Various Strategies for the Immobilization of a Phospholipase C from Bacillus cereus for the Modulation of Its Biochemical Properties.

In this study, the effect of various immobilization methods on the biochemical properties of phospholipase C (PLC) from Bacillus cereus obtained from the oily soil located in Sfax, Tunisia, was described. Different supports were checked: octyl sepharose, glyoxyl agarose in the presence of N-acetyl cysteine, and Q-sepharose. In the immobilization by hydrophobic adsorption, a hyperactivation of the PLCBc was obtained with a fold of around 2 times. The recovery activity after immobilization on Q-sepharose and glyoxyl agarose in the presence of N-acetyl cysteine was 80% and 58%, respectively. Furthermore, the biochemical characterization showed an important improvement in the three immobilized enzymes. The performance of the various immobilized PLCBc was compared with the soluble enzyme. The derivatives acquired using Q-sepharose, octyl sepharose, and glyoxyl agarose were stable at 50 °C, 60 °C, and 70 °C. Nevertheless, the three derivatives were more stable in a large range of pH than the soluble enzyme. The three derivatives and the free enzyme were stable in 50% (v/v) ethanol, hexane, methanol, and acetone. The glyoxyl agarose derivative showed high long-term storage at 4 °C, with an activity of 60% after 19 days. These results suggest the sustainable biotechnological application of the developed immobilized enzyme.

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. In this study, a glycosyltransferase and a sucrose synthase (as an UDP-regenerating enzyme) were co-immobilized onto porous agarose-based supports coated with polycationic polymers: polyethylenimine and polyallylamine. In addition, the UDP cofactor was strongly ionically adsorbed and co-immobilized with the enzymes, eliminating the need to add it separately. Thus, the optimal self-sufficient heterogeneous biocatalyst was able to catalyze the glycosylation of three polyphenolic compounds (piceid, phloretin and quercetin) with in situ regeneration of the UDP-glucose, allowing multiple consecutive reaction cycles without the addition of exogenous cofactor. A TTN value of 50 (theoretical maximum) was obtained in the reaction of piceid glycosylation, after 5 reaction cycles, using the self-sufficient biocatalyst based on an improved sucrose synthase variant. This result was 5-fold higher than the obtained using soluble cofactor and the co-immobilized enzymes, and much higher than those reported in the literature for similar processes.

Enzymatic synthesis of mono- and disubstituted phospholipids by direct condensation of oleic acid and glycerophosphocholine with immobilized lipases and phospholipase.

Lysophospholipids which contain polyunsaturated fatty acids play a key role in food and cosmetic industries because of their bioactivity. Therefore, the formation of mono- and disubstituted phospholipids is quite interesting as they could be used for the formation of different natural liposomes. Using immobilized derivatives of lipases and phospholipases, the esterification of oleic acid with glycerophosphocholine (GPC) has been studied. Thus, derivatives were quite active in completely anhydrous media and in solvent-free reaction systems where the reaction takes place. CALB biocatalyst was able to successfully form oleoyl-LPC at 60 °C in the presence of 30 % butanone, where the synthesis rate was 100 times higher than in the absence of solvents at 40 °C. On the other hand, the best synthesis rate for dioleoyl-PC was achieved with immobilized Lecitase in a solvent-free process at 60 °C, an 83 % synthesis yield was achieved with an initial synthesis rate of 4.32 mg/mL × h × g.

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. Under the manipulation of the oxidation variables, MNP-conjugated antibody showed the highest Fab accessibility, of 1.32 µmol analyte per µmol antibody, corresponding to 139 µmol aldehyde per gram of nanocarrier (5 mM NaIO4, 4 h). Demonstrating anti-interference advantage up to 10% serum, colorimetric immunoassay gave a detection limit (LOD) of 300 ng mL-1, while electrochemical transduction led to a considerable (680 times) improvement, with a LOD of 0.44 ng mL-1. In addition, polyaldehyde-dextran showed priority over polycarboxylated-dextran as the multivalent antibody crosslinker for MNPs in terms of sensitivity and LOD value, while immunosensors constructed with carboxylated magnetic microbeads (HOOC-MBs) outperformed MNPs-based immunoplatforms. This work sheds light on the importance of surface chemistry (type and density of functional groups) and the dimension (nanosize vs micrometer) of magnetic carriers to conjugate antibodies with better directional orientation and improve the analytical performance of the resulting immunosensors.

