Bacteria-Polymer Composite Material for Glycerol Valorization.

Bacterial immobilization is regarded as an enabling technology to improve the stability and reusability of biocatalysts. Natural polymers are often used as immobilization matrices but present certain drawbacks, such as biocatalyst leakage and loss of physical integrity upon utilization in bioprocesses. Herein, we prepared a hybrid polymeric matrix that included silica nanoparticles for the unprecedented immobilization of the industrially relevant Gluconobacter frateurii (Gfr). This biocatalyst can valorize glycerol, an abundant by-product of the biodiesel industry, into glyceric acid (GA) and dihydroxyacetone (DHA). Different concentrations of siliceous nanosized materials, such as biomimetic Si nanoparticles (SiNps) and montmorillonite (MT), were added to alginate. These hybrid materials were significantly more resistant by texture analysis and presented a more compact structure as seen by scanning electron microscopy. The preparation including 4% alginate with 4% SiNps proved to be the most resistant material, with a homogeneous distribution of the biocatalyst in the beads as seen by confocal microscopy using a fluorescent mutant of Gfr. It produced the highest amounts of GA and DHA and could be reused for up to eight consecutive 24 h reactions with no loss of physical integrity and negligible bacterial leakage. Overall, our results indicate a new approach to generating biocatalysts using hybrid biopolymer supports.

Remote Activation of Enzyme Nanohybrids for Cancer Prodrug Therapy Controlled by Magnetic Heating.

Herein, we have developed nanohybrids (nHs) to remotely activate a therapeutic enzyme for its use in Directed Enzyme Prodrug Therapy (DEPT). The coencapsulation of magnetic nanoparticles (MNPs) with horseradish peroxidase (HRP) using biomimetic silica as an entrapment matrix was optimized to obtain nanosized hybrids (∼150 nm) for remote activation of the therapeutic enzyme. HRP converts indole-3-acetic acid (3IAA) into peroxylated radicals, whereas MNPs respond to alternating magnetic fields (AMFs) becoming local hotspots. The AMF application triggered an increase in the bioconversion rate of HRP matching the activity displayed at the optimal temperature of the nHs (Topt = 50 °C) without altering the temperature of the reaction media. This showed that enzyme nanoactuation is possible with MNPs even if they are not covalently bound. After an extensive physicochemical/magnetic characterization, the spatial location of each component of the nH was deciphered, and an insulating role of the silica matrix was suggested as critical for introducing remote control over HRP. In vitro assays, using a human pancreatic cancer cell line (MIA PaCa-2), showed that only upon exposure to AMF and in the presence of the prodrug, the enzyme-loaded nHs triggered cell death. Moreover, in vivo experiments showed higher reductions in the tumor volume growth in those animals treated with nHs in the presence of 3IAA when exposed to AMF. Thus, this work demonstrates the feasibility of developing a spatiotemporally controlled DEPT strategy to overcome unwanted off-target effects.

New perspectives into Gluconobacter-catalysed biotransformations.

Different from other aerobic microorganisms that oxidise carbon sources to water and carbon dioxide, Gluconobacter catalyses the incomplete oxidation of various substrates with regio- and stereoselectivity. This ability, as well as its capacity to release the resulting products into the reaction media, place Gluconobacter as a privileged member of a non-model microorganism class that may boost industrial biotechnology. Knowledge of new technologies applied to Gluconobacter has been piling up in recent years. Advancements in its genetic modification, application of immobilisation tools and careful designs of the transformations, have improved productivities and stabilities of Gluconobacter strains or enabled new bioconversions for the production of valuable marketable chemicals. In this work, the latest advancements applied to Gluconobacter-catalysed biotransformations are summarised with a special focus on recent available tools to improve them. From genetic and metabolic engineering to bioreactor design, the most recent works on the topic are analysed in depth to provide a comprehensive resource not only for scientists and technologists working on/with Gluconobacter, but for the general biotechnologist.

Increased paclitaxel recovery from Taxus baccata vascular stem cells using novel in situ product recovery approaches.

