INVESTIGADORES
RODRIGUEZ TALOU Julian
congresos y reuniones científicas
Título:
New strategies for the production of medicinal compounds: Tropane alkaloids.
Autor/es:
CARDILLO AB; RODRÍGUEZ TALOU J; GIULIETTI AM
Lugar:
Giardini Naxos
Reunión:
Congreso; Biotrans 2011; 2011
Resumen:
Plants are able to
produce a large diversity of natural products of vast interest for
pharmaceutical purposes. Tropane alkaloids, such as hyoscyamine and
scopolamine, are secondary metabolites traditionally applied in medicine
according to their anticholinergic activity [1]. Hyoscyamine is converted by Hyoscyamine 6b-hydroxylase (H6H) into anisodamine and
scopolamine [2]. Recently, potential medical applications were
also described for anisodamine [3]. Nowadays, these compounds are obtained from
natural producer plants due to the cost and complexity of the chemical
synthesis of them. For this reason, tropane alkaloids production by
biotransformation processes is an attractive strategy for the pharmaceutical
industry [4]. In the present work we explored the
development of an alternative strategy for the production of the most valuable
alkaloids, anisodamine and scopolamine, using the H6H as biocatalyst. For this
purpose the h6h gene was amplified
from total RNA preparations obtained from immature anthers of the South
American tropane alkaloid producer plant,
Brugmansia candida and cloned into different vectors in order to produce
tagged and untagged enzymes. The H6H enzyme was expressed fused at its
C-terminus to a V5 epitope and a His-tag (H6H-V5-6His) [4]. On the other hand, it was fused at its
N-terminus to a cellulose binding domain (CBD-H6H) in order to combine the
purification step with the enzyme immobilization on cellulose, a low cost
matrix [5]. The constructions were introduced by chemical
transformation in S. cerevisiae CEN
PK2. In order to explore the different strategies, crude protein extracts of
the induced yeast strains were assayed for the enzyme activity at 30ºC for 15hs. The analysis of
the alkaloids was carried out by HPLC with UV detection. The mobile phase used
was octanesulfonic acid 0.01M pH3/methanol (65:35), flow rate 1ml/min [4]. The results showed that the tagged and
untagged enzymes were able to transform hyoscymine, showing a functional
expression of the h6hcDNA. However,
the products obtained were different when the reaction was performed by the
different enzyme constructions. The untagged H6H and the CBD-H6H were able to
convert hyoscyamine into anisodamine and scopolamine while H6H-V5-6His only produced
anisodamine. Surprisingly, the cellulose binding domain did not negatively
affect the catalytic activity of the H6H as the V5 epitope and the Histidine
tag affected it. These facts are encouraging for the development of a
biocatalytic process using immobilized enzymes