Difficulties and solutions for the assays of the key enzymes of a new enzymatic glycerol bioconversion

Abstract

For years our research group has been working on developing a new enzymatic method for the bioconversion of glycerol (a byproduct of biodiesel production) to 1,3-propanediol (PD) and 1,3-dihydroxyacetone (DHA) simultaneously. Our bioconversion method applies three key enzymes in a membrane reactor with coenzyme regeneration: glycerol-dehydratase (GDHt) that produces 3-hydroxy-propionaldehyde (HPA) from glycerol; 1,3-propanediol-oxydoreductase (PDOR) that converts HPA into PD using NADH_2, that is then reoxydized by glycerol-dehydrogenase (GDH) during glycerol-DHA conversion. From an economical point of view crude enzyme solutions of sonicated K. pneumoniae and C. butyricum cells were used. In such a crude enzyme solution it was impossible to determine the GDHt activity with the assays known from the literature, thus a new method for GDHt activity determination was developed based on the measurment of HPA (formed by GDHt) with tryptophan in acidic conditions. Furthermore Lin´s assay for PDOR was also modified, and then successfully adapted to GDH as well. In both cases, NADH₂ formation should be followed photometrically, but in crude enzyme solutions a lot of NAD⁺NADH₂ dependent side-reactions disturb the activity determinations. In our improved method, the absorbance change should be recorded without substrate addition until the absorbance becomes stable, then adding the actual substrate the changing readings should be recorded further.