Natural killer (NK) cells are a subpopulation of effector lymphocytes with cytotoxic activity and cytokine-producing functions considered as an integral part of the innate immune response. Functions of NK cells include tumour elimination, engagement and regulation of antiviral immune responses and regulation of immune cells by production and secretion of chemokines and cytokines. CD69 is a C-type lectin-like transmembrane receptor expressed in NK cells. CD69 is an activating receptor and acts also as a very early marker of lymphocyte activation. Putative protein ligands have been described for CD69 in the last years: Galectin-1, S1P1, S100A8/S100A9 and Myl9/12. Galectin-1 is a prototypical lectin characterized by the presence of a common lectin structural fold and a carbohydrate recognition domain involved in carbohydrate binding. Galectin-1 was identified as a binding partner for CD69 based on biological and functional studies, but structural details about the complex are still missing. This thesis describes the successful establishment of an expression protocol for a tag-less cysteine-less mutant of galectin-1 and the study of the interaction between galectin-1 and NK cell receptors. The interaction was studied using microscale thermophoresis and confirmed as dependent on the presence of a...

Amicoumacin A (Ami) halts bacterial growth by inhibiting the ribosome during translation. The Ami binding site locates in the vicinity of the E-site codon of mRNA. However, Ami does not clash with mRNA, rather stabilizes it, which is relatively unusual and implies a unique way of translation inhibition. In this work, we performed a kinetic and thermodynamic investigation of Ami influence on the main steps of polypeptide synthesis. We show that Ami reduces the rate of the functional canonical 70S initiation complex (IC) formation by 30-fold. Additionally, our results indicate that Ami promotes the formation of erroneous 30S ICs; however, IF3 prevents them from progressing towards translation initiation. During early elongation steps, Ami does not compromise EF-Tu-dependent A-site binding or peptide bond formation. On the other hand, Ami reduces the rate of peptidyl-tRNA movement from the A to the P site and significantly decreases the amount of the ribosomes capable of polypeptide synthesis. Our data indicate that Ami progressively decreases the activity of translating ribosomes that may appear to be the main inhibitory mechanism of Ami. Indeed, the use of EF-G mutants that confer resistance to Ami (G542V, G581A, or ins544V) leads to a complete restoration of the ribosome functionality. It is possible that the changes in translocation induced by EF-G mutants compensate for the activity loss caused by Ami. / Russian Foundation for Basic Research / Revisión por pares

The development of a biologic protein involves different stages and becomes a highly complex process which can be costly and time consuming to scale up for industrial production. Therefore, optimization is a necessary part of the production process development to lower the production expenses.An on-going project is working on upscaling the production of a protein derived from mussel adhesive proteins (MAPs) which has great properties to be used as a pharmaceutical drug or in medical devices. The protein is expressed in a bacterial host cell and the necessary post translational modifications (PTMs) are done in-vitro using enzymes. The work presented in this report was done to optimize both the protein production in lab scale bioreactors and the enzymatic reaction using an immobilized prolyl-4-hydroxylase (P4H) which does a post translational modification on prolyl-residues. Additionally, an interaction study was conducted to better understand the hydroxylation using the prolyl-4-hydroxylase.For the bioreactor optimization four initial trials were performed testing different growth and induction temperatures and also comparing exponential to linear feeding. From these trials it appeared that having 30 ℃ growth overnight and induction at the same temperature in combination with an exponential feeding rate gave the best results. The modifications done by the prolyl-4-hydroxylase were analysed by LC-MS and suggest that longer incubation time and more immobilized protein gives more modifications in the tested ranges and the possibilities of reusing the immobilized proteins looks promising. No conclusive data was discovered for the optimal substrate concentration. The interaction study revealed the importance of reagents used for catalysis with the enzyme to be present for interaction to occur, however more work needs to be done to discover an accurate KD for the interaction.

No description available.

