- Kinetic resolution of 3-fluoroalanine using a fusion protein of D-amino acid oxidase with Vitroscilla hemoglobin
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In this study, a fusion protein (VHb-DAAO) of D-amino acid oxidase (DAAO) with Vitreoscilla hemoglobin (VHb) was functionally expressed in Escherichia coli and purified. The kcat value VHb-DAAO (47.1 s-1) towards rac-3-flouroalanine was about 2-fold higher than that of DAAO (21.9 s -1). rac-3-Flouroalanine (500 mm) was kinetically resolved into (R)-3-fluoroalanine with high enatiomeric excess (>99%) by VHb-DAAO with about 52% conversion.
- Seo, Young-Man,Khang, Yong-Ho,Yun, Hyungdon
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p. 820 - 822
(2011/11/30)
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- Thermodynamics and kinetic aspects involved in the enzymatic resolution of (R,S)-3-fluoroalanine in a coupled system of redox reactions catalyzed by dehydrogenases
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Two systems of redox enzymatic reactions were tested, looking forward to the preparation of (S)-3-fluoroalanine, a potent antibiotic, by kinetic resolution of rac-3-fluoroalanine. This starting material was the main substrate for the deaminative oxidation reaction catalyzed by L-alanine dehydrogenase (L-AlaDH) in the presence of NAD+. One system was formed by coupling this reaction (main reaction) to the reduction of 3-fluoropyruvate (a cascade system) produced in the main reaction catalyzed by L-lactate dehydrogenase (L-LDH) in the presence of NADH, also formed in the main reaction. This system, that was able to achieve 92% of conversion, allows the accumulation of NH 4+, one of the secondary products of the main reaction. The other coupled redox system involved the coupling to the L-AlaDH reaction to the aminative reduction reaction of α-ketoglutarate in the presence of NADH and NH4+ (both side products of the main reaction) catalyzed by L-glutamate dehydrogenase (L-GluDH), that allows accumulation of 3-fluoropyruvate. With this system, the extent of the reaction in the coupled system was only 22%. This big difference in the efficiency of both systems was identified as being the result of a different potency of the products that accumulates in both systems, acting as inhibitors of L-AlaDH. It was demonstrated that 3-fluoropyruvate is a much stronger inhibitor of L-AlaDH than NH4+. This fact, and not thermodynamic considerations, explains the results obtained with both systems.
- Goncalves, Luciana P. B.,Antunes,Oestreicher, Enrique G.
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p. 673 - 677
(2012/12/22)
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