Properties of l-amino acid deaminase: En route to optimize bioconversion reactions

Abstract

Interest is rising in the agrochemical and pharmaceutical industries concerning the use of enantiomerically pure amino acids. l-Amino acids are easily produced by deracemization of D,L-mixtures or by stereoinversion of d-amino acids, employing the flavoenzyme d-amino acid oxidase. On the other hand, the production of the D-enantiomers is hampered by the lack of a suitable enzyme with reversed stereoselectivity. In recent years, the enzyme l-amino acid deaminase has been proposed as an alternative to l-amino acid oxidase. l-Amino acid deaminase from Proteus myxofaciens (PmaLAAD) is a membrane-bound flavoprotein that catalyzes the deamination of l-amino acids to the corresponding α-keto acids and ammonia without producing hydrogen peroxide since the electrons are transferred from the reduced cofactor to a b-type cytochrome. For this reason, purified PmaLAAD has no significant enzymatic activity; this can be recovered by adding exogenous E. coli membranes. In order to circumvent the use of membranes, we analyzed the ability of PmaLAAD to use alternative electron acceptors, as well as detergents, to reproduce the hydrophobic environment. With phenazine methosulfate (PMS) and anionic detergents, at concentrations lower than the critical micellar concentration, higher enzymatic activity can be reached than with membranes. The effect on stability, protein conformation, oligomeric state and activity of temperature, pH, ionic strength, and detergents was also investigated. By optimizing the reaction conditions (namely, using 0.8 mM PMS and 0.1 mM SDS) the rate of l-leucine bioconversion was improved.

Keywords: Bioconversion; Conformational change; Membrane-bound enzyme; Phenazine methosulfate; Sodium dodecyl sulfate; l-amino acid deaminase.