote an additional intramolecular reaction between Lys residues of enzyme and epoxy groups of the support. The establishment of at least three covalent attachments per PGA molecule was determined for all immobilized enzyme variants. The different derivatives exhibited diverse stability against several distorting agents and different selectivity in two interesting reactions. The derivative of the PGA variant obtained by replacement of GlnB380 by Cys was the most stable against heat and organic cosolvents: it preserved 90% of the initial activity and was 30-fold more stable than soluble PGA. This derivative also exhibited an improved enantioselectivity in the hydrolysis of chiral esters (E was improved from 8 to 16) and in kinetically controlled synthesis of amides (synthetic yields were increased from 31 to 49%). © 2009 Elsevier Ltd. All rights reserved.


Lomba M.,University of Zaragoza | Oriol L.,University of Zaragoza | Sanchez-Somolinos C.,University of Zaragoza | Grazu V.,Instituto Universitario Of Nanociencia Of Aragon | And 3 more authors.
Reactive and Functional Polymers | Year: 2013

Aliphatic hyperbranched polyesters of different generation, having different nucleophilic groups (such as hydroxyl or sulfonate) in the periphery, have been photocrosslinked by using a trifunctional low molecular weight diazonium salt. The photocrosslinking process, that does not require photoinitiator, allows to prepare polymeric materials that have shown not to cause cytotoxicity when used as substrate for cell growth. Furthermore, cell adhesion studies carried out in polymeric micropatterns generated in glass by direct laser writing, show that cells preferentially proliferate on the structure demonstrating cell affinity for the material. © 2012 Elsevier Ltd. All rights reserved.


PubMed | University Institute of Health Sciences, University of Zaragoza and Instituto Universitario Of Nanociencia Of Aragon
Type: Journal Article | Journal: Carbohydrate polymers | Year: 2014

Chemically unmodified sodium hyaluronate has been crosslinked by photoinduced decomposition of a trifunctional diazonium salt to generate new biomaterials. In addition, the photocrosslinking process does not require a photoinitiator. Thin films of formulations of sodium hyaluronate and the photocrosslinker at different percentages have been processed. Cytotoxicity has been explored and toxicity was not observed with the selected cell lines. 2D patterns of controlled geometry have been generated by direct laser writing to perform cell adhesion studies. Different adhesion behavior of the cell lines, as assessed by vinculin immunostaining and scanning electron microscopy, has been observed in the polymeric films depending on the degree of photocrosslinking.

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