Effect of the support alkyl chain nature in the functional properties of the immobilized lipases.

Supports coated with amino-hexyl and amino octyl have been prepared from glyoxyl agarose beads and compared in their performance with octyl-agarose to immobilize lipases A and B from Candida antarctica (CALA and CALB). Immobilization courses were similar using all supports, but enzyme release was more difficult using the amino-alkyl supports suggesting a mixed interfacial activation/ionic exchange immobilization. The enzyme activity and specificity (using p-nitrophenyl propionate, triacetin and both isomers of methyl mandelate) greatly depended on the support.

Enzyme loading in the support and medium composition during immobilization alter activity, specificity and stability of octyl agarose-immobilized Eversa Transform.

Eversa Transform (ETL) was immobilized on octyl agarose beads at two different enzymes loadings (1 mg/g and 15 mg/g) under 18 different conditions, including different pH values, buffers, additives (different solvents, Ca, NaCl). Their activity was analyzed at pH 5 and 7 with p-nitrophenyl butyrate and at pH 5 with triacetin, determining also its stability at pH 5 and 7 (in different media). Ca stabilized ETL biocatalysts while phosphate destabilized them.

Optimizing the activation of agarose beads with divinyl sulfone for enzyme immobilization and stabilization.

The focus of the present work is to find the optimal conditions for the activation of agarose beads with divinyl sulfone (DVS). The reactivity of the vinyl sulfone groups in the support was checked by the support capacity to react with ethylamine; via elemental analysis. In addition, trypsin was used as a model enzyme to test the immobilization and stabilization capabilities of the different supports.

Designing mixed cationic/anionic supports to covalently immobilize/stabilize enzymes with high isoelectric point by enzyme adsorption and support-enzyme glutaraldehyde crosslinking.

Ficin fully immobilized on Asp-agarose beads at pH 7 but not on an aminated support. This made enzyme adsorption plus glutaraldehyde modification non-viable for this enzyme. Modifying glyoxyl-agarose beads with mixtures of Asp and 1,6-hexamethylenediamine (HA) at different ratios, mixed anion/cation exchanger supports were built.

Changes in ficin specificity by different substrate proteins promoted by enzyme immobilization.

Ficin extract has been immobilized using different supports: glyoxyl and Aspartic/1,6 hexamethylenediamine (Asp/HA) agarose beads. The latter was later submitted to glutaraldehyde modification to get covalent immobilization. The activities of these 3 kinds of biocatalysts were compared utilizing 4 different substrates, casein, hemoglobin and bovine serum albumin and benzoyl-arginine-p-nitroanilide at pH 7 and 5.

Designing tailor-made steric matters to improve the immobilized ficin specificity for small versus large proteins.

The development of strategies that can permit to adjust the size specificity of immobilized proteases by the generation of steric hindrances may enlarge its applicability. Using as a model ficin immobilized on glyoxyl agarose, two strategies were assayed to generate tailor made steric hindrances. First, ficin has been coimmobilized on supports coated with large proteins (hemoglobin or bovine serum albumin (BSA)).

Support Enzyme Loading Influences the Effect of Aldehyde Dextran Modification on the Specificity of Immobilized Ficin for Large Proteins.

It has been reported that the modification of immobilized glyoxyl-ficin with aldehyde dextran can promote steric hindrances that greatly reduce the activity of the immobilized protease against hemoglobin, while the protease still maintained a reasonable level of activity against casein. In this paper, we studied if this effect may be different depending on the amount of ficin loaded on the support. For this purpose, both the moderately loaded and the overloaded glyoxyl-ficin biocatalysts were prepared and modified with aldehyde dextran.

Tailoring the specificity of ficin versus large hemoglobin and small casein by co-immobilizing inert proteins on the immobilized enzyme layer and further modification with aldehyde dextran.

Ficin has been immobilized at full loading on glyoxyl agarose beads. Then, ficin was blocked with 2,2'-dipyridyldisulfide. To be effective, the modification must be performed in the presence of 0.

Tuning almond lipase features by the buffer used during immobilization: The apparent biocatalysts stability depends on the immobilization and inactivation buffers and the substrate utilized.

