Frank Hollmann

The use of enzymes in the chemical industry in Europe.

Many European chemical industries are in a phase of reorganization resulting in a general opening towards life sciences. Traditional chemical markets are served increasingly with products derived from bioprocesses or hybrid chemical/biocatalytic processes. Biocatalytic steps are already being used to produce a wide range of products, including agricultural chemicals, organics, drugs and plastic materials, to name but a few. Apart from the rapidly growing number of commercialized bioprocesses, a partial survey of exploratory activities points to future applications of enzymes in the European chemical industry, which will bring new products and technologies and, in some cases, replace traditional syntheses.

[Cp*Rh(bpy)(H2O)]2+: a versatile tool for efficient and non-enzymatic regeneration of nicotinamide and flavin coenzymes

Abstract [Cp ∗ Rh(bpy)(H 2 O)] 2+ is a versatile tool for the non-enzymatic regeneration of oxidoreductase coenzymes such as NAD(P)H, NAD(P) + , and FADH 2 . The complex shows high stability and activity over a very broad range of pH (more than 60% activity between pH 3.5 and 9.7) and temperature (exponential increase of activity until at least 69xa0°C). The regeneration concept is not limited to sacrificial electron donors such as formate or alcohols for regeneration of reduced coenzymes; cheap electrical power as a reagent less source of reduction equivalents can also be used. At 30xa0°C, the complex has a specific activity of 1.2xa0Uxa0mg −1 for NAD(P)H regeneration and about 1xa0Uxa0mg −1 for FADH 2 regeneration with identical K M values for both NAD + and NADP + of about 9xa0μM. In addition, a [Cp ∗ Rh(bpy)(H 2 O)] 2+ /FAD based concept for the regeneration of oxidized nicotinamide coenzymes is proposed.

Integrated Biocatalytic Synthesis on Gram Scale: The Highly Enantioselective Preparation of Chiral Oxiranes with Styrene Monooxygenase

Enantiopure oxiranes have been prepared on a gram scale by recombinant E.xa0coli JM101 (pSPZ10) containing styrene monooxygenase in an emulsion process including facile and efficient downstream processing. This biocatalyst was compared to the chemical catalysts introduced by Jacobsen et al. with respect to its performance for epoxidations of vinyl groups.

Electrochemical regeneration of oxidoreductases for cell-free biocatalytic redox reactions

Oxidoreductases represent a highly interesting and versatile class of biocatalysts for specific reduction, oxidation, and oxyfunctionalization reactions. Since oxidoreductases depend on cofactors and coenzymes to supply or withdraw redox equivalents released during the catalytic process, their application in cell-free environments requires external supply with these redox equivalents. Next to enzymatic approaches, a variety of non-enzymatic regeneration strategies have been developed. This review focuses on electrochemical methods for the in situ regeneration of nicotinamide cofactors as well as flavin- and heme-coenzymes, developed for synthetic application. The fields of electrochemical biosensors as well as biofuel cells are not discussed in detail. Electrochemical approaches bear much promise and in some cases are more efficient and more versatile than enzymatic regeneration approaches.

Light-Driven Biocatalytic Oxidation and Reduction Reactions: Scope and Limitations

The quest for practical regeneration concepts for nicotinamide‐dependent oxidoreductases continues. Recently we proposed the use of visible light to promote the direct reductive regeneration of a flavin‐dependent monooxygenase. With this enzyme (PAMO‐P3) light‐driven enantioselective Baeyer–Villiger oxidations were performed. In spite of the significant reduction in the complexity achieved, catalytic performance of the novel approach did not meet the requirements for an efficient biocatalytic oxygenation system. Driven by this ultimate goal, we further investigated the limiting factors of our particular system. We discovered that oxidative uncoupling of the flavin‐regeneration reaction from enzymatic O2‐activation accounts for the futile consumption of approximately 95u2009% of the reducing equivalents provided by the sacrificial electron donor, EDTA. Furthermore, it was found that the apparent turnover frequency (TOF) for PAMO‐P3 in the present setup is approximately two orders of magnitude lower than in conventional setups that use NADPH as reductant. This finding was traced to sluggish electron transfer kinetics that arose from an impeded interaction between PAMO‐P3‐bound FAD and the reducing catalyst. The limiting factors and potential approaches for their circumvention are discussed. Furthermore, we broadened the light‐driven regeneration approach to the class of flavin‐dependent reductases. By using the Old Yellow Enzyme homologue YqjM as a model system, a significantly higher catalytic turnover for the enzyme catalyst was achieved, which we assign to a higher accessibility of the prosthetic group as well as to the absence of oxidative uncoupling.

