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The nanoscale arrangement of ligands can have a major effect on the activation of membrane receptor proteins and thus cellular communication mechanisms. Here we report on the technological development and use of tailored DNA origami-based molecular rulers to fabricate "Multiscale Origami Structures As Interface for Cells" (MOSAIC), to enable the systematic investigation of the effect of the nanoscale spacing of epidermal growth factor (EGF) ligands on the activation of the EGF receptor (EGFR). MOSAIC-based analyses revealed that EGF distances of about 30-40 nm led to the highest response in EGFR activation of adherent MCF7 and Hela cells. Our study emphasizes the significance of DNA-based platforms for the detailed investigation of the molecular mechanisms of cellular signaling cascades.
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Factor de Crecimiento Epidérmico , Receptores ErbB , Humanos , ADN/química , Factor de Crecimiento Epidérmico/química , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Células HeLa , Ligandos , Transducción de SeñalRESUMEN
Artificial reconstruction of naturally evolved principles, such as compartmentalization and cascading of multienzyme complexes, offers enormous potential for the development of biocatalytic materials and processes. Due to their unique addressability at the nanoscale, DNA origami nanostructures (DON) have proven to be an exceptionally powerful tool for studying the fundamental processes in biocatalytic cascades. To systematically investigate the diffusion-reaction network of (co)substrate transfer in enzyme cascades, a model system of stereoselective ketoreductase (KRED) with cofactor regenerating enzyme is assembled in different spatial arrangements on DNA nanostructures and is located in the sphere of microbeads (MB) as a spatially confining nano- and microenvironment, respectively. The results, obtained through the use of highly sensitive analytical methods, Western blot-based quantification of the enzymes, and mass spectrometric (MS) product detection, along with theoretical modeling, provide strong evidence for the presence of two interacting compartments, the diffusion layers around the microbead and the DNA scaffold, which influence the catalytic efficiency of the cascade. It is shown that the microscale compartment exerts a strong influence on the productivity of the cascade, whereas the nanoscale arrangement of enzymes has no influence but can be modulated by the insertion of a diffusion barrier.
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ADN , Nanoestructuras , ADN/química , Nanoestructuras/química , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Biocatálisis , CatálisisRESUMEN
DNA hydrogels hold significant promise for biomedical applications and can be synthesized through enzymatic Rolling Circle Amplification (RCA). Due to the exploratory nature of this emerging field, standardized RCA protocols specifying the impact of reaction parameters are currently lacking. This study varied template sequences and reagent concentrations, evaluating RCA synthesis efficiency and hydrogel mechanical properties through quantitative PCR (qPCR) and indentation measurements, respectively. Primer concentration and stabilizing additives showed minimal impact on RCA efficiency, while changes in polymerase and nucleotide concentrations had a stronger effect. Concentration of the circular template exerted the greatest influence on RCA productivity. An exponential correlation between hydrogel viscosity and DNA amplicon concentration was observed, with nucleobase sequence significantly affecting both amplification efficiency and material properties, particularly through secondary structures. This study suggests that combining high-throughput experimental methods with structural folding prediction offers a viable approach for systematically establishing structure-property relationships, aiding the rational design of DNA hydrogel material systems.
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Microfluidic systems have fundamentally transformed the realm of adaptive laboratory evolution (ALE) for microorganisms by offering unparalleled control over environmental conditions, thereby optimizing mutant generation and desired trait selection. This review summarizes the substantial influence of microfluidic technologies and their design paradigms on microbial adaptation, with a primary focus on leveraging spatial stressor concentration gradients to enhance microbial growth in challenging environments. Specifically, microfluidic platforms tailored for scaled-down ALE processes not only enable highly autonomous and precise setups but also incorporate novel functionalities. These capabilities encompass fostering the growth of biofilms alongside planktonic cells, refining selection gradient profiles, and simulating adaptation dynamics akin to natural habitats. The integration of these aspects enables shaping phenotypes under pressure, presenting an unprecedented avenue for developing robust, stress-resistant strains, a feat not easily attainable using conventional ALE setups. The versatility of these microfluidic systems is not limited to fundamental research but also offers promising applications in various areas of stress resistance. As microfluidic technologies continue to evolve and merge with cutting-edge methodologies, they possess the potential not only to redefine the landscape of microbial adaptation studies but also to expedite advancements in various biotechnological areas. KEY POINTS: ⢠Microfluidics enable precise microbial adaptation in controlled gradients. ⢠Microfluidic ALE offers insights into stress resistance and distinguishes between resistance and persistence. ⢠Integration of adaptation-influencing factors in microfluidic setups facilitates efficient generation of stress-resistant strains.
