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Creation of a robust and R-selective ω-amine transaminase for the asymmetric synthesis of sitagliptin intermediate on a kilogram scale.
- Source :
-
Enzyme and microbial technology [Enzyme Microb Technol] 2020 Nov; Vol. 141, pp. 109655. Date of Electronic Publication: 2020 Sep 07. - Publication Year :
- 2020
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Abstract
- The creation of an R-selective ω-amine transaminase (ω-ATA) as biocatalyst is crucial for the asymmetric amination of prochiral ketones to produce sitagliptin intermediates because rare ω-ATAs are R-selective in nature and most of them suffer from poor stability and low activity toward bulky prochiral ketones. Here, the gene of an R-selective ω-ATA was cloned from Arthrobacter cumminsii ZJUT212 (AcATA) and expressed in Escherichia coli. The best variants (M1 + M122H and M1+T134 G) were obtained using a semi-rational protein design after screening. These variants not only exhibited improved activity and substrate affinity but also enhanced stability in aqueous phase containing 20 % dimethyl sulfoxide. The conversion of asymmetric amination on 50 g/L pro-sitagliptin ketone PTfpB (1-[1-piperidinyl]-4-[2,4,5-trifluorophenyl]-1,3-butanedione) achieved 92 %, with an extremely high e.e. of >99 %, using 2 g <subscript>DCW</subscript> /L E. coli cells harboring M1 + M122H as biocatalyst. In the kilogram-scale experiment, approximately 40 kg of (R)-APTfpB (e.e. >99 %) was produced within 30 h when 50 kg PTfpB was used as the substrate. Furthermore, the space-time yield reached ≈32 g/(L·d).<br /> (Copyright © 2020 Elsevier Inc. All rights reserved.)
- Subjects :
- Amination
Amines chemistry
Biocatalysis
Enzyme Stability
Escherichia coli genetics
Ketones chemistry
Ketones metabolism
Kinetics
Micrococcaceae enzymology
Micrococcaceae genetics
Molecular Dynamics Simulation
Mutagenesis
Protein Engineering
Recombinant Proteins chemistry
Recombinant Proteins genetics
Recombinant Proteins metabolism
Sitagliptin Phosphate chemistry
Stereoisomerism
Substrate Specificity
Transaminases chemistry
Transaminases genetics
Amines metabolism
Sitagliptin Phosphate metabolism
Transaminases metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1879-0909
- Volume :
- 141
- Database :
- MEDLINE
- Journal :
- Enzyme and microbial technology
- Publication Type :
- Academic Journal
- Accession number :
- 33051014
- Full Text :
- https://doi.org/10.1016/j.enzmictec.2020.109655