Discovery of carboxylic acid reductase (CAR) from Thermothelomyces thermophila and its evaluation for vanillin synthesis

香兰素 化学 生物转化 香兰素酸 基质(水族馆) 产量(工程) 还原酶 生物化学 生物催化 肉桂酸 立体化学 有机化学 发酵 催化作用 生物 反应机理 生态学 材料科学 冶金
作者
Melissa Horvat,Giuseppe Fiume,Susanne Fritsche,Margit Winkler
出处
期刊:Journal of Biotechnology [Elsevier BV]
卷期号:304: 44-51 被引量:26
标识
DOI:10.1016/j.jbiotec.2019.08.007
摘要

A novel type III fungal CAR was identified from the organism Thermothelomyces thermophila. High expression levels were observed in E. coli using the pETDuet-1 plasmid system in combination with an autoinduction protocol. A broad substrate scope ranging from aromatic to aliphatic carboxylic acids was tested and TtCAR showed activity for all substrates. High specific activities for aromatic substrates and short chain aliphatic substrates were observed, comparable to those of NcCAR, the first type III fungal CAR. TtCAR's pH and temperature optima were at 6.5 and 30 °C, respectively. Up to 20% (v/v) cosolvents did not show a decrease in specific activity of TtCAR using (E)-cinnamic acid as a substrate. Its half-life at 40 °C was determined to be 8.25 h and its melting temperature (Tm) was 56 °C. In vitro reactions with TtCAR reduced 95.2% of 10 mM vanillic acid, which correlated to a titer of 1.4 g L−1 of vanillin. The space time yield of 0.029 g L-1 h-1 indicates that further improvements would be necessary for an industrially relevant application. This would be especially important when competing against de novo synthesis of bio vanillin by microbial strains producing >30 g L-1. In de novo and in vivo biosynthesis systems, by-products are fairly common. By contrast, we were pleased to observe less than 0.7% of vanillyl alcohol formed, making the cell-free acid reduction in the envisaged sequential two-step bioconversion from eugenol to vanillin very attractive.
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