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.

Optimizing the enzymatic production of biolubricants by the Taguchi method: Esterification of the free fatty acids from castor oil with 2-ethyl-1-hexanol catalyzed by Eversa Transform 2.0.

The solvent-free esterification of the free fatty acids (FFAs) obtained by the hydrolysis of castor oil (a non-edible vegetable oil) with 2-ethyl-1-hexanol (a branched fatty alcohol) was catalyzed by different free lipases. Eversa Transform 2.0 (ETL) features surpassed most commercial lipases. Some process parameters were optimized by the Taguchi method (L16'). As a result, a conversion over 95% of the FFAs of castor oil into esters with lubricants properties was achieved under optimized reaction conditions (15 wt% of biocatalyst content, 1:4 molar ratio (FFAs/alcohol), 30 °C, 180 rpm, 96 h). The substrates molar ratio had the highest influence on the dependent variable (conversion at 24 h). FFAs/2-ethyl-1-hexanol esters were characterized regarding the physicochemical and tribological properties. Interestingly, the modification of the FFAs with 2-ethyl-1-hexanol by ETL increased the oxidative stability of the FFAs feedstock from 0.18 h to 16.83 h. The biolubricants presented a lower friction coefficient than the reference commercial mineral lubricant (0.052 ± 0.07 against 0.078 ± 0.04). Under these conditions, ETL catalyzed the oligomerization of ricinoleic acid (a hydroxyl fatty acid) into estolides, reaching a conversion of 25.15% of the initial FFAs (for the first time).

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.

Effect of Support Matrix and Crosslinking Agents on Nutritional Properties of Orange Juice during Enzyme Clarification: A Comparative Study.

This study investigated the impact of a support matrix and active group on the support to the nutritional properties of orange juice after juice clarification. Pectinase was immobilized on chitosan and aminated silica supports, activated with genipin or glutaraldehyde, and applied for juice clarification. The effects on various juice properties, including reducing sugars, total soluble solids, vitamin C, and phenolic compounds, juice color, and pH, were evaluated. The results revealed that the immobilization on chitosan activated using genipin resulted in the highest biocatalyst activity (1211.21 U·g-1). The juice treatments using the biocatalysts led to turbidity reduction in the juice (up to 90%), with the highest reductions observed in treatments involving immobilized enzyme on chitosan. Importantly, the enzymatic treatments preserved the natural sugar content, total soluble solids, and pH of the juice. Color differences between treated and raw juice samples were especially relevant for those treated using enzymes, with significant differences in L* and b*, showing loss of yellow vivid color. Analysis of phenolic compounds and vitamin C showed no significant alterations after the enzymatic treatment of the raw juice. According to our results, the clarification of orange juice using immobilized enzymes can be a compromise in turbidity reduction and color reduction to maintain juice quality.

Peptides with biological and technofunctional properties produced by bromelain hydrolysis of proteins from different sources: A review.

Bromelains are cysteine peptidases with endopeptidase action (a subfamily of papains), obtained from different parts of vegetable belonging to the Bromeliaceae family. They have some intrinsic medical activity, but this review is focused on their application (individually or mixed with other proteases) to produce bioactive peptides. When compared to other proteases, perhaps due to the fact that they are commercialized as an extract containing several proteases, the hydrolysates produced by this enzyme tends to have higher bioactivities than other common proteases. The peptides and the intensity of their final properties depend on the substrate protein and reaction conditions, being the degree of hydrolysis a determining parameter (but not always positive or negative). The produced peptides may have diverse activities such as antioxidant, antitumoral, antihypertensive or antimicrobial ones, among others or they may be utilized to improve the organoleptic properties of foods and feeds. Evolution of the use of this enzyme in this application is proposed to be based on a more intense direct application of Bromeliaceae extract, without the cost associated to enzyme purification, and the use of immobilized biocatalysts of the enzyme by simplifying the enzyme recovery and reuse, and also making the sequential hydrolysis using diverse proteases possible.