The lipase from Prunus dulcis almonds was inactivated under different conditions. At pH 5 and 9, enzyme stability remained similar under the different studied buffers. However, when the inactivation was performed at pH 7, there were some clear differences on enzyme stability depending on the buffer used.

Green Enzymatic Synthesis of Geranyl Butyrate: Process Optimization and Mechanistic Insights.

Flavor esters are organic compounds widely used in the food industry to enhance the aroma and taste of products. However, most chemical processes for the production of these flavoring compounds use toxic organic solvents. Some organic solvents derived from petroleum can leave behind residual traces in food products, which may raise concerns about potential health risks and contamination.

Adsorption features of reduced aminated supports modified with glutaraldehyde: Understanding the heterofunctional features of these supports.

Immobilization of enzymes on aminated supports using the glutaraldehyde chemistry may involve three different interactions, cationic, hydrophobic, and covalent interactions. To try to understand the impact this heterofunctionality, we study the physical adsorption of the beta-galactosidase from Aspergillus niger, on aminated supports (MANAE) and aminated supports with one (MANAE-GLU) or two molecules of glutaraldehyde (MANAE-GLU-GLU). To eliminate the chemical reactivity of the glutaraldehyde, the supports were reduced using sodium borohydride.

The effects of the chemical modification on immobilized lipase features are affected by the enzyme crowding in the support.

In this article, we have analyzed the interactions between enzyme crowding on a given support and its chemical modification (ethylenediamine modification via the carbodiimide route and picryl sulfonic (TNBS) modification of the primary amino groups) on the enzyme activity and stability. Lipase from Thermomyces lanuginosus (TLL) and lipase B from Candida antarctica (CALB) were immobilized on octyl-agarose beads at two very different enzyme loadings, one of them exceeding the capacity of the support, one well under this capacity. Chemical modifications of the highly loaded and lowly loaded biocatalysts gave very different results in terms of activity and stability, which could increase or decrease enzyme activity depending on the enzyme support loading.

Glycosylation of polyphenolic compounds: Design of a self-sufficient biocatalyst by co-immobilization of a glycosyltransferase, a sucrose synthase and the cofactor UDP.

Glycosyltransferases catalyze the regioselective glycosylation of polyphenolic compounds, increasing their solubility without altering their antioxidant properties. Leloir-type glycosyltransferases require UDP-glucose as a cofactor to glycosylate a hydroxyl of the polyphenol, which is expensive and unstable. To simplify these processes for industrial implementation, the preparation of self-sufficient heterogeneous biocatalysts is needed.

Biocatalytic production of biolubricants: Strategies, problems and future trends.

The increasing worries by the inadequate use of energy and the preservation of nature are promoting an increasing interest in the production of biolubricants. After discussing the necessity of producing biolubricants, this review focuses on the production of these interesting molecules through the use of lipases, discussing the different possibilities (esterification of free fatty acids, hydroesterification or transesterification of oils and fats, transesterification of biodiesel with more adequate alcohols, estolides production, modification of fatty acids). The utilization of discarded substrates has special interest due to the double positive ecological impact (e.

Glutaraldehyde modification of lipases immobilized on octyl agarose beads: Roles of the support enzyme loading and chemical amination of the enzyme on the final enzyme features.

Lipase B from Candida antarctica (CALB) and lipase from Thermomyces lanuginosus (TLL) have been immobilized on octyl agarose at low loading and at a loading exceeding the maximum support capacity. Then, the enzymes have been treated with glutaraldehyde and inactivated at pH 7.0 in Tris-HCl, sodium phosphate and HEPES, giving different stabilities.

Immobilization of Penicillin G Acylase on Vinyl Sulfone-Agarose: An Unexpected Effect of the Ionic Strength on the Performance of the Immobilization Process.

Penicillin G acylase (PGA) from was immobilized on vinyl sulfone (VS) agarose. The immobilization of the enzyme failed at all pH values using 50 mM of buffer, while the progressive increase of ionic strength permitted its rapid immobilization under all studied pH values. This suggests that the moderate hydrophobicity of VS groups is enough to transform the VS-agarose in a heterofunctional support, that is, a support bearing hydrophobic features (able to adsorb the proteins) and chemical reactivity (able to give covalent bonds).

