RESUMO
The inâ vitro synthesis of Coenzyme A (CoA)-thioester intermediates opens new avenues to transform simple molecules into more complex and multifunctional ones by assembling cell-free biosynthetic cascades. In this review, we have systematically cataloged known CoA-dependent enzyme reactions that have been successfully implemented inâ vitro. To faciliate their identification, we provide their UniProt ID when available. Based on this catalog, we have organized enzymes into three modules: activation, modification, and removal. i) The activation module includes enzymes capable of fusing CoA with organic molecules. ii) The modification module includes enzymes capable of catalyzing chemical modifications in the structure of acyl-CoA intermediates. And iii) the removal module includes enzymes able to remove the CoA and release an organic molecule different from the one activated in the upstream. Based on these reactions, we constructed a reaction network that summarizes the most relevant CoA-dependent biosynthetic pathways reported until today. From the information available in the articles, we have plotted the total turnover number of CoA as a function of the product titer, observing a positive correlation between both parameters. Therefore, the success of a CoA-dependent inâ vitro pathway depends on its ability to regenerate CoA, but also to regenerate other cofactors such as NAD(P)H and ATP.
Assuntos
Acil Coenzima A , NAD , Acil Coenzima A/metabolismo , NAD/metabolismo , Coenzima A/metabolismoRESUMO
Heterogeneous biocatalysis is highly relevant in biotechnology as it offers several benefits and practical uses. To leverage the full potential of heterogeneous biocatalysts, the establishment of well-crafted protocols, and a deeper comprehension of enzyme immobilization on solid substrates are essential. These endeavors seek to optimize immobilized biocatalysts, ensuring maximal enzyme performance within confined spaces. For this aim, multidimensional characterization of heterogeneous biocatalysts is required. In this context, spectroscopic and microscopic methodologies conducted at different space and temporal scales can inform about the intraparticle enzyme kinetics, the enzyme spatial distribution, and the mass transport issues. In this Minireview, we identify enzyme immobilization, enzyme catalysis, and enzyme inactivation as the three main processes for which advanced characterization tools unveil fundamental information. Recent advances in operando characterization of immobilized enzymes at the single-particle (SP) and single-molecule (SM) levels inform about their functional properties, unlocking the full potential of heterogeneous biocatalysis toward biotechnological applications.
Assuntos
Biocatálise , Enzimas Imobilizadas , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , CinéticaRESUMO
Immobilization is a key enabling technology in applied biocatalysis that facilitates the separation, recovery, and reuse of heterogeneous biocatalysts. However, finding a consensus immobilization protocol for several enzymes forming a multi-enzyme system is extremely difficult and relies on a combinatorial trial-and-error approach. Herein, we describe a protocol in which 17 different carriers functionalized with different reactive groups are tested in a 96-well microtiter plate to screen up to 21 immobilization protocols for up to 18 enzymes. This screening includes an activity and stability assay to select the optimal immobilization chemistry to achieve the most active and stable heterogeneous biocatalysts. The information retrieved from the screening can be rationalized using a Python-based application CapiPy. Finally, through scoring the screening results, we find the consensus immobilization protocol to assemble an immobilized four-enzyme system to transform vinyl acetate into (S)-3-hydroxybutyric acid. This methodology opens a path to speed up the prototyping of immobilized multi-enzyme pathways for chemical manufacturing.
RESUMO
The development of methods to engineer and immobilize amine transaminases (ATAs) to improve their functionality and operational stability is gaining momentum. The quest for robust, fast, and easy-to-use methods to screen the activity of large collections of transaminases, is essential. This work presents a novel and multiplex fluorescence-based kinetic assay to assess ATA activity using 4-dimethylamino-1-naphthaldehyde as an amine acceptor. The developed assay allowed us to screen a battery of amine donors using free and immobilized ATAs from different microbial sources as biocatalysts. As a result, using chromatographic methods, 4-hydroxybenzylamine was identified as the best amine donor for the amination of 5-(hydroxymethyl)furfural. Finally, we adapted this method to determine the apparent Michaelis-Menten parameters of a model immobilized ATA at the microscopic (single-particle) level. Our studies promote the use of this multiplex, multidimensional assay to screen ATAs for further improvement.
