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Third-party-funded project

Use of selen containng biocatalysts for selective oxidoreductions

Project management at the University of Würzburg:

Participating scientists:

Subtilisin is an intensively studied serine protease which catalyzes the hydrolysis or the formation of peptides, amides and esters. The enzyme is commercially available, shows high stability and the catalyzed reactions proceed with high stereoselectivity. Serine 221 in the active center of subtilisin Carlsberg and subtilisin BPN' was transformed chemically into selenocysteine, resulting in the semisynthetic peroxidase seleno-subtilisin. Owing to the identity of the substrate binding pockets in the protease and the peroxidase similar enantioselectivity of both the enzymes was expected. In a seleno-subtilisin catalyzed kinetic resolution ten chiral hydroperoxides were studied in detail. The enantioselectivities reached 99% ee of the optically active hydroperoxides; the preferred absolute configuration of the products correponded to the working hypothesis.

In kinetic studies the semisynthetic enzyme showed catalytic efficiency and enantioselectivity comparable to a native enzyme. The substrate affinity seleno-subtilisin to alkyl aryl hydroperoxides was analogous to that of subtilisin to arylboronic acid inhibitors, allowing predictions of substrate acceptance of the semisynthetic enzyme.

After enzyme production in semi-preparative scale and subsequent glutardialdehyde treatment, the cross-linked enzyme exhibited excellent stability against high temperature (10 days at 60°C) and organic solvents (10 days in 50% DMF at 40°C). Tenfold re-use in semi-preparative scale was reached without any loss of activity or enantioselectivity.

Analogously, seleno-proteinase K, seleno-savinase, seleno-esperase and seleno-panstimase were produced and characterized by comprehensive substrate studies. Differences in the enantioselectivities could be related to the geometry of the S1-binding pocket of the seleno-proteases. The hypothesis that the substrate binding in the active center of seleno-proteases occurs nearby the catalytic triade was supported by molecular modelling studies. Thus, the geometry of the S1-pocket and the amino acid His-64 play an important role for the stereoselectivity of seleno-proteases.

Using seleno-trypsine, various seleno-lipases as well as glutathione peroxidase in kinetic racemate resolution studies, no chiral recognition of alkyl aryl hydroperoxides was observed.

Key words:
    Semisynthetic enzymes
    Kinetic resolution

Projekt period: from 01.1996 to 12.1998

Funding institution:
DFG ,Granting date: 01.10.1995


Research area