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. 2020 Apr 2;5(14):7745-7750.
doi: 10.1021/acsomega.9b03416. eCollection 2020 Apr 14.

An Aminotransferase from Enhydrobacter aerosaccus to Obtain Optically Pure β-Phenylalanine

Xinming Feng  1   2 , Jing Guo  1 , Rubing Zhang  1 , Wei Liu  1 , Yujin Cao  1 , Mo Xian  1 , Huizhou Liu  1
Affiliations

An Aminotransferase from Enhydrobacter aerosaccus to Obtain Optically Pure β-Phenylalanine

Xinming Feng et al. ACS Omega. .

Abstract

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)-β-phe, 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%.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Effect of (A) temperature and (B) pH on the enzyme activity. The reaction was carried out in 100 mM Tris–HCl buffer containing 0.1 mM PLP, 0.025 mg/mL ω-TAEn, 10% (v/v) organic solvent, 20 mM rac-β-phe, and 20 mM pyruvate.
Figure 2
Figure 2
Effect of (A) organic solvent and (B) metal ion on enzyme activity. (A) The reaction was carried out in 100 mM Tris–HCl buffer containing 0.1 mM PLP, 0.025 mg/mL ω-TAEn, 20 mM rac-β-phe, and 20 mM pyruvate at 50 °C for 50 min. (B) The enzymatic reactions of ω-TA were performed in the presence of a metal ion (1 mM) for 50 min.
Figure 3
Figure 3
Enzymatic thermal stability. The enzyme was incubated at a specific temperature for 10 min in Tris buffer (100 mM, pH 8) before the reaction.
Figure 4
Figure 4
Michaelis–Menten plot of the reaction of ω-TAEn on rac-β-phe. The enzyme reaction for Michaelis–Menten plot was carried out in a reaction volume of 1 mL containing 10 mM pyruvate, rac-β-phe (0–4 mM), enzyme (0.025 mg/mL), and 100 mM Tris–HCl buffer (pH 8.0), and the mixture was incubated at 37 °C for 5 min.
Figure 5
Figure 5
Reaction profile for ω-TAEn catalyzed by chiral resolution of rac-β-phe. The enzyme reaction was carried out in a reaction volume of 1 mL of Tris–HCl buffer (100 mM, pH 8.0) containing pyruvate (20 mM), rac-β-phe (10 mM), enzyme (0.045 mg/mL), and DMSO (10%, v/v), and the mixture was incubated at 50 °C.
Figure 6
Figure 6
Asymmetric synthesis of (S)-β-phe by ω-TAEn. The reaction was carried out in Tris–HCl buffer (100 mM, pH 8) containing ethyl phenylpyruvate (10 mM), PLP (0.1 mM), DMSO (5%, v/v), dl-3-aminobutyric acid (80 mM), lipase (1 mg/mL), and ω-TA (0.045 mg/mL) at 37 °C.
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