Biocatalysed redox reactions in aqueous and organic media

Detta är en avhandling från Department of Biotechnology, Lund University

Sammanfattning: This thesis deals mainly with biocatalysed methods for stereoselective reduction. D-(S)-chlorolactic acid was produced by both microbial and electromicrobial reduction of chloropyruvate using whole cells of Proteus vulgaris and an artificial electron mediator. The electron donor was formate, hydrogen gas or the cathode in an electrochemical cell. Since chloropyruvate was susceptible to mediator dependent non-enzymatic dehalogenation the choice of mediator was of importance for the obtainable yield of chlorolactic acid. The best results were obtained using 1,1'-carbamoylmethyl viologen. For large scale (mmol) synthesis, formate and formate dehydrogenase was the best method to regenerate the mediator and it was beneficial to add the chloropyruvic acid continuously during the reaction so as to avoid side reactions. A productivity of 8300 mmol kg-1 h-1 and a space time yield of 68 g l-1 d-1 was obtained. Hydrogenase driven NADH regeneration using permeabilised whole cells of Alcaligenes eutrophus was shown possible and used for lactate dehydrogenase and horse liver alcohol dehydrogenase (HLADH) catalysed reductions in aqueous media. The immobilisation of the cells in alginate and addition of reducing agents as dithiothreitol improved the stability. The permeabilised whole cell approach was also suitable as NADH regenerating catalyst in aqueous organic two-phase media (i.e. ¾10 % water). The permeabilised cells were co-immobilised together with HLADH and NAD by deposition on Celite. Hydrophobic solvents such as heptane and toluene were found to provide high activity. A turnover of the NAD of 200 and a spacetime yield of 10 g l-1 d-1 was obtained in heptane with 10 % water. Furthermore, the stability of HLADH in dry organic solvents could be well correlated to solvent descriptors by PLS (projection to latent structure) analysis of the data. Finally, Celite immobilised chloroperoxidase from Caldariomyces fumago was shown to be able to catalyse the oxidation of bensyl alcohol using tert-butyl hydroperoxide as oxidant in organic media at water activities above 0.6. Highest enzymatic rate was obtained in water saturated cyclohexane. The apparent Km for bensyl alcohol was 13 mM.

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