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Feruloyl esterases synthesize butyl hydroxycinnamates, molecules possessing interesting biological properties, nonetheless, they exhibit a low stability under synthesis conditions in organic solvents, restricting its use. To enhance its operational stability in synthesis, we immobilized type A feruloyl esterase from Aspergillus niger (AnFAEA) using several carrier-bound and carrier-free strategies. The most active biocatalysts were: 1) AnFAEA immobilized on epoxy-activated carriers (protein load of 0.6 mgenzyme x mg−1carrier) that recovered 91 % of the initial hydrolytic activity, and 2) AnFAEA aggregated and cross-linked in the presence of 5 mg of BSA and 15 mM of glutaraldehyde (AnFAEA-amino-CLEAs), which exhibited 385 % of its initial hydrolytic activity; both using 4-nitrophenyl butyrate as substrate. The AnFAEA-amino-CLEAs were 12.7 times more thermostable at 60 °C than the AnFAEA immobilized on epoxy-activated carrier, thus AnFAEA-amino-CLEAs were selected for further characterization. Interestingly, during methyl sinapate hydrolysis (pH 7.2 and 30 °C), AnFAEA-amino-CLEAs KM was 15 % higher, while during butyl sinapate synthesis the KM was reduced in 63 %, both compared with the soluble enzyme. The direct esterification of butyl sinapate at solvent free conditions using sinapic acid 50 mM, reached 95 % conversion after 24 h employing AnFAEA-amino-CLEAs, which could be used for 10 cycles without significant activity losses, demonstrating their outstanding operational stability., We acknowledge the funding of Fundación Hergar (F), Foundation ARAID, Aragon Government, Spain (E37-17R) for funding the work by Fernando López Gallego. This work was supported by the programa Atención a Problemas Nacionales from CONACyT-México [project number 2017-01-6267]. Daniel Grajales and Susana Velasco acknowledge the scholarships received from CONACyT-México.

Alcohol dehydrogenase from Bacillus (Geobacillus) stearothermophilus (BsADH) is a NADH-dependent enzyme catalyzing the oxidation of alcohols, however its thermal and operational stabilities are too low for its long-term use under non-physiological conditions. Enzyme immobilizations emerges as an attractive tool to enhance the stability of this enzyme. In this work, we have screened a battery of porous carriers and immobilization chemistries to enhance the robustness of a His-tagged variant of BsADH. The selected carriers recovered close to 50% of the immobilized activity and increased enzyme stability from 3 to 9 times compared to the free enzyme. We found a trade-off between the half-life time and the specific activity as a function of the relative anisotropy values of the immobilized enzymes, suggesting that both properties are oppositely related to the enzyme mobility (rotational tumbling). The most thermally stable heterogeneous biocatalysts were coupled with a NADH oxidase/catalase pair co-immobilized on porous agarose beads to perform the batch oxidation of five different 1,ω-diols with in situ recycling of NAD+. Only when His-tagged BsADH was immobilized on porous glass functionalized with Fe3+, the heterogeneous biocatalyst oxidized 1, 5-pentanediol with a conversion higher than 50% after five batch cycles. This immobilized multi-enzyme system presented promising enzymatic productivities towards the oxidation of three different diols. Hence, this strategical study accompanied by a functional and structural characterization of the resulting immobilized enzymes, allowed us selecting an optimal heterogeneous biocatalyst and their integration into a fully heterogeneous multi-enzyme system.

β-Hydroxyesters are essential building blocks utilised by the pharmaceutical and food industries in the synthesis of functional products. Beyond the conventional production methods based on chemical catalysis or whole-cell synthesis, the asymmetric reduction of β-ketoesters with cell-free enzymes is gaining relevance. To this end, a novel thermophilic (S)-3-hydroxybutyryl-CoA dehydrogenase from Thermus thermophilus HB27 (Tt27-HBDH) has been expressed, purified and biochemically characterised, determining its substrate specificity towards β-ketoesters and its dependence on NADH as a cofactor. The immobilization of Tt27-HBDH on agarose macroporous beads and its subsequent coating with polyethyleneimine has been found the best strategy to increase the stability and workability of the heterogeneous biocatalyst. Furthermore, we have embedded NADH in the cationic layer attached to the porous surface of the carrier. Since Tt27-HBDH catalyses cofactor recycling through 2-propanol oxidation, we achieve a self-sufficient heterogeneous biocatalyst where NADH is available for the immobilised enzymes but its lixiviation to the reaction bulk is avoided. Taking advantage of the autofluorescence of NADH, we demonstrate the activity of the enzyme towards the immobilised cofactor through single-particle analysis. Finally, we tested the operational stability in the asymmetric reduction of β-ketoesters in batch, succeeding in the reuse of both the enzyme and the co-immobilised cofactor up to 10 reaction cycles., A highly robust and productive self-sufficient heterogeneous biocatalysts to asymmetrically reduce β-ketoesters.

