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

MINOIA J; VILLANUEVA ME; COPELLO GJ; RODRIGUEZ TALOU J; CARDILLO AB

APPLIED MICROBIOLOGY AND BIOTECHNOLOGY

SPRINGER

Lugar: Berlin; Año: 2023 vol. Jun p. 3459 - 3478

0175-7598

The tropane alkaloids hyoscyamine, anisodamine and scopolamine are extensively used medicines. The last one is the most valuable due to its pharmacological profile. Hence, strategies to enhance its production have been explored as an alternative to traditional field-plant cultivation. In this work, we developed consecutive reaction-based biocatalytic strategies for the transformation of hyoscyamine into its products utilizing a recombinant fusion protein that combines the enzyme Hyoscyamine 6β-hydroxylase (H6H) from Brugmansia candida and the chitin-binding domain of the chitinase A1 from Bacillus subtilis (ChBD-H6H). Catalysis and recycling of H6H constructions was studied using macroporous chitin matrices, chitin nanowhiskers and chitosan beads, following different approaches such as affinity-immobilization, glutaraldehyde crosslinking and adsorption-desorption of the enzyme. Chitin particles (ChP) 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 respectively 49.8% and 22.2% of anisodamine and 0.7% and 0.3% of scopolamine. While glutaraldehyde crosslinking decreased enzymatic activity, the adsorption desorption approach equaled the maximal conversion of the free enzyme in the first cycle and retained higher enzymatic activity along the consecutive cycles than the carrier-bound strategy. That is, in four cycle bioprocess anisodamine and scopolamine production was 85.5% and 14.1% in the first cycle and 25.4% and 0.7% in the fourth cycle, respectively.