Mutagenesis of the l-Amino Acid Ligase RizA Increased the Production of Bioactive Dipeptides

The l-amino acid ligase RizA from B. subtilis selectively synthesizes dipeptides containing an N-terminal arginine. Many arginyl have salt-taste enhancing properties while Arg-Phe has been found to antihypertensive effect. A total of 21 variants were created by site-directed mutagenesis eight amino acids in the substrate binding pocket. recombinantly produced E. coli and purified affinity chromatography. Biocatalytic reactions set up with arginine four differing size polarity (aspartic acid, serine, alanine, phenylalanine) analyzed RP-HPLC fluorescence detection. Variant T81F significantly improved yield comparison wild type for aspartic (7 17%), serine (33 47%) alanine (12 17%). S84F increased product similarly 17%) 42%). D376E 19%) phenylalanine (11 26%). largest change was observed S156A, which showed a 40% corresponding 270% increase concentration. This study expands knowledge about positions governing specificity may help inform future protein engineering endeavors.