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.

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.

Oriented immobilization of antibodies onto sensing platforms - A critical review.

The immunosensor has been proven a versatile tool to detect various analytes, such as food contaminants, pathogenic bacteria, antibiotics and biomarkers related to cancer. To fabricate robust and reproducible immunosensors with high sensitivity, the covalent immobilization of immunoglobulins (IgGs) in a site-specific manner contributes to better performance. Instead of the random IgG orientations result from the direct yet non-selective immobilization techniques, this review for the first time introduces the advances of stepwise yet site-selective conjugation strategies to give better biosensing efficiency. Noncovalently adsorbing IgGs is the first but decisive step to interact specifically with the Fc fragment, then following covalent conjugate can fix this uniform and antigens-favorable orientation irreversibly. In this review, we first categorized this stepwise strategy into two parts based on the different noncovalent interactions, namely adhesive layer-mediated interaction onto homofunctional support and layer-free interaction onto heterofunctional support (which displays several different functionalities on its surface that are capable to interact with IgGs). Further, the influence of ligands characteristics (synthesis strategies, spacer requirements and matrices selection) on the heterofunctional support has also been discussed. Finally, conclusions and future perspectives for the real-world application of stepwise covalent conjugation are discussed. This review provides more insights into the fabrication of high-efficiency immunosensor, and special attention has been devoted to the well-orientation of full-length IgGs onto the sensing platform.

Self-sufficient asymmetric reduction of ß-ketoesters catalysed by a novel and robust thermophilic alcohol dehydrogenase co-immobilised with NADH.

ß-Hydroxyesters are essential building blocks utilised by the pharmaceutical and food industries in the synthesis of functional products. Beyond the conventional production methods based on chemical catalysis or whole-cell synthesis, the asymmetric reduction of ß-ketoesters with cell-free enzymes is gaining relevance. To this end, a novel thermophilic (S)-3-hydroxybutyryl-CoA dehydrogenase from Thermus thermophilus HB27 (Tt27-HBDH) has been expressed, purified and biochemically characterised, determining its substrate specificity towards ß-ketoesters and its dependence on NADH as a cofactor. The immobilization of Tt27-HBDH on agarose macroporous beads and its subsequent coating with polyethyleneimine has been found the best strategy to increase the stability and workability of the heterogeneous biocatalyst. Furthermore, we have embedded NADH in the cationic layer attached to the porous surface of the carrier. Since Tt27-HBDH catalyses cofactor recycling through 2-propanol oxidation, we achieve a self-sufficient heterogeneous biocatalyst where NADH is available for the immobilised enzymes but its lixiviation to the reaction bulk is avoided. Taking advantage of the autofluorescence of NADH, we demonstrate the activity of the enzyme towards the immobilised cofactor through single-particle analysis. Finally, we tested the operational stability in the asymmetric reduction of ß-ketoesters in batch, succeeding in the reuse of both the enzyme and the co-immobilised cofactor up to 10 reaction cycles.

Oriented immobilization of antibodies through different surface regions containing amino groups: Selective immobilization through the bottom of the Fc region.

