An aminotransferase ω-TAEn was identified from Enhydrobacter aerosaccus. The ω-TAEn was successfully expressed in Escherichia coli and the obtained enzyme showed activity toward β-phenylalanine (β-phe) at optimal conditions. For optically pure (R)-β-phe, 50% yield was observed by kinetic resolution of racemic amino with pyruvate as the amino acceptor. To obtain (S)-βphe, the lipase/ω-TAEn catalytic system was adopted. The ω-TAEn showed strict stereoselectivity to the amino donor. The formation of (S)-β-phe was observed using 3-aminobutyric acid as the amino donor, and (S)-β-phe was obtained by asymmetric synthesis with a yield of 82%.
Background
Fluorinases play a unique role in the production of fluorine-containing organic molecules by biological methods. Whole-cell catalysis is a better choice in the large-scale fermentation processes, and over 60% of industrial biocatalysis uses this method. However, the in vivo catalytic efficiency of fluorinases is stuck with the mass transfer of the substrates.
Results
A gene sequence encoding a protein with fluorinase function was fused to the N-terminal of ice nucleation protein, and the fused fluorinase was expressed in Escherichia coli BL21(DE3) cells. SDS-PAGE and immunofluorescence microscopy were used to demonstrate the surface localization of the fusion protein. The fluorinase displayed on the surface showed good stability while retaining the catalytic activity. The engineered E.coli with surface-displayed fluorinase could be cultured to obtain a larger cell density, which was beneficial for industrial application. And 55% yield of 5′-fluorodeoxyadenosine (5′-FDA) from S-adenosyl-L-methionine (SAM) was achieved by using the whole-cell catalyst.
Conclusions
Here, we created the fluorinase-containing surface display system on E.coli cells for the first time. The fluorinase was successfully displayed on the surface of E.coli and maintained its catalytic activity. The surface display provides a new solution for the industrial application of biological fluorination.
Graphical Abstract
Fluoride plays an important role in the fields of materials and medicine. Compared with chemical synthesis, fluorinases are natural catalysts with more application potential, which provide a green and effective way to obtain organofluorine. However, the application of fluorinases is limited by certain factors, such as the limited number of enzymes and their low activity. In this work, two new fluorinases from Amycolatopsis sp. CA-128772 and Methanosaeta sp. PtaU1.Bin055 were identified by gene mining and named Fam and Fme, respectively. The activities of these two enzymes were reported for the first time, and Fme showed good thermal stability, which was different from the reported fluorinases. In addition, the activity toward natural substrate of Fam was improved by site-directed mutagenesis, the catalytic efficiency (kcat/Km) of the best mutant containing two amino acid substitutions (T72A and S164G) toward the substrate S-adenosyl-L-methionine was improved by 2.2-fold compared to the wild-type. Structural modeling analysis revealed that the main reason for the increased enzyme activity might be the formation of a new substrate channel. Experimental evidence suggests that the substrate channel may indeed play a key role in regulating the function of the fluorinases.
BackgroundFluorinases play a unique role in producing fluorinated organic molecules through a biological method. Whole-cell catalysis is a better choice in the large-scale fermentation processes, and over 60% of industrial biocatalysis uses this method. However, the in vivo catalytic efficiency of fluorinases is stuck with the mass transfer of the substrates.ResultsA gene sequence encoding a protein with fluorinase function was fused to the N-terminal of ice nucleation protein, and the fused protein was expressed in Escherichia coli BL21(DE3) cells. SDS-PAGE and Immunofluorescence microscopy were used to demonstrate the surface localization of the fusion protein. The fluorinase-containing surface display system with improved whole-cell catalytic efficiency and stability showed low growth pressure on the protein expressing host. The conversion rate of 5′-fluorodeoxyadenosine (5′-FDA) from S-adenosyl-L-methionine (SAM) achieved 55%.ConclusionsHere, we created the fluorinase-containing surface display system on E.coli cells for the first time. The fluorinase was successfully displayed on the surface of Escherichia coli and maintained its catalytic activity. The surface display offers a new solution for the industrial application of biological fluorination.
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