Assuntos
Aminas , Enzimas Imobilizadas , Aminas/química , Biocatálise , Aminação , Enzimas Imobilizadas/metabolismo , Transaminases/metabolismoRESUMO
Invited for the cover of this issue are the groups of Gonzalo Jiménez-Osés and Fernando López-Gallego at CIC bioGUNE and CIC biomaGUNE, respectively. The image depicts the substrate scope of an engineered acyl transferases for the synthesis of statin derivatives. Read the full text of the article at 10.1002/chem.202300911.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases , AciltransferasesRESUMO
This study identifies new acyl donors for manufacturing statin analogues through the acylation of monacolin J acid by the laboratory evolved acyltransferase LovD9. Vinyl and p-nitrophenyl esters have emerged as alternate substrates for LovD9-catalyzed acylation. While vinyl esters can reach product yields as high as the ones obtained by α-dimethyl butyryl-S-methyl-3-mercaptopropionate (DMB-SMMP), the thioester for which LovD9 was evolved, p-nitrophenyl esters display a reactivity even higher than DMB-SMMP for the first acylation step yet the acylation product yield is lower. The reaction mechanisms were elucidated through quantum mechanics (QM) calculations.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases , Aciltransferases/metabolismo , Biocatálise , Acilação , Ésteres , Especificidade por SubstratoRESUMO
ß-Glucosidase (BG) catalyzes the hydrolysis of cellobiose to glucose, a substrate for fermentation to produce the carbon-neutral fuel bioethanol. Enzyme thermal stability and reusability can be improved through immobilization onto insoluble supports. Moreover, nanoscaled matrixes allow for preserving high reaction rates. In this work, BG was physically immobilized onto wrinkled SiO2 nanoparticles (WSNs). The adsorption procedure was tuned by varying the BG:WSNs weight ratio to achieve the maximum controllability and maximize the yield of immobilization, while different times of immobilization were monitored. Results show that a BG:WSNs ratio equal to 1:6 wt/wt provides for the highest colloidal stability, whereas an immobilization time of 24 h results in the highest enzyme loading (135 mg/g of support) corresponding to 80% yield of immobilization. An enzyme corona is formed in 2 h, which gradually disappears as the protein diffuses within the pores. The adsorption into the silica structure causes little change in the protein secondary structure. Furthermore, supported enzyme exhibits a remarkable gain in thermal stability, retaining complete folding up to 90 °C. Catalytic tests assessed that immobilized BG achieves 100% cellobiose conversion. The improved adsorption protocol provides simultaneously high glucose production, enhanced yield of immobilization, and good reusability, resulting in considerable reduction of enzyme waste in the immobilization stage.
Assuntos
Enzimas Imobilizadas , Nanopartículas , Adsorção , beta-Glucosidase/metabolismo , Celobiose , Estabilidade Enzimática , Enzimas Imobilizadas/química , Glucose , Concentração de Íons de Hidrogênio , Dióxido de Silício/química , Temperatura , BiocatáliseRESUMO
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.
Assuntos
Aldeídos , Enzimas Imobilizadas , Enzimas Imobilizadas/química , Biocatálise , CatáliseRESUMO
In vitro biosynthetic pathways that condense and reduce molecules through coenzyme A (CoASH) activation demand energy and redox power in the form of ATP and NAD(P)H, respectively. These coenzymes must be orthogonally recycled by ancillary reactions that consume chemicals, electricity, or light, impacting the atom economy and/or the energy consumption of the biosystem. In this work, we have exploited vinyl esters as dual acyl and electron donor substrates to synthesize ß-hydroxy acids through a non-decarboxylating Claisen condensation, reduction and hydrolysis stepwise cascade, including a NADH recycling step, catalyzed by a total of 4 enzymes. Herein, the chemical energy to activate the acyl group with CoASH and the redox power for the reduction are embedded into the vinyl esters. Upon optimization, this self-sustaining cascade reached a titer of (S)-3-hydroxy butyrate of 24â mM without requiring ATP and simultaneously recycling CoASH and NADH. This work illustrates the potential of in vitro biocatalysis to transform simple molecules into multi-functional ones.
