Plant enzymes as Pharmaceutical biocatalysts

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

Viña del Mar - Valparaíso

Congreso; REDBIO 2007 VI Encuentro Latinoamericano y del Caribe de Biotecnología Agropecuaria; 2007

Several plant compounds are interesting for the pharmaceutical industry due to their medicinal properties. The South American native plant, Brugmansia candida, produces hyoscyamine and scopolamine, the most important tropane alkaloids. The latter is the more valuable, with a demand 10 times higher than that of hyoscyamine. The possible reasons for this are the scopolamine anticholinergic properties widely used in medicine and fewer side effects of this alkaloid comparing to its precursor. Hyoscyamine 6b-hydroxylase (H6H, EC 1.14.11.11) is the enzyme responsible for the conversion of hyoscyamine into scopolamine. The enzyme is a 2-oxoglutarate dependent dioxygenase that performs the hydroxylation and epoxidation of hyoscyamine leading to 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. Crude protein extracts performed by mechanical lysis with acid washed glass beads and protease inhibitors have been purified by IMAC allowing the concentration of the H6H. However, it was found that the preparation still has low quantities of yeast proteins. Nowadays this preparation is been purified for sequencing purposes by HPLC using a gradient Acetonitrile + TFA : Water + TFA (5 % to 95 % of Acetonitrile) and monitoring at 210 nm and 280 nm. In parallel with the purification, the enzyme in vitro activity assay is being carried out according to the bibliography described methods. For that 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. The retention times for scopolamine and hyoscyamine were 5.3 min and 7.5 min respectively with a 2.6 resolution factor. Several plant compounds are interesting for the pharmaceutical industry due to their medicinal properties. The South American native plant, Brugmansia candida, produces hyoscyamine and scopolamine, the most important tropane alkaloids. The latter is the more valuable, with a demand 10 times higher than that of hyoscyamine. The possible reasons for this are the scopolamine anticholinergic properties widely used in medicine and fewer side effects of this alkaloid comparing to its precursor. Hyoscyamine 6b-hydroxylase (H6H, EC 1.14.11.11) is the enzyme responsible for the conversion of hyoscyamine into scopolamine. The enzyme is a 2-oxoglutarate dependent dioxygenase that performs the hydroxylation and epoxidation of hyoscyamine leading to 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. Crude protein extracts performed by mechanical lysis with acid washed glass beads and protease inhibitors have been purified by IMAC allowing the concentration of the H6H. However, it was found that the preparation still has low quantities of yeast proteins. Nowadays this preparation is been purified for sequencing purposes by HPLC using a gradient Acetonitrile + TFA : Water + TFA (5 % to 95 % of Acetonitrile) and monitoring at 210 nm and 280 nm. In parallel with the purification, the enzyme in vitro activity assay is being carried out according to the bibliography described methods. For that 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. The retention times for scopolamine and hyoscyamine were 5.3 min and 7.5 min respectively with a 2.6 resolution factor. Several plant compounds are interesting for the pharmaceutical industry due to their medicinal properties. The South American native plant, Brugmansia candida, produces hyoscyamine and scopolamine, the most important tropane alkaloids. The latter is the more valuable, with a demand 10 times higher than that of hyoscyamine. The possible reasons for this are the scopolamine anticholinergic properties widely used in medicine and fewer side effects of this alkaloid comparing to its precursor. Hyoscyamine 6b-hydroxylase (H6H, EC 1.14.11.11) is the enzyme responsible for the conversion of hyoscyamine into scopolamine. The enzyme is a 2-oxoglutarate dependent dioxygenase that performs the hydroxylation and epoxidation of hyoscyamine leading to 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. Crude protein extracts performed by mechanical lysis with acid washed glass beads and protease inhibitors have been purified by IMAC allowing the concentration of the H6H. However, it was found that the preparation still has low quantities of yeast proteins. Nowadays this preparation is been purified for sequencing purposes by HPLC using a gradient Acetonitrile + TFA : Water + TFA (5 % to 95 % of Acetonitrile) and monitoring at 210 nm and 280 nm. In parallel with the purification, the enzyme in vitro activity assay is being carried out according to the bibliography described methods. For that 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. The retention times for scopolamine and hyoscyamine were 5.3 min and 7.5 min respectively with a 2.6 resolution factor. Key words: Biotransformation, scopolamine, Saccharomyces cerevisiae, Hyoscyamine 6b-hydroxylase