Membrane proteins play a key role in numerous physiological processes such as transport, energy transduction in respiratory and photosynthetic systems, and signal transduction, and are of great pharmaceutical interest, comprising more than 60% of known drug targets. However, crystallisation of membrane proteins, and G protein-coupled receptors (GPCRs) in particular, still relies heavily on the use of protein engineering strategies, which have been shown to hamper protein activity. Here, a range of biophysical methods were used to study the structure and function of two membrane proteins, a prokaryotic peptide transporter, PepT_So and a GPCR, neurotensin receptor 1 (NTS1), using different membrane reconstitution methods to study the proteins in a native-like environment. Firstly, using the pulsed electron paramagnetic resonance (EPR) method of double electron-electron resonance (DEER) the conformation of PepT_So reconstituted into lipid bilayers was assessed and compared to previous structural data obtained from crystallography and modelling. The influence of the membrane potential and the presence of substrate on the conformational heterogeneity of this proton-coupled transporter were investigated. Secondly, NTS1 purification was optimized for biophysical study. Cysteine mutants were created and a labelling protocol was developed and optimized for fluorophore and nitroxide labelling studies. NTS1 was then studied by continuous-wave EPR, to assess the influence of ligand on local protein dynamics, and to assess the structure of a receptor segment known as helix 8, that was proposed to be an α-helix, but was only observed to be helical in one of the NTS1 crystallographic studies. Ensemble and single-molecule Förster resonance energy transfer (FRET), and DEER were combined to study the dimerisation behaviour of NTS1, showing novel dynamics of the interfacial associations. Finally, the signalling mechanism of NTS1 was also investigated using microscale thermophoresis (MST) to assess the affinity of the receptor for G protein in vitro in the absence of ligand, or in the presence of agonist or antagonist. MST measurements were performed in detergent and in nanodiscs of different lipid compositions, to assess the influence of the lipid environment on receptor function. In summary, this thesis demonstrates the potential of biophysical techniques to study various aspects of membrane protein structure and function in native-like lipid systems, complementing e.g. structural data obtained from crystallographic studies with functional data for membrane proteins in more native environments, as well as shedding light on protein dynamics. The work presented here provides novel insights into PepTSo transport, and in particular into NTS1 structure, signalling, and oligomerisation, opening up several avenues for future research.

Intracellular transport is provided by two major types of molecular motors kinesins and cytoplasmic dynein. Kinesin-1 is a molecular motor that transports molecules and organelles along microtubule tracks anterogradely. Specific protein-protein interactions are required to activate kinesin-1 as the free kinesin exist in an autoinhibited state. The activation of kinesin-1 induces its conformational change, enables microtubule binding and ATP hydrolysis necessary for the directional cargo transport. HDAC6 is a multifunctional protein composed of several domains. It plays an important role in many microtubule dependent processes as HDAC6 is a major tubulin deacetylase. It has been shown that HDAC6 manipulation (inhibition/genetic ablation) affects transport along microtubules but the exact mechanisms are unknown. The effect can be caused either by deacetylation microtubules or direct interaction with molecular motors. This thesis is focused on characterization of interactions between kinesin-1 and HDAC6 that have not been described so far. To this end, we expressed and purified various constructs of kinesin-1 and HDAC6 and tested their interactions by microscale thermophoresis (MST) and hydrogen deuterium exchange (HDX) to determine affinity and interaction sites, respectively. MST data revealed that...

Das Influenza A Virus (IAV) stellt weltweit eine ernstzunehmende Bedrohung für Gesundheit und Wirtschaft der Menschheit dar. Ein universeller und langanhaltender Impfstoff konnte noch nicht entwickelt werden und klinisch zugelassene Medikamente verlieren durch die rasante Entstehung von resistenten Stämmen zunehmend ihre Wirkung. Aus diesem Grund gewinnt die Erforschung neuer antiviraler Strategien zur Bekämpfung des Influenzavirus an Bedeutung zum Schutze unserer Gesellschaft. Eine vielversprechende Zielstruktur für die Entwicklung neuer antiviraler Medikamente stellt das virale Hämagglutinin (HA) dar. Das HA liegt in hoher Dichte auf Influenzaviren vor und ermöglicht die Bindung an Sialinsäuren (SA) auf Wirtszellen und die Verschmelzung mit deren Lipidmembran. HA-bindende