Potential role of Hyoscyamine 6b-hydroxylase plant enzyme in Pharmaceutical Industry

CARDILLO, ALEJANDRA BEATRIZ; RODRIGUEZ TALOU, JULIÁN; GIULIETTI, ANA MARÍA

Mar del plata

Congreso; SAIB 2007; 2007

Plants produce compounds of interest in medicine. The South American native plant, Brugmansia candida, produces tropane alkaloids including hyoscyamine and scopolamine. The anticholinergic properties of them made both alkaloids the most important of the group. Scopolamine is widely used due to the fewer side effects generated. Hyoscyamine 6b-hydroxylase (H6H, EC 1.14.11.11) is the enzyme responsible for the conversion of hyoscyamine into scopolamine. In this work we report the expression of H6H enzyme in Saccharomyces cerevisiae as a potential tool for the industrial production of scopolamine. The h6h cDNA was cloned into the pYES2 and the pYES2.1-TOPO TA vectors. The constructions were introduced by chemical transformation in S. cerevisiae CEN PK2. The enzyme was expressed 4 hs after induction with galactose as it was observed in Western blot analysis. The enzyme in vitro activity assay is being carried out according to the bibliography described methods using crude and IMAC semi-purified enzyme preparations. For the assay analysis it was developed an HPLC method able to detect and quantify low concentrations of alkaloids. It was possible to detect less than 2 ppm of both alkaloids with a 2.6 resolution factor.