Amino groups on the antibody surface (amino terminus and Lys) are very interesting conjugation targets due to their substantial quantities and selectivity toward various reactive groups. Oriented immobilization of antibodies via amino moieties on the Fc region instead of the antigen-binding fragment (Fab) is highly appreciated to conserve antigen-binding capacity. In this paper, targeting amino moieties on distinct regions, three antibody immobilization strategies were compared with the recognition ability of corresponding adsorbents. Our results demonstrate that oriented immobilization of antibodies onto heterofunctional chelate-epoxy support selectively involving Lys residues placed at the bottom of the Fc region, thus preserved the highest antigen recognition capacity (over 75% functionality). For homofunctional aldehyde support, immobilization at pH 10 demonstrates 50% remaining functionality due to the random orientation of tethered antibodies; while only 10% functionality remained when N-terminus were specifically conjugated at pH 8.5. With the rationalization of moieties density onto heterofunctional support, 2-fold recognition capacity was exhibited over randomly immobilization for antigens with higher size (ß-galactosidase, 425 kDa vs. horseradish peroxidase, 40 kDa). Meanwhile, at least 97% of antigens with a varied concentration in diluted human serum were efficiently captured by the optimized chelate-epoxy support. Therefore, our antibody immobilization protocol proved the potential to be utilized as a promising candidate to capture voluminous antigens (large proteins and cells) in real samples.

Optimization of theoretical maximal quantity of cells to immobilize on solid supports in the rational design of immobilized derivatives strategy.

Current worldwide challenges are to increase the food production and decrease the environmental contamination by industrial emissions. For this, bacteria can produce plant growth promoter phytohormones and mediate the bioremediation of sewage by heavy metals removal. We developed a Rational Design of Immobilized Derivatives (RDID) strategy, applicable for protein, spore and cell immobilization and implemented in the RDID1.0 software. In this work, we propose new algorithms to optimize the theoretical maximal quantity of cells to immobilize (tMQCell) on solid supports, implemented in the RDIDCell software. The main modifications to the preexisting algorithms are related to the sphere packing theory and exclusive immobilization on the support surface. We experimentally validated the new tMQCell parameter by electrostatic immobilization of ten microbial strains on AMBERJET® 4200 Cl- porous solid support. All predicted tMQCell match the practical maximal quantity of cells to immobilize with a 10% confidence. The values predicted by the RDIDCell software are more accurate than the values predicted by the RDID1.0 software. 3-indolacetic acid (IAA) production by one bacterial immobilized derivative was higher (~ 2.6 µg IAA-like indoles/108 cells) than that of the cell suspension (1.5 µg IAA-like indoles/108 cells), and higher than the tryptophan amount added as indole precursor. Another bacterial immobilized derivative was more active (22 µg Cr(III)/108 cells) than the resuspended cells (14.5 µg Cr(III)/108 cells) in bioconversion of Cr(VI) to Cr(III). Optimized RDID strategy can be used to synthesize bacterial immobilized derivatives with useful biotechnological applications.

Capture of enzyme aggregates by covalent immobilization on solid supports. Relevant stabilization of enzymes by aggregation.

In this paper, a novel procedure for the immobilization and stabilization of enzymes is proposed: the multipoint covalent attachment of bi-molecular enzyme aggregates. This immobilization protocol allows the "capture" and fixation of the enzyme aggregate on the support surface. In addition to stabilization by multipoint attachment, enzyme aggregation promotes very interesting stabilizing effects. In the presence of low concentrations of polyethylene glycol (30 %) the dimeric amine oxidase from Pisum sativum forms soluble bi-molecular aggregates. Enzyme aggregates were analyzed by Dynamic Light Scattering and by full chemical loading of a mesoporous support (10 % agarose gels activated with glyoxyl groups). The soluble aggregate was immobilized by multipoint attachment on glyoxyl- agarose at pH 8.5 though the four amino termini of the two dimeric molecules (Lys residues are not reactive at this pH). The immobilized aggregated structure cannot undergo any movement (translational or rotational) after multipoint attachment and the aggregate is "fixed" on the support surface even after the removal of PEG. The immobilized aggregate was further incubated at pH 10 in order to allow the Lys residues to react with the glyoxyl groups on the support. Enzyme aggregation has an important effect on enzyme stabilization: the aggregated derivative was 40 fold more stable than a similar derivative of the isolated enzyme and 200 fold more than native enzymes in experiments of thermal inactivation.

Production of new nanobiocatalysts via immobilization of lipase B from C. antarctica on polyurethane nanosupports for application on food and pharmaceutical industries.

