Gabriela Irazoqui

Stabilization of multimeric enzymes via immobilization and post-immobilization techniques

Abstract Controlled and directed immobilization plus post-immobilization techniques are proposed to get full stabilization of the quaternary structure of most multimeric industrial enzymes. The sequential utilization of two stabilization approaches is proposed: (a) Multi-subunit immobilization: a very intense multi-subunit covalent immobilization has been achieved by performing very long immobilization processes between multimeric enzymes and porous supports composed by large internal surfaces and covered by a very dense layer of reactive groups secluded from the support surface through very short spacer arms. (b) Additional cross-linking with poly-functional macromolecules: additional chemical modification of multi-subunit immobilized derivatives with polyfunctional macromolecules promotes an additional cross-linking of all subunits of most of multimeric enzymes. A number of homo and hetero-dimeric enzymes has been stabilized by the simple application of multi-subunit immobilization but more complex multimeric enzymes (e.g., tetrameric ones) were only fully stabilized after the sequential application of both strategies. After such stabilization of the quaternary structure these three features were observed: no subunits were desorbed from derivatives after boiling them in SDS, thermal inactivation becomes independent from enzyme concentration and derivatives became much more stable than soluble enzymes as well as than non-stabilized derivatives. For example, thermal stability of d -amino acid oxidase from Rhodotorula gracilis was increased 7.000 fold after stabilization of its quaternary structure.

Characterization of Galactosyl Derivatives Obtained by Transgalactosylation of Lactose and Different Polyols Using Immobilized β-Galactosidase from Aspergillus oryzae

The synthesis of novel galactosides is interesting because of their important role in several biological processes. Their properties greatly depend upon the configuration and type of galactoside. Therefore, to study biological activity, it is essential to elucidate the structure of the products. Glycosidases are capable of catalyzing glycosidic linkages with absolute stereoselectivity of the anomeric center. We report the enzymatic synthesis of galactosyl-ethylene glycol, galactosyl-glycerol, and galactosyl-erythritol by immobilized beta-galactosidase from Aspegillus oryzae. The obtained galactosides were isolated and fully characterized by an extensive nuclear magnetic resonance (NMR) study. Complete structure elucidation and full proton and carbon assignments were carried out using 1D ((1)H and (13)C) and 2D (gCOSY, TOCSY, multiplicity-edited gHSQC, and gHMBC) NMR experiments. The beta-galactosidase from A. oryzae showed a strong preference for primary alcohols. For galactosyl-glycerol and galactosyl-erythritol, this preference generated one and two chiral centers, respectively, and a mixture of stereoisomers was obtained as a consequence.

Enzymatic synthesis of galactosyl-xylose by Aspergillus oryzae β-galactosidase

In aqueous medium, the reaction catalyzed by Aspergillus oryzae β-galactosidase with O-nitrophenyl-β-D-galactopyranoside (ONPG) in the presence of an acceptor leads to the synthesis of transglycosylation compounds in addition to the hydrolysis products (ONP and galactose). Our goal was to develop a simple system for the synthesis of galactosyl-xylose, a disaccharide of possible application to diagnostics. To maximize synthesis yields, we have studied the effect of several conditions: increase of acceptor concentration (0.05-2.7 M xylose), organic co-solvents (dimethylformamide, acetone) and reaction time. In the absence of co-solvents ONPG was completely consumed in 2 h; with 0.5 M xylose the maximum yield of galactosyl-xylose (16%) was attained at 60 min, while with 2.7 M xylose the yield reached 21%. Both co-solvents tested decreased the kinetics of ONPG convertion into products and 50% (v/v) dimethylformamide was deleterious to the synthesis. However, in 50% (v/v) acetone the synthesis yield was 12% and interestingly, the proportion of transglycosylation with respect to the reacted substrate was higher than in buffer. The synthesis of galactosyl-ethyleneglycol was also studied; it was achieved with extremely high yield and no detectable hydrolysis products. This proves that other acceptor alcohols can be preferred over water in some conditions.

