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Revista Espanola de Quimioterapia :... Feb 2022
Topics: Adult; Female; Humans; Micrococcaceae; RNA, Ribosomal, 16S; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Urine
PubMed: 34839653
DOI: 10.37201/req/088.2021 -
New Microbes and New Infections Jun 2023
PubMed: 37216022
DOI: 10.1016/j.nmni.2023.101133 -
Enzyme and Microbial Technology Nov 2020The creation of an R-selective ω-amine transaminase (ω-ATA) as biocatalyst is crucial for the asymmetric amination of prochiral ketones to produce sitagliptin...
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/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).
Topics: Amination; Amines; Biocatalysis; Enzyme Stability; Escherichia coli; Ketones; Kinetics; Micrococcaceae; Molecular Dynamics Simulation; Mutagenesis; Protein Engineering; Recombinant Proteins; Sitagliptin Phosphate; Stereoisomerism; Substrate Specificity; Transaminases
PubMed: 33051014
DOI: 10.1016/j.enzmictec.2020.109655