Riet Hilhorst

Enzymatic conversion of apolar compounds in organic media using an NADH regenerating system and dihydrogen as reductant

A combined enzyme system, consisting of hydrogenase, lipoamide dehydrogenase and 20β‐hydroxysteroid dehydrogenase has been enclosed in reversed micelles. This system catalyzes the stereo‐ and site‐specific enzymatic reduction of apolar, poorly water‐soluble ketosteroids to their corresponding 20β‐hydroxyform using an in situ NADH‐regenerating enzyme system and H2 as ultimate reductant.

Mass transfer rate of protein extraction with reversed micelles.

The rate of mass transfer in the liquid—liquid extraction of the enzyme α-amylase between an aqueous phase and a reversed micellar phase has been investigated. Mass transfer rate coefficients have been measured in a mixer/settler and in a stirred cell. The pH of the aqueous phase determines the distribution coefficient of the enzyme and thus the direction of transfer. Forward transfer of the enzyme from the aqueous to the reversed micellar phase (cationic surfactant) occurs at pH 10.0. The mass transfer rate of this process was found to be controlled by the diffusion of the enzyme in the aqueous phase boundary layer. Back transfer from the reversed micellar phase to the aqueous phase occurs in the pH range 4–6. In contrast to the forward transfer, this process was found to be controlled by the interfacial process of enzyme release from the reversed micelles instead of the boundary layer diffusion. The same effects have been observed for the transfer of the enzyme ribonuclease A to and from a reversed micellar phase with an anionic surfactant. A mechanism to explain the different mass transfer behaviour in forward and back transfer is suggested.

Effect of temperature on the reversed micellar extraction of enzymes

Abstract In “conventional” liquid-liquid extraction of protein solutions with a reversed micellar phase, the protein is transferred from an aqueous phase to the reversed micellar phase by forward extraction, and subsequently to a second aqueous phase during back extraction. This back transfer is a relatively slow process, owing to a large interfacial resistance to mass transfer. In this paper an alternative procedure is described for the back extraction, using the effect of temperature. By increasing the temperature of the reversed micellar phase, after it has been saturated with the aqueous phase during the forward extraction, a separate aqueous phase is formed in which most of the enzyme is concentrated. This excess aqueous phase can be separated easily from the reversed micellar phase by centrifugation. The enzyme can be recovered in this expelled phase at extremely high concentrations (up to 2000 times the initial concentration). This process has been performed continuously using two centrifugal separators, with the reversed micellar phase circulating between the two units. For α-amylase a recovery rate of 73% of the enzyme activity was achieved.

Peptide microarrays for detailed, high-throughput substrate identification, kinetic characterization, and inhibition studies on protein kinase A

A microarray-based mix-and-measure, nonradioactive multiplex method with real-time detection was used for substrate identification, assay development, assay optimisation, and kinetic characterization of protein kinase A (PKA). The peptide arrays included either up to 140 serine/threonine-containing peptides or a concentration series of a smaller number of peptides. In comparison with existing singleplex assays, data quality was high, variation in assay conditions and reagent consumption were reduced considerably, and assay development could be accelerated because phosphorylation kinetics were monitored simultaneously on 4, 12, or 96 arrays. PKA was shown to phosphorylate many peptides containing known PKA phosphorylation sites as well as some new substrates. The kinetic behavior of the enzyme and the mechanism of inhibition by AMP-PNP, staurosporin, and PKA inhibitor peptide on the peptide microarray correlated well with data from homogeneous assays. Using this multiplex setup, we showed that the kinetic parameters of PKA and the potency of PKA inhibitors can be affected by the sequence of the peptide substrate. The technology enables kinetic monitoring of kinase activity in a multiplex setting such as a cell or tissue lysate. Finally, this high-throughput method allows fast identification of peptide substrates for serine/threonine kinases that are still uncharacterized.

Fluorescence Detection of Enzymatically Formed Hydrogen Peroxide in Aqueous Solution and in Reversed Micelles

A sensitive enzymatic assay for oxidase reactions both in aqueous solution and in hexadecyltrimethylammoniumbromide (CTAB) reversed micelles has been developed. The assay is based on the fluorescence detection of dichlorofluorescein, which is formed by hydrogen peroxide oxidation of the nonfluorescent precursor dichlorofluorescin. Hydrogen peroxide as product of the reaction catalyzed by glucose oxidase served to select the reaction conditions. The reaction rate is distinctly enhanced in CTAB reversed micelles as compared to the rate in aqueous solution. This effect, combined with the high sensitivity owing to the strong fluorescence of dichlorofluorescein, makes the assay attractive for the detection of low enzyme, substrate, or peroxide concentrations.

Protein extraction using reversed micelles

Proteins can be extracted from an aqueous phase into reversed micelles. This transfer is governed by electrostatic interactions, for protein and surfactant have to bear opposite charges. The presence of high salt concentrations diminishes the attractive interactions and can lead to expulsion of the solubilized proteins. This is used to recover extracted proteins. Larger proteins require a larger number of charged residues on their surface in order to be transferred into reversed micelles, so the larger the protein, the further the pH of maximal transfer is removed from the isoelectric point. The transfer profiles can be manipulated by micellar size and charge density at the interface. When the charge density at the interface is modified by variation of the type of head group of the cosurfactant, shifts of transfer profiles to higher or lower pH values are observed. Variation of the number and length of the tails of quaternary ammonium surfactants revealed that of the 16 surfactants tested, only didecyldimethyl ammonium chloride and trioctylmethylammonium chloride enabled transfer. There was no relation between the water content of the organic phase and the transfer properties of the surfactants tested. Because for application not only forward transfer is important, an alternative method for back transfer was tested. Exposing an enzyme containing reversed micellar solution to a temperature increase led to expulsion of aqueous phase and enzyme, yielding a highly concentrated enzyme preparation.

