Thomas Schmidt

Mechanistic kinetic model for symmetric carboligations using benzaldehyde lyase

For reactions using thiamine diphosphate (ThDP)‐dependent enzymes many empirically‐derived kinetic models exist. However, there is a lack of mechanistic kinetic models. This is especially true for the synthesis of symmetric 2‐hydroxy ketones from two identical aldehydes, with one substrate acting as the donor and the other as the acceptor. In this contribution, a systematic approach for deriving such a kinetic model for thiamine diphosphate (ThDP)‐dependent enzymes is presented. The derived mechanistic kinetic model takes this donor–acceptor principle into account by containing two Km‐values even for identical substrate molecules. As example the stereoselective carbon–carbon coupling of two 3,5‐dimethoxy‐benzaldehyde molecules to (R)‐3,3′,5,5′‐tetramethoxy‐benzoin using benzaldehyde lyase (EC 4.1.2.38) from Pseudomonas fluorescens is studied. The model is derived using a model‐based experimental analysis method which includes parameter estimation, model analysis, optimal experimental design, in silico experiments, sensitivity analysis and model revision. It is shown that this approach leads to a robust kinetic model with accurate parameter estimates and an excellent prediction capability. Biotechnol. Biotechnol. Bioeng. 2008;101: 27–38.

An efficient process for the saccharification of wood chips by combined ionic liquid pretreatment and enzymatic hydrolysis

A process concept combining pretreatment of wood in ionic liquids and subsequent enzymatic hydrolysis to sugars is herein investigated to identify operating conditions which allow for (i) the processing of larger wood chips of 10 mm length, (ii) low temperature, (iii) high sugar yield, and (iv) short processing time. A careful quantitative study of the interaction of pretreatment and hydrolysis reveals that hydrolysis is most effective if beech chips are first disintegrated in [EMIM][Ac] at 115 °C for 1.5 h. The cellulose conversion varies between 70.5 wt% and 90.2wt% for hydrolysis times between 5 h and 72 h. A complete recovery of cellulose and xylan resulting in a total saccharification of 65 wt% of the wood chips could be demonstrated. It is shown that short pretreatment times are required to enable high sugar yield as well as to limit product degradation.

Biphasic mini-reactor for characterization of biocatalyst performance

Biphasic reaction media are extending the scope of technical biocatalysis. Thorough investigation of the factors affecting catalyst performance under these conditions is of key importance for the successful implementation of catalytic processes. Here, we present a reactor setup suitable for comprehensive systematic characterization and optimization of biocatalyzed reactions in biphasic systems with distinct phases. It is distinguished by small volumes allowing reproducible experimentation with minimum amounts of solvent and catalyst. The interfacial area is kept constant and independent stirring of both phases is allowed in order to minimize superimposing effects. Evaporation of low‐volatile organic solvents is prevented by use of airtight construction. The broad applicability of this mini‐reactor is demonstrated with regard to determination of mass transfer, enzyme productivity, and enzyme stability in both batch and continuous mode.

Mechanistic model for prediction of formate dehydrogenase kinetics under industrially relevant conditions

Formate dehydrogenase (FDH) from Candida boidinii is an important biocatalyst for the regeneration of the cofactor NADH in industrial enzyme‐catalyzed reductions. The mathematical model that is currently applied to predict progress curves during (semi‐)batch reactions has been derived from initial rate studies. Here, it is demonstrated that such extrapolation from initial reaction rates to performance during a complete batch leads to considerable prediction errors. This observation can be attributed to an invalid simplification during the development of the literature model. A novel mechanistic model that describes the course and performance of FDH‐catalyzed NADH regeneration under industrially relevant process conditions is introduced and evaluated. Based on progress curve instead of initial reaction rate measurements, it was discriminated from a comprehensive set of mechanistic model candidates. For the prediction of reaction courses on long time horizons (>1 h), decomposition of NADH has to be considered. The model accurately describes the regeneration reaction under all conditions, even at high concentrations of the substrate formate and thus is clearly superior to the existing model. As a result, for the first time, course and performance of NADH regeneration in industrial enzyme‐catalyzed reductions can be accurately predicted and used to optimize the cost efficiency of the respective processes.

Biochemical peculiarities of benzaldehyde lyase from Pseudomonas fluorescens Biovar I in the dependency on pH and cosolvent concentration.

Benzaldehyde lyase from Pseudomonas fluorescens (BAL, EC 4.1.2.38) is a versatile catalyst for stereoselective carboligations. Nevertheless, rather inconsistent data about its biochemical properties are reported in literature. In this study, the dependency of BAL activity on ionic strength, pH, and concentration of DMSO was for the first time systematically investigated and interpreted. It was found that the activity of BAL strongly depends on all three parameters, and a correlation exists between the dependency on pH and DMSO concentration. This correlation could be explained by an interaction of DMSO with an ionic amino acid in the catalytic site. A model-based analysis indicated that the pK(a) of this residue shifts to the alkaline milieu upon addition of DMSO. Consequently, the optimum pH also shifts to alkaline values when DMSO is present. Potentiometric experiments confirmed that the pK(a) can most probably be attributed to Glu50 which governs the activity increase of BAL on the acidic limb of its pH-activity profile. With these findings, the apparently contradicting data from literature become comprehensible and optimal reaction conditions for synthesis can easily be deduced.

Optimized knowledge-based motion correction of fMRI time series using parallel algorithms

The structure of an fMRI time series coregistration algorithm can be divided into modules (preprocessing, minimization procedure, interpolation method, cost function), for each of which there are many different approaches. In our study we implemented some of the most recent techniques and compared their combinations with regard to both registration accuracy and runtime performance. Bidirectional inconsistency and difference image analysis served as quality measures. The result shows that with an appropriate choice of methods realignment results can be improved by far compared with standard solutions. Finally, an automatic parameter adaptation method was incorporated. Additionally, the algorithm was implemented to run on a distributed 48 processor PC cluster, surpassing the performance of conventional applications running on high end workstations.

Hospital integrated parallel cluster for fast and cost-efficient image analysis: clinical experience and research evaluation

In the last few years more and more University Hospitals as well as private hospitals changed to digital information systems for patient record, diagnostic files and digital images. Not only that patient management becomes easier, it is also very remarkable how clinical research can profit from Picture Archiving and Communication Systems (PACS) and diagnostic databases, especially from image databases. Since images are available on the finger tip, difficulties arise when image data needs to be processed, e.g. segmented, classified or co-registered, which usually demands a lot computational power. Todays clinical environment does support PACS very well, but real image processing is still under-developed. The purpose of this paper is to introduce a parallel cluster of standard distributed systems and its software components and how such a system can be integrated into a hospital environment. To demonstrate the cluster technique we present our clinical experience with the crucial but cost-intensive motion correction of clinical routine and research functional MRI (fMRI) data, as it is processed in our Lab on a daily basis.