Moleküle entfalten eine hemmende Wirkung bereits bei dem ersten Kontakt mit Zellen, sodass eine Infektion erst gar nicht stattfinden kann. Aufgrund einer hohen HA-Dichte auf der Virusoberfläche eignen sich besonders multivalente SA tragende Nanopartikel für die Hemmung einer viralen Infektion. Aufbauend auf diesen Erkenntnissen, wurden in der vorliegenden Arbeit neue multivalente Binder gegenüber dem viralen Hämagglutinin (HA) entwickelt und studiert. Im Gegensatz zu bereits bekannten multivalenten Sialosiden, die in einer undefinierten räumlichen Orientierung auf Polymergerüsten präsentiert wurden, konnten in der vorliegenden Arbeit strukturelle Aspekte identifiziert werden, um Gerüstsysteme mit optimaler Rezeptorpräsentation gegenüber der Influenza A Virusoberfläche zu generieren. Neben SA-basierten Polymersystemen wurde auch ein gegen HA gerichtetes Peptid aus einem Antikörper identifiziert, welches sich auch für eine multivalente Interaktion mit IAV eignet. Diese Arbeit ermöglicht neue Einblicke in die Auswahl geeigneter Trägersysteme, eines optimalen Rezeptorabstandes und der Verwendung alternativer Rezeptoren mit dem Ziel einer Infektionshemmung von IAV. / Influenza A virus (IAV) still poses a serious threat to global health and economy of mankind. So far, a universal, long-lasting vaccine could not be developed, and clinically approved drugs are prone to lose activity due to the fast development of resistant strains. Because of this, research on new antiviral compounds and strategies to combat influenza viruses is of great importance for the protection of our society. A promising candidate for the development of novel antiviral drugs is the viral hemagglutinin (HA) protein. HA is present at high density on the viral envelope, which allows binding to sialic acid (SA) molecules on host cells and fusion with their membrane. Following, HA binding molecules have an inhibitory effect at the very first step of the infection cycle, leading to the inability of an infection. Based on a high HA density on the viral surface, SA carrying nanoparticles qualify for the inhibition of a viral infection. Based on this knowledge the study at hand demonstrates the development of new multivalent binders against viral HA and discusses them critically. In contrast to published multivalent sialosides, which are displayed in an undefined fashion on polymer scaffolds, the results of this thesis support the identification of structural requirements for the design of new scaffold systems with an optimal match to the viral surface. Beside sialoside based polymer systems, completely new peptide based systems, based on an HA binding antibody, were developed. Similar to polyglycerolsialosides, such multivalent peptide-decorated polymers were able to achieve nanomolar binding inhibition constants, too. In summary, this thesis enables new insights into the choice of a suitable carrier system, the optimal receptor spacing, and the use of alternative receptors with the ultimate goal of virus neutralization.

Thymidylate kinase (TMK) is a key enzyme for DNA synthesis. It occurs at the junction of the de novo and salvage pathways for the synthesis of deoxythymidine triphosphate (dTTP). Its inhibition affects cell viability, thereby making it an important target for the development of anticancer, antibacterial and antiparasitic drugs. This thesis describes the analyses of the sequence, structure and dynamics of thymidylate kinase to obtain insights into its function. Two thermophilic variants of the enzyme were chosen for our studies. The studies provide valuable insights about the active site residues and the mechanism of catalysis, which have implications in protein engineering and design of specific inhibitors. Following is a chapter-wise description of the overall layout of the thesis. Chapter 1 | Introduction: This chapter provides a brief survey of the literature on TMKs and the scope of the work presented in the thesis. TMK belongs to the nucleoside monophosphate kinase (NMPK) family of enzymes, which includes adenylate kinase (AMK), guanylate kinase (GMK), uridylate kinase (UMK) and cytidylate kinase (CMK). The NMPK family of enzymes is associated with the reversible transfer of the terminal phosphoryl group from a nucleoside triphosphate (NTP) (usually adenosine triphosphate, i.e., ATP) to a nucleoside monophosphate (NMP). The identity of the NMP substrate varies among different enzymes. NMPKs share a common Rossmann fold and are comprised of a conserved P-loop, Lid region, CORE and NMP domains. The enzymes in the NMPK family also contain structurally similar active site architecture. Besides the three signature motifs, there are other conserved residues at the active site of TMK which are involved in interactions with the substrates ATP and dTMP. Despite the overall similarity, TMKs exhibit significant variations in sequence, residue conformation, substrate specificity and