The functionalization of the internal surface of macroporous carriers with glyoxyl groups has proven to highly stabilize a large variety of enzymes through multipoint covalent immobilization. In this work, we have translated the surface chemistry developed for the fabrication of glyoxyl-agarose carriers to macroporous cellulose (CEL). To that aim, CEL-based microbeads were functionalized with glyoxyl groups through a stepwise alkoxylation (or alkylation)/oxidation synthetic scheme. This functionalization sequence was analyzed by solid-state NMR, while the scanning electron miscroscopy of CEL microbeads reveals that the mild oxidation conditions negligibly affect the morphological properties of the material. Through the optimal functionalization protocol using rac-glycidol, we introduce up to 200 μmols of aldehyde groups per gram of wet CEL, a similar density to the one obtained for the benchmarked agarose-glyoxyl carrier. This novel CEL-based carrier succeeds to immobilize and stabilize industrially relevant enzymes such as d-amino acid oxidase from Trigonopsis variabilis and xylanases from Trichoderma reseei. Remarkably, the xylanases immobilized on the optimal CEL-based materials present a half-life time of 51 h at 60 °C and convert up to 90% of the xylan after four operation cycles for the synthesis of xylooligosaccharides., The authors acknowledge funding from the National Council for Scientific and Technological Development (CNPq) for financial support for the PhD scholarships of S.M.O (Process CsF 201683/2014–2008). J.M.G thanks the funding from the EU FP7 project Lignofood (Ingredients for Food and Beverage Industry from a Lignocellulosic Source, grant agreement no 606073). F.L-G acknowledges the funding of IKERBASQUE. This work was performed under the Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency—grant no. MDM-2017-0720. J.M.F., thanks to the funding from the Spanish Ministerio de Ciencia e Innovación (grant RTI2018-093431-BI00).

Biocatalytic cascades afford the development of economically sustainable and green processes. Herein we examined the unprecedented coupling of co-immobilised Gluconobacter oxydans and an isolated transaminase to synthesise serinol from glycerol. Through this approach, we manufactured up to 36 mM serinol, the highest titer ever reported for a non-fermentative biosynthesis. More importantly, similar productivities are obtained starting from the industrial by-product crude glycerol, demonstrating the possibilities of this hybrid heterogenenous biocatalyst for valorising bio-based raw materials., S. Velasco acknowledges CONACyT for the granted postdoctoral fellowship. L. Betancor, E. Jackson and M. Ripoll acknowledge PEDECIBA, National Research and Innovation Agency of Uruguay (ANII) (POS_NAC_2019_1_158182) and Universidad ORT Uruguay. Fernando López acknowledges the funding of IKERBASQUE and Spanish Government (BIO2015-69887-R). This work was performed under the Maria de Maeztu Units of Excellence Programme – Grant No. MDM-2017-0720 Ministry of Science, Innovation and Universities.

Sustainable electricity generation is one of the major current challenges for our society. In this context, the evolution of nanomaterials and nanotechnologies has enabled the fabrication of microscopic devices to produce clean energy from a great variety of renewable sources. To expand the possibilities of energy generation, we have designed and fabricated bioinorganic generators capable to produce electricity by conversion of chemical energy from renewable fuel sources. Unlike traditional generators, the systems described herein produce mechanical energy through enzyme-driven gas production which generates vibration and pressure that are thus converted into electricity by the action of a piezoelectric component properly integrated into the device. Our generators are able to produce an electric ernergy from different renewable sources like glucose, ethanol, and amino acids, attaining energy outputs around 250 nJ cm–2 and reaching maximum open-circuit voltages of up to 1 V. In addition, the produced energy can be easily regulated by adjusting both enzyme and fuel concentration which can tune the electrical output according to the application. The systems described herein propose a new concept for self-sufficient energy harvesting that bridges biocatalysis and piezoelectricity, where the energy production is based on the piezoelectric effect triggered by enzymatic action rather than on the enzyme-driven electron transfer that governs biofuel cells. Although the electric output is too low yet to be considered an alternative for energy production, this technology opens the door to power small devices. We envision the utilization of this technology in such remote locations where mechanical energy is lacking but there are chemical energy reservoirs., We would like to acknowledge Marie-Curie Actions (NANOBIENER project), IKERBASQUE foundation for funding F.L.-G., and the support of COST Action CM1303 Systems Biocatalysis. We also acknowledge HERGAR foundation for the funding.