Tuning Immobilized Enzyme Features by Combining Solid-Phase Physicochemical Modification and Mineralization.

Lipase B from (CALB) and lipase from (TLL) were immobilized on octyl agarose. Then, the biocatalysts were chemically modified using glutaraldehyde, trinitrobenzenesulfonic acid or ethylenediamine and carbodiimide, or physically coated with ionic polymers, such as polyethylenimine (PEI) and dextran sulfate. These produced alterations of the enzyme activities have, in most cases, negative effects with some substrates and positive with other ones (e.

The immobilization protocol greatly alters the effects of metal phosphate modification on the activity/stability of immobilized lipases.

Mineralization of immobilized enzymes has showed to couple the advantages of both processes. Here, the influence of the immobilization protocol on the effects of mineralization has been investigated. The lipases from Thermomyces lanuginosus and Candida rugosa were immobilized on octyl-, vinyl sulfone (VS) octyl (blocked with different nucleophiles) and glutaraldehyde- (at different pH values) agarose beads.

Tuning Immobilized Commercial Lipase Preparations Features by Simple Treatment with Metallic Phosphate Salts.

Four commercial immobilized lipases biocatalysts have been submitted to modifications with different metal (zinc, cobalt or copper) phosphates to check the effects of this modification on enzyme features. The lipase preparations were LipozymeTL (TLL-IM) (lipase from ), Lipozyme435 (L435) (lipase B from ), LipozymeRM (RML-IM), and LipuraSelect (LS-IM) (both from lipase from ). The modifications greatly altered enzyme specificity, increasing the activity versus some substrates (e.

Stabilization of immobilized lipases by treatment with metallic phosphate salts.

Lipases from Thermomyces lanuginosus (TLL), Rhizomucor miehei (RML), Candida rugosa (CRL), forms A and B of lipase from Candida antarctica (CALA and CALB) and Eversa Transform 2.0 have been immobilized on octyl-agarose beads at two different loads (1 mg/g and saturated support) and treated with phosphate and/or some metallic salts (Zn, Co, Cu). They have been also immobilized on the support modified by the metallic phosphate, usually driving to biocatalyst with lower stability or marginal improvements.

Dextran-coated nanoparticles as immunosensing platforms: Consideration of polyaldehyde density, nanoparticle size and functionality.

Magnetic nanoparticles (MNPs) can be used as antibody carriers in a wide range of immunosensing applications. The conjugation chemistry for preparing antibody-MNP bionanohybrids should assure the nanoparticle's colloidal dispersity, directional conformation and high biofunctionality retention of attached antibodies. In this work, peroxidase (HRP) was selected as model target analyte, and stable antibody-MNP conjugates were prepared using polyaldehyde-dextrans as multivalent linkers, also to prevent nanoparticles agglomeration and steric shielding of non-specific proteins.

Chemical amination of immobilized enzymes for enzyme coimmobilization: Reuse of the most stable immobilized and modified enzyme.

Although Lecitase and the lipase from Thermomyces lanuginosus (TLL) could be coimmobilized on octyl-agarose, the stability of Lecitase was lower than that of TLL causing the user to discard active immobilized TLL when Lecitase was inactivated. Here, we propose the chemical amination of immobilized TLL to ionically exchange Lecitase on immobilized TLL, which should be released to the medium after its inactivation by incubation at high ionic strength. Using conditions where Lecitase was only adsorbed on immobilized TLL after its amination, the combibiocatalyst was produced.

Coimmobilization of lipases exhibiting three very different stability ranges. Reuse of the active enzymes and selective discarding of the inactivated ones.

Lipase B from Candida antarctica (CALB) and lipases from Candida rugosa (CRL) and Rhizomucor miehei (RML) have been coimmobilized on octyl and octyl-Asp agarose beads. CALB was much more stable than CRL, that was significantly more stable than RML. This forces the user to discard immobilized CALB and CRL when only RML has been inactivated, or immobilized CALB when CRL have been inactivated.