- Biocatalytic Racemization Employing TeSADH: Substrate Scope and Organic Solvent Compatibility for Dynamic Kinetic Resolution
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Racemization in combination with a kinetic resolution is the base for a dynamic kinetic resolution (DKR). Biocatalytic racemization was successfully performed for a broad scope of sec-alcohols by employing a single alcohol dehydrogenase (ADH) variant from Thermoanaerobacter pseudoethanolicus (formerly T. ethanolicus; TeSADH W110A I86A C295A). The catalyst employed as a lyophilized whole cell preparation or cell free extract, which tolerated various non-water miscible organic solvents under micro-aqueous or two-phase conditions, whereby cyclohexane and n-hexane suited best. Various concepts for combining the enzymatic racemization with an enzymatic kinetic resolution to achieve overall a bis-enzymatic DKR were evaluated. A proof of concept showed a successful DKR with racemization in aqueous phase combined with acylation in the organic phase.
- Pop?oński, Jaros?aw,Reiter, Tamara,Kroutil, Wolfgang
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p. 763 - 768
(2018/02/27)
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- Fungal mediated kinetic resolution of racemic acetates to (R)-alcohols using Fusarium proliferatum
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Fungal mediated kinetic resolution of seven acyclic/aromatic acetates was achieved using Fusarium proliferatum to furnish (R)-alcohols in high enantiomeric excess (>95%). The kinetic resolution was established as one-pot two-step de-esterification/oxidation biocatalytic process. Further, the preparative scale synthesis of (R)-(+)-1-phenylethanol was accomplished through de-esterification/oxidation of (±)-1-phenylethyl acetate using the whole cell of F. proliferatum NCIM 1105.
- Jadhav, Dipesh D.,Patil, Harshal S.,Chaya, Patil S.,Thulasiram, Hirekodathakallu V.
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p. 4563 - 4567
(2016/09/23)
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- Screening, Molecular Cloning, and Biochemical Characterization of an Alcohol Dehydrogenase from Pichia pastoris Useful for the Kinetic Resolution of a Racemic β-Hydroxy-β-trifluoromethyl Ketone
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The stereoselective synthesis of chiral 1,3-diols with the aid of biocatalysts is an attractive tool in organic chemistry. Besides the reduction of diketones, an alternative approach consists of the stereoselective reduction of β-hydroxy ketones (aldols). Thus, we screened for an alcohol dehydrogenase (ADH) that would selectively reduce a β-hydroxy-β-trifluoromethyl ketone. One potential starting material for this process is readily available by aldol addition of acetone to 2,2,2-trifluoroacetophenone. Over 200 strains were screened, and only a few yeast strains showed stereoselective reduction activities. The enzyme responsible for the reduction of the β-hydroxy-β-trifluoromethyl ketone was identified after purification and subsequent MALDI-TOF mass spectrometric analysis. As a result, a new NADP+-dependent ADH from Pichia pastoris (PPADH) was identified and confirmed to be capable of stereospecific and diastereoselective reduction of the β-hydroxy-β-trifluoromethyl ketone to its corresponding 1,3-diol. The gene encoding PPADH was cloned and heterologously expressed in Escherichia coli BL21(DE3). To determine the influence of an N- or C-terminal His-tag fusion, three different recombinant plasmids were constructed. Interestingly, the variant with the N-terminal His-tag showed the highest activity; consequently, this variant was purified and characterized. Kinetic parameters and the dependency of activity on pH and temperature were determined. PPADH shows a substrate preference for the reduction of linear and branched aliphatic aldehydes. Surprisingly, the enzyme shows no comparable activity towards ketones other than the β-hydroxy-β-trifluoromethyl ketone.
- Bulut, Dalia,Hummel, Werner,Gr?ger, Harald,Duangdee, Nongnaphat,Berkessel, Albrecht
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p. 1349 - 1358
(2016/12/24)
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- ALKANE OXIDATION BY MODIFIED HYDROXYLASES
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This invention relates to modified hydroxylases. The invention further relates to cells expressing such modified hydroxylases and methods of producing hydroxylated alkanes by contacting a suitable substrate with such cells.
