Enzyme biocatalysis

The use of isolated enzymes in a bioreactor offers several advantages over the use of whole cells. For example, substrate and enzyme concentration can be set to optimal values in an enzyme reactor while at the same time, transport limitations by cellular membranes can be excluded. Furthermore, yield reductions by side reactions of the cellular metabolism are avoided. However, it is crucial to address typical drawbacks such as efficient enzyme production, in vitro stability or cofactor dependency at process engineering as well as at biochemical levels.

Carotenoid cleavage dioxygenases

In nature, carotenoids are cleaved highly specifically by the family of carotenoid cleavage dioxygenase (CCD) enzymes using molecular oxygen. Due to their different substrate and regiospecificities, these enzymes represent the key element in the initial biosynthesis steps of apocarotenoids which can have a wide range of biological functions. Since they require no cofactors besides iron in their active centre, CCDs are highly attractive for research in applied in vitro biocatalysis.

Our model enzyme AtCCD1 from Arabidopsis thaliana cleaves carotenoids at the C9-C10- and C9'-C10' double bonds, yielding volatile C13 norisoprenoid substances such as β-ionone, a natural high value violet-like flavour compound. The conversion of the chromophoric carotenoids is accompanied by a color change which was used for the development of a photometric method of measurement for carotenoid cleavage reactions. This method of measurement now allows for a high throughput characterization and optimization of CCDs for in vitro applications. An important prerequisite for it is the solubilisation of the hydrophobic substrates in the aqueous reaction system using non-ionic detergent micelles. Broadening our understanding of the reaction mechanisms of this new group of enzymes and developing concepts for their technical application are the focus of current research projects in this area. Particular emphasis is put on the delivery of the highly hydrophobic substrates to the enzyme and on improving in vitro enzyme stability.

Chloroperoxidase

The enzyme chloroperoxidase (CPO) from the filamentous fungus Caldariomyces fumago is considered a promising biocatalyst for selective oxidations. In contrast to many oxygenases, this enzyme which uses hydrogen peroxide as oxidant does not require any cofactors, making it particularly suitable for in vitro use. Recently, it was discovered that under certain conditions such as the presence of organic cosolvents, CPO is capable of selectively oxidizing or halohydroxylating monoterpenes. The products of such reactions are attractive flavor compounds and chiral synthons.

Our group developed a novel cultivation method resulting in improved biomass and enzyme formation by the filamentous fungus C. fumago . By adding Al₂ O₃ microparticles to submersed cultures, their morphology changes from pellets to single hyphae, resulting in improved nutrient supply, increased biomass and increased enzyme yields. The patented process was termed MPEC (microparticle enhanced cultivation) and is also applicable to other filamentous microorganisms. Current research focuses on further improvements of enzyme production with C. fumago and on technical systems for the in vitro application of CPO.

P450 Monooxygenases

For terpene biotechnology, cytochrome P450 monooxygenases are of utmost importance.During biosynthesis, ethy play a key role in functionalisaiton of principal terpene hydrocarbon structures. These regio- and stereoselective hydroxylations set the course for the diversity of terpenoid secondary metabolites. Therefore our group is dealing with P450 monooxygenases, not only in whole cell biotransformations but also investigating in vitro biocatalysis. Here one of the challenges is the dependence of P450 monooxygenases on the cellular cofactor NAD(P)H and frequently on other electron transfer proteins and the fact that they are usually membrane bound. In this regard enzymes dissolved in the cytosol, such as

• P450BM-3 (from B. megaterium ; P450BM-3 muteins can oxidize terpenes),
• P450cam (from Pseudomonas putida ; P450cam oxidizes camphor) and
• P450cin (from Citrobacter braakii ; P450cin oxidizes cineole)

are an exception. With these model enzymes in vitro biocatalysis is studied using electrochemistry to substitute NAD(P)H as the source of reduction equivalents. Thus the expensive cofactor NAD(P)H could be avoided in technical applications. First experiments with P450BM-3, which was immobilized onto the electrode surface by entrapment in a conductive polymer, showed that electrochemically driven biocatalysis is possible. Current work is focusing on the optimization of the electron transfer between enzyme and electrode as well as the improvement of volumetric productivities. For this purpose, redox mediators are used which shuttle electrons between the electrode and the active site of the enzyme.   

For the future microtiterplates with electrochemical contacts (eMTP) will facilitate the medium-throughput characterization and optimization of the artificial electron transfer towards cofactor dependent oxygenases in parallelized assays.