Metals in Biotechnology: Cr-Driven Stereoselective Reduction of Conjugated C=C Double Bonds.

Elemental metals are shown to be suitable sacrificial electron donors to drive the stereoselective reduction of conjugated C=C double bonds using Old Yellow Enzymes as catalysts. Both direct electron transfer from the metal to the enzyme as well as mediated electron transfer is feasible, although the latter excels by higher reaction rates. The general applicability of this new chemoenzymatic reduction method is demonstrated, and current limitations are outlined.

A Photoenzymatic NADH Regeneration System.

A photoenzymatic NADH regeneration system was established. The combination of deazariboflavin as a photocatalyst with putidaredoxin reductase enabled the selective reduction of NAD+ into the enzyme-active 1,4-NADH to promote an alcohol dehydrogenase catalysed stereospecific reduction reaction. The catalytic turnover of all the reaction components was demonstrated. Factors influencing the efficiency of the overall system were identified.

Latest development in the synthesis of ursodeoxycholic acid (UDCA): a critical review.

Ursodeoxycholic acid (UDCA) is a pharmaceutical ingredient widely used in clinics. As bile acid it solubilizes cholesterol gallstones and improves the liver function in case of cholestatic diseases. UDCA can be obtained from cholic acid (CA), which is the most abundant and least expensive bile acid available. The now available chemical routes for the obtainment of UDCA yield about 30% of final product. For these syntheses several protection and deprotection steps requiring toxic and dangerous reagents have to be performed, leading to the production of a series of waste products. In many cases the cholic acid itself first needs to be prepared from its taurinated and glycilated derivatives in the bile, thus adding to the complexity and multitude of steps involved of the synthetic process. For these reasons, several studies have been performed towards the development of microbial transformations or chemoenzymatic procedures for the synthesis of UDCA starting from CA or chenodeoxycholic acid (CDCA). This promising approach led several research groups to focus their attention on the development of biotransformations with non-pathogenic, easy-to-manage microorganisms, and their enzymes. In particular, the enzymatic reactions involved are selective hydrolysis, epimerization of the hydroxy functions (by oxidation and subsequent reduction) and the specific hydroxylation and dehydroxylation of suitable positions in the steroid rings. In this minireview, we critically analyze the state of the art of the production of UDCA by several chemical, chemoenzymatic and enzymatic routes reported, highlighting the bottlenecks of each production step. Particular attention is placed on the precursors availability as well as the substrate loading in the process. Potential new routes and recent developments are discussed, in particular on the employment of flow-reactors. The latter technology allows to develop processes with shorter reaction times and lower costs for the chemical and enzymatic reactions involved.

Biocatalytic synthesis of the Green Note trans-2-hexenal in a continuous-flow microreactor.

The biocatalytic preparation of trans-hex-2-enal from trans-hex-2-enol using a novel aryl alcohol oxidase from Pleurotus eryngii (PeAAOx) is reported. As O2-dependent enzyme PeAAOx-dependent reactions are generally plagued by the poor solubility of O2 in aqueous media and mass transfer limitations resulting in poor reaction rates. These limitations were efficiently overcome by conducting the reaction in a flow-reactor setup reaching unpreceded catalytic activities for the enzyme in terms of turnover frequency (up to 38 s-1) and turnover numbers (more than 300000) pointing towards preparative usefulness of the proposed reaction scheme.

Identification of catalytically important residues of the carotenoid 1,2-hydratases from Rubrivivax gelatinosus and Thiocapsa roseopersicina.

Carotenoid 1,2-hydratases (CrtC) catalyze the selective addition of water to an isolated carbon-carbon double bond. Although their involvement in the carotenoid biosynthetic pathway is well understood, little is known about the mechanism by which these hydratases transform carotenoids such as lycopene into the corresponding hydroxyl compounds. Key residues were identified at positions His239, Trp241, Tyr266, and Asp268 in CrtC from Rubrivivax gelatinosus (and corresponding positions in Thiocapsa roseopersicina). Alanine mutants at these positions were found to be completely inactive, suggesting their direct involvement in the catalytic reaction. Our resulting mechanistic hypothesis is in analogy with the recently studied class of terpenoid cyclase enzymes containing a highly acidic aspartic residue in their active site. We propose that a similar aspartic acid residue, which is conserved through all putative CrtCs, is involved in initial protonation of the double bond in lycopene.

Enhancement of the substrate scope of transketolase.