oligomerization mode. However, the residues responsible for these differences have not been studied. This thesis describes a comprehensive analysis of the sequence space of TMKs to detect the residues involved in such diversity. Subsequently, TMKs from a thermophilic archaeon (Sulfolobus tokodaii) and a hyperthermophilic bacterium (Aquifex aeolicus) were chosen for biochemical characterization and structural studies. Of these, the Sulfolobus tokodaii TMK (StTMK) has low sequence identity to the other TMKs of known three dimensional (3D) structures. Crystal structure analyses depicted the presence of some novel structural features and provided insights into the role of a conserved Arginine residue in function, which was verified through computational studies and mutagenesis experiments. Finally, the study on Aquifex aeolicus TMK resulted in multiple crystal structures of the apo form and different holo forms. These helped us to understand the mechanistic details of TMK-mediated catalysis, namely, the order of substrate binding and the reaction mechanism for phosphate transfer. Chapter 2 | Materials and Methods: This chapter provides a brief description of the procedures used to carry out the thesis work. The protein samples were purified to a high degree using column chromatography, and the purity was assessed using SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) analysis. Circular dichroism (CD) spectroscopy was employed to assess if the purified protein was well-folded. The pure and properly folded protein samples were used in further experiments. Differential scanning fluorimetry (DSF) was performed to determine the melting temperature of the thermophilic protein. MicroScale Thermophoresis (MST) and Surface Plasmon Resonance (SPR) were carried out to detect protein-substrate interactions. The protein samples were crystallized using the hanging drop vapour diffusion and microbatch under-oil techniques using commercially available crystallization screens, and the conditions which gave crystals were further optimized. Diffraction data, collected at either the home source or the synchrotron, were processed and scaled. Subsequently, phase information was obtained using the molecular replacement (MR) calculations. The MR solution was refined till convergence and its geometry was validated using different softwares. Finally, molecular dynamics (MD) simulations were performed to study the functionally important motions in the protein. Chapter 3 Insights into substrate specificity and oligomerization mode of Thymidylate Kinases from sequence evolution and conformational dynamics: Thymidylate kinase homologs exhibit significant variations in sequence, residue conformation, substrate specificity and oligomerization mode. However, the influence of sequence evolution and conformational dynamics on its quaternary structure and function has not been studied before. Based on extensive sequence and structure analyses, our study detected several non-conserved residues which are linked by co-evolution and are implicated in the observed variations in flexibility, oligomeric assembly and substrate specificity among the homologs. These lead to differences in the pattern of interactions at the active site in TMKs of different specificity. The method was further tested on thymidylate kinase from Sulfolobus tokodaii (StTMK) which has substantial differences in sequence and structure compared to other TMKs. Our sequence analyses pointed to a more flexible dTMP-binding site in StTMK compared to the other homologs, which was also indicated in MD simulations on the protein 3D structure. Binding assays proved that the protein can accommodate both purine and pyrimidine nucleotides at the dTMP binding site with comparable affinity. Additionally, the residues responsible for the narrow specificity of Brugia malayi TMK, whose three dimensional structure is unavailable, were detected. Our study provides a residue-level understanding of the differences observed among TMK homologs in previous experiments. It also illustrates the correlation among sequence evolution, conformational dynamics, oligomerization mode and substrate recognition in thymidylate kinases and detects co-evolving residues that affect binding, which should be taken into account while designing novel inhibitors. Chapter 4 | Biochemical and Structural characterization of a thermophilic variant of thymidylate kinase: This chapter reports the biochemical characterization and crystal structure determination of thymidylate kinase from the hyperthermophilic organism Sulfolobus tokodaii (StTMK) in its apo and ADP-bound forms. Our study describes the first three-dimensional structure of an archaeal TMK. The different structures had resolution ranging between 1.60 Å and 2.40 Å. StTMK is a thermostable enzyme with a melting temperature of 85.3 °C, as observed from thermal unfolding studies. The protein exists as a dimer in solution. A coupled