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Background

D-phenylglycine aminotransferase (D-PhgAT) of Pseudomonas stutzeri ST-201 catalyzes the reversible stereo-inverting transamination potentially useful in the application for synthesis of D-phenylglycine and D-4-hydroxyphenylglycine using L-glutamate as a low cost amino donor substrate in one single step. The enzyme is a relatively hydrophobic homodimeric intracellular protein difficult to express in the soluble functionally active form. Over-expression of the dpgA gene in E. coli resulted in the majority of the D-PhgAT aggregated into insoluble inclusion bodies that failed to be re-natured. Expression in Pichia pastoris was explored as an alternative route for high level production of the D-PhgAT.

Background

The D-phenylglycine aminotransferase (D-PhgAT) from a soil bacterium Pseudomonas stutzeri ST-201 [], catalyzes the reversible transamination specific for D-phenylglycine or D-4-hydroxyphenylglycine in which 2-oxoglutarate is an amino-group acceptor that are converted into benzoylformate or 4-hydroxybenzoylformate and L-glutamate (Figure ). Both D-phenylglycine and D-4-hydroxyphenylglycine are important side chain moieties in high demand for production of semisynthetic penicillin and cephalosporin antibiotics such as ampicillin, amoxicillin, cephalexine and cephadroxyl. Currently, D-phenylglycine and D-4-hydroxyphenylglycine are industrially produced by a process using two enzymes, the hydantoinase and carbamoylase []. However, the reaction rates are low since the solubilities of the substrates, (D,L) phenylhydantoin and (D,L)-4-hydroxyphenylhydantoin, are very poor. Alternatively, a single-step enzymatic synthesis using a D-amino acid aminotransferase is possible. But known D-amino acid aminotransferases have very low transamination activity towards D-phenylglycine [] and they accept only D-amino acids as the amino-group donors. Due to the high cost of D-amino acids (such as D-alanine, D-glutamic acid), their direct application as amino-group donors is impractical. Using an L-amino acid (such as L-alanine or L-glutamic acid) together with an amino acid racemase to convert it to the corresponding D-amino acid to serve as the amino-group donor for the D-amino acid aminotransferase will increase the production cost. Additionally, the racemase may interfere with the transamination reaction. By contrast, with the D-PhgAT, due to its characteristic “stereo-inverting” transamination activity, a low cost substrate L-glutamic acid can be used as an amino-group donor for the synthesis of enantiomerically pure D-phenylglycine or D-4-hydroxyphenylglycine in a single transamination step without the need for any amino acid racemase [].