Nanobiocatalysts were produced via immobilization of CalB lipase on polyurethane (PU) based nanoparticles and their application on the synthesis of important industrial products was evaluated. Nanoparticles of polyurethane functionalized with poly(ethylene glycol) (PU-PEG) were synthetized through miniemulsion polymerization and the addition of crosslinking agents were evaluated. The nanoparticles were employed as support for CalB and the kinetic parameters were reported. The performance of new biocatalysts was evaluated on the hydrolysis reaction of p-NPB and on the enantioselective hydrolysis of (R,S)-mandelic acid. The esterification reaction was evaluated on the production of ethyl esters of Omega-3. The effect of poly(ethylene glycol) molar mass (400, 4000 or 6000 Da)on the biocatalyst activity was also analyzed. The PU-PEG6000-CalB showed the highest value of the kinetic parameters, highlighting the high reaction rate. The addition of trehalose as crosslinking agent improved the thermal stability of the biocatalysts. PU-PEG400-CalB was the most active nanobiocatalyst, exhibiting a ethyl esters production of 43.72 and 16.83 mM.U -1 using EPA and DHA, respectively. The nanobiocatalyst was also applied in enantiomeric resolution of mandelic acid, showing promising enantiomeric ratios. The results obtained in this work present alternative and sustainable routes for the synthesis of important compounds used on food and pharmaceutical industries.

Stabilization of Glycosylated ß-Glucosidase by Intramolecular Crosslinking Between Oxidized Glycosidic Chains and Lysine Residues.

Many industrial enzymes can be highly glycosylated, including the ß-glucosidase enzymes. Although glycosylation plays an important role in many biological processes, such chains can cause problems in the multipoint immobilization techniques of the enzymes, since the glycosylated chains can cover the reactive groups of the protein (e.g., Lys) and do not allow those groups to react with reactive groups of the support (e.g., aldehyde and epoxy groups). Nevertheless, the activated glycosylated chains can be used as excellent crosslinking agents. The glycosylated chains when oxidized with periodate can generate aldehyde groups capable of reacting with the amino groups of the protein itself. Such intramolecular crosslinks may have significant stabilizing effects. In this study, we investigated if the intramolecular crosslinking occurs in the oxidized ß-glucosidase and its effect on the stability of the enzyme. For this, the oxidation of glycosidic chains of ß-glucosidase was carried out, allowing to demonstrate the formation of aldehyde groups and subsequent interaction with the amine groups and to verify the stability of the different forms of free enzyme (glycosylated and oxidized). Furthermore, we verified the influence of the glycosidic chains on the immobilization of ß-glucosidase from Aspergillus niger and on the consequent stabilization. The results suggest that intramolecular crosslinking occurred and consequently the oxidized enzyme showed a much greater stabilization than the native enzyme (glycosylated). When the multipoint immobilization was performed in amino-epoxy-agarose supports, the stabilization of the oxidized enzyme increases by a 6-fold factor. The overall stabilization strategy was capable to promote an enzyme stabilization of 120-fold regarding to the soluble unmodified enzyme.

A mild intensity of the enzyme-support multi-point attachment promotes the optimal stabilization of mesophilic multimeric enzymes: Amine oxidase from Pisum sativum.

Stabilization of dimeric enzymes requires the stabilization of the quaternary structure as well as the 3D one. Both subunits may be easily immobilized on a highly activated support. Additional stabilization of the 3D structure may be achieved via multipoint covalent attachment (MCA) on highly activated supports. In the case of monomeric enzymes or thermophilic dimeric ones, the optimal stabilization is obtained via the most intense MCA and it is associated to a small loss of catalytic activity. However, in the case of mesophilic enzymes, a very intense MCA of both subunits may promote negative effects, e.g., associated to distortions of the assembly between subunits and a subsequent very important loss of catalytic activity. A dimeric mesophilic amine oxidase from P.sativum was stabilized by MCA on glyoxyl-agarose. Both subunits were covalently immobilized on the support through the region with the highest density in Lys residues. In addition to that, an interesting activity/stabilization binomial was obtained after only 3 h of enzyme-support multiinteraction (50 % of activity/350 fold stabilization). However, after 24 h of enzyme-support multi-interaction this binomial activity-stabilization decreased down to 30/150. A moderate multiinteraction seems to be the optimal strategy for immobilization-stabilization of mesophilic dimeric enzymes and it promotes moderate losses of activity and interesting stabilizations against the combined effect of heat, acid pH and ethanol. The control of the intensity of enzyme-support multi-interactions becomes now strictly necessary.