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.

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

The increasing worries by the inadequate use of energy and the preservation of nature are promoting an increasing interest in the production of biolubricants. After discussing the necessity of producing biolubricants, this review focuses on the production of these interesting molecules through the use of lipases, discussing the different possibilities (esterification of free fatty acids, hydroesterification or transesterification of oils and fats, transesterification of biodiesel with more adequate alcohols, estolides production, modification of fatty acids). The utilization of discarded substrates has special interest due to the double positive ecological impact (e.g., oil distillated, overused oils). Pros and cons of all these possibilities, together with general considerations to optimize the different processes will be outlined. Some possibilities to overcome some of the problems detected in the production of these interesting compounds will be also discussed.

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.

A Novel, Highly Potent NADPH-Dependent Cytochrome P450 Reductase from Waste Liza klunzingeri Liver.

The use of marine enzymes as catalysts for biotechnological applications is a topical subject. Marine enzymes usually display better operational properties than their animal, plant or bacterial counterparts, enlarging the range of possible biotechnological applications. Due to the fact that cytochrome P450 enzymes can degrade many different toxic environmental compounds, these enzymes have emerged as valuable tools in bioremediation processes. The present work describes the isolation, purification and biochemical characterization of a liver NADPH-dependent cytochrome P450 reductase (CPR) from the marine fish Liza klunzingeri (LkCPR). Experimental results revealed that LkCPR is a monomer of approximately 75 kDa that is active in a wide range of pH values (6-9) and temperatures (40-60 °C), showing the highest catalytic activity at pH 8 and 50 °C. The activation energy of the enzyme reaction was 16.3 kcal mol-1 K-1. The KM values for cytochrome C and NADPH were 8.83 µM and 7.26 µM, and the kcat values were 206.79 s-1 and 202.93 s-1, respectively. LkCPR displayed a specific activity versus cytochrome C of 402.07 µmol min-1 mg1, the highest activity value described for a CPR up to date (3.2-4.7 times higher than the most active reported CPRs) and showed the highest thermostability described for a CPR. Taking into account all these remarkable catalytic features, LkCPR offers great potential to be used as a suitable biocatalyst.

Enzyme Immobilization.

The development of enzyme immobilization started in the middle of the previous century as a potential answer to the problem of the enzyme recovery and reuse [...].

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

Penicillin G acylase (PGA) from Escherichia coli was immobilized on vinyl sulfone (VS) agarose. The immobilization of the enzyme failed at all pH values using 50 mM of buffer, while the progressive increase of ionic strength permitted its rapid immobilization under all studied pH values. This suggests that the moderate hydrophobicity of VS groups is enough to transform the VS-agarose in a heterofunctional support, that is, a support bearing hydrophobic features (able to adsorb the proteins) and chemical reactivity (able to give covalent bonds). Once PGA was immobilized on this support, the PGA immobilization on VS-agarose was optimized with the purpose of obtaining a stable and active biocatalyst, optimizing the immobilization, incubation and blocking steps characteristics of this immobilization protocol. Optimal conditions were immobilization in 1 M of sodium sulfate at pH 7.0, incubation at pH 10.0 for 3 h in the presence of glycerol and phenyl acetic acid, and final blocking with glycine or ethanolamine. This produced biocatalysts with stabilities similar to that of the glyoxyl-PGA (the most stable biocatalyst of this enzyme described in literature), although presenting just over 55% of the initially offered enzyme activity versus the 80% that is recovered using the glyoxyl-PGA. This heterofuncionality of agarose VS beads opens new possibilities for enzyme immobilization on this support.

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.

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.

Biosynthesis of alkanes/alkenes from fatty acids or derivatives (triacylglycerols or fatty aldehydes).