To enhance the activity of transketolase towards nonphosphorylated substrates and enlarge the scope of its substrates, notably to long polyol aldehyde acceptors (D-ribose or D-glucose), a rational design-supported evolution strategy was applied. By using docking experiments, an in silico library, and iterative mutagenesis, libraries of single- and double-point mutants were designed and generated. A double-screening approach was implemented, coupling a preselection activity assay (HPLC method) and a selective assay (GC method) to find the best enzymes. Several mutants (R526N, R526Q, R526Q/S525T, R526K/S525T) showed improved activities towards nonphosphorylated substrates as the coupled products of lithium hydroxypyruvate (HPA) with glycolaldehyde (GO), D-ribose or D-glucose. These mutated enzymes were further characterised. They were shown to be up to four times more active than the wild-type (mutant R526Q/S525T) for nonphosphorylated substrates LiHPA/GO (V(m) /K(m) for LiHPA = 92.4 instead of 28.8×10(-3) min(-1) for the wild-type) and 2.6 times more active for substrates LiHPA/rib.

Towards recyclable NAD(P)H regeneration catalysts.

Rh(III)-TsDPEN, an immobilized analog of the well-known [Cp*Rh(bpy)(H(2)O)](2+) was evaluated as a heterogeneous, recyclable regeneration catalyst for reduced oxidoreductase cofactors [NAD(P)H]. Repeated use of this catalyst was established and the catalytic properties were initially investigated. Apparently, Rh(III)-TsDPEN is prone to severe diffusion limitations, necessitating further developments. Overall, a promising concept for chemoenzymatic redox catalysis is proposed, which may overcome some of the current limitations such as catalyst cost and incompatibility of Rh with some biocatalysts.

Enantioselective oxidation of aldehydes catalyzed by alcohol dehydrogenase.

Teaching old dogs new tricks: Alcohol dehydrogenases (ADHs) may be established redox biocatalysts but they still are good for a few surprises. ADHs can be used to oxidize aldehydes, and this was demonstrated by the oxidative dynamic kinetic resolution of profens. In the presence of a suitable cofactor regeneration system, this reaction can occur with high selectivity.

Biochemical characterization of the carotenoid 1,2-hydratases (CrtC) from Rubrivivax gelatinosus and Thiocapsa roseopersicina.

Two carotenoid 1,2-hydratase (CrtC) genes from the photosynthetic bacteria Rubrivivax gelatinosus and Thiocapsa roseopersicina were cloned and expressed in Escherichia coli in an active form and purified by affinity chromatography. The biochemical properties of the recombinant enzymes and their substrate specificities were studied. The purified CrtCs catalyze cofactor independently the conversion of lycopene to 1-HO- and 1,1'-(HO)(2)-lycopene. The optimal pH and temperature for hydratase activity was 8.0 and 30°C, respectively. The apparent K (m) and V (max) values obtained for the hydration of lycopene were 24 µM and 0.31 nmol h(-1) mg(-1) for RgCrtC and 9.5 µM and 0.15 nmol h(-1) mg(-1) for TrCrtC, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed two protein bands of 44 and 38 kDa for TrCrtC, which indicate protein processing. Both hydratases are also able to convert the unnatural substrate geranylgeraniol (C20 substrate), which functionally resembles the natural substrate lycopene.

Chemoenzymatic Halocyclization of 4-Pentenoic Acid at Preparative Scale.

The scale-up of chemoenzymatic bromolactonization to 100 g scale is presented, together with an identification of current limitations. The preparative-scale reaction also allowed for meaningful mass balances identifying current bottlenecks of the chemoenzymatic reaction.

Diphenyldiselenide-catalyzed selective oxidation of activated alcohols with tert-butyl hydroperoxide: new mechanistic insights.

Diselenides are known precatalysts for catalytic oxidations. The use of these widely available compounds as catalysts for the oxidation of alcohols was investigated, still leaving many mechanistic questions to be answered. By using a range of analytical techniques, we found evidence for the unexpected involvement of seleninic anhydride in the catalytic mechanism. The activation time of the diselenide and its influence on the oxidation reaction itself was also investigated. On the basis of these findings, an improved protocol for the selective oxidation of activated alcohols was devised resulting in significantly decreased catalyst loadings (<1%).

NAD+ -Dependent Enzymatic Route for the Epimerization of Hydroxysteroids.

Epimerization of cholic and chenodeoxycholic acid (CA and CDCA, respectively) is a notable conversion for the production of ursodeoxycholic acid (UDCA). Two enantiocomplementary hydroxysteroid dehydrogenases (7α- and 7ß-HSDHs) can carry out this transformation fully selectively by specific oxidation of the 7α-OH group of the substrate and subsequent reduction of the keto intermediate to the final product (7ß-OH). With a view to developing robust and active biocatalysts, novel NADH-active 7ß-HSDH species are necessary to enable a solely NAD+ -dependent redox-neutral cascade for UDCA production. A wild-type NADH-dependent 7ß-HSDH from Lactobacillus spicheri (Ls7ß-HSDH) was identified, recombinantly expressed, purified, and biochemically characterized. Using this novel NAD+ -dependent 7ß-HSDH enzyme in combination with 7α-HSDH from Stenotrophomonas maltophilia permitted the biotransformations of CA and CDCA in the presence of catalytic amounts of NAD+ , resulting in high yields (>90 %) of UCA and UDCA.

