TY - JOUR
T1 - A high-throughput and generic assay method for the determination of substrate specificities of thermophilic α-aminotransferases
AU - Sawai, Toshiya
AU - Koma, Daisuke
AU - Hara, Ryotaro
AU - Kino, Kuniki
AU - Harayama, Shigeaki
N1 - Funding Information:
We thank Professor Isao Shimizu, Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, for providing us keto-acids, and Ms. Ayako Hashimoto for technical assistance. This study was supported in part by Waseda University Grant for Special Research Projects (2004A-141).
PY - 2007/10
Y1 - 2007/10
N2 - For the determination of substrate specificities of thermophilic α-aminotransferases (AATs), a novel high-throughput assay method was developed. In this method, a thermophilic ω-aminotransferase (OAT) and a thermophilic aldehyde dehydrogenase (ALDH) are coupled to the AAT reaction. Glutamic acid is used as an amino group donor for the AAT reaction yielding 2-oxoglutalic acid. 2-Oxoglutalic acid produced by the AAT reaction is used as an amino group acceptor in the OAT reaction regenerating glutamic acid. The amino group donor of the OAT reaction is 5-aminopentanoic acid yielding pentanedioic acid semialdehyde which is oxidized by ALDH to pentanedioic acid with concomitant reduction of NADP+ to NADPH. NADPH thus produced then reduces colorless tetrazolium salt into colored formazan. To construct such a reaction system, the genes for a thermophilic AAT, a thermophilic OAT and a thermophilic ALDH were cloned and expressed in Escherichia coli. These enzymes were subsequently purified and used to determine the activities of AAT for the synthesis of unnatural amino acids. This method allowed the clear detection of the AAT activities as it measures the increase in the absorbance on a low background absorbance reading.
AB - For the determination of substrate specificities of thermophilic α-aminotransferases (AATs), a novel high-throughput assay method was developed. In this method, a thermophilic ω-aminotransferase (OAT) and a thermophilic aldehyde dehydrogenase (ALDH) are coupled to the AAT reaction. Glutamic acid is used as an amino group donor for the AAT reaction yielding 2-oxoglutalic acid. 2-Oxoglutalic acid produced by the AAT reaction is used as an amino group acceptor in the OAT reaction regenerating glutamic acid. The amino group donor of the OAT reaction is 5-aminopentanoic acid yielding pentanedioic acid semialdehyde which is oxidized by ALDH to pentanedioic acid with concomitant reduction of NADP+ to NADPH. NADPH thus produced then reduces colorless tetrazolium salt into colored formazan. To construct such a reaction system, the genes for a thermophilic AAT, a thermophilic OAT and a thermophilic ALDH were cloned and expressed in Escherichia coli. These enzymes were subsequently purified and used to determine the activities of AAT for the synthesis of unnatural amino acids. This method allowed the clear detection of the AAT activities as it measures the increase in the absorbance on a low background absorbance reading.
KW - Aldehyde dehydrogenase
KW - Biocatalysis
KW - Enzyme assay
KW - Thermophilic enzyme
KW - Unnatural amino acids
KW - α-aminotransferase
KW - ω-aminotransferase
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U2 - 10.1016/j.mimet.2007.07.006
DO - 10.1016/j.mimet.2007.07.006
M3 - Article
C2 - 17719665
AN - SCOPUS:34548832233
VL - 71
SP - 32
EP - 38
JO - Journal of Microbiological Methods
JF - Journal of Microbiological Methods
SN - 0167-7012
IS - 1
ER -