In this study, several approaches were tested to optimise the production and recovery of the widely used anticancer drug Taxol® (paclitaxel) from culturable vascular stem cells (VSCs) of Taxus baccata, which is currently used as a successful cell line for paclitaxel production. An in situ product recovery (ISPR) technique was employed, which involved combining three commercial macro-porous resin beads (HP-20, XAD7HP and HP-2MG) with batch and semi-continuous cultivations of the T. baccata VSCs after adding methyl jasmonate (Me-JA) as an elicitor. The optimal resin combination resulted in 234 ± 23 mg of paclitaxel per kg of fresh-weight cells, indicating a 13-fold improved yield compared to the control (with no resins) in batch cultivation. This resin treatment was further studied to evaluate the resins' removal capacity of reactive oxygen species (ROS), which can cause poor cell growth or reduce product synthesis. It was observed that the ISPR cultivations had fourfold less intracellular ROS concentration than that of the control; thus, a reduced ROS concentration established by the resin contributed to increased paclitaxel yield, contrary to previous studies. These paclitaxel yields are the highest reported to date using VSCs, and this scalable production method could be applied for a diverse range of similar compounds utilising plant cell culture.

Oryza sativa as a Tool for Assessing Arsenic Efficacy of Arsenic Remediation of Agricultural Soils by Sulfidated Zerovalent Iron Nanoparticles.

Arsenic (As) is highly toxic in its inorganic form. It is naturally presented at elevated levels in the groundwater of a number of countries and contaminates drinking water sources, generating numerous health and environmental problems. Current methodologies for its remediation have deficiencies which fuel the constant exploration of new alternatives. Therefore, the development of robust methodologies for the evaluation of potential remediation technologies are not only timely but also highly needed. In this study we have investigated the use of a rice plant species as a means to evaluate the efficacy of As remediation using sulfidated zerovalent iron nanoparticles (S-nZVI). The obtained results show that addition of S-nZVI to soils had a beneficial impact to plant growth in the presence of As(V) and As(III) concentrations between 10 and 50 ppm. Positive effects were also found for plant biomass and chlorophyll content in the plants. Moreover, evaluation of As uptake by plants showed that the application of S-nZVI reduced the amount of both As(V) and As(III) in shoots and increased the amount of As in the roots. Studies on the Fe and P content in shoot and root after exposure to As with and without the nanoparticles demonstrated that nanoparticles remain mainly in the roots and that P uptake by plants was not significantly affected, suggesting that S-nZVI treatment is safe for plants at the assayed doses. These results overall confirm the method as robust and reliable for demonstrating the reduction of the bioavailability of As in soil by S-nZVI sequestration.

Dihydroxyacetone production via heterogeneous biotransformations of crude glycerol.

In this work, several immobilization strategies for Gluconobacter oxydans NBRC 14819 (Gox) were tested in the bioconversion of crude glycerol to dihydroxyacetone (DHA). Agar, agarose and polyacrylamide were evaluated as immobilization matrixes. Glutaraldehyde crosslinked versions of the agar and agarose preparations were also tested. Agar immobilized Gox proved to be the best heterogeneous biocatalyst in the bioconversion of crude glycerol reaching a quantitative production of 50 g/L glycerol into DHA solely in water. Immobilization allowed reutilization for at least eight cycles, reaching four times more DHA than the amount obtained by a single batch of free cells which cannot be reutilized. An increase in scale of 34 times had no impact on DHA productivity. The results obtained herein constitute a contribution to the microbiological production of DHA as they not only attain unprecedented productivities for the reaction with immobilized biocatalysts but also proved that it is feasible to do it in a clean background of solely water that alleviates the cost of downstream processing.

Green Production of Cladribine by Using Immobilized 2'-Deoxyribosyltransferase from Lactobacillus delbrueckii Stabilized through a Double Covalent/Entrapment Technology.

Nowadays, enzyme-mediated processes offer an eco-friendly and efficient alternative to the traditional multistep and environmentally harmful chemical processes. Herein we report the enzymatic synthesis of cladribine by a novel 2'-deoxyribosyltransferase (NDT)-based combined biocatalyst. To this end, Lactobacillus delbrueckii NDT (LdNDT) was successfully immobilized through a two-step immobilization methodology, including a covalent immobilization onto glutaraldehyde-activated biomimetic silica nanoparticles followed by biocatalyst entrapment in calcium alginate. The resulting immobilized derivative, SiGPEI 25000-LdNDT-Alg, displayed 98% retained activity and was shown to be active and stable in a broad range of pH (5-9) and temperature (30-60 °C), but also displayed an extremely high reusability (up to 2100 reuses without negligible loss of activity) in the enzymatic production of cladribine. Finally, as a proof of concept, SiGPEI 25000-LdNDT-Alg was successfully employed in the green production of cladribine at mg scale.

Stabilization of ω-transaminase from Pseudomonas fluorescens by immobilization techniques.