Influence of the immobilization chemistry on the properties of immobilized β-galactosidases

Neutral b-galactosidases from E. coli and K. lactis were bound to glutaraldehyde-agarose Glut-agarose through . amino groups, and to thiolsulfinate-agarose TSI-agarose through thiol groups. In general, TSI-gels exhibited higher yields . . after immobilization 60-85% than Glut-gels 36-40% . The kinetic parameters of the enzymes bound to TSI-gels .particularly those with lower concentration of active groups were less affected than those of the Glut-gels. This might indicate that the binding to TSI-agarose is more conservative of the protein conformation. However, the Glut-derivatives exhibited in general better thermal and solvent stabilities than TSI-derivatives. The stability of the derivatives was studied in . the presence of ethanol, dioxane and acetone 18% vrv . The stabilization of the immobilized enzymes, for some of the solvents assayed, was evidenced by the existence of final very stable enzyme states with high residual activities, thus allowing the utilization of the derivatives in the presence of organic cosolvents. q 2001 Elsevier Science B.V. All rights reserved.

Covalent immobilization of tobacco-etch-virus NIa protease: a useful tool for cleavage of the histidine tag of recombinant proteins

Addition of tags [such as His (histidine) tags] is extremely helpful for the affinity purification of recombinant proteins. In several cases, these tags must be removed before performing functional and structural studies. The enzyme most frequently used to cleave tags of recombinant proteins is the TEV‐protease (tobacco‐etch‐virus NIa protease). The continuous production of this enzyme in soluble form is quite an expensive process and not easily accessible to many laboratories. Thus an interesting alternative is the use of TEV‐protease in an immobilized form, which may be reutilized several times. The main objective of the present study was to obtain a TEV‐protease in an immobilized form, by covalent immobilization on to solid supports through selective use of different amino acid residues, lysine or cysteine. High protein immobilization yields (75–97%) were obtained with both strategies. The TEV‐protease immobilized through its exposed cysteine thiol groups maintained its ability for cleaving a 20 kDa substrate. While the activity of the immobilized TEV‐protease maintained only 30% of the activity of the enzyme in soluble form, its stability at 4 °C was improved three times. Moreover, this enzyme could be reutilized in at least five cycles of cleavage without loss of performance. The present results indicate that the use of a TEV‐protease in an immobilized form is a potentially useful tool for the cleavage of His tags of recombinant proteins and may be useful for reducing the cost of the total process of cleavage.

Enzymatic generation of chitooligosaccharides from chitosan using soluble and immobilized glycosyltransferase (Branchzyme).

Chitooligosaccharides possessing remarkable biological properties can be obtained by enzymatic hydrolysis of chitin. In this work, the chitosanase activity of soluble and immobilized glycosyltransferase (Branchzyme) toward chitosan and biochemical characterization are described for the first time. This enzyme was found to be homotetrameric with a molecular weight of 256 kDa, an isoelectric point of 5.3, and an optimal temperature range of between 50 and 60 °C. It was covalently immobilized to glutaraldehyde-agarose with protein and activity immobilization yields of 67% and 17%, respectively. Immobilization improved enzyme stability, increasing its half-life 5-fold, and allowed enzyme reuse for at least 25 consecutive cycles. The chitosanase activity of Branchzyme on chitosan was similar for the soluble and immobilized forms. The reaction mixture was constituted by chitooligosaccharides with degrees of polymerization of between 2 and 20, with a higher concentration having degrees of polymerization of 3-8.