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Biopelículas , Microfluídica , Biotecnología , Laboratorios , FenotipoRESUMEN
Sustainable approaches to circular economy in animal agriculture are still poorly developed. Here, we report an approach to reduce gaseous emissions of CO2 and NH3 from animal housing while simultaneously using them to produce value-added biomass. To this end, a cone-shaped, helical photobioreactor was developed that can be integrated into animal housing by being freely suspended, thereby combining a small footprint with a physically robust design. The photobioreactor was coupled with the exhaust air of a chicken house to allow continuous cultivation of a mixed culture of Arthrospira spec. (Spirulina). Continuous quantification of CO2 and NH3 concentration showed that the coupled algae reactor effectively purifies the exhaust air from the chicken house while producing algal biomass. Typical production rates of greater than 0.3 g/l*day dry mass were obtained, and continuous operation was possible for several weeks. Morphological, biochemical, and genomic characterization of Spirulina cultures yielded insights into the dynamics and metabolic processes of the microbial community. We anticipate that further optimization of this approach will provide new opportunities for the generation of value-added products from gaseous CO2 and NH3 waste emissions, linking resource-efficient production of microalgae with simultaneous sequestration of animal emissions. KEY POINTS: ⢠Coupling a bioreactor with exhaust gases of chicken coop for production of biomass. ⢠Spirulina mixed culture removes CO2 and NH3 from chicken house emissions. ⢠High growth rates and biodiversity adaptation for nitrogen metabolism. Towards a sustainable circular economy in livestock farming. The functional coupling of a helical tube photobioreactor with exhaust air from a chicken house enabled the efficient cultivation of Spirulina microalgae while simultaneously sequestering the animals' CO2 and NH3 emissions.
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Microalgas , Spirulina , Animales , Gases/metabolismo , Dióxido de Carbono/metabolismo , Fotobiorreactores , Biomasa , Vivienda para Animales , Pollos , Microalgas/metabolismoRESUMEN
To research the effect of the COVID-19 pandemic on mental health, the prevalence and characteristics of all completed suicides in the city of Frankfurt am Main were compared for a 10-month period before the pandemic (March 2019-December 2019) with one during the early pandemic (March 2020-December 2020). Medicolegal data collected in the context of the FraPPE suicide prevention project were evaluated using descriptive statistical methods. In total, there were 81 suicides during the early pandemic period, as opposed to 86 in the pre-pandemic period. Though statistically not significant, the proportion of male suicides (73%) was higher during the early pandemic period than before (63%). The age-at-death was comparable in the pre-pandemic and pandemic periods (average, 54.8 vs. 53.1 years). Between these two periods, there was no difference in respect to the three most commonly used suicide methods by men: fall from a height (26% vs. 22%), intoxication, and strangulation (each 24% vs. 19%). For women, there was, however, a shift in methods from strangulation (38%), intoxication (28%), and fall from a height (19%) to fall from a height (50%), strangulation (18%), intoxication, and collision with a rail vehicle (14% each). There was a trend towards more suicides among non-German nationals during the early pandemic (suicide rate/100,000 inhabitants: German, 14.3 vs. 11.5; non-German, 4.4 vs. 8.8). Before the pandemic, 54% of the suicides were known to have a mental illness in contrast to 44% during the early pandemic. Overall, no increase in completed suicides could be observed in Frankfurt am Main during the early pandemic.