enzyme assay, performed using thermo-stable lactate dehydrogenase (TtLdh) and pyruvate kinase (TtPk) from Thermus thermophilus, showed that StTMK has optimum activity at 80 °C. Despite the overall similarity to homologous TMKs, StTMK structures revealed several residue substitutions at the active site. However, enzyme assays demonstrated specificity to its natural substrates ATP and dTMP. A novel insertion (9 residues long) is observed in the C-terminal stretch of the Lid region. However, it is relatively rigid, which may be attributed to the presence of two proline residues and a hydrogen bond with an arginine residue in the α4/α5 loop. The C-terminus of the α2 helix points away from the Lid region in StTMK to avoid steric clashes with the Lid insertion. The main chain dihedral angles of the conserved Arg in the DRX motif are in the disallowed region of the Ramachandran plot in all holo TMK structures, wherein it forms several conserved hydrogen bonds with residues in the P-loop, α4 helix and α7 helix, as well as with the phosphate groups of both the substrates. A similar feature is observed in some of the StTMK structures. However, torsion angles in the allowed region of the Ramachandran plot are observed in one chain each in two of the apo structures. Further, conformational rearrangements of this Arg and its neighboring residues at the binding site of the second substrate are observed. The functional implication of this variation is described in the next chapter (chapter 5). Chapter 5 | Role of a conserved active site Arginine residue in Thymidylate kinase: Analysis of the structures of StTMK revealed multiple conformational states of Arg93 which is located at the reaction centre and is a part of the highly conserved DRX motif. Conformational heterogeneity of Arg can also be observed in some structures of Staphylococcus aureus and human TMK. However, the functional implication of this feature has not been probed before. The rearrangements of Arg93 are accompanied by related changes in the conformations of its neighbouring residues at the active site. This leads to three distinct conformational states in the dTMP-binding site, namely ‘Arg in’, ‘Arg intermediate’ and ‘Arg out’. Only the ‘Arg in’ state was found to be suitable for the proper positioning of the α-phosphate group of dTMP at the active site. This is hindered in the ‘Arg out’ and ‘Arg intermediate’ states. MD simulations showed that the torsion angles of the DRX Arg can sample between allowed and disallowed values in the apo-protein, with a preference for the catalytically suitable disallowed conformation in the holo-protein. Computational alanine scanning and MM/PBSA binding energy calculation further revealed the importance of Arg93 side chain in substrate binding. Subsequent site directed mutagenesis at this position to an Ala resulted in the loss of activity. Our work provides the first experimental evidence for the functional importance of Arg93 and gives insight into its regulatory role in the catalytically competent placement of dTMP. Our study also has implications for the development of potent inhibitors to lock the enzyme in the catalytically non-productive state. Chapter 6 | Characterizing active site dynamics from structural studies on the Intermediates along the reaction coordinate of a hyperthermophilic Thymidylate Kinase: TMK belongs to the family of nucleoside monophosphate kinases (NMPKs), several of which undergo structure-encoded conformational changes to perform their function. However, the absence of three dimensional structures for all the different reaction intermediates of a single TMK homolog hinders a clear understanding of its functional mechanism. We herein report the different conformational states along the reaction coordinate of a hyperthermophilic TMK from Aquifex aeolicus, determined via X-ray diffraction and further validated through normal mode studies. The analyses implicate an arginine residue in the Lid region in catalysis, which was confirmed through site-directed mutagenesis and subsequent enzyme assays on the wild type protein and mutants. Further, the enzyme was found to exhibit broad specificity towards phosphate group acceptor nucleotides. Our comprehensive analyses of the conformational landscape of TMK, together with associated biochemical experiments, provide insights into the mechanistic details of TMK-driven catalysis, for example, the order of substrate binding and the reaction mechanism for phosphate transfer. Such a study has utility in the design of potent inhibitors for these enzymes. Finally, the implications of the work described in this thesis and its future applications have been discussed in the section titled ‘Future prospects’. The work described in chapters 3 – 6 have been published in peer reviewed journals. Additionally, the author was involved in several collaborative projects which also resulted in publications (reprints attached in appendix).