A novel thermophilic and halophilic esterase from Janibacter sp. R02, the first member of a new lipase family (Family XVII)

Janibacter sp. strain R02 (BNM 560) was isolated in our laboratory from an Antarctic soil sample. A remarkable trait of the strain was its high lipolytic activity, detected in Rhodamine-olive oil supplemented plates. Supernatants of Janibacter sp. R02 displayed superb activity on transesterification of acyl glycerols, thus being a good candidate for lipase prospection. Considering the lack of information concerning lipases of the genus Janibacter, we focused on the identification, cloning, expression and characterization of the extracellular lipases of this strain. By means of sequence alignment and clustering of consensus nucleotide sequences, a DNA fragment of 1272bp was amplified, cloned and expressed in E. coli. The resulting recombinant enzyme, named LipJ2, showed preference for short to medium chain-length substrates, and displayed maximum activity at 80°C and pH 8-9, being strongly activated by a mixture of Na+ and K+. The enzyme presented an outstanding stability regarding both pH and temperature. Bioinformatics analysis of the amino acid sequence of LipJ2 revealed the presence of a consensus catalytic triad and a canonical pentapeptide. However, two additional rare motifs were found in LipJ2: an SXXL β-lactamase motif and two putative Y-type oxyanion holes (YAP). Although some of the previous features could allow assigning LipJ2 to the bacterial lipase families VIII or X, the phylogenetic analysis showed that LipJ2 clusters apart from other members of known lipase families, indicating that the newly isolated Janibacter esterase LipJ2 would be the first characterized member of a new family of bacterial lipases.

Activity and stability of Escherichia coli β-galactosidase in cosolvent systems

The kinetic parameters of E.coli β-galactosidase were not altered by the addition of 2-propanol or ethyl acetate (1.6% v/v). While ethylene glycol (1.6% v/v) doubled the values of both KM (0.29 mM) and kcat (1393 s−), tetraethyleneglycol-dimethylether (Tetraglyme,1.6% v/v) preserved KM, but decreased kcat. At 50°C all the cosolvents dramatically shortened the enzymatic half life, and so did Tetraglyme and 2-propanol at 28°C. At 28°C, both ethyl acetate and ethylene glycol stabilised the enzyme 9- and 6-fold respectively. This fact, together with the activation effect of ethylene glycol may lead to practical applications.

Enzymatic synthesis of 2-aminoethyl β-D-galactopyranoside catalyzed by Aspergillus oryzae β-galactosidase.

Glycosidases provide a powerful resource for in vitro synthesis of novel anomerically pure glycosides. Generation of new low molecular weight galactosides is of interest since they are potential galectin inhibitors. Galectins are molecular targets for cancer therapy and thus their inhibitors are potential antitumor agents. Here we report the enzymatic synthesis and structural characterization of 2-aminoethyl β-D-galactopyranoside. Critical parameters for transgalactosylation using either soluble or immobilized enzyme were investigated and optimized for the galactoside synthesis. We found that 0.2 M lactose, and 0.5 M 2-aminoethanol at 50 °C for 30 min were the optimal conditions for synthesis. 2-Aminoethanol proved to be an enzyme inhibitor, fitting a mixed inhibition model with inhibition constants, K(ic)=0.31±0.04 M and K(iu)=0.604±0.035 M.

Chemical thiolation strategy: A determining factor in the properties of thiol-bound biocatalysts

Immobilization of enzymes on thiolsulphinate-agarose, a thiol-reactive support, is a unique method which allows reversible covalent immobilization under mild conditions, so excellent immobilization and activity yields are obtained. It allows both the formation of stable bonds as well as enzyme desorption and matrix regeneration. The impact of the source of the enzymes thiol group involved in the immobilization (native, reduced disulphide or chemically introduced) on the properties of the resulting biocatalysts was studied using three β-galactosidases from Escherichia coli, Kluyveromices lactis and Aspergillus oryzae as a model. Chemical thiolation, which generates changes at surface exposed lysines, produced derivatives similar to their soluble counterparts. However, the reduction of native disulphide bonds prior to immobilization lead to very variable activity and stability of the derivatives depending on the accessibility and location of the disulphide bonds in the enzyme structure.