Engineering bio-brick protein scaffolds for organizing enzyme assemblies.

Enzyme scaffolding is an emerging approach for enhancing the catalytic efficiency of multi-enzymatic cascades by controlling their spatial organization and stoichiometry. This study introduces a novel family of engineered SCAffolding Bricks, named SCABs, utilizing the consensus tetratricopeptide repeat (CTPR) domain for organized multi-enzyme systems. Two SCAB systems are developed, one employing head-to-tail interactions with reversible covalent disulfide bonds, the other relying on non-covalent metal-driven assembly via engineered metal coordinating interfaces. Enzymes are directly fused to SCAB modules, triggering assembly in a non-reducing environment or by metal presence. A proof-of-concept with formate dehydrogenase (FDH) and L-alanine dehydrogenase (AlaDH) shows enhanced specific productivity by 3.6-fold compared to free enzymes, with the covalent stapling outperforming the metal-driven assembly. This enhancement likely stems from higher-order supramolecular assembly and improved NADH cofactor regeneration, resulting in more efficient cascades. This study underscores the potential of protein engineering to tailor scaffolds, leveraging supramolecular spatial-organizing tools, for more efficient enzymatic cascade reactions.

Mechanistic studies of a lipase unveil effect of pH on hydrolysis products of small PET modules.

Biocatalysis is a key technology enabling plastic recycling. However, despite advances done in the development of plastic-degrading enzymes, the molecular mechanisms that govern their catalytic performance are poorly understood, hampering the engineering of more efficient enzyme-based technologies. In this work, we study the hydrolysis of PET-derived diesters and PET trimers catalyzed by the highly promiscuous lipase B from Candida antarctica (CALB) through QM/MM molecular dynamics simulations supported by experimental Michaelis-Menten kinetics. The computational studies reveal the role of the pH on the CALB regioselectivity toward the hydrolysis of bis-(hydroxyethyl) terephthalate (BHET). We exploit this insight to perform a pH-controlled biotransformation that selectively hydrolyzes BHET to either its corresponding diacid or monoesters using both soluble and immobilized CALB. The discoveries presented here can be exploited for the valorization of BHET resulting from the organocatalytic depolymerization of PET.

Engineered repeat proteins as scaffolds to assemble multi-enzyme systems for efficient cell-free biosynthesis.

Multi-enzymatic cascades with enzymes arranged in close-proximity through a protein scaffold can trigger a substrate channeling effect, allowing for efficient cofactor reuse with industrial potential. However, precise nanometric organization of enzymes challenges the design of scaffolds. In this study, we create a nanometrically organized multi-enzymatic system exploiting engineered Tetrapeptide Repeat Affinity Proteins (TRAPs) as scaffolding for biocatalysis. We genetically fuse TRAP domains and program them to selectively and orthogonally recognize peptide-tags fused to enzymes, which upon binding form spatially organized metabolomes. In addition, the scaffold encodes binding sites to selectively and reversibly sequester reaction intermediates like cofactors via electrostatic interactions, increasing their local concentration and, consequently, the catalytic efficiency. This concept is demonstrated for the biosynthesis of amino acids and amines using up to three enzymes. Scaffolded multi-enzyme systems present up to 5-fold higher specific productivity than the non-scaffolded ones. In-depth analysis suggests that channeling of NADH cofactor between the assembled enzymes enhances the overall cascade throughput and the product yield. Moreover, we immobilize this biomolecular scaffold on solid supports, creating reusable heterogeneous multi-functional biocatalysts for consecutive operational batch cycles. Our results demonstrate the potential of TRAP-scaffolding systems as spatial-organizing tools to increase the efficiency of cell-free biosynthetic pathways.

Multienzyme Coimmobilization on Triheterofunctional Supports.

