CARDILLO Alejandra Beatriz
congresos y reuniones científicas
Hyoscyamine 6-beta-hidroxilase, a recombinant biocatalyst for the industrial production of scopolamine
CARDILLO, ALEJANDRA BEATRIZ; RODRIGUEZ TALOU, JULIÁN; GIULIETTI, ANA MARÍA
Congreso; IBS 2008, Thirteenth International Biotechnology Symposium and Exhibition; 2008
Abstract: 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 [1, 2, 3]. Brugmansia candida (syn Datura candida) is a South American native plant and a tropane alkaloid producer . The conversion of hyoscyamine into scopolamine is carried out by Hyoscyamine 6-b hydroxylase (H6H, EC 22.214.171.124). The development of a recombinant cell harbouring the plant enzyme constitutes an interesting and 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 total RNA preparations obtained from B. candida immature 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 by chemical transformation 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 hs after induction. Crude protein extracts of the induced strains were assayed for the enzyme activity according to Hashimoto and Liu [5, 6]. The activity assay was incubated 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. The results showed that the tagged and untagged enzymes were able to transform hyoscymine, 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 6β-hydroxyhyoscyamine in the incubation times assayed. According to the results obtained in this work it can be concluded that the recombinant S. cerevisiae strains obtained are promissory for technological applications in the production of scopolamine by biotransformation.