Design of Artificial Enzymes Bearing Several Active Centers: New Trends, Opportunities and Problems.

Harnessing enzymes which possess several catalytic activities is a topic where intense research has been carried out, mainly coupled with the development of cascade reactions. This review tries to cover the different possibilities to reach this goal: enzymes with promiscuous activities, fusion enzymes, enzymes + metal catalysts (including metal nanoparticles or site-directed attached organometallic catalyst), enzymes bearing non-canonical amino acids + metal catalysts, design of enzymes bearing a second biological but artificial active center (plurizymes) by coupling enzyme modelling and directed mutagenesis and plurizymes that have been site directed modified in both or in just one active center with an irreversible inhibitor attached to an organometallic catalyst. Some examples of cascade reactions catalyzed by the enzymes bearing several catalytic activities are also described. Finally, some foreseen problems of the use of these multi-activity enzymes are described (mainly related to the balance of the catalytic activities, necessary in many instances, or the different operational stabilities of the different catalytic activities). The design of new multi-activity enzymes (e.g., plurizymes or modified plurizymes) seems to be a topic with unarguable interest, as this may link biological and non-biological activities to establish new combo-catalysis routes.

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

Lipase B from Candida antarctica (CALB) and lipase from Thermomyces lanuginosus (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.g., amination of immobilized TLL increases the activity versus p-nitro phenyl butyrate (p-NPB), reduces the activity with R-methyl mandate by half and maintains the activity with S-isomer). The modification with PEI increased the biocatalyst activity 8-fold versus R-methyl mandelate. Enzyme stability was also modified, usually showing an improvement (e.g., the modification of immobilized TLL with PEI or glutaraldehyde enabled to maintain more than 70% of the initial activity, while the unmodified enzyme maintained less than 50%). The immobilized enzymes were also mineralized by using phosphate metals (Zn2+, Co2+, Cu2+, Ni2+ or Mg2+), and this affected also the enzyme activity, specificity (e.g., immobilized TLL increased its activity after zinc mineralization versus triacetin, while decreased its activity versus all the other assayed substrates) and stability (e.g., the same modification increase the residual stability from almost 0 to more than 60%). Depending on the enzyme, a metal could be positively, neutrally or negatively affected for a specific feature. Finally, we analyzed if the chemical modification could, somehow, tune the effects of the mineralization. Effectively, the same mineralization could have very different effects on the same immobilized enzyme if it was previously submitted to different physicochemical modifications. The same mineralization could present different effects on the enzyme activity, specificity or stability, depending on the previous modification performed on the enzyme, showing that these previous enzyme modifications alter the effects of the mineralization on enzyme features. For example, TLL modified with glutaraldehyde and treated with zinc salts increased its activity using R-methyl mandelate, while almost maintaining its activity versus the other unaltered substrates, whereas the aminated TLL maintained its activity with both methyl mandelate isomers, while it decreased with p-NPB and triacetin. TLL was found to be easier to tune than CALB by the strategies used in this paper. In this way, the combination of chemical or physical modifications of enzymes before their mineralization increases the range of modification of features that the immobilized enzyme can experienced, enabling to enlarge the biocatalyst library.

Immobilization of Lipase B from Candida antarctica in Octyl-Vinyl Sulfone Agarose: Effect of the Enzyme-Support Interactions on Enzyme Activity, Specificity, Structure and Inactivation Pathway.

Lipase B from Candida antarctica was immobilized on heterofunctional support octyl agarose activated with vinyl sulfone to prevent enzyme release under drastic conditions. Covalent attachment was established, but the blocking step using hexylamine, ethylenediamine or the amino acids glycine (Gly) and aspartic acid (Asp) altered the results. The activities were lower than those observed using the octyl biocatalyst, except when using ethylenediamine as blocking reagent and p-nitrophenol butyrate (pNPB) as substrate. The enzyme stability increased using these new biocatalysts at pH 7 and 9 using all blocking agents (much more significantly at pH 9), while it decreased at pH 5 except when using Gly as blocking agent. The stress inactivation of the biocatalysts decreased the enzyme activity versus three different substrates (pNPB, S-methyl mandelate and triacetin) in a relatively similar fashion. The tryptophane (Trp) fluorescence spectra were different for the biocatalysts, suggesting different enzyme conformations. However, the fluorescence spectra changes during the inactivation were not too different except for the biocatalyst blocked with Asp, suggesting that, except for this biocatalyst, the inactivation pathways may not be so different.

