Enhanced catalytic activity of recombinant transaminase by molecular modification to improve L-phosphinothricin production

Abstract

Transaminases catalyze the transfer of an amino group from a donor to a keto group of an acceptor substrate and are applicable to the asymmetric synthesis of herbicide L-phosphinothricin (L-PPT). Here, the important residue sites (C390, I22, V52, R141, Y138 and D239) of transaminase from Salmonella enterica (SeTA) were modified at the adjacency of the substrate-binding pocket to improve the enzyme activity. Among the constructed mutant library, the SeTA-Y138F mutant displayed higher activity than the wild-type enzyme. Compared to the wild-type, SeTA-Y138F showed improved catalytic efficiency with a 4.36-fold increase. The Km and kcat of SeTA -Y138F toward 4-(hydroxy(methyl) phosphoryl)-2-oxobutanoic acid (PPO) were 26.39 mM and 34.28 s−1, respectively. Subsequently, the three-enzyme co-expression system of E. coli BL21 (DE3)/pACYCDuet-SeTA-Y138F/pETDuet-AlaDH-BsGDH was developed by combining a alanine dehydrogenase (AlaDH) to recycle the byproduct of amino donor, a glucose dehydrogenase (BsGDH) for cofactor recycling. Under the optimized conditions, an excellent L-PPT yield of 90.8% was achieved by the whole-cell biotransformation with 500 mM PPO. It exhibited the tri-enzymatic coupling system was potential for effective production of target L-PPT.