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Paragraph 0323; 0324
(2016/02/16)
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- Discrimination of the prochiral hydrogens at the C-2 position of n-alkanes by the methane/ammonia monooxygenase family proteins
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The selectivity of ammonia monooxygenase from Nitrosomonas europaea (AMO-Ne) for the oxidation of C4-C8n-alkanes to the corresponding alcohol isomers was examined to show the ability of AMO-Ne to recognize the n-alkane orientation within the catalytic site. AMO-Ne in whole cells produces 1- and 2-alcohols from C4-C8n-alkanes, and the regioselectivity is dependent on the length of the carbon chain. 2-Alcohols produced from C4-C7n-alkanes were predominantly either the R- or S-enantiomers, while 2-octanol produced from n-octane was racemic. These results indicate that AMO-Ne can discriminate between the prochiral hydrogens at the C-2 position, with the degree of discrimination varying according to the n-alkane. Compared to the particulate methane monooxygenase (pMMO) of Methylococcus capsulatus (Bath) and that of Methylosinus trichosporium OB3b, AMO-Ne showed a distinct ability to discriminate between the orientation of n-butane and n-pentane in the catalytic site.
- Miyaji, Akimitsu,Miyoshi, Teppei,Motokura, Ken,Baba, Toshihide
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p. 8261 - 8270
(2015/08/03)
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- A novel P450-based biocatalyst for the selective production of chiral 2-alkanols
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A P450 monooxygenase from Nocardia farcinica (CYP154A8) catalyses the stereo- and regioselective hydroxylation of n-alkanes, still a challenging task in chemical catalysis. In a biphasic reaction system, the regioselectivity for the C2-position of C7-C9 alkanes was over 90%. The enzyme showed strict S-selectivity for all tested substrates, with enantiomeric excess (ee) of up to 91%. This journal is the Partner Organisations 2014.
- Von Bühler, Clemens J.,Urlacher, Vlada B.
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supporting information
p. 4089 - 4091
(2014/04/03)
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- In vitro double oxidation of n-heptane with direct cofactor regeneration
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A novel concept for the direct oxidation of cycloalkanes to the corresponding cyclic ketones in a one-pot synthesis in water with molecular oxygen as sole oxidizing agent was reported recently. Based on this concept we have developed a new strategy for the double oxidation of n-heptane to enable a biocatalytic resolution for the direct synthesis of heptanone and (R)-heptanols in a one-pot reaction. The bicatalytic cascade employs an NADH driven P450 BM3 monooxygenase variant (WTNADH, 19A12NADH or CM1 NADH) and an (S)-enantioselective alcohol dehydrogenase (RE-ADH). In the initial step n-heptane is hydroxylated under consumption of NADH to produce (R/S)-heptanol. In the second oxidation step the (S)-heptanol enantiomers are transformed to the corresponding ketones, reducing and thereby regenerating the cofactor. Characterization of initial hydroxylation step revealed high turnover frequencies (TOF) of up to 600 min-1, as well as high coupling efficiencies using NADH as cofactor (up to 44%). In the cascade reaction a nearly 2-fold improved product formation was achieved, compared to the single hydroxylation reaction. The total product concentration reached 1.1 mM, corresponding to a total turnover number (TTN) of 2500. Implementation of an additional cofactor regeneration system (D-glucose/glucose dehydrogenase) enabled a further enhancement in product formation with a total product concentration of 1.8 mM and a TTN of 3500. Copyright
- Mueller, Christina A.,Akkapurathu, Beneeta,Winkler, Till,Staudt, Svenja,Hummel, Werner,Groeger, Harald,Schwaneberg, Ulrich
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supporting information
p. 1787 - 1798
(2013/07/19)
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- A thermodynamic study of the ketoreductase-catalyzed reduction of 2-alkanones in non-aqueous solvents