Transaminases are a class of enzymes with promising applications for the preparation and resolution of a vast diversity of valued amines. Their poor operational stability has fueled many investigations on its stabilization due to their biotechnological relevance. In this work, we screened the stabilization of the tetrameric ω-transaminase from Pseudomonas fluorescens (PfωTA) through both carrier-bound and carrier-free immobilization techniques. The best heterogeneous biocatalyst was the PfωTA immobilized as cross-linked enzyme aggregates (PfωTA-CLEA) which resulted after studying different parameters as the precipitant, additives and glutaraldehyde concentrations. The best conditions for maximum recovered activity (29 %) and maximum thermostability at 60 ºC and 70 ºC (100 % and 71 % residual activity after 1 h, respectively) were achieved by enzyme precipitation with 90% acetone or ethanol, in presence of BSA (100 mg/mL) and employing glutaraldehyde (100 mM) as cross-linker. Studies on different conditions for PfωTA-CLEA preparation yielded a biocatalyst that exhibited 31 and 4.6 times enhanced thermal stability at 60 °C and 70 °C, respectively, compared to its soluble counterpart. The PfωTA-CLEA was successfully used in the bioamination of 4-hydroxybenzaldehyde to 4-hydroxybenzylamine. To the best of our knowledge, this is the first report describing a transaminase cross-linked enzyme aggregates as immobilization strategy to generate a biocatalyst with outstanding thermostability.

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.

Very Strong but Reversible Immobilization of Enzymes on Supports Coated with Ionic Polymers.

In this chapter, the properties of tailor-made anionic exchanger resins based on films of large polyethylenimine polymers (e.g., molecular weight 25,000) as supports for strong but reversible immobilization of proteins are shown. The polymer is completely coated, via covalent immobilization, the surface of different porous supports. Proteins can interact with this polymeric bed, involving a large percentage of the protein surface in the adsorption. Different enzymes have been very strongly adsorbed on these supports, retaining enzyme activities. On the other hand, adsorption is very strong and the derivatives may be used under a wide range of pH and ionic strengths. These supports may be useful even to stabilize multimeric enzymes, by involving several enzyme subunits in the immobilization.

Stabilization of Multimeric Enzymes via Immobilization and Further Cross-Linking with Aldehyde-Dextran.

Subunit dissociation of multimeric proteins is one of the most important causes of inactivation of proteins having quaternary structure, making these proteins very unstable under diluted conditions. A sequential two-step protocol for the stabilization of this protein is proposed. A multisubunit covalent immobilization may be achieved by performing very long immobilization processes between multimeric enzymes and porous supports composed of large internal surfaces and covered by a very dense layer of reactive groups. Additional cross-linking with polyfunctional macromolecules promotes the complete cross-linking of the subunits to fully prevent enzyme dissociation. Full stabilization of multimeric structures has been physically shown because no subunits were desorbed from derivatives after boiling them in SDS. As a functional improvement, these immobilized preparations no longer depend on the enzyme.

In Situ Immobilization of Enzymes in Biomimetic Silica.

In this chapter we describe different strategies for enzyme immobilization in biomimetic silica nanoparticles. Synthesis of this type of support is performed under mild and biocompatible conditions and has been proven suitable for the immobilization and stabilization of a range of enzymes and enzymatic systems in nanostructured particles. Immobilization occurs by entrapment while the silica matrix is formed via catalysis of a polyamine molecule and the presence of silicic acid. Parameters such as enzyme, polyamine molecule, or source of Si concentration have been tailored in order to maximize enzymatic loads, stabilities, and specific activities of the catalysts. We provide different approaches for the immobilization and co-immobilization of enzymes that could be potentially extensible to other biocatalysts.

Efficient glycerol transformation by resting Gluconobacter cells.

In the present work, glycerol biotransformation using Gluconobacter strains was studied with a process intensification perspective that facilitated the development of a cleaner and more efficient technology from those previously reported. Starting from the industrial by-product, crude glycerol, resting cells of Gluconobacter frateurii and Gluconobacter oxydans were able to convert glycerol under batch reactor conditions in water with no other additive but for the substrate. The study of strains, biomass:solution ratio, pH, growth stage, and simplification of media composition in crude glycerol bioconversions facilitated productivities of glyceric acid of 0.03 g/L.h and 2.07 g/L.h (71.5 g/g % pure by NMR) of dihydroxyacetone (DHA). Productivities surmounted recent reported fermentative bioconversions of crude glycerol and were unprecedented for the use of cell suspended solely in water. This work proposes a novel approach that allows higher productivities, cleaner production, and reduction in water and energy consumption, and demonstrates the applicability of the proposed approach.