The immobilization of multi-enzyme systems on advanced materials is an emerging technology inspired by the spatial localization found in Nature. These systems harness the high chemo-, regio- and stereoselectivity of the enzymes and the heterogeneous nature of the resulting biocatalyst. This synergy allows more efficient and selective synthetic schemes which reduce waste production and simplify downstream processing. The revolution of the nanotechnology has contributed to design advanced materials that allow precisely controlling the spatial distribution of the different catalytic modules forming a multi-enzyme system. Outstandingly, this fact has boosted the development and the improvement of more complex cascade reactions catalyzed in vitro by heterogeneous multi-functional biocatalysts. In this review, we have discussed the different challenges that must be faced during the immobilization of multi-enzyme systems; from the carrier surface to the incorporation of cofactors into the solid-phase. We...

The manufacturing of aminoalcohols is an appealing transformation for industrial biocatalysis due to their high value as building blocks in synthetic chemistry. By co‐immobilizing dehydrogenases, ω‐transaminases and oxidases on porous carriers, we fabricated and characterized a multi‐functional heterogeneous biocatalyst that was further applied to the cell‐free biosynthesis of amines and aminoalcohols from alcohols and diols, respectively. This immobilized cascade integrates both redox cofactor and amine donor recycling systems into the amino alcohol biosynthesis. Spatial co‐localization of the multi‐enzyme system significantly increases the cofactor recycling efficiency, the system productivity and the product conversion. Using this multi‐functional heterogeneous biocatalyst, we achieved 80 % conversion of alcohols into amines and accessed a palette of up to 5 aminoalcohols starting from their corresponding diols in one‐pot. The results herein presented contribute to understanding the effects of the confinement of co‐immobilized enzymes on the cascade processes., This work was performed under the Maria de Maeztu Units of Excellence Programme – Grant No. MDM‐2017‐0720 Ministry of Science, Innovation and Universities. S. Velasco thanks the Mexican Council of Science and Technology (CONACyT) for the received postdoctoral fellowship. ARAID and IKERBASQUE foundations, Spanish government (BIO2015‐69887‐R), Era‐CoBiotech (Project ID: 61 HOMBIOCAT/ PCI2018‐092984) have funded the contribution of FLG and Aragón Government (Group E37_17R) cofounded by FEDER 2014–2020 “Construyendo Europa desde Aragón”.

Among carrier-free immobilization techniques, cross-linked enzyme aggregates (CLEA) have been extensively described for a great number of diverse enzymes. During the last two decades, numerous efforts have been devoted to identify and understand the main variables involved in CLEA's preparation process leading to robust immobilized biocatalysts. Since every enzyme immobilized as CLEA requires specific conditions and protocols, herein we provide a general preparation strategy where main parameters are highlighted and correlated with a possible desired improved enzyme feature.

The biomineralisation of metal phosphates is a promising approach to develop more efficient nanobiocatalysts; however, the interactions between the protein and the inorganic mineral are poorly understood. Elucidating which protein regions most likely participate in the mineral formation will guide the fabrication of more efficient biocatalysts based on metal-phosphate nanoflowers. We have biomineralised the lipase from Thermomyces lanuginosus using three calcium, zinc and copper phosphates to fabricate different types of bio-inorganic nanoflowers. To better understand how the biomineralisation process affects the enzyme properties, we have computationally predicted the protein regions with a higher propensity for binding Ca2+, Cu2+ and Zn2+. These binding sites can be considered as presumable nucleation points where the biomineralisation process starts and explain why different metals can form bio-inorganic nanoflowers of the same enzyme with different functional properties. The formation of calcium, copper and zinc phosphates in the presence of this lipase gives rise to nanoflowers with different morphologies and different enzymatic properties such as activity, stability, hyperactivation and activity–pH profile; these functional differences are supported by structural studies based on fluorescence spectroscopy and can be explained by the different locations of the predicted nucleation sites for the different metals. Among the three metals used herein, the mineralisation of this lipase with zinc-phosphate enables the fabrication of bio-inorganic nanoflowers 34 times more stable than the soluble enzyme. These bio-inorganic nanoflowers were reused for 8 reaction cycles achieving 100% yield in the hydrolysis of p-nitrophenol butyrate but losing more than 50% of their initial activity after 6 operational cycles. Finally, this heterogeneous biocatalyst was more active and enantioselective than the soluble enzyme (ee = 79%(R)) towards the kinetic resolution of rac-1-phenylethyl acetate yielding the R enantiomer with ee = 84%.