Immobilized multienzyme systems are gaining momentum in applied biocatalysis; however, the coimmobilization of several enzymes on one carrier is still challenging. In this work, we exploited a heterofunctional support activated with three different chemical functionalities to immobilize a wide variety of different enzymes. This support is based on agarose microbeads activated with aldehyde, amino, and cobalt chelate moieties that allow a fast and irreversible immobilization of enzymes, enhancing the thermostability of most of the heterogeneous biocatalysts (up to 21-fold higher than the soluble one). Furthermore, this trifunctional support serves to efficiently coimmobilize a multienzyme system composed of an alcohol dehydrogenase, a reduced nicotinamide adenine dinucleotide (NADH) oxidase, and a catalase. The confined multienzymatic system demonstrates higher performance than its free counterpart, achieving a total turnover number (TTN) of 1 × 105 during five batch consecutive cycles. We envision this solid material as a platform for coimmobilizing multienzyme systems with enhanced properties to catalyze stepwise biotransformations.

Feruloyl Esterases Protein Engineering to Enhance Their Performance as Biocatalysts: A Review.

Feruloyl esterases (FAEs) are versatile enzymes able to release hydroxycinnamic acids or synthesize their ester derivatives, both molecules with interesting biological activities such as: antioxidants, antifungals, antivirals, antifibrotic, anti-inflammatory, among others. The importance of these molecules in medicine, food or cosmetic industries provides FAEs with several biotechnological applications as key industrial biocatalysts. However, FAEs have some operational limitations that must be overcome, which can be addressed through different protein engineering approaches to enhance their thermal stability, catalytic efficiencies, and selectivity. This review aims to present a brief historical tour through the mutagenesis strategies employed to improve enzymes performance and analyze the current protein engineering strategies applied to FAEs as interesting biocatalysts. Finally, an outlook of the future of FAEs protein engineering approaches to achieve successful industrial biocatalysts is given.

Cell-Free Biosynthesis of ω-Hydroxy Acids Boosted by a Synergistic Combination of Alcohol Dehydrogenases.

The activity orchestration of an unprecedented cell-free enzyme system with self-sufficient cofactor recycling enables the stepwise transformation of aliphatic diols into ω-hydroxy acids at the expense of molecular oxygen as electron acceptor. The efficiency of the biosynthetic route was maximized when two compatible alcohol dehydrogenases were selected as specialist biocatalysts for each one of the oxidative steps required for the oxidative lactonization of diols. The cell-free system reached up to 100 % conversion using 100 mM of linear C5 diols and performed the desymmetrization of prochiral branched diols into the corresponding ω-hydroxy acids with an exquisite enantioselectivity (ee>99 %). Green metrics demonstrate superior sustainability of this system compared to traditional metal catalysts and even to whole cells for the synthesis of 5-hydroxypetanoic acid. Finally, the cell-free system was assembled into a consortium of heterogeneous biocatalysts that allowed the enzyme reutilization. This cascade illustrates the potential of systems biocatalysis to access new heterofunctional molecules such as ω-hydroxy acids.

Self-sufficient asymmetric reduction of ß-ketoesters catalysed by a novel and robust thermophilic alcohol dehydrogenase co-immobilised with NADH.

ß-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 ß-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 ß-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 ß-ketoesters in batch, succeeding in the reuse of both the enzyme and the co-immobilised cofactor up to 10 reaction cycles.

Functionalization of Porous Cellulose with Glyoxyl Groups as a Carrier for Enzyme Immobilization and Stabilization.

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.

Metal substrate catalysis in the confined space for platinum drug delivery.

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.

Chitosan-based CLEAs from Aspergillus niger type A feruloyl esterase: high-productivity biocatalyst for alkyl ferulate synthesis.

