Minoia Juan M, Villanueva María E, Copello Guillermo J, Rodríguez Talou Julián, Cardillo Alejandra B
Facultad de Farmacia Y Bioquímica, Departamento de Microbiología, Inmunología, Biotecnología y Genética, Cátedra de Biotecnología, Universidad de Buenos Aires, Buenos Aires, Argentina.
CONICET - Universidad de Buenos Aires, Instituto de Nanobiotecnología (NANOBIOTEC), Buenos Aires, Argentina.
Appl Microbiol Biotechnol. 2023 Jun;107(11):3459-3478. doi: 10.1007/s00253-023-12537-w. Epub 2023 Apr 26.
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 6β-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.
托烷生物碱阿托品、山莨菪碱和东莨菪碱是广泛使用的药物。特别是东莨菪碱在市场上价值最高。因此，人们探索了提高其产量的策略，作为传统田间种植的替代方法。在这项工作中，我们开发了生物催化策略，利用重组的与枯草芽孢杆菌几丁质酶A1的几丁质结合结构域融合的阿托品6β-羟化酶（H6H）融合蛋白（ChBD-H6H），将阿托品转化为其产物。催化反应以分批方式进行，通过亲和固定、戊二醛交联以及酶在不同几丁质基质上的吸附-解吸实现H6H构建体的循环利用。作为游离酶使用的ChBD-H6H在3小时和22小时的生物过程中实现了阿托品的完全转化。几丁质颗粒被证明是ChBD-H6H固定化和循环利用最方便的载体。在三轮生物过程（30℃，每轮3小时）中运行的亲和固定化ChBD-H6H，在第一个和第三个反应循环中分别产生了49.8%和22.2%的山莨菪碱以及0.7%和0.3%的东莨菪碱。然而，戊二醛交联在广泛的浓度范围内降低了酶活性。相反，吸附-解吸方法在第一个循环中达到了游离酶的最大转化率，并且在连续循环中比载体结合策略保留了更高的酶活性。吸附-解吸策略允许以简单且经济的方式重复利用酶，同时利用游离酶显示的最大转化活性。这种方法是有效的，因为大肠杆菌裂解物中存在的其他酶不会干扰反应。要点：• 开发了一种用于生产山莨菪碱和东莨菪碱的生物催化系统。• ChP中亲和固定化的ChBD-H6H保留了催化活性。• 通过吸附-解吸策略进行酶循环提高了产物产量。