Enzyme cofactors play a major role in biocatalysis, as many enzymes require them to catalyze highly valuable reactions in organic synthesis. However, the cofactor recycling is often a hurdle to implement enzymes at the industrial level. The fabrication of heterogeneous biocatalysts co‐immobilizing phosphorylated cofactors (PLP, FAD+, and NAD+) and enzymes onto the same solid material is reported to perform chemical reactions without exogeneous addition of cofactors in aqueous media. In these self‐sufficient heterogeneous biocatalysts, the immobilized enzymes are catalytically active and the immobilized cofactors catalytically available and retained into the solid phase for several reaction cycles. Finally, we have applied a NAD+‐dependent heterogeneous biocatalyst to continuous flow asymmetric reduction of prochiral ketones, thus demonstrating the robustness of this approach for large scale biotransformations.

The enantioselectivity of carrier-bound and carrier-free immobilized lipase from Candida rugosa (CRL) was studied. CRL was immobilized in six agarose-based carriers functionalized with different reactive groups and in two different CRL cross-linked aggregates. Both, activity and enantioselectivity of all the immobilized lipase preparations were evaluated with different racemic esters under different reaction conditions (temperature, pH and solvent polarity). A strong effect of reaction media and immobilization protocol on enzyme activity and selectivity was found. Enzyme immobilization and reaction engineering allowed us obtaining the best immobilization protocol and reaction conditions to achieve high activity and enantioselectivty of CRL as heterogeneous catalyst. CRL immobilized on an agarose-based carrier activated with primary amino groups preferentially hydrolyzed (S)-phenylethyl acetate with E > 200 under pH 7, 4 °C and 30% of acetonitrile. On the other hand, CRL aggregated and cross-linked through their carboxylic groups preferentially hydrolyzed the (S)-isomer of ethyl 2-hydroxy-4-phenylbutyrate with an E = 39 under pH 5, 4 °C and 30% of acetonitrile. This work demonstrates the success of the combinatorial enzyme engineering for the production of highly enantioselective heterogeneous biocatalysts by screening different immobilization protocols and reaction media conditions.

One-pot multi-step conversions are desirable to achieve more efficient and sustainable chemical processes. In this context, the immobilization of multi-enzyme systems allows the reusability of several stabilized biocatalysts working in cascade and orthogonal reactions to access more complex synthetic schemes. Herein, we have shown the one-pot tandem hydrolysis and oxidation of racemic esters (1-phenylethyl acetate) to yield quantitative conversion of prochiral ketones (acetophenone) catalyzed by a consortium of immobilized enzymes. Eukaryotic lipase and catalase, and microbial thermophilic alcohol dehydrogenase and NADH oxidase are covalently, irreversibly and individually immobilized onto four different carriers, achieving high immobilization yields (>95%) for all the enzymes, and residual activities >50% for both thermophilic alcohol dehydrogenase and NADH oxidase, 18% for the catalase and 10% for the lipase. This heterogeneous system efficiently recycles NAD+ with a maximum turnover frequency (TOF) of 294 h−1 and can be reused for up to 10 operational cycles, retaining more than 80% of its initial activity.

