A novel one-step expression and immobilization method for the production of biocatalytic preparations.

BACKGROUND: Whole cell biocatalysts and isolated enzymes are considered as state of the art in biocatalytic preparations for industrial applications. Whole cells as biocatalysts are disadvantageous if substrate or products are toxic to the cells or undesired byproducts are formed due to the cellular metabolism. The use of isolated enzymes in comparison is more expensive due to the required downstream processing. Immobilization of enzymes after purification increases preparation costs for biocatalysts significantly, but allows for the efficient reuse of the enzymes in the biocatalytic process. For a more rapid processing one-step expression and immobilization is desirable. RESULTS: This study focused on the development of a new one-step expression and immobilization technique for enzymes on the example of the ß-galactosidase from Escherichia coli K12. The enzyme was expressed in E. coli with a C-terminal membrane anchor originating from cytochrome b5 from rabbit liver and was thus in situ immobilized to the inner surface of the cytosolic membrane. Then, the expression of a lytic phage protein (gene E from PhiX174) caused the formation of a pore in the cell wall of E. coli, which resulted in release of the cytosol. The cellular envelopes with immobilized enzymes were retained. Batch and fed-batch processes were developed for efficient production of these biocatalysts. It was possible to obtain cellular envelopes with up to 27,200 ± 10,460 immobilized enzyme molecules per cellular envelope (753 ± 190 U/gdry weight). A thorough characterization of the effects of membrane immobilization was performed. Comparison to whole cells showed that mass transfer limitation was reduced in cellular envelopes due to the pore formation. CONCLUSION: In this study the feasibility of a new one-step expression and immobilization technique for the generation of biocatalytic preparations was demonstrated. The technique could be a useful tool especially for enzyme systems, which are not suitable for whole-cell biocatalysts due to severe mass transfer limitations or undesired side reactions mediated by cytosolic enzymes.

Engineering of formate dehydrogenase: synergistic effect of mutations affecting cofactor specificity and chemical stability.

Formate dehydrogenases (FDHs) are frequently used for the regeneration of cofactors in biotransformations employing NAD(P)H-dependent oxidoreductases. Major drawbacks of most native FDHs are their strong preference for NAD(+) and their low operational stability in the presence of reactive organic compounds such as α-haloketones. In this study, the FDH from Mycobacterium vaccae N10 (MycFDH) was engineered in order to obtain an enzyme that is not only capable of regenerating NADPH but also stable toward the α-haloketone ethyl 4-chloroacetoacetate (ECAA). To change the cofactor specificity, amino acids in the conserved NAD(+) binding motif were mutated. Among these mutants, MycFDH A198G/D221Q had the highest catalytic efficiency (k cat/K m) with NADP(+). The additional replacement of two cysteines (C145S/C255V) not only conferred a high resistance to ECAA but also enhanced the catalytic efficiency 6-fold. The resulting quadruple mutant MycFDH C145S/A198G/D221Q/C255V had a specific activity of 4.00 ± 0.13 U mg(-1) and a K m, NADP(+) of 0.147 ± 0.020 mM at 30 °C, pH 7. The A198G replacement had a major impact on the kinetic constants of the enzyme. The corresponding triple mutant, MycFDH C145S/D221Q/C255V, showed the highest specific activity reported to date for a NADP(+)-accepting FDH (v max, 10.25 ± 1.63 U mg(-1)). However, the half-saturation constant for NADP(+) (K m, NADP(+) , 0.92 ± 0.10 mM) was about one order of magnitude higher than the one of the quadruple mutant. Depending on the reaction setup, both novel MycFDH variants could be useful for the production of the chiral synthon ethyl (S)-4-chloro-3-hydroxybutyrate [(S)-ECHB] by asymmetric reduction of ECAA with NADPH-dependent ketoreductases.