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  • The orientation of the immobilized enzyme

    2021-09-14

    The orientation of the immobilized enzyme may affect its activity and stability and therefore the final biocatalyst performance. Since proteins present areas more prone to unfolding in their surface or near to it [36], the immobilization through these areas may improve the final biocatalyst stability. One strategy for modifying the orientation of the enzyme is the immobilization in a two-step process using heterofunctional supports [[37], [38], [39], [40], [41], [42]]. The orientation of the enzyme depends on a first step of physical or chemical intermolecular interaction of its surface and then the immobilization conditions are changed for favoring an intense intramolecular multipoint covalent reaction between the nucleophilic groups of the already immobilized enzyme and the other functional groups of the support [[37], [38], [39], [40], [41], [42]]. This strategy of immobilization has been utilized using agarose [38,40,42] and different commercial supports composed by a methacrylic polymer matrix as carriers [37]. To the best of our knowledge, this strategy of immobilization based on the surface chemistry of chitosan has not been reported before. However, the heterofunctionality of the polymer may be used for the immobilization of stat 3 inhibitor in a two-step process, requiring only the introduction stat 3 inhibitor of one functional group for the formation of covalent bonds between the enzyme and the carrier, since the amino groups of chitosan may be used for a first step of adsorption, even though they may be bonded to the aldehyde groups (Fig. S1, supplementary material). A low ionic strength and a pH lower than the pKa of the carrier amino groups, but higher than the isoelectric point of the enzyme, should be used for favoring this first step of adsorption. In the case of amino-glyoxyl agarose, triethylamine (pKa 10.67 [43]) was utilized for the functionalization of the support with aldehyde and amino groups directly bonded to the matrix, using pH 7 for the enzyme adsorption [38]. In the case of chitosan, a pH lower than 6.5 (pKa of the amino groups ~6.5 [11]) is necessary. After the adsorption of the enzyme, the pH should be changed for favoring the intramolecular covalent reaction between the adsorbed enzyme and the aldehyde or epoxy groups of chitosan. If a chitosan carrier activated only with aldehyde groups is utilized, increasing the pH to 10 should favor the multipoint covalent reaction between the ε-amino groups of the lysine residues of the enzyme and the aldehyde groups of the support, as has been reported previously for amino-glyoxyl agarose [38]. This strategy of immobilization in chitosan reflects the high versatility of this polymer as support for enzyme immobilization; even though, it has not been explored yet. β-Galactosidase has been used for a long time in the dairy industry for hydrolyzing lactose [44], but in recent years it has also been used as catalyst for the synthesis of galacto-oligosaccharides (GOS), which are products with recognized prebiotic condition [45,46]. Transgalactosylation is a kinetically controlled reaction, caused by the competition between the reactions of hydrolysis and synthesis. β-Galactosidase forms a galactosyl-enzyme complex after attacking the anomeric center of the galactose residue in lactose, releasing a glucose molecule to the medium [[47], [48], [49]]. The second step of the reaction depends on the acceptor substrate. If it is water, the galactosyl-enzyme complex undergoes hydrolysis, releasing a galactose molecule to the medium. If the acceptor is lactose, transgalactosylation occurs resulting in the production of GOS, a mixture of disaccharides, trisaccharides, and even higher oligosaccharides characterized by the presence of a terminal glucose with the remaining saccharide units being galactose, and disaccharides comprising two units of galactose [[49], [50], [51]]. The predominance of synthesis over hydrolysis depends mainly on the origin of the β-galactosidase [49,[52], [53], [54]], the initial sugar concentration [55], and the water thermodynamic activity [56,57].