Design of stable magnetic hybrid nanoparticles of Si-entrapped HRP.

Hybrid and composite nanoparticles represent an attractive material for enzyme integration due to possible synergic advantages of the structural builders in the properties of the nanobiocatalyst. In this study, we report the synthesis of a new stable hybrid nanobiocatalyst formed by biomimetic silica (Si) nanoparticles entrapping both Horseradish Peroxidase (HRP) (EC 1.11.1.7) and magnetic nanoparticles (MNPs). We have demonstrated that tailoring of the synthetic reagents and post immobilization treatments greatly impacted physical and biocatalytic properties such as an unprecedented ~280 times increase in the half-life time in thermal stability experiments. The optimized nanohybrid biocatalyst that showed superparamagnetic behaviour, was effective in the batch conversion of indole-3-acetic acid, a prodrug used in Direct Enzyme Prodrug Therapy (DEPT). Our system, that was not cytotoxic per se, showed enhanced cytotoxic activity in the presence of the prodrug towards HCT-116, a colorectal cancer cell line. The strategy developed proved to be effective in obtaining a stabilized nanobiocatalyst combining three different organic/inorganic materials with potential in DEPT and other biotechnological applications.

Heterogeneous Systems Biocatalysis: The Path to the Fabrication of Self-Sufficient Artificial Metabolic Cells.

Industrial biocatalysis is playing a key role in the development of the global bio-economy that must change our current productive model to pair the socio-economical development with the preservation of our already harmed planet. The exploitation of isolated multi-enzyme systems and the discovery of novel biocatalytic activities are leading us to manufacture chemicals that were inaccessible through biological routes in the early past. These endeavors have been grouped under the concept of systems biocatalysis. However, by using isolated biological machineries, fundamental features underlying the protein confinement found inside the living cells are missed. To re-gain these properties, such concepts can be expanded to a new concept; heterogeneous systems biocatalysis. This new concept is based on the fabrication of heterogeneous biocatalysts inspired by the spatial organization and compartmentalization that orchestrate metabolic pathways within cells. By assembling biological machineries (including enzymes and cofactors) into artificial solid chassis, one can fabricate self-sufficient and robust cell-free systems able to catalyze orchestrated chemical processes. Furthermore, the confinement of enzymes and and "artificial cofactor" inside solid materials has also attracted our attention because these self-sufficient systems exert de novo and non-natural functionalities. Here, we intend to go beyond immobilization of multi-enzyme systems, discussing only those enzymatic systems that have been co-immobilized with their cofactor or exogenous partners to enhance their cooperative action. In this article, we review the latest architectures developed to fabricate self-sufficient heterogeneous biocatalysts with application in chemical manufacturing, biosensing or energy production.

Protein-templated biomimetic silica nanoparticles.

Biomimetic silica particles can be synthesized as a nanosized material within minutes in a process mimicked from living organisms such as diatoms and sponges. In this work, we have studied the effect of bovine serum albumin (BSA) as a template to direct the synthesis of silica nanoparticles (NPs) with the potential to associate proteins on its surface. Our approach enables the formation of spheres with different physicochemical properties. Particles using BSA as a protein template were smaller (∼250-380 nm) and were more monodisperse than those lacking the proteic core (∼700-1000 nm) as seen by dynamic light scattering (DLS), scanning electron microscopy (SEM), and environmental scanning electron microscopy (ESEM) analysis. The absence of BSA during synthesis produced silica nanoparticles without any porosity that was detectable by nitrogen adsorption, whereas particles containing BSA developed porosity in the range of 4 to 5 nm which collapsed on the removal of BSA, thus producing smaller pores. These results were in accordance with the pore size calculated by high-resolution transmission electron microscopy (HTEM). The reproducibility of the BSA-templated nanoparticle properties was determined by analyzing four batches of independent synthesizing experiments that maintained their properties. The high positive superficial charge of the nanoparticles facilitated adsorption under mild conditions of a range of proteins from an E. coli extract and a commercial preparation of laccase from Trametes versicolor. All of the proteins were quantitatively desorbed. Experiments conducted showed the reusability of the particles as supports for the ionic adsorption of the biomolecules. The protein loading capacity of the BSA-based biomimetic particles was determined using laccase as 98.7 ± 6.6 mg·g(-1) of particles.

