Semi-Rational Engineering of Leucine Dehydrogenase for L-2-Aminobutyric Acid Production

L -2-aminobutyric acid ( L -ABA) as a precursor for the anticonvulsant and the antituberculotic is a key intermediate in the chemical and pharmaceutical industries. Recently, leucine dehydrogenase (LeuDH) with NAD + regeneration was developed for L -ABA production on a large scale. Previously, the L -ABA yield was improved by optimizing conversion conditions, including cofactor regeneration and enzyme immobilization but not protein engineering on LeuDH due to lacking an applicable high-throughput screening (HTS) method. Recently, an HTS assay was developed by us, which enables researchers to engineer LeuDH in a relatively short period of time. Herein, a semirational engineering was performed on LeuDH to increase the catalytic efficiency of BcLeuDH. Firstly, the structure of wild-type (WT) BcLeuDH was modeled and seven potentially beneficial positions were selected for mutation. Five beneficial variants were then identified from the seven site-saturation mutagenesis (SSM) libraries by HTS and confirmed by rescreening via amino acid analyzer. The “best” variant M5 (WT + Q358N) showed 44.5-fold higher catalytic efficiency ( k cat / K M ) than BcLeuDH WT, which suggested that BcLeuDH M5 is an attractive candidate for L -ABA production on a large scale. Furthermore, the structure-functional relationship was investigated based on the docking and kinetic results.