A Robust Protein Host for Anchoring Chelating Ligands and Organocatalysts

In order to put the previously proposed concept of directed evolution of hybrid catalysts (proteins that harbor synthetic transition‐metal catalysts or organocatalysts) into practice, several prerequisites must be met. The availability of a robust host protein that can be expressed in sufficiently large amounts, and that can be purified in a simple manner is crucial. The thermostable enzyme tHisF from Thermotoga maritima, which constitutes the synthase subunit of a bi‐enzyme complex that is instrumental in the biosynthesis of histidine, fulfills these requirements. In the present study, fermentation has been miniaturized and parallelized, as has purification of the protein by simple heat treatment. Several mutants with strategically placed cysteines for subsequent bioconjugation have been produced. One of the tHisF mutants, Cys9Ala/Asp11Cys, was subjected to bioconjugation by the introduction of a variety of ligands for potential metal ligation, of a ligand/metal moiety, and of several organocatalytic entities that comprise a flavin or thiazolium salts. Characterization by mass spectrometry and tryptic digestion was achieved. As a result of this study, a platform for performing future directed evolution of these hybrid catalysts is now available.

Towards practical biocatalytic Baeyer-Villiger reactions: applying a thermostable enzyme in the gram-scale synthesis of optically-active lactones in a two-liquid-phase system

Baeyer-Villiger monooxygenases (BVMOs) are extremely promising catalysts useful for enantioselective oxidation reactions of ketones, but organic chemists have not used them widely due to several reasons. These include instability of the enzymes in the case of in vitro and even in vivo systems, reactant/product inhibition, problems with upscaling and the necessity of using specialized equipment. The present study shows that the thermally stable phenylacetone monooxygenase (PAMO) and recently engineered mutants can be used as a practical catalysts for enantioselective Baeyer-Villiger oxidations of several ketones on a preparative scale under in vitro conditions. For this purpose several parameters such as buffer composition, the nature of the solvent system and the co-factor regeneration system were optimized. Overall a fairly versatile and efficient catalytic system for enantioselective laboratory scale BV-oxidations of ketones was developed, which can easily be applied even by those organic chemists who are not well versed in the use of enzymes.

Deazaflavins as mediators in light-driven cytochrome P450 catalyzed hydroxylations

A light-driven deazaflavin-dependent direct enzyme regeneration system has been developed for a P450-BM3 catalyzed CH-activating hydroxylation, thereby avoiding the need for the expensive NADPH cofactor.

Towards [Cp∗Rh(bpy)(H2O)]2+-promoted P450 catalysis: Direct regeneration of CytC

The organometallic complex pentamethylcyclopentadienyl rhodium 2,2-bipyridin ([Cp(*)Rh(bpy)(H(2)O)](2+)) was applied as regeneration catalyst for cytochrome C (CytC). Direct reduction of CytC-bound Fe(III) was achieved in this model system pointing towards a potential usefulness of this concept to promote cell-free P450 catalysis. In addition, controlled in situ provision with hydrogen peroxide was performed using [Cp(*)Rh(bpy)(H(2)O)](2+) resulting in improved CytC-catalyzed sulfoxidation of thioanisol This work represents the first step towards the direct-[Cp(*)Rh(bpy)(H(2)O)](2+) catalyzed regeneration of P450 monooxygenases and peroxidases.

Diplexers for Power Combination and Switching in High Power ECRH Systems

Electron Cyclotron Resonance Heating (ECRH) systems for next step large fusion-devices operate at a Continuous Wave (CW) power well beyond 10 MW generated by a large number of gyrotrons with typically 1 MW power per unit. The combination of the power of two (or more) gyrotrons and switching of the power between different launchers for different physics applications is an attractive feature for such systems. The combination of beams from different gyrotrons would reduce the number of transmission lines and the requirements on port space. Fast switching between two antennas synchronously with the Magneto-Hydro Dynamic (MHD) modes frequency would increase the efficiency of mode stabilization. Both combination and switching as well as power sharing between different ports can be performed with high-power four-port diplexers using small frequency differences or small frequency-shift keying of the gyrotrons, respectively. Fast directional switches (FADIS) and beam combiners (BC) can be designed on the basis of different physical mechanisms: some selected design variants were investigated and the results are presented. Considerations on the integration of FADIS/BCs into large ECRH systems and their use in test arrangements are presented.