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 Lipozyme®TL (TLL-IM) (lipase from Thermomyces lanuginose), Lipozyme®435 (L435) (lipase B from Candida antarctica), Lipozyme®RM (RML-IM), and LipuraSelect (LS-IM) (both from lipase from Rhizomucor miehei). The modifications greatly altered enzyme specificity, increasing the activity versus some substrates (e.g., TLL-IM modified with zinc phosphate in hydrolysis of triacetin) while decreasing the activity versus other substrates (the same preparation in activity versus R- or S- methyl mandelate). Enantiospecificity was also drastically altered after these modifications, e.g., LS-IM increased the activity versus the R isomer while decreasing the activity versus the S isomer when treated with copper phosphate. Regarding the enzyme stability, it was significantly improved using octyl-agarose-lipases. Using all these commercial biocatalysts, no significant positive effects were found; in fact, a decrease in enzyme stability was usually detected. The results point towards the possibility of a battery of biocatalysts, including many different metal phosphates and immobilization protocols, being a good opportunity to tune enzyme features, increasing the possibilities of having biocatalysts that may be suitable for a specific process.

Effect of Concentrated Salts Solutions on the Stability of Immobilized Enzymes: Influence of Inactivation Conditions and Immobilization Protocol.

This paper aims to investigate the effects of some salts (NaCl, (NH4)2SO4 and Na2SO4) at pH 5.0, 7.0 and 9.0 on the stability of 13 different immobilized enzymes: five lipases, three proteases, two glycosidases, and one laccase, penicillin G acylase and catalase. The enzymes were immobilized to prevent their aggregation. Lipases were immobilized via interfacial activation on octyl agarose or on glutaraldehyde-amino agarose beads, proteases on glyoxyl agarose or glutaraldehyde-amino agarose beads. The use of high concentrations of salts usually has some effects on enzyme stability, but the intensity and nature of these effects depends on the inactivation pH, nature and concentration of the salt, enzyme and immobilization protocol. The same salt can be a stabilizing or a destabilizing agent for a specific enzyme depending on its concentration, inactivation pH and immobilization protocol. Using lipases, (NH4)2SO4 generally permits the highest stabilities (although this is not a universal rule), but using the other enzymes this salt is in many instances a destabilizing agent. At pH 9.0, it is more likely to find a salt destabilizing effect than at pH 7.0. Results confirm the difficulty of foreseeing the effect of high concentrations of salts in a specific immobilized enzyme.

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. For example, both lowly loaded biocatalysts increased their activity after modification while the effect was the opposite for the highly loaded biocatalysts. Additionally, the modification with TNBS of highly loaded CALB biocatalyst increased its stability while decrease the activity.

Proteases immobilized on nanomaterials for biocatalytic, environmental and biomedical applications: Advantages and drawbacks.

Proteases have gained significant scientific and industrial interest due to their unique biocatalytic characteristics and broad-spectrum applications in different industries. The development of robust nanobiocatalytic systems by attaching proteases onto various nanostructured materials as fascinating and novel nanocarriers has demonstrated exceptional biocatalytic performance, substantial stability, and ease of recyclability over multiple reaction cycles under different chemical and physical conditions. Proteases immobilized on nanocarriers may be much more resistant to denaturation caused by extreme temperatures or pH values, detergents, organic solvents, and other protein denaturants than free enzymes. Immobilized proteases may present a lower inhibition. The use of non-porous materials in the immobilization prevents diffusion and steric hindrances during the binding of the substrate to the active sites of enzymes compared to immobilization onto porous materials; when using very large or solid substrates, orientation of the enzyme must always be adequate. The advantages and problems of the immobilization of proteases on nanoparticles are discussed in this review. The continuous and batch reactor operations of nanocarrier-immobilized proteases have been successfully investigated for a variety of applications in the leather, detergent, biomedical, food, and pharmaceutical industries. Information about immobilized proteases on various nanocarriers and nanomaterials has been systematically compiled here. Furthermore, different industrial applications of immobilized proteases have also been highlighted in this review.

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. After enzyme adsorption, the release of the enzyme from the supports using different NaCl concentrations, Triton X100, ionic detergents (SDS and CTAB), or different temperatures (4 °C to 55 °C) was studied. Using MANAE support, at 0.3 M NaCl almost all the immobilized enzyme was released. Using MANAE-GLU, 0.3 M, and 0.6 M NaCl similar results were obtained. However, incubation at 1 M or 2 M NaCl, many enzyme molecules were not released from the support. For the MANAE-GLU-GLU support, none of the tested concentrations of NaCl was sufficient to release all enzyme bound to the support. Only using high temperatures, 0.6 M NaCl, and 1 % CTAB or SDS, could the totality of the proteins be released from the support. The results shown in this paper confirm the heterofunctional character of aminated supports modified with glutaraldehyde.

