Biocatalytic Production of Bioactive Dipeptides

Abstract

Dipeptides are a promising substance class with a large variety of bioactivities. N‐terminal ar‐ ginyl dipeptides (Arg‐X) have been shown to exhibit both salt‐taste enhancing (Arg‐Ser among others) and antihypertensive properties (Arg‐Phe). A novel biocatalytic strategy for the specific synthesis of these dipeptides is the application of L‐amino acid ligases like RizA from B. subtilis, which enables production of Arg‐X dipeptides directly from their amino acids. However, challenges for an industrial application are the dependence on the expensive cofactor ATP (adenosine triphosphate), low product yields, formation of the undesired side product Arg Arg and the potentially high enzyme costs.In order to reduce the need for ATP, a strategy for ATP regeneration from acetyl phosphate was established using acetate kinase from E. coli (AckA). Up to 5.9 g/L Arg‐Ser were produced, corresponding to an ATP efficiency of 23 g Arg‐Ser per gram ATP (without regeneration max. 0.5 g Arg‐Ser/g ATP). Next, variants of RizA were created to alter the substrate specificity. A total of 21 RizA variants were created by site‐directed mutagenesis of eight positions in the substrate binding pocket. Additional 14 variants were created by combination of different mutations. Variants with improved activity and specificity were identified for all five examined amino acid combinations (Arg + Arg, Asp, Ser, Ala, Phe). The largest change was observed for K83F_S156A, which improved the product formation of Arg‐Phe six‐fold and improved specificity more than tenfold. Finally, co immobilization of RizA and selected variants with AckA was performed to enable reusability of this biocatalytic system. Immobilisates of T81F_A158S produced up to 3.0 g/L Arg Ser and immobilisates of K83F_S156A produced up to 3.8 g/L Arg‐Phe and could both be reused several times.This thesis represents the first extensive scientific investigation into establishing ATP regeneration for an L‐amino acid ligase and co‐immobilization of such a biocatalytic system, and substantially expanded the knowledge about engineering the substrate specificity of L amino acid ligases.