The enzymatic synthesis of alkyl ferulates is an important reaction in cosmetic and pharmaceutical chemistries, since it may allow to expand the biorefinery concept valorizing biomass wastes enriched in ferulic acid. However, robust biocatalysts for that purpose are scarce. Herein, we have immobilized the type A feruloyl esterase from Aspergillus niger (AnFaeA) as cross-linked enzyme aggregates, employing chitosan as co-feeder (ChCLEAs). High immobilization yields and relative activity recovery were attained in all assessed conditions (> 93%). Furthermore, we enhanced the thermal stability of the soluble enzyme 32-fold. AnFaeA-ChCLEAs were capable to quantitatively perform the solvent-free direct esterification of short- to medium-chain alkyl ferulates (C4-C12) in less than 24 h. By raising the operational temperature to 50 °C, AnFaeA-ChCLEAs transformed 350 mM ferulic acid into isopentyl ferulate with a space-time yield of 46.1 g of product × L-1 × day-1, 73-fold higher than previously reported. The overall sustainability of this alkyl ferulate production bioprocess is supported by the high total turnover number (TTN 7 × 105) and the calculated green metrics (E factor = 30). Therefore, we herein present a robust, efficient, and versatile heterogeneous biocatalyst useful for the synthesis of a wide diversity of alkyl ferulates. KEY POINTS: • CLEAs of feruloyl esterase A from A. niger using chitosan as co-feeder were obtained. • Microenvironment of the biocatalysts allowed to obtain C1 to C18 alkyl ferulates. • Biocatalyst at boundary conditions showed a high productivity of 46 g/L day. Graphical Abstract.

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.

Carrier-bound and carrier-free immobilization of type A feruloyl esterase from Aspergillus niger: Searching for an operationally stable heterogeneous biocatalyst for the synthesis of butyl hydroxycinnamates.

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.

Immobilization of Enzymes as Cross-Linked Enzyme Aggregates: General Strategy to Obtain Robust Biocatalysts.

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.

Selective oxidation of alkyl and aryl glyceryl monoethers catalysed by an engineered and immobilised glycerol dehydrogenase.

Enzymes acting over glyceryl ethers are scarce in living cells, and consequently biocatalytic transformations of these molecules are rare despite their interest for industrial chemistry. In this work, we have engineered and immobilised a glycerol dehydrogenase from Bacillus stearothermophilus (BsGlyDH) to accept a battery of alkyl/aryl glyceryl monoethers and catalyse their enantioselective oxidation to yield the corresponding 3-alkoxy/aryloxy-1-hydroxyacetones. QM/MM computational studies decipher the key role of D123 in the oxidation catalytic mechanism, and reveal that this enzyme is highly enantioselective towards S-isomers (ee > 99%). Through structure-guided site-selective mutagenesis, we find that the mutation L252A sculpts the active site to accommodate a productive configuration of 3-monoalkyl glycerols. This mutation enhances the k cat 163-fold towards 3-ethoxypropan-1,2-diol, resulting in a specific activity similar to the one found for the wild-type towards glycerol. Furthermore, we immobilised the L252A variant to intensify the process, demonstrating the reusability and increasing the operational stability of the resulting heterogeneous biocatalyst. Finally, we manage to integrate this immobilised enzyme into a one-pot chemoenzymatic process to convert glycidol and ethanol into 3-ethoxy-1-hydroxyacetone and (R)-3-ethoxypropan-1,2-diol, without affecting the oxidation activity. These results thus expand the uses of engineered glycerol dehydrogenases in applied biocatalysis for the kinetic resolution of glycerol ethers and the manufacturing of substituted hydroxyacetones.

Selective Immobilization of Fluorescent Proteins for the Fabrication of Photoactive Materials.

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.

Biocatalytic Protein-Based Materials for Integration into Energy Devices.

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.

Sustainable and Continuous Synthesis of Enantiopure l-Amino Acids by Using a Versatile Immobilised Multienzyme System.

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.

Different Covalent Immobilizations Modulate Lipase Activities of Hypocrea pseudokoningii.

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.

Understanding the functional properties of bio-inorganic nanoflowers as biocatalysts by deciphering the metal-binding sites of enzymes.

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%.