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The production of unnatural amino acids is important in chemical industry due to an increase in demand for optically pure non-proteinogenic amino acids as building blocks for pharmaceutical compounds. Given the significance of the unnatural amino acid, its efficient synthesis to optically pure form has become an attractive challenge to organic chemists and biologists. Among the different unnatural amino acids, l-homoalanine is of high pharmacological significance as it is used as a key chiral intermediate for the synthesis of several important drugs such as brivaracetam, levetiracetam and ethambutol. We developed a deracemization method to generate optically pure l-homoalanine from racemic homoalanine using d-amino acid oxidase and ω-transaminase. A whole cell reaction using biphasic system converted 500 mM racemic homoalanine to 485 mM l-homoalanine (> 99% ee).