Enzymes, increasingly important in the synthesis of fine chemicals and pharmaceutical intermediates, are often insufficiently stable under reacting conditions. We have investigated the stability, in homogeneous aqueous solution and at gas-liquid interfaces, of formate dehydrogenase (FDH), important for cofactor regeneration, from Candida boidinii and overexpressed in E. coli. When exposed to mechanical stress, residual activity, [E](t)/[E](0), and residual protein were found to scale proportionally with gas-liquid surface area in the bubble column, verifying a surface-driven process, and with time and total throughput in a gear pump, but did not seem to be influenced much by shear in a Couette viscometer. All FDH variants are deactivated by chaotropes but not kosmotropes: the first-order deactivation constant k(d) correlates well with the Jones-Dole coefficient B but not well with the surface tension increment deltasigma of various concentrated ammonium salt solutions. This finding might provide guidance for focusing the search for quantitative theories of Hofmeister effects.

The performance of fluidized bed adsorption is strongly influenced by the hydrodynamics of the fluidization process. Especially axial mixing in the liquid and solid phase may lead to reduced capacity and resolution. In this article axial mixing in the liquid phase of a classified fluidized bed based on porous glass granules is presented. Axial mixing was analyzed by measurements of residence time distributions in a fluidized bed, showing a reduction of mixing at increased ratio of bed height to diameter as well as at increased linear velocity of the liquid stream. These results were transferred to two real adsorption systems on two different scales: In a bench scale (up to 15 mL of adsorbent) the purification of monoclonal antibodies from hybridoma supernatant was performed with a cation exchanger, in a larger scale (up to 750 mL of matrix) the adsorption of bovine serum albumin (BSA) on the same matrix was investigated. The results showed an increase of capacity at increased bed height-to-diameter ratio; with regard to linear velocity a broad range of only slightly changed capacity was found. A shift from dispersion controlled to diffusion controlled adsorption at intermediate linear velocity was proposed by isolating the effect of pore diffusion from the effect of dispersion.

Microbial physiology plays a crucial role in whole-cell biotransformation, especially for redox reactions that depend on carbon and energy metabolism. In this study, regio-and enantio-selective proline hydroxylation with recombinant Escherichia coli expressing proline-4-hydroxylase (P4H) was investigated with respect to its interconnectivity to microbial physiology and metabolism. P4H production was found to depend on extracellular proline availability and on codon usage. Medium supplementation with proline did not alter p4h mRNA levels, indicating that P4H production depends on the availability of charged prolyl-tRNAs. Increasing the intracellular levels of soluble P4H did not result in an increase in resting cell activities above a certain threshold (depending on growth and assay temperature). Activities up to 5-fold higher were reached with permeabilized cells, confirming that host physiology and not the intracellular level of active P4H determines the achievable whole-cell proline hydroxylation activity. Metabolic flux analysis revealed that tricarboxylic acid cycle fluxes in growing biocatalytically active cells were significantly higher than proline hydroxylation rates. Remarkably, a catalysis-induced reduction of substrate uptake was observed, which correlated with reduced transcription of putA and putP, encoding proline dehydrogenase and the major proline transporter, respectively. These results provide evidence for a strong interference of catalytic activity with the regulation of proline uptake and metabolism. In terms of whole-cell biocatalyst efficiency, proline uptake and competition of P4H with proline catabolism are considered the most critical factors. I n redox biocatalysis, especially when activation of molecular oxygen is involved, the whole cell still remains the preferred catalyst. Besides the capability to continuously regenerate redox cofactors via central carbon metabolism, the whole-cell approach provides advantages with respect to operational catalyst stability, e.g., via continuous (multicomponent) enzyme synthesis, assembly, and stabilization, as well as degradation of reactive oxygen species (1-3).Oxygenases are of special interest for industrial applications, since they catalyze the highly specific oxyfunctionalization of unactivated C-H bonds under mild conditions, with molecular oxygen as the oxygen donor (4). The efficiency of an oxygenase-containing whole-cell biocatalyst depends on both enzyme and host cell performance. Hence, biocatalyst engineering must encompass both factors and be integrated with biochemical process engineering (5, 6). The technological progress in enzyme engineering of the recent decades now allows fast generation of enzyme variants with improved properties (7). For in vivo applications, intracellular conditions have to be considered, and host cell engineering often is necessary to allow the exploitation of the catalytic capability of (engineered) enzymes. The benefits of metabolic engineering have especially become evident in fermentation processes, as ex...

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