This review summarizes the most relevant advances in the biological transformation of fatty acids (or derivatives) into hydrocarbons to be used as biofuels (biogasoline, green diesel and jet biofuel). Among the used enzymes, the fatty acid decarboxylase from Jeotgalicoccus sp. ATCC 8456 (OleTJE) stands out as a promising enzyme. OleTJE may be coupled in cascade reactions with metalloenzymes or reductases from the Old Yellow Enzymes (OYE) family to perform the hydrogenation of α-olefins into paraffins. The photodecarboxylase from Chlorella variabilis NC64A (CvFAP) is an example of coupling biocatalysis and photocatalysis to produce alkanes. Besides the (photo)decarboxylation of free fatty acids and/or triacyclglycerols to produce alkanes/alkenes, by enzymes has also been employed. The cyanobacterial aldehyde decarbonylase (cAD) from Nostoc punctiforme is an outstanding example of this kind of enzymes used to produce alkanes. Overall, these kinds of enzymes open up new possibilities to the production of biofuels from renewable sources, even if they have many limitations on the current situation. The possibilities of improving enzymes features via immobilization or coimmobilization, as well as the utilization of whole cells haves been also reviewed.

Switch off/switch on of a cysteinyl protease as a way to preserve the active catalytic group by modification with a reversible covalent thiol modifier: Immobilization of ficin on vinyl-sulfone activated supports.

The immobilization of ficin (a cysteinyl proteases) on vinyl sulfone agarose produced its almost full inactivation. It was observed that the incubation of the free and immobilized enzyme in ß-mercaptoethanol produced a 20 % of enzyme activity recovery, suggesting that the inactivation due to the immobilization could be a consequence of the modification of the catalytic Cys. To prevent the enzyme inactivation during the immobilization, switching off of ficin via Cys reaction with dipyridyl-disulfide was implemented, giving a reversible disulfide bond that produced a fully inactive enzyme. The switch on of ficin activity was implemented by incubation in 1 M ß-mercaptoethanol. Using this strategy to immobilize the enzyme on vinyl sulfone agarose beads, the expressed activity of the immobilized ficin could be boosted up to 80 %. The immobilized enzyme presented a thermal stabilization similar to that obtained using ficin-glyoxyl-agarose beads. This procedure may be extended to many enzymes containing critical Cys, to permit their immobilization or chemical modification.

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.

Is enzyme immobilization a mature discipline? Some critical considerations to capitalize on the benefits of immobilization.

Enzyme immobilization has been developing since the 1960s and although many industrial biocatalytic processes use the technology to improve enzyme performance, still today we are far from full exploitation of the field. One clear reason is that many evaluate immobilization based on only a few experiments that are not always well-designed. In contrast to many other reviews on the subject, here we highlight the pitfalls of using incorrectly designed immobilization protocols and explain why in many cases sub-optimal results are obtained. We also describe solutions to overcome these challenges and come to the conclusion that recent developments in material science, bioprocess engineering and protein science continue to open new opportunities for the future. In this way, enzyme immobilization, far from being a mature discipline, remains as a subject of high interest and where intense research is still necessary to take full advantage of the possibilities.

Lipase immobilization via cross-linked enzyme aggregates: Problems and prospects - A review.

In this review we have focused on the preparation of cross-linked enzyme aggregates (CLEAs) from lipases, as these are among the most used enzyme in bioprocesses. This immobilization method is considered very attractive due to preparation simplicity, non-use of supports and the possibility of using crude enzyme extracts. CLEAs provide lipase stabilization under extreme temperature or pH conditions or in the presence of organic solvents, in addition to preventing enzyme leaching in aqueous medium. However, it presents some problems in the preparation and limitations in their use. The problems in preparation refer mainly to the crosslinking step, and may be solved using an aminated feeder. The problems in handling have been tackled designing magnetic-CLEAs or trapping the CLEAs in particles with better mechanical properties, the substrate diffusion problems has been reduced by producing more porous-CLEAs, etc. The enzyme co-immobilization using combi-CLEAs is also a new tendency. Therefore, this review explores the CLEAs methodology aimed at lipase immobilization and its applications.