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.

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.

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.

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.