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News from an old acquaintance: The streptavidin (STV)-biotin binding system is frequently used for the decoration of DNA origami nanostructures (DON) to study biological systems. Here, a surprisingly high dynamic of the STV/DON interaction is reported, which is affected by the structure of the DNA linker system. Analysis of different mono- or bi-dentate linker architectures on DON with a novel high-speed atomic force microscope (HS-AFM) enabling acquisition times as short as 50 ms per frame gave detailed insights into the dynamics of the DON/STV interaction, revealing dwell times in the sub-100 millisecond range. The linker systems are also used to present biotinylated epidermal growth factor on DON to study the activation of the epidermal growth factor receptor signaling cascade in HeLa cells. The studies confirm that cellular activation correlated with the binding properties of linker-specific STV/DON interactions observed by HS-AFM. This work sheds more light on the commonly used STV/DON system and will help to further standardize the use of DNA nanostructures for the study of biological processes.
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ADN , Nanoestructuras , ADN/química , Células HeLa , Humanos , Ligandos , Microscopía de Fuerza Atómica , Nanoestructuras/química , Estreptavidina/químicaRESUMEN
Comparison of different membrane anchor motifs for the surface display of a protein of interest (passenger) is crucial for achieving the best possible performance. However, generating genetic fusions of the passenger to various membrane anchors is time-consuming. We herein employ a recently developed modular display system, in which the membrane anchor and the passenger are expressed separately and assembled inâ situ via SpyCatcher and SpyTag interaction, to readily combine a model passenger cytochrome P450 BM3 (BM3) with four different membrane anchors (Lpp-OmpA, PgsA, INP and AIDA-I). This approach has the significant advantage that passengers and membrane anchors can be freely combined in a modular fashion without the need to generate direct genetic fusion constructs in each case. We demonstrate that the membrane anchors impact not only cell growth and membrane integrity, but also the BM3 surface display capacity and whole-cell biocatalytic activity. The previously used Lpp-OmpA as well as PgsA were found to be efficient for the display of BM3 via SpyCatcher/SpyTag interaction. Our strategy can be transferred to other user-defined anchor and passenger combinations and could thus be used for acceleration and improvement of various applications involving cell surface display.
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Escherichia coli/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/metabolismoRESUMEN
Enantiopure α-hydroxy ketones are important building blocks of active pharmaceutical ingredients (APIs), which can be produced by thiamine-diphosphate-dependent lyases, such as benzaldehyde lyase. Here we report the discovery of a novel thermostable benzaldehyde lyase from Rhodococcus erythropolis R138 (ReBAL). While the overall sequence identity to the only experimentally confirmed benzaldehyde lyase from Pseudomonas fluorescens Biovar I (PfBAL) was only 65 %, comparison of a structural model of ReBAL with the crystal structure of PfBAL revealed only four divergent amino acids in the substrate binding cavity. Based on rational design, we generated two ReBAL variants, which were characterized along with the wild-type enzyme in terms of their substrate spectrum, thermostability and biocatalytic performance in the presence of different co-solvents. We found that the new enzyme variants have a significantly higher thermostability (up to 22 °C increase in T50 ) and a different co-solvent-dependent activity. Using the most stable variant immobilized in packed-bed reactors via the SpyCatcher/SpyTag system, (R)-benzoin was synthesized from benzaldehyde over a period of seven days with a stable space-time-yield of 9.3â mmol â L-1 â d-1 . Our work expands the important class of benzaldehyde lyases and therefore contributes to the development of continuous biocatalytic processes for the production of α-hydroxy ketones and APIs.
