Enzyme evolution: a promising method for obtaining new enzymes for carbohydrate synthesis [Rhodobacter sphaeroides]
1995
Stein, M. | Morfova, M. | Schneider, K.H. | Jaekel, G. | Huwig, A. | Giffhorn, F. (Universitaet des Saarlandes, Saarbruecken (Germany). Lehrstuhl fuer Angewandte Mikrobiologie)
Using the methodology of in vivo enzyme evolution a gain of function mutant of Rhodobacter sphaeroides Si4, capable of growing on galactitol, was isolated from a chemostat culture. The mutant strain, designated R. sphaeroides D, grew slowly on galacitol but produced constitutively high levels of a so far unrecognized galactitol dehydrogenase (GDH). In order to study whether it was a newly evolved enzyme or an improved side activity of one of the preexisting R. sphaeroides Si4 polyol dehydrogenases (PDH), the GDH was purified to homogeneity. Biochemical characterization of GDH revealed that it was different to mannitol dehydrogenase (MDH), ribitol dehydrogenase (RDH) and sorbitol dehydrogenase (SDH) present in the wild type strain. On the other hand, there was a significant identity among the N-terminal amino acid seqences of GDH and those of SDH and MDH, suggesting that these PDHs had evolved from a common ancestor protein. A striking characteristic of GDH was its broad substrate specificity. In addition to polyols GDH catalysed the NAD-linked oxidation of a variety of diols and secondary aliphatic alcohols. Using galactitol and xylitol as starting materials in bioconversions the regioselective oxidation of galactitol at C-5 and of xylitol at C-4 to the corresponding L-ketoses L-tagatose and L-xylulose, respectively, was demonstrated with HPLC, NMR-spectrometry and polarimetry. On the basis of an efficient cosubstrate regeneration system L-tagatose and L-xylulose were prepared with yields of 90-97 per cent and 65-77, respectively
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