Glutaraldehyde-mediated protein immobilization.

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 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 proteic 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 DAAO on different amino-activated supports.

Hydrolysis of fish oil by hyperactivated Rhizomucor miehei lipase immobilized by multipoint anion exchange.

Rhizomucor miehei lipase (RML) is greatly hyperactivated (around 20- to 25-fold toward small substrates) in the presence of sucrose laurate. Hyperactivation appears to be an intramolecular process because it is very similar for soluble enzymes and covalently immobilized derivatives. The hyperactivated enzyme was immobilized (in the presence of sucrose laurate) on cyanogen bromide-activated Sepharose (very mild covalent immobilization through the amino terminal residue), on glyoxyl Sepharose (intense multipoint covalent immobilization through the region with the highest amount of Lys residues), and on different anion exchangers (by multipoint anionic exchange through the region with the highest density of negative charges). Covalent immobilization does not promote the fixation of the hyperactivated enzyme, but immobilization on Sepharose Q retains the hyperactivated enzyme even in the absence of a detergent. The hydrolysis of fish oils by these hyperactivated enzyme derivatives was sevenfold faster than by covalently immobilized derivatives and three and a half times faster than by the enzyme hyperactivated on octyl-Sepharose. The open structure of the hyperactivated lipase is fairly exposed to the medium, and no steric hindrance should interfere with the hydrolysis of large substrates. These new hyperactivated derivatives seem to be more suitable for hydrolysis of oils by RML immobilized inside porous supports. In addition, the hyperactivated derivatives are fairly stable against heat and organic cosolvents.

Protein hydrolysis by immobilized and stabilized trypsin.

The preparation of novel immobilized and stabilized derivatives of trypsin is reported here. The new derivatives preserved 80% of the initial catalytic activity toward synthetic substrates [benzoyl-arginine p-nitroanilide (BAPNA)] and were 50,000-fold more thermally stable than the diluted soluble enzyme in the absence of autolysis. Trypsin was immobilized on highly activated glyoxyl-Sepharose following a two-step immobilization strategy: (a) first, a multipoint covalent immobilization at pH 8.5 that only involves low pK(a) amino groups (e.g., those derived from the activation of trypsin from trypsinogen) is performed and (b) next, an additional alkaline incubation at pH 10 is performed to favor an intense, additional multipoint immobilization between the high concentration of proximate aldehyde groups on the support surface and the high pK(a) amino groups at the enzyme surface region that participated in the first immobilization step. Interestingly, the new, highly stable trypsin derivatives were also much more active in the proteolysis of high molecular weight proteins when compared with a nonstabilized derivative prepared on CNBr-activated Sepharose. In fact, all the proteins contained a cheese whey extract had been completely proteolyzed after 6 h at pH 9 and 50°C, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Under these experimental conditions, the immobilized biocatalysts preserve more than 90% of their initial activity after 20 days. Analysis of the three-dimensional (3D) structure of the best immobilized trypsin derivative showed a surface region containing two amino terminal groups and five lysine (Lys) residues that may be responsible for this novel and interesting immobilization and stabilization. Moreover, this region is relatively far from the active site of the enzyme, which could explain the good results obtained for the hydrolysis of high-molecular weight proteins.

Hydrolysis of tannic acid catalyzed by immobilized-stabilized derivatives of Tannase from Lactobacillus plantarum.

A recombinant tannase from Lactobacillus plantarum , overexpressed in Escherichia coli , was purified in a single step by metal chelate affinity chromatography on poorly activated nickel supports. It was possible to obtain 0.9 g of a pure enzyme by using only 20 mL of chromatographic support. The pure enzyme was immobilized and stabilized by multipoint covalent immobilization on highly activated glyoxyl agarose. Derivatives obtained by multipoint and multisubunit immobilization were 500- and 1000-fold more stable than both the soluble enzyme and the one-point-immobilized enzyme in experiments of thermal and cosolvent inactivation, respectively. In addition, up to 70 mg of pure enzyme was immobilized on 1 g of wet support. The hydrolysis of tannic acid was optimized by using the new immobilized tannase derivative. The optimal reaction conditions were 30% diglyme at pH 5.0 and 4 degrees C. Under these conditions, it was possible to obtain 47.5 mM gallic acid from 5 mM tannic acid as substrate. The product was pure as proved by HPLC. On the other hand, the immobilized biocatalyst preserved >95% of its initial activity after 1 month of incubation under the optimal reaction conditions.