Effect of amine length in the interference of the multipoint covalent immobilization of enzymes on glyoxyl agarose beads

Morellon‐Sterling, Roberto; Siar, El-Hocine; Braham, Sabrina Ait; Andrades, Diandra de; Pedroche, Justo; Millán, Ma del Carmen; Fernández‐Lafuente, Roberto

doi:10.1016/j.jbiotec.2021.02.005

Cited by 23 publications

(5 citation statements)

References 80 publications

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“…For example, it has recently been shown that the presence of compounds bearing primary amino groups may alter the immobilization rate and the final stability of enzymes immobilized in glyoxyl agarose. 439 The problems were more significant when the aminated compounds were larger and their concentrations significant, but even 1 mM of these compounds may affect the immobilized enzyme properties. The aminated compounds may be peptides, amino acids, or amino saccharides that remain in the crude enzyme mixture or even Tris buffer used to store the enzyme.…”

Section: Enzyme Release From the Supportmentioning

confidence: 99%

Is enzyme immobilization a mature discipline? Some critical considerations to capitalize on the benefits of immobilization

2022

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Section: Enzyme Release From the Supportmentioning

confidence: 99%

Is enzyme immobilization a mature discipline? Some critical considerations to capitalize on the benefits of immobilization

2022

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“…Looking to the structure of the enzyme (Figure 1), it can be seen that although the enzyme is rich in Lys residues areas, the polysaccharide chains may hinder the simultaneous interaction of several primary amino groups of the enzyme with the support, thus avoiding the enzyme immobilization in glyoxyl-agarose. The considerable dilution of the enzyme solution (almost 1000 folds) discards the possibility of some aminated compounds on the enzyme solution that could prevent the enzyme immobilization [101].…”

Section: Covalent Immobilization Of Raaeupomentioning

confidence: 99%

Immobilization of the Peroxygenase from Agrocybe aegerita. The Effect of the Immobilization pH on the Features of an Ionically Exchanged Dimeric Peroxygenase

Carballares

Morellon‐Sterling

et al. 2021

Catalysts

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This paper outlines the immobilization of the recombinant dimeric unspecific peroxygenase from Agrocybe aegerita (rAaeUPO). The enzyme was quite stable (remaining unaltered its activity after 35 h at 47 °C and pH 7.0). Phosphate destabilized the enzyme, while glycerol stabilized it. The enzyme was not immobilized on glyoxyl-agarose supports, while it was immobilized albeit in inactive form on vinyl-sulfone-activated supports. rAaeUPO immobilization on glutaraldehyde pre-activated supports gave almost quantitative immobilization yield and retained some activity, but the biocatalyst was very unstable. Its immobilization via anion exchange on PEI supports also produced good immobilization yields, but the rAaeUPO stability dropped. However, using aminated agarose, the enzyme retained stability and activity. The stability of the immobilized enzyme strongly depended on the immobilization pH, being much less stable when rAaeUPO was adsorbed at pH 9.0 than when it was immobilized at pH 7.0 or pH 5.0 (residual activity was almost 0 for the former and 80% for the other preparations), presenting stability very similar to that of the free enzyme. This is a very clear example of how the immobilization pH greatly affects the final biocatalyst performance.

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“…This is an excellent method for improving enzyme stability (1000 fold compared with immobilized derivatives obtained by conventional methods) by multipoint covalent attachment on a pre-existing carrier . Immobilization of a large number of enzymes, viz., trypsin, trypsin, chymotrypsin, and carboxypeptidase A, alcalase, pullulanase, catalases, ACE, glucoamylase, ManDH, PGA and ADH2, Novo-Pro D, D-amino acid oxidase and Xylanase, and Angiotensin-Converting Enzyme, etc., on glyoxal-activated support have been made using protein’s primary amino groups, mostly the ϵ-amino groups in lysine residues. But, the immobilization of pepsin on glyoxal-activated support was scanty; as in its molecular sequence, it contains only one Lys residue, and the reaction needs a pH over 10.5, where the enzyme itself suffers from instability.…”

Section: Introductionmentioning

confidence: 99%

Chemically Bonded Pepsin via Its Inert Center to Diazo Functionalized Silica Gel through Multipoint Attachment Mode: A Way of Restoring Biocatalytic Sustainability over “Wider pH” Range

Hansda,

Mishra,

Ghosh

et al. 2024

Langmuir

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Proteolytic enzymes play a pivotal role in the industry. Still, because of denaturation, the extensive applicability at their level of best catalytic efficiency over a more comprehensive pH range, particularly in alkaline conditions over pH 8, has not been fully developed. On the other hand, enzyme immobilization following a suitable protocol is a long pending issue that determines the conformational stability, specificity, selectivity, enantioselectivity, and activity of the native enzymes at longrange pH. As a bridge between these two findings, in an attempt at a freezing temperature 273−278 K at an alkaline pH, the diazofunctionalized silica gel (SG) surface has been used to rapidly diazo couple pepsin through its inert center, the O-carbon of the phenolic −OH of surface-occupied Tyr residues in a multipoint mode: when all the various protein groups, viz., amino, thiol, phenol, imidazole, carboxy, etc., in the molecular sequence including those belonging to the active sites, remain intact, the inherent inbuilt interactions among themselves remain. Thereby, the macromolecule's global conformation and helicity preserve the status quo. The dimension of the SG-enzyme conjugate confirms as {Si (OSi) where the values of n and y have been determined respectively as 347 and 188. The material performs the catalytic activity much better at 7−8.5 than at pH 2−3.5 and continues for up to six months without any appreciable change.

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Effect of amine length in the interference of the multipoint covalent immobilization of enzymes on glyoxyl agarose beads

Cited by 23 publications

References 80 publications

Is enzyme immobilization a mature discipline? Some critical considerations to capitalize on the benefits of immobilization

Is enzyme immobilization a mature discipline? Some critical considerations to capitalize on the benefits of immobilization

Immobilization of the Peroxygenase from Agrocybe aegerita. The Effect of the Immobilization pH on the Features of an Ionically Exchanged Dimeric Peroxygenase

Chemically Bonded Pepsin via Its Inert Center to Diazo Functionalized Silica Gel through Multipoint Attachment Mode: A Way of Restoring Biocatalytic Sustainability over “Wider pH” Range

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