Coimmobilization and colocalization of a glycosyltransferase and a sucrose synthase greatly improves the recycling of UDP-glucose: Glycosylation of resveratrol 3-O-ß-D-glucoside.

Glycosylation is one of the most efficient biocompatible methodologies to enhance the water solubility of natural products, and therefore their bioavailability. The excellent regio- and stereoselectivity of nucleotide sugar-dependent glycosyltransferases enables single-step glycosylations at specific positions of a broad variety of acceptor molecules without the requirement of protection/deprotection steps. However, the need for stoichiometric quantities of high-cost substrates, UDP-sugars, is a limiting factor for its use at an industrial scale. To overcome this challenge, here we report tailor-made coimmobilization and colocalization procedures to assemble a bi-enzymatic cascade composed of a glycosyltransferase and a sucrose synthase for the regioselective 5-O-ß-D-glycosylation of piceid with in situ cofactor regeneration. Coimmobilization and colocalization of enzymes was achieved by performing slow immobilization of both enzymes inside the porous support. The colocalization of both enzymes within the porous structure of a solid support promoted an increase in the overall stability of the bi-enzymatic system and improved 50-fold the efficiency of piceid glycosylation compared with the non-colocalized biocatalyst. Finally, piceid conversion to resveratrol 3,5-diglucoside was over 90% after 6 cycles using the optimal biocatalyst and was reused in up to 10 batch reaction cycles accumulating a TTN of 91.7 for the UDP recycling.

Correction to: Immobilization of Enzymes and Cells.

Chapter 12 was inadvertently published with the contributing authors listed as Mihaela Badea, Akhtar Hayat, and Jean-Louis Marty, whereas it should have been printed as Audrey Sassolas, Akhtar Hayat, and Jean-Louis Marty. This correction has been updated in the book.

The Science of Enzyme Immobilization.

Protocols for simple immobilization of unstable enzymes are plenty, but the vast majority of them, unfortunately, have not reached their massive implementation for the preparation of improved heterogeneous biocatalyst. In this context, the science of enzyme immobilization demands new protocols capable of fabricating heterogeneous biocatalysts with better properties than the soluble enzymes. The preparation of very stable immobilized biocatalysts enables the following: (1) higher operational times of enzyme, increasing their total turnover numbers; (2) the use of enzymes under non-conventional media (temperatures, solvents, etc.) in order to increase the concentrations of substrates for intensification of processes or in order to shift reaction equilibria; (3) the design of solvent-free reaction systems; and (4) the prevention of microbial contaminations. These benefits gained with the immobilization are critical to scale up chemical processes like the synthesis of biodiesel, synthesis of food additives or soil decontamination, where the cost of the catalysts has an enormous impact on their economic feasibility. The science of enzyme immobilization requires a multidisciplinary focus that involves several areas of knowledge such as, material science, surface chemistry, protein chemistry, biophysics, molecular biology, biocatalysis, and chemical engineering. In this chapter, we will discuss the most relevant aspects to do "the science of enzyme immobilization." We will emphasize the immobilization techniques that promote multivalent attachments between the surface of the enzymes and the porous carriers. Finally, we will discuss the effect that the structural rigidification promotes at different protein regions on the functional properties of the immobilized enzymes.

One-Point Covalent Immobilization of Enzymes on Glyoxyl Agarose with Minimal Physico-Chemical Modification: Immobilized "Native Enzymes".