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. The enzyme was more stable in Gly than when Tris was employed for inactivation. Then, the enzyme was immobilized on methacrylate beads coated with octadecyl groups at pH 7 in the presence of Gly, Tris, phosphate and HEPES. Its activity was assayed versus triacetin and S-methyl mandelate. The biocatalyst prepared in phosphate was more active versus S-methyl mandelate, while the other ones were more active versus triacetin. The immobilized enzyme stability at pH 7 depends on the buffer used for enzyme immobilization. The buffer used in the inactivation and the substrate used determined the activity. For example, glycine was the buffer that promoted the lowest or the highest stabilities depending on the substrate used to quantify the activities.

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. Stabilization (depending on the buffer) of the highly loaded biocatalysts was found, very likely as a consequence of the detected intermolecular crosslinkings. This did not occur for the lowly loaded biocatalysts. Next, the enzymes were chemically aminated and then treated with glutaraldehyde. In the case of TLL, the intramolecular crosslinkings (visible by the apparent reduction of the protein size) increased enzyme stability of the lowly loaded biocatalysts, an effect that was further increased for the highly loaded biocatalysts due to intermolecular crosslinkings. Using CALB, the intramolecular crosslinkings were less intense, and the stabilization was lower, even though the intermolecular crosslinkings were quite intense for the highly loaded biocatalyst. The stabilization detected depended on the inactivation buffer. The interactions between enzyme loading and inactivating buffer on the effects of the chemical modifications suggest that the modification and inactivation studies must be performed under the target biocatalysts and conditions.

Additively Manufactured 3D Micro-bioelectrodes for Enhanced Bioelectrocatalytic Operation.

The drive toward miniaturization of enzyme-based bioelectronics established a need for three-dimensional (3D) microstructured electrodes, which are difficult to implement using conventional manufacturing processes. Additive manufacturing coupled with electroless metal plating enables the production of 3D conductive microarchitectures with high surface area for potential applications in such devices. However, interfacial delamination between the metal layer and the polymer structure is a major reliability concern, which leads to device performance degradation and eventually device failure. This work demonstrates a method to produce a highly conductive and robust metal layer on a 3D printed polymer microstructure with strong adhesion by introducing an interfacial adhesion layer. Prior to 3D printing, multifunctional acrylate monomers with alkoxysilane (-Si-(OCH3)3) were synthesized via the thiol-Michael addition reaction between pentaerythritol tetraacrylate (PETA) and 3-mercaptopropyltrimethoxysilane (MPTMS) with a 1:1 stoichiometric ratio. Alkoxysilane functionality remains intact during photopolymerization in a projection micro-stereolithography (PµSLA) system and is utilized for the sol-gel reaction with MPTMS during postfunctionalization of the 3D printed microstructure to build an interfacial adhesion layer. This leads to the implementation of abundant thiol functional groups on the surface of the 3D printed microstructure, which can act as a strong binding site for gold during electroless plating to improve interfacial adhesion. The 3D conductive microelectrode prepared by this technique exhibited excellent conductivity of 2.2 × 107 S/m (53% of bulk gold) with strong adhesion between a gold layer and a polymer structure even after harsh sonication and an adhesion tape test. As a proof-of-concept, we examined the 3D gold diamond lattice microelectrode modified with glucose oxidase as a bioanode for a single enzymatic biofuel cell. The lattice-structured enzymatic electrode with high catalytic surface area was able to generate a current density of 2.5 µA/cm2 at 0.35 V, which is an about 10 times increase in current output compared to a cube-shaped microelectrode.

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. To solve this problem, a new strategy has been proposed using three different immobilization protocols. CALB was covalently immobilized on octyl-vinyl sulfone agarose and blocked with Asp. Then, CRL was immobilized via interfacial activation. After coating both immobilized enzymes with polyethylenimine, RML could be immobilized via ion exchange. That way, by incubating in ammonium sulfate solutions, inactivated RML could be released enabling the reuse of coimmobilized CRL and CALB to build a new combi-lipase. Incubating in triton and ammonium sulfate solutions, it was possible to release inactivated CRL and RML, enabling the reuse of immobilized CALB when CRL was inactivated. These cycles could be repeated for 3 full cycles, maintaining the activity of the active and immobilized enzymes.