This article belongs to the Special Issue Advances in the Design and Characterization of Heterogeneous Biocatalysts., Understanding the functionality of immobilized enzymes with spatiotemporal resolution and under operando conditions is an unmet need in applied biocatalysis, as well as priceless information to guide the optimization of heterogeneous biocatalysts for industrial purposes. Unfortunately, enzyme immobilization still relies on trial-and-error approximations that prevail over rational designs. Hence, a modern fabrication process to achieve efficient and robust heterogeneous biocatalysts demands comprehensive characterization techniques to track and understand the immobilization process at the protein–material interface. Recently, our group has developed a new generation of self-sufficient heterogeneous biocatalysts based on alcohol dehydrogenases co-immobilized with nicotinamide cofactors on agarose porous microbeads. Harnessing the autofluorescence of NAD+(P)H and using time-lapse fluorescence microscopy, enzyme activity toward the redox cofactors can be monitored inside the beads. To analyze these data, herein we present an image analytical tool to quantify the apparent Michaelis–Menten parameters of alcohol dehydrogenases co-immobilized with NAD(P)+/H at the single-particle level. Using this tool, we found a strong negative correlation between the apparent catalytic performance of the immobilized enzymes and the bead radius when using exogenous bulky substrates in reduction reactions. Therefore, applying image analytics routines to microscopy studies, we can directly unravel the functional heterogeneity of different heterogeneous biocatalyst samples tested under different reaction conditions., A.I.B.-M. and F.L.-G. are grateful to MINECO (BIO2015-69887-R, BIO2014-61838-EXP and PCI2018-092984) and HOMBIOCAT ERA-CoBioTech project for funding their research. We also thank ARAID and IKERBASQUE foundations for funding F.L.-G. and P.R.-C., and S.V.-L. thanks the Mexican Council of Science and Technology (CONACyT) for the postdoctoral fellowship she received.

The immobilization of fluorescent proteins is a key technology enabling to fabricate a new generation of photoactive materials with potential technological applications. Herein we have exploited superfolder green (sGFP) and red (RFP) fluorescent proteins expressed with different polypeptide tags. We fused these fluorescent proteins to His-tags to immobilize them on graphene 3D hydrogels, and Cys-tags to immobilize them on porous microparticles activated with either epoxy or disulfide groups and with Lys-tags to immobilize them on upconverting nanoparticles functionalized with carboxylic groups. Genetically programming sGFP and RFP with Cys-tag and His-tag, respectively, allowed tuning the protein spatial organization either across the porous structure of two microbeads with different functional groups (agarose-based materials activated with metal chelates and epoxy-methacrylate materials) or across the surface of a single microbead functionalized with both metal-chelates and disulfide groups. By using different polypeptide tags, we can control the attachment chemistry but also the localization of the fluorescent proteins across the material surfaces. The resulting photoactive material formed by His-RFP immobilized on graphene hydrogels has been tested as pH indicator to measure pH changes in the alkaline region, although the immobilized fluorescent protein exhibited a narrower dynamic range to measure pH than the soluble fluorescent protein. Likewise, the immobilization of Lys-sGFP on alginate-coated upconverting nanoparticles enabled the infrared excitation of the fluorescent protein to be used as a green light emitter. These novel photoactive biomaterials open new avenues for innovative technological developments towards the fabrication of biosensors and photonic devices., A.I.B.-M. and F.L.-G. are grateful to MINECO (BIO2015-69887-R and BIO2014-61838-EXP) for funding them. We also thank ARAID foundation for funding F.L.-G., and S.V.-L. thanks the Mexican Council of Science and Technology (CONACyT) for the postdoctoral fellowship she received. L.S. thanks MINECO for grant CTQ2012-39315.

There is a current need to fabricate new biobased functional materials. Bottom-up approaches to assemble simple molecular units have shown promise for biomaterial fabrication due to their tunability and versatility for the incorporation of functionalities. Herein, the fabrication of catalytic protein thin films by the entrapment of catalase into protein films composed of a scaffolding protein is demonstrated. Extensive structural and functional characterization of the films provide evidence of the structural integrity, order, stability, catalytic activity, and reusability of the biocatalytic materials. Finally, these functional biomaterials are coupled with piezoelectric disks to fabricate a second generation of bio-inorganic generators. These devices are capable of producing electricity from renewable fuels through catalase-driven gas production that mechanically stimulates the piezoelectric material., This work was partially supported by the European Research Council ERC‐CoG‐648071‐ProNANO (ALC), the Spanish Ministry of Economy and Competitiveness (BIO2016‐77367‐R) (ALC); PCI2018‐092984, and the Basque Government (Elkartek KK‐2017/00008). This work was performed under the Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency—grant no. MDM‐2017‐0720 (CIC biomaGUNE). We also acknowledge funding from the Fundación Hergar (FLG), Foundation ARAID, and Aragon Government (E37‐17R) for funding F.L.G., and Marie‐Curie Actions (NANOBIENER project) for funding S.V.L.

