Continuous Synthesis of (R)-(+)-Benzaldehydecyanohydrin Using (R)-Oxynitrilase in Lyotropic Liquid Crystals

The possibility of using the enzyme (R)-Oxynitrilase in a biphasic lyotropic liquid crystal/dibutylether system has been demonstrated. This reaction system is applicable for the continuous production of (R)-benzaldehydecyanohydrin in a fixed bed reactor. The optical purity was between 94 and 96% ee and independent of the flow rate. The space time yield was maximal (2650 g/(1*d)) at a flow rate of 1.6 ml/min.     

Biocatalytic synthesis of hydroxylated natural products using aldolases and related enzymes

Synthetic building blocks bearing hydroxylated chiral centers are important targets for biocatalysis. Many C-C bond forming enzymes have recently been investigated for new applications and new strategies towards the synthesis of natural products and related oxygenated compounds. Several old catalysts have been studied to increase our functional knowledge of natural aldolase-type enzymes, and new mutated catalysts or catalytic antibodies have been tested for their synthetic utility.     

A new, mild cross-linking methodology to prepare cross-linked enzyme aggregates

Cross-linked enzyme aggregates (CLEAs) were prepared from several enzymes (penicillin G acylase, hydroxynitrile lyase, alcohol dehydrogenase, and two different nitrilases) by precipitation and subsequent cross-linking using dextran polyaldehyde. In most cases, higher immobilization yields were obtained using the latter cross-linker as compared with the commonly used glutaraldehyde. Active site titration of penicillin acylase CLEAs showed that the higher activity originated from a significantly lower loss in active sites using dextran polyaldehyde as a cross-linking agent. It is proposed that macromolecular cross-linkers are too large to penetrate the protein active site and react with catalytically essential amino acid residues.     

Three-dimensional structures of enzyme-substrate complexes of the hydroxynitrile lyase from Hevea brasiliensis.

The 3D structures of complexes between the hydroxynitrile lyase from Hevea brasiliensis (Hb-HNL) and several substrate and/or inhibitor molecules, including trichloracetaldehyde, hexafluoracetone, acetone, and rhodanide, were determined by X-ray crystallography. The complex with trichloracetaldehyde showed a covalent linkage between the protein and the inhibitor, which had apparently resulted from nucleophilic attack of the catalytic Ser80-Ogamma. All other complexes showed the substrate or inhibitor molecule merely hydrogen bonded to the protein. In addition, the native crystal structure of Hb-HNL was redetermined at cryo-temperature and at room temperature, eliminating previous uncertainties concerning residual electron density within the active site, and leading to the observation of two conserved water molecules. One of them was found to be conserved in all complex structures and appears to have mainly structural significance. The other water molecule is conserved in all structures except for the complex with rhodanide; it is hydrogen bonded to the imidazole of the catalytic His235 and appears to affect the Hb-HNL catalyzed reaction. The observed 3D structural data suggest implications for the enzyme mechanism. It appears that the enzyme-catalyzed cyanohydrin formation is unlikely to proceed via a hemiacetal or hemiketal intermediate covalently attached to the enzyme, despite the observation of such an intermediate for the complex with trichloracetaldehyde. Instead, the data are consistent with a mechanism where the incoming substrate is activated by hydrogen bonding with its carbonyl oxygen to the Ser80 and Thr11 hydroxy groups. A hydrogen cyanide molecule subsequently replaces a water molecule and is deprotonated presumably by the His235 base. Deprotonation is facilitated by the proximity of the positive charge of the Lys236 side chain.