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. Unfortunately, the release of Lecitase was not possible using just high ionic strength solutions, and if detergent was added, TLL was also released from the support. This occurred when using 0.25 M ammonium sulfate, Lecitase did not immobilize on aminated TLL. That makes the use octyl-vinylsulfone supports necessary to irreversibly immobilize TLL, and after blocking with ethylendiamine, the immobilized TLL was aminated. Lecitase immobilized and released from this biocatalyst using 0.25 M ammonium sulfate and 0.1% Triton X-100. That way, a coimmobilized TLL and Lecitase biocatalyst could be produced, and after Lecitase inactivation, it could be released and the immobilized, aminated, and fully active TLL could be utilized to build a new combibiocatalyst.

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 (Zn2+, Co2+, Cu2+). They have been also immobilized on the support modified by the metallic phosphate, usually driving to biocatalyst with lower stability or marginal improvements. The effects of the phosphate/metal modification on enzyme features depended on the loading of the support. Some enzymes (TLL, CRL or CALA), mainly using the highly loaded biocatalysts, showed very significant improvement on enzyme stability after the treatment with some of the metal phosphates (next to a 20-fold factor), improvements that were not justified by the presence of metallic or phosphate ions in solution, as they had negative effects on enzyme stabilities. In some other cases, a significant increase in enzyme activity was detected (e.g., CALB). This could be explained by the modification of the nucleation places of the enzymes by the metallic phosphate, and this could help to explain the good results obtained in the nanoflower immobilization of many enzymes.

The combination of covalent and ionic exchange immobilizations enables the coimmobilization on vinyl sulfone activated supports and the reuse of the most stable immobilized enzyme.

The coimmobilization of lipases from Rhizomucor miehei (RML) and Candida antarctica (CALB) has been intended using agarose beads activated with divinyl sulfone. CALB could be immobilized on this support, while RML was not. However, RML was ionically exchanged on this support blocked with ethylendiamine. Therefore, both enzymes could be coimmobilized on the same particle, CALB covalently using the vinyl sulfone groups, and RML via anionic exchange on the aminated blocked support. However, immobilized RML was far less stable than immobilized CALB. To avoid the discarding of CALB (that maintained 90% of the initial activity after RML inactivation), a strategy was developed. Inactivated RML was desorbed from the support using ammonium sulfate and 1% Triton X-100 at pH 7.0. That way, 5 cycles of RML thermal inactivation, discharge of the inactivated enzyme and re-immobilization of a fresh sample of RML could be performed. In the last cycle, immobilized CALB activity was still over 90% of the initial one. Thus, the strategy permits that enzymes can be coimmobilized on vinyl sulfone supports even if one of them cannot be immobilized on it, and also permits the reuse of the most stable enzyme (if it is irreversibly attached to the support).

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. The stability, activity and specificity of the biocatalysts were very different, both the differently blocked VS-biocatalysts and the glutaraldehyde biocatalysts prepared at different pH. All biocatalysts were submitted to mineralization using different metals. The activity, specificity and stability effects of the mineralization strongly depended on the enzyme and on the immobilization protocol. For the same enzyme, a mineralization protocol could be negative, positive or present no effect depending on the enzyme immobilization procedure and substrate. In the best cases, activity could be increased by a two-fold factor, while stability was significantly improved in many instances. These results highlight the great potential of mineralization of immobilized enzymes to improve their properties, as well as the great interactions that immobilization protocol and mineralization can exhibit. The combination of both methodologies greatly increases the possibilities to find a biocatalyst that can be suitable for a specific process.

Stabilization of enzymes via immobilization: Multipoint covalent attachment and other stabilization strategies.

The use of enzymes in industrial processes requires the improvement of their features in many instances. Enzyme immobilization, a requirement to facilitate the recovery and reuse of these water-soluble catalysts, is one of the tools that researchers may utilize to improve many of their properties. This review is focused on how enzyme immobilization may improve enzyme stability. Starting from the stabilization effects that an enzyme may experience by the mere fact of being inside a solid particle, we detail other possibilities to stabilize enzymes: generation of favorable enzyme environments, prevention of enzyme subunit dissociation in multimeric enzymes, generation of more stable enzyme conformations, or enzyme rigidification via multipoint covalent attachment. In this last point, we will discuss the features of an "ideal" immobilization protocol to maximize the intensity of the enzyme-support interactions. The most interesting active groups in the support (glutaraldehyde, epoxide, glyoxyl and vinyl sulfone) will be also presented, discussing their main properties and uses. Some instances in which the number of enzyme-support bonds is not directly related to a higher stabilization will be also presented. Finally, the possibility of coupling site-directed mutagenesis or chemical modification to get a more intense multipoint covalent immobilization will be discussed.

