Abstract
In order to provide more alternative epoxide hydrolases for industrial production, a novel cDNA gene Rpeh-encoding epoxide hydrolase (RpEH) of Rhodotorula paludigena JNU001 identified by 26S rDNA sequence analysis was amplified by RT-PCR. The open-reading frame (ORF) of Rpeh was 1236 bp encoding RpEH of 411 amino acids and was heterologously expressed in Escherichia coli BL21(DE3). The substrate spectrum of expressed RpEH showed that the transformant E. coli/Rpeh had excellent enantioselectivity to 2a, 3a, and 5a–10a, among which E. coli/Rpeh had the highest activity (2473 U/g wet cells) and wonderful enantioselectivity (E = 101) for 8a, and its regioselectivity coefficients, αR and βS, toward (R)- and (S)-8a were 99.7 and 83.2%, respectively. Using only 10 mg wet cells/mL of E. coli/Rpeh, the near-perfect kinetic resolution of rac-8a at a high concentration (1000 mM) was achieved within 2.5 h, giving (R)-8a with more than 99% enantiomeric excess (ees) and 46.7% yield and producing (S)-8b with 93.2% eep and 51.4% yield with high space-time yield (STY) for (R)-8a and (S)-8b were 30.6 and 37.3 g/L/h.
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Acknowledgments
The authors are grateful to Prof. Xianzhang Wu (School of Biotechnology, Jiangnan University, Jiangsu, China) for providing technical assistance.
Funding
This work was financially supported by the China Postdoctoral Science Foundation (No. 2018M630522) and the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX18_1804).
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Xu, XF., Hu, D., Hu, BC. et al. Near-perfect kinetic resolution of o-methylphenyl glycidyl ether by RpEH, a novel epoxide hydrolase from Rhodotorula paludigena JNU001 with high stereoselectivity. Appl Microbiol Biotechnol 104, 6199–6210 (2020). https://doi.org/10.1007/s00253-020-10694-w
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DOI: https://doi.org/10.1007/s00253-020-10694-w