Stabilitäten der Benzaldehydlyase aus Pseudomonas fluorescens und der Carbonylreduktase aus Candida parapsilosis in wässrig-organischen Zweiphasensystemen

Benzaldehyde lyase from Pseudomonas fluorescens (BAL) and carbonylreductase from Candida parapsilosis (CPCR) are versatile catalysts for the production of various chiral intermediates, which are of interest for the pharmaceutical industry. Due to the mostly hydrophobic reactants only low productivities can be achieved in aqueous reaction systems and thus the use of alternative reaction media is desirable. Especially the use of aqueous-organic biphasic systems is promising, since the extraction of products and the spatial separation from the enzyme facilitate efficient processes. However, both enzymes show only insufficient stability in these systems and therefore a stabilisation is indispensable. Stability of both enzymes was determined in three different biphasic systems as well as the respective solvent-saturated aqueous monophasic systems (BAL: MTBE, 2 octanone, toluene; CPCR: MTBE, cyclohexane, n heptane). It was shown that BAL was already significantly deactivated by the solvent molecules within the aqueous phase. In the respective biphasic systems especially the interfaces towards 2-octanone and toluene led to considerable additional deactivation. On the molecular level CPCR was deactivated mainly by dissolved MTBE-molecules. In contrast, dissolved cyclohexane and n-heptane molecules only had minor effects on CPCR stability, whereas the respective biphasic systems led to significant deactivation of CPCR. The deactivation by the solvents is probably a result of interactions between the solvents and the proteins’ surfaces. Therefore the surface characteristics of both enzymes were altered in order to prevent these interactions. Chemical modification of free amino groups was applied to introduce new chemical functionalities to the enzymes’ surfaces. Acetylation with cyclic anhydrides decreased activity of both enzymes significantly. In contrast, modification with mPEG750 and mPEG2000 resulted in good residual activities of BAL (60-70%), while CPCR retained less than 10% residual activity. Gel electrophoresis showed that apparently only few amino groups of BAL were accessible for mPEG-modification, whereas CPCR was modified to great extent. Storage stability measurements with the mPEG-BAL-variants revealed a considerable increase of stability towards dissolved molecules in the solvent systems with MTBE and toluene. Site-directed mutagenesis was applied to exchange unpolar surface amino acid residues against polar amino acid residues in order to decrease interactions between the enzymes and the solvents. Most of the resulting BAL-variants showed similar activities as the native BAL, whereas many CPCR-variants lost most of their activity. For both enzymes enantioselectivity was unaffected by the introduced mutations. Kinetic constants of BAL-variants differed only marginally from the ones of native BAL. CPCR-variants exhibited higher kcat-values than native CPCR in both reduction- and oxidation reaction. Determination of storage stability in the different mono-and biphasic systems showed that most variants exhibited decreased stability against dissolved solvent molecules compared to native enzymes. However, additional deactivation at the interface towards toluene (BAL) and MTBE, cyclohexane und n-heptane (CPCR) respectively, was decreased. For the technical application enzyme stability under relevant process conditions is of importance. Therefore a reactor set-up was established, that allows stability determination in reactive, aqueous-organic biphasic systems. Respective measurements with BAL indicated that deactivation by the aldehyde substrate superimposed the deactivation by the solvents, thus resulting in similar BAL stability in all tested solvent systems. Neither the mPEG-modification nor the introduced mutations prevented this deactivation by the substrate. CPCR showed a strong stability decrease under process conditions in the MTBE-system, probably due to a combined effect of dissolved MTBE-molecules and isopropanol which was used for substrate-coupled cofactor regeneration. CPCR stability in reactive systems with cyclohexane and n-heptane was comparable to stability under non-reactive conditions. Variant CPCR-I79T,V83S showed increased process stability in all three biphasic systems.