Peroxygenase-Catalysed Epoxidation of Styrene Derivatives in Neat Reaction Media.

Biocatalytic oxyfunctionalisation reactions are traditionally conducted in aqueous media limiting their production yield. Here we report the application of a peroxygenase in neat reaction conditions reaching product concentrations of up to 360 mM.

Photoenzymatic epoxidation of styrenes.

Two-component-diffusible-flavomonooxygenases are versatile biocatalysts for selective epoxidation-, hydroxylation- or halogenation reactions. Their complicated molecular architecture can be simplified using photochemical regeneration of the catalytically active, reduced FADH2 prosthetic group. In this contribution we provide the proof-of-concept and characterization for the direct regeneration of the styrene monooxygenase from Pseudomonas.

Expanding the Spectrum of Light-Driven Peroxygenase Reactions.

Peroxygenases require a controlled supply of H2O2 to operate efficiently. Here, we propose a photocatalytic system for the reductive activation of ambient O2 to produce H2O2 which uses the energy provided by visible light more efficiently based on the combination of wavelength-complementary photosensitizers. This approach was coupled to an enzymatic system to make formate available as a sacrificial electron donor. The scope and current limitations of this approach are reported and discussed.

Vanadate substituted phytase: immobilization, structural characterization and performance for sulfoxidations.

A cross-linked enzyme aggregate (CLEA) of 3-phytase (EC 3.1.3.8) was synthesised, which was incubated with vanadate and tested as a biocatalyst in the asymmetric sulfoxidation of thioanisole using hydrogen peroxide as the oxidant. The results show that the 3-phytase-CLEA demonstrates a similar efficiency (ca. 95% conversion) and asymmetric induction (ca. 60%) as the free enzyme. Moreover, the 3-phytase-CLEA can be reused at least three times without significant loss of activity. The activity of the 3-phytase in the presence of organic solvents is however still limited. Studies were undertaken to elucidate the role of vanadate on the active site and on the effect of organic solvents on the conformation of the enzyme. The incorporation of vanadate in the active sites of two different phytases could be followed using (51)V NMR and circular dichroism (CD) spectroscopies. (51)V NMR spectra show the incorporation of vanadate into the active site at pH 5.0 and 7.6, and suggest coordination to oxygen functions at two different binding sites, which probably explains the poor enantioselectivity found in the catalytic studies. After addition of H(2)O(2), only at pH 5.0 and with the 3-phytase a V-phytase-peroxide complex could be observed, which is the active species responsible for the oxidation reactions. CD studies showed that the alpha-helical content of the enzyme decreased upon coordination of vanadate, but in the concentration range used in the catalytic studies (<30 microM) the secondary conformation of the enzyme was unchanged. Acetonitrile decreases the alpha-helical content of both phytases from 59% to 51% and from 42% to 34%, in the 3- and 6-phytases, respectively, this being in agreement with the activity loss in the catalytic experiments.

Study on the isomerism in meso-amavadin and an amavadin analogue.

An X-ray crystallographic study of 'meso-amavadin' revealed that in the crystal the negatively charged anionic species of the title compound join into infinite hydrogen-bonded chains, counterbalanced by cationic hydronium species. Along with water of crystallization a three-dimensional hydrogen-bonded network is formed. Based on NMR- and X-ray data of amavadin and 'meso-amavadin', a model was developed that accounts for the structure of amavadin-type complexes, i.e. vanadium(IV) non-oxo complexes that contain two ligands with a tridentate N-hydroxyiminodiacetate backbone. The model describes the different arrangements of the two ligands around the vanadium and it accounts for eventual symmetry in the complex. The model was used for the interpretation of NMR-data of an amavadin analogue with a benzyl group at the ligand backbone.

Selective Photooxidation Reactions using Water-Soluble Anthraquinone Photocatalysts.

The aerobic organocatalytic oxidation of alcohols was achieved by using water-soluble sodium anthraquinone sulfonate. Under visible-light activation, this catalyst mediated the aerobic oxidation of alcohols to aldehydes and ketones. The photo-oxyfunctionalization of alkanes was also possible under these conditions.

Nanoparticles of lanthanide oxysulfate/oxysulfide for improved oxygen storage/release.

Lanthanide oxysulfates have the ability to store and release large volumes of oxygen under oxidizing/reducing conditions, rendering them interesting as automotive catalysts. Herein we demonstrate a remarkable improvement of both processes by utilization of nanoparticles compared to the bulk materials. A further improvement of the catalytic activity was achieved by cost-effective doping with 1.9 wt% of Ni.