RODRIGUEZ TALOU Julian
congresos y reuniones científicas
Strategies for the anisodamine production by a recombinant Saccharomyces cerevisiae strain
CARDILLO AB; QUEVEDO CV; PERASSOLO M; MARTÍNEZ CA; BUSTO VD; RODRÍGUEZ TALOU J; GIULIETTI AM
Carlos Paz, Cordoba
Congreso; XLIV Congreso de SAIB; 2008
The tropane alkaloids, hyoscyamine and scopolamine are widely used as pharmaceuticals due to their anticholinergic activity. Scopolamine is the more valuable, with a demand 10 times higher than that of hyoscyamine (Palazón et al., 2003; Hashimoto and Yamada, 1986). Brugmansia candida is a South American native plant and a tropane alkaloid producer. The conversion of hyoscyamine into scopolamine is carried out by hyoscyamine 6-_-hydroxylase (H6H, EC 18.104.22.168). The development of a recombinant cell harbouring the plant enzyme constitutes a useful strategy for performing pharmaceutical processes. This work reports the functional expression of H6H enzyme in Saccharomyces cerevisiae as a potential tool for the industrial production of scopolamine. The gene that codifies for the H6H enzyme was amplified from B. candida mmature anthers. The h6h cDNA obtained was cloned into the pYES2 and the pYES2.1-TOPO TA vectors to produce an untagged and a tagged enzyme, respectively. The constructions were introduced in S. cerevisiae CEN PK2. The recombinant yeast strains obtained were induced for the expression with galactose. It was seen that the expression of the recombinant protein starts 4 h after induction. Crude protein extracts of the induced strains were assayed for the enzyme activity (Hashimoto and Yamada, 1987; Liu et al., 2005). The activity assay was incubated at 30 ◦C for 15 h. The analysis of the alkaloids was carried out by HPLC. The mobile phase used was octanesulfonic acid 0.01M pH 3/methanol (65:35), flow rate 1 ml/min. The results showed that the tagged and untagged enzymeswere able to transform hyoscyamine, showing a functional expression of the h6hcDNA. The untagged enzyme presented a higher rate of conversion of hyoscyamine than the tagged enzyme and was able to produce scopolamine and not only 6-_-hydroxyhyoscyamine in the incubation times assayed. It can be concluded that the recombinant S. cerevisiae strains obtained are promissory for technological applications in the production of scopolamine by biotransformation.