Chitosan flocculation: an effective method for immobilization of E. coli for biocatalytic processes.

Immobilization of Escherichia coli cells containing a ω-transaminase was carried out by flocculation with chitosan and the preparation was used in asymmetric synthesis of (S)-4'-cyano-α-methylbenzylamine, recycled in five consecutive batches. Chitosans with different molecular weights and degrees of acetylation were compared and effects of varying the chitosan properties, cell concentration and ratio of cells to chitosan were studied. Immobilization was achieved by increasing the pH to slightly alkaline, which induced the formation of large fast sedimenting flocs.

Transaminations with isopropyl amine: equilibrium displacement with yeast alcohol dehydrogenase coupled to in situ cofactor regeneration.

Enantiopure chiral amines synthesis using omega-transaminases is hindered by an unfavourable equilibrium, but when using isopropylamine as the amine donor the equilibrium can be completely displaced by using a specific dehydrogenase in situ for removal of formed acetone.

Rational design of Pseudozyma antarctica lipase B yielding a general esterification catalyst.

Pseudozyma antarctica lipase B (CALB) shows activity in the acrylation of hydroxypropylcarbamate, a racemic mixture of enantiomers of primary and secondary alcohols. However, full conversion is hampered by the slowly reacting S enantiomer of the secondary alcohol. The same is true for a wide range of secondary alcohols, for example, octan-2- and -3-ol.

Direct epoxidation in Candida antarctica lipase B studied by experiment and theory.

Candida antarctica lipase B (CALB) is a promiscuous serine hydrolase that, besides its native function, catalyzes different side reactions, such as direct epoxidation. A single-point mutant of CALB demonstrated a direct epoxidation reaction mechanism for the epoxidation of alpha,beta-unsaturated aldehydes by hydrogen peroxide in aqueous and organic solution. Mutation of the catalytically active Ser105 to alanine made the previously assumed indirect epoxidation reaction mechanism impossible.

Silica-immobilized His6-tagged enzyme: alanine racemase in hydrophobic solvent.

A new immobilization method for enzymes is presented to facilitate synthetic applications in aqueous as well as organic media. The enzyme Alanine racemase (AlaR) from Geobacillus stearothermophilus was cloned, overexpressed and then immobilized on a silica-coated thin-layer chromatography plate to create an enzyme surface. The enzyme, fused to a His(6)-tag at its N-terminal, was tethered to the chemically modified silica-coated TLC plate through cobalt ions.

Exploring the active-site of a rationally redesigned lipase for catalysis of Michael-type additions.

Michael-type additions of various thiols and alpha,beta-unsaturated carbonyl compounds were performed in organic solvent catalyzed by wild-type and a rationally redesigned mutant of Candida antarctica lipase B. The mutant lacks the nucleophilic serine 105 in the active-site; this results in a changed catalytic mechanism of the enzyme. The possibility of utilizing this mutant for Michael-type additions was initially explored by quantum-chemical calculations on the reaction between acrolein and methanethiol in a model system.

Carbon-carbon bonds by hydrolytic enzymes.

Enzymes are efficient catalysts in synthetic chemistry, and their catalytic activity with unnatural substrates in organic reaction media is an area attracting much attention. Protein engineering has opened the possibility to change the reaction specificity of enzymes and allow for new reactions to take place in their active sites. We have used this strategy on the well-studied active-site scaffold offered by the serine hydrolase Candida antarctica lipase B (CALB, EC 3.