Assuntos
Hidroxiácidos , NAD , NAD/metabolismo , Ésteres , Coenzima A/metabolismo , Trifosfato de Adenosina/metabolismoRESUMO
The contactless heating capacity of magnetic nanoparticles (MNPs) has been exploited in fields such as hyperthermia cancer therapy, catalysis, and enzymatic thermal regulation. Herein, we propose an advanced technology to generate multiple local temperatures in a single-pot reactor by exploiting the unique nanoheating features of iron oxide MNPs exposed to alternating magnetic fields (AMFs). The heating power of the MNPs depends on their magnetic features but also on the intensity and frequency conditions of the AMF. Using a mixture of diluted colloids of MNPs we were able to generate a multi-hot-spot reactor in which each population of MNPs can be selectively activated by adjusting the AMF conditions. The maximum temperature reached at the surface of each MNP was registered using independent fluorescent thermometers that mimic the molecular link between enzymes and MNPs. This technology paves the path for the implementation of a selective regulation of multienzymatic reactions.
Assuntos
Hipertermia Induzida , Nanopartículas de Magnetita , Nanopartículas , Campos Magnéticos , Nanopartículas Magnéticas de Óxido de Ferro , MagnetismoRESUMO
Regulation of enzymes is highly relevant toward orchestrating cell-free and stepwise biotransformations, thereby maximizing their overall performance. Plasmonic nanomaterials offer a great opportunity to tune the functionality of enzymes through their remarkable optical properties. Localized surface plasmon resonances (LSPR) can be used to modify chemical transformations at the nanomaterial's surface, upon light irradiation. Incident light can promote energetic processes, which may be related to an increase of local temperature (photothermal effects) but also to effects triggered by generated hotspots or hot electrons (photoelectronic effects). As a consequence, light irradiation of the protein-nanomaterial interface affects enzyme functionality. To harness these effects to finely and remotely regulate enzyme activity, the physicochemical features of the nanomaterial, properties of the incident light, and parameters governing molecular interactions must be optimized. In this Perspective, we discuss relevant examples that illustrate the use of plasmonic nanoparticles to control enzyme function through LSPR excitation. Finally, we also highlight the importance of expanding the use of plasmonic nanomaterials to the immobilization of multienzyme systems for light-driven regulation of cell-free biosynthetic pathways. Although this concept is living its infancy, we encourage the scientific community to advance in the development of novel light-controlled biocatalytic plasmonic nanoconjugates and explore their application in biosensing, applied biocatalysis, and biomedicine.
Assuntos
Biocatálise , Enzimas/metabolismo , Luz , Nanoestruturas/química , Nanotecnologia , Ressonância de Plasmônio de Superfície , TemperaturaRESUMO
We herein describe a bioinspired solid-phase assembly of a multienzyme system scaffolded on an artificial cellulosome. An alcohol dehydrogenase and an ω-transaminase were fused to cohesin and dockerin domains to drive their sequential and ordered coimmobilization on agarose porous microbeads. The resulting immobilized scaffolded enzymatic cellulosome was characterized through quartz crystal microbalance with dissipation and confocal laser scanning microscopy to demonstrate that both enzymes interact with each other and physically colocalize within the microbeads. Finally, the assembled multifunctional heterogeneous biocatalyst was tested for the one-pot conversion of alcohols into amines. By using the physically colocalized enzymatic system confined into porous microbeads, the yield of the corresponding amine was 1.3 and 10 times higher than the spatially segregated immobilized system and the free enzymes, respectively. This work establishes the basis of a new concept to organize multienzyme systems at the nanoscale within solid and porous immobilization carriers.
Assuntos
Celulossomas , Sequência de Aminoácidos , Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , CoesinasRESUMO
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.
Assuntos
Celulose , Enzimas Imobilizadas , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Concentração de Íons de Hidrogênio , Porosidade , Saccharomycetales , SefaroseRESUMO
Alkyl hydroxycinnamates (AHs) is a group of molecules of biotechnological interest due to their cosmetic, food, and pharmaceutical applications. Among their most interesting uses are as UV protectants, skin depigmentation agents, and antioxidant ingredients which are often claimed for their antitumoral potential. Nowadays, many sustainable enzymatic approaches using low-cost starting materials are available and interesting immobilization techniques are helping to increase the reuse of the biocatalysts, allowing the intensification of the processes and increasing AHs accessibility. Here a convenient summary of AHs most interesting biological activities and possible applications is presented. A deeper analysis of the art state to obtain AHs, focusing on most employed enzymatic synthesis approaches, their sustainability, acyl donors relevance, and most interesting enzyme immobilization strategies is provided.Key points⢠Most interesting alkyl hydroxycinnamates applications are summarized.⢠Enzymatic approaches to obtain alkyl hydroxycinnamates are critically discussed.⢠Outlook of enzyme immobilization strategies to attain alkyl hydroxycinnamates.