Bacterial DNA replication can stall at DNA lesions, leading to cell death if the damage fails to be repaired. To circumvent this, bacteria possess a mechanism called translesion DNA synthesis (TLS) to allow DNA damage bypass. The ImuABC TLS mutasome comprises the RecA domain-containing protein ImuA, the inactive polymerase ImuB, and the error-prone polymerase ImuC. ImuA and ImuB are necessary for the mutational function of ImuC that can lead to antimicrobial resistance (AMR) as seen in high-priority pathogens Pseudomonas aeruginosa and Mycobacterium tuberculosis. Understanding how ImuA and ImuB contribute to this function can lead to new targets for antimicrobial development. This research aims to discover the molecular functions of ImuA and ImuB homologs from Myxococcus xanthus through structural modelling and biochemical analyses. ImuA was discovered to be an ATPase whose activity is enhanced by DNA. Based on predicted structural models of the ATPase active site, I identified the critical residues needed for ATP hydrolysis, and found that the ImuA C-terminus regulates ATPase activity. Further, ImuA and ImuBNΔ34 (a soluble truncation of ImuB) display a preference for longer single-stranded DNA and overhang DNA substrates, and their affinity for DNA was quantified in vitro. To better understand how ImuA and ImuB assemble in the TLS mutasome, bacterial two-hybrid assays determined that ImuA and ImuB can self-interact and bind one another. Mass photometry revealed that ImuA is a monomer and ImuBNΔ34 is a trimer in vitro. ImuA and ImuBNΔ34 binding affinity was quantified in vitro at 1.69 μM ± 0.21 by microscale thermophoresis, and removal of the ImuA C-terminus weakens this interaction. Lastly, ImuA and ImuBNΔ34 secondary structures were quantified using circular dichroism spectroscopy, and ImuA was modified to enable crystallization for future structural studies. Together, this research provides a better understanding of ImuABC-mediated TLS, potentially leading to novel antibiotics to reduce the clinical burden of AMR. / Thesis / Master of Science (MSc) / The antimicrobial resistance (AMR) crisis is fueled by the emergence of multi-drug resistant microbes, posing a major threat to global health and disease treatment. Bacteria can develop resistance to antibiotics through mutations in the genome. When the genome becomes damaged, bacteria can acquire these mutations by an error-prone replication mechanism called translesion DNA synthesis (TLS). In some bacteria, TLS involves a specialized enzyme complex, consisting of proteins ImuA, ImuB and ImuC, allowing replication past bulky DNA damage and lesions. The goal of this thesis is to investigate how the ImuA and ImuB proteins contribute to the functioning of this mistake-making machinery. I used biochemical and biophysical methods to identify ImuA and ImuB interactions with each other and themselves. I discovered that ImuA is an enzyme that uses energy to enhance its binding to DNA, and determined the specific amino acids involved in this function.

Le vieillissement de la peau est l'un des signes extérieurs du passage du temps. Avec l’âge, la peau devient plus sèche et se ride suite à la dégradation des macromolécules de la matrice extracellulaire par des enzymes cutanées telles que l’élastase, l’hyaluronidase et la collagénase. L’objectif de ce travail de thèse est de développer des tests enzymatiques miniaturisés en électrophorèse capillaire pour cribler des extraits de plantes et identifier de nouveaux bioactifs pour la cosmétique et le bienêtre de la peau. Ces essais ont été développés soit en dehors du capillaire (qui sert uniquement de milieu de séparation) ou dans le capillaire (qui sert alors de nanoréacteur enzymatique), puis optimisés pour permettre la détermination des constantes cinétiques (Km, Vmax et IC₅₀). La diffusion transversale des réactifs (TDLFP) a été appliquée pour mélanger les créneaux de réactifs injectés dans le capillaire. Des détecteurs tels que la fluorescence induite par laser ou la spectrométrie de masse à haute résolution ont été couplés à l’électrophorèse capillaire afin d’atteindre de fortes sensibilités de détection et la possibilité d’identifier les produits de la réaction enzymatique. Ces essais miniaturisés ont été appliqués à des algues extraites par électroporation ou à des plantes régionales extraites par des technologies vertes afin d’évaluer leur activité biologique vis-à-vis des enzymes de la peau. Les essais développés sont fiables, robustes et économes en réactifs. Enfin, l’utilisation d’une nouvelle technique d’analyse, la thermophorèse à micro-échelle, s’est montrée très utile et pleine d’espoir pour l’étude des interactions enzyme-effecteur. / Skin aging is one of the exterior/external signs of the passage of time. With age, the skin becomes drier and gets wrinkled due to the degradation of macromolecules of the extracellular matrix by skin enzymes such as elastase, hyaluronidase and collagenase. The aim of this thesis is to develop miniaturized enzymatic assays by capillary electrophoresis to screen plant extracts and identify new bioactives for cosmetics and skin wellbeing. These assays were developed either outside the capillary (which serves only as a separation tool) or in the capillary (which then serves as an enzymatic nanoreactor) then optimized to allow the determination of kinetic constants (Km, Vmax and IC₅₀). Tranvserse diffusion of laminar flow profiles (TDLFP) was applied to mix the reactants injected into the capillary. Detectors such as laser-induced fluorescence or high-resolution mass spectrometry have been coupled to capillary electrophoresis to achieve high sensitivities of detection and the possibility of identifying the products of the enzymatic reaction. These miniaturized assays were applied to algae extracted by electroporation or to regional plants extracted by green technologies in order to evaluate their biological activity towards skin enzymes. The assays developed are reliable, robust and economic in reactants consumption. Finally, the use of a new analytical technique, microscale thermophoresis, was shown to be very useful and hopeful for the study of enzyme-effector interactions.