Immobilization of Proteins on Glyoxyl Activated Supports: Dramatic Stabilization of Enzymes by Multipoint Covalent Attachment on Pre-Existing Supports

Fernández‐Lorente, Gloria; López‐Gallego, Fernando; Bolívar, Juan M.; Rocha-Martín, Javier; Moreno-Pérez, Sonia; Guisán, José M.

doi:10.2174/1385272819666150429232725

Cited by 32 publications

(32 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…At high temperatures, free enzymes suffer a slight unfolding, so hydrophobic interphases can interact and the enzymes are inactivated by aggregation. When the proteins are covalently immobilized, they do not undergo this inactivation [19]. This increase in the optimum temperature is an advantage when the substrates are poorly soluble and a slight increase in temperature is necessary to facilitate their dissolution.…”

Section: Efficiency Of the System: Substrate Spectra And Reaction Cyclesmentioning

confidence: 99%

l-Amino Acid Production by a Immobilized Double-Racemase Hydantoinase Process: Improvement and Comparison with a Free Protein System

et al. 2017

View full text Add to dashboard Cite

Protein immobilization is proving to be an environmentally friendly strategy for manufacturing biochemicals at high yields and low production costs. This work describes the optimization of the so-called "double-racemase hydantoinase process," a system of four enzymes used to produce optically pure L-amino acids from a racemic mixture of hydantoins. The four proteins were immobilized separately, and, based on their specific activity, the optimal whole relation was determined. The first enzyme, D,L-hydantoinase, preferably hydrolyzes D-hydantoins from D,L-hydantoins to N-carbamoyl-D-amino acids. The remaining L-hydantoins are racemized by the second enzyme, hydantoin racemase, and continue supplying substrate D-hydantoins to the first enzyme. N-carbamoyl-D-amino acid is racemized in turn to N-carbamoyl-L-amino acid by the third enzyme, carbamoyl racemase. Finally, the N-carbamoyl-L-amino acid is transformed to L-amino acid by the fourth enzyme, L-carbamoylase. Therefore, the product of one enzyme is the substrate of another. Perfect coordination of the four activities is necessary to avoid the accumulation of reaction intermediates and to achieve an adequate rate for commercial purposes. The system has shown a broad pH optimum of 7-9, with a maximum activity at 8 and an optimal temperature of 60 • C. Comparison of the immobilized system with the free protein system showed that the reaction velocity increased for the production of norvaline, norleucine, ABA, and homophenylalanine, while it decreased for L-valine and remained unchanged for L-methionine.

show abstract

Section: Efficiency Of the System: Substrate Spectra And Reaction Cyclesmentioning

confidence: 99%

l-Amino Acid Production by a Immobilized Double-Racemase Hydantoinase Process: Improvement and Comparison with a Free Protein System

et al. 2017

View full text Add to dashboard Cite

show abstract

“…Protein immobilization is a physicochemical strategy that has proven to be a simple and cost-efficient methodology to generate improved biocatalysts [7,8]. The immobilized proteins are usually more stable than the soluble enzyme [9]. Moreover, a well-designed immobilization strategy 2 of 15 could alter the selectivity or specificity of the enzymes [10], and could even remove the substrate or product inhibitory effects [11].…”

Section: Introductionmentioning

confidence: 99%

“…Different methods for enzyme immobilization have been described in the scientific literature in recent years, e.g., encapsulation, entrapment, cross-linking enzyme aggregates or crystals, adsorption and covalent attachment [8,[13][14][15][16]. Among the covalent methods, one of the most effective approaches are enzyme immobilization by multipoint covalent attachment on supports functionalized with glyoxyl (short aliphatic aldehydes) groups [8,9]. This attachment consists of the irreversible immobilization of proteins to an insoluble support such as silica [17], agarose [18], or methacrylic polymers [19,20], magnetic nanoparticles [21], even lignocellulosic wastes [22,23].…”

Section: Introductionmentioning

confidence: 99%

“…One of these supports is cross-linked agarose, which consists of macroporous agarose beads functionalized with glyoxyl groups (GA). This immobilization chemistry promotes a very intense multipoint covalent attachment through Schiff base formation between the aldehydes of the support surface and the non-protonated amino groups of the enzyme surface [9,18]. Moreover, this immobilization protocol can be applied to commercial polyacrylic supports (e.g., Sepabeads and Purolite) containing epoxy groups [20].…”

