Alejandro Vian

Complutense University of Madrid | UCM · Department of Chemical Engineering

The gene of a thermostable alpha-galactosidase, aglA, from Thermus sp. strain T2 was sequenced, cloned and overexpressed in Escherichia coli (strain MC 2508. The purified enzyme proved to be quite thermostable, retaining high levels of activity even after incubation at 70°C. The optimal T was 65°C at pH 7. The highest activity was achieved at acid...

Kinetic modelling of the thermal and pH inactivation of the free and an immobilized form of the β-galactosidase from Thermus sp. strain T2 has been carried out. A lacteous buffer containing 50 g L−1 lactose and a commonly used phosphate buffer 50 mM pH 7.2 were employed for the thermal and the pH inactivation studies, respectively. Temperatures cha...

β-Galactosidase from Thermus sp. strain T2 is a promising thermostable enzyme for the hydrolysis of lactose at temperatures from 60 to 70 °C. In this work, thermal and pH stability of such enzyme were investigated in a lacteous buffer containing 50 g L−1 lactose. Temperatures ranged from 60 to 110 °C while pH values cover the pH range for the poten...

A strategy to selectively adsorb large proteins on immobilized metal ion affinity chromatography supports is presented. It is based on the fact that large proteins have a large surface that permits the long distance interaction with groups placed quite far apart (very dispersed onto the support surface) in the support, therefore, even using lowly a...

The heating of protein preparations of mesophilic organism (e.g., E. coli) produces the obliteration of all soluble multimeric proteins from this organism. In this way, if a multimeric enzyme from a thermophilic microorganism is expressed in these mesophilic hosts, the only large protein remaining soluble in the preparation after heating is the the...

Ion-exchange chromatography using commercial ionic supports is a commonly used technique for protein purification. However, selective adsorption of a target protein from a given extract onto commercial ion exchangers seems to be quite complex since they are designed to adsorb the maximum percentage of proteins with the opposite charge. In this pape...

This work exemplifies the advantages of using a battery of new heterofunctional epoxy supports to immobilize enzymes. We have compared the performance of a standard Sepabeads-epoxy support with other Sepabeads-epoxy supports partially modified with boronate, iminodiacetic, metal chelates, and ethylenediamine in the immobilization of the thermostabl...

The β-galactosidase from Thermus sp. T2 (Htag-BgaA) is competitively inhibited by galactose (3.1 mM) and non-competitively inhibited by glucose (49.9 mM). These inhibitions were strongly reduced by immobilization on heterofunctional epoxy Sepabeads (boronate-epoxy-Sepabeads and chelate-epoxy-Sepabeads). The immobilized preparations displayed increa...

A novel thermostable chimeric β-galactosidase was constructed by fusing a poly-His tag to the N-terminal region of the β-galactosidase from Thermus sp. strain T2 to facilitate its overexpression in Escherichia coli and its purification by immobilized metal-ion affinity chromatography (IMAC). The poly-His tag fusion did not affect the activation, ki...

The immobilization of the enzyme β-galactosidase from Thermus sp. T2 was performed via ionic adsorption onto two different supports: a new anionic exchanger resin, based on the coating of Sepabeads internal surfaces with polyethylenimine (PEI) polymers (Mw 25,000), and conventional DEAE-agarose. Immobilization proceeded very rapidly in both cases,...

Using the poly-His-tagged-beta-galactosidase from Thermus sp. strain T2 overexpressed in Escherichia coli (MC1116) as a model enzyme, we have developed a strategy to purify and immobilize proteins in a single step, combining the excellent properties of epoxy groups for enzyme immobilization with the good performance of immobilized metal-chelate aff...

We show that the conformational features of the molecular complexes of E. coli beta-galactosidase and O-glycosides may differ from those formed with closely related compounds in their chemical nature, such as C- and S-glycosyl analogues. In the particular case presented here, NMR and ab initio quantum mechanical results show that the 3D-shapes of t...

New immobilized metal ion affinity chromatography (IMAC) matrices containing a high concentration of metal–chelate moieties and completely coated with inert flexible and hydrophilic dextrans are here proposed to improve the purification of polyhistidine (poly-His) tagged proteins. The purification of an interesting recombinant multimeric enzyme (a...

New immobilized metal ion affinity chromatography (IMAC) matrices containing a high concentration of metal–chelate moieties and completely coated with inert flexible and hydrophilic dextrans are here proposed to improve the purification of polyhistidine (poly-His) tagged proteins. The purification of an interesting recombinant multimeric enzyme (a...

The nucleotide sequence of both the bgaA gene, coding for a thermostable beta-galactosidase of Thermus sp. strain T2, and its flanking regions was determined. The deduced amino acid sequence of the enzyme predicts a polypeptide of 645 amino acids (Mr, 73,595). Comparative analysis of the open reading frames located in the flanking regions of the bg...

The enzyme-bound conformation of C-lactose, an Escherichia coli β-galactosidase inhibitor has been determined by NMR spectroscopy. It is demonstrated that the enzyme selects a high-energy conformation of this closely related structural analogue of the natural substrate, lactose. In addition, a molecular modeling protocol has been performed in order...

The enzyme-bound conformation of C-lactose, an Escherichia coli-galactosidase inhibitor has been determined by NMR spectroscopy. It is demonstrated that the enzyme selects a high-energy conformation of this closely related structural analogue of the natural substrate, lactose. In addition, a molecular modeling protocol has been performed in order t...