Werner Hummel

Large-scale applications of NAD(P)-dependent oxidoreductases: recent developments

NAD(P)-dependent dehydrogenases are useful catalysts for the synthesis of chiral compounds. Many active and stable enzymes are available that (with high enantioselectivity) reduce ketones or keto acids to chiral alcohols, hydroxy acids or amino acids. For economic reasons, these reactions need coupling to the simultaneous regeneration of NAD(P)H. For preparative applications, three components have to be combined: an appropriate enzyme, an efficient coenzyme-regenerating step and a suitable reaction-engineering technique.

Enzyme engineering aspects of biocatalysis: Cofactor regeneration as example

Reaction engineering is an important tool in the case of cofactor depending enzyme-catalyzed reactions. It allows the establishment of conditions resulting in lower product specific cofactor costs as compared with product-specific enzyme costs. This is shown for the stereospecific reduction of carbonyl compounds yielding chiral amino acids and alcohols. In continuous processes, cofactor costs can be reduced if the cofactor can be retained within the bioreactor or recycled into it after separation of the product. In case of readily water-soluble substrates it is even possible to recycle the cofactor during a single pass through a continuously operated reactor more than 4000 times because normally very low cofactor concentrations are sufficient to saturate the enzymes involved. L-tert-Leucine has been produced by reductive amination with a space-time yield of up to 366 g L(-1) d(-1) in a single continuously operated enzyme membrane reactor and a two-stage cascade. Total turnover number of the cofactor NAD(+) increased to 4230. (S)-1-Phenyl-2-propanol was obtained by reduction of the corresponding ketone in an membrane reactor with integrated extraction of the product. A new alcohol dehydrogenase from Rhodococcus erythropolis was used. A space-time yield of 63 g L(-1) d(-1) and a total turnover number of 1350 have been reached. L-Leucine has been produced using polymer-enlarged NADH. The total turnover number was 80,000 at a space-time yield of 214 g L(-1) d(-1).

Cloning, expression, and characterization of an (R)-specific alcohol dehydrogenase from Lactobacillus kefir

Lactobacillus kefir DSM 20587 produces an (R)-specific NADP-dependent alcohol dehydrogenase (ADH) with a broad substrate specificity. The gene of this ADH was isolated and the complete nucleotide sequence determined. The adh gene comprises 759 bp and encodes a protein of 252 amino acids with a calculated molecular weight of 26 781 Da. The deduced amino acid sequence indicated a high degree of similarity to short-chain dehydrogenases. After cloning and expression in Escherichia coli the enzyme was purified and characterized. For the reduction of acetophenone the specific activity of the homogeneous recombinant ADH was 558 U mg−1. The enzyme shows its maximum activity at 50°C while the pH optimum was at pH 7.0. In order to demonstrate its preparative application, purified ADH was used for the stereoselective reduction of several aliphatic and aromatic ketones as well as β-keto esters. Glucose dehydrogenase was added for the regeneration of NADPH. All prochiral ketones were stereoselectively reduced to the corresponding alcohols with >99% ee and in the case of diketones >99% de.

Reduction of acetophenone to R(+)-phenylethanol by a new alcohol dehydrogenase from Lactobacillus kefir

SummaryA new alcohol dehydrogenase catalysing the enantioselective reduction of acetophenone to R(+)-phenylethanol was found in a strain of Lactobacillus kefir. A 70-fold enrichment of the enzyme with an overall yield of 76% was obtained in two steps. The addition of Mg2+ ions was found to be necessary to prevent rapid deactivation. The enzyme depends essentially on NADPH and was inactive when supplied with NADH as the coenzyme. Important enzymological data of the dehydrogenase are: Km (acetophenone) 0.6 mM, Km (NADPH) 0.14 mM, and a pH optimum for acetophenone reduction at 7.0. Addition of EDTA leads to complete deactivation of the enzyme activity. Added iodoacetamide or p-hydroxymercuribenzoate cause only slight inhibition, revealing that the active centre of the enzyme contains no essential SH-group. Besides acetophenone several other aromatic and long-chain aliphatic secondary ketones are substrates for this enzyme. Batch production of phenylethanol was examined using three different methods for the regeneration of NADPH: glucose/glucose dehydrogenase, glucose-6-phosphate/glucose-6-phosphate dehydrogenase, and isopropanol.

A novel, efficient regenerating method of NADPH using a new formate dehydrogenase

Abstract A NADPH regenerating system using a new, protein engineered formate dehydrogenase (FDH) is investigated. The new enzyme is the first known NAD(P)H dependent FDH. It can be successfully employed in synthesis with other enzymes requiring a NADPH regeneration. All advantages of the known NAD(H) dependent FDHs in these enzyme coupled synthesis can now be transferred to NADP(H) dependent systems.

Isolation of l-phenylalanine dehydrogenase from Rhodococcus sp. M4 and its application for the production of l-phenylalanine

Summaryl-Phenylalanine dehydrogenase [l-phenylalanine: NAD+-oxidoreductase (deaminating)] of Rhodococcus sp. strain M4 was studied emphasizing its application for the production of l-phenylalanine. A high enzyme level (30,000 U·l-1, 25–30 U·mg-1 in the crude extract) could be reached during aerob degradation of l-phenylalanine (10 g·l-1) under optimized growth coditions. A partial purification of the intracellular enzyme by liquid-liquid extraction, and DEAE-cellulose led to a specific activity of more than 1300 U·mg-1. The continuous production of l-phenylalanine in an enzyme-membrane-reactor for 350h resulted in a space-time yield of 456 g·l-1·d-1 with a mean substrate conversion of 95%. Consumption of phenylalanine dehydrogenase was 1,500 U·kg Phe-1.

l-2-hydroxyisocaproate dehydrogenase—A new enzyme from Lactobacillus confusus for the stereospecific reduction of 2-ketocarboxylic acids

SummaryA new dehydrogenase from Lactobacillus confusus has been purified. The following reactions are catalyzed by this enzyme:

An enzyme cascade synthesis of ε-caprolactone and its oligomers.

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