Enzyme-support interactions and inactivation conditions determine Thermomyces lanuginosus lipase inactivation pathways: Functional and florescence studies.

Lipase from Thermomyces lanuginosus (TLL) has been covalently immobilized on heterofunctional octyl-vinyl agarose. That way, the covalently immobilized enzymes will have identical orientation. Then, it has blocked using hexyl amine (HEX), ethylenediamine (EDA), Gly and Asp. The initial activity/stability of the different biocatalysts was very different, being the most stable the biocatalyst blocked with Gly. These biocatalysts had been utilized to analyze if the enzyme activity could decrease differently along thermal inactivation courses depending on the utilized substrate (that is, if the enzyme specificity was altered during its inactivation using 4 different substrates to determine the activity), and if this can be altered by the nature of the blocking agent and the inactivation conditions (we use pH 5, 7 and 9). Results show great changes in the enzyme specificity during inactivation (e.g., activity versus triacetin was much more quickly lost than versus the other substrates), and how this was modulated by the immobilization protocol and inactivation conditions. The difference in the changes induced by immobilization and inactivation were confirmed by fluorescence studies. That is, the functional and structural analysis of partially inactivated immobilized enzyme showed that their inactivation pathway is strongly depended on the support features and inactivation conditions.

Enzyme co-immobilization: Always the biocatalyst designers' choice or not?

The increasing relevance of cascade reactions in biocatalysis has sparked a great interest for enzyme co-immobilization. Enzyme co-immobilization allows access to some kinetic advantages that in some instances are necessary to get the desired product, avoiding side-reactions. However, the kinetic effect is very relevant mainly at the initial reaction rates, while it may be less relevant in the whole reaction course, depending on the kinetic parameters of the involved enzymes. This review not only critically discusses the advantages but also the drawbacks of enzymes co-immobilization: immobilization on the same support and surface, under similar conditions, discarding the whole biocatalyst when one of the co-immobilized enzymes is inactivated. We will discuss when co-immobilization is almost compulsory, when the advantages of co-immobilization may not be enough to compensate their problems and when it should be fully discarded. The co-immobilization of cofactors and enzymes bears special interest, as this can open up the opportunity to the building of artificial cells and extremely complex one-pot transformations. Finally, some recent strategies to overcome some the co-immobilization problems will be presented.

Effects of Enzyme Loading and Immobilization Conditions on the Catalytic Features of Lipase From Pseudomonas fluorescens Immobilized on Octyl-Agarose Beads.

The lipase from Pseudomonas fluorescens (PFL) has been immobilized on octyl-agarose beads under 16 different conditions (varying pH, ionic strength, buffer, adding some additives) at two different loadings, 1 and 60 mg of enzyme/g of support with the objective of check if this can alter the biocatalyst features. The activity of the biocatalysts versus p-nitrophenyl butyrate and triacetin and their thermal stability were studied. The different immobilization conditions produced biocatalysts with very different features. Considering the extreme cases, using 1 mg/g preparations, PFL stability changed more than fourfolds, while their activities versus pNPB or triacetin varied a 50-60%. Curiously, PFL specific activity versus triacetin was higher using highly enzyme loaded biocatalysts than using lowly loaded biocatalysts (even by a twofold factor). Moreover, stability of the highly loaded preparations was higher than that of the lowly loaded preparations, in many instances even when using 5°C higher temperatures (e.g., immobilized in the presence of calcium, the highly loaded biocatalysts maintained after 24 h at 75°c a 85% of the initial activity, while the lowly loaded preparation maintained only 27% at 70°C). Using the highly loaded preparations, activity of the different biocatalysts versus pNPB varied almost 1.7-folds and versus triacetin 1.9-folds. In this instance, the changes in stability caused by the immobilization conditions were much more significant, some preparations were almost fully inactivated under conditions where the most stable one maintained more than 80% of the initial activity. Results suggested that immobilization conditions greatly affected the properties of the immobilized PFL, partially by individual molecule different conformation (observed using lowly loaded preparations) but much more relevantly using highly loaded preparations, very likely by altering some enzyme-enzyme intermolecular interactions. There is not an optimal biocatalyst considering all parameters. That way, preparation of biocatalysts using this support may be a powerful tool to tune enzyme features, if carefully controlled.