Section: Introductionmentioning

confidence: 99%

“…and structures (monomeric or multimeric enzymes, cofactor-dependent or cofactor-independent enzymes, etc.) [9,[28][29][30][31][32]. However, one of the main disadvantages of using this immobilization methodology is the reduction step with sodium borohydride (NaBH4) that may adversely affect the enzyme activity.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Stabilization of Enzymes by Multipoint Covalent Attachment on Aldehyde-Supports: 2-Picoline Borane as an Alternative Reducing Agent

et al. 2018

Self Cite

View full text Add to dashboard Cite

Enzyme immobilization by multipoint covalent attachment on supports activated with aliphatic aldehyde groups (e.g., glyoxyl agarose) has proven to be an excellent immobilization technique for enzyme stabilization. Borohydride reduction of immobilized enzymes is necessary to convert enzyme–support linkages into stable secondary amino groups and to convert the remaining aldehyde groups on the support into hydroxy groups. However, the use of borohydride can adversely affect the structure–activity of some immobilized enzymes. For this reason, 2-picoline borane is proposed here as an alternative milder reducing agent, especially, for those enzymes sensitive to borohydride reduction. The immobilization-stabilization parameters of five enzymes from different sources and nature (from monomeric to multimeric enzymes) were compared with those obtained by conventional methodology. The most interesting results were obtained for bacterial (R)-mandelate dehydrogenase (ManDH). Immobilized ManDH reduced with borohydride almost completely lost its catalytic activity (1.5% of expressed activity). In contrast, using 2-picoline borane and blocking the remaining aldehyde groups on the support with glycine allowed for a conjugate with a significant activity of 19.5%. This improved biocatalyst was 357-fold more stable than the soluble enzyme at 50 °C and pH 7. The results show that this alternative methodology can lead to more stable and active biocatalysts.

show abstract

Modeling and experimental validation of covalent immobilization of Trametes maxima laccase on glyoxyl and MANA‐Sepharose CL 4B supports, for the use in bioconversion of residual colorants

Cutiño-Avila

Sánchez-López

Cárdenas-Moreno

et al. 2021

Biotech and App Biochem

View full text Add to dashboard Cite

Our novel strategy for the rational design of immobilized derivatives (RDID) is directed to predict the behavior of the protein immobilized derivative before its synthesis, by the usage of mathematic algorithms and bioinformatics tools. However, this approach needs to be validated for each target enzyme. The objective of this work was to validate the RDID strategy for covalent immobilization of the enzyme laccase from Trametes maxima MUCL 44155 on glyoxyl‐ and monoaminoethyl‐N‐aminoethyl (MANA)‐Sepharose CL 4B supports. Protein surface clusters, more probable configurations of the protein–supports systems at immobilization pHs, immobilized enzyme activity, and protein load were predicted by RDID1.0 software. Afterward, immobilization was performed and predictions were experimentally confirmed. As a result, the laccase‐MANA‐Sepharose CL 4B immobilized derivative is better than laccase‐glyoxyl‐Sepharose CL 4B in predicted immobilized derivative activity (63.6% vs. 29.5%). Activity prediction was confirmed by an experimentally expressed enzymatic activity of 68%, using 2,6‐dimethoxyphenol as substrate. Experimental maximum protein load matches the estimated value (11.2 ± 1.3 vs. 12.1 protein mg/support mL). The laccase‐MANA‐Sepharose CL 4B biocatalyst has a high specificity for the acid blue 62 colorant. The results obtained in this work suggest the possibility of using this biocatalyst for wastewater treatment.

show abstract

Immobilization of Proteins on Glyoxyl Activated Supports: Dramatic Stabilization of Enzymes by Multipoint Covalent Attachment on Pre-Existing Supports

Cited by 32 publications

References 0 publications

l-Amino Acid Production by a Immobilized Double-Racemase Hydantoinase Process: Improvement and Comparison with a Free Protein System

l-Amino Acid Production by a Immobilized Double-Racemase Hydantoinase Process: Improvement and Comparison with a Free Protein System

Stabilization of Enzymes by Multipoint Covalent Attachment on Aldehyde-Supports: 2-Picoline Borane as an Alternative Reducing Agent

Modeling and experimental validation of covalent immobilization of Trametes maxima laccase on glyoxyl and MANA‐Sepharose CL 4B supports, for the use in bioconversion of residual colorants

Contact Info

Product

Resources

About