We expanded the application of self-sufficient heterogeneous biocatalysts containing coimmobilized ω-transaminases and pyridoxal 5′-phosphate (PLP) to efficiently operate packed-bed reactors in continuous flow. Using a ω-transaminase from Halomonas elongata coimmobilized with PLP onto porous methacrylate-based carriers coated with polyethylenimine, we operated a packed-bed reactor continuously for up to 50 column volumes at 1.45 mL × min–1 in the enantioselective deamination of model amines (α-methylbenzyl amine), yielding >90% conversion in all cycles without exogenous addition of cofactor. In this work, we expanded the concept of self-sufficient heterogeneous biocatalysts to other ω-transaminases such as the ones from Chromobacterium violaceum and Pseudomonas fluorescens. We found that enzymes with lower affinities toward PLP present lower operational stabilities in flow, even when coimmobilizing PLP. Furthermore, ω-transaminases coimmobilized with PLP were successfully implemented for the continuous synthesis of amines and the sustainable metrics were assessed. These results give some clues to exploit PLP-dependent ω-transaminases under industrially relevant continuous operations in a more cost-effective and environmentally friendly process., We want to acknowledge ARAID foundation for funding F.L.G. and to MINECO (BIO2015-69887-R) for funding A.I.B.M and F.L.G. This work was also supported for M.L.C. and F.P. by BBSRC [grant number BB/P002536/1]. F.L.G. and F.P. thank the support of COST action CM1303-System biocatalysis that made possible this collaborative research funding A.I.B.M. short-stay at University of Nottingham. S.V.L. is grateful for the postdoctoral fellowship received from the Mexican Council for Science and Technology (CONACyT).

The enzymatic synthesis of α-amino acids is a sustainable and efficient alternative to chemical processes, through which achieving enantiopure products is difficult. To more address this synthesis efficiently, a hierarchical architecture that irreversibly co-immobilises an amino acid dehydrogenase with polyethyleneimine on porous agarose beads has been designed and fabricated. The cationic polymer acts as an irreversible anchoring layer for the formate dehydrogenase. In this architecture, the two enzymes and polymer colocalise across the whole microstructure of the porous carrier. This multifunctional heterogeneous biocatalyst was kinetically characterised and applied to the enantioselective synthesis of a variety of canonical and noncanonical α-amino acids in both discontinuous (batch) and continuous modes. The co-immobilised bienzymatic system conserves more than 50 % of its initial effectiveness after five batch cycles and 8 days of continuous operation. Additionally, the environmental impact of this process has been semiquantitatively calculated and compared with the state of the art.

This article belongs to the Special Issue Lipases and Lipases Modification., Enzyme immobilization can promote several advantages for their industrial application. In this work, a lipase from Hypocrea pseudokoningii was efficiently linked to four chemical supports: agarose activated with cyanogen bromide (CNBr), glyoxyl-agarose (GX), MANAE-agarose activated with glutaraldehyde (GA) and GA-crosslinked with glutaraldehyde. Results showed a more stable lipase with both the GA-crosslinked and GA derivatives, compared to the control (CNBr), at 50 °C, 60 °C and 70 °C. Moreover, all derivatives were stabilized when incubated with organic solvents at 50%, such as ethanol, methanol, n-propanol and cyclohexane. Furthermore, lipase was highly activated (4-fold) in the presence of cyclohexane. GA-crosslinked and GA derivatives were more stable than the CNBr one in the presence of organic solvents. All derivatives were able to hydrolyze sardine, açaí (Euterpe oleracea), cotton seed and grape seed oils. However, during the hydrolysis of sardine oil, GX derivative showed to be 2.3-fold more selectivity (eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) ratio) than the control. Additionally, the types of immobilization interfered with the lipase enantiomeric preference. Unlike the control, the other three derivatives preferably hydrolyzed the R-isomer of 2-hydroxy-4-phenylbutanoic acid ethyl ester and the S-isomer of 1-phenylethanol acetate racemic mixtures. On the other hand, GX and CNBr derivatives preferably hydrolyzed the S-isomer of butyryl-2-phenylacetic acid racemic mixture while the GA and GA-crosslink derivatives preferably hydrolyzed the R-isomer. However, all derivatives, including the control, preferably hydrolyzed the methyl mandelate S-isomer. Moreover, the derivatives could be used for eight consecutive cycles retaining more than 50% of their residual activity. This work shows the importance of immobilization as a tool to increase the lipase stability to temperature and organic solvents, thus enabling the possibility of their application at large scale processes., This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, process No. 2013/50892-5; 2010/52322-3), Conselho de Desenvolvimento Científico e Tecnológico (CNPq process No. 406838/2013-5; 563260/2010-6). This project is also part of the National Institute of Science and Technology of the Bioethanol (No. 574002/2008-1), CNPq—Ciência sem Fronteira (No. 242775/2012-8). JAJ and MLTMP are Research Fellows of CNPq. MGP and ACV are supported by CNPq. FDAF was recipient of a FAPESP Fellowship.

