Immobilization Protocol Research Articles

Study and deployment of methacrylate-based polymer resins for immobilized lipase catalyzed triglyceride hydrolysis

The performance of microbial lipase from the organism Candida rugosa immobilized onto three methacrylate-based polymer resins was studied. Two of the resins are surface functionalized with epoxy & butyl (ECR8285) and octadecyl (ECR8806M) groups and were compared with an un-functionalized resin (ECR1030M). Sizing of protein using dynamic light scattering technique suggested immobilization was surface dominant. Lipase was immobilized from 0.1 M, pH 7.4 phosphate buffer, at 22 °C for a contact time of 20 h. The resins were characterized using FT-IR spectrometry, N2 adsorption, contact angle measurements and scanning electron microscopy. Continuously stirred-tank batch reactors operated at 100 RPM were used to compare the performance of the immobilized lipase using triglyceride hydrolysis. Octadecyl functionalized methacrylate resins showed superior performance and showed a maximum of 62% yield of FFA on a 100-h time course study. Comparing octadecyl functionalized resins with un-functionalized resins, a four-fold increase in activity retention was observed. Epoxy & butyl functionalized resins showed lower performance compared to octadecyl functionalized resins but higher than un-functionalized resins. Performance in the presence of chemical modifying agents, glutaraldehyde, (3-aminopropyl) triethoxysilane and itaconic acid, applied during immobilization protocol was measured. Superior performance was observed in the case of itaconic acid for functionalized resins.

Modifying alcalase activity and stability by immobilization onto chitosan aiming at the production of bioactive peptides by hydrolysis of tilapia skin gelatin

The protease from Bacillus licheniformis, commercially known as Alcalase®, was insolubilized and stabilized by immobilization onto activated chitosan. Activation with different agents, such as glutaraldehyde (GLU-Chi), glyoxyl (GLY-Chi) and divinyl sulfone (DVS-Chi) was investigated. The effect of the immobilization protocol, for instance different pH and times, were also evaluated. GLU-Chi showed the highest activity (35.6UNPA/g) with the smallest substrate (N-Boc-l-alanine p-nitrophenyl-ester, NPA), while GLY-Chi showed the highest activity (1.5 UAzocasein/g) using the greatest substrate (azocasein). A 24-h immobilization period was enough to stabilize the enzyme using the three supports under almost all conditions. Operational stability in azocasein hydrolysis was assayed and GLU-Chi showed no activity loss during 5 cycles. DVS-Chi retained around 70 % of its initial activity after the fifth cycle, whereas GLY-Chi activity retained only 10 %. Finally, the biocatalysts were used in the hydrolysis of tilapia skin gelatin aiming the production of peptides with antioxidant activity. The protein hydrolysates obtained using GLU-Chi presented the highest antioxidant activity (36.7 μM Trolox Eq). However, the best results of operational stability were obtained using DVS-Chi, which did not lose its initial activity after 3 consecutive cycles of gelatin hydrolysis.

Recent Trends in Biomaterials for Immobilization of Lipases for Application in Non-Conventional Media

The utilization of biomaterials as novel carrier materials for lipase immobilization has been investigated by many research groups over recent years. Biomaterials such as agarose, starch, chitin, chitosan, cellulose, and their derivatives have been extensively studied since they are non-toxic materials, can be obtained from a wide range of sources and are easy to modify, due to the high variety of functional groups on their surfaces. However, although many lipases have been immobilized on biomaterials and have shown potential for application in biocatalysis, special features are required when the biocatalyst is used in non-conventional media, for example, in organic solvents, which are required for most reactions in organic synthesis. In this article, we discuss the use of biomaterials for lipase immobilization, highlighting recent developments in the synthesis and functionalization of biomaterials using different methods. Examples of effective strategies designed to result in improved activity and stability and drawbacks of the different immobilization protocols are discussed. Furthermore, the versatility of different biocatalysts for the production of compounds of interest in organic synthesis is also described.