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Cetonas , Rhodococcus , Aldehído-Liasas/metabolismo , BenzaldehídosRESUMEN
All-enzyme hydrogel (AEH) particles with a hydrodynamic diameter of up to 120â nm were produced intracellularly with an Escherichia coli-based inâ vivo system. The inCell-AEH nanoparticles were generated from polycistronic vectors enabling simultaneous expression of two interacting enzymes, the Lactobacillus brevis alcohol dehydrogenase (ADH) and the Bacillus subtilis glucose-1-dehydrogenase (GDH), fused with a SpyCatcher or SpyTag, respectively. Formation of inCell-AEH was analyzed by dynamic light scattering and atomic force microscopy. Using the stereoselective two-step reduction of a prochiral diketone substrate, we show that the inCell-AEH approach can be advantageously used in whole-cell flow biocatalysis, by which flow reactors could be operated for >4â days under constant substrate perfusion. More importantly, the inCell-AEH concept enables the recovery of efficient catalyst materials for stable flow bioreactors in a simple and economical one-step procedure from crude bacterial lysates. We believe that our method will contribute to further optimization of sustainable biocatalytic processes.
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Alcohol Deshidrogenasa , Nanopartículas , Biocatálisis , Alcohol Deshidrogenasa/metabolismo , Escherichia coli/metabolismo , Reactores Biológicos , Enzimas Inmovilizadas/metabolismoRESUMEN
Glycolipids can be synthetized in deep eutectic solvents (DESs) as they possess low water content allowing a reversed lipase activity and thus enables ester formation. Based on this principle, honey can also serve as a media for glycolipid synthesis. Indeed, this supersaturated sugar solution is comparable in terms of physicochemical properties to the sugar-based DESs. Honey-based products being commercially available for therapeutic applications, it appears interesting to enhance its bioactivity. In the current work, we investigate if enriching medical grade honey with in situ enzymatically-synthetized glycolipids can improve the antimicrobial property of the mixture. The tested mixtures are composed of Manuka honey that is enriched with octanoate, decanoate, laurate, and myristate sugar esters, respectively dubbed GOH, GDH, GLH, and GMH. To characterize the bioactivity of those mixtures, first a qualitative screening using an agar well diffusion assay has been performed with methicillin-resistant Staphylococcus aureus, Bacillus subtilis, Candida bombicola, Escherichia coli, and Pseudomonas putida which confirmed considerably enhanced susceptibility of these micro-organisms to the different glycolipid enriched honey mixtures. Then, a designed biosensor E. coli strain that displays a stress reporter system consisting of three stress-specific inducible, red, green, and blue fluorescent proteins which respectively translate to physiological stress, genotoxicity, and cytotoxicity was used. Bioactivity was, therefore, characterized, and a six-fold enhancement of the physiological stress that was caused by GOH compared to regular Manuka honey at a 1.6% (v/v) concentration was observed. An antibacterial agar well diffusion assay with E. coli was performed as well and demonstrated an improved inhibitory potential with GOH upon 20% (v/v) concentration.
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Antiinfecciosos , Miel , Staphylococcus aureus Resistente a Meticilina , Agar , Antibacterianos/análisis , Antibacterianos/farmacología , Antiinfecciosos/farmacología , Caprilatos , Decanoatos , Escherichia coli , Ésteres , Glucolípidos/farmacología , Lauratos , Lipasa , Pruebas de Sensibilidad Microbiana , Miristatos , Azúcares , AguaRESUMEN
Fully exploiting the potential of enzymes in cell-free biocatalysis requires stabilization of the catalytically active proteins and their integration into efficient reactor systems. Although in recent years initial steps towards the immobilization of such biomolecules in metal-organic frameworks (MOFs) have been taken, these demonstrations have been limited to batch experiments and to aqueous conditions. Here we demonstrate a MOF-based continuous flow enzyme reactor system, with high productivity and stability, which is also suitable for organic solvents. Under aqueous conditions, the stability of the enzyme was increased 30-fold, and the space-time yield exceeded that obtained with other enzyme immobilization strategies by an order of magnitude. Importantly, the infiltration of the proteins into the MOF did not require additional functionalization, thus allowing for time- and cost-efficient fabrication of the biocatalysts using label-free enzymes.
