Kinetic stability of the water-forming NADH oxidase from Giardia lamblia: implications for biotechnological processes

Abstract

NAD(P)H oxidases (NOXs) catalyze the oxidation of NAD(P)H by reducing molecular O2 to H2O2 or H2O. Water-forming NOXs have been highlighted as promising biocatalyst systems for regeneration of NAD+ or NADP+ in industrial applications. The NADH oxidase of Giardia lamblia (GlNOX) uses naturally both nicotinamide cofactors producing only water as byproduct; therefore, it can be considered a multipurpose and innocuous regeneration system for redox reactions using either NAD+ or NADP+. From a biotechnological perspective, kinetic stability (and not thermodynamic stability) must be the most important aspect to consider for enzymes involved in industrial applications; therefore, understanding the kinetic stability of a technologically interesting enzyme is an indispensable step toward its application. In this work, we analyze the thermal inactivation kinetics of GlNOX and the influence of diverse additives on it. A combination of 1,4-Dithiothreitol as reducing agent and the disaccharide trehalose protects remarkably GlNOX from inactivation. Structural studies indicate that inactivation is not related to denaturation, whereas overoxidation of the active site cysteine seems to be the main factor related to loss of the enzyme activity. It is concluded that understanding the factors that influence the protein inactivation of GlNOX allowed to improve its kinetic stability, making it biotechnologically valuable.

Keywords

• thermal inactivation
• redox reactions
• cofactor regeneration
• water-forming nox