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11.
Ciprian G. Crismaru Gjalt G. Wybenga Wiktor Szymanski Hein J. Wijma Bian Wu Sebastian Bartsch Stefaan de Wildeman Gerrit J. Poelarends Ben L. Feringa Bauke W. Dijkstra Dick B. Janssen 《Applied and environmental microbiology》2013,79(1):185-195
By selective enrichment, we isolated a bacterium that can use β-phenylalanine as a sole nitrogen source. It was identified by 16S rRNA gene sequencing as a strain of Variovorax paradoxus. Enzyme assays revealed an aminotransferase activity. Partial genome sequencing and screening of a cosmid DNA library resulted in the identification of a 1,302-bp aminotransferase gene, which encodes a 46,416-Da protein. The gene was cloned and overexpressed in Escherichia coli. The recombinant enzyme was purified and showed a specific activity of 17.5 U mg−1 for (S)-β-phenylalanine at 30°C and 33 U mg−1 at the optimum temperature of 55°C. The β-specific aminotransferase exhibits a broad substrate range, accepting ortho-, meta-, and para-substituted β-phenylalanine derivatives as amino donors and 2-oxoglutarate and pyruvate as amino acceptors. The enzyme is highly enantioselective toward (S)-β-phenylalanine (enantioselectivity [E], >100) and derivatives thereof with different substituents on the phenyl ring, allowing the kinetic resolution of various racemic β-amino acids to yield (R)-β-amino acids with >95% enantiomeric excess (ee). The crystal structures of the holoenzyme and of the enzyme in complex with the inhibitor 2-aminooxyacetate revealed structural similarity to the β-phenylalanine aminotransferase from Mesorhizobium sp. strain LUK. The crystal structure was used to rationalize the stereo- and regioselectivity of V. paradoxus aminotransferase and to define a sequence motif with which new aromatic β-amino acid-converting aminotransferases may be identified. 相似文献
12.
An asymmetric synthesis route towards (3S,3'S)-(M,M)-(E)-(+)-1,1',2, 2',3,3',4,4'-octahydro-3,3',7,7'-tetramethyl-4,4'-biphenanthrylidene was developed using the Evans procedure as a key step. The absolute configurations of the title compound and of its parent ketone were determined by CD spectroscopy and could be correlated with the stereochemical results of the asymmetric alkylation. Furthermore, a comparison was made with the known (3R,3'R)-(P,P)-(E)-(-)-1,1',2,2', 3,3',4,4'-octahydro-3,3',7,7'-dimethyl-4,4'-biphenanthrylidene. Finally, the X-ray crystallographic analysis of (3S,3'S)-(M, M)-(E)-(+)-1,1',2,2',3,3',4,4'-octahydro-3,3',7,7'-tetramethyl-4, 4'-biphenanthrylidene is presented. 相似文献
13.
Raj H Puthan Veetil V Szymanski W Dekker FJ Quax WJ Feringa BL Janssen DB Poelarends GJ 《Applied microbiology and biotechnology》2012,94(2):385-397
Methylaspartate ammonia lyase (MAL; EC 4.3.1.2) catalyzes the reversible addition of ammonia to mesaconate to give (2S,3S)-3-methylaspartate and (2S,3R)-3-methylaspartate as products. MAL is of considerable biocatalytic interest because of its potential use for the asymmetric
synthesis of substituted aspartic acids, which are important building blocks for synthetic enzymes, peptides, chemicals, and
pharmaceuticals. Here, we have cloned the gene encoding MAL from the thermophilic bacterium Carboxydothermus hydrogenoformans Z-2901. The enzyme (named Ch-MAL) was overproduced in Escherichia coli and purified to homogeneity by immobilized metal affinity chromatography. Ch-MAL is a dimer in solution, consisting of two identical subunits (∼49 kDa each), and requires Mg2+ and K+ ions for maximum activity. The optimum pH and temperature for the deamination of (2S,3S)-3-methylaspartic acid are 9.0 and 70°C (k
cat = 78 s−1 and K
m = 16 mM). Heat inactivation assays showed that Ch-MAL is stable at 50°C for >4 h, which is the highest thermal stability observed among known MALs. Ch-MAL accepts fumarate, mesaconate, ethylfumarate, and propylfumarate as substrates in the ammonia addition reaction. The enzyme
also processes methylamine, ethylamine, hydrazine, hydroxylamine, and methoxylamine as nucleophiles that can replace ammonia
in the addition to mesaconate, resulting in the corresponding N-substituted methylaspartic acids with excellent diastereomeric excess (>98% de). This newly identified thermostable MAL appears
to be a potentially attractive biocatalyst for the stereoselective synthesis of aspartic acid derivatives on large (industrial)
scale. 相似文献
14.
Marten Hornsveld Femke M. Feringa Lenno Krenning Jeroen van den Berg Lydia M.M. Smits Nguyen B.T. Nguyen Maria J. Rodríguez-Colman Tobias B. Dansen René H. Medema Boudewijn M.T. Burgering 《Cell reports》2021,34(4):108675
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15.
This paper describes the first examples of asymmetric induction in the oxidative coupling of phenols using chiral oxidants. When chiral cupric-amine complexes were used as oxidants, low asymmetric induction was achieved in the coupling of naphthols. The formation of optically active d-dehydrogriseofulvin and l-Licarin A using the cupric-l-a-phenylethylamine complex perhaps mimics the action of copper-containing enzymes known to catalyze phenol coupling. 相似文献
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