The immobilization of soluble enzymes inside the porous structure of a preexisting support is one of the most interesting techniques to prepare heterogeneous biocatalysts. The main cause of inactivation of these biocatalysts is the distortion of the tridimensional structure of the immobilized enzymes. In some cases, immobilization of enzymes on preexisting supports can be used in order to improve its functional properties: stabilization by multipoint covalent immobilization, hyper-activation, and stabilization of lipases by interfacial adsorption on hydrophobic supports, etc. In other cases, the properties of the enzyme can be modified by additional interactions of the enzyme surface with the support surface: hydrophobic or electrostatic interactions.In all cases, it would be very interesting to evaluate the intrinsic tridimensional stability of native industrial enzymes. Under drastic experimental conditions, soluble enzymes may undergo undesirable aggregations, and the tridimensional stability of one enzyme is more accurately evaluated by using immobilized native enzymes. That is, immobilized derivatives associated to a minimal chemical modification of the enzyme surface placed in the proximity of a fully hydrophilic and inert support surfaces. In this chapter, the immobilization of enzymes with minimal physicochemical modification on glyoxyl agarose supports is proposed. At pH 8.5, the unique reactive amino group on the enzyme surface is the N-terminus. At the end of the immobilization, mild borohydride reduction, the primary amino terminus is simply converted into a secondary amino group, with similar physical properties, and aldehyde groups on the supports are converted into fully inert hydroxyl groups. The preparation of immobilized derivatives of penicillin G acylase (PGA) with identical properties (activity and stability) that one of the soluble enzyme is reported: preparation of immobilized native PGA.

Multi-Point Covalent Immobilization of Enzymes on Glyoxyl Agarose with Minimal Physico-Chemical Modification: Stabilization of Industrial Enzymes.

Stabilization of enzymes via immobilization techniques is a valuable approach in order to convert a necessary protocol (immobilization) into a very interesting tool to improve key enzyme properties (stabilization). Multipoint covalent attachment of each immobilized enzyme molecule may promote a very interesting stabilizing effect. The relative distances among all enzyme residues involved in immobilization have to remain unaltered during any conformational change induced by any distorting agent. Amino groups are very interesting nucleophiles placed on protein surfaces. The immobilization of enzyme through the region having the highest amount of amino groups (Lys residues) is key for a successful stabilization. Glyoxyl groups are small aliphatic aldehydes that form very unstable Schiff's bases with amino groups, and they do not seem to be useful for enzyme immobilization at neutral pH. However, under alkaline conditions, glyoxyl supports are able to immobilize enzymes via a first multipoint covalent immobilization through the region having the highest amount of lysine groups. Activation of supports with a high surface density of glyoxyl groups and the performance of very intense enzyme-support multipoint covalent attachments are here described.

Multi-Point Covalent Immobilization of Enzymes on Supports Activated with Epoxy Groups: Stabilization of Industrial Enzymes.

Commercial epoxy supports may be very useful tools to stabilize proteins via multipoint covalent attachment if the immobilization is properly designed. In this chapter, a protocol to take full advantage of the support's possibilities is described. The basics of the protocol are as follows: (1) the enzymes are hydrophobically adsorbed on the supports at high ionic strength. (2) There is an "intermolecular" covalent reaction between the adsorbed protein and the supports. (3) The immobilized protein is incubated at alkaline pH to increase the multipoint covalent attachment, thereby stabilizing the enzyme. (4) The hydrophobic surface of the support is hydrophylized by reaction of the remaining groups with amino acids in order to reduce the unfavorable enzyme-support hydrophobic interactions. This strategy has produced a significant increase in the stability of penicillin G acylase compared with the stability achieved using conventional protocols.

Immobilization of Enzymes on Supports Activated with Glutaraldehyde: A Very Simple Immobilization Protocol.

In this chapter, we describe different approaches for the utilization of glutaraldehyde in protein immobilization. First, we focus on the covalent attachment of proteins to glutaraldehyde-activated matrixes. We describe conditions for the synthesis of such supports and provide an example of the immobilization and stabilization of a fructosyltransferase. We also describe how glutaraldehyde may be used for the cross-linking of protein-protein aggregates and protein adsorbed onto amino-activated matrixes. In these cases, glutaraldehyde bridges either two lysine groups from different protein molecules or a lysine from the protein structure and an amine group from the support. Examples of cross-linking are given for the immobilization of a D-amino acid oxidase on different amino-activated supports.