Assuntos
Biotecnologia , Enzimas Imobilizadas , Enzimas Imobilizadas/metabolismo , EsterificaçãoRESUMO
Hybrid bioinorganic biocatalysts have received much attention due to their simple synthesis, high efficiency, and structural features that favor enzyme activity and stability. The present work introduces a biomineralization strategy for the formation of hybrid nanocrystals from ß-galactosidase. The effects of the immobilization conditions were studied, identifying the important effect of metal ions and pH on the immobilization yield and the recovered activity. For a deeper understanding of the biomineralization process, an in silico study was carried out to identify the ion binding sites at the different conditions. The selected ß-galactosidase nanocrystals showed high specific activity (35,000 IU/g biocatalyst) and remarkable thermal stability with a half-life 11 times higher than the soluble enzyme. The nanobiocatalyst was successfully tested for the synthesis of galacto-oligosaccharides, achieving an outstanding performance, showing no signs of diffusional limitations. Thus, a new, simple, biocompatible and inexpensive nanobiocatalyst was produced with high enzyme recovery (82%), exhibiting high specific activity and high stability, with promising industrial applications.
Assuntos
Enzimas Imobilizadas/química , Enzimas/química , beta-Galactosidase/química , Sítios de Ligação/fisiologia , Biomineralização/fisiologia , Simulação por Computador , Estabilidade Enzimática , Enzimas/metabolismo , Enzimas Imobilizadas/metabolismo , Galactose/química , Concentração de Íons de Hidrogênio , Nanopartículas/química , Oligossacarídeos/química , Temperatura , beta-Galactosidase/metabolismoRESUMO
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.
Assuntos
Aspergillus niger , Quitosana , Hidrolases de Éster CarboxílicoRESUMO
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.
Assuntos
Catalase/metabolismo , Fontes de Energia Elétrica , Biocatálise , Catalase/química , Eletricidade , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Modelos MolecularesRESUMO
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.
Assuntos
Grafite/química , Proteínas de Fluorescência Verde/química , Hidrogéis/química , Proteínas Imobilizadas/química , Proteínas Luminescentes/química , Proteínas Recombinantes de Fusão/química , Alginatos/química , Técnicas Biossensoriais , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histidina/química , Histidina/genética , Histidina/metabolismo , Concentração de Íons de Hidrogênio , Proteínas Imobilizadas/genética , Proteínas Imobilizadas/metabolismo , Luz , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Metacrilatos/química , Nanopartículas/química , Oligopeptídeos/química , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Processos Fotoquímicos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sefarose/química , Proteína Vermelha FluorescenteRESUMO
Thiolases catalyze the condensation of acyl-CoA thioesters through the Claisen condensation reaction. The best described enzymes usually yield linear condensation products. Using a combined computational/experimental approach, and guided by structural information, we have studied the potential of thiolases to synthesize branched compounds. We have identified a bulky residue located at the active site that blocks proper accommodation of substrates longer than acetyl-CoA. Amino acid replacements at such a position exert effects on the activity and product selectivity of the enzymes that are highly dependent on a protein scaffold. Among the set of five thiolases studied, Erg10 thiolase from Saccharomyces cerevisiae showed no acetyl-CoA/butyryl-CoA branched condensation activity, but variants at position F293 resulted the most active and selective biocatalysts for this reaction. This is the first time that a thiolase has been engineered to synthesize branched compounds. These novel enzymes enrich the toolbox of combinatorial (bio)chemistry, paving the way for manufacturing a variety of α-substituted synthons. As a proof of concept, we have engineered Clostridium's 1-butanol pathway to obtain 2-ethyl-1-butanol, an alcohol that is interesting as a branched model compound.
Assuntos
Acetil-CoA C-Acetiltransferase/metabolismo , Acil Coenzima A/metabolismo , Hexanóis/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetil-CoA C-Acetiltransferase/química , Acetil-CoA C-Acetiltransferase/genética , Domínio Catalítico , Redes e Vias Metabólicas , Modelos Moleculares , Engenharia de Proteínas/métodos , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
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 8â days of continuous operation. Additionally, the environmental impact of this process has been semiquantitatively calculated and compared with the state of the art.