Expression of L-amino acid oxidases for biocatalytic applications

L-Amino acid oxidases (LAAOs) are flavoproteins, which use oxygen to deaminate L-amino acids and produce the corresponding a-keto acids, ammonia and hydrogen peroxide. LAAOs with broad substrate spectra are of interest for biotechnological applications, but could not be expressed in sufficient amounts so far. We succeded in expressing an LAAO from the fungus Rhizoctonia solani (details). The recombinant enzyme could be activated by the detergent SDS and undergoes a conformational change (details). The L-AAO4 from the fungus Hebeloma cylindrosporum was also expressed and characterized. It is activated by exposure to acidic pH, SDS and freezing. hcLAAO4 has a broader substrate spectrum and higher specific activity than rsLAAO1. An aldehyde tag was added to site-specifically immobilize the enzyme (details). The crystal structure of hcLAAO4 was solved and used as starting point for enzyme engineering (details).  hcLAAO4 was used for co-substrate recycling in (S)-selective transaminase-catalyzed kinetic resolutions together with catalase to remove the reactive side product hydrogen peroxide (details). This enzyme cascade was immobilized to improve reuse of the enzymes (details) .