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

MINOIA, JUAN MAURICIO; VILLANUEVA, MARÍA EMILIA; COPELLO, GUILLERMO; RODRIGUEZ TALOU, JULIAN; CARDILLO, ALEJANDRA BEATRIZ

APPLIED MICROBIOLOGY AND BIOTECHNOLOGY

SPRINGER

Lugar: Berlin; Año: 2023

0175-7598

The tropane alkaloids hyoscyamine, anisodamine and scopolamine are extensivelyused medicines. In particular, scopolamine has the greatest value in the market.Hence, strategies to enhance its production have been explored as an alternative totraditional field-plant cultivation. In this work, we developed biocatalytic strategies forthe transformation of hyoscyamine into its products utilizing a recombinantHyoscyamine 6β-hydroxylase (H6H) fusion protein to the chitin-binding domain of thechitinase A1 from Bacillus subtilis (ChBD-H6H). Catalysis was carried out in batch andrecycling of H6H constructions was performed via affinity-immobilization,glutaraldehyde crosslinking and adsorption-desorption of the enzyme to different chitinmatrices. ChBD-H6H utilized as free enzyme achieved complete conversion ofhyoscyamine in 3- and 22-hours bioprocesses. Chitin particles demonstrated to be themost convenient support for ChBD-H6H immobilization and recycling. AffinityimmobilizedChBD-H6H operated in a three-cycle bioprocess (3 h/cycle, 30°C) yieldedin the first and third reaction cycle 49.8% and 22.2% of anisodamine and 0.7% and0.3% of scopolamine, respectively. However, glutaraldehyde crosslinking decreasedenzymatic activity in a broad range of concentrations. Instead, the adsorption-desorption approach equaled the maximal conversion of the free enzyme in the firstcycle and retained higher enzymatic activity than the carrier-bound strategy along theconsecutive cycles. The adsorption-desorption strategy permitted the reutilization ofthe enzyme in a simple and economical manner while exploiting the maximalconversion activity displayed by the free enzyme. This approach is valid since otherenzymes present in the E. coli lysate do not interfere with the reaction.