Enhanced thermal and alkaline stability of L-lysine decarboxylase CadA by combining directed evolution and computation-guided virtual screening

Abstract As an important monomer for bio-based nylons PA5X, cadaverine is mainly produced by enzymatic decarboxylation of L-lysine. A key issue with this process the instability L-lysine decarboxylase (CadA) during reaction due to dissociation CadA subunits accumulation alkaline cadaverine. In work, we attempted improve thermal and stability combining directed evolution computation-guided virtual screening. Interestingly, site 477 residue located at protein surface not decamer interface was found as a hotspot in evolution. By combinatorial mutagenesis positive mutations obtained screening previously reported T88S mutation, K477R/E445Q/T88S/F102V generated best mutant, delivering 37% improvement yield 50 ºC pH 8.0. Molecular dynamics simulations suggested improved rigidity regional structures, increased number salt bridges, enhancement hydrogen bonds multimeric possible origins mutant. Using four-point 160.7 g/L from 2.0 M Lysine hydrochloride °C without regulation, conversion 78.5%, whereas wild type 143.7 cadaverine, corresponding 70% conversion. This work shows combination efficient engineering strategy. Graphical