Thesis for Master of Science
Carla da Conceição Caramujo Rocha de Carvalho
”Produção
e recuperação de limoneno-1,2-diol e resolução simultânea de uma mistura
diastereomérica de 1,2-epóxido de limoneno com células inteiras de
Rhodococcus erythropolis DCL14.”
(Production
and recovery of limonene-1,2-diol and simultaneous resolution of a
diastereomeric mixture of 1,2-limonene epoxide with whole cells of Rhodococcus
erythropolis)
MSc.
Biotechnology, IST, 1999
Supervisor: M. Manuela R. da Fonseca
Abstract:
The biotransformation of terpenes allows the production of fragrances and flavours under mild reaction conditions with high stereo-specificity and selectivity. These facts are leading to an increasing economical interest for these biotransformations.
One
of the purposes of the European project BIO4-CT95-0049, in which the present
work is inserted, was the screening of microorganisms able to convert limonene
into carvone. Until the end of the project that was not achieved. Nevertheless,
at the
The epoxide is unstable and has a low solubility in water solutions. In aqueous buffer at pH below 7, cis-epoxide is readily hydrolysed (at a rate of 141.37 and 40.65 nmol/min at pH 5 and 6, respectively, for an initial epoxide concentration of 2.5 mM) and even at higher pH values it’s not negligible (7.14 nmol/min at pH 8, for the same initial concentration). The aim was the development of a reaction system in which hydrolysis would be carried out solely by the action of the biocatalyst, chemical hydrolysis being prevented.
A
biphasic system was developed to overcome substrate instability and low
solubility. The presence of an organic solvent allowed high substrate
concentrations, which resulted in high diol production rates (about 280 nmol/min.mgprot
for epoxide concentrations higher than 50 mM). Several relatively cheap solvents
were tested without significant loss of LEH activity. It was assessed that the
best aqueous phase: organic phase ratio is 1:5 (it allows good conversion rates
and allows the “storage” of an acceptable amount of diol in the aqueous
phase), and, that the enzyme activity in whole cells increases with an increase
in temperature (in the range 30-50ºC, but with a decrease in the number of cell
reutilizations) and with stirring speed. R.
erythropolis DCL14 cells reveal to be highly hydrophobic, what resulted in
the migration of cells towards the aqueous/organic interface, being the contact
area between cells and the organic phase soluble substrate strongly dependent of
stirring (a 4 times increase in the stirring speed resulted approximately in a
2.5 fold increase in the reaction rate).
Several reactor configurations were tested. Using a 500 ml fed-batch mechanically stirred reactor it was possible to obtain a trans-epoxide yield of 98.2% and of 67.9% for the diol. Diol and trans-epoxide yields obtained using a magnetically stirred reactor with an external aqueous phase recirculation for product separation, were 94.1 and 98.5%, respectively. With a membrane reactor the diol and trans-epoxide yields were 98.4 and 76.6%, respectively.
A simple downstream process, based both on the preference of the substrate for organic solvents and that of the product for aqueous phases, allowed the recovery of limonene-1,2-diol, as well as trans-limonene-1,2-epoxide, with a purity higher than 99 %.
Keywords: terpenes, monoterpenes, epoxide hydrolase, aqueous/organic system, biotransformation, Rhodococcus erythropolis