Sunday, August 10, 2008
P25
Optical resolution of β-amino acids using microbial enzymes
Hisashi Kawasaki1, Shun'ichi Suzuki2, Yusuke Asami1, Yuki Imabayashi2, Kunihiko Watanabe2, Kunisuke Izawa2, and Tsuyoshi Nakamatsu1. (1) Green and Sustainable Chemistry, Tokyo Denki University, 2-2, Nishiki-cho, Kanda, Chiyoda-ku, Tokyo, 101-8457, Japan, (2) Process Research, Ajinomoto Co., Inc., 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki, 210-8681, Japan
There is increasing interest in enantiomerically pure β-amino acids due to their biological activities and their importance as key compounds in the synthesis of pharmaceuticals. 3-Amino-3-phenylpropionic acid (β-Phe) is one of the most sought-after β-amino acids because it is an important ingredient in the synthesis of taxol. Therefore, microorganisms capable of amidohydrolyzing (R,S)-N-acetyl-β-Phe in an enantiomer-specific manner were screened. A microorganism with (R)-enantiomer-specific amidohydrolyzing activity and another with both (R)-enantiomer- and (S)-enantiomer-specific amidohydrolyzing activities were isolated from soil samples. The former organism was identified as Variovorax sp. and the latter as Burkholderia sp. Enantiomerically pure (R)-β-Phe (>99% ee) and (S)-β-Phe (>99% ee) with a high molar conversion yield were obtained from the racemic substrate by using these organisms.
Three genes, which encode respective enzymes, were cloned and sequenced. The amino acid sequence of the (R)-enantiomer-specific amidohydrolyzing enzyme from Variovorax sp. showed some similarity to a hypothetical protein from Sphingomonas wittichii (19%) and to N,N-dimethylformamidase from Alcaligenes sp. (15%). The (R)-enantiomer-specific enzyme from Burkholderia sp. was homologous to hypothetical proteins (35%) and N,N-dimethylformamidase (31%). The (S)-enantiomer-specific enzyme from Burkholderia sp. was homologous to phosphotriesterase (40%).
The 3 cloned genes were successfully expressed in Escherichia coli, and the 3 enzymes encoded by these 3 genes expressed in a crude extract prepared from E. coli cells were characterized. It was found that besides N-acetyl-β-Phe, the 3 enzymes amidohydrolyzed other N-acetyl-β-amino acid enantiomer-specifically. These data suggest that these enzymes can be used in the production of a variety of enantiomerically pure β-amino acids.
Three genes, which encode respective enzymes, were cloned and sequenced. The amino acid sequence of the (R)-enantiomer-specific amidohydrolyzing enzyme from Variovorax sp. showed some similarity to a hypothetical protein from Sphingomonas wittichii (19%) and to N,N-dimethylformamidase from Alcaligenes sp. (15%). The (R)-enantiomer-specific enzyme from Burkholderia sp. was homologous to hypothetical proteins (35%) and N,N-dimethylformamidase (31%). The (S)-enantiomer-specific enzyme from Burkholderia sp. was homologous to phosphotriesterase (40%).
The 3 cloned genes were successfully expressed in Escherichia coli, and the 3 enzymes encoded by these 3 genes expressed in a crude extract prepared from E. coli cells were characterized. It was found that besides N-acetyl-β-Phe, the 3 enzymes amidohydrolyzed other N-acetyl-β-amino acid enantiomer-specifically. These data suggest that these enzymes can be used in the production of a variety of enantiomerically pure β-amino acids.
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