Design of reversed micellar media for the enzymatic synthesis of apolar compounds

Publisher Summary This chapter focuses on the reversed micellar enzymology of 20 β-hydroxysteroid dehydrogenase that catalyzes the reduction of various ketosteroids to the corresponding 20 β-hydroxysteroids at the expense of NADH. Reversed micelles are tiny oases of water stabilized in an organic solvent with the aid of surfactants. It shows that enzymes entrapped in these pools are capable of synthesizing apolar compounds—such as steroids, oxidized polyunsaturated fatty acids, and peptides. A reversed micellar medium consists of at least three components: an aqueous solution, a water-immiscible organic solvent, and a surfactant. The mechanism leading to the formation of reversed micelles is the tendency to extend the interfacial area until the concentrations of surfactants are sufficiently low to achieve a nonnegative interfacial tension. The results demonstrate that the reaction rate declines when the conversion is almost complete. Furthermore, at higher concentrations the reaction rate increases and the reaction proceeds steadily for >10 hr, indicating that the multienzyme system is stable for a considerable length of time.

Development of Selective Bisubstrate-Based Inhibitors Against Protein Kinase C (PKC) Isozymes By Using Dynamic Peptide Microarrays

Kinase inhibitors are increasingly important in drug development. Because the majority of current inhibitors target the conserved ATP‐binding site, selectivity might become an important issue. This could be particularly problematic for the potential drug target protein kinase C (PKC), of which twelve isoforms with high homology exist in humans. A strategy to increase selectivity is to prepare bisubstrate‐based inhibitors that target the more selective peptide‐binding site in addition to the ATP‐binding site. In this paper a generally applicable, rapid methodology is presented to discover such bisubstrate‐based leads. Dynamic peptide microarrays were used to find peptide‐binding site inhibitors. These were linked with chemoselective click chemistry to an ATP‐binding site inhibitor, and this led to novel bisubstrate structures. The peptide microarrays were used to evaluate the resulting inhibitors. Thus, novel bisubstrate‐based inhibitors were obtained that were both more potent and selective compared to their constituent parts. The most promising inhibitor has nanomolar affinity and selectivity towards PKCθ amongst three isozymes.

Purification and characterization of β-amylase from Curculigo pilosa

Curculigo pilosa is traditionally used in the manufacture of sorghum beer in West Africa. β-Amylase was purified 100-fold with 38% yield from a crude extract, giving final specific activities of 4850 U/mg and 5650 U/mg using soluble starch and p-nitrophenyl maltopentaoside, respectively, as substrates. The molecular mass of the monomeric enzyme was 64 kDa and its pI 4.2. Both activity and thermostability are higher than reported for other plant β-amylases. The catalytic efficiency was lower for amylose than for starches and amylopectin. In contrast to other plant amylases, the β-amylase from C. pilosa is able to degrade raw starches from wheat, corn, potato and rice. In this respect, it resembles β-amylases from microbial origin. This property, and its high activity and stability, explain its traditional use in the manufacture of infant food and sorghum beer in Burkina Faso and could make it applicable for other biotechnological purposes.

Analysis of jak2 catalytic function by peptide microarrays: The role of the JH2 domain and V617F mutation

Janus kinase 2 (JAK2) initiates signaling from several cytokine receptors and is required for biological responses such as erythropoiesis. JAK2 activity is controlled by regulatory proteins such as Suppressor of Cytokine Signaling (SOCS) proteins and protein tyrosine phosphatases. JAK2 activity is also intrinsically controlled by regulatory domains, where the pseudokinase (JAK homology 2, JH2) domain has been shown to play an essential role. The physiological role of the JH2 domain in the regulation of JAK2 activity was highlighted by the discovery of the acquired missense point mutation V617F in myeloproliferative neoplasms (MPN). Hence, determining the precise role of this domain is critical for understanding disease pathogenesis and design of new treatment modalities. Here, we have evaluated the effect of inter-domain interactions in kinase activity and substrate specificity. By using for the first time purified recombinant JAK2 proteins and a novel peptide micro-array platform, we have determined initial phosphorylation rates and peptide substrate preference for the recombinant kinase domain (JH1) of JAK2, and two constructs comprising both the kinase and pseudokinase domains (JH1-JH2) of JAK2. The data demonstrate that (i) JH2 drastically decreases the activity of the JAK2 JH1 domain, (ii) JH2 increased the Km for ATP (iii) JH2 modulates the peptide preference of JAK2 (iv) the V617F mutation partially releases this inhibitory mechanism but does not significantly affect substrate preference or Km for ATP. These results provide the biochemical basis for understanding the interaction between the kinase and the pseudokinase domain of JAK2 and identify a novel regulatory role for the JAK2 pseudokinase domain. Additionally, this method can be used to identify new regulatory mechanisms for protein kinases that provide a better platform for designing specific strategies for therapeutic approaches.