The gene of the NAD-dependent formate dehydrogenase (FDH) from the yeast Candida boidinii was cloned by PCR using genomic DNA as a template. Expression of the gene in Escherichia coli yielded functional FDH with about 20% of the soluble cell protein. To confirm the hypothesis of a thiol-coupled inactivation process, both cysteine residues in the primary structure of the enzyme have been exchanged by site-directed mutagenesis using a homology model based on the 3D structure of FDH from Pseudomonas sp. 101 and from related dehydrogenases. Compared to the wt enzyme, most of the mutants were significantly more stable towards oxidative stress in the presence of Cu(II) ions, whereas the temperature optima and kinetic constants of the enzymatic reaction are not significantly altered by the mutations. Determination of the T m values revealed that the stability at temperatures above 50 8C is optimal for the native and the recombinant wt enzyme (T m 57 8C), whereas the T m values of the mutant enzymes vary in the range 44±52 8C. Best results in initial tests concerning the application of the enzyme for regeneration of NADH in biotransformation of trimethyl pyruvate to l-tert leucine were obtained with two mutants, FDHC23S and FDHC23S/C262A, which are significantly more stable than the wt enzyme.

We report the partition coefficient, K(p') at the isoelectric point of lysozyme, chymotrypsinogen A, albumin, transferrin, and catalase in 64 different polyethylene(PEG)/ dextran(Dx)/water systems. We study the trends of the partition coefficient with protein type, polymer concentration, and polymer molecular weight. We find that the partition coefficient decreases with increasing tie line length for lysozyme, albumin, transferrin, and catalase for which K(p) is less than 1, but increases for chymotrysinogen for which K(p) is larger than 1. The effect of the tie line length on the partition coefficient is larger for the large proteins than for the small proteins. The partition coefficient decreases with increasing protein molecular weight except for lysozyme suggesting that lysozyme is present as a dimer or a trimer. The partition coefficient decreases with increasing PEG molecular weight, but the magnitude of the increase is larger for the smaller PEG molecular eights and tends to level of at high PEG molecular weight. The partition coefficient increases with increasing dextran (Dx) molecular weight for chymotrypsinogen but decreases for catalase. The partition coefficients of lysozyme, albumin, and transferrin increase with increasing Dx molecular weight from Dx 10(4) to Dx 1.1 x 10(5) and then slightly decrease from Dx 1.1 x 10(5) to Dx 5 x 10(5). The experimental results are analyzed using a statistical thermodynamics model. The experimental results are analyzed using a statistical thermodynamics model. The experiments suggest that protein partitioning at the isoelectric point in aqueous two-phase systems is strongly related to the size of the proteins and polymers. Finally, the impossibility of obtaining data completely independent of polymer concentration is emphasized.

Benzoylformate decarboxylase (BFD) from Pseudomonas putida was subjected to directed molecular evolution to generate mutants with increased carboligase activity which is a side reaction of the enzyme. After a single round of random mutagenesis mutants were isolated which exhibited a 5-fold increased carboligase activity in aqueous buffer compared to the wild-type enzyme with a high enantiomeric excess of the product (S)-2-hydroxy-1-phenyl-propanone. From the same library, mutants with enhanced carboligase activity in water-miscible organic solvents have been isolated. The selected mutants have been characterized by sequencing, revealing that all mutants carry a mutation at Leu476, which is close to the active site but does not directly interact with the active center. BFD-L476Q has a 5-fold higher carboligase activity than the wild-type enzyme. L476 was subjected to saturation mutagenesis yielding eight different mutants with up to 5-fold increased carboligase activity. Surprisingly, all L476 mutants catalyze the formation of 2-hydroxy-1-phenyl-propanone with significantly higher enantioselectivity than the wild-type enzyme although enantioselectivity was not a selection parameter. Leu476 potentially plays the role of a gatekeeper of the active site of BFD, possibly by controlling the release of the product. The biocatalyst could be significantly improved for its side reaction, the C-C bond formation and for application under conditions that are not optimized in nature.

Details of the process development and a flow diagram for the isolation of formate dehydrogenase from Candida boidinii are presented. Formate dehydrogenase was isolated from 30-50 kg of wet cells of C. boidinii to produce a technical-grade enzyme, purity about 2-3 units mg-1 with yields of up to 75 %. The preparation is suitable for NADH-regeneration in a membrane reactor. A summary and comparison of different possible routes for the isolation and purification are given and analysed with regard to process time and volumes handled. It is shown, that the time required is much smaller and the resulting productivity is much higher, when aqueous two-phase systems are fully utilised in the process.

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SummaryTechnical aspects of the separation of aqueous two-phase systems in a commercial separator were studied in detail. For the Gyrotester B, the smallest available separator, a flow rate of 200 mYmin and a length of the regulating screw in the outlet port of 13.5 mm were found as optimal operation parameters for the separation of a poly(ethy1ene glycol) (PEG)/dextran two-phase system. In the presence of cells and cell debris the characteristics of the carrier two-phase systems are changed, most notably the phase ratio. Nevertheless good separation and high throughput can be maintained up to 30% wet cell material in the complete system. Using this method the enzyme pullulanase was extracted from 6.65 kg Klebsiella pneumoniae in 88% yield in a single step in less than 2 hr. A yield of 90% was predicted for this step based upon laboratory data, indicating that the performance of the extraction and separation can be calculated with the necessary accuracy and the further scale-up of the process should be accomplished quite easily. The hydrophilic polymers constituting the phase system will often stabilize the enzymes, so that the separation can be carried out at room temperature without extensive cooling. The method of enzyme solubilization or cell disruption is not decisive for the successful extraction of the enzymes, the only limitation being the necessity to find a suitable two-phase system where the desired product and the cells or cell debris will partition in opposite phases. This is shown for a-glucosidase from Succharorn.vcrs c.ur/sbc,rgcJnsis and three aminoacyl-tRNA-synthetases from Eschrrichia coli. The results obtained demonstrate that aqueous two-phase systems can be separated in commercially available separators with high capacity and efficiency. It can be expected that the advanced separation technology available from chemical engineering studies can also be used for the development of large-scale isolation processes for enzymes involving liquidliquid partitions.