Structural and functional catalytic characteristics of cross-linked enzyme aggregates (CLEA) are reviewed. Firstly, advantages of enzyme immobilization and existing types of immobilization are described. Then, a wide description of the factors that modify CLEA activity, selectivity and stability is presented. Nowadays CLEA offers an economic, simple and easy tool to reuse biocatalysts, improving their catalytic properties and stability. This immobilization methodology has been widely and satisfactorily tested with a great variety of enzymes and has demonstrated its potential as a future tool to optimize biocatalytic processes.

Optimal immobilization of enzymes on porous microbeads enables the fabrication of highly active and stable heterogeneous biocatalysts to implement biocatalysis in synthetic and analytical chemistry. However, empirical procedures for enzyme immobilization still prevail over rational ones because there is an unmet need for more comprehensive characterization techniques that aid to understand and trace the immobilization process. Here, we present the use of atomic force spectroscopy (AFS) as an innovative solution to indirectly characterize immobilized proteins on porous materials and monitor the immobilization process in real time. We investigate the mechanical properties of porous agarose microbeads immobilizing proteins by indenting a colloidal probe (silica microparticle) into a single bead. AFS demonstrates that the binding of proteins to the solid matrix of an agarose microbead alters its stiffness. Interestingly, we discovered that irreversible and multivalent immobilizations that make microbeads stiffer also stabilize the immobilized proteins against the temperature. Hence, we propose atomic force spectroscopy as a useful technique to indirectly unravel the stability of the immobilized enzymes investigating the mechanics of the heterogenous biocatalysts as a solid biomaterial beyond the intrinsic mechanics of the proteins., We acknowledge COST action CM103-System biocatalysis and IKERBASQUE for the funding to FLG. DG and SEM acknowledge the project MAT2013-48169-R from the Spanish Ministry of Economy (MINECO) and LV thanks financial support from CAM (project NANOAVANSENS, Ref. S2013/MIT-3029) and FIS2012-38866-C05-05 from MINECO. FLG also acknowledges the Spanish Ministry of Economy and Marie-Curie actions for funding the BIO2014-61838-EXP and NANOBIOENER, respectively.

Catalysis-based approaches for the activation of anticancer agents hold considerable promise. These principally rely on the use of metal catalysts capable of deprotecting inactive precursors of organic drugs or transforming key biomolecules available in the cellular environment. Nevertheless, the efficiency of most of the schemes described so far is rather low, limiting the benefits of catalytic amplification as strategy for controlling the therapeutic effects of anticancer compounds. In the work presented here, we show that flavin reactivity within a hydrogel matrix provides a viable solution for the efficient catalytic activation and delivery of cisplatin, a worldwide clinically-approved inorganic chemotherapy agent. This is achieved by ionically adsorbing a flavin catalyst and a Pt(iv) prodrug as substrate into porous amino-functionalized agarose beads. The hydrogel chassis supplies high local concentrations of electron donating groups/molecules in the surrounding of the catalyst, ultimately boosting substrate conversion rates (TOF >200 min−1) and enabling controlled liberation of the drug by light or chemical stimuli. Overall, this approach can afford platforms for the efficient delivery of platinum drugs as demonstrated herein by using a transdermal diffusion model simulating the human skin., Loading of a flavin catalyst and Pt prodrug onto a hydrogel affords biomaterials for the catalytic generation and delivery of cisplatin upon light irradiation or addition of electron donors. Confinement boosts the turnover frequency of the flavin.