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Enzimas Inmovilizadas , Estructuras Metalorgánicas , Biocatálisis , Catálisis , Enzimas/metabolismo , Enzimas Inmovilizadas/metabolismo , Estructuras Metalorgánicas/metabolismo , Proteínas/metabolismo , SolventesRESUMEN
Combining structural DNA nanotechnology with the virtually unlimited variety of enzymes offers unique opportunities for generating novel biocatalytic devices. However, the immobilization of enzymes is still restricted by a lack of efficient covalent coupling techniques. The rational re-engineering of the genetically fusible SNAP-tag linker is reported here. By replacing five amino acids that alter the electrostatic properties of the SNAP_R5 variant, up to 11-fold increased coupling efficiency with benzylguanine-modified oligonucleotides and DNA origami nanostructures (DON) was achieved, resulting in typical occupancy densities of 75%. The novel SNAP_R5 linker can be combined with the equally efficient Halo-based oligonucleotide binding tag (HOB). Since both linkers exhibit neither cross-reactivity nor non-specific binding, they allowed orthogonal assembly of an enzyme cascade consisting of the stereoselective ketoreductase Gre2p and the cofactor-regenerating isocitrate dehydrogenase on DON. The cascade showed approximately 1.6-fold higher activity in a stereoselective cascade reaction than the corresponding free solubilized enzymes. The connector system presented here and the methods used to validate it represent important tools for further development of DON-based multienzyme systems to investigate mechanistic effects of substrate channeling and compartmentalization relevant for exploitation in biosensing and catalysis.
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Nanoestructuras , Biocatálisis , ADN/metabolismo , Nanotecnología , OligonucleótidosRESUMEN
Microfluidic devices can mimic naturally occurring microenvironments and create microbial population heterogeneities ranging from planktonic cells to biofilm states. The exposure of such populations to spatially organized stress gradients can promote their adaptation into complex phenotypes, which are otherwise difficult to achieve with conventional experimental setups. Here a microfluidic chip that employs precise chemical gradients in consecutive microcompartments to perform microbial adaptive laboratory evolution (ALE), a key tool to study evolution in fundamental and applied contexts is described. In the chip developed here, microbial cells can be exposed to a defined profile of stressors such as antibiotics. By modulating this profile, stress adaptation in the chip through resistance or persistence can be specifically controlled. Importantly, chip-based ALE leads to the discovery of previously unknown mutations in Escherichia coli that confer resistance to nalidixic acid. The microfluidic device presented here can enhance the occurrence of mutations employing defined micro-environmental conditions to generate data to better understand the parameters that influence the mechanisms of antibiotic resistance.
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Dispositivos Laboratorio en un Chip , Microfluídica , Antibacterianos/farmacología , Biopelículas , Farmacorresistencia Microbiana/genética , Escherichia coli/genética , MutaciónRESUMEN
We here report the application of a machine-based microfluidic biofilm cultivation and analysis platform for studying the performance of biocatalytically active biofilms. By using robotic sampling, we succeeded in spatially resolving the productivity of three microfluidic reactors containing biocatalytically active biofilms that inducibly overexpress recombinant enzymes. Escherichia coli biofilms expressing two stereoselective oxidoreductases, the (R)-selective alcohol dehydrogenase LbADH and the (S)-selective ketoreductase Gre2p, as well as the phenolic acid decarboxylase EsPAD were used. The excellent reproducibility of the cultivation and analysis methods observed for all three systems underlines the usefulness of the new technical platform for the investigation of biofilms. In addition, we demonstrated that the analytical platform also opens up new opportunities to perform in-depth spatially resolved studies on the biomass growth in a reactor channel and its biochemical productivity. Since the platform not only offers the detailed biochemical characterization but also broad capabilities for the morphological study of living biofilms, we believe that our approach can also be performed on many other natural and artificial biofilms to systematically investigate a wide range of process parameters in a highly parallel manner using miniaturized model systems, thus advancing the harnessing of microbial communities for technical purposes.
