Recycling of hyoscyamine 6ß-hydroxylase for the in vitro production of anisodamine and scopolamine.

The tropane alkaloids hyoscyamine, anisodamine, and scopolamine are extensively used medicines. In particular, scopolamine has the greatest value in the market. Hence, strategies to enhance its production have been explored as an alternative to traditional field-plant cultivation. In this work, we developed biocatalytic strategies for the transformation of hyoscyamine into its products utilizing a recombinant Hyoscyamine 6ß-hydroxylase (H6H) fusion protein to the chitin-binding domain of the chitinase A1 from Bacillus subtilis (ChBD-H6H). Catalysis was carried out in batch, and recycling of H6H constructions was performed via affinity-immobilization, glutaraldehyde crosslinking, and adsorption-desorption of the enzyme to different chitin matrices. ChBD-H6H utilized as free enzyme achieved complete conversion of hyoscyamine in 3- and 22-h bioprocesses. Chitin particles demonstrated to be the most convenient support for ChBD-H6H immobilization and recycling. Affinity-immobilized ChBD-H6H operated in a three-cycle bioprocess (3 h/cycle, 30 °C) yielded in the first and third reaction cycle 49.8% and 22.2% of anisodamine and 0.7% and 0.3% of scopolamine, respectively. However, glutaraldehyde crosslinking decreased enzymatic activity in a broad range of concentrations. Instead, the adsorption-desorption approach equaled the maximal conversion of the free enzyme in the first cycle and retained higher enzymatic activity than the carrier-bound strategy along the consecutive cycles. The adsorption-desorption strategy permitted the reutilization of the enzyme in a simple and economical manner while exploiting the maximal conversion activity displayed by the free enzyme. This approach is valid since other enzymes present in the E. coli lysate do not interfere with the reaction. KEY POINTS: • A biocatalytic system for anisodamine and scopolamine production was developed. • Affinity-immobilized ChBD-H6H in ChP retained catalytic activity. • Enzyme-recycling by adsorption-desorption strategies improves product yields.

Expression of Brugmansia candida Hyoscyamine 6beta-Hydroxylase gene in Saccharomyces cerevisiae and its potential use as biocatalyst.

BACKGROUND: Tropane alkaloids, mainly hyoscyamine and scopolamine, are widely used in medicine due to their anticholinergic activity. Scopolamine has a higher demand being the more valuable alkaloid due to its fewer side effects and higher physiological activity. Anisodamine (6beta-hydroxyhyoscyamine) is the intermediate in the conversion of hyoscyamine into scopolamine. Current studies report that this alkaloid is potentially applicable in medicine. The gene that codifies for Hyoscyamine 6-beta hydroxylase, the enzyme responsible for hyoscyamine hydroxylation and epoxidation, leading to scopolamine was isolated from Brugmansia candida. RESULTS: The h6hcDNA was cloned into pYES2.1 and pYES2.1/V5-His-TOPO vectors to produce an untagged and a tagged protein, respectively. The H6H enzyme was produced in Saccharomyces cerevisiae in order to obtain a biological catalyst for potential industrial applications. Protein extracts of the induced yeast were analyzed by Western blot. The expression was detected 4 h after induction and no degradation was observed during the period assayed. The tagged and the untagged proteins were able to transform hyoscyamine, showing a functional expression of the h6hcDNA. CONCLUSION: The strains obtained in this work are promising and potentially applicable in biocatalytic processes.

Analysis and sequencing of h6hmRNA, last enzyme in the tropane alkaloids pathway from anthers and hairy root cultures of Brugmansia candida (Solanaceae)

Brugmansia candida (Solanaceae) is a native tree distributed across South-American and produces the pharmacologically- important group of tropane alkaloids including scopolamine. This biocompound is synthesised from hyoscyamine by action of Hyoscyamine 6-â hydroxylase (H6H, EC 1.14.11.11) at the end of the tropane alkaloid pathway. Here are reported the tissue and organ-specific expression of h6hmRNA by RT-PCR analyses and the isolation, cloning and sequencing of the cDNA obtained from B. candida anthers and hairy root transformed cultures. Bioinformatic analysis of the nucleotide sequence revealed an uninterrupted ORF of 1038 bp and the predicted aminoacid sequence could be 344 aminoacid long. A database search showed that this sequence has high homology (97 percent identity) to Hyoscyamus niger H6H protein (Genbank accession number AAA33387.1).