Thesis for Doctor of Philosophy

Frederik van Keulen
“Xanthobacter Py2 growth on various alkenes and the respective cell activity towards the degradation of epoxides”
Ph. D. Biotechnology, IST, 2000
Supervisor: M. M. R. da Fonseca

Abstract

This thesis addresses the enantiomeric resolution of epoxides by resting 1-pentene grown Xanthobacter Py2 cells.

The strain had been originally isolated on propene. Growth on 1-pentene as sole carbon source was obtained after a lengthy adaptation period, during which cells were repeatedly subcultured with the addition of yeast extract at the intermediate stages. Batch production of the biocatalyst was optimised, resulting in a culture density of 0.63 gdw/L with 19% of the sealed flask occupied by broth (1-pentene is gaseous at 30°C), using an initial 1-pentene concentration of 0.1% (v/v). The biomass yield on 1-pentene was 0.49 Ceq/Ceq.

Problems associated with the toxicity of the epoxides towards the cells and also with the low solubility and instability of epoxides in water were solved by implementing a two phase biotransformation system, consisting of phosphate buffer containing the freely suspended cells, and a biocompatible organic substrate reservoir (iso-octane or n-dodecane). A maximum specific 1,2-epoxyhexane conversion rate of 25 nmol/min.mg protein was attained when cells harvested from a batch fermentation in the late exponential phase were used. In this biphasic system, the epoxide conversion capacity of the whole cells was limited, even in the presence of co-substrates. Epoxide conversion activity could temporarily be restored by the addition of (NH4)2SO4. Substitution of the buffer by mineral medium resulted in growth of the strain (using the epoxide as carbon and energy source) and in an enhanced epoxide conversion capacity of the system. Under these conditions, re-use of the biocatalyst was successfully achieved.

The strain was also adapted to grow on 1-hexene and 1-octene. The resulting whole cells showed a substrate specificity similar to those grown on 1-pentene.

Keywords: 1,2-Epoxyalkanes, 1-Alkenes, Organic/aqueous system, Enantiomeric resolution, Xanthobacter Py2, Biotransformation.