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Biopelículas/crecimiento & desarrollo , Escherichia coli/fisiología , Dispositivos Laboratorio en un Chip , Técnicas Analíticas MicrofluídicasRESUMEN
This study aimed at characterizing the effects of dietary l-carnitine supplementation on hepatic fatty acid (FA) metabolism during inflammation in mid-lactating cows. Fifty-three pluriparous Holstein dairy cows were randomly assigned to either a control (CON, n = 26) or an l-carnitine supplemented (CAR; n = 27) group. The CAR cows received 125 g of a rumen-protected l-carnitine product per cow per day (corresponding to 25 g of l-carnitine/cow per day) from d 42 antepartum (AP) until the end of the trial on d 126 postpartum (PP). Aside from the supplementation, the same basal diets were fed in the dry period and during lactation to all cows. In mid lactation, each cow was immune-challenged by a single intravenous injection of 0.5 µg of LPS/kg of BW at d 111 PP. Blood samples were collected before and after LPS administration. The mRNA abundance of in total 39 genes related to FA metabolism was assessed in liver biopsies taken at d -11, 1, and 14 relative to LPS (d 111 PP) and also on d 42 AP as an individual covariate using microfluidics integrated fluidic circuit chips (96.96 dynamic arrays). In addition to the concentrations of 3 selected proteins related to FA metabolism, acetyl-CoA carboxylase α (ACACA), 5' AMP-activated protein kinase (AMPK), and solute carrier family 25 member 20 (SLC25A20) were assessed by a capillary Western blot method in liver biopsies from d -11 and 1 relative to LPS from 11 cows each of CAR and CON. On d -11 relative to LPS, differences between the mRNA abundance in CON and CAR were limited to acyl-CoA dehydrogenase (ACAD) very-long-chain (ACADVL) with greater mRNA abundance in the CAR than in the CON group. The liver fat content decreased from d -11 to d 1 relative to the LPS injection and remained at the lower level until d 14 in both groups. One day after the LPS challenge, lower mRNA abundance of carnitine palmitoyltransferase 1 (CPT1), CPT2, ACADVL, ACAD short-chain (ACADS), and solute carrier family 22 member 5 (SLC22A5) were observed in the CAR group as compared with the CON group. However, the mRNA abundance of protein kinase AMP-activated noncatalytic subunit gamma 1 (PRKAG1), ACAD medium-chain (ACADM), ACACA, and FA binding protein 1 (FABP1) were greater in the CAR group than in the CON group on d 1 relative to LPS. Two weeks after the LPS challenge, differences between the groups were no longer detectable. The altered mRNA abundance before and 1 d after LPS pointed to increased transport of FA into hepatic mitochondria during systemic inflammation in both groups. The protein abundance of AMPK was lower in CAR than in CON before the LPS administration. The protein abundance of SLC25A20 was neither changing with time nor treatment and the ACACA protein abundance was only affected by time. In conclusion, l-carnitine supplementation temporally altered the hepatic mRNA abundance of some genes related to mitochondrial biogenesis and very-low-density lipoprotein export in response to an inflammatory challenge, but with largely lacking effects before and 2 wk after LPS.
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Lactancia , Leche , Animales , Carnitina , Bovinos , Suplementos Dietéticos , Ácidos Grasos , Femenino , Hígado , ARN MensajeroRESUMEN
The display of complex proteins on the surface of cells is of great importance for protein engineering and other fields of biotechnology. Herein, we describe a modular approach, in which the membrane anchor protein Lpp-OmpA and a protein of interest (passenger) are expressed independently as genetically fused SpyCatcher and SpyTag units and assembled inâ situ by post-translational coupling. Using fluorescent proteins, we first demonstrate that this strategy allows the construct to be installed on the surface of E. coli cells. The scope of our approach was then demonstrated by using three different functional enzymes, the stereoselective ketoreductase Gre2p, the homotetrameric glucose 1-dehydrogenase GDH, and the bulky heme- and diflavin-containing cytochrome P450 BM3 (BM3). In all cases, the SpyCatcher-SpyTag method enabled the generation of functional whole-cell biocatalysts, even for the bulky BM3, which could not be displayed by conventional fusion with Lpp-OmpA. Furthermore, by using a GDH variant carrying an internal SpyTag, the system could be used to display an enzyme with unmodified N- and C-termini.