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Equilibrium constants K have been measured for the reactions (2-alkanone + 2-propanol = 2-alkanol + acetone), where 2-alkanone = 2-butanone, 2-pentanone, 2-hexanone, 2-heptanone, and 2-octanone and 2-alkanol = 2-butanol, 2-pentanol, 2-hexanol, 2-heptanol, and 2-octanol. The solvents used were n-hexane, toluene, methyl tert-butyl ether (MTBE), and supercritical carbon dioxide SCCO 2 (pressure P - 10.0 MPa). The temperature range was T - (288.15 to 308.27) K. Chiral analysis of the reaction products showed that the enzyme used in this study was stereoselective for the 2-octanone reaction system, i.e. only (S)-(+)-2-octanol was formed. For the reactions involving butanone, pentanone, and hexanone, the products were racemic mixtures of the respective (S)-(+)-2-alkanol and the (R)-(-)-2-alkanol. Chiral analysis showed that for the 2-heptanone reaction system, the 2-alkanol product was a mixture of (S)-(+)-2-heptanol and (R)-(-)-2-heptanol, at the respective mole fractions of 0.95 and 0.05. The equilibrium constant for the reaction system involving 2-butanone carried out in n-hexane was measured at several temperatures. For this reaction, the values for the thermodynamic reaction quantities at T= 298. 15 K are: K= 0.838±0.013; the standard molar Gibbs free energy change ΔrgHm° = (0.44±0.040) kJ · mol-1; the standard molar enthalpy change ΔrgHm° = -(1.2±1.7) kJ mol-1, and the standard molar entropy change ΔrgHm° = -(5.5±5.7) J K-1 mol-1. Interestingly, inspection of the values of the equilibrium constants for these reactions carried out in n-hexane, toluene, MTBE, and SCCO2 shows that these values are comparable and have little dependence on the solvent used to carry out the reaction. The values of the equilibrium constants decrease monotonically with increasing value of the number of carbons Nc and trend towards a limiting value of ≈0.30 for Nc > 8. Published by Elsevier Ltd.
- Tewari, Yadu B.,Schantz, Michele M.,Phinney, Karen W.,Rozzell, J. David
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- Biocatalytic asymmetric hydrogen transfer employing Rhodococcus ruber DSM 44541
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Nonracemic sec-alcohols of opposite absolute configuration were obtained either by asymmetric reduction of the corresponding ketone using 2-propanol as hydrogen donor or by enantioselective oxidation through kinetic resolution of the rac-alcohol using acetone as hydrogen acceptor employing whole lyophilized cells of Rhodococcus ruber DSM 44541. The microbial oxidation/reduction system exhibits not only excellent stereo- and enantioselectivity but also a broad substrate spectrum. Due to the exceptional tolerance of the biocatalyst toward elevated concentrations of organic materials (solvents, substrates and cosubstrates), the process is highly efficient. The simple preparation of the biocatalyst and its ease of handling turns this system into a versatile tool for organic synthesis.
- Stampfer, Wolfgang,Kosjek, Birgit,Faber, Kurt,Kroutil, Wolfgang
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p. 402 - 406
(2007/10/03)
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- Deracemization of (±)-cis-2,3-epoxyheptane via enantioconvergent biocatalytic hydrolysis using Nocardia EH1-epoxide hydrolase
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Biocatalytic hydrolysis of (±)-cis-2,3-epoxyheptane using lyophilized bacterial cells of Nocardia EH1 gave (2R,3R)-heptane-2,3-dil as the sole product in 79% yield and 91% e.e. at 100% conversion. The pathway of this deracemization was elucidated by 18OH2-labelling experiments using a partially purified epoxide hydrolase preparation and was shown to proceed in an enantioconvergent manner. Thus both substrate enantiomers were hydrolyzed via attack of [OH-] at the respective (S)-configurated C-atom with concomitant inversion of configuration.
- Kroutil, Wolfgang,Mischitz, Martin,Plachota, Peter,Faber, Kurt
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p. 8379 - 8382
(2007/10/03)
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- Efficient Stereocontrolled Total Syntheses of Racemic and Natural Brefeldin-A
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Brefeldin-A, an antiobiotic fungal metabolite, has been obtained in both racemic and natural forms through a direct approach that employs norbornenone and 6-heptyn-2-ol as the basic starting materials.
- Drian, Claude Le,Greene, Andrew E.
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p. 5473 - 5483
(2007/10/02)
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