Journal of Biotechnology ( IF 4.1 ) Pub Date : 2024-01-22 , DOI: 10.1016/j.jbiotec.2024.01.008
Dominic Goj 1 , Stella Ebner 1 , Melissa Horvat 1 , Simon Arhar 2 , Ludmila Martínková 3 , Margit Winkler 4
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Mycobacterium marinum CAR (MmCAR) is one of the most widely used CARs as the key enzyme for the synthesis of aldehydes, alcohols and further products from the respective carboxylic acids. Herein, we describe the first functionally secreted 131 kDa CAR and its isolated A-domain using Komagataella phaffii and a methanol-free constitutive expression strategy. Precipitated and lyophilized MmCAR (500 µg) was isolated from the culture supernatant and showed no decrease in activity for piperonylic acid (80% conversion), even when stored for up to 3 weeks at 4°C. Lyophilized MmCAR precipitate gave 48% yield of E/Z-nonanal-4-nitrobenzoyloxime from the reduction of nonanoic acid and in-situ derivatization with O-4-nitrobenzoyl-hydroxylamine. Furthermore, K. phaffii could successfully secrete the MmCAR adenylation domain. Its activity was confirmed by the amidation of benzoic acid with n-hexylamine. Neither enzyme variant was glycosylated by the yeast. In summary, functional CAR can be secreted by K. phaffii and used for cell free conversion of carboxylic acids to various products.
中文翻译:
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用分泌的羧酸还原酶对羧酸进行无细胞还原
马里纳分枝杆菌CAR (MmCAR) 是使用最广泛的 CAR 之一,是用各自的羧酸合成醛、醇和其他产物的关键酶。在此,我们描述了第一个功能分泌的 131 kDa CAR 及其分离的 A 结构域,使用 Komagataella phaffii 和无甲醇组成型表达策略。从培养上清液中分离沉淀和冻干的 MmCAR (500 μg),即使在 4°C 下储存长达 3 周,对胡椒酸的活性也没有降低(转化率为 80%)。 冻干的 MmCAR 沉淀物通过还原壬酸和用 O-4-硝基苯甲酰羟胺原位衍生化,得到 E/Z-壬醛-4-硝基苯甲酰肟的产率为 48%。此外,K. phaffii 可以成功分泌 MmCAR 腺苷酸化结构域。其活性通过苯甲酸与 n-己胺的酰胺化得到证实。两种酶变体均未被酵母糖基化。总之,功能性 CAR 可由 K. phaffii 分泌,并用于将羧酸无细胞转化为各种产物。