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Técnicas de Visualización de Superficie Celular/métodos , Enzimas/metabolismo , Membrana Celular/metabolismo , Enzimas/genética , Escherichia coli/citología , Escherichia coli/genética , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Microfluidic water-in-oil droplets are a versatile tool for biological and biochemical applications due to the advantages of extremely small monodisperse reaction vessels in the pL-nL range. A key factor for the successful dissemination of this technology to life science laboratory users is the ability to produce microfluidic droplet generators and related accessories by low-entry barrier methods, which enable rapid prototyping and manufacturing of devices with low instrument and material costs. The direct, experimental side-by-side comparison of three commonly used additive manufacturing (AM) methods, namely fused deposition modeling (FDM), inkjet printing (InkJ), and stereolithography (SLA), is reported. As a benchmark, micromilling (MM) is used as an established method. To demonstrate which of these methods can be easily applied by the non-expert to realize applications in topical fields of biochemistry and microbiology, the methods are evaluated with regard to their limits for the minimum structure resolution in all three spatial directions. The suitability of functional SLA and MM chips to replace classic SU-8 prototypes is demonstrated on the basis of representative application cases.
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Disciplinas de las Ciencias Biológicas , Dispositivos Laboratorio en un Chip , Enzimas/metabolismo , Diseño de Equipo , Cinética , Impresión Tridimensional , EstereolitografíaRESUMEN
The optimization of enzyme properties for specific reaction conditions enables their tailored use in biotechnology. Predictions using established computer-based methods, however, remain challenging, especially regarding physical parameters such as thermostability without concurrent loss of activity. Employing established computational methods such as energy calculations using FoldX can lead to the identification of beneficial single amino acid substitutions for the thermostabilization of enzymes. However, these methods require a three-dimensional (3D)-structure of the enzyme. In contrast, coevolutionary analysis is a computational method, which is solely based on sequence data. To enable a comparison, we employed coevolutionary analysis together with structure-based approaches to identify mutations, which stabilize an enzyme while retaining its activity. As an example, we used the delicate dimeric, thiamine pyrophosphate dependent enzyme ketoisovalerate decarboxylase (Kivd) and experimentally determined its stability represented by a T50 value indicating the temperature where 50% of enzymatic activity remained after incubation for 10 min. Coevolutionary analysis suggested 12 beneficial mutations, which were not identified by previously established methods, out of which four mutations led to a functional Kivd with an increased T50 value of up to 3.9°C.
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Aminoácidos/análisis , Carboxiliasas/metabolismo , Temperatura , Sustitución de Aminoácidos , Aminoácidos/genética , Aminoácidos/metabolismo , Carboxiliasas/química , Estabilidad de Enzimas , Modelos Moleculares , Mutación , Conformación ProteicaRESUMEN
We present a facile method for the combined synthesis and purification of protein-decorated DNA origami nanostructures (DONs). DONs bearing reductively cleavable biotin groups in addition to ligands for ligation of recombinant proteins are bound to magnetic beads. Protein immobilization is conducted with a large protein excess to achieve high ligation yields. Subsequent to cleavage from the solid support, pure sample solutions are obtained which are suitable for direct AFM analysis of occupation patterns. We demonstrate the method's utility using three different orthogonal ligation methods, the "halo-based oligonucleotide binder" (HOB), a variant of Halo-tag, the "SpyTag/SpyCatcher" (ST/SC) system, and the enzymatic "ybbR tag" coupling. We find surprisingly low efficiency for ST/SC ligation, presumably due to electrostatic repulsion and steric hindrance, whereas the ybbR method, despite its ternary nature, shows good ligation yields. Our method is particularly useful for the development of novel ligation methods and the synthesis of mechanically fragile DONs that present protein patterns for surface-based cell assays.