Combination of intramuscular alfaxalone, butorphanol, and midazolam as a novel immobilization protocol in 3 ring-tailed lemurs (Lemur catta)

BackgroundThere is very little data on the optimal anaesthetic management of ring-tailed lemurs, and the available information is mostly based on extrapolation from other species. In addition, a thorough pre-anaesthetic assessment of lemurs might not be possible without prior chemical immobilization, making a safe immobilization protocol essential.Case presentationThree ring-tailed lemurs (Lemur catta) were immobilized using a combination of intramuscular alfaxalone (5 mg/kg), butorphanol (0.2 mg/kg), and midazolam (0.2 mg/kg), at the University College Dublin Veterinary Hospital. One lemur was anaesthetised once, two lemurs twice, amounting to five anaesthetic events. Conversion to general anaesthesia was warranted in all five occasions, and anaesthesia was maintained with either sevoflurane in oxygen or alfaxalone infusion. The immobilization protocol provided an adequate duration of deep sedation for diagnostic procedures and in some occasions allowed the intubation of the trachea. Analgesia was also provided for minor procedures. No major complications were noted with the protocol used.ConclusionsThe combination of intramuscular alfaxalone, butorphanol and midazolam provided a clinically useful sedation/immobilization in ring-tailed lemurs with only minor complications such as mild hypothermia, hypotension, hypoventilation and bradycardia. This protocol could be considered in ring-tailed lemurs that need to be immobilized for minor procedures, or as a pre-anaesthetic premedication, especially if a full pre-anaesthetic clinical exam is not possible.

One-pot, direct glucose detection in human whole blood without using a dilution factor by a magnetic nanozyme with dual enzymatic activity

The necessity of sample pretreatment, the limited absorbance variation in the concentration range of calibration plot are the common restrictions of the colorimetric glucose sensors used with the complex samples. A calibration plot in a concentration range far below the common level of complex biological sample mostly involves to use a high dilution factor for finding the actual glucose level in the complex sample. Glucose oxidase immobilized-Au nanoparticle attached-magnetic SiO2 (GOx@Au@MagSiO2) microspheres were proposed as a new magnetic nanozyme, allowing one-pot and direct determination of glucose concentration in human whole blood without using a sample pretreatment and a high dilution factor. As an unusual approach which is not coherent with the common strategy followed for colorimetric determination of blood glucose level, a calibration plot based on the common range of human whole blood (i.e. 2.8–38.9 mM) is generated with the synthesized nanozyme. GOx was directly linked onto Au nanoparticles (Au NPs) immobilized on the magnetic SiO2 microspheres via a simple protocol, using the thiol groups of its cysteine residues, as a unique property of GOx’s, for the first time. The activity of free GOx could be highly preserved by the selected immobilization protocol. GOx@Au@MagSiO2 microspheres exhibited peroxidase-like activity due to Au NPs immobilized on the magnetic microspheres, together with the glucose oxidation ability originated from GOx linked to Au NPs. The nanozyme was efficiently used for one-pot, direct determination of glucose level in human whole blood in the concentration range of 50–700 mg/dL (i.e. 2.8–38.9 mM).

Robust Magnetized Oil Palm Leaves Ash Nanosilica Composite as Lipase Support: Immobilization Protocol and Efficacy Study.

Strategies to immobilize the individual enzymes are crucial for enhancing catalytic applicability and require a controlled immobilization process. Herein, protocol for immobilizing Candida rugosa lipase (CRL) onto modified magnetic silica derived from oil palm leaves ash (OPLA) was optimized for the effects of concentration of CRL, immobilization time, and temperature, monitored by titrimetric and spectrometric methods. XRD and TGA-DTG spectrometric observations indicated that OPLA-silica was well coated over magnetite (SiO2-MNPs) and CRLs were uniformly bound by covalent bonds to SiO2-MNPs (CRL/Gl-A-SiO2-MNPs). The optimized immobilization protocol showed that in the preparation of CRL/Gl-A-SiO2-MNPs, CRL with 68.3 mg/g protein loading and 74.6 U/g specific activity was achieved using 5 mg/mL of CRL, with an immobilization time of 12 h at 25 °C. The present work also demonstrated that acid-pretreated OPLA is a potential source of renewable silica, envisioning its applicability for practical use in enzymatic catalysis on solid support.

Impact of Silanization Parameters and Antibody Immobilization Strategy on Binding Capacity of Photonic Ring Resonators.

Ring resonator-based biosensors have found widespread application as the transducing principle in “lab-on-a-chip” platforms due to their sensitivity, small size and support for multiplexed sensing. Their sensitivity is, however, not inherently selective towards biomarkers, and surface functionalization of the sensors is key in transforming the sensitivity to be specific for a particular biomarker. There is currently no consensus on process parameters for optimized functionalization of these sensors. Moreover, the procedures are typically optimized on flat silicon oxide substrates as test systems prior to applying the procedure to the actual sensor. Here we present what is, to our knowledge, the first comparison of optimization of silanization on flat silicon oxide substrates to results of protein capture on sensors where all parameters of two conjugation protocols are tested on both platforms. The conjugation protocols differed in the chosen silanization solvents and protein immobilization strategy. The data show that selection of acetic acid as the solvent in the silanization step generally yields a higher protein binding capacity for C-reactive protein (CRP) onto anti-CRP functionalized ring resonator sensors than using ethanol as the solvent. Furthermore, using the BS3 linker resulted in more consistent protein binding capacity across the silanization parameters tested. Overall, the data indicate that selection of parameters in the silanization and immobilization protocols harbor potential for improved biosensor binding capacity and should therefore be included as an essential part of the biosensor development process.

Organic-based field effect transistors for protein detection fabricated by inkjet-printing

Abstract Biosensors based on Organic Field-Effect Transistors (OFETs) have attracted increasing attention due to the possibility of rapid, label-free, and inexpensive detection. Among all the different possibilities, inkjet-printed top-gate organic Field Effect Transistors-Based Biosensors (BioFETs) using a polymeric gate insulator have been seldom reported. In this work, a systematic investigation in terms of topographical and electrical characterization was carried out in order to find the optimal fabrication process for obtaining a reliable polymer insulator. Previous studies have demonstrated that the best electrical performance arises from the use of the perfluoropolymer Cytop™[12,13,14]. Consequently, a simple immobilization protocol was used to ensure the proper attachment of a model biomolecule onto the Cytop's hydrophobic surface whilst keeping its remarkable insulating properties with gate current in the range of dozens of pico-amperes. The top-gate inkjet-printed BioFETs presented in this study operate at threshold voltages in the range of 1–2 V and show durability even when exposed to oxygen plasma, wet amine functionalization treatments, and aqueous media. As a preliminary application, the inkjet-printed top-gate BioFETs is used for monitoring an immunoreaction by measuring changes in the drain current, paving the way for further use of this device in the immunosensing field.

In vitro attenuation of astrocyte activation and neuroinflammation through ibuprofen-doping of poly(3,4-ethylenedioxypyrrole) formulations

Neuroinflammation is often associated with poor functional recovery and may contribute to or initiate the development of severe neurological disorders, such as epilepsy, Parkinson’s disease or Alzheimer’s disease. Ibuprofen (IBU), being one of the most commonly used non-steroidal anti-inflammatory drugs, is known to possess neuroprotective activity and serve as a promising therapeutic for the treatment of neuroinflammation. In this study, the potential of an IBU-loaded poly(3,4-ethylenedioxypyrrole) (PEDOP) matrix has been assessed as a neural interface material with an aim to control astrocyte activation and suppress neuroinflammation in vitro. Three types of drug immobilization protocols were investigated, leading to the fabrication of IBU-loaded PEDOP matrices exhibiting a broad spectrum of electrical characteristics, drug release profiles, as well as biological responses. Among all investigated PEDOP formulations, PEDOP matrices formed through a three-step immobilization protocol exhibited the highest charge storage capacity (30 ± 1 mC/cm2) as well as a double layer capacitance of 645.0 ± 51.1 µF, associated with a relatively enlarged surface area. Demonstrating a total drug loading capacity of 150 µg/ml and a release rate constant of 0.15 1/h, this coating formulation may be employed as a safe electrical conducting drug eluting system.

Abstract 40: Post-Traumatic Limb Immobilization Alters Mesenchymal Stem Cell Fate

Purpose: Traumatic heterotopic ossification (HO) is a debilitating condition where aberrant bone is formed outside the skeleton due to a fate switch of tissue resident mesenchymal/progenitor cells (MSCs). HO can occur after extremity trauma, burns, and extremity surgeries including amputations and joint replacements. No effective preventive strategies exist as the underlying mechanisms have not been elucidated. Though HO forms at sites of mechanical stress, the role of joint mobilization during extremity trauma, healing, and HO formation has not been clearly defined. We hypothesize that movement is central to HO formation via mechanotransductive signaling, and can provide a basis for improving post-trauma guidelines to prevent HO. Methods: HO was induced in mice via a dorsal partial thickness burn with concomitant Achilles tenotomy (B/T). Single cell RNA (scRNA) sequencing was performed prior to injury and on 3, 7, and 21 days post-B/T tissue using 10X genomics and downstream analysis with Seurat R package. scRNA sequencing was performed on immobilized mice 7 days post B/T and compared to that of mobile mice at the same time point. Scores were generated for each cell based on correlations with either osteogenic or adipogenic gene signatures in MSCs from mobile or immobilized mice. B/T was performed in mice of 4 groups (n=3/group): forced run, exercised passive range of motion (ROM), ambulated normally (mobile), or immobilized, and hindlimb bone volume was assessed at 9 weeks post-B/T by MicroCT (uCT). Immunofluorescent (IF) labeling for PDGFRα and pFAK, TAZ, or Perilipin-1 was done on 1 week cross sections and quantified (n=3/group). Results: Single cell clustering showed there are 15 unique clusters, 3 of which are MSC populations with increased expression of mechanotransductive markers such as Ptk2 (FAK), Yap1 (YAP) and Wwtr1 (TAZ). Joint immobilization of the ankle completely inhibited HO formation, therefore, the comparison of mobile and immobile mice was explored. Histology of immobilized mice demonstrated there is decreased mechanotransductive signaling (pFAK and nuclear TAZ) compared to mobile group. Interestingly, we noted increased adipocytes in the immobilized group at 1 week. Comparing scRNA sequencing revealed that MSCs (clusters 2, 3, and 14) from immobile mice correlated with an adipogenic signature compared to mobile MSCs that favored osteogenesis. This finding suggests a cell fate shift towards adipogenesis with joint immobilization. Conclusion: Hindlimb immobilization plays a significant role altering mechanotransductive pathways which we demonstrate results in an shift in MSC differentiation programming from endochondral ossification to adipogenesis. Immobilization protocols should be considered in patients at high HO risk.

SPR Analysis of Protein-Protein Interactions Involving Cytochromes P450 and Cytochrome b5 Integrated into Lipid Membrane

Identification of new protein-protein interactions (PPI) and characterization of quantitative parameters of complex formation represent one of central tasks of modern protein interactomics. This study is a logical continuation of the cycle of our previous works aimed at the PPIs between components of the cytochrome P450-dependent monooxygenase system. Using an optical biosensor employing surface plasmon resonance (SPR biosensor), a comparative analysis characterizing kinetic and equilibrium parameters of complex formation between the membrane-bound hemoprotein cytochrome b5 and cytochrome P450s was performed using two different protocols for protein immobilization: (1) covalent non-oriented immobilization on the carboxymethyl dextran chip and (2) non-covalent oriented immobilization in the lipid environment. In the case of the second protocol, PPIs were characterized by 2.5-fold higher affinity due to a decrease in rate dissociation constants values of the studied complexes.

Influence of phosphate anions on the stability of immobilized enzymes. Effect of enzyme nature, immobilization protocol and inactivation conditions

A destabilizing effect at pH 7 of sodium phosphate on several lipases immobilized via interfacial activation is shown in this work. This paper investigates if this destabilizing effect is extended to other inactivation conditions, immobilization protocols or even other immobilized enzymes (ficin, trypsin, β-galactosidase, β-glucosidase, laccase, glucose oxidase and catalase). As lipases, those from Candida antarctica (A and B), Candida rugosa and Rhizomucor miehei have been used. Results confirm the very negative effect of 100 mM sodium phosphate at pH 7.0 for the stability of all studied lipases immobilized on octyl agarose, while using glutaraldehyde-support the effect is smaller (still very significant using CALA) and in some cases the effect disappeared (e.g., using CALB). The change of the pH to 5.0 or 9.0, or the addition of 1 M NaCl reduced the negative effect of the phosphate in some instances (e.g., at pH 5.0, this negative effect is only relevant for CALB). Regarding the other enzymes, only the monomeric β-galactosidase from Aspergillus oryzae is strongly destabilized by the phosphate buffer. This way, the immobilization protocol and the inactivation conditions strongly modulate the negative effect of sodium phosphate on the stability of immobilized lipases, and this effect is not extended to other enzymes.

Hydroxyapatite nanoparticles modified with metal ions for xylanase immobilization

Hydroxyapatite (HA) nanoparticles are promising materials for enzyme immobilization, since they provide a high specific surface area for enzyme loading and can also be modified with metal ions (HA-Me2+) to enable interaction with proteins. The adsorption of proteins on HA-Me2+ has been explored for purification purposes, while the use of this material as a support for the immobilization of enzymes remains to be further investigated. Xylanase is an enzyme of considerable industrial interest, being used in the biofuel, pharmaceutical, pulp, and food & beverage sectors, among others. The immobilization of xylanase can enable recovery of the enzyme after biocatalysis, so that it can be reused several times, hence reducing the costs of industrial processes. Here, a systematic study was performed of the immobilization of xylanase on HA nanoparticles modified with metal ions (Cu2+ and Ni2+). A simple, fast, and efficient immobilization protocol was established using statistical experimental design as a tool, generating derivatives by interactions involving complexation of metals of the support with electron donor groups of the enzyme. The affinity towards xylanase was higher for the HA-Cu2+ support, compared to HA and HA-Ni2+. The pH and temperature profiles for the immobilized enzyme activity remained the same as for the soluble enzyme, indicating that the xylanase did not undergo major changes in its conformational state after immobilization. The HA-Cu2+ support was the most effective in reuse assays, retaining up to 80% activity in the second cycle. The results showed that xylanase could be immobilized on HA nanoparticles modified with Cu2+ and Ni2+ metal ions, using a simple and effective method, indicating the promising potential of the system for applications in different industrial processes.

Fine Modulation of the Catalytic Properties of Rhizomucor miehei Lipase Driven by Different Immobilization Strategies for the Selective Hydrolysis of Fish Oil.

Functional properties of each enzyme strictly depend on immobilization protocol used for linking enzyme and carrier. Different strategies were applied to prepare the immobilized derivatives of Rhizomucor miehei lipase (RML) and chemically aminated RML (NH2-RML). Both RML and NH2-RML forms were covalently immobilized on glyoxyl sepharose (Gx-RML and Gx-NH2-RML), glyoxyl sepharose dithiothreitol (Gx-DTT-RML and Gx-DTT-NH2-RML), activated sepharose with cyanogen bromide (CNBr-RML and CNBr-NH2-RML) and heterofunctional epoxy support partially modified with iminodiacetic acid (epoxy-IDA-RML and epoxy-IDA-NH2-RML). Immobilization varied from 11% up to 88% yields producing specific activities ranging from 0.5 up to 1.9 UI/mg. Great improvement in thermal stability for Gx-DTT-NH2-RML and epoxy-IDA-NH2-RML derivatives was obtained by retaining 49% and 37% of their initial activities at 70 °C, respectively. The regioselectivity of each derivative was also examined in hydrolysis of fish oil at three different conditions. All the derivatives were selective between cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) in favor of EPA. The highest selectivity (32.9 folds) was observed for epoxy-IDA-NH2-RML derivative in the hydrolysis reaction performed at pH 5 and 4 °C. Recyclability study showed good capability of the immobilized biocatalysts to be used repeatedly, retaining 50–91% of their initial activities after five cycles of the reaction.

Lipase Immobilized on a Novel Rigid–Flexible Dendrimer-Grafted Hierarchically Porous Magnetic Microspheres for Effective Resolution of (R,S)-1-Phenylethanol

With the rapid development of biotechnological industry, there is an urgent need for exploiting new materials to immobilize enzymes to improve the performance of biocatalysts. In this paper, hierarchically porous magnetic microspheres (PFMMs) were prepared through solvothermal method and rapidly grafted with a novel rigid-flexible dendrimer first synthesized from monomers of trimesoyl chloride (TMC) and 1,6-hexanediamine (HDA) via interfacial polymerization process for covalent immobilization of Pseudomonas fluorescens lipase (PFL). The maximum PFL loading of the synthesized support reaches 87.5 mgprotein/gsupport, and 864% activity recovery of PFMMs-G3.0-PFL can be achieved at pH 9.0. Then, it was used to catalyze the resolution of (R,S)-1-phenylethanol with vinyl acetate. Under the optimized conditions, 50.0% conversion with 99.0% ees can be reached within 1.5 h. In addition, a conversion of 49.2% and ees of 96.9% can be retained after 10 batches of running, displaying an excellent operational stability. Importantly, a further investigation shows that the obviously improved reusability of the immobilized PFL is ascribed to the increased rigidity in comparison to fully flexible dendrimer. Thus, the newly constructed protocol for lipase immobilization exhibits a great prospect in biochemical engineering.

Covalent Immobilization of β-Glucosidase into Mesoporous Silica Nanoparticles from Anhydrous Acetone Enhances Its Catalytic Performance.

An immobilization protocol of a model enzyme into silica nanoparticles was applied. This protocol exploited the use of the bifunctional molecule triethoxysilylpropylisocyanate (TEPI) for covalent binding through a linker of suitable length. The enzyme β-glucosidase (BG) was anchored onto wrinkled silica nanoparticles (WSNs). BG represents a bottleneck in the conversion of lignocellulosic biomass into biofuels through cellulose hydrolysis and fermentation. The key aspect of the procedure was the use of an organic solvent (anhydrous acetone) in which the enzyme was not soluble. This aimed to restrict its conformational changes and thus preserve its native structure. This approach led to a biocatalyst with improved thermal stability, characterized by high immobilization efficiency and yield. It was found that the apparent KM value was about half of that of the free enzyme. The Vmax was about the same than that of the free enzyme. The biocatalyst showed a high operational stability, losing only 30% of its activity after seven reuses.

Immobilization of Cells on Polyurethane Foam.

In this chapter, protocols and details for the immobilization of a model cell onto polyurethane foam carriers are provided in order to facilitate the use of such systems in laboratory or industrial reactors. Polyurethane foam has recently acquired great relevance as a carrier for its good mechanical properties, high porosity, and large adsorption surface. In addition, it has a very low commercial cost. Two different immobilization protocols have been described, differing in the flow regime or the possibilities for the reactor: immobilization in a stirred tank reactor working in a discontinuous regime (by cycles) and immobilization in a packed column working in continuous operation mode. Protocols for carrier sterilization, analytical methodology, and immobilization are described.

Dexmedetomidine and ketamine simultaneous administration in tigers (Panthera tigris): pharmacokinetics and clinical effects.

BackgroundThe study determines the pharmacokinetic profiles of dexmedetomidine (DEX), ketamine (KET) and its active metabolite, norketamine (NORKET), after simultaneous administration. Moreover, the study evaluates the sedative effects of this protocol, its influence on the main physiological variables and the occurrence of adverse effects.MethodsEighteen captive tigers were initially administered with a mixture of DEX (10 µg/kg) and KET (2 mg/kg) by remote intramuscular injection. In case of individual and specific needs, the protocol was modified and tigers could receive general anaesthesia, propofol or additional doses of DEX and KET.ResultsBased on the immobilisation protocol, nine animals were assigned to the standard protocol group and the other nine to the non-standard protocol group. Higher area under the first moment curve (AUMC0-last) and longer mean residence time (MRT0-last) (P<0.05) were observed in the non-standard protocol group for DEX, KET and NORKET, and higher area under the concentration-time curve from administration to the last measurable concentration (AUC0-last) only for KET. The KET metabolisation rate was similar (P=0.296) between groups. No differences between groups were detected in terms of stages of sedation and recoveries. All physiological variables remained within normality ranges during the whole observation period. During the hospitalisation period, no severe adverse reactions and signs of resedation were observed.ConclusionThe simultaneous administration of 10 µg/kg of DEX and 2 mg/kg of KET can be considered an effective protocol for chemical immobilisation of captive tigers, along with dosage adjusments or when other drugs are needed.

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.

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.