Chiral amines and unnatural amino acids have been widely used in diverse sectors such as pharmaceutical, chemical, cosmetic, food and agricultural industries. ω-Transaminase (ω-TA), due to its advantages such as high stereoselectivity, rapid reaction rate, broad substrate specificity, and no requirement for external cofactor, has increasingly been used for the production of chiral amines and unnatural amino acids.
Recently, the researchers at Laboratory of Biocatalysis and Green chemistry at Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences identified a new (S)-selective ω-TA ((S)-TA) and five new (R)-selective ω-TAs ((R)-TAs) using in silico method. These enzymes showed unique catalytic properties and may serve as useful catalysts for the production of optically pure amines and unnatural amino acids.
The (S)-TA HBV from Burkholderia vietnamiensis was found to have a broad substrate profile. Especially, it catalyzed the amino transfer of (S)-1-methylbenzylamine to ethyl acetoacetate, ethyl propionylacetate and levulinic acid to afford the corresponding (S)-configurated amino acids or esters with 99% ee. It was the first report that optically pure (S)-3-aminopentanoic acid ethyl ester and (S)-4-aminovaleric acid were produced with transaminase as catalyst. In the kinetic resolution of racemic amines using HBV as catalyst and glyoxylate as amino acceptor, optically pure amines such as (R)-1-methylbenzylamine (conv= 50%, ee>99%), (R)-1-phenylpropylamine (conv=50%, ee>99%), (R)-2-amino-4-phenylbutane (conv=53%, ee>99%) and (R)-1-aminotetraline (conv=51%, ee>99%), were obtained.
The five new (R)-TAs (HFO, HNH, HTA, HTR and HTV) were from Fusarium oxysporum, Nectria haematococca, Trichoderma atroviride, Trichoderma reesei and Trichoderma virens, respectively. The (R)-TAs showed activity toward a series of keto acids and keto esters including pyruvic acid, 2-oxobutyric acid, 2-ketovaleric acid, 3-mercaptopyruvate, ethyl acetoacetate, ethyl propionylacetate and levulinic acid. All of the corresponding products were unnatural amino acids or esters (ee>99%). For example, both pyruvic acid and 2-oxobutyric acid had high conversions even at a high concentration of 50 mM. The ee values of all the corresponding products exceeded 99% (R). The feasibility of (R)-TAs (HFO and HTR) for the kinetic resolution of racemic amines was tested with pyruvate (40 mM) as amino accepter. All the racemic amines (50 mM) tested were successfully resolved and optically pure (S)-enantiomers (ee>99%) were obtained with good conversions. The lyophilized crude cell-free extract of HFO showed high thermostability in organic solvent (ethyl acetate), 95% catalytic activity remained after incubation at 60 °C for 5 h. The lyophilized enzyme powder (HFO) can efficiently catalyze the asymmetric amination in organic solvent. For example, in water saturated ethyl acetate containing (R)-1-methylbenzylamine (100 mM) and lyophilized enzyme powder (5 mg/mL), the conversion of pyruvate ethyl ester (100 mM) reached 98% and ee value of the product exceeded 99% (R) after the reaction mixture was shaken at 200 rpm for 28 h at 30 °C.
The research articles entitled “Characterization of (R)-selective amine transaminases identified by in silico motif sequence blast” and “Substrate profile of an ω-transaminase from Burkholderia vietnamiensis and its potential for the production of optically pure amines and unnatural amino acids” were published in Applied Microbiology and Biotechnology, and Journal of Molecular Catalysis B: Enzymatic. JIANG Jinju, a PhD graduate from TIB, is the first author of these papers. Two Chinese patents were also applied (201310556932.X and 201310556825.7). This research were supported by the National Basic Research Program of China (973 Program, No. 2011CB710801), National Natural Science Foundation of China (grant no. 21072151) and the CAS Special Grant for